Functional Food Ingredients and Nutraceuticals - Processing Technologies Volume in Functional Foods and Nutraceuticals (2nd Ed.) - CRC-Taylor & Francis (PDFDrive)

SECOND EDITION
Functional Food
Ingredients
and Nutraceuticals
Processing Technologies
© 2016 by Taylor & Francis Group, LLC

Series Editor
John Shi, Ph.D.
Guelph Food Research Center, Canada
Functional Food Ingredients and Nutraceuticals: (2015)

Processing Technologies, Second Edition
Edited by John Shi, Ph.D.
Marine Products for Healthcare: Functional and Bioactive (2009)

Nutraceutical Compounds from the Ocean
Vazhiyil Venugopal, Ph.D.
Methods of Analysis for Functional Foods and Nutraceuticals, (2008)

Second Edition
Edited by W. Jeffrey Hurst, Ph.D.
Handbook of Fermented Functional Foods, Second Edition (2008)

Edited by Edward R. Farnworth, Ph.D.
Functional Food Carbohydrates (2007)

Costas G. Biliaderis, Ph.D. and Marta S. Izydorczyk, Ph.D.
Dictionary of Nutraceuticals and Functional Foods (2006)

N. A. Michael Eskin, Ph.D. and Snait Tamir, Ph.D.
Handbook of Functional Lipids (2006)

Edited by Casimir C. Akoh, Ph.D.
Handbook of Functional Dairy Products (2004)

Edited by Collete Short and John O’Brien
Herbs, Botanicals, and Teas (2002)

Edited by G. Mazza, Ph.D. and B.D. Oomah, Ph.D.
Functional Foods: Biochemical and Processing Aspects, Volume 2 (2002)

Edited by John Shi, Ph.D., G. Mazza, Ph.D., and Marc Le Maguer, Ph.D.
Functional Foods: Biochemical and Processing Aspects, Volume 1 (1998)

Edited by G. Mazza, Ph.D.

SECOND EDITION
Functional Food
Ingredients
and Nutraceuticals
Processing Technologies
EDITED BY
John Shi
Boca Raton London New York
CRC Press is an imprint of the

Taylor & Francis Group, an informa business

CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works

Version Date: 20150827
International Standard Book Number-13: 978-1-4822-4065-8 (eBook - PDF)
This book contains information obtained from authentic and highly regarded sources. Reasonable
efforts have been made to publish reliable data and information, but the author and publisher cannot
assume responsibility for the validity of all materials or the consequences of their use. The authors and
publishers have attempted to trace the copyright holders of all material reproduced in this publication
and apologize to copyright holders if permission to publish in this form has not been obtained. If any
copyright material has not been acknowledged please write and let us know so we may rectify in any
future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced,
transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or
hereafter invented, including photocopying, microfilming, and recording, or in any information stor-
age or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copy-
right.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222
Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that pro-
vides licenses and registration for a variety of users. For organizations that have been granted a photo-
copy license by the CCC, a separate system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are
used only for identification and explanation without intent to infringe.
Visit the Taylor & Francis Web site at
http://www.taylorandfrancis.com
and the CRC Press Web site at
http://www.crcpress.com

Contents
Foreword..................................................................................................................ix
Preface.......................................................................................................................xi
Acknowledgments............................................................................................... xiii
Editor.......................................................................................................................xv
Contributors......................................................................................................... xvii
Section I “Green” Separation/Extraction/

Concentration Process and Technology
1. Extraction of Health-Promoting Components by
Supercritical-CO2 Fluid Process...................................................................3
John Shi, Sophia Jun Xue, Lamin S. Kassama, and Xingqian Ye
2. Solubility of Food Components in the Supercritical-CO2

Fluid Process................................................................................................... 53
John Shi, Sophia Jun Xue, and Siew Young Quek
3. Mathematical Modeling of Supercritical Fluid Extraction................. 105

Helena Sovová
4. Biochemical Reactions in Supercritical Fluids...................................... 127

Željko Knez, Maja Leitgeb, and Mateja Primožič
5. Mass Transfer Coefficient of Plant Oil in Supercritical-CO2

Fluid Extraction............................................................................................ 159
John Shi and Sophia Jun Xue
6. Pressurized Low-Polarity Water Extraction of Biologically

Active Compounds from Plant Products................................................ 177
Juan Eduardo Cacace and Giuseppe (Joe) Mazza
7. Purification of Orange Peel Oil and Oil Phase as Functional

Foods by Vacuum Distillation.................................................................. 199
Mércia de Fátima M. Bettini
8. Membrane Technology for Production of Nutraceuticals................... 217

Marie-Pierre Belleville and Fabrice Vaillant
v
vi Contents
9. Extraction of Functional Food Ingredients and Nutraceuticals

from Dairy..................................................................................................... 235
Geneviève Gésan-Guiziou
Section II Nano-Microencapsulation, Delivery

System, and Packaging Technologies
10. Microencapsulation of Food Ingredients for Functional Foods........ 267
Siew Young Quek, Qiong Chen, and John Shi
11. Nano-Microencapsulation Technology and Applications in

Fortified and Functional Foods................................................................. 319
Yao Olive Li
12. Microencapsulation and Delivery of Omega-3 Fatty Acids............... 373

Luz Sanguansri and Mary Ann Augustin
13. Packaging Functional Foods: From Basic Requirements to Nano

Perspectives.................................................................................................. 409
Louise Deschênes
14. High-Pressure Processing of Foods toward Their

Industrialization and Commercialization: An Up-to-Date
Overview....................................................................................................... 427
Giovanna Ferrentino, Sara Spilimbergo, and Alberto Bertucco
15. Spray-Drying of Nano- and Microcapsules of Nutraceuticals........... 455

Xiang Li, Nicolas Anton, and Thierry F. Vandamme
Section III Bioprocessing Technology
16. Bioprocessing Technology for Production of Nutraceutical

Compounds................................................................................................... 481
Terry H. Walker, Caye M. Drapcho, and Feng Chen
17. Microbial Modeling as Basis for Bioreactor Design for

Nutraceutical Production........................................................................... 509
Caye M. Drapcho and Darryl B. Jones

Contents vii
Section IV Stability and Bioactivity of Antioxidative

Components during Food Processing
18. Dehydration Technologies for Functional Foods
and Nutraceuticals.......................................................................................545
Robert V. Parsons and Stefan Cenkowski
19. Biological Antioxidation Mechanisms: Quenching

of Peroxynitrite............................................................................................ 589
Takashi Maoka and Hideo Etoh
20. Bioactive Stability and Antioxidative Property of Lycopene

from Tomatoes during Processing........................................................... 609
John Shi, Sophia Jun Xue, Lishui Chen, Wenliang Wang, Hetong Lin,
Ying Ma, and Gauri S. Mittal
Index...................................................................................................................... 639

Foreword
Food and nutrition science has advanced significantly over the years, pro-
gressing from the introduction of fortified foods to the construction of foods
that promote health. Consumer demand for foods with benefits beyond basic
nutrition has created commercialization opportunities for food manufactur-
ers; functional foods containing bioactive ingredients and nutraceuticals are
becoming more prominent in the marketplace.
The creation and application of functional food ingredients and nutra-
ceuticals require knowledge and understanding of complex physiochemical
processes. Food scientists, nutritionists, functional food designers, and man-
ufacturers are all confronted with issues related to consumer expectations
and confidence. These include challenges regarding the stability and safety
of functional foods and dietary supplements that are associated with claims
to health-promoting benefits. This edition of Functional Food Ingredients and
Nutraceuticals: Processing Technologies helps address some of these challenges
in a structured way.
The editor, Dr. John Shi (Agriculture and Agri-Food Canada, Guelph
Food Research Centre, Ontario), has once again assembled leading experts
from 12 countries to update this valuable resource. Thousands of research
papers have been published on the health benefits of bioactive components
from natural resources; many books on functional foods are related to
chemical properties or medical functions. With only a few books capturing
the related processing technologies, the first edition of this book has been
in high demand by those in the food industry, research, and education
fields. This resource has become a valuable tool to help transform results
from the lab to industrial applications.
The second edition incorporates many new and emerging technologies
that were not present when the first edition was published in 2004. This
includes an emphasis on nanotechnology in packaging processes and nano-
microencapsulation technology to protect and stabilize the bioactivity of
health-promoting components. The section on green separation technolo-
gies contains revised information on industrial applications as well as some
new processes and stabilization technologies that have been developed to
address consumer concerns regarding quality and safety.
Congratulations to Dr. Shi and his colleagues for pursuing the second edi-
tion. It will serve as a unique reference for food science professionals and
food companies involved in research and development of functional foods
ix
x Foreword
and food ingredients, as well as college and university students majoring in

food science and technology and nutrition science.
Siddika Mithani, PhD

Assistant Deputy Minister, Science and Technology Branch
Agriculture and Agri-Food Canada
Gilles Saindon, PhD

Associate Assistant Deputy Minister, Science and Technology
Branch, Agriculture and Agri-Food Canada

Preface
Since the publication of the book Functional Food Ingredients and Nutraceuticals:
Processing Technologies in 2004, many new and innovative technologies for
the processing of functional foods and nutraceuticals have emerged that
show potential for academic use and broad industrial applications. Hence,
the editor felt obligated to update the original version commensurate with
the new developments in the area of functional foods and nutraceuticals.
Furthermore, a number of “green” separation and stabilization technologies
have also been developed to address consumers’ concerns about quality and
safety issues. For example, nano-microencapsulation field has witnessed
substantial technological advancements in enhancing the solubility, bioac-
tivity and bioavailability, and the preservation of health-promoting bioactive
components in functional food products.
The second edition of Functional Food Ingredients and Nutraceuticals: Process
ing Technologies has been extensively revised and expanded considerably to
reflect recent developments in the different areas of processing technologies.
These include theoretical approaches in mass transfer modeling, solubility
properties, and simulation in the extraction process, as well as the practi-
cal review of new application technologies. The incorporation of these new
emerging technologies is aimed to strengthen the second edition without
compromising the contextual integrity of the original publication. In this new
edition, the innovative nanotechnologies in packaging process and nano-
microencapsulation technology to protect bioactivity and enhance solubil-
ity and bioavailability of health-promoting bioactive components have been
emphasized. The green separation technologies have been updated with
more information on industrial applications.
This book can serve as a reference for food science professionals in either
government or industry pursuing functional food, and in food ingredient
development and for R&D staff in food companies. The book is also appropri-
ate for academic use, as it makes a good scientific reference source for food sci-
ence and technology and nutrition science and pharmaceutical science faculty
and students. Readers will obtain current and sound scientific knowledge of
engineering techniques, and information on the quality of functional foods.
It is our hope that the scientific community will appreciate our efforts in
preparing this book and the impact it will have on advancing the frontiers of
functional foods and nutraceuticals.
John Shi, PhD

Senior Research Scientist
Guelph Food Research Center
Agriculture and Agri-Food Canada
xi
Acknowledgments
This book is a product of an extensive multidisciplinary collaborative

effort among scientists and engineers, academicians, and government and
industry personnel. Therefore, the editor wishes to express his sincere
appreciation to Assistant Deputy Minister Dr. Siddika Mithani, Associate
Assistant Deputy Minister Dr. Gilles Saindon, Director General Dr. Denis
Petitclerc, and Science Directors Dr. Michele Marcotte and Dr. Gabriel Piette
at the Science & Technology Branch, Agriculture and Agri-Food Canada for
their assistance, support, and direction in my career accomplishments at
Agriculture and Agri-Food Canada.
The editor would also like to express his profound appreciation to
Professor Yukio Kakud and Professor Gauri S. Mittal (University of Guelph,
Canada), Professor James H. Moy (University of Hawaii at Manoa, USA),
Professor Sam K.C. Chang (Mississippi State University, USA), Dr. Asbjørn
Gildberg (Norwegian Institute of Fisheries and Aquaculture Research,
Norway), Dr. Giuseppe Joe Mazza (former CRC Press series editor and
senior scientist at Agriculture and Agri-Food Canada, current CEO of
MAZZA Innovation Ltd., Canada), Professor Pedro Fito and Professor
Amparo Chiralt (Polytechnic University of Valencia, Spain), Professor
Albert Ibarz (University of Lleida, Spain), and Stephen Zollo (CRC Press/
Taylor & Francis) for their valuable scientific comments and support and
encouragement during the editing process.
xiii
Editor
Dr. John Shi is a senior research scientist with the Federal Department
of Agriculture and Agri-Food Canada and an adjunct professor at the
University of Guelph, Canada. He is coeditor of four books: Functional Foods
II—Biochemical and Processing Aspects; Asian Functional Foods; Functional Food
Ingredients and Nutraceuticals: Processing Technology; and Functional Foods of
the East, published by CRC Press, USA. Recently, he was invited to serve
as the book series editor for the Functional Foods and Nutraceuticals book
series for CRC Press, as a guest editor for the special issue of Food Innovation
in China, and as an editorial member of LWT-Food Science and Technology.
Dr. Shi graduated from Zhejiang University, China, with a master’s in
1985, and Polytechnic University of Valencia, Spain, with a PhD in 1994. As
a postdoctoral research fellow, he conducted a USDA research project at
North Dakota State University, USA. As a visiting professor he conducted
international collaborative research at the Norwegian Institute of Fishery
and Aquaculture, Norway, and Lleida University, Spain. He has been an
invited keynote speaker at numerous international conferences in the United
States, Canada, France, Portugal, Japan, China, Korea, Italy, Thailand, Spain,
Argentina, Columbia, Brazil, etc. He has published more than 180 peer-
reviewed research papers in international scientific journals and 47 book
chapters. His current research interests are focused on innovative “green”
processes and technology for health-promoting food ingredients, includ-
ing innovative green separation technology for recovery of functional food
ingredients and nano-microencapsulation and micronization technologies to
protect bioactivity and enhance the solubility and bioavailability of health-
promoting bioactive components in functional foods for better health benefits.
xv
Contributors
Nicolas Anton Lishui Chen

Faculté de Pharmacie Food R&D Centre
Laboratoire de Conception et COFCO Institute of Nutrition
d’Application de Molécules and Health
Bioactives Beijing, China
Université de Strasbourg
Strasbourg, France Qiong Chen
Food Science and Nutrition
Mary Ann Augustin School of Chemical Sciences
CSIRO Food and Nutrition Flagship The University of Auckland
Werribee, Victoria, Australia Auckland, New Zealand
Marie-Pierre Belleville Louise Deschênes
Institut Européen des Membranes Food Research and Development
Université Montpellier 2 Centre
Montpellier, France Agriculture and Agri-Food Canada
Alberto Bertucco St. Hyacinthe, Quebec, Canada
Department of Industrial Engineering
University of Padova Caye M. Drapcho
Padova, Italy Department of Environmental
Engineering and Earth
Mércia de Fátima M. Bettini Sciences
Flavor Tec – Aromas de Frutas Ltd. Clemson University
Pindorama, Brazil Clemson, South Carolina
Juan Eduardo Cacace Hideo Etoh
Pacific Agri-Food Research Center Faculty of Agriculture
Agriculture and Agri-Food Canada Shizuoka University
Summerland, British Columbia, Shizuoka, Japan
Canada
Stefan Cenkowski Giovanna Ferrentino
Department of Biosystems Department of Industrial
Engineering Engineering
University of Manitoba University of Trento
Winnipeg, Manitoba, Canada Trento, Italy
Feng Chen Geneviève Gésan-Guiziou

Department of Food Science INRA-Agrocampus Ouest
and Human Nutrition Science et Technologie du Lait et de
Clemson University l’Oeuf
Clemson, South Carolina Rennes, France
xvii
xviii Contributors
Darryl B. Jones Ying Ma

Department of Environmental School of Food Science and
Engineering and Earth Sciences Engineering
Clemson University Harbin Institute of Technology
Clemson, South Carolina Heilongjian, China
Lamin S. Kassama
Department of Food and Animal Takashi Maoka
Sciences Research Institute for Production
Alabama A&M University Development
Normal, Alabama Kyoto, Japan
Željko Knez Giuseppe (Joe) Mazza

Faculty of Chemistry and Chemical MAZZA Innovation Ltd.
Engineering Summerland, British Columbia,
University of Maribor Canada
Maribor, Slovenia
Maja Leitgeb Gauri S. Mittal

Faculty of Chemistry and Chemical School of Engineering
Engineering University of Guelph
University of Maribor Guelph, Ontario, Canada
Maribor, Slovenia
Robert V. Parsons
Xiang Li Department of Biosystems
Faculté de Pharmacie Engineering
Laboratoire de Conception et University of Manitoba
d’Application de Molécules Winnipeg, Manitoba, Canada
Bioactives
Université de Strasbourg Mateja Primožič
Strasbourg, France Faculty of Chemistry and Chemical
Engineering
Yao Olive Li
University of Maribor
Food Science/Engineering Human
Maribor, Slovenia
Nutrition and Food Science
Department
California State Polytechnic Siew Young Quek
University Food Science and Nutrition
Pomona, California School of Chemical Sciences
The University of Auckland
Hetong Lin Auckland, New Zealand
College of Food Science
Fujian Agriculture and Forestry Luz Sanguansri
University CSIRO Food and Nutrition Flagship
Fujian, China Werribee, Victoria, Australia

Contributors xix
John Shi Terry H. Walker

Guelph Food Research Center Department of Biosystems
Agriculture and Agri-Food Canada Engineering
Guelph, Ontario, Canada Clemson University
Clemson, South Carolina
Helena Sovová
Institute of Chemical Process Wenliang Wang
Fundamentals Institute of Agro-Food Science
Academy of Sciences of the Czech and Technology
Republic Shandong Institute of Agricultural
Prague, Czech Republic Sciences
Jinan, China
Sara Spilimbergo Sophia Jun Xue
Department of Industrial Guelph Food Research Center
Engineering Agriculture and Agri-Food Canada
University of Trento Guelph, Ontario, Canada
Trento, Italy
Xingqian Ye
Fabrice Vaillant College of Biosystems and Food
Research Center of Food Technology Science
University of Costa Rica Zhejiang University
San José, Costa Rica Zhejiang, China
Thierry F. Vandamme
Faculté de Pharmacie
Laboratoire de Conception et
d’Application de Molécules
Bioactives
Université de Strasbourg
Strasbourg, France

Section I
“Green” Separation/
Extraction/Concentration
Process and Technology

1
Extraction of Health-Promoting Components
by Supercritical-CO2 Fluid Process
John Shi, Sophia Jun Xue, Lamin S. Kassama, and Xingqian Ye
CONTENTS
1.1 Introduction.....................................................................................................4
1.2 Principle of Supercritical-CO2 Fluid Extraction Technology....................5
1.3 Process Concept Schemes and Systems.......................................................8
1.3.1 Process Principle.................................................................................9
1.3.2 Process System.................................................................................. 10
1.3.3 Single-Stage Extraction Process...................................................... 13
1.3.4 Multistage Extraction Process......................................................... 13
1.4 Physicochemical Properties of Supercritical-CO2 Fluids........................ 14
1.4.1 Phase Diagram.................................................................................. 14
1.4.2 Physical Properties........................................................................... 15
1.5 Factors Affecting Extraction Yield............................................................. 15
1.5.1 Pressure.............................................................................................. 16
1.5.2 Temperature....................................................................................... 17
1.5.3 Moisture Content of Raw Materials............................................... 18
1.5.4 Cosolvent............................................................................................ 19
1.5.5 Particle Size........................................................................................ 21
1.5.6 Flow Rate............................................................................................ 23
1.5.7 Effect of Time on Yield..................................................................... 24
1.6 Applications in the Food Industry............................................................. 24
1.6.1 Extraction of Bioactive Compounds............................................... 29
1.6.2 Fractionation of Flavors and Fragrances.......................................30
1.6.3 Cholesterol-Free Food Products...................................................... 32
1.6.4 Separation of Spices and Essential Oils......................................... 33
1.6.5 Decaffeination of Coffee and Tea................................................... 36
1.6.6 Fish Oil Concentration..................................................................... 39
1.7 Summary........................................................................................................ 40
References................................................................................................................43
3
4 Functional Food Ingredients and Nutraceuticals
1.1 Introduction
Extraction of health-promoting components from raw materials has usually
been accomplished by conventional extraction processes such as solid–liquid
extractions, employing organic solvents such as methanol, ethanol, acetone,
or hexane, and also through steam distillation. An additional process to
evaporate these solvents from extracts is required when organic solvents are
used, and the disposal of the effluent raises environmental and safety con-
cerns. There is increasing public awareness of the hazards associated with
the use of organic solvents in food processing with regard to the possible
contamination of the final products.
The possibility of toxic solvent residues remaining in the final product has
been a growing concern to consumers, thus warranting stringent regulations.
The demand for ultrapure and high value-added products is redirecting the
focus of the food and pharmaceutical industries into seeking the develop-
ment of new and clean separation technology to obtain natural compounds
with excellent quality. One of the most important aspects in developing new
extraction processes is safety. Thus, there has been increasing interest in the
use of environmentally friendly “green” separation technologies that are
able to provide high-quality and high bioactive extracts while precluding the
toxicity associated with solvents. The reasons to employ “green” separation
technologies as a viable separation technique are: (a) tightening government
regulations on toxic-chemical solvent residues in food and environmental pol-
lution control, (b) consumers’ concern over the use of toxic-chemical solvents
in the processing of food commodities, and (c) increased demand for higher-
quality products which traditional processing techniques cannot meet.
The supercritical fluid extraction technology provides an excellent alterna-
tive to the conventional organic solvent extraction methods. It is considered
clean and safe, thus generally recognized as “green” separation technol-
ogy (Herrero et al., 2006; Wu et al., 2007; Chang et al., 2008). Similar to other
innovative emerging separation technologies that meet the food quality and
safety requirements, it will also ameliorate some of the problems associated
with conventional organic solvent-oriented separation processes. Recent
changes in the food-processing practices and the new opportunities that
exist for innovative food product development have prompted much interest
in the supercritical-CO2 fluid extraction technology.
Although the technology is known for many years, its application in the
food and pharmaceutical industry began only three decades ago (Sihvonen
et al., 1999). Since then, more than 100 plants of different capacities have been
built globally for extraction of desired solutes from solid materials (Brunner,
2005; Oliveira et al., 2013; Zulkafli et al., 2014). Supercritical-CO2 fluid technol-
ogy has been widely used to extract essential oils, functional fatty acids, and
bioactive compounds, and also recently been applied in the extraction and
fractionation of carbohydrates (Glisic et al., 2007; Shi et al., 2007a; Montañés

Extraction of Health-Promoting Components 5
et al., 2008, 2009; Mitra et al., 2009; Sahena et al., 2009; Sanchez-Vicente et al.,
2009; Shi et al., 2010a,b). Therefore, supercritical-CO2 fluid extraction is an
excellent “green” separation process for health-promoting components
because of its nontoxic and environmentally friendly attributes and it does
not leave any traces of toxic residues in foods.
1.2 Principle of Supercritical-CO2 Fluid Extraction Technology

Supercritical-CO2 fluid extraction is a clean technology and a novel sepa-
ration process that utilizes the solvent properties of fluids near their ther-
modynamic critical points. Although many different types of supercritical
fluids exist for many industrial applications, CO2 as supercritical solvent is
the most desirable fluid for the extraction of bioactive components because
of its generally recognized as safe (GRAS) status. The advantages of CO2
as solvent are its easy accessibility and good technological process stabil-
ity, and it prevents degradation of thermosensitive compounds. It can easily
be separated from the extract by altering the system pressure under stan-
dard atmosphere or controlled pressure and temperature conditions (Abbas
et al., 2008). Table 1.1 shows the physical properties of compressed (20 MPa)
supercritical CO2 at 55°C when compared with some condensed liquids that
are commonly used as extraction solvents at 25°C. It should be noticed that
supercritical-CO2 exhibits density similar to that of the liquid solvents, but
it is less viscous and hence highly diffusive. This fluid-like attribute of CO2
coupled with its ideal transport properties and other quality attributes out-
lined above make it a better choice over other solvents.
The specific heat capacity (Cp) of CO2 increases rapidly as the critical
point (31.1°C temperature, 7.37 MPa pressure, and at 467.7 g/L flow rate) is
approached. Like enthalpy and entropy, the heat capacity also is a function
TABLE 1.1
Comparison of Physical Properties of Supercritical CO2 at 20 MPa and 55°C with
Some Selected Liquid Solvents at 25°C
Properties CO2 n-Hexane Methylene Chloride Methanol
Density (g/mL) 0.75 0.66 1.33 0.79
Kinematic viscosity (m2/s) 1.0 4.45 3.09 6.91
Diffusivity (m2/s) 6.0 × 109 4.0 × 109 2.9 × 109 1.8 × 109
Cohesive energy density, 10.8 7.24 9.93 14.28
δ (cal/cm3)
Source: Modified from King, J.W., Hill Jr., H.H., Lee, M.L. 1993. In Supplement and Cumulative
Index Anonymous. New York, John Wiley & Sons, pp. 1–83. Copyright Wiley-VCH
Verlag GmbH & Co. KGaA.

of temperature, pressure, and density (Mukhopadhyay, 2000). Under constant

temperature, both the enthalpy and entropy of supercritical-CO2 decreases
with increased pressure and increases with increased temperature at con-
stant pressure. The change in specific heat as a result of varying the pressure
and temperature is also dependent on density. For example, under constant
temperature, specific heat increases with increasing density up to a certain
critical level. Above this critical level, any further increase in density reduces
the specific heat. When CO2 fluid is pressurized above its critical pressure
and temperature (Figure 1.1), it exhibits supercritical solvent behavior. Under
these conditions, various characteristic features of the fluid are neither gas
nor liquid but in between the two. Although the density of a supercritical-
CO2 fluid is similar to that of a liquid, its viscosity is similar to a gas, and
hence the diffusivity is intermediate between the two states. Thus, the super-
critical state of a fluid has been defined as a state in which the liquid and
gas are indistinguishable from each other, or as a state in which the fluid
is compressible (i.e., similar behavior to a gas), even though possessing a
density similar to that of a liquid and a similar solvating power. Owing to
its different physicochemical properties, supercritical-CO2 provides several
operational advantages over traditional extraction methods. Due to their low
viscosity and relatively high diffusivity, supercritical-CO2 fluids have bet-
ter transport properties than liquids. They can diffuse easily through solid
materials and therefore give faster extraction yields. One of the main charac-
teristics of a supercritical fluid is the possibility of modifying the density of
the fluid by changing its pressure and/or temperature. As density is directly
related to solubility, the solvent strength of the fluid can be simply modified
by altering the extraction pressure (Raventós et al., 2002; Shi and Zhou, 2007;
Shi et al., 2009a).
FIGURE 1.1
Supercritical carbon dioxide pressure–temperature phase diagram.

The significant characteristic traits of CO2 are its inertness, nonflammabil-

ity, noncorrosiveness, nontoxicity, inexpensiveness, easy availability, odor-
lessness, tastelessness, and environmentally friendly and GRAS status. Its
near-ambient critical temperature makes it ideal for thermolabile natural
products (Mendiola et al., 2007; Oliveira et al., 2013). Carbon dioxide has low
selectivity for some polar components, and therefore changing selectivity by
the addition of a relatively low amount of modifier (cosolvent) such as etha-
nol and other polar solvents (water) improves the extraction efficacy. It may
be considered the most desirable supercritical fluid for extracting natural
products for food and medicinal uses (Shi et al., 2007b, 2009b; Kassama et al.,
2008; Yi et al., 2009; Vargas et al., 2013). Other supercritical fluids, such as
ethane, propane, butane, pentane, ethylene, ammonia, sulfur dioxide, water,
chlorodifluoromethane, etc., are also used as fluid for extraction processes.
The supercritical-CO2 fluid has a solvating power similar to organic liquid
solvents and a much higher diffusivity because of its low surface tension
and viscosity. The physicochemical properties of supercritical fluids, such as
the density, diffusivity, viscosity, and dielectric constant, can be controlled
by varying the operating conditions such as the pressure and temperature
or both in combination (Tena et al., 1997; Shi et al., 2007a,b,c, 2009b; Kassama
et al., 2008). The separation process can be affected by simply changing the
operating pressure and temperature to alter the solvating power of the sol-
vent. After modifying CO2 with a cosolvent, the extraction process can sig-
nificantly augment the selective and separation power and in some cases
extend its solvating powers to polar components (Shi et al., 2009a).
In short, the supercritical-CO2 fluid has many advantages over other sol-
vents: (a) it has a solvating power similar to organic liquid solvents and higher
diffusivity, lower surface tension, and viscosity; (b) separation can be affected
by simply changing the operating pressure and/or temperature to alter the
solvating power of the solvent; and (c) modifying CO2 with a cosolvent can
significantly augment the selective and separation power, and in some cases
extend its solvating powers to polar components. Supercritical-CO2 is being
given a great deal of attention as an alternative to industrial solvents because
of (a) increased governmental scrutiny and new regulations restricting the
use of common industrial solvents such as chlorinated hydrocarbons; (b) its
nontoxic and environment-friendly attributes, given that it leaves no traces
of toxic solvent residue in final products; (c) sharp increase in energy cost,
which increased the cost of traditional energy-intensive separation tech-
nique, such as distillation; (d) carbon dioxide is cheap, safe to use, recyclable,
and with minimum disposal cost required; (e) stringent pollution-control
legislation prompting industries to seek alternative means of waste treat-
ment and utilization; and (f) increased performance demands on materials,
which traditional processing techniques cannot meet.
Supercritical-CO2 fluid extraction is particularly relevant to food and
pharmaceutical applications because of the processing and handling of
complex, thermo-sensitive bioactive components, increased application

in the areas of nutraceuticals, flavors, and other high-value items such as

fats and oils (Sahena et al., 2009; Oliveira et al., 2013; Carla et al., 2014; Zhao
and Zhang, 2014), purification of a solid matrix, separation of tocopherols
and antioxidants, removal of pesticide residues from herbs, medicines, and
food products, the detoxification of shellfish, the concentration of fermented
broth, fruit juices, essential oils, spices, coffee, and the separation of caf-
feine, etc. (Perrut, 2000; González et al., 2002; Quancheng et al., 2004; Liu
et al., 2008, 2009; Martinez et al., 2008; Miyawaki et al., 2008; Bashipour and
Ghoreishi, 2014; Solana et al., 2014).
The supercritical extraction fluid systems are designed such that an inti-
mate contact exists between packed beds formed by a ground solid substra-
tum (fixed-bed of extractable material) with supercritical fluid (Ferreira and
Meireles, 2002). During the supercritical extraction process, the solid phase
comprises of the solute and the insoluble residuum (matrix) and is brought
into contact with the fluid phase which is the solution of the solute in the
supercritical fluid (solvent). The extracted material is then conveyed to a
separation unit. This technology has been successfully applied in the extrac-
tion of bioactive components (antioxidants, flavonoids, lycopene, essential
oils, lectins, carotenoids, etc.) from a variety of biological materials such as
hops, spices, tomato skins, and other raw or waste agricultural materials
(Kassama et al., 2008; Shi et al., 2009a, 2010a,b; Yi et al., 2009; Huang et al.,
2010; Xiao et al., 2010). The scale up of some supercritical extraction processes
has already been proved in industrial use.
It must, however, be stated that commercial applications of the
supercritical-CO2 fluid extraction technology remained limited to a few high-
value products due to high initial capital investment, its novelty, and com-
plex operating system (Perrut, 2000; Rosa and Meireles, 2005). Adoption of
the technology is on the rise as a result of advances in processing equipment
and the realization of producing high-value products with high profitability
(Rosa and Meireles, 2005).
1.3 Process Concept Schemes and Systems

The supercritical fluid extraction technology is conceptualized on the basis
of obtaining pure extracts without detrimental residues in food and pharma-
ceutical products desired by consumers. Extraction is a unit process used to
separate and isolate a targeted component from substances. The success of
the process depends on the distribution of the analytes between two phases,
the separation and stationary phases (King et al., 1993). In the phase equilib-
rium between the liquid and gas, the partition of the liquid phase increases
with increasing pressure and decreases with increasing temperature. If
the temperature and pressure are simultaneously increased, the transport

properties of both liquid and gas increase and thus convergence occurs.
When a supercritical extraction system is set to work under pressure and
temperature of 5–50 MPa and ambient to 300°C, respectively, the solubility
properties of the supercritical fluid are greatly influenced by its density, dif-
fusivity, and viscosity. The supercritical-CO2 fluid becomes liquid-like with
higher extraction potential than organic liquid solvents. King et al. (1993)
stated that CO2 at high densities has solvent properties similar to chloroform
and acetone, and if intermediately compressed, it behaves like nonpolar
hydrocarbons such as n-pentane or diethyl ether.
The separation phase is the dynamic extraction period when the fluid is in
direct contact with the sample, whereas the stationary phase is the sample
material loaded as a fixed-bed in the extraction column. Supercritical fluid
extraction involves the use of compressed gases at or above their critical tem-
perature (Tc) and pressure (Pc). It utilizes the potentials of these special fluids
as excellent solvents to solvate certain solutes (bioactive components) from a
solid matrix (Rozzi and Singh, 2002). The solute extraction stream from the
sample matrix is directly proportional to the product’s solubility and dif-
fusivity in the supercritical medium. Hence, the solutes solubility increases
with pressure, whereas its corresponding diffusivity is expected to decrease
by two orders of magnitude. The solvent capacity is mainly the function of
density and can be improved with the addition of a cosolvent to modify the
density and increase the polarity of the supercritical fluid, and thus signifi-
cantly increase the yield.
The supercritical fluid extraction power is dependent on the solubility and
phase equilibrium of substances in the compressed gas. Hence, the targeted
bioactive components being extracted must be soluble in the supercritical
fluid. Controlling the pressure and temperature of supercritical fluid varies
the solubility and phase equilibria. The extraction of pure and high-value
extract is accomplished without the risk of toxic residual solvent contamina-
tion in the final products and environmental pollution of the effluent.
1.3.1 Process Principle

Supercritical fluid extraction is a novel separation technique that utilizes the
solvent properties of fluids near their thermodynamic critical point (Uquiche
et al., 2004). The physicochemical properties of the supercritical fluids are
crucial to the understanding of the process design, calculation and model-
ing of the extraction process, and optimization of the operating conditions.
Therefore, selectivity of solvents to discriminate solutes is a key property
of great significance to process engineers. Physical characteristics such as
density and interfacial tension are important properties to manipulate for
separation to proceed. The density and interfacial properties of the extracts
must vary from that of the raffinate to influence coalescence, a step that must
occur if the extract and raffinate phases are separate. The supercritical state of
the fluid is influenced by temperature and pressure above the critical point.

The critical point is the end of the vapor–liquid coexistence curve as shown

in the pressure–temperature curve in Figure 1.1, where a single gaseous
phase is generated. When pressure and temperature are further increased
beyond this critical point, it enters a supercritical state. At this state, no phase
transition occurs, regardless of any increase in pressure or temperature, nor
will it transit into the liquid phase. Hence, diffusion and mass transfer rates
during supercritical extraction are about two orders of magnitude greater
than organic/liquid solvents.
Substances that have similar polarities are easily soluble in solutions, but
with increased deviation in polarity, make solubility increasingly difficult.
Intermolecular polarities exist as a result of van der Waals forces. Although
the solubility behaviors depend on the degree of intermolecular attraction
between molecules, the discriminations between different types of polarities
are also important. Substances dissolve in each other if their intermolecular
forces are similar, or if the composite forces are manifest in the same manner.
The physical properties such as the density, diffusivity, dielectric constant,
viscosity, and solubility are paramount to supercritical extraction process
design.
A variety of processes involving extractions with supercritical fluids have
been developed and are regarded as a viable extraction technology that meets
the food quality and safety requirements. The physicochemical properties of
supercritical fluids can be easily varied by altering the operating conditions
of pressure and temperature individually or in combination (Brunner, 2005).
Many supercritical fluids (carbon dioxide, ethane, propane, butane, pentane,
ethylene, ammonia, sulfur dioxide, water, chlorodifluoromethane, etc.) are
used in supercritical extraction processes. Brunner (2005) and Rozzi and
Singh (2002) have recommended CO2 because of its favorable properties and
the ease of changing selectivity by the addition of modifiers such as ethanol
and or other polar solvents.
1.3.2 Process System

The supercritical fluid extraction process is governed by four key steps:
extraction, expansion, separation, and solvent conditioning. The steps are
accompanied by four generic primary components: extractor column (high-
pressure vessel), pressure control valves, separator column, and pressure
intensifier (pump) (Figure 1.2) for the recyclable solvent (Reverchon, 1997).
The system has other built-in accessories such as heat exchangers for provid-
ing a source of heating and condensers for condensing supercritical fluids
to liquid, storage vessels, and a supercritical fluid source. Raw materials are
usually ground and charged into a temperature-controlled extractor column
(forming a fixed bed), which is usually the case for a batch and single stage
extractors (Shi et al., 2007a,b; Kassama et al., 2008).
The supercritical-CO2 fluid is fed at high pressure by means of a pump,
which pressurizes the extraction column and also circulates the supercritical

Pressure Dry test

regulator Three-stage separator columns meter
Extracts
Extractor column
Ice water trap

CO2 inlet
CO2 pump
Mixer
Cosolvent inlet Cosolvent pump
FIGURE 1.2
Schematic diagram of the supercritical-CO2 fluid extraction system used to fractionate bioac-
tive components from plant matrix.
medium throughout the system. Figure 1.3 shows an example of a typi-

cal single-stage supercritical-CO2 fluid extraction system. Once the
supercritical-CO2 and the raffinate reach equilibrium in the extraction vessel
by manipulation of p ressure and temperature to achieve the ideal operating
conditions, the extraction process proceeds. The mobile phase consisting of
the supercritical-CO2 fluid and the solubilized components is transferred
to the separator where the solvating power of the fluid is reduced by increas-
ing the temperature and/or decreasing the pressure of the system. The extract
precipitates in the separator, whereas the supercritical-CO2 fluid is either
released to the atmosphere or recycled back to the extractor. In the case where
highly volatile components are being extracted, a multistage configuration is
employed as shown in Figure 1.4 (Shi et al., 2007a,b; Kassama et al., 2008).
CO2 pump Back pressure valve

Extractor column
CO2 outlet
exchanger
Separator
Heat
Heat exchanger
Cooling
bath
Pressure valve Extracts outlet

CO2 source
FIGURE 1.3
Schematic diagram of a typical single-stage supercritical-CO2 fluid extraction system.

2 stage separation vessels

3 stage extraction columns
Storage tank
CO2 inlet Cosolvent inlet
Pumps
FIGURE 1.4
Schematic diagram of a commercial-scale, multistage supercritical fluid extraction system
used to fractionate bioactive components. The symbol “ ” is the pressure valves and “ ” is
heat exchangers.
The processes described above are semibatch continuous processes

where the supercritical-CO2 flows in a continuous mode, whereas the
extractable solid feed is charged into the extraction vessel in batches. In
commercial-scale processing plants, multiple extraction vessels are sequen-
tially used to enhance process performance and output. Although the sys-
tem is interrupted at the end of the extraction period, when the process
is switched to another vessel prepared for extraction, the unloading and/
or loading of the spent vessels can be carried out while extraction is in
progress, reducing the downtime and improving the production efficiency
(Kassama et al., 2008).
A semicontinuous approach on a commercial scale uses multistage
extraction processes, which involve running the system concurrently by
harnessing a series of extraction vessels in tandem as shown in Figure 1.4.
In this system, the process is not interrupted at the end of extraction period
for each vessel, because the process is switched to the next prepared ves-
sel by control valves for extraction, while unloading and/or loading the
spent vessels. Thus, supercritical-CO2 technology is available in the form
of single-stage batch that could be upgraded to multistage semicontinu-
ous batch operations coupled with a multiseparation process. The needs
of improving the design into truly continuous modes are growing.
Supercritical-CO2 fluid extraction could be cost-effective under large-scale
production.

1.3.3 Single-Stage Extraction Process

The supercritical fluid extraction system utilizes a prime mover, a pump
which provides a constant pressure on the system, thus circulating the super-
critical medium from the tank/cylinder throughout the system. Figure 1.3 is
an example of a typical single-stage supercritical extraction system. Once
operating parameters are preset, the system reaches equilibrium in the single
extraction vessel under dynamic condition. The extracts are precipitates in
the separator, whereas the supercritical fluids are either released or recycled
back to the extractor. In the case where highly volatile components are being
extracted, a multistage configuration may have to be employed as shown in
Figure 1.4.
As the solution leaves the extractor, it flows to the first separation vessel
via a pressure regulator. The pasty oleoresins settle to the bottom as they
separate due to density difference and collected, whereas the remaining
solution goes to the second-stage separator where the fractionation of the
volatile components takes place. For more sensitive products, the third stage
of separation would be required for the complete isolation of pure volatile
components. Saltzman et al. (1993) presented a design (Figure 1.2) where
the solution flows through a heated valve and precipitates into a preweight
U-tube in an ice water bath. The glass wool on the U-tube exit trap entrains
the solutes in the gasses, which in turn flows through a flow meter that mon-
itors the flow rate. Oszagyan et al. (1996) used a similar system as illustrated
in Figure 1.2 to extract essential oil from Lavandula intermedia Emeric ex
aloisel and herb of Thymus vulgaris L., and further fractionated volatile com-
ponents (ρ-cymene, γ-terpinene, thymol, and carvacrol). Similarly, Ozcan
et al. (2003) used it to fractionate volatile components from Turkish herbal
tea (Salvia aucheri Bentham var. canaescen Boiss and Heldr.). Duquesnoy et al.
(2004) and Boutekedjiret et al. (2003) extracted and fractionated volatile com-
pounds from plant materials using the supercritical-CO2 fluid with a similar
multistage fractionation method.
The processes described above are semibatch continuous processes where
the supercritical-CO2 fluid is in a continuous mode, whereas the extractable
solid samples are charged into the extraction vessel in batches. In commer-
cial processing plants, multiple extraction vessels are sequentially used to
enhance the process performance and output. Although the system is inter-
rupted at the end of the extraction period, when the process is switched to
a prepared vessel for extraction, the unloading and/or loading of the spent
vessels can be carried out while extraction is in progress, reducing the down-
time and improving the production efficiency.
1.3.4 Multistage Extraction Process

A semicontinuous approach on a commercial scale uses multistage extrac-
tion processes that involve running the system concurrently by harnessing

a series of extraction vessels in tandem as shown in Figure 1.4. The system

provides an option to continue the extraction process at the end of the extrac-
tion period for each vessel by switching to the next prepared vessel by con-
trol valves for extraction while unloading and/or loading the spent vessels,
although imperfect, continuity is attained.
The primary extraction stages operate in a similar mode to the ones
depicted in Figures 1.3 and 1.4. The raffinate from the premier stage enters
the first separation vessel, whereas separation and fractionation of different
compounds occur based on their relative solubility. The options of cosolvent
are available to enhance the solvent power of separation of specific compo-
nents. This is effective for cases where more than one targeted component
is to be extracted, giving the flexibility to vary the extraction parameters
such as pressure and temperature to achieve different solubilities for differ-
ent components being extracted at each stage of the operation. Gamse (2003)
suggested that highly soluble substances could be extracted at the initial
stages at low supercritical-CO2 fluid density, and by increasing the density
in the subsequent stages removes the less soluble substances. The supercriti-
cal pressure, temperature, and flow rate at each stage could be controlled
independently.
1.4 Physicochemical Properties of Supercritical-CO2 Fluids

Supercritical fluids combine unique properties of the gas and liquid phases
to enhance their functionality. The values of the supercritical fluid density,
diffusivity, and viscosity lie between those of conventional liquids and gases.
For example, the supercritical density is less than that of a liquid organic
solvent but greater than that of a gas. This distinct property provides super-
critical fluid extraction a higher extraction efficacy compared with liquid
solvents because of its higher mass transfer rate.
1.4.1 Phase Diagram

Supercritical state of fluids is influenced by temperature and pressure above
their thermodynamic critical points. The critical point is the interface of the
saturated vapor–liquid curves as shown in Figure 1.1 for a single gas. Fluids
are said to be in their supercritical state by increasing their relative pres-
sure and temperature beyond the critical point. When fluids enter the super-
critical region, no phase transition will occur even with further increase in
pressure and/or temperature. Supercritical fluids are about two orders of
magnitude greater than in the liquid state because of their diffusion and
mass-transfer characteristics.

1.4.2 Physical Properties

Physical properties are critical to the functionality of supercritical fluids.
Density, diffusivity, dielectric constant, viscosity, and solubility are impor-
tant properties vital to the successful extraction process design. The physical
properties of supercritical fluids such as high diffusivity, low viscosity, and
interfacial tension may improve the dissolving power of supercritical fluids,
consequently enhancing the extraction efficacy. Substances that have similar
polarities are easily extracted because they are readily soluble in the selected
solvent, hence with slight deviation in polarity decrease the solubility of the
targeted components. The intermolecular polarities between the extraction
components are influenced by the van der Waals forces, and the solubility
behaviors depend on the degree of intermolecular attraction between mol-
ecules. Unique intermolecular forces of substances easily dissolve in each
other and hence enhance extraction efficacy.
Although many different types of supercritical fluids are available for
industrial applications, CO2 is the most desired for extraction of bioac-
tive components. Table 1.1 shows some physical properties of compressed
supercritical-CO2 at pressure (20 MPa) and temperature (55°C) compared
with condensed liquids commonly used as extraction solvents at 25°C. It
should be noticed that supercritical-CO2 exhibited density similar to that of
the liquid solvents, but it is less viscous and highly diffusive. This fluid-like
attribute of CO2 coupled with its ideal transport properties and other quality
attributes outlined above make it a better choice over other solvents.
The heat capacity of supercritical fluids is a function of temperature, pres-
sure, and density. The specific heat capacity (Cp) of CO2 increases rapidly
as the critical point (31.1°C temperature, 7.37 MPa pressure, and at 467.7 g/L
flow rate) is approached. This implies an increase in enthalpy per unit mass
of supercritical fluid at any given unit change in temperature, thus having
significant effect on the rate of heat transfer. Therefore, the effects of tem-
perature and specific heat on energy gain/loss and on the target bioactive
compounds must be well evaluated.
Sample matrix is an important parameter that influences solubility and
mass-transfer process during supercritical-CO2 fluid extraction. Properties
such as particle shape and size distribution, porosity and pore size distribu-
tions, surface area, and moisture content influence solubility and mass trans-
fer. The presence of water (moisture content) in the sample matrix during
supercritical extraction also has an effect on the extraction outcome.
1.5 Factors Affecting Extraction Yield

Several bioactive components were extracted successfully by the
supercritical-CO2 fluid extraction method as outlined in the preceding

sections. Optimization of yield is a function of various independent param-

eters. Process parameters such as solvent flow rate, resident time, moisture
content, particle sizes, and particle size distribution in conjunction with
supercritical pressures and temperatures are key parameters for achieving
optimum results. Most of these parameters can have individual or combined
effects on the rate of extraction, for example, the resident time can have an
immense influence on the composition of the extracted compound.
1.5.1 Pressure
Figure 1.5 is a typical extraction yield rate curve. It is apparent from the
curve that pressure significantly influences the rate of extraction, likewise
the extraction time. Extrapolating the normalized yield at the point where
the yield curve becomes asymptotic gives significantly different normalized
yields of 15%, 11%, and 4% for pressures of 10, 9, and 8 MPa, respectively
(Marongiu et al., 2003). Macias-Sanchez et al. (2005) observed similar trends
in the supercritical-CO2 fluid extraction of carotenoids and chlorophyll a
from Nannochloropsis gaditana, although, as pressure increased beyond a criti-
cal point, the yields dropped as a result of increased density. Higher den-
sity manifests a double effect, causing an increase in salvation power and
a decrease in interaction between the fluids and matrix thus decreasing the
diffusion coefficient. Excessive pressure may also increase the compactness
of the sample matrix in the extraction column, thus reducing the interpar-
ticle porosity hence reducing the mass transport through the matrix which
eventually contributes to diminish the yield.
The selectivity of solutes is a function of pressure, and an increase in
the extraction pressure enhances the extraction ability of different solutes.
16
12
Yield (%)
0
0 100 200 300 400 500 600 700
Time (min)
8 MPa 9 MPa 10 MPa
FIGURE 1.5
The change in bioactive compound yield against time during supercritical-CO2 fluid extraction
of essential oil from J. oxycedrus on extraction rate at a flow rate of 1.5 kg/h and a temperature
of 50°C. (Modified from Marongiu, B. et al. 2003. Flavour and Fragrance Journal, 18: 390–397.)

D’Andrea et al. (1994) also found optimum yield at a working pressure
of 25 MPa and temperature of 55°C for the extraction of azadirachtin and
3-tigloylazadirachtol from neem seeds. Similarly, Tonthubthimthong et al.
(2001) reported optimum yield at a pressure of 23 MPa and a temperature
of 55°C.
1.5.2 Temperature
Manipulating temperature may have a significant influence on yield dur-
ing supercritical-CO2 fluid extraction. Figure 1.6 shows a general trend
of increase in extraction yield as process temperature increases relative
to the pressure (Ge et al., 2002). Tonthubthimthong et al. (2001) reported
similar trends for extracting nimbin from neem seeds at 20 MPa and at
a CO2 flow rate of 0.62 mL/min, and the optimum yield was found at
35°C. Ge et al. (2002) indicated that at a temperature of 35°C, the highest
yield was obtained in the first 45 min of a prolonged extraction period of
120 min (Figure 1.6). Although many literatures reported that a correlation
is established between increasing temperature and extraction yield (Spanos
et al., 1993), others showed no particular trend as far as temperature was
concerned (Gomez et al., 1996; Kassama et al., 2008; Yi et al., 2009). Some
researchers reported that yield was inversely proportional to temperature
at 15 MPa.
The combined effect of pressure and temperature on cholesterol extrac-
tion was studied by Chao et al. (1993). At a pressure–temperature setting
of 34 MPa and 50°C, respectively, cholesterol yield of 160 mg/100 g was
realized compared with 430 mg/100 g when the temperature drops to 40°C
and 2.5 kg of CO2 is used. As the CO2 mass increases, yields decrease, but
the lowest temperature still maintains the highest yield as shown in Figure
2600
Yield (mg/100 g)
2200
1800
1400
10 15 20 25 30 35 40
Pressure (MPa)
35°C 40°C 45°C 50°C
FIGURE 1.6
The change in bioactive compound yield against pressure during supercritical-CO2 fluid
extraction of wheat germ on extraction, time 120 min; rate at flow: 2.0 mL/min and sam-
ple size 5 g. (Modified from Ge, Y. et al. 2002. Journal of Agricultural and Food Chemistry,
50: 686–689.)

500
Cholesterol (mg/100 g) 400
300
200
100
0
0 5 10 15 20 25
Carbon dioxide (kg)
34.5 MPa/50°C 13.8 MPa/50°C
34.5 MPa/40°C 24.1 MPa/40°C
FIGURE 1.7
Supercritical-CO2 fluid extraction of cholesterol against carbon dioxide mass in beef tallow
at different pressures and temperatures. (Modified from Chao, R.R. et al. 1993. Journal of the
American Oil Chemists’ Society, 70: 139–143; Chow, C.K. 2000. Fatty Acids in Foods and Their Health
Implications, second edn. (revised and expanded). Marcel Dekker Inc., New York, Basel.)
1.7. Also under constant temperature, yield increase was achieved when
pressure decreased. The results demonstrated that higher selectivity is
possible at lower pressures and higher temperatures. Froning et al. (1994)
corroborated this fact based on their experiment with lipid and cholesterol
extraction from dehydrated chicken meat. The combination of pressure and
temperature, 38.6 MPa and 55°C, respectively, yielded 89% lipid and 90%
cholesterol, whereas a pressure and temperature combination of 30.3 MPa
and 45°C, respectively, produces a much lower yield. Yi et al. (2009) also
reported that increases in lycopene yield could be achieved by raising both
the temperature and pressure. As expected, the temperature dependence
of the lycopene yield was higher than was the pressure dependence. It is
because higher temperatures promote solubility of the solute and increase
mass transfer of solute from matrix to the supercritical fluid, thus increasing
the yield of extracts.
1.5.3 Moisture Content of Raw Materials

Moisture content is a factor that influences extraction of bioactive com-
pound yields as shown in Table 1.2. A maxima yield of 1678 mg/100 g was
achieved with 5% moisture content and any further increase or decrease
in moisture reduced the yield (Ge et al., 2002). Therefore, it is important to
establish an optimum moisture content to maximize yield in the supercriti-
cal-CO2 fluid extraction process. The effect of sample pretreatment is crucial
in attaining the optimum condition. High-moisture samples inhibit the flow
of the supercritical-CO2 fluid by changing the surface tension and contact
angles as a result of phase interaction between the three components (water,

TABLE 1.2
Effect of Moisture Variation on Bioactive Compound
Yield during Supercritical-CO2 Fluid Extraction of
Wheat Germ
Water Content Yield
(% Wet Basis) (mg/100 g)
4 1470
5 1678
8 1352
12 1290
Source: Modified from Ge, Y. et al. 2002. Journal of
Agricultural and Food Chemistry, 50: 686–689.
sample matrix, and supercritical-CO2 fluid). However, the removal of excess

water frees up the interparticle pores and thus increases the mass transport
intensity during extraction. For example, the higher the moisture content,
the higher the probability for the formation of a thin film of water between
the sample matrix and the supercritical fluid phase. Water has a small but
finite solubility in the supercritical-CO2 fluid, and as a result it can also be
extracted with the targeted components and its separation can be done at
the end of the process.
1.5.4 Cosolvent
The use of cosolvent (entrainers) during supercritical-CO2 fluid extraction
is key to enhancing the extraction efficiency and cost-effectiveness of the
processes. Joslin et al. (1996) indicated two significant attributes of cosol-
vents: the interaction between the cosolvent and the solute (direct effect)
and the cosolvent–solvent interactions (indirect effect). Cosolvents used in
small doses normally at a range of 1%–15% in the supercritical-CO2 fluid can
change the overall characteristics of the extraction fluid such as polarity, sol-
vent strength, and specific interactions. These changes in turn can signifi-
cantly alter the density and compressibility of the supercritical-CO2 fluid.
Furthermore, cosolvents improve the selectivity of the desired compo-
nents and facilitate selective fractional separations. Water and ethanol
are GRAS products, an environmental benign, and can therefore be used
in food-extraction processes. In consequence, the use of these cosolvents
enables the extraction of polar compounds without losing the supercritical-
CO2 fluid advantages. The use of cosolvent results in the development of
an environment-friendly and safe process in food and pharmaceutical uses.
Table 1.3 summarizes the results of different cosolvents, ethanol, methylene
chloride, and methanol (Cygnarowicz et al., 1990).
Ethanol seems to be the most used cosolvent and it was selected in 53%
of the supercritical fluid extraction studies on vegetable matrices involving

TABLE 1.3
Fractionation Data of β-Carotene from Supercritical Carbon
Dioxide with Cosolvent Mixtures at the Temperature of 70°C and
the Enhancement Factor Based on Fluid Density (17 mol−1)
Pressure CO2 Density Yields
(MPa) (mol−1) (×107)
β-Carotene CO 2
21.2 15.71 1.95
24.9 16.73 3.33
28.7 17.65 6.23
32.8 18.43 10.00
35.8 18.91 12.50
40.0 19.49 19.10
43.9 19.95 25.4
β-Carotene CO 2 + 1 wt% Ethanol (Enhancement Factor = 4.7)

22.3 15.92 9.6
24.9 16.73 19.5
31.6 18.22 25.2
37.4 19.14 37.5
β-Carotene CO 2 + 1 wt% Methylene Chloride (Enhancement Factor = 3.5)

23.4 16.28 12.8
24.7 16.67 13.3
31.2 18.16 21.7
37.0 19.08 27.7
β-Carotene CO 2 + 1 wt% Methanol (Enhancement Factor = 2.1)

18.0 13.92 3.98
26.8 17.26 9.62
33.0 18.47 15.60
37.3 19.12 30.60
Source: Modified from Cygnarowicz, M.L., Maxwell, R.J., Seider, W.D.
1990. Fluid Phase Equilibria, 59: 57–71.
entrainers (de Melo et al., 2014). The solubility enhancement with ethanol
resulted in the complex interaction between β-carotene and the supercritical-
CO2 fluid and the cosolvent. Joslin et al. (1996) also reported an enhancement
factor of 64, 63, and 29 for extracting palmitic acid, stearic, and behenic fatty
acids, respectively. Baysal et al. (2000) used ethanol at different concentra-
tions (5%, 10%, and 15%) to recover β-carotene and lycopene from tomato
paste. Although they observed that with a high ethanol concentration, the
extraction was hindered due to a decrease in the homogeneity of the extrac-
tion mixture, and no statistically significant differences were found between
the 10% and 15% concentrations.

Water as cosolvent has recently been gaining lots of attention. For

instance, the addition of water to supercritical-CO2 has been reported to
be more effective than adding ethanol to extract phenolic compounds from
grape pomace (Da Porto et al., 2014). Water as cosolvent has also been dem-
onstrated to be more efficient than ethanol on the supercritical fluid extrac-
tion of ro-grapholide from Andrographis paniculata (Burm. F) Nees leaves
(Chen and Yin, 2009) and the removal of caffeine from green tea (Kim et al.,
2008). Nevertheless, despite the promising results, experiments reported
involving water as cosolvent is not widespread, hence only represents 5%
of the supercritical fluid extractions in vegetable matrices (de Melo et al.,
2014).
1.5.5 Particle Size

Particle sizes have significant impact on the flow behavior of the
supercritical-CO2 fluid in the sample matrix. The mechanism of sample
pretreatment, for example, the methods of drying (air-, oven-, vacuum-, or
freeze-drying), would influence particle sizes when subjected to attrition or
size reduction. The sizes of particles, shapes, and their random layout (size
distribution) would determine what goes through the medium and how fast.
The layout would influence the type of pore, either open or blind pores, and
their degree of interconnectedness.
Process parameters such as pressure influence particle size distributions.
Pressure tends to create compactness, and thus decreases the intergranu-
lar porosity resulting in increased solid density. The smaller the particle
size, the larger is the surface area, and as a result bioactive components are
released easily. Coelho et al. (2003) observed no significant effect of particle
sizes on the extraction yield as a function of extraction time on a fixed flow
rate. The oxygenated compounds increased from 81% to 85% as particle size
decreases as shown in Table 1.4. However, the findings of Ge et al. (2002)
were contrary to those of Coelho et al. (2003) in their study of the effect of
particle sizes on wheat germ (Table 1.5). Papamichail et al. (2000) extracted
essential oil from celery with the supercritical-CO2 fluid. They experienced
increased yield (more oil released) as the particle sizes of the seed decrease
and attributed that to the pretreatment milling and sieving. A maximum
yield of 1838 mg/100 g was obtained with an optimum particle size of
0.505 mm. They observed that very fine and big particle sizes have low
extraction yield probably due to higher/too low resistances to mass transfer
because of the compact tendency, reflecting reduced pore sizes in finer par-
ticle sizes while less interactions with the supercritical fluids in the case for
the latter. Likewise, larger particles contain undamaged cell walls rendering
them impervious.
Particle size reduction is essential for lipoprotein matrix in order to
release the embedded lipids in fish during supercritical-CO2 fluid extraction,

TABLE 1.4
Supercritical-CO2 Fluid Data Compared to Hydrodistillation Extraction of Volatile
Components from Fennel (F. vulgare) Fruits of Different Particle Sizes
Hydrodistillation (%) Supercritical-CO2 (%)
Volatile
Components Stalks Fruits (0.5 mm) Fruits (0.55 mm) Fruits (0.35 mm)
Canfene 0.2 0.2 0.2 0.1
Sebinene 0.1 0.2 0.2 0.2
Myrcene 1.2 1.4 1.4 1.3
α-Phellandrene 2.0 2.2 2.2 1.9
Limonene 2.1 3.6 3.5 3.1
γ-Terpinene Tr 0.1 Tr Tr
Terpinolene 0.5 0.6 0.6 0.6
Fenchone 15.8 16.8 16.2 17.1
Estragol 18.9 20.9 21.0 21.9
(E)-Anethole 42.5 42.2 42.5 44.6
Piperitenone oxide 0.2 0.2 0.3 0.3
Unknowns 4.8 3.4 5.5 0.3
Waxes 0.6
Source: Modified from Coelho, J.A.P. et al. 2003. Flavour and Fragrance Journal, 18: 316–319.
Note: Tr (Trace < 0.05).
although some believed that the higher supercritical-CO2 fluid flow rate is
capable of degrading protein structure to release the targeted bioactive com-
ponents (Femenia et al., 2001). Papamichail et al. (2000) reported that it was
possible to extract more essential oil per kg of CO2 at a lower flow rate due to
low intraparticle porosity and high diffusion resistance. Therefore, a thresh-
old level for each product has to be experimentally determined in pursuance
to pilot plant prior to industrial scale-up processing.
TABLE 1.5
Particle Size Data of Bioactive Compound Yield during
Supercritical-CO2 Fluid Extraction of Wheat Germ
Sieving Particle Size Yield
(Mesh) (mm) (mg/100 g)
No grinding 2.1 1610
20 0.86 1710
30 0.51 1838
40 0.40 1550
60 0.22 1070
80 0.18 890
100 0.13 742
Source: Modified from Ge, Y. et al. 2002. Journal of Agricultural and
Food Chemistry, 50: 686–689.

1.5.6 Flow Rate

Coelho et al. (2003) presented three mass flow rate scenarios of 2.3, 1.5, and
0.85 kg/h as a function of extraction time and concluded that the highest
flow rate of 2.3 kg/h gave the higher rate of extraction of Foeniculum vulgare
volatile oil with the supercritical-CO2 fluid at 9 MPa and 40°C. A similar
trend was observed by Ge et al. (2002) who reported that a flow rate of 3 mL/
min yielded 1927 mg/100 g of bioactive component from wheat germ. The
higher flow rate could produce faster extractions and higher recoveries as the
higher flow rate could overcome the interface resistance for targeted com-
pounds transported from the solid matrix to the CO2 fluid.
Yi et al. (2009) studied the effect of supercritical fluid extraction param-
eters on lycopene yield and antioxidant activity. Their results showed that
increasing the flow rate from 1.0 to 2.0 mL/min did not show any significant
(P > 0.05) change on the yield of lycopene at constant pressure (30 MPa) or
temperature (70°C). However, the increase in flow rate from 1.5 to 2.5 mL/
min could enhance the outcome of lycopene, but a further increase from
2.5 to 4.5 mL/min resulted in a decrease in the lycopene amount. Some of
these facts were also corroborated by Peker et al. (1992) in their experimental
study on extraction rates of coffee beans with the supercritical-CO2 fluid.
They indicated the need for long extraction time in conditions where low
flow rates are used.
Figure 1.8 shows an apparent yield at high flow rates for supercritical extrac-
tion of celery oil (Papamichail et al., 2000) and Juniperus oxycedrus essential
oil (Marongiu et al., 2003). Summarized in Table 1.6 are the results of Baysal
et al. (2000), where a flow rate of 4 kg/h was identified as the optimum condi-
tion for attaining the highest yield. Similar trend was observed by Ferreira
and Meireles (2002) for extracting essential oil from black p epper. They
observed larger yield at 30 MPa using the upper level flow rate (10.54 kg/s).
0.18
Yield (kg/kg feed)
0.12
0.06
0
0 50 100 150 200 250
Time (min)
1.1 kg/h 3 kg/h
FIGURE 1.8
The supercritical-CO2 fluid extraction yield against extraction time of essential oil from celery
at pressure (15 MPa) and temperature (45°C). (Modified from Papamichail, I. et al. 2000. Journal
of Supercritical Fluids, 18: 213–226.)

TABLE 1.6
Flow Rate Data on the Supercritical-CO2 Fluid Extraction of Lycopene
and β-Carotene from Tomato Paste
Flow Rate Extraction Lycopene β-Carotene
(kg/h) Time (h) (%) (%)
2 4 14 30
4 2 22 43
8 1 20 34
Source: Modified from Baysal, T., Ersus, S., Starmans, D.A.J. 2000. Journal of Agricultural
and Food Chemistry, 48: 5507–5511.
When maximum solubility is attained, the highest CO2 flow rate would offer
the highest recovery in extracting lipids from fish.
1.5.7 Effect of Time on Yield

Several factors have direct or indirect implications on yield during
s upercritical-CO2 fluid extraction. Resident time is an important factor that
influences yield and the economic viability of the process. Other factors such as
temperature and pressure could have individual or combined effects. Cherchi
et al. (2001) performed detailed analysis of flavor compounds in essential oil
extracted from Santolina insularis by supercritical-CO2 fluid extraction and
they reported that a change in concentration exhibited a reduced concentra-
tion of monoterpenes from 50% in the fraction collected after 30 min to 10% in
the fraction collected after 240 min (Figure 1.9a) under optimum conditions of
9 MPa and 50°C in a two-stage separation process. The first-stage separation
was accomplished under 9 MPa and 12°C, whereas the final stage used 2 MPa
and 15°C. The yield becomes asymptotic at 1.75% with an increase in extrac-
tion time, whereas the rate decreases (Figure 1.9b). Hawthorne et al. (1992)
studied the extraction rate on basil conducted at 30 MPa and 45°C for 10 min
and identified 1,8-cineole, estragole, eugenol, and selinene and the yield were
reportedly dependent on time. Temelli et al. (1995) considered 3–4 h as suf-
ficient time to extract all extractable lipids from freeze-dried krill and 6 h for
rainbow trout regardless of the extraction conditions.
1.6 Applications in the Food Industry

One of the most important trends in the food industry today is the demand
for all-natural food ingredients that are free of chemical additives. Natural
antioxidants for food are derivatives of plant by-products. A quantum leap in
the supercritical-CO2 fluid extraction technology is made by its applications
in decaffeinating coffee, tea, and other bioactive (essential oils from spices)

(a) 60
Content (%) 50
40
30
20
10
0
0 50 100 150 200 250 300 350
Time (min)
HM OM HS OS
(b)
2
Cumulative quantity (g)
1.5
0.5
0
0 50 100 150 200 250 300 350
Time (min)
HM OM HS OS Overall
FIGURE 1.9
Families of flavor compounds extracted from S. insularis at different supercritical extraction
times. (a) Percentage of the various compound families at different extraction times, (b) cumu-
lative quantities, expressed as grams of extracted compound families at various extraction
times. HM, hydrocarbon monoterpenes; OM, oxygenated monoterpenes; HS, hydrocarbon ses-
quiterpenes; OS, oxygenated sesquiterpenes. (Modified from Cherchi, G. et al. 2001. Flavour and
Fragrance Journal, 16: 35–43.)
components used as ingredients in foods. Likewise, supercritical-CO2 fluid

extraction is used to extract flavor and fragrance, and high-value compounds
used as ingredients in the food, pharmaceuticals, and neutraceutical products.
Large-scale supercritical-CO2 fluid extraction has become a reality for
the extraction of high-value products from natural materials. The solvating
power of supercritical-CO2 fluids is sensitive to temperature and pressure
changes, and thus the extraction parameters may be optimized to provide
the highest possible extraction yields with maximum antioxidant activ-
ity for health-promoting bioactive components (Kassama et al., 2008; Chen
et al., 2009; Yi et al., 2009). With this innovative technology, a process could
be designed to extract natural nutrients without the fear of organic solvent
residues. A compendium of process parameters used for different product
applications is listed in Table 1.7.

26
TABLE 1.7
Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by Supercritical-CO2
Fluid Extraction
Component CO2 Flow
Product Raw Raw Material Concentration Temp. Pressure Rate Time MC Recovery
Extracted Material Pretreatment (%) (°C) (MPa) (L/h) (h) (%) Cosolvent (%) Source
Sunflower oil Sunflower Ground 40–50 32–35 2.5 Ethanol 36 Cocero and Calvo
seed (1996)
Soybean oil Soybean Flaked, 20.1 50 7.58–8.27 900–1080 9.8 Friedrich et al. (1982)
0.38–5.1 mm
Corn germ oil Corn germ Dried, milled 23.4 50 55.2 3.5 Ethanol 50 Ronyai et al. (1998)
Seed oil Avocado Dried, ground 70 75.8 1140 24 3.4 58.2 Friedrich and Pryde
Cottonseed (<0.25 mm) 50 55.2 8 8.6 30.8 (1984)
Functional Food Ingredients and Nutraceuticals

Paprika 50 55.2 7 5 7.2
Peanuts 70–75 68.9 8 9.0 48.0
Peanut hearts 50 68.9 11 2.8 42.6
Rice bran 70 65.5 6 6.3 19.2
Sorghum 78 66.2 11 6.1 5.0
bran 68.9 7 9.4 16.8
Sorghum 70 55.2 8 11.4 19.4
germ 55.2 5 11.4 4.0
Soybean 50 55.2 6 10.1 7.0
Wheat bran 50
Wheat germ 50
Essential oil Dried ground 23–40 9–10 60 6.7 60 Sovava et al. (1994)
Limonene Sweet orange Sieved (1, 0.7, 0.4,
Carvone Caraway seed 0.2, 0.08 mm)
Anethole Anise seed
Eugenol and Cloves
caryophyllene Spikenard
Yaleranone
Wheat germ oil Wheat germ Milled, powder 10.2 35–50 13–41 0.12 2 4.3– 98.7 Ge et al. (2002)
(20 μm diameter) 11.5
(Continued)
TABLE 1.7 (Continued)
Extraction of Health-Promoting Components

Fluid Extraction
Component CO2 Flow
Defatted Mustard seed Dried, fully 16 40 30.4 300–400 3 80 Taniguchi et al.

mustard pressed (1 mm) (1987)
Canola oil Canola seed Flaking (0.2– 45–70 41–62 180 3 Ethanol 44 Dunford and Temelli
0.5 mm) cooking (1995)
(90°C)
Bergaptene Bergamot Peel dried ground 0.15 40–60 8–10 2.6 2 17–19 85 Poiana et al. (1993)
citrus
Carotene lutein Alfalfa Leaf protein 40 30–70 300–360 3 70–90 Favati et al. (1988)
concentration
Defatted and Dehydrated Spray dried 45–55 23–3927.6 300–600 1.4– 87 Wehling et al. (1992)
decholesterol beef powder + air- 3.2
of muscle dried chunk
Oil Antarctic krill Freeze-dried and 16.2 80 24.5 51.4 3–4 7.83 99 Yamaguchi et al.
meal ground (1986)
Bixin pigment Annatto seed Seed, 3.92 mm 50 34.5 117.2– 1–2 Chao et al. (1991a)
60 31 144.8
Defatted and Fresh frozen Ground 19 35 31 117.2– 4–8 56-lipid Chao et al. (1991b)
decholesterol beef 144.8 26-cholesterol
beef
Bixin pigment Annatto seed Whole seed 1.3 50 29.6 1.2 Soy-bean Degnan et al. (1991)
(11.6 oil) oil 20% v
Evening Evening Dried, ground, 21.9 60 70 64.8 1.7–5 <8 97 Favati et al. (1991)
primrose oil primrose 0.355 mm
seed
Cardamom oil Cardamom Freshly ground 40 10 3 10 85–95 Gopalakrishnan and
seeds seeds, 40–60 mesh Narayanan (1991)
Decholesterol Crude beef 0.15–0.2 40 34.5 226.7–250 4–6 60–70 Chao et al. (1993)
beef tallow tallow
27
(Continued)
28
Fluid Extraction
Component CO2 Flow
β-Carotene Sweet potato Freeze dried 94 48 41.4 840–1080 80 Spanos et al. (1993)
ground (0.25 mm)
Lanolin Wool-grease 80 38 396–2880 15.5 Cygnarowicz-
Provost et al. (1994)
Tocopherol Soybean 80 25 300 60-Soybean King et al. (1996)
enrichment flakes 70-Rice bran
Rice bran 80-wheat germ

Carotene Carrot Frozen puree 500 ppm 55 20.7 90 81 Ethanol Vega et al. (1996)
(0.93 mm) 10%
Essential oil Black pepper Dried ground 1.5 40 9–15 2.55 9 18 Perakis et al. (2005)
Piperine 5.7
Essential oil Eucalyptus Air drying 50 9 1.1 2.5 9.5 2.4 Porta et al. (1999)
leaves
Essential oils Ginger Dried ground 30 40 30 138 2 8.8 8.4 Catchpole et al.
gingerols rhizomes (2003)
Triglycerides Palm oil fiber Oven dried 45–55 20–30 1.45 2.25 5 7 França and Meirele
carotenoid (2000)
Lycopene Tomatoes Dried skin ground 65 40–80 17–28 30 0.5 Chloro- Cadoni et al. (2000)
β-Carotene 35 form
Essential oil Peppermint Cut leaves 24–43 6–18 3.42 4–9 14–82 76 Barton et al. (1992)
Essential oil Chili pepper Dried ground 2 40 30 2 8.8 Catchpole et al.
Capsaicin (2003)
alkaloids
Essential oil Coriander Dried grounds 96 50 15 3 3 0.61 Anitescu et al. (1997)
(0.4 mm)
Antioxidants Sweet Thai Dried Ground 35–80 10–30 0.3 5 Ethanol Luengthanaphol
Tamarinds (mesh 40–70) et al. (2004)
Essential oil Grape seed Wash dry ground 80 35–75 20–47 0.75 10 2% Ethanol 77 Lee et al. (2000)
1.6.1 Extraction of Bioactive Compounds

Phenolics or polyphenolics are bioactive compounds of plant origins and
they interfere with the formation of free radicals, thus preventing the for-
mation of hydroperoxides. However, during food processing, especially
conventional thermal processing of food products, these bioactive compo-
nents undergo considerable degradation. In order to restore these compo-
nents in products, it necessitates the fortification with high concentration
of bioactive compounds. Some of the most common bioactive compounds
include lycopene, flavonoids, tocopherol, lecithin, ascorbic acid, citric acid,
polyphenols, etc.
Most of the separation procedures involve physical and chemical processes
such as centrifugation, filtration, membrane separation, precipitation, chro-
matography, solvent extraction, crystallization, evaporation, molecular dis-
tillation, and supercritical-CO2 fluid extraction. To overcome the detrimental
effects of conventional extraction techniques, a rapid separation process is
needed to avoid any significant loss in quality of the natural components and
their stability. Most bioactive components used as functional food additives
are used in concentrated form. Appropriate extraction procedures are con-
sequently required when preparing them from their original matrices. Some
compounds in the concentrated form are thermolabile, volatile, and prone to
degradation when subject to intensive heat.
Supercritical extraction with CO2 is the most viable method for food
applications. Baysal et al. (2000) extracted lycopene and β-carotene from
tomato using the supercritical-CO2 fluid. The processing conditions used
were extraction pressures of 20, 25, and 30 MPa; temperatures of 35°C, 45°C,
55°C, and 65°C; resident time of 1, 2, and 3 h; and CO2 flow rates of 2, 4, and
8 kg/h. The best conditions for lycopene extraction were: 2 h at a flow rate
of 4 kg/h, pressure of 30 MPa, temperature of 55°C, and with the addition
of 5% c osolvent (ethanol). They noted that if too much ethanol is used, it
decreased the homogeneity of the extraction mixture and reduced the sepa-
ration efficiency.
Yi et al. (2009) investigated the effects of the supercritical-CO2 fluid extrac-
tion parameters on the antioxidant activities of lycopene extracts from tomato
skin and found that the activity of lycoepne extracts differed with the yield.
For each unit of lycopene extract, the antioxidant activity level was constant
below 70°C, but then gradually decreased above 70°C due to isomerization of
the lycopene which occurs as a result of the higher temperature. The ratio of
all-trans-lycopene to the cis-isomers changed from 1.70 to 1.32 when the oper-
ating temperature was adjusted from 40°C to 100°C. No significant effects of
pressure or flow rate of the supercritical-CO2 fluid on the antioxidant activity
were observed.
Tsuda et al. (1995), Vega et al. (1996), and Luengthanaphol et al. (2004) com-
pared the supercritical-CO2 fluid extraction to other extraction methods and
their effects on the bioactivity of the extracted bioactive compounds. Their

TABLE 1.8
Yields and Bioactivities Data of Antioxidants Extracted by Different Methods
from Sweet Thai Tamarind Seed Using Supercritical-CO2 Fluid Extraction at
30 MPa and 80°C, and Organic Solvent at Room Temperature
Epicatechin Yield PV after 24 h (meq/kg
(mg/100 g Dry Weight) Dry Weight)
Tsuda Tsuda
Luengthanaphol et al. Luengthanaphol et al.
Extraction et al. (2004) (1995) et al. (2004) (1995)
Supercritical-CO2 0.022 0.336 – –
Supercritical-CO2 + cosolvent 13 26 231 ≈26
(10% ethanol)
Ethanol 25 32 –
α-Tocopherol 157 ≈25
Source: Modified from Luengthanaphol, S. et al. 2004. Journal of Food Engineering, 63: 247–
252; Tsuda, T. et al. 1995. Journal of Agriculture and Food Chemistry, 42: 2671–2674.
results indicated the superiority of the supercritical-CO2 fluid with cosolvent

as shown in Table 1.8. The studies show the superiority of the antioxidant
extracted with the supercritical-CO2 fluid and modifiers, although some dis-
parity occurred which could have been caused by the varieties used. Macias-
Sanchez et al. (2005) extracted carotenoids and chlorophyll from N. gaditana
and achieved the highest yield at 20 MPa and 60°C, the optimal pressure and
temperature, respectively. Wang et al. (2005) also reported that the antioxi-
dant activity of Bupleurum kaoi fractionated with the supercritical-CO2 fluid
gave the highest yield of phenol and the strongest antioxidant capacities.
1.6.2 Fractionation of Flavors and Fragrances

The extraction of flavor compounds and fragrances by supercritical-CO2 is
paramount in the food industry. Mother nature is a splendid synthesizer
of flavors and fragrances in natural products. The cleaner and safer attri-
butes make supercritical technology an ideal candidate for extracting such
valuable and heat-sensitive products in contrast to toxic organic solvents.
The high value-added natural products are good for use in soft drinks. An
example is ginger extract, which gives the pungency and flavor in ginger ale
drinks (Fasoli et al., 2012).
Bhattacharjee et al. (2003) compared the Likens–Nickerson extraction
and the supercritical-CO2 fluid extraction methods on Basmati rice. They
reported that the supercritical fluid extraction technique was more superior
and the extract of the flavor components purer and bore the closest resem-
blance to the original Basmati flavor (Table 1.9) than its counterpart (Likens–
Nickerson method).

A desired fragrance isolated from concentrates extracted from flowers

using several stage processing. The process consists of initial solvent extrac-
tion usually with organic solvent (hexane), which yields an intermediate
product called concrete (Reverchon and Poletto, 1996). This product contains
fragrances and other components such as paraffin, fatty acids, fatty acids
methyl ester, di- and tri-terpenic compounds, pigments, etc. The postpro-
cessing of the concrete can be done using supercritical-CO2 fluid extraction.
Reverchon et al. (1992) used a single-step supercritical-CO2 fluid extraction at
a pressure of 8 MPa and temperature of 40°C followed by a two-stage frac-
tional separation procedure, setting the first separator at 9 MPa and −5°C
TABLE 1.9
Results of Gas Chromatography and Mass Spectroscopy of Flavor Compounds
Extracted by the Likens–Nickerson Extraction and the Supercritical CO2 Fluid
Extraction Methods of Basmati Rice
Likens–Nickenson Extraction Supercritical-CO2 Fluid Extraction
Peak Retention Peak Retention
No. Time (min) Compound No. Time (min) Compound
2 7.51 Hexamethyl disiloxane a
3 8.11 Silicate anion tetramera

5 9.51 Dibutyl phthalatea
8 14.52 Hexane cyclotrisiloxanea
13 21.98 Pentanal 12 22.11 Pentanal
14 22.68 Butan-2-one-3-Me 13 22.56 Butan-2-one-
3-Me
19 27.71 Heptanol 19 28.32 Heptanol
21 28.53 2-Heptanone 20 29.27 2-Heptanone
22 30.45 Octanol 21 30.92 Octanol
24 32.14 Octanal 23 32.26 Octanal
29 35.19 2-Octenal
28 35.14 2-Octanone 30 35.44 2-Octanone
30 36.12 Nonanal 31 36.13 Nonanal
31 37.34 Decanol 32 37.42 Decanol
34 40.22 2-Decenal 34 40.31 2-Decenal
35 42.77 Undecane 37 42.52 Undecane
37 43.82 Benzoic acid 2,5-bis 40 45.11 Dodecane
(trimethylsiloxy)
benzenea
44 45.24 Tetradecane
39 46.28
Source: Modified from Bhattacharjee, P. et al. 2003. Journal of the Science of Food and Agriculture,
83: 880–883.
a Compounds identified as artifacts.

and the second one at 1.5 MPa and 10°C. These conditions allowed a very
efficient fractionation. The first stage was used to remove cuticular waxes.
Under these optimum conditions, the extracted volatile rose oil contains 50%
2-phenylethanol. When a cosolvent (ethanol) is mixed with the supercritical-
CO2 fluid, a yield of 50%–60% was obtained (Sastry and Mukhopadhyay,
1994). Jasmine fragrance extracted at 12 MPa and 40°C gave superior results
compared with other solvents. Sastry and Mukhopadhyay (1994) experienced
an increased yield from 45% to 53% with the use of cosolvents. Similarly, the
supercritical-CO2 fluid has been used effectively to extract fragrances from
orange, marigold, sandalwood, vetiver, etc.
1.6.3 Cholesterol-Free Food Products

Cholesterol is an inevitable substance required for the daily maintenance
of human body. Lipoproteins are vehicles that transport cholesterol to vari-
ous body tissues to be used, stored, or excreted. High-density lipoprotein
(HDL) termed good cholesterol transports cholesterol back to the liver,
where endogenous metabolism prevents cholesterol buildup and reduces
the risk of heart disease. Low-density lipoprotein (LDL) termed bad cho-
lesterol causes fat buildup in the arteries increasing the risk of coronary
heart disease (atherosclerosis). The indiscriminate consumption of saturated
fats in our diet may raise the total LDL (>100 mg/dL) level and decrease the
HDL (<35 mg/dL) level, thus increasing the risk of heart disease. The rec-
ommended daily intake of cholesterol is about 300 mg (James and Ralph,
2000). The correlation between the serum cholesterol level and the mortality
rate associated to cardiovascular disease has been reported in many studies
(Griffin, 1999).
Pork meat has cholesterol content ranging from 30 to 450 mg/100 g, poul-
try (70 mg), fish (35–70 mg/100 g), and beef (65–331 mg/100 g). One common
source of cholesterol is from the consumption of fried fast-food products. The
fast-food industry uses hydrogenated fats for their deep fat-frying processes
because of its stability and high economic turnover. The hydrogenated fat
is the potential source of trans-fatty acids, which are taken up by the fried
foods during cooking (French fries, onion ring, chicken nuggets, etc.) and
ultimately ingested by the consumer. Trans-fats have been shown to increase
the LDL cholesterol levels and decrease the HDL cholesterol levels, thus rais-
ing the risk of heart disease. Public health initiatives such as the National
Cholesterol Education programs have raised consumer awareness, resulting
in the advocate for healthy foods with low cholesterol. Thus, the food indus-
try is under tremendous pressure to address this consumer concern.
Supercritical-CO2 fluid extraction is a great potential to revolutionize the
oil/fat industry. Many researchers (Dunford and Temelli, 1995) reported the
feasibility of supercritical fluid extraction of lipids from food without compro-
mising their organoleptic quality. Chao et al. (1993) used a similar extractor

TABLE 1.10
Solubility Data of Cholesterol in Supercritical-CO2 under Different
Operating Conditions
Run Pressure Temperature Density Solubility of
No. (MPa) (°C) of CO2 (g/L) Cholesterol (mg/L)
1 10 40 602.4 146.9
2 10 45 494.4 82.6
3 10 50 408.1 47.2
4 10 55 342.3 28.5
5 13 40 723.4 289.2
6 13 45 655.8 234.8
7 13 50 588.5 182.7
8 13 55 527.7 141.1
Source: Modified from Yeh, A., Liang, J.H., Hwang, L.S. 1991. Journal of the
American Oil Chemist Society, 68: 224–229.
configuration (Figure 1.4) as discussed earlier with three-stage separations,

to remove cholesterol. They applied pressure at each stage sequentially from
17, 11, to 4 MPa and were able to achieve higher selectivity for cholesterol
at the lower pressures. The results also showed that the fractions collected
from the third separator at 4 MPa contained concentrated cholesterol rang-
ing from 272 to 433 mg/100 g lipid. Furthermore, Chao et al. (1993) used an
operating pressure of 10–30 MPa and the temperature range of 30–50°C to
reduce the cholesterol level in ground beef.
Hardardottir and Kinsella (1988) also explored the removal of lipids and
cholesterol from fish muscle with the supercritical-CO2 fluid. They removed
more than 80%–99% cholesterol using fluid pressures of 14–35 MPa and
temperatures of 40–50°C. Although the authors noted limited effect on
lipid/cholesterol yield with increased extraction pressure and temperature,
increased extraction time from 3 to 9 h significantly (P < 0.05) increased the
yield. Yeh et al. (1991) used eight operating conditions shown in Table 1.10
to optimize their process and observed that at 10.3 MPa and 55°C operating
pressure and temperature, respectively, and the cholesterol level was reduced
from 2867 mg/100 g to 14.1 mg/100 g. The supercritical-CO2 fluid technology
was used to fractionate milk fat, which is an excellent raw material with
specific functionalities used in many products (Rizvi and Bhaskar, 1995).
Extracted cholesterol from anhydrous milk fat with the supercritical-CO2
fluid is used in conjunction with adsorbents (silica gel) to maximize yield, as
demonstrated by Huber et al. (1996).
1.6.4 Separation of Spices and Essential Oils

Spices have strongly flavored or aromatic components that can be used
in small quantities in food as a preservative or flavoring ingredient. Chili

(Capsicium species), ginger (Zingiber officinalis), and pepper (Piper nigrum L.) are
classic pungent flavorings, while ginger and chili have additional neutraceu-
tical values. These products have high economic value in their concentrated
form. The extraction of spices is usually carried out in two stages: the first
stage separates the pungent oleoresins and the second stage the essential oil
fractions. Essentials oils are typically volatile terpenes and esters. Essential
oils are concentrated from pure plant extracts that have long been revered
for their therapeutic applications and are derivatives from flowers, leaves,
stems, berries, rinds, resins, or roots of plants (Lee et al., 2000; Liu et al., 2009;
Sanchez-Vicente et al., 2009). These are very important ingredients and food
additives of high value.
Catchpole et al. (2003) performed a detailed study on the extraction of spices
and essential oils using supercritical-CO2, propane, and dimethylether flu-
ids. They reported ginger to be the easiest of all spices in terms of optimized
yield relative to pressure and temperature, whereas capsaicin in chili could
be extracted at moderate pressure and temperature especially with the use of
modifiers. Chili oil fraction contains fatty oil and carotenoids and is specu-
lated that the fatty oil acts as a modifier for capsaicins (Peusch et al., 1997).
Perakis et al. (2005) extracted black pepper oil with much duress, because of
its viscous characteristics, thus requiring higher pressure and moderate-to-
high temperatures. The use of supercritical propane for extracting spices was
reported by Illes et al. (2000). They found propane to adequately extract fatty
oils, tocopherols, and carotenoid but was inadequate for capsaicins, whereas
CO2 was adequate for capsaicinoids, fatty oils, and tocopherols but not for
carotenoids.
Figure 1.10 shows the supercritical extraction of ginger with three extrac-
tion fluids (CO2, propane, and dimethyl ether) (Catchpole et al., 2003). Propane
gave the lowest yields, whereas dimethyl ether gave the highest yield. They
160
120
Yield (g/kg)
80
40
0
0 5 10 15 20 25
Solvent (kg/kg ginger )
CO2, 313 K Propane, 323 K Dimethylether, 313 K
FIGURE 1.10
Supercritical extraction yield against solvent mass of ginger using CO2, propane, and dimethyl
ether. (Modified from Catchpole, O.J. et al. 2003. Journal of Agriculture and Chemistry, 51:
4853–4860.)

reported dimethyl ether to have mutual solubility with water. Ginger con-
tains high amount of volatiles, and CO2 extraction offers the advantage of
dividing the extract into oleoresins and essential oil fractions by using a
two-stage separation procedure with sequential pressure reduction.
Similarly, if propane or dimethylether is used, considerable heating is
required which ultimately results in thermal degradation, and a larger
energy requirement in the form of cooling, depressurization, and boiling
to recover the essential oils. In the case of ginger, the oxygenated frac-
tion was much greater than the steam-distilled oils, and the gingerols
in the oleoresin were extracted without decomposition. Oleoresins and
piperine from peppers were extracted with insignificant losses although
a longer processing time was required. Similar trends were observed for
chili and pepper (Figures 1.11 and 1.12). The extracts contained carotenoid
pigments, and those obtained with the supercritical-CO2 fluid were bright
red with pink residues, whereas those from propane and dimethyl ether
were dark red. The extract obtained from chili with the supercritical-CO2
fluid was a yellow viscous pastry semisolids, whereas those extracted with
dimethyl ether were yellow/black and liquid at room temperature with a
high quantity of water resulting in the dilution of the essential oil and pip-
erine content. Nguyen (1991) described the extraction of antioxidants from
Labiatae herbs (rosemary, sage, oregano, and thymus) with the supercriti-
cal-CO2 fluid at pressures in the vicinity of 50 MPa and temperature rang-
ing from 80°C to 100°C. The extracted oleoresin was precipitated into two
fractions at various pressures and temperatures. The first fraction consisted
of a greenish-brown, oil-soluble, heat-stable, resin containing less than 2%
essential oil and exhibiting remarkable antioxidant properties. The second
fraction was the essential oil containing more than 95 mL steam-distilled
oil per 100 g.
160
120
Yield (g/kg)
80
40
0
0 5 10 15 20 25
Solvent (kg/kg chili)
FIGURE 1.11
Supercritical extraction yield against solvent mass of chili using CO2, propane, and dimethyl
ether. (Modified from Catchpole, O.J. et al. 2003. Journal of Agriculture and Chemistry, 51:
4853–4860.)

200
150
Yield (g/kg)
100
50
0
0 5 10 15 20 25 30 35
Solvent (kg/kg pepper)
FIGURE 1.12
Supercritical extraction yield against solvent mass of pepper using CO2, propane, and
dimethyl ether. (Modified from Catchpole, O.J. et al. 2003. Journal of Agriculture and Chemistry,
51: 4853–4860.)
The use of the supercritical-CO2 fluid for the production of essential oils or
oleoresins from spices is possible by selecting suitable combination of pres-
sure and temperature. The oils extracted with supercritical technology were
found to be more valuable quality terms of their chemical composition and
higher percentage of sesquiterpene compounds. Supercritical fluid extrac-
tions with CO2 and hydrodistillation extraction methods were used to extract
essential oil (Juniperus communis L.) (Pourmortazavi et al., 2004) (Table 1.11).
Oils obtained by the supercritical-CO2 fluid and hydrodistillation showed
significant differences (P < 0.05), and the former was more selective and par-
ticularly efficient for the isolation of α-thoujone and limonene. Anitescu et al.
(1997) did a comparative analysis of coriander oil with supercritical-CO2 and
stream distillation (Table 1.12). They concluded that oils obtained by super-
critical extraction gave far better aroma compared with both the commercial
and hydrodistillated extracted oils.
1.6.5 Decaffeination of Coffee and Tea

Caffeine (1,3,7-trimethylxanthine) is a bioactive plant component com-
monly found in popular beverages such as teas (Camellia sinensis), coffees
(Coffee arabica, canephora, liberica) (since the 1820s), and soft drinks (Ashihara
and Crozier, 2001). Caffeine is a secondary metabolite, a product of nucleic
acid catabolism, and belongs to the group of compounds known as purine
alkaloids. Excessive ingestion of caffeine may cause certain health prob-
lems such as palpitations, gastrointestinal disturbance, anxiety, tremor,
increased blood pressure, dizziness, and insomnia (Ogita et al., 2002;
Jameel, 2003).

TABLE 1.11
Result of Comparative Analysis of the Supercritical Extraction to HD of Essential
Oil (Juniperus communis)
Pressure (MPa) 20 20 20 35 35 35 35 HD
Temperature (°C) 45 45 55 45 55 55 55
Dynamic time (min) 20 30 30 30 30 30 30
Modifier (μL) – – – – – 80 400
No. Compound RIa 1 2 3 4 5 6 7

1 α-Thoujone 928 25.1 26.2 26.9 17.0 13.0 22.0 4.0 –
2 α-Pinene 943 1.3 1.4 1.4 1.8 – 1.9 22.4 24.5
3 Sabinene 972 2.3 2.1 2.3 2.1 1.8 1.9 34.6 0.4
4 Myrcene 987 2.8 3.8 3.8 1.3 1.2 2.4 4.2 3.4
5 3-Carene 1007 35.0 36.8 37.1 19.9 17.1 29.1 2.6 39.4
6 Limonene 1020 22.0 24.2 22.9 9.0 8.6 15.6 6.1 –
7 Terpinolene 1071 2.6 – 2.6 0.8 2.1 2.7 2.7 3.1
Source: Modified from Pourmortazavi, S.M., Baghaee, P., Mirhosseini, M.A. 2004. Flavour and
Fragrance Journal, 19: 417–420.
a Retention index.
The aroma and flavor coupled with the stimulant effects come from
c affeine. Coffee beans have about 2%–3% caffeine, whereas tea leaves have
about 5% caffeine, depending on the variety and species (Jameel, 2003).
Decaffeinated coffee must contain less than 0.1% caffeine on a dry weight
basis, as specified by European Economic Commission (EEC) regulations.
Therefore, decaffeination of coffees and tea poses significant challenges to
both the producers and processors. The demand for decaffeinated coffee is
high on the world market. It accounts for more than 20% of coffee sales in
the United States, with a 50% growing demand among the adult population
(Jameel, 2003).
Research in genetics engineering to produce transgenic tea and coffee
plants deficient in caffeine is in progress (Uefuji et al., 2003). However, the
consumption of genetically modified products is still contentious globally,
and the supercritical-CO2 fluid extraction technology gives the best option
in combating these critical issues. The decaffeination of coffee and tea using
the supercritical-CO2 fluid extraction technology is among the first-known
commercial operation in the food industry.
In the past, methylene chloride was used for decaffeination of coffee
with one cycle of production lasting from 24 to 36 h, whereas the end prod-
ucts usually contain toxic residues, thus posing more harm than caffeine.
Owing to its suspected carcinogenic effect, the FDA placed regulations
against methylene chloride used. However, decaffeination process with
the supercritical-CO2 fluid can be accomplished on green coffee and/or
roasted coffee beans or tea leaves without deleterious effects on the flavor

TABLE 1.12
Results of Comparative Analysis of Essential (Coriander) Oil between
Supercritical-CO2 Data to Steam Distillation Processes
Comm. Steam Supercritical-CO2
No. Compounds Kovats RI Oil Distillation Oil Oil
1 α-Thujene 928 Trace (Tr) Tr 0.1
2 α-Pinene 936 3.3 2.3 2.8
3 Camphene 951 0.6 0.4 1.5
4 Sabinene 975 0.1 0.3 0.9
5 β-Pinene 980 1.0 0.3 0.9
6 β-Myrcene 990 1.2 0.8 1.0
7 Δ3-Carene 1006 1.1 0.3 0.3
8 Limonene 1030 2.4 2.3 2.7
9 1,8-Cineole 1033 Tr 0.1 0.1
10 Linalol 1103 63.8 62.8 61.9
11 Camphor 1147 5.5 5.6 5.6
12 Menthol 1174 Tr 0.1 0.1
13 ρ-Cymen-8-ol 1184 Tr 0.1 0.3
14 cis-Hex-3-enyl 1186 Tr 0.1 0.2
butyrate
15 α-Terpineol 1192 1.0 0.9 0.6
16 β-Citronellol 1226 0.1 0.3 0.2
17 Neral 1241 0.1 0.1 0.2
18 Carvone 1245 0.5 1.0 1.0
19 Anethole 1287 0.7 0.4 0.4
20 Carvacrol 1299 Tr 0.1 0.2
21 Neryl acetate 1363 0.1 0.1 0.2
22 Geranyl acetate 1382 1.0 1.8 2.4
23 β-Caryophylene 1428 Tr 2.1 0.8
24 α-Humulene 1463 Tr 0.3 0.2
25 Eugenyl acetate 1526 – Tr 0.2
26 β-Caryophylene 1594 Tr Tr 0.2
27 Unidentified 3.8 2.9 1.7
Compounds
Source: Anitescu, G., Doneanu, C., and Radulescu, V. 1997. Flavour and Fragrance Journal,
12: 173–176.
even after 10 h of processing, and many patents already exist for such
processes.
The process requires charging the extraction vessel containing the c offee
beans with CO2 at a pressure of 7–22 MPa and a temperature of 31°C. Caffeine
is dissolved in the supercritical-CO2 fluid stream, which subsequently enters
a washing tower, or alternatively activated carbon scrubbers, distillation,
recrystallization, or reverse osmosis are used in some instances to entrain
caffeine. The method can strip coffee of its caffeine content (0.7%–3%) by

71%–97% (Caragay and Little, 1981). The caffeine recovered is sold for medic-
inal purpose and/or for use in soft drinks. Peker et al. (1992) reported that
soaking raw coffee beans in water prior to processing enhances the rate of
decaffeination.
1.6.6 Fish Oil Concentration

Fish oils are characterized by a high percentage of unsaturated, straight-
chain fatty acids ranging from C14 to C22 with one to six double bonds. They
contain essential fatty acids (EFAs) and polyunsaturated fatty acids (PUFAs),
grouped into omega-6 and omega-3 EFAs. The main sources of omega-3 are
flaxseed, walnut, and marine plankton and fish. This review would focus
on omega-3 oils derived from fish. Eicosapentaenoic acid (EPA) and decosa-
hexaenoic acid (DHA) are predominant in fish oil and have been reported
to contribute to the prevention of atherosclerosis, heart attack, depression,
and cancer if consumed in sufficient quantities (Chow, 2000). Fish oil deriva-
tives in the form of omega-3 oils are in high demands as food additives. For
example, Ocean Nutrition’s ME-3TM Omega-3 powder is currently used in
several breads. In the United States, Wegman’s Food Markets, Rochester, NY,
launched breads fortified with MEG omega-3 fats, two slices of which offer
80–90 mg of omega-3 (Ohr, 2005). Encapsulated omega-3 fatty acid forms are
available for fortified bakery products.
Fish oils are processed as fatty acids or as methyl or ethyl esters which
are more stable than the free acids form (Espinosa et al., 2002). Fatty acids
are highly soluble in CO2 and as a result supercritical-CO2 fluid extraction
is a preferred method of fractionation. With this technology, it is possible to
separate heat-sensitive compounds (omega-3 fatty acids) and avoid toxic sol-
vent residues in the final product. The isolation and fractionation of omega-3
PUFA from fish, fish oil, and esters using the supercritical-CO2 fluid have
been studied by several researchers (Letisse et al., 2006; Rubio-Rodríguez
et al., 2008; Amiguet et al., 2012).
Amiguet et al. (2012) extracted omega-3 PUFA-rich oil from by-products
by supercritical-CO2 extraction at 35 MPa and 40°C, and produced 137 mg
of oil/g of dried by-products with 7.8 ± 0.06% EPA and 8.0 ± 0.07% of DHA.
Eisenbach (1984) fractionated the ethyl esters from cold fish oil using the
supercritical-CO2 fluid at a pressure of 15 MPa and an extracting temperature
of 50°C. Alkio et al. (2000) produced EPA and DHA with 50% and 90% purity,
respectively, from trans-esterified tuna oil using carbon dioxide. Temelli
et al. (1995) obtained the highest yield of omega-3 fatty acids at 35 MPa and
35°C without denaturing the protein during supercritical-CO2 fluid extrac-
tion. They also compared solvent (hexane) extraction to the supercritical-CO2
fluid as shown in Table 1.13. A higher concentration of omega-3 was achieved
with the supercritical-CO2 fluid (Table 1.13). At 25 MPa pressure and tem-
perature from 40°C to 80°C, no significant effect on yield was observed in
oil extraction from krill (Yamaguchi et al., 1986). Hardardottir and Kinsella

TABLE 1.13
Fatty Acid Composition of Fall Atlantic Mackerel Oil Concentrate Extracted with
Hexane and Supercritical-CO2 Fluid Extraction at a Pressure of 34.5 MPa and
a Temperature of 35°C and Fractionated by Gas Chromatography
GC Retention Time Supercritical-CO2
Fatty Acid (min) Hexane Extract Fluid Extract
C16:0 8.07 15.86 16.66
C16:1 8.53 6.65 7.94
C18:0 12.49 3.53 3.16
C18:1 13.09 3.59 3.16
C18:3 15.58 0.88 1.04
C20:0 18.05 11.22 7.84
C20:1 18.14 1.20 1.20
C20:5 (EPA, ω-3) 22.79 6.07 8.76
C22:5 30.08 1.34 1.38
C20:5 (DHA, ω-3) 31.43 8.58 8.97
EPA + DHA (ω-3) 14.65 17.73
Source: Modified from Tamelli, F., LeBlanc, E., Fu, L. 1995. Journal of Food Science,
60: 703–706.
(1988) did not see any yield change on the recovery of fatty acids in rain-
bow trout at operating pressure ranging from 13 to 35 MPa and temperature
range of 40–50°C.
1.7 Summary
The growing interests in natural foods have raised the demand for health-
promoting products of nonsynthetic origin. High-value functional substances
can be obtained from biological materials by using various purification and
separation methods. Many different commercially processes have been
developed for extracting bioactive components from agricultural materials.
High-value functional ingredients can be obtained from biological materials
by various purification and separation methods for developing nutraceutical
products. Extraction with organic solvents is a well-established method of
selective separation of specific constituents from food and semiluxury prod-
ucts. Extractants with a low-boiling point such as ethyl acetate, methanol,
dichloromethane, etc., are suitable for isolating valuable constituents from
products such as hops, spices, and oil seeds, and also for removing or reduc-
ing the level of less desirable accompanying substances such as nicotine and
caffeine.

Among the separation processes being used or developed are physical

and chemical processes such as centrifugation, filtration, membrane sepa-
ration, precipitation, chromatography, solvent extraction, supercritical fluid
extraction, crystallization, evaporation, and distillation. Extractions with
organic solvents are limited by numerous disadvantages, the solvents used
are also not very selective for the bioactive components, and consequently,
the resulting extracts are not as pure as synthetic chemical compounds. The
most organic solvents such as hexane, acetone, and methyl chloride are pro-
duced as by-products from hydrocarbons and petroleum that are known to
be highly toxic. The danger of high concentration of toxic residues in the food
products is prohibited in some instances by regulatory bodies, hence compli-
cating their application in foods as discussed above. A very important step in
the process is the complete removal of the solvents from extracts and its resi-
due as they are in part toxicologically objectionable. In addition, organic sol-
vents have a low selectivity. The challenge is to develop an efficient separation
economical system that meets food regulatory requirements. Nutraceutical
products must be purified in compliance to regulatory requirements and or
eliminate/minimize risk of adverse effects. So, many researchers applied the
supercritical-CO2 fluid extraction technology for the extraction of bioactive
components.
A supercritical-CO2 fluid extraction process offers a unique advantage of
adding value to agricultural material by extracting bioactive compounds
from agricultural raw material and by-products which are then used for func-
tional food development. The drawback of supercritical fluids is the difficul-
ties in extracting polar compounds and extracting compounds from complex
matrix where the phase interaction with the intrinsic properties of the prod-
uct inhibits its effectiveness. Some of these drawbacks can be ameliorated by
using small amounts of food-grade cosolvents (<10%) to achieve high extrac-
tion efficiency. However, much investigation is required to understand the
solvation effects on the targeted bioactive components being extracted.
The CO2 density, pressure, and temperature have been noted to have great
impacts on the results of the extraction process. By understanding the effect
of the parameters that influence the extraction process, the conditions can be
set to optimize yield and improve cost efficiency. Optimizing the parameters
that should be used to maximize yields and solubility of the targeted compo-
nents, many researchers attempted to use conditions that can be applicable
in large-scale applications. For example, nontoxic cosolvents and modifiers
could be acceptable for food processing, and therefore a number of research-
ers have opted to use food-grade cosolvents and modifiers in extraction
processes. The nature of the material used as a source for high-value com-
ponents, such as health-promoting components, governs the availability of
the compounds for the extraction process. The presence of other components
such as lipids may impede the process or elevate costs due to a prolonged
extraction time.

Although a high temperature in the extraction process generally increases

the solubility of the components in supercritical-CO2 fluids, the conditions
under which thermally labile-targeted compounds are negatively affected
should be considered. The intensity and the length of heat treatment affect
the health-promoting properties of the bioactive compounds. Therefore, ide-
ally, the extraction time and temperature should be optimum. Minimizing
such conditions also leads to a more economically viable process. Excessively
high flow rates may reduce the contact time between the solute and the sol-
vent and restrict the fluid flow in the sample if it becomes compacted. The
optimal flow rate appears to vary with the targeted molecule, and relatively
high flow rates have a negative effect on some components.
Sample matrix is also an important parameter that influences the solu-
bility and mass-transfer process during supercritical-CO2 fluid extraction.
Properties such as particle shape and size distribution, porosity and pore
size distributions, surface area, and moisture content influence solubility
and mass transfer. The presence of water (moisture content) in the sample
matrix during supercritical-CO2 fluid extraction also has an effect on the
extraction outcome. In order to improve the yield and quality of the high-
value extracted food components from raw material, a pretreatment of the
raw material is essential. Cell disruption is the most important pretreatment,
and this procedure can be conducted by several processes such as mechani-
cal, ultrasonic, pulses electric field, and nonmechanical treatments. With
improved processing conditions and reduced cost, high-value components
extracted from natural materials by supercritical-CO2 extraction process will
become even more economical at high throughput.
Supercritical-CO2 fluid extraction has been shown to be a viable a lternative
to the conventional solvent extraction technique to extract bioactive compo-
nents from agricultural materials. It offers a unique advantage of a dding
value to agricultural waste by extracting antioxidants and flavonoids
(lycopene from tomato skin, essential oils, flavor and fragrances, etc.), which
are then used for the fortification of foods and other applications.
The supercritical-CO2 fluid extraction technology can be utilized to pro-
vide healthy snack foods. The snack food industry is facing problems with
regard to fat/oil contents in their products which is becoming a greater
public health concern. A lot of cost is incurred reformulating products to
devoid products (French fries, onion ring, and other snack foods) of trans-
fats and high cholesterol content. Defatting and decholesterol treatment with
supercritical-CO2 fluid extraction have been demonstrated as applicable to
food products. Although most of the tests were conducted on dehydrated
products, research has shown successful application of the supercritical-CO2
fluid on high-moisture products where extraction could be accomplished
without compromising the organoleptic characteristics.
The supercritical-CO2 fluid extraction technology is available in the form
of single-stage batch process and could be augmented to multistage semicon-
tinuous batch coupled with multiseparation process. Although significant

accomplishments were obtained in the couple of years that should not war-
rant complacency, batch modes render the supercritical-CO2 fluid extraction
technology cumbersome for certain industrial applications, which has been
the drawback for broader commercial adaptations. However, the needs to
improve the design into continuous modes are growing. One possibility is
to integrate membrane technology into supercritical process. Study on the
membrane-supercritical-CO2 fluid has been attempted and needs an aggres-
sive pursuance. This concept would make feasible the extraction of bioactive
components from aqueous, less viscous, and pumpable substances in a con-
tinuous mode.
Supercritical-CO2 extraction could be cost-effective only under large-scale
production, which made it ideal for decaffeination of coffee, tea, and hops. It
is expensive but much more economical when compared with conventional
solvent extraction at high production scale. With improved processing condi-
tions and reduced cost, supercritical-CO2 fluid extraction will become even
more economical at low throughput. The demand for ultrapure and high
value-added bioactive compounds is redirecting the focus of the food and
pharmaceutical industries into seeking the development of “green” technol-
ogies for their products. Extracts from natural sources are key elements in
the manufacturing of health-promoting functional foods and ingredients.
Improving the efficacy of “green” separation processes and technologies is
critical to the use of bioactive components in health-promoting functional
foods and in nutritional supplements.
References
Abbas, K.A., Mohamed, A., Abdulamir, A.S., Abas, H.A. 2008. A review on supercriti-
cal fluid extraction as new analytical method. American Journal of Biochemistry
and Biotechnology 4: 345–353.
Alkio, M., Gonzalez, C., Jantii, M., Altonen, O. 2000. Purification of polyunsaturated
fatty acid ester from tuna oil with supercritical fluid chromatography. Journal of
the American Oil Chemists’ Society 77: 315.
Amiguet, V.T., Kramp, K.L., Maoa, J., McRae, C., Goulah, A., Kimpe, L.E., Blais, J.M.,
Arnason, J.T. 2012. Supercritical carbon dioxide extraction of polyunsaturated
fatty acids from Northern shrimp (Pandalus borealis Kreyer) processing by-
products. Food Chemistry 130: 853–858.
Anitescu, G., Doneanu, C., Radulescu, V. 1997. Isolation of coriander oil: Comparison
between steam distillation and supercritical CO2 extraction. Flavour and
Fragrance Journal 12: 173–176.
Ashihara, H., Crozier, A. 2001. Caffeine: A well-known but little mentioned com-
pound in plant science. Trends in Plant Science 6: 407–413.
Bashipour, F., Ghoreishi, S.M. 2014. Response surface optimization of supercritical
CO2 extraction of α-tocopherol from gel and skin of Aloe vera and almond
leaves. The Journal of Supercritical Fluids 95: 348–354.

Baysal, T., Ersus, S., Starmans, D.A.J. 2000. Supercritical CO2 extraction of beta-
carotene and lycopene from tomato paste waste. Journal of Agricultural and Food
Chemistry 48: 5507–5511.
Barton, P., Hughes, R.E., Hussein, M.M. 1992. Supercritical carbon dioxide extraction
of peppermint and spearmint. Journal of Supercritical Fluids 5: 157–161.
Bhattacharjee, P., Ranganathan, T.V., Singhal, R.S., Kulkarni, P.R. 2003. Comparative
aroma profiles using supercritical carbon dioxide and Likens–Nickerson extrac-
tion from a commercial brand of basmati rice. Journal of the Science of Food and
Agriculture 83: 880–883.
Boutekedjiret, C., Bentahar, F., Belabbes, R., Bessiere, J.M. 2003. Extraction of rosemary
essential oil by steam distillation and hydrodistillation. Flavour and Fragrance
Journal 18: 481–484.
Brunner, G. 2005. Supercritical fluids: Technology and application to food processing.
Journal of Food Engineering 67: 21–33.
Cadoni, E., De Giorgi, M.R., Medda, E., Poma, G. 2000. Supercritical CO2 extraction of
lycopene and β-carotene from ripe tomatoes. Dyes and Pigments 44:27–32.
Caragay, A.B., Little, A.D. 1981. Supercritical fluids for extraction of flavours and fra-
grances from natural products. Perfumer & Flavorsito 43: 54.
Carla, D.P., Deborha, D., Andrea, N. 2014. Separation of aroma compounds from
industrial hemp inflorescences (Cannabis sativa L.) by supercritical CO2 extrac-
tion and on-line fractionation. Industrial Crops and Products 58: 99–103.
Catchpole, O.J., Grey, J.B., Perry, N.B., Burgess, E.J., Redmond, W.A., Porter, N.G.
2003. Extraction of chili, pepper and ginger with near critical CO2, propane and
dimethyl ether: Analysis of the extracts by quantitative nuclear magnetic reso-
nance. Journal of Agriculture and Chemistry 51: 4853–4860.
Chang, C.H., Chyau, C.C., Hsieh, C.L., Wu, Y.Y., Ker, Y.B., Tsen, H.Y., Peng, R.Y. 2008.
Relevance of phenolic diterpene constituents to antioxidant activity of supercriti-
cal CO2 extract from the leaves of rosemary. Natural Product Research 22: 76–90.
Chao, R.R., Mulvaney, S.J., Bailey, M.E., Fernando, L.N. 1991b. Supercritical CO2 con-
ditions affecting extraction of lipid and cholesterol from ground beef. Journal of
Food Science 56: 183.
Chao, R.R., Mulvaney, S.J., Hanah, H. 1993. Effects of extraction and fractionation
pressures on supercritical extraction of cholesterol from beef tallow. Journal of
the American Oil Chemists’ Society 70: 139–143.
Chao, R.R., Mulvaney, S.J., Sanson, D.R., Hsieh, F.H., Tempesta, M. 1991a. Super
critical CO2 extraction of annatto (Bixa orellana) pigments and some character-
istics of the color extracts. Journal of Food Science 56(1): 80.
Chen, J., Shi, J., Yi, C., Sophia Xue, S., Jiang, Y., Ye, X. 2009. Effects of supercritical CO2
fluid parameters on chemical composition and yield of carotenoids extracted
from pumpkin. LWT-Food Science and Technology 43: 39–44.
Chen, K., Yin, W. 2009. Effects of entrainers on the purity of andrographolide extracted
from andrographolide raw material by SC-CO2. Separation and Purification
Technology 70: 207–211.
Cherchi, G., Deidda, D., De Gioannis, B., Marongiu, B., Pompei, R., Porcedda, S. 2001.
Extraction of Santolina insularis essential oil by supercritical carbon d ioxide:
Influence of some process parameters and biological activity. Flavour and
Fragrance Journal 16: 35–43.
Chow, C.K., 2000. Fatty Acids in Foods and Their Health Implications, Second edn.
(revised and expanded). Marcel Dekker Inc., New York, Basel.

Cocero, M.J., Calvo, L. 1996. Supercritical fluid extraction of sunflower seed oil with
CO2-ethanol mixtures. Journal of the American Oil Chemists’ Society 73: 1573–1574.
Coelho, J.A.P., Pereira, A.P., Mendes, R.L., Palavra, A.M.F. 2003. Supercritical carbon
dioxide extraction of Foeniculum vulgare volatile oil. Flavour and Fragrance Journal
18: 316–319.
Cygnarowicz-Provost, M., King, J.W., Marmer, W.N., Magidman, P. 1994. Extraction
of wool grease with supercritical carbon dioxide. Journal of the American Oil
Chemists’ Society 71: 223–225.
Cygnarowicz, M.L., Maxwell, R.J., Seider, W.D. 1990. Equilibrium solubilities of
β-carotene in supercritical carbon dioxide. Fluid Phase Equilibria 59: 57–71.
Da Porto, C., Decorti, D., Natolino, A. 2014. Water and ethanol as co-solvent in super-
critical fluid extraction of proanthocyanidins from grape marc: A comparison
and a proposal. The Journal of Supercritical Fluids 87: 1–8.
D’Andrea, A., Ferri, D., Maccioni, O., Van der Esch, S.A., Vitali, F. 1994. Application of
SFE Technology to the extraction of Active Substances from Azadirachtaindica
(A. juss) Seeds. In: H. Kleeburg (Ed.), Practice Oriented Results on the Use and
Production of Neem Ingredients and Pheromones. Drug and Graphic, Giessen.
de Melo, M.M.R., Silvestre, J.D., Silva, C.M. 2014. Supercritical fluid extraction of veg-
etable matrices: Applications, trends and future perspectives of a convincing
green technology. The Journal of Supercritical Fluids 92: 115–176.
Degnan, A.J., Elbe, J.H., Hartel, R.W. 1991. Extraction of annatto seed pigment by
supercritical carbon dioxide. Journal of Food Science 56(6): 1655.
Dunford, N.T., Temelli, F. 1995. Extraction and fractionation of canola phospholipids
with supercritical carbon dioxide and ethanol mixture. Journal of the American
Oil Chemists’ Society 70: 1009–1015.
Duquesnoy, E., Marongiu, B., Castola, V., Piras, A., Porcedda, S., Casanova, J. 2004.
Analysis of the Volatile Concentrate of the Leaves of Pinus nigra subsp. Chimie et
Biomas, Univeriste de Corse, France.
Eisenbach, W. 1984. Supercritical extraction. Physical Chemistry Chemical Physics 88: 882.
Espinosa, S., Diaz, S., Brignole, E.A. 2002. Thermodynamic modeling and process
optimization of supercritical fluid fractionation of fish oil fatty acid ethyl esters.
Industrial & Engineering Chemistry Research 41: 1516–1527.
Fasoli, E., D’Amato, A., Citterio, A., Righetti, P.G. 2012. Ginger rogers? No, ginger ale
and its invisible proteome. Journal of Proteomics 75: 1960–1965.
Favati, F., King, J.W., Friedrich, J.P., Eskins, K. 1988. Supercritical CO2 extraction of
carotene and lutein from leaf protein concentrates. Journal of Food Science 53: 1532.
Favati, F., King, J.W., Mazzanti, M. 1991. Supercritical carbon dioxide extraction of
evening primrose oil. Journal of the American Oil Chemists’ Society 68: 422.
Femenia, A., Garcia-Marin, M., Simal, S., Rossello, C., Blasco, M. 2001. Effects of
supercritical carbon dioxide (SC-CO2) oil extraction on the cell walls composi-
tion of Almond fruits. Journal of Agriculture and Food Chemistry 49: 5828–5834.
Ferreira, S.R.S., Meireles, M.A.A. 2002. Modeling the supercritical fluid extraction
of black pepper (Piper nigrum L.) essential oil. Journal of Food Engineering 54:
263–269.
França, L.F., Meireles, M.A.A. 2000. Modeling the extraction of carotene and lipids
from pressed palm oil (Elaes Guineensis) fibers using supercritical CO2. Journal
of Supercritical Fluids 18: 35–47.
Friedrich, J.P., List, G.R., Heakin, A.J. 1982. Petroleum-free extraction of oil from soy-
beans with supercritical CO2. Journal of the American Oil Chemists’ Society 59: 288.

Friedrich, J.P., Pryde, E.H. 1984. Supercritical CO2 extraction of lipid-bearing materi-
als and characterization of the products. Journal of the American Oil Chemists’
Society 61: 223.
Froning, G.W., Fieman, F., Wehling, R.L., Cuppett, S.L. 1994. Niemann. Supercritical
carbon dioxide extraction of lipids and cholesterol from dehydrated chicken
meat. Poultry Science 73: 571–575.
Gamse, T. 2003. Supercritical extraction of liquid mixtures. Lecture series Socrates
intensive course: High pressure chemical engineering process: Basic applica-
tions. Institut fur Thermische Verfahrenstechnik und Umwelttechnik, Graz,
Austria.
Ge, Y., Yan, H., Hui, B., Ni, Y., Wang, S., Cai, T. 2002. Extraction of natural vitamin E
from wheat germ by supercritical carbon dioxide. Journal of Agricultural and Food
Chemistry 50: 686–689.
Glisic, S.B., Misic, D.R., Stamenic, D., Zizovic, I.T., Asamin, R.M., Skala, D.U. 2007.
Supercritical carbon dioxide extraction of carrot fruit essential oil: Chemical
composition and antimicrobial activity. Food Chemistry 105: 346–352.
Gomez, A.M., Lopez, C.P., de la Ossa, E.M. 1996. Recovery of grape seed oil by liquid
and supercritical carbon dioxide extraction: A comparison with conventional
solvent extraction. The Chemical Engineering Journal 61: 227–231.
González, J.C., Fontal, O.I., Mercedes, R., Vieytes, J., Vieites, M., Botana, L.M. 2002.
Basis for a new procedure to eliminate diarrheic shellfish toxins from a contami-
nated Matrix. Journal of Agriculture and Food Chemistry 50: 400–405.
Gopalakrishnan, N., Narayanan, C.S. 1991. Supercritical carbon dioxide extraction of
cardamom. Journal of Agriculture and Food Chemistry 39(11): 1976–1978.
Griffin, B. 1999. Lipoprotein atherogenicity: An overview of current mechanism.
Proceedings of the Nutrition Society 58: 163–169.
Hardardottir, I., Kinsella, J.E. 1988. Extraction of lipid and cholesterol from fish mus-
cle with supercritical fluids. Journal of Food Science 53: 1656–1661.
Hawthorne, S.B., Langenfeld, J.J., Miller, D.J., Burford, M.D. 1992. Comparison of
supercritical CHClF2, N2O, and CO2 for the extraction of polychlorinated biphe-
nyls and polycyclic aromatic hydrocarbons. Analytical Chemistry 64: 1614–1622.
Herrero, M., Cifuentes, A., Ibáñez, E. 2006. Sub- and supercritical fluid extraction of
functional ingredients from different natural sources: Plants, food-by-products,
algae and microalgae. A review. Food Chemistry 98: 136–148.
Huang, W., Deng, Q., Xie, B., Shi, J., Huang, F., Tian, B., Liu, R., Huang, Q., Xue, S.
2010. Purification and characterization of an antioxidant protein from ginkgo
biloba seeds. Food Research International 43: 86–94.
Huber, W., Molero, A., Pereyra, C., de la Ossa, E.M. 1996. Dynamic supercritical CO2
extraction of cholesterol from anhydrous milk fat. International Journal of Food
Science 31: 143–151.
Illes, V., Daood, H.G., Perneczki, S., Szokonya, L., Then, M. 2000. Extraction of corian-
der seed oil by CO2 and propane at super- and subcritical conditions. Journal of
Supercritical Fluids 17: 177–186.
Jameel, S. 2003. Genetically decaffeinated coffee. Journal of Biosciences 28: 529–531.
James, W.P.T., Ralph, A. 2000. Policy and a prudent diet. In: Garrow, J.S., James,
W.T.P., Ralph, A. (Eds.), Human Nutrition and Dietetics. Churchill Livingstone,
Edinburgh, Great Britain.
Joslin, C.G., Gray, C.G., Goldman, S. 1996. Solubility in supercritical fluids from the
virial equation of state. Molecular Physics 89: 489–503.

Kassama, L., Shi, J., Mittal, G. 2008. Response surface analysis of lycopene yield
from tomato using supercritical CO2 fluid extraction. Separation and Purification
Kim, W.J., Kim, J.D., Kim, J., Oh, S.G., Lee, Y.W. 2008. Selective caffeine removal
from green tea using supercritical carbon dioxide extraction. Journal of Food
Engineering 89: 3–309.
King, J.W., Favati, F., and Taylor, S.L. 1996. Production of tocopherol concentrates
by supercritical fluid extraction and chromatography. Separation Science and
King, J.W., Hill Jr., H.H., Lee, M.L. 1993. Analytical supercritical fluid chromatogra-
phy and extraction, 2nd edn. In: R.C. Baetzold, B.W. Rossiter (Eds.), Supplement
and Cumulative Index Anonymous. John Wiley and Sons, Inc., New York. pp. 1–83.
Lee, W.Y., Cho, Y.J., Oh, S.L., Park, J.H., Cha, W.S., Jung, J.Y., Choi, Y.H. 2000. Extraction
of grape seed oil by supercritical CO2 and ethanol modifier. Food Science and
Biotechnology. 9(3): 174–78.
Letisse, M., Rozieres, M., Hiol, A., Sergent, M., Comeau, L. 2006. Enrichment of EPA
and DHA from sardine by supercritical fluid extraction without organic modifier.
I. Optimization of extraction conditions. Journal of Supercritical Fluids 38: 27–36.
Liu, D., Shi, J., Rodríguez Posada, L., Xue, J., Kakuda, Y. 2008. Separation of tocotri-
enols from palm oil by molecular distillation process. Food Review International
24: 376–391.
Liu, G., Xu, X., Hao, Q., Gao. 2009. Supercritical CO2 extraction optimization of
pomegranate (Punica granatum L.) seed oil using response surface methodology.
LWT—Food Science and Technology 42: 1491–1495.
Luengthanaphol, S., Mongkholkhajornsilp, D., Douglas, S., Douglas, P.L., Pengsopa,
L.I., Pongamphai, S. 2004. Extraction of antioxidants from sweet Thai tamarind
seed coat—Preliminary experiments. Journal of Food Engineering 63: 247–252.
Macias-Sanchez, M.D., Mantell, C., Rodriguez, M., Martinez de la Ossa, E., Lubian,
L.M., Montero, O. 2005. Supercritical fluid extraction of carotenoids and chlo-
rophyll from Nannochloropsis gaditana. Journal of Food Engineering 66: 247–251.
Marongiu, B., Porcedda, S., Caredda, A., De Gioannis, B., Vargiu, L., La Colla, P. 2003.
Extraction of Juniperus oxycedrus of Oxycedrus essential oil by supercritical
carbon dioxide: Influence of some process parameters and biological activity.
Flavour and Fragrance Journal 18: 390–397.
Martinez, M.L., Mattea, M.A., Maestri, D.M. 2008. Pressing and supercritical carbon
dioxide extraction of walnut oil. Journal of Food Engineering 88: 399–404.
Mendiola, J.A., Herrero, M., Cifuentes, A., Ibáñez, E. 2007. Use of compressed fluids for
sample preparation: Food applications. Journal of Chromatography A 1152: 234–246.
Mitra, P., Ramaswamy, G.S., Chang, K.S. 2009. Pumpkin (Cucurbita maxima) seed oil
extraction using supercritical carbon dioxide and physicochemical properties of
the oil. Journal of Food Engineering 95: 208–213.
Miyawaki, T., Kawashima, A., Honda, K. 2008. Development of supercritical carbon
dioxide extraction with a solid phase trap for dioxins in soils and sediments.
Chemosphere 70: 648–655.
Montañés, F., Corzo, N., Olano, A., Regiero, G., Ibáñez, E., Fornari, T. 2008. Selective
fractionation of carbohydrate complex mixtures by supercritical extraction with
CO2 and different co-solvents. Selective fractionation of carbohydrate complex
mixtures by supercritical extraction with CO2 and different co-solvents. Journal
of Supercritical Fluids 45(2): 189–194.

Montañés, F., Olano, A., Regiero, G., Ibáñez, E., Fornari, T. 2009. Supercritical technol-
ogy as an alternative to fractionate prebiotic galactooligosaccharides. Separation
and Purification Technology 66(2): 383–389.
Mukhopadhyay, M. 2000. Natural Extracts Using Supercritical Carbon dioxide. CRC
Press, Boca Raton, FL.
Nguyen, U. 1991. Process for extracting antioxidants from Labiatae herb. US Patent
No. 5017397.
Ogita, S., Uefuji, H., Choi, Y.E., Hatanaka, T., Ogawa, M., Yamaguchi, Y., Koizumi, N.,
Sana, H. 2002. Genetic modification of coffee plants. Journal of Plant Biotechnology
4: 91–94.
Ohr, L.M. 2005. Nutraceuticals and functional foods. Food Technology 59: 63–65.
Oliveira, A.L., Pozza, L.N.L., Santos, D.N., Kamimura, E.S.E., Vicente, E., Cabral, F.A.
2013. Supercritical extraction of coumarin from guaco (Mikania laevigata and
Mikania glomerata) for pharmaceutical applications A. The Journal of Supercritical
Fluids 83: 65–71.
Oszagyan, M., Simandi, B., Sawinsky, J. 1996. Supercritical fluid extraction of vola-
tile compounds from Lavandin and Thyme. Flavour and Fragrance Journal 11:
157–165.
Ozcan, M., Tzakou, O., Couladis, M. 2003. Essential oil composition of Turkish herbal
tea (Salvia aucheri Bentham var. canescens Boiss & Heldr.). Flavour and Fragrance
Journal 18: 213–226.
Papamichail, I., Loili, V., Magoulas, K. 2000. Supercritical fluid extraction of celery
seed oil. Journal of Supercritical Fluids 18: 213–226.
Peker, H., Srinivasan, M.P., Smith, J.M., McCoy, B.J. 1992. Caffeine extraction rates
from coffee beans with supercritical carbon dioxide. AIChE Journal 38: 761–770.
Perakis, C., Louli, V., Magoulas, K. 2005. Supercritical Fluid extraction of black pep-
per oil. Journal of Food Engineering 71(4): 386–393.
Perrut, M. 2000. Supercritical fluid applications: Industrial developments and eco-
nomic issues. Industry and Engineering Chemistry Research 39(12): 4531–4535.
Peusch, M., Mueller, S.E., Petz, M., Mueller, A., Anklam, E. 1997. Extraction of cap-
saicinoids from chillies (Capsicum frutescens L.) and paprika (Capsicum annuum
L.) using supercritical fluids and organic solvents. Zeitschrift für Lebensmittel-
Untersuchung und -Forschung 17: 177–186.
Poiana, M., Crispo, F., Manziu, E., Sicari, V., and Mincione, B. 1993. Supercritical car-
bon dioxide extraction of bergamot oil: Bergaptene content in the extracts, In
E. Reverchon, A. Schiraldi (Eds.), Atti de1 II Congresso i Fluidi Supercritici e le Loro
Applicazioni, Ravello, June 20–22, 1993, p. 113.
Porta, D.G., Porcedda, S., Marongiu, B., Reverchon, E. 1999. Isolation of eucalyptus
oil by supercritical fluid extraction. Flavour and Fragrance Journal 14: 214–218.
Pourmortazavi, S.M., Baghaee, P., Mirhosseini, M.A. 2004. Extraction of volatile
compounds from Juniperus communis L. leaves with supercritical fluid carbon
dioxide: Comparison with hydrodistillation. Flavour and Fragrance Journal 19:
417–420.
Quancheng, Z., Guihua, S., Hong, J. 2004. Concentration of tocopherols by supercriti-
cal carbon dioxide with cosolvents. European Food Research and Technology 219:
398–402.
Raventós, M., Duarte, S., Alarcón, R. 2002. Application and possibilities of supercriti-
cal CO2 extraction in food processing industry: An overview. Food Science and
Technology International 8: 269–284.

Ronyai, E., Simandi, B., Tomoskozi, S., Deak, A., Vigh, L., Weinbrenner, Zs. 1998.
Supercritical fluid extraction of corn germ with carbon dioxide-ethyl alcohol
mixture. The Journal of Supercritical Fluids 14: 75–81.
Reverchon, E. 1997. Supercritical fluid extraction and fractionation of essential oils
and related products. Journal of Supercritical Fluids 10: 1–37.
Reverchon, E., Poletto, M. 1996. Mathematical modelling of supercritical CO2 frac-
tionation of flower concretes. Chemical Engineering Science 51: 3741–3753.
Reverchon, E., Sonsi, G., Pota, F. 1992. Extraction of essential oils using supercritical
CO2: Effects of some process and preprocess parameters. Italian Journal of Food
Science 3: 187–194.
Rizvi, S.S.H., Bhaskar, A.R. 1995. Supercritical fluid processing of milk fat:
Fractionation, scale-up, and economics. Food Technologies 49(2): 90–97.
Rosa, P.T.V., Meireles, A.A.A. 2005. Rapid estimation of the manufacturing cost of
extracts obtained by supercritical fluid extraction. Journal of Food Engineering 67:
235–240.
Rozzi, N.L., Singh, R.K. 2002. Supercritical fluids and the food industry: Comprehensive
review. Food Science and Food Safety 1: 33–34.
Rubio-Rodríguez, N., de-Diego-Rupérez, S., Beltrán, S., Jaime, I., Sanz, M.T., Rovira,
J. 2008. Supercritical fluid extraction of the omega-3 rich oil contained in hake
(Merluccius capensis–Merluccius paradoxus) byproducts: Study of the influence of
process parameters on the extraction yield and oil quality. Journal of Supercritical
Fluids 47: 215–226.
Sahena, F., Zaidul, I.S.M., Jinap, S., Karim, A.A., Abbas, K.A., Norulaini, N.A.N.,
Omar, A.K.M. 2009. Application of supercritical CO2 in lipid extraction—A
review. Journal of Food Engineering 95: 240–253.
Saltzman, M.W., Sheppard, N.F., McHugh, M.A., Dause, R.B. 1993. Controlled anti-
body release from matrix of poly (ethylene-covinyl acetate) fractionated with a
supercritical fluid. Journal of Applied Polymer Science 48: 1793–1800.
Sanchez-Vicente, Y., Cabañas, A., Renuncio, J.A.R., Pando, C. 2009. Supercritical fluid
extraction of peach (Prunus persica) seed oil using carbon dioxide and ethanol.
Journal of Supercritical Fluids 49: 167–173.
Sastry, S.V.G., Mukhopadhyay, M. 1994. Substrate hindrance in supercritical extrac-
tion of fragrance from jasmine flowers. 3rd International Symposium Supercritical
Fluids, France.
Shi, J., Kakuda, Y., Zhou, X., Mittal, G., Pan, Q. 2007a. Correlation of mass transfer
coefficient in the extraction of plant oil in a fixed bed for supercritical CO2.
Journal of Food Engineering 78: 33–40.
Shi, J., Khatri, M., Xue, J., Mittal, G., Ma, Y., Li, D. 2009a. Solubility of lycopene in
supercritical CO2 fluid affected by temperature and pressure. Separation and
Purification Technology 66: 322–328.
Shi, J., Mittal, G., Kim, E., Xue, S. 2007b. Solubility of carotenoids in supercritical CO2.
Food Review International 23: 341–371.
Shi, J., Xue, S., Jiang, Y., Ye, X. 2010a. Supercritical-fluid extraction of lycopene from
tomatoes. In: S. Rizvi (Ed.), Separation, Extraction and Concentration Processes in
the Food, Beverage and Nutraceutical Industries. Woodhead Publishing Limited,
UK, pp. 619–639.
Shi, J., Yi, C., Xue, J., Jiang, Y., Ma, Y., Li, D. 2009b. Effects of modifier on lycopene
extract profile from tomato skin using supercritical-CO2 fluid. Journal of Food

Shi, J., Yi, C., Ye, X., Xue, S., Jiang, Y., Ma, Y., Liu, D. 2010b. Effects of supercritical CO2
fluid parameters on chemical composition and yield of carotenoids extracted
from pumpkin. LWT-Food Science and Technology 43(1): 39–44.
Shi, J., Zhou, X. 2007. Solubility property of bioactive components on recovery yield
in separation process by supercritical fluid. In: John Shi (Ed.), Functional Food
Ingredients and Nutraceuticals: Processing Technology. CRC Press, USA, pp. 45–74.
Shi, J., Zhou, X., Kassama, L. 2007c. Correlation of mass transfer coefficient in separa-
tion process with supercritical CO2. Drying Technology International 25: 335–339.
Sihvonen, M., Jarvenpaa, E., Hietaniemi, V., Huopalahti, R. 1999. Advances in super-
critical carbon dioxide technologies. Trends in Food Science and Technology 10:
217–222.
Solana, M., Boschiero, I., Dall’Acqua, S., Bertucco, A. 2014. Extraction of bioactive
enriched fractions from Eruca sativa leaves by supercritical CO2 technology
using different co-solvents. The Journal of Supercritical Fluids 94: 245–251.
Sovava, H., Komers, R., Kucera, J., Jez, J. 1994. Supercritical carbon dioxide extraction
of caraway essential oil. Chemical Engineering Science 49: 2499–2505.
Spanos, G., Chen, H., Schwartz, S. 1993. Supercritical CO2 extraction of β-carotene
from sweet potatoes. Journal of Food Science 58: 817–820.
Taniguchi, M., Nomura, R., Kijima, I., Kobayashi, T. 1987. Preparation of defat-
ted mustard by extraction with supercritical carbon dioxide. Agricultural and
Biological Chemistry 51(2): 413–417.
Temelli, F., LeBlanc, E., Fu, L. 1995. Supercritical CO2 extraction of oil from Atlantic
Mackerel (Scomber scombrus) and protein functionality. Journal of Food Science 60:
703–706.
Tena, M.T., Valcarcel, M., Hidalogo, P., Ubera, J.L. 1997. Supercritical fluid extraction
of natural antioxidants from rosemary: Comparison with liquid solvent sonica-
tion. Analytical Chemistry 69: 521–526.
Tonthubthimthong, P., Chuapraset, S., Douglas, P., Luewisutthicat, W. 2001.
Supercritical CO2 extraction of nimbin from neem seeds—An experimental
study. Journal of Food Engineering 47: 289–293.
Tsuda, T., Mizuno, K., Oshima, K., Kawakishi, S., Osawa, T. 1995. Supercritical carbon
dioxide extraction if antioxidative components from tamarind (Tamarindus
indica L.) seed coat. Journal of Agriculture and Food Chemistry 42: 2671–2674.
Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., Sano, H. 2003. Molecular c loning
and functional characterization of three distinct N-methyltransferases involved
in the caffeine biosynthetic pathway in coffee plants. Plant Physiology 132:
372–380.
Uquiche, E., del Valle, J.M., Ortiz, J. 2004. Supercritical carbon dioxide extraction of
red pepper (Capsicum annuum L.) oleorisin. Journal of Food Engineering 65: 55–66.
Vega, P.J., Balaban, M.O., Keefe, S.F.O., Cornell, J.A. 1996. Supercritical carbon dioxide
extraction efficiency for carotenes from carrots by RSM. Journal of Food Science
61(4): 757–765.
Vargas, R.M.F., Barroso, M.S.T., Neto, R.G., Scopel, R., Falcão, M.A., de Silva, C.F.,
Cassel, E. 2013. Natural products obtained by subcritical and supercritical fluid
extraction from Achyrocline satureioides (Lam) D.C. using CO2. Industrial Crops
and Products 50: 430–435.
Wang, B.J., Liu, C.T., Tseng, C.Y., Yu, Z.R. 2005. Antioxidant activity of Bulpeurum
kaoi (Chao and Chuang) fractions fractionated by supercritical CO2. LWT-Food
Science and Technology 38: 281–287.

Wehling, R.L., Froning, G.W., Cuppett, S.L., Niemann, L. 1992. Extraction of choles-
terol and other lipids from dehydrated beef using supercritical carbon dioxide.
Journal of Agriculture and Food Chemistry 40: 1204–1207.
Wu, Z., Wu, S., Shi, X. 2007. Supercritical fluid extraction and determination of
lutein inheterotrophically cultivated Chlorella pyrenoidosa. Journal of Food Process
Xiao, J., Tian, B., Xie, B., Yang, E., Shi, J., Sun, Z. 2010. Supercritical fluid extraction and
identification of isoquinoline alkaloids from leaves of Nelumbo nucifera Gaertn.
European Food Research and Technology 231(3): 407–414.
Yamaguchi, K., Murakami, M., Nakano, H., Konosu, S., Kokura, T., Yamamoto, H.,
Kosaka, M., Hata, K. 1986. Supercritical carbon dioxide extraction of oil from
Antarctic krill. Journal of Agricultural and Food Chemistry 34: 904–907.
Yeh, A., Liang, J.H., Hwang, L.S. 1991. Separation of fatty acids esters from choles-
terol in esterified natural and synthetic mixtures by supercritical carbon diox-
ide. Journal of the American Oil Chemists’ Society 68: 224–229.
Yi, C., Shi, J., Xue, J., Jiang, Y., Li, D. 2009. Effects of supercritical fluid extraction
parameters on lycopene yield and antioxidant activity. Food Chemistry 113(4):
1088–1094.
Zhao, S., Zhang, D. 2014. Supercritical CO2 extraction of Eucalyptus leaves oil and
comparison with Soxhlet extraction and hydro-distillation methods. Separation
and Purification Technology 133: 443–451.
Zulkafli, Z.D., Wang, H., Miyashita, F., Utsumi, N., Tamura, K. 2014. Cosolvent-
modified supercritical carbon dioxide extraction of phenolic compounds from
bamboo leaves (Sasa palmata). The Journal of Supercritical Fluids 94: 123–129.

2
Solubility of Food Components in the
Supercritical-CO2 Fluid Process
John Shi, Sophia Jun Xue, and Siew Young Quek
CONTENTS
2.1 Introduction................................................................................................... 53
2.2 Solubility of Food Components in Supercritical Fluid............................ 55
2.3 Factors Affecting Solubility in Supercritical-CO2 Fluid.......................... 59
2.3.1 Solvent Selectivity............................................................................. 61
2.3.2 Polar and Nonpolar Solvents..........................................................63
2.3.3 Cosolvent Modification of Supercritical-CO2 Fluid
Phase Behavior..................................................................................64
2.3.4 Pressure Effects................................................................................. 73
2.3.5 Effect of Water Content in Material................................................77
2.3.6 Temperature....................................................................................... 81
2.4 Solubility Prediction.....................................................................................84
2.5 Equation of State........................................................................................... 88
2.6 Verification of Solubility of Lycopene in the Supercritical-CO2 Fluid....... 90
2.7 Summary........................................................................................................ 94
References................................................................................................................ 96
2.1 Introduction
Supercritical-CO2 fluid extraction has been used in the food industry for
three decades (Rizvi et al., 1986) and has accrued two decades of experi-
ence in the extraction of oils from plant materials. Initially, it is mainly
used in the decaffeination of coffee and tea and the extraction of spices
and hops. Those had to be in large-scale processing operations in order
to make them economically feasible and cost-effective. However, with the
development and advancements in processing conditions and equipment,
the cost-effectiveness improved allowing the small-scale productions of
ultrahigh-quality or high-margin product using supercritical-CO2 fluid
extraction for commercial distribution. The hottest area for supercritical
extraction application is for nutraceutical development, as supercritical
53
fluid extraction can be operated at mild conditions and will retain high
bioactivity of health-promoting components.
When a CO2 fluid is subjected to conditions exceeding supercritical tem-
perature and pressure, it is referred to as a supercritical-CO2 fluid. When the
fluid enters this phase, it adopts an array of gas and liquid properties as well
as increased solvent capacity and parameter sensitivity. A major difficulty
in utilizing supercritical fluid extraction for biomolecules is to measure and
predict their solubility in supercritical-CO2 as solvents at various pressures
and temperatures for process optimization. Lack of data for intermolecular
energy parameters, critical properties, and molecular refractions limit that
use for modeling and prediction of their solubility in the supercritical fluid
(Chimowitz and Pennisi, 1986). A series of commonly used supercritical flu-
ids as solvents and their respective critical points are listed in Table 2.1. The
solubility is the amount of a substance dissolved by the solvent at thermo-
dynamic equilibrium. For a multicomponent system (different components
in the analyzed matrix and a mixture of solvents), the solubility of various
components determines the selectivity and gives the opportunity of solv-
ing a separation problem. The critical temperature and critical pressure are
characterized by the point where the phase transition is no longer possible,
regardless of changes in pressure. Physical characteristics including diffu-
sivity, viscosity, and surface tension attribute to the increased solvent capac-
ity of the fluid, which may be exploited for extraction applications (Foster
et al., 1991).
TABLE 2.1
Critical Properties of Commonly Used Supercritical Fluids
Molecular Critical Critical
Fluid Weight (g/mol) Temperature (K) Pressure (MPa)
Carbon dioxide 44.01 304.1 7.38

Water 18.02 647.3 22.12
Methane 16.04 190.4 4.60
Ethane 30.07 305.3 4.87
Propane 44.09 369.8 4.25
Ethylene 28.05 282.4 5.04
Propylene 42.08 364.9 4.60
Methanol 32.04 512.6 8.09
Ethanol 46.07 513.9 6.14
Acetone 58.08 508.1 4.70
Ammonia 17.031 405.6 11.3
Chlorotrifluoromethane 104.46 302 3.92
Diethyl ether 74.12 467.7 3.64
n-Pentane 72.15 469.6 3.37
Source: Adapted from Liong, K.K., Wells, P.A., Foster, N.R. 1991. The Journal of
Supercritical Fluids, 4: 91–108.

Solubility of Food Components in the Supercritical-CO2 Fluid Process 55
To design a supercritical fluid extraction process, the solubility of solutes

in the supercritical fluid is fundamental. It influences the ideal operating
conditions of the extractor and recovery unit. Also, it controls the minimum
amount of supercritical fluid required to complete the extraction. In addition,
the primary characteristic of a supercritical fluid which makes it so attrac-
tive is its continuously changing solvating power. As the solvating power is
sensitive to temperature and pressure in the supercritical region, it can be
finely adjusted by varying temperature and pressure. The goal of optimiz-
ing temperature and pressure of a supercritical fluid extraction system is to
maximize the solubility of solutes and increase their upper limit of yield.
Over the last years, studies have focused on the experimental solubility data
in the supercritical region, solubility prediction, and improvement (modifying
solvent). As the molecular structures of the food components in the natural
material become more complex, the interaction between solutes, solvents, and
the solubility of biocomponents in supercritical fluid become more complex.
2.2 Solubility of Food Components in Supercritical Fluid

Because solubility is important to the process design of a supercritical fluid, a
lot of experimental work have been done on the measurements of solubilities
of a food component in supercritical fluids. Typical solubility behavior of a
solid solute in a supercritical fluid solvent is shown in Figure 2.1.
T3
T2
T1
T3 > T2 > T1
Mole fraction
P PL Pressure PU
min
FIGURE 2.1
Solubility behavior of a solid solute in the supercritical fluid-CO2 solvent.

In Figure 2.1, two convergence points are shown at PL and PU, and one
minima (Pmin). At low-pressure region (<Pmin), the solubility decreases as
pressure increases, whereas in the region of pressure Pmin < P < PU, the solu-
bility increases sharply with the increase in pressure. This region is usually
observed in the near critical and high compressed region of a supercritical
fluid solvent. It indicates that in the supercritical fluid extraction, the solubil-
ity can be controlled by pressure. In other words, it is related to the state of
the supercritical fluid. Hence the prediction of solubility is usually based on
the equation of state (EOS) of the solvent.
The crossover points PL and PU are attracting some interest as a method of
separating components with small difference in selectivity, such as isomers.
For a multicomponent system, the crossover point of each component may
not overlap, so there is a “crossover region,” where most of the crossover
points are located. At this point or in this region, the solubility of compo-
nents are similar. Johnston et al. (1987) pointed out that the crossover point,
in fact, is the turning point of the isotherm line of solubility:
∂(ln y 2 )
=0 (2.1)
∂T
where y2 is the mole fraction of the solute in gas phase and T is the tempera-
ture. For one component, a slight increase in temperature at PU will cause the
solubility to increase above the crossover pressure and decrease below the
crossover pressure. The results are reversed at PL. Under these conditions,
a retrograde region is formed. For multicomponent systems, at the cross-
over point of one component, its solubility will not change with temperature,
whereas others do, so the selectivity of those components increases. Through
several cycling of retrograde crystallization or solvation, the components
are separated. The operation becomes similar to distillation and requires a
temperature gradient. Foster et al. (1991) gave a detailed description of the
operation in the crossover pressure region. Because fewer data available in
the critical region, this “distillation” operation runs more often in the upper
crossover pressure than in the lower crossover pressure.
The other interesting observation is the sharp change in solubility with
pressure. In view of the macroscopic thermodynamics, the influence of
the pressure to the solubility can be explained by partial molar volume V2
directly:
∂(ln y 2 ) V2s − V2
= (2.2)
∂P RT[1 + (∂ ln φ 2 /∂ ln y 2 )T ,P ]

P
1

ln φ 2 =
RT ∫
V2 dp
0
(2.3)

where V2s is the saturated volume, ϕ2 is the fugacity coefficient, and R is the gas
constant. The partial molar volume is a differential quantity that describes
the solution behavior at a particular pressure. Here, fugacity coefficient ϕ2 is
the pressure integral of partial molar volume (Equation 2.3). Using the above
equation, the solubility behavior vs. pressure can be easily explained.
When the pressure is much lower than the critical point, the partial molar
volume is not a function of composition, so the equation is simplified to
∂(ln y 2 ) V2s − V2
= (2.4)
∂P RT
At low pressure, V2s V2 , hence the solubility decreases with pressure,

and as the pressure increases, V2s decreases more slowly than V2 . When the
partial molar volume equals the saturate volume, the solubility reaches its
minimum at a certain pressure. At high pressures, that are significantly
above the critical pressure, V2 increases slowly, when V2 exceeds V2s , because
of the repulsive force, the solubility will reach a maximum and decrease
slowly with the increase in pressure. In this region, the solubility does not
change much. The quickest increase in solubility occurs at a pressure cor-
responding to the minimum V2 . This theory explains the solubility behavior
in the supercritical region (Figure 2.1) and is supported by some experimen-
tal data (Patton and Luks, 1995). Unfortunately, due to the lack of adequate
experimental data for biosubstances, the practical application of the partial
molar volume model is limited. So, under these circumstances, the factors
that influence solubility are usually analyzed phenomenologically.
Biocompounds are usually present as a mixture in natural tissues
and are often not used in their pure form, thus they are regarded as one
pseudo-component in research. Some researchers will measure the pure
substance and others just measure the oil solubility. Staby and Mollerup
(1993) published a list for fish oil-related component. Bartle (1991) com-
piled solubility, which included solubility in the supercritical-CO2 fluid
of not only bioactive components, but also other chemical components.
Güçlü-Üstündağ and Temelli (2000) collected data on pure lipids in the
supercritical-CO2 fluid. Foster et al. (1991) collected some solubility data of
various chemical substances in various supercritical fluids. Recent solubil-
ity information for bioactive components from different sources is listed
in Table 2.1.
The extraction of natural products using supercritical fluids typically
occurs in three distinct phases (Brunner, 1987; Dobbs et al., 1987). Initially,
a straight line characterizes the constant extraction rate period and then a
falling rate period occurs followed by a diffusion-controlled rate period.
The straight line in the constant rate period likely occurs because compo-
nents that are easily transferred from the solid to fluid phase such as oil are
situated near the surface of the particles. Samples are often ground before

analysis, rupturing cells and causing oil to cover the surface of the resultant
particles. Therefore, the mass transfer during the constant extraction rate
period is mostly between the oil covering the particles and the solvent, until
solubility limits are reached. The falling extraction rate period is influ-
enced by diffusion in the solid phase (Smith et al., 1985). A combination
of the solvent’s ability of diffusion through the solids and back-diffusion
of any dissolved components to the surface characterize the phase of the
extraction process, as well as interactions of readily available components
that are still on the particle surface. After these components are removed,
the diffusion-controlled period commences. Particles found deeper within
the solid particles can now be obtained due to the absence of the compo-
nents that controlled the mass-transfer rates during the first two phases.
This typical behavior is observed in the extraction of buriti fruit in
Figure 2.2, along with the increase in solvating power at an elevated pres-
sure. Results at 20 and 30 MPa indicated that, as the pressure increases, the
slope of the constant extraction rate period increases, corresponding to the
rise in solute solubility for the components at the surface of the particles
are dissolved rapidly due to the increase in solvation power. Components
that are readily available on the surface of the particles, such as lipids and
oils, are relatively less energy-intensive than compounds that are found
deep within the particles, such as β-carotene. This is because the yield
obtained in the latter part of the extraction does not eclipse the drastic
increase in the necessary solvent compression costs (Dobbs et al., 1987).
6
Amount of oil (g)
3
20 MPa
2 30 MPa
0
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Amount of CO2 (g)
FIGURE 2.2
Extraction data of buriti fruit at 313 K and pressures of 20 and 30 MPa. (Modified from de
França, L.F. et al. 1999. The Journal of Supercritical Fluids, 14: 247–256.)

2.3 Factors Affecting Solubility in Supercritical-CO2 Fluid

There are numerous factors that affect the solubility of carotenoids in super-
critical fluids. As previously discussed, the solvating power of fluids in the
supercritical phase is very sensitive to changes in temperature and pressure.
However, other parameters including the flow rate of the fluid, the presence
of cosolvents and modifiers, storage conditions, extraction times, moisture
content of the samples, and compound morphology should also be consid-
ered. In addition, one must be cautious when dealing with labile compounds
that may degrade or isomerize when parameters reach or exceed certain
thresholds. A compilation of studies investigating the solubility of carot-
enoids in supercritical fluids is presented in Table 2.2. Issues such as discrep-
ancies and lack of data in certain categories may limit the acceptability of
some of the data. Discrepancies may arise from numerous factors including
sample impurities, different analytical approaches, and a lack of uniformity
in sample preparation.
The most common fluid for supercritical fluid extraction is supercritical-
CO2, because it is safe, cheap, nonflammable, and its critical state is easy to
obtain (31°C, 7.8 MPa). This critical temperature is close to the room tempera-
ture and it preserves the bioactivity of biocompounds. In food processing
and nutraceutical fields, it is approved as safe without declaration.
In the liquid and supercritical states, CO2 behaves very similarly to hydro-
carbon solvents with very low polarizability (Hyatt, 1984). In general, the
extractability of compounds with supercritical CO2 depends on the occur-
rence of the individual functional groups in these compounds, their molecu-
lar weights, and polarity. The nonpolar, lipophilic solutes have the greatest
solubility in the supercritical-CO2 fluid, whereas the introduction of polar
function groups will decrease the solubility. Extensive experiments have
revealed the following (Brunner, 1987):
• Nonpolar and slightly polar components with low-molecular weight

such as hydrocarbons and some typical lipophilic compounds can
be extracted at lower pressure.
• Solvent power of the supercritical-CO2 fluid for low-molecular-
weight compounds (<250) is high and decreases with increase in
molecular weight. Organics with the molecular weights greater than
400 are almost insoluble.
• The supercritical-CO2 fluid has high affinity with oxygenated
organic compounds of medium-molecular weight.
• Free fatty acids and their glycerides exhibit low solubilities.
• Pigments are even less soluble.
• Water has a low solubility (<0.5%, w/w) at temperatures below 100°C.

TABLE 2.2
Solubility of Bioactive Components in Supercritical CO2
Temperature Pressure
Substance (°C) (bar) Resources
Sterols Cholesterol 40, 50, 60 70–190 Singh et al. (1993)
Phenolic Syringic acid, vanillic acid 40–60 100–500 Murga et al. (2004)
compounds Echium, borage, and lunaria 10–55 60–300 Gaspar et al. (2003)
seed oils
Essential oil from black 16–20 60–350 Ferreira et al. (1993)
pepper
Fish oil 20, 40, 80, 60–650 Botch-Jensen and
120 Mollerup (1997)
Hazelnut oil 40–60 150–600 Ozkal et al. (2005)
Hippophae rhamnoides L. seed 30–45 150–300 Yin et al. (2002)
oils
Palm kernal oil 40–80 207–483 Hassan et al. (2000)
Tomato seeds oil 40–70 108–245 Roy et al. (1996)
Lipids and Fatty acid (C6, C12, C16) 40–80 25–300 Bharath et al.
fatty acids and triglyceride (C24, C36, (1993)
C48)
Triglycerides with acryl 40 150–350 Hammam (1992)
chain, phospholipid and
monoglyceride
Fatty acid Lauric acid 40 345–483 Norulaini et al.
(2004b)
Lauric acid and oleic acid 80 276–483 Norulaini et al.
(2004a)
Lauric, linoleic, myristic, 30–60 130–400 Maheshwari et al.
oleic, palmitic, and stearic (1992)
acid
Fatty acid 2-Ethyl-1-hexanol + 40–100 138 Ghaziaskar et al.
esters 2-ethylhexanoic acid (2003)
Fish oil fatty acid ethyl 10, 40, 70 20–220 Staby et al. (1993)
esters
Artemisinin 37–65 00–270 Xing et al. (2003)
α-Carotene in SC-CO2 or 40–70 60–340 Hansen et al.
halocarbon (2001)
β-Carotene 35–50 96–300 Sakaki (1992)
Caffeine, uracil, and 40–60 300 Burgos-Solorzano
erythromycin et al. (2004)
Capsaicin in SC-CO2 or 35–70 60–332 Hansen et al.
halocarbon (2001)
Fat-soluble vitamins A, D, E, 40, 60, 80 200–350 Johannsen and
and K Brunner (1997)
Limonene + linalool 40% 41, 42 70–100 Chafer et al. (2001)
limonene or 60% limonene
(Continued)


Solubility of Bioactive Components in Supercritical CO2
Temperature Pressure
Substance (°C) (bar) Resources
Limonene + linalool 45, 55 69–111 Berna et al. (2000)

Nitroaromatics and 50–100 100–450 Lewin-
adamantane Kretzschmar
et al. (2002)
Poly(α-hydroxybutyrate) 35–75 122–355 Khosravi-Darani
et al. (2003)
p-t-Butylcalix[n] arenes 40–80 150–300 Graham et al.
(1998)
Pure limonene and linalool 40, 50 69–111 Berna et al. (2000)
• Compounds with strong polar functional group such as ─OH,

COOH, are more difficult to extract. For instance, phenols with one
carboxyl and three or more hydroxyl groups cannot be extracted by
the supercritical-CO2 fluid.
• When pressure increases, less volatile, high-molecular weight, and
more polar components may be dissolved.
• The highly polar substances such as sugar and amino acids need
pressure up to 40 MPa to be extracted. Proteins, polysaccharides,
and mineral salts are insoluble.
2.3.1 Solvent Selectivity

An important feature of supercritical fluid solvent is that it can selectively
dissolve more of a certain compound from a mixture of compounds hav-
ing similar volatility but different chemical structures. Strictly speaking,
the selectivity of a solvent is the ratio of partition coefficient (K 2, K 2′ ) of two
components (Equation 2.5). For a solid–fluid system, it is often approxi-
mated as the ratio of binary solubility (y2, y 2′ ) in the supercritical-CO2 fluid
(Equation 2.6):
K2
S= (2.5)
K 2′
y2
S= (2.6)
y 2′

Two key factors that influence the selectivity are the volatilities of the
solids and the strength of solute–solvent interactions in the supercritical
phase. They are described in the following sections. Generally, for many

high-molecular-weight compounds, the selectivity increases with pres-

sure. For low-molecular-weight compounds, at low pressure, the selectivity
attains a maximum and then decreases with pressure, and a high level of
selectivity is usually reached at higher temperatures. As a nonpolar solvent,
supercritical-CO2 fluid has high selectivity on lipids, due to strong solute–
solvent interactions.
A cosolvent may increase the selectivity of polar components, as it increases
the solubility of components in the mixture at different folds. Usually, the
quantity of cosolvent is small, so the effect on selectivity is mainly due to the
change of interaction between the solute and the solvent. This effect is lim-
ited, but if the partial pressure of the solute is changed by the cosolvent, the
effect on selectivity is greater. Wong et al. (2001) showed that the selectivity
of terpinen-4-ol and other components increased at least twofold with water
(Figure 2.3).
A general rule is that the solubility increases with increasing pressure and
decreasing molecular weight. Molecular weight mainly influences the vapor
pressure of the components. High molecular weight substances are usually
not volatile. Hence, increased molecular weight leads to decreased solubility
of a homologous series in the supercritical-CO2 fluid. Materials with molecu-
lar weights above 500 Da have limited solubility. Low-to-medium molecular-
weight compounds such as aldehydes, ketones, esters, alcohols, and ethers
are very soluble. Low-molecular weight, nonpolar, aliphatic hydrocarbons
with up to 20 carbons and small aromatic hydrocarbons are soluble. If the
molecular weight and structure of two components are similar, a great
deal of more comprehensive analysis is required to determine selectivity.
60.0
50.0
40.0
% Composition
α-Pinene
α-Terpinene
30.0
γ-Terpine
Terpene-4-ol
20.0
10.0
0.0
Whole dried leaves Rehydrated whole dried leaves
FIGURE 2.3
Recovery of target analytes from tea leaves. Comparison of whole dried leaves and rehydrated
whole dried leaves. (Modified from Wong, V. et al. 2001. Molecules, 6: 92–103.)

Hammam (1992) investigated the different molecular weight of lipids and

found that, above 20 MPa, dilaurin was less soluble than trilaurin, reflecting
its more polar characteristic, whereas below 20 MPa, the effect is opposite
due to the lower-molecular weight of dilaurin.
2.3.2 Polar and Nonpolar Solvents

Besides vapor pressure, solubility is strongly affected by the interaction of
the solvent and the solute. A biocomponent usually has an aromatic group
that has high polarity or other functional groups which have specific force
such as electron donor–acceptor properties. Obviously, these compounds
will have high solubility in polar solvent. Primary supercritical solvents are
listed in Table 2.1. Organic solvents are usually used in polymer devaporiza-
tion and petrol purification procedure.
The polarizability of CO2 is 2.65 × 10−24 cm3, less than that of all the hydro-
carbons other than methane (Dobbs et al., 1987). Under this condition, the
solubility of biocomponents (mostly hydrocarbons) in the supercritical-CO2
fluid will decrease several orders of magnitude below the ideal solubility
of a solid in a liquid. Xenon possesses a polarizability of 4.01 × 10−24 cm3
(Castellan, 1964), which is slightly greater than CO2 and close to the polariz-
ability of lower alkanes, such as ethane and propane which are used widely
in the petroleum field. It is suitable for solvating some polymers whose
molecular weights are approximately 100,000 Da (Krukonis et al., 1984).
Because of its convenient critical properties (Tc = 289.7 K, Pc = 5.8 MPa),
safety, and larger polarity, it may be well suited for extraction of bioactive
components. However, xenon is much more expensive than CO2, so its appli-
cation is limited.
Halocarbons (CFCs) are very good solvents in supercritical fluid extrac-
tion due to their high density, which is a good indication of strong solvat-
ing power. Their polarities are similar to diethyl ether (Abbott et al., 2000),
a solvent that is able to dissolve many solutes. Because of their effect on
the ozonosphere, the use of chlorofluorohydrocarbons is restricted by the
Montreal Protocol. Recently, some modified halocarbons with zero ozone-
depletion potential, such as difluoromethane (R-32), 1,1,1,2-tetra-fluoroethane
(R-134a), and 1,1,1-trifluoroethane (R-143a), have been used for extracting
biocomponents (Abbott et al., 2002). The solubility of these polar biocompo-
nents was increased by 10–20 times when compared with their solubility in
the supercritical-CO2 fluid under the same conditions. However, the critical
temperatures of these halocarbons range from 70°C to 100°C. The extraction
of biocomponents has to proceed near or under supercritical conditions to
avoid destroying the bioactivity.
Polar solvents, such as acetone, have strong solvating powers, but due to
the safety problems in food, they are not used. Generally, the common safe
polar solvents are water, ethanol, and sometimes oil. They have high critical
pressure and temperature and are often used as cosolvent.

2.3.3 Cosolvent Modification of Supercritical-CO2 Fluid Phase Behavior

Adding a cosolvent (entrainer or modifier) will dramatically increase solu-
bility. Usually, the amount of additive is 1–5 mol%. A quantity of 3.5 mol%
methanol in the supercritical-CO2 fluid will improve the solubility of cho-
lesterol sevenfold compared with pure CO2 at 15 MPa (Wong and Johnston,
1986) and increase the solubility of 2-aminobenzoic acid sixfold. Chandra
and Nair (1997) observed much higher extraction yields of carotene from car-
rots with the addition of hexane or chloroform to the supercritical-CO2 fluid,
and this higher yield results from a combined effect of increased solubility
and increased availability of the carotene by competing with sorption sites
within the matrix. Table 2.3 lists some material systems that show the effect
of a cosolvent. Generally, the role of a cosolvent in the supercritical fluid is
to increase the polarity and solvent strength of the pure supercritical-CO2
fluid while retaining its solubility sensitivity to temperature and pressure.
A suitable cosolvent is one that as a liquid can normally dissolve the solutes.
When considering safety, ethanol, ethyl acetate, and water are the best nat-
ural cosolvent for food-grade products and they are “generally recognized
as safe.” Recently, some new compounds have come into use as entrainers,
but they are cosolutes. They act as surfactants in the supercritical fluid-CO2
because of their ability to increase solubility. These include some modified
sugars (peracylated sugars) which have hydroxyl groups, will help those
philize hydroxylated compounds come into CO2 (Raveendran and Wallen,
2003).
The effect of cosolvents is based on the interaction between the solute and
the solvent or the solvent and the cosolvent. For the supercritical fluid extrac-
tion process, there exists an extraction stage and a regeneration stage (recycle
of the solvent). In the cosolvent, solute, and solvent system, the interaction
of the solute and the cosolvent is a key in the extraction stage, whereas the
solvent and the cosolvent interaction becomes important in the regenera-
tion stage. In the extraction stage, the solubility of the solute in the cosolvent
can be used to make the initial selection. In the regenerating stage, raising
temperature at a constant pressure is preferred, so the solvent and cosolvent
binary should have a wide two-phase region at that temperature and pres-
sure (Sunol et al., 1985).
The quantity of the cosolvent is usually small, as large quantity may cause
the critical points to move and may form multiphases. This is not expected
in the operation. Due to lack of multicomponent phase equilibrium data, the
mixture-phase behavior at different ratios cannot be predicted. It is usually
determined experimentally. Empirically, the quantities of a cosolvent are
controlled at about 5 wt%. That requires a flexible cosolvent system to obtain
the operation flexibility and efficiency. In addition, large quantities of cosol-
vent may coextract other components as by-products, which is not desired.
Teberikler et al. (2001) observed that 5 wt% ethanol made the deoiling pro-
cess for soybean lecithin possible at 17 MPa, whereas no oil was extracted

TABLE 2.3
Solubility Data and Cosolvent Effects
Cosolvents Results Resources
Cholesterol (1.75–6.0 mol%) Ethane-acetone produced the best Foster et al.

of acetone and solvate power (1993)
hexane
Cotton seed Ethanol or IPA Gossypol can be dissolved; Kuk and
phosphorus content doubled Hron (1994)
Fatty acids (palmitic 1–10 mol% Stearic acid: 4 mol% ethanol, 10–15 Koga et al.
acid and stearic ethanol and fold; 4 mol% octane, threefold, (1996)
acid) and higher octane, at 308.2 palmitic acid: 5 mol% ethanol,
alcohols (cetyl K, 9.9–19.7 12–13-fold, 5 mol% octane,
alcohol and stearyl MPa 8–11-fold, stearyl alcohol: 5 mol%
alcohol) ethanol, fourfold, mol% octane,
4–6-fold, cetyl alcohol: 3 mol%
ethanol, 2–3-fold 3 mol% octane,
threefold
Glucose Ethanol and Glucose dissolved and ternary and Dohrn et al.
water quaternary phase diagram was (1993)
obtained
Grape seeds Ethanol Phenolic compounds, 25%, highest Chafer et al.
5–30 wt% solubility (2004)
Maize Methanol 500 μL Final yield doubled and comparable Ambrosino
with organic solvent extraction et al. (2004)
o- and 3.5 mol% The solubility was increased Gurdial et al.
m-Hydroxybenzoic acetone or significantly and exhibited a (1993)
acid 3.5 mol% retrograde vaporization
methanol phenomenon and a common upper
crossover pressure for each ternary
system
Soybean lecithin 5 wt% ethanol Made deoiling process possible at Teberikler
or 10 wt% 170 bar et al. (2001)
acetone
Sterols 3.5 mol% Cholesterol–CO2–acetone (T = 35°C) Wong and
cosolvent: increase 6–7-fold, cholesterol–CO2– Johnston
acetone, methanol (T = 35°C) 8–9-fold, (1986)
ethanol, or stigmasterol–CO2–ethanol (T = 35°C)
methanol 6–9-fold
Sunflower seed oil Ethanol, up to Oil solubility in SC-CO2 greatly Cocero and
20%, 150–350 increases over the whole range of Calvo (1996)
bar and pressure and temperature conditions.
42–80°C Some phospholipids are coextracted
at levels directly proportional to the
added ethanol. A large amount of
them was recovered in the ethanolic
phase. Acidity of the extracted oil
was lower than that without alcohol.
Part of the free fatty acids was found
in the ethanolic phase
(Continued)


Solubility Data and Cosolvent Effects
Cosolvents Results Resources
Xanthines and Ethanol, Targeted component got higher Li and

cocoa butter 20–25 wt% solubility (twofold), and selectivity; Hartland
caffeine no change (1996)
Various binary, 3.5 mol% Increased 3–7.2 Dobbs et al.
ternary, and cosolvent (1987)
quaternary systems
when pure supercritical fluid-CO2 was used at the same pressure. On the
other hand, 5 wt% ethanol did not result in any coextraction of phospholip-
ids. However, when ethanol was increased to 10 wt% to accelerate the deoil-
ing process, the phospholipids were coextracted.
Yuan et al. (1997) pointed out that the Lennard–Jones energy param-
eter and the dipole moment of a cosolvent influenced the solute chemical
potential significantly, which is related to solubility directly. However, that
research is focused on qualitative analysis. If solubility is increased signifi-
cantly by the addition of small amounts of cosolvent, the economics of exist-
ing or proposed processes through reduction in the pressure and/or recycle
ratio could be improved (Dobbs et al., 1987). Furthermore, cosolvents could
lead to more selective extraction of components which have similar vola-
tility but different type of chemical forces and for compounds which have
extremely limited solubility in pure fluids, for example, biomolecules. Some
detailed studies have shown that the cosolvent may work differently on dif-
ferent solutes. For example, Wong and Johnston (1986) found that in a sterol
mixture, 3.5 mol% acetone will improve the solubility of cholesterol 5.7-fold,
stigmaterol 1.8- fold, and almost no effect on ergosterol at 35°C and 12.3 MPa.
Obviously, the selectivity of cholesterol increases. Dobbs and Johnston (1987)
observed the selectivity of benzoic acid–hexamethylbenzene increased
from 1.9- to 2.5-fold with the addition of 3.5 mol% methanol and the yield
also increased. However, Brunner (1983) found that the selectivity always
decreased with an increase in yield for hexadecanol–octadecane–CO2 sys-
tem using acetone as cosolvent. Generally, if a pair of nonpolar solutes is
chosen, a nonpolar cosolvent would increase each solubility about the same,
thus causing little change in the selectivity (Dobbs et al., 1986). Similarly,
a nonpolar cosolvent will not change the selectivity of a pair consisting of
either a nonpolar and a polar solute or two polar solutes.
Although the nature of CO2 drastically reduces or eliminates the solubility
of many polar molecules, this is easily remedied with the addition of polar
entrainers. Modifiers, such as methanol, increase the solubility of polar com-
pounds, but may also increase the critical temperature of the solvent. This
may be an issue for compounds that are thermally labile, thus entrainers
must be used with caution. Another possible liability of addition of modifiers

is product contamination. This may be solved with the addition of a separa-

tion step subsequent to the extraction process (Hansen et al., 2001).
Several studies have been conducted to observe the effects of entrain-
ers. Results from Chandra and Nair (1997) indicate that the addition of sol-
vent modifiers to the supercritical-CO2 fluid increases the solvating power
(Table 2.4a and b). Modifiers were hexane and chloroform, where chloroform
had a higher increase of the power. Chang and Randolph (1989) observed
that the solubility of β-carotene increased from the addition of toluene. The
highest solubility reported (Table 2.4b) was more than nine times higher
than the extraction without toluene under the same conditions, thus show-
ing a large β-carotene solubility enhancement factor. The influence of vari-
ous modifiers at a constant temperature, pressure, and solvent flow rate was
studied by Ollanketo et al. (2001). Figure 2.4 shows that the optimal results
from this particular study were obtained from the addition of acetone, which
provided a relative recovery of 94% in 15 min, thus saving time, energy, and
costs. In reality, however, ethanol is the only viable modifier that may be
used in large-scale food applications. Cygnarowicz et al. (1990) also reported
that ethanol functioned as a more effective entrainer than methanol and
methylene chloride. It appears that modifiers which are miscible with water,
such as acetone and methanol, provide higher recoveries than those which
are not, including hexane and dichloromethane. However, a disadvantage of
100
90
80
70
Relative recovery (%)
60
50
Acetone
40
Methanol
30 Ethanol
Hexane
20 Dichloromethane
No addition
10
0
0 10 20 30 40 50 60 70 80
Extraction time (min)
FIGURE 2.4
Relative recoveries of lycopene from powdered tomato skins at 110°C, 400 atm, and a CO2
flow rate of 1.5 mL/min with various modifiers. An 100% relative recovery represents the best
recovery result. (Modified from Ollanketo, M. et al. 2001. European Food Research and Technology,
212: 561–565.)

68
TABLE 2.4A
α-Carotene Solubility and Yield in Supercritical Fluids
Supercritical-CO2 Yield (g/g Raw Temp. Pressure CO2 Flow Rate
Extraction Solid Material Cosolvent(s) Material) × 10 6 (K) (MPa) (m3/h) × 10 3 Ref.
CO2 Carrot powder CO2 82.2 313 40.4 30–36 Chandra

and Nair
(1997)

5% hexane + CO2 92.7 313 60.6 48–51
5% chloroform + CO2 111.2 313 60.6 42–48
CO2 Carrots, ground, and CO2 + 5% ethanol 215a 303 3 – Barth et al.
dried (1995)
CO2 + 10% ethanol 250a 303 3 –
CO2 Carrot, freeze-dried, CO2 181.7 313 27.6 60 Sun and
and ground Temelli
(2006)
CO2 + 2.5% (w/w) canola oil 369.1 313 27.6 60
CO2 + 5% (w/w) canola oil 443 313 27.6 60
Note: –, not reported.

Values estimated from the graph.
a
Solubility of Food Components in the Supercritical-CO2 Fluid Process

TABLE 2.4B
β-Carotene Solubility and Yield in Supercritical Fluids
Yield (g/g Raw Solubility (g/g Temp. Pressure Density
Solvent Solid Material Cosolvent Material) × 10 6 Solvent) × 10 6 (K) (MPa) (kg/m3) Ref.
CO2 Crystalline synthetic – 1.6 298 21 921.2 Škerget et al. (1995)

trans-β-carotene, 95%
purity
N 2O β-Carotene, 97% purity – 6 288 5.1b 880 Jay and Steytler
(1992)
C2H6 β-Carotene, 97% purity – 13.4 288 25 452 Jay and Steytler
(1992)
C2H4 β-Carotene, 97% purity – 9.2 308 20 384 Jay and Steytler
(1992)
Xe β-Carotene, 97% purity – 59 300 27.3 2270 Jay and Steytler
(1992)
SF6 β-Carotene, 97% purity – 0.01 323 3 1800 Jay and Steytler
(1992)
C3H8 β-Carotene, 97% purity – 79.3 288 7.3b 510 Jay and Steytler
(1992)
CHClF2 β-Carotene, 97% purity – 31.3 288 8.0b 1231 Jay and Steytler
(1992)
NH3 β-Carotene, 97% purity – 0 288 7.3b 700 Jay and Steytler
(1992)
CCl2F2 β-Carotene, 97% purity – 41.7 288 4.8b 1345 Jay and Steytler
(1992)
n-C4H10 β-Carotene, 97% purity – 108.7 288 1.5b 570 Jay and Steytler
(1992)
(Continued)
69
70
TABLE 2.4B (Continued)
CO2 β-Carotene standard, – 2.6 313 27.6 897.1 Mendes et al. (1999)
95% purity
CO2 Carrot powder – – 313 40.4 – Chandre and Nair
(1997)
5% hexane + CO2 – – 313 60.6 –
CO2 Synthetic trans-β- – 0.4 313.2 12 718.9 Sabio et al. (2003)

carotene, 97% purity
CO2 + ethanol – 6 313.2 15 –
CO2 Tomato paste waste, 100.9 – 308 30 – Baysal et al. (2000)
dried, and ground
CO2 + 5% ethanol 114.8 – 308 30 –
CO2 + 10% ethanol 115.5 – 308 30 –
CO2 + 15% ethanol 120.6 – 308 30 –
CO2 Carrots, ground, and CO2 + 5% ethanol 150a – 303 3 – Barth et al. (1995)
dried
CO2 + 10% ethanol 193a – 303 3 –
CO2 β-Carotene standard from – 0.02a 313 6.8 190a Hansen et al. (2001)
carrots
CO2 β-Carotene standard, – 0.3 308 10 700 Sakaki (1992)
95% purity
CO2 β-Carotene standard, – 1.1 313 20 840.2 Johannsen and
95% purity Brunner (1997)
(Continued)

CO2 β-Carotene standard, – 1.9 313 30 910 Cocero et al. (2000)
98% purity
CO2 Tomato seeds and skin, 154.5 – 313 27.6 – Cadoni et al. (2000)
dried, and ground
CO2 β-Carotene standard and – 0.2 310 9.7 673 Subra et al. (1997)
synthetic β-carotene
CO2 Carrot, freeze dried, and 220.3 – 313 27.6 – Sun and Temelli
ground (2006)
CO2 Carrot, freeze dried, and CO2 + 2.5% (w/w) 387.1 – 313 27.6 –
ground canola oil
CO2 β-Carotene standard – 5.3 313 12 – Saldaña et al. (2006)
CO2 Carrots, dried, and – 0.3 313 12 – Saldaña et al. (2006)
ground
CO2 Pure crystalline – 23.3 343 40 853.8 Cygnarowicz et al.
β-carotene (1990)
CO2 + 1 wt% ethanol – 11.7 343 22.3 700.6
CO2 + 1 wt% – 15.6 343 23.4 716.5
methylene chloride
CO2 + 1 wt% – 4.9 343 18 612.6
methanol
CO2 Trans-β-carotene vegetable – 11.8 333 30 – Ambrogi et al. (2002)
oil (14.8%, w/w)
CO2 Paprika oleoresin – 0.7 333 30 – Ambrogi et al. (2002)
(Continued)
71
72
CO2 Red pepper flakes, 640 – 313 32 922 Uquiche et al. (2004)
ground, and dried
CO2 Ground paprika 57 – 308 20 – Gnayfeed et al. (2001)
CO2 All trans-β-carotene 0.1 307.6 8 490.9 Kraska et al. (2002)

synthetic, amorphous,
purity 97%
CCIF3 All trans-β-carotene – 0.4 308.9 12 1584.2 Kraska et al. (2002)
synthetic, amorphous,
purity 97%
CO2 All-trans-β-carotene, – 0.2 288 5b 825 L’air liquid (1976)
purity 97.1%
N 2O Synthetic, crystalline – 2.3 307.6 8 693.4 Poling et al. (2000)
all-trans-β-carotene
N 2O Synthetic, crystalline – 8.6 322.9 15 731.1
all-trans-β-carotene
Note: –, not reported.

a Values estimated from the graph.
b Saturated vapor pressure of the liquid.

using ethanol as a cosolvent is the fact that an additional step must be added
to the process in order to remove it from the final product. This procedure
requires the use of heat, thus posing the risk of isomerization of the targeted
compound (Sun and Temelli, 2006).
One of the major advantages of supercritical-CO2 fluid extraction is its
ability to produce nontoxic extracts. However, the addition of toxic organic
solvents essentially negates this rationale. Natural entrainers used in the
extraction of carotenoids include vegetable oils and waxes. However, Sovová
et al. (2001) indicated that the entrainer effect of vegetable oil was not as
great as ethanol, where the increase in β-carotene solubility being four times
smaller. The effect of vegetable oil as a modifier is limited due to its low
solubility in CO2. Canola oil has been suggested as a natural cosolvent, as
it is highly soluble in supercritical-CO2 fluids in comparison to β-carotene
and the results show that the carotenoid concentration nearly doubled from
canola oil addition. In addition, the effect of increasing the pressure is height-
ened by this modifier. Sun and Temelli (2006) hypothesized that this effect
may have occurred due to interactions between the modifier and the solid
material. Penetration of the oil into the vegetable matrix may swell it, thus
making it easier for the supercritical-CO2 fluid to penetrate the matrix. In
addition, the oil may help release carotenoids in the matrix by loosening the
cell structures and increasing solute exposure.
2.3.4 Pressure Effects

An increase in pressure at a constant temperature increases the solvating power
of the fluid. This allows extraction of a wider variety of carotenoids and thus
results in an increase in solvent density. Data from Johannsen and Brunner
(1997) demonstrated that solvating power of fluids such as CO2 increases as the
density increases. Figure 2.5 illustrates the increase in solubility of β-carotene
in supercritical-CO2 fluids would occur when the pressure of the system is
increased. Corresponding to this trend, the amount of required CO2 reduces
as the extraction pressure increases. An increase in pressure results in an
increase in fluid density, which might lead to a quick increase in the solubil-
ity of the targeted component. The solvating power of the supercritical fluid
would increase, and due to a decrease in the diffusion coefficient at higher
density, the interaction between the fluid and the matrix would be reduced
(Gordon and Bauernfeind, 1982). Sabio et al. (2003) noted that after a certain
point, yield changes when increase in the pressure/density was insignificant.
Results from Subra et al. (1997) indicate that, at lower pressures, the uncer-
tainty of solubility measurements increases. However, low solubility of the con-
densed phase, particularly those of β-carotene, occurs at pressures close to the
critical pressure of the solvent. The errors may be larger at lower pressures due
to the decreased absorbance (Sakaki, 1992). Discrepancies between data from
literature may be attributed to the different experimental techniques used as
well as the assortment of carotenoids and CO2 purities applied in the extraction

1.8
1.6
1.4
β-Carotene (mg)
1.2
0.8
0.6
12 MPa
0.4 15 MPa
20 MPa
0.2
0
0 200 400 600 800 1000 1200
CO2 (g)
FIGURE 2.5
β-Carotene extraction yields at various pressures and 323 K as a function of the amount of
carbon dioxide used in the extraction process. (Modified from Saldaña, M.D.A. et al. 2006.
The Journal of Supercritical Fluids, 37: 342–349.)
processes. The effect of pressure is enhanced by the addition of entrainers such

as canola oil (Sun and Temelli, 2006). Carotenoid yield data from an experiment
conducted by Sun and Temelli (2006) provided in Table 2.5 demonstrates that
the solubility of carotenoids increases as the pressure increases, however an
entrainer will enhance this effect under the same experimental conditions. The
authors of the study hypothesized that interaction between the plant material
matrix and the canola oil led to increased carotenoid yields. They proposed
that solvent’s ability to diffuse through the matrix may have been facilitated by
the canola oil, which may have caused the cellular structures within the source
material to swell and also release the carotenoids from the matrix. The nonpo-
lar nature of the supercritical-CO2 fluid and the canola oil may also influence
the results. Lastly, competition from the canola oil for adsorption sites with the
carotenoids may also enhance carotenoid release.
Cosolvents change the interaction of the solute and the solvent, and this
phenomenon is often called the “chemical effect.” Another effect that signifi-
cantly affects the solubility is the “physical effect” of temperature and pres-
sure. In supercritical-CO2 fluid extraction, the solubilities are often extremely
sensitive to pressure and temperature. That results in an important feature
of supercritical fluid extraction that allows the yield and the selectivity of
the solvent to be finely tuned by temperature and pressure above the critical
temperature. Usually, pressure is preferred as a control factor. The advantage
of pressure control is that the solubility can be changed on a nearly instanta-
neous basis, whereas the temperature control suffers from thermal lag and


TABLE 2.5
Data for Carotenoid Solubility and Yield in Supercritical Fluids
Yield (g/g Raw Solubility (g/g Temp. Pressure CO2 Flow Rate
Solvent Solid Material Cosolvent(s) Material) × 10 6 Solvent) × 10 6 (K) (MPa) (m3/h) × 10 3 Ref.
CO2 Microalga C. vulgaris, CO2 760 – 313 30 20.40 Gouveia et al.

slightly crushed (2007)
CO2 + soybean oil 840 – 313 30 20.40
CO2 + 5% ethanol 880 – 313 30 20.40
CO2 Carrot, freeze-dried, CO2 432.1 – 313 27.6 60 Sun and Temelli
and ground (2006)
CO2 + 2.5% 754.3 – 313 41.3 60
(w/w) canola oil
CO2 + 5% (w/w) 989.8 – 313 27.6 60
canola oil
CO2 Sweet potato tissue, fresh 43.6 – 311 41.4 840–1080 Spanos et al. (1993)
CO2 Sweet potato tissue, 77.3 – 311 41.4 840–1080
freeze-dried, and
ground (fine)
CO2 Sweet potato tissue, 47.0 – 311 41.4 840–1080
freeze-dried, and
ground (coarse)
CO2 Microalga CO2 + Methanol 152 – 313 20 11.9 Macías-Sánchez
Nannochloropsis gaditana et al. (2005)
CO2 Paprika powder CO2 – 0.6 333 30 5102.0 Ambrogi et al.
(2002)
CO2 Cyanobacterium, 0.7 – 313 20 6.1 Montero et al.
Synecoccus species (2005)
75
3.0
Tr = T/Tc = 0.8
0.9
2.0 1.0
Reduced density
1.1
1.2
1.0 CP
1.55
0.0
0.1 1.0 10.0
Reduced pressure Pr = P/Pc
FIGURE 2.6
Variation of the reduced density (ρr) of a pure component near its critical point.
the temperature gradients that limit the maximum useful speed of tempera-
ture increase, because the gradient elution requires the time for complete
mixing of two liquid streams (Smith et al., 1985). Therefore, in most cases, the
“physical effect” is usually referred to as the “pressure effect.” This extreme
pressure sensitivity of solubility can be explained by its compressibility,
which is expressed as a gradient of density over pressure. As the solubility
parameters are proportional to the density of the solvent–supercritical-CO2
fluid, the sharp variance of density near the critical region (Figure 2.6) shows
why solubility changes greatly at different temperatures and pressures. At
the critical point, the slope of density–pressure is almost vertical, which
means that the rate of change is close to infinite and results in extreme pres-
sure sensitivity.
To further understand the impact of pressure on the solubility, an enhance-
ment factor E was derived:
P
E = y2 (2.7)
P2sat
where P2sat is the solute saturated pressure. This equation removes the effect
of pressure and focuses on the solvent–solute interaction in the supercritical
fluid phase. For the sterols in the supercritical-CO2 fluid, at 35°C and 20 MPa,
the enhancement factors have almost the same value. In fact, the enhance-
ment factor varies very little for many organic solids. As seen in Figure 2.7, the
E values vary within a range of only 1.5 orders of magnitude for substances
with a variety of polar functional groups, whose actual solubilities may vary

6.5
Acridine
Antracence
6 Hexamethylbenzene
5.5
Log E
4.5
4
16 17 18 19 20 21 22
Density (mol/L)
FIGURE 2.7
Enhancement factors at various in supercritical fluid-CO2 solvent densities. (Modified from
Dobbs, J.M., Johnston, K.P. 1987. Industrial & Engineering Chemistry Research, 26: 1476–1482.)
by many orders of magnitude (Dobbs et al., 1987). These results indicate that
in supercritical-CO2 fluid extraction, pressure is primarily responsible for
the solubility, not the solute–solvent interaction.
Furthermore, vapor pressures of the components in the mixture have sig-
nificant impact on the selectivity. Vapor pressure measures the volatility of
solutes. If the components in the mixture have similar chemical structures,
their selectivity are mainly determined by their vapor pressure. A typical
example is the components in a sterol mixture that contains three sterols:
cholesterol, stigmaterol, and ergosterol. CO2 is surprisingly more selective
for cholesterol than ergosterol, despite the fact that both of them have very
similar chemical structures. The solubilities of these two components differ
by two orders of magnitude. The reason is that their vapor pressures are
different. Although their vapor pressure is as low as 10−11 MPa, which is neg-
ligibly small, the selectivity follows the ratio of pressure.
It should be mentioned that if an inert component is added to a mixture
which does not strongly interact with the extracted component but is also
volatile, that volatile component will decrease the solubility of the desired
component as its partial pressure is decreased by the added volatile compo-
nent. Water behaves in this manner with many biocomponents.
2.3.5 Effect of Water Content in Material

Approximately 80%–90% of the total weight of plant materials is attrib-
uted to water content. However, water content influences the efficacy of
supercritical-CO2 fluid extraction processes. Therefore, the drying process is
an important step in supercritical fluid extraction (Hopper and King, 1991).

A drying step is implemented prior to the extraction process to increase

efficiency. Many compounds such as carotenoids are sensitive to light, heat,
and oxygen, and therefore freeze-drying is an effective method to remove
moisture from samples while avoiding degradation. A disadvantage to this
process, however, is the cost. Therefore, optimizing the level of drying may
improve the efficiency of the process; alternatives to freeze-drying include
vacuum-drying, air-drying, and drum-drying. However, the drying pro-
cess should be carefully chosen to minimize any negative effect on the tar-
geted components, material matrix, or any other factors in the extraction
process. Oven-drying of raw material provided Spanos et al. (1993) a higher
yield, oxidative degeneration, and case hardening. In the case of drum-
drying, severe case hardening prevented the extraction of carotenoids. The
best pretreatment method found by Spanos et al. (1993) was the freeze-
drying process.
Depending on the polarity of the compound, the initial moisture con-
tent may improve or impede the yield results. Nonpolar compounds such
as α-carotene and β-carotene produce smaller yields with higher moisture
content (Weathers et al., 1999); however, yields of polar compounds such
as lutein increase with moisture. Figure 2.8 shows the effect of various
moisture levels on the results obtained for β-carotene. Possible effects of
water include an increase in distance that carotenoids must travel to reach
the solvent, and swelling of the cell matrix, which have negative and posi-
tive effects on the process, respectively. Data indicate that the presence of
water aided in the extraction of carotenoids, particularly in the beginning
900
800
β-Carotene (μg/g feed material)
700
600
500
400
300
0.80%
200
17.50%
100 48.10%
84.60%
0
0 50 100 150 200 250
Time (min)
FIGURE 2.8
The amount of β-carotene (μg) extracted per g carrot for moisture levels at 343 K, 55.1 MPa,
and 5% (w/w) canola oil concentration. (Modified from Sun, M., Temelli, F. 2006. The Journal of
Supercritical Fluids, 37: 397–408.)

of the process. The total carotene recovery from materials that were previ-
ously dried was quite similar to that from starting material which did not
undergo a drying process (Ambrogi et al., 2003).
Many natural products contain water and its solubility is close to oil, at
normal temperature (30°C) and pressure (10 MPa or so). Wiebe and Gaddy
(1941) measured the vapor phase composition of carbon dioxide–water
mixtures at various temperatures and pressures up to 70 MPa. Their mea-
surements revealed the similar solubility behavior of the water–supercriti-
cal-CO2 fluid system with the oil–supercritical-CO2 fluid system (Figure 2.9).
There is a minimum solubility at a certain pressure. Near this pressure,
water solubility changes greatly, and at high pressures and above the super-
critical region, water solubility increases slowly. King et al. (1992) obtained
more mutual solubility data on water–supercritical-CO2 fluid at lower tem-
peratures (down to 15°C) and at lower pressures (a little over 20 MPa), which
is the liquid and supercritical region. According to their data, over the tem-
perature range 15–40°C and pressure range 5.1–20.3 MPa, the solubility of
water in liquid and supercritical-CO2 fluid varies from 2.2 × 10−3 to 5.8 × 10−3
mole fraction. It is close to the oil solubility and that may be the reason why
water is extracted along with the oil. Further, as discussed above, if water
comes into the supercritical-CO2 fluid phase in large quantities, the partial
pressure of oil in the supercritical-CO2 fluid phase will decrease. That will
influence the solubility of oil in the supercritical fluid. The binary phase
0.008
0.007
Solubility (g  water/L. CO2 S.T.P)
0.006
0.005
0.004
0.003
0.002
25°C
0.001 31.04°C (Tc)
50°C
0
0 100 200 300 400 500 600 700 800
Totoal pressure, atm
FIGURE 2.9
Composition of phase rich in carbon dioxide. (Modified from Wiebe, R., Gaddy, V.L. 1941.
Journal of the American Chemical Society, 63: 475–477.)

(a) (b)
Pressure Pressure
0 Water rich CO2 rich 1 0 1

Mole fraction of CO2 Mole fraction of CO2
FIGURE 2.10
Pressure-composition diagram for CO2/water system. (a) Temperature below critical point of
CO2. (b) Temperature above critical point of CO2.
diagram is shown in Figure 2.10 (King et al., 1992). The turning point D in
Figure 2.10 corresponds to the minimum solubility. Dunford et al. (1998)
observed that the solubility of Atlantic mackerel oil decreases significantly
at moisture levels >40.5%.
However, for some proteins or other substances with hydroxyl groups,
their water-binding potential may help them dissolve in the supercritical-
CO2 fluid, like a cosolvent. Owing to its dual effects, the moisture content
needs to be optimized. Dunford et al. (1997) reported an optimum moisture
content of 10.2% for the extraction of oil and residual proteins from Atlantic
Mackerel. Figure 2.11 shows the typical effect of water on supercritical fluid
extraction of oil. Figure 2.11 also shows that the optimized moisture level,
from C1 to C2, for Atlantic Mackerel, is 0%–26% of moisture; the solubility
does not decrease significantly. Figure 2.11 proves again that the effect of
pressure is much larger than that of the solute–solvent interaction. If the
extracted component has no or weak interaction with water, before the par-
tial pressure of water changed significantly, water has almost no effect on the
solubility and extract yield of oil. Snyder et al. (1984) observed that moisture
level between 3% and 12% had little effect on the extraction of soybean oil at
a pressure of 1.7 MPa and a temperature of 50°C.
In terms of an industrial process, the low sensitivity to water content has
some advantages. The fact that in some regions the solubility is not sensitive
to water content will reduce the need for a stronger drying process. Samples
that do not require extensive drying will have lower drying cost, because it
is usually an energy-intensive process.

ymax
Oil solubility
C1 C2 C3 Water content
FIGURE 2.11
Oil solubility versus water concentration. (Plotted according to data of Dunford, N.T., Temelli,
F., Leblanc, E. 1997. Journal of Food Science, 62: 289–294; Dunford, N.T., Goto, M., Temelli, F. 1998.
Journal of Supercritical Fluids, 13: 303–309.)
2.3.6 Temperature
It is important to observe the temperature at which the targeted compo-
nents decompose and to keep the parameters realistic for use in the food
industry. Lycopene, for example, becomes unstable at 393°K, therefore
Ollanketo et al. (2001) ensured that experimental conditions did not exceed
383°K. Saldaña et al. (2006) noted that isomerization of β-carotene may not
occur at temperatures less than 343°K, although it was observed at this
temperature after approximately 2 h. Therefore, despite the fact that it may
be tempting to perform the extraction at a high temperature to increase the
solubility of the targeted component, the effect of the temperature and the
extraction time should be evaluated in terms of degradation. The crystal-
linity of solids such as β-carotene is not well preserved under high temper-
atures. However, this has an impact on the solubility because a crystalline
solid has a lower solubility than an amorphous solid due to the difference
in free energy and a relatively higher enthalpy of fusion (Hansen et al.,
2001).
The effects of temperature are harder to assess than that of pressure, for
an increase in temperature leads to an augmentation in solute vapor pres-
sure and a decrease in supercritical-CO2 fluid density, but the magnitude of
the change in density becomes smaller at elevated temperatures (Marentis,
1998). Spanos et al. (1993), using the supercritical-CO2 fluid, showed favor-
able results at lower temperatures when the pressure was low (13.8 MPa)

and also concluded that at a high pressure (41.4 MPa) the opposite effect
was observed where an increase in temperature produced higher extrac-
tion yields. Experimental data taken at an intermediate pressure (27.6 MPa)
indicates that temperature increases have no apparent effect. This behavior
may be explained by a complex balance effect between the density of the
solvent and the solute vapor pressure as the temperature is increased. A
decrease in density would lead to a reduced yield, however the increase in
vapor pressure of the pigments as the temperature increases should lead
to higher solubility (Macías-Sánchez et al., 2005). For example, at low pres-
sure (13.8 MPa), it is suggested that the increase in solute vapor pressure
could not compensate the relatively large drop in solvent density from the
increase in temperature. The opposite effect may have occurred at high
pressures, where the effect of the increase in solute vapor pressure over-
came the relatively small change in density when the temperature was
increased. The intermediate pressure would have a balance between the
two (Spanos et al., 1993).
At a constant density, solubility increases alongside a rise in tempera-
ture, due to increase in vapor pressure of the solid (Johannsen and Brunner,
1997). Figure 2.12 shows the relative extraction of lycopene at varying tem-
peratures when the pressure and CO2 flow rate were kept constant. A similar
relationship is observed from work done by Rozzi et al. (2002) in Table 2.6.
Experimental data from Hansen et al. (2001) suggest that the uncertainty of
solubility measurements increase as the temperature decreases.
100
90
80
70
Relative recovery (%)
60°C
60 85ºC
50 110ºC
40
30
20
10
0
0 10 20 30 40 50 60 70 80
Extraction time (min)
FIGURE 2.12
Relative recoveries of lycopene from tomato skins at a constant pressure of 40.5 MPa and a CO2
flow rate of 1.5 mL/min. (Modified from Ollanketo, M. et al. 2001. European Food Research and
Technology, 212: 561–565.)


TABLE 2.6
Data for Lycopene Solubility and Yield in Supercritical Fluids
Yield (g/g Raw Solubility (g/g Temp. Pressure CO2 Flow Rate
Solvent Solid Material Cosolvent(s) Material) × 10 6 Solvent) × 10 6 (K) (MPa) (m3/h) × 10 3 Ref.
CO2 Tomato paste waste, CO2 2.2 – 308 30 2040.8 Baysal et al.
dried, and ground (2000)
CO2 + 5% ethanol 6.8 – 308 30 2040.8
CO2 + 10% ethanol 4 – 308 30 2040.8
CO2 + 15% ethanol 4.1 – 308 30 2040.8
CO2 Tomato seeds and CO2 0.6a – 305 13.8 0.15 Rozzi et al.
skin (2002)
CO2 Tomato seeds and CO2 124.2 – 323 27.6 3 Cadoni et al.
skin, dried, and (2000)
ground
CO2 Purified lycopene CO2 – 3.9 313 30.4 – de la Fuente
from tomato paste et al. (2006)
CO2 Purified lycopene CO2 – 20.1 333 40.3 –
from tomato paste
Note: –, Not reported.

a Values estimated from the graph.
83
2.4 Solubility Prediction

Obtaining solubility data requires a lot of experimental work, so the ability
to predict solubility is important for design. The solubility can be predicted
through properties of pure substances. Its foundation is built on the thermo-
dynamics of solvent equilibrium state, which is described by phase equilib-
rium and chemical potential equilibrium. In the view of phase equilibrium,
the fugacity of species i in the supercritical phase is equal to the fugacity
of the pure solid. The compressed gas model (Prausnitz, 1969) is used to
describe this equilibrium on the solid–gas interface:
 Vi (P − Pisat ) 
yi Pφi = Pisat φsat
i exp   (2.8)
 RT 

Pisat exp[Vi (P − Pisat )/RT ]

yi = (2.9)
Pφi
where the subscript i means the component i and Vi is the volume of the
pure substance. The fugacity coefficient ϕ is calculated by the EOS. The most
widely used EOS is Peng–Robinson (P–R) equation and Redlich–Kwong
(R–K) equation.
Cygnarowicz et al. (1990) used this method to estimate the solubility of
β-carotene in supercritical carbon dioxide at temperature ranging from
40°C to 70°C and pressure ranging from 20 to 45 MPa, and the result agreed
well with the experimental data. Soave (2000) applied the R–K EOS and
Huron–Vidal mixing rules to calculate the fugacity coefficient. This method
contained two adjustable parameters and agreed well with the isothermal
experimental data.
In the view of chemical potential equilibrium, if the chemical potential
of component i is the same in each phase, then the following equation is
obtained to calculate the solubility:
Pivap exp(− ∆µ i /kT )exp[Vi (P − Pivap )/kT ]

yi = (2.10)
ρkT
where Δμ is the excess chemical potential, ρ is the density, and k is the

Boltzmann constant. Calculating the excess chemical potential Δμ is a great
challenge. Goldman et al. (1996) developed a method based on the Redlich–
Kwong EOS, and Lennard–Jones intermolecular potential (Equation 2.11):
 σ ij  12  σ ij  6 
µ ij = 4ε ij   −    (2.11)
 r   r  


where εij is the potential well depth for the interactions of components i and j
and σij measure their range. They obtained an expression for chemical poten-
tial directly in terms of the intermolecular potentials at infinite dilution of
components for ternary system:
∆µ 2
= − log(1 − bρ) − γz( x1 , x3 )log(1 + bρ) + y( x1 , x3 )
kT
 bρ  bρ  
 + γ  log(1 + bρ) −  (2.12)
 1 − bρ  1 + bρ  

where
3/ 2
 ε 
γ = 7.45  
 kT 
σ 323 σ3
y( x1 , x3 ) = 2x1 3
+ 2x3 233 − 1
σ σ
3
σ 12 σ 323
z( x1 , x3 ) = 2x1 3 + 2x3 3 − 1
σ σ
and ε and σ are the counterpart parameters of mixture, b is an adjustable

parameter, and x is the mole fraction of each component. This model passed
a simulation test and agreed well with the theory. The obvious advantage is
that it can be used to directly demonstrate the cosolvent effect through solute
chemical potential which is strongly dependent on the molecular interac-
tion. In the view of solute property, Politzer et al. (1993) built relationships
of molecular structure and solubility in the supercritical-CO2 fluid through
surface potential. The logarithm solubility shows a linear relationship with
the square of surface potential. Their work encompassed 22 solutes in CO2 at
308 K and two pressures: 14 and 20 MPa.
The above methods based on the thermodynamic equilibrium have a
strong theoretical background. However, the inherent weakness is that they
need extensive chemical physical data to characterize the solvent and the
solute. Therefore, they are usually applied to simple structures where prop-
erties have been fully investigated substance. That limits their application in
the bioproducts as those substances usually have complicated structures and
only a limited amount of physical data available for calculation.
Since solubility does not directly influence the mass transfer, sometimes a
qualitative analysis is used. The solubility parameter concept is a good tool
for representing the solubility level. It can be easily obtained from thermo-
dynamic properties. Even if there is a lack of thermodynamic data, it still can
be approximated by the UNIFAC model, which is based on summation of all
the functional group contributions. The obvious advantage of this method

is a simpler analysis that estimates the solubility based on the molecular

structure.
Giddings et al. (1968) obtained the correlation of solubility parameters δ by
extending the theory of the solubility power and using it to rapidly estimate
a solubility level in the condensed solvent:
V1(δ 1 − δ 2 )2
χ= + χs
RT
(2.13)
ρ 
δ = 1.25 Pc 0.5  r , SF 
 ρr.L 

where the subscript r stands for the reduced property and 1 is the solvent,
2 is the solute, χ is the total interaction parameter, and χs is the entropic inter-
action parameter. Fedors (1974) correlated chemical structure and solubility
parameter. They collected many thermodynamic properties of typical atoms
and function a group contributions. With this knowledge, the solubility
parameter and the molar volume for both solvent and solute were estimated.
In addition, a very simple correlation is obtained for temperature depen-
dence of solubility parameter as following:
R   V1  
2.27 1.27
V 
δ 22 = δ 12  1  + T1   − T2  (2.14)
 V2  V2   V2  

It is suitable for both polar and nonpolar liquids. When |T1 − T2| < 150°C

and both are at or below the normal boiling point of the liquid, this equation
can be simplified as
1.13 1.13
V  ρ 
δ 2 = δ1  1  = δ1  2  (2.15)
 V2   ρ1 

It is built on the relationship between the solubility temperature depen-

dency and the temperature dependency of density. When |T1 − T2| < 50, it
can be further simplified as
V2 = V1[1 + α(T2 − T1 )] (2.16)

δ 2 = δ 1[1 + 1.13α(T1 − T2 )] (2.17)

where α is the cubic coefficient of expansion and the prediction accuracy is

around 2%.

To further utilize the advantage of solubility parameters, Prausnitz et al.

(1986) built the relationship between solubility parameters and solubility,
where ΔHf is the change in enthalpy of fluid. It is valid for a nonpolar solute
in a nonpolar solvent.
∆H f  1 1  ν ϕ 2 (δ − δ 2 )2
ln y 2 =  − − 2 1 1 (2.18)
R  Tf T  RT

Later the reduced solubility parameter concept was developed by King

and Friedrich (1990):
δ1
∆= (2.19)
δ2
where δ1 and δ2 are the solubility parameters of the solvent and the solute,
respectively. It described solubility parameters in a more general way, and
even the relationship with partition coefficient can be built through the Δ
term. When plotting the data of various extraction systems, it showed appar-
ent correlation between partition coefficient and reduced solubility param-
eter, which was confirmed in one equation, that was formalized by Giddings
et al. (1968) as follows:
RT K 
ln  normal  = (2 − ∆ )∆ (2.20)
V2δ 22  K 

where subscript “normal” means low-pressure reference state. This implied

that the partition coefficients can be predicted by other well-known solutes
under similar conditions if their reduced solubility parameters were calculated.
All of the above correlations were based on thermodynamics and EOS, so
they have complicated forms. To simplify the calculations, the empirical mod-
els are still accepted by researchers. The empirical models of Chrastil (1982)
and Mitra and Wilson (1991) are based on the phenomenological method,
which builds the correlation of solubility with their effect factors directly.
One of the most widely used empirical models was made by Chrastil (1982).
This is a simple empirical correlation:
a1
ln C2 = k ln ρ1 + + a0 (2.21)
T
where
∆H
a1 = /R
R (2.22)
a0 = ln( M2 + kM1 ) + q − k ln M1


where M1 and M2 is the molecular weight of the component 1 and 2 is an

adjustable parameter. This correlation describes the fact that the solubility
is proportional to the solvent density. Because it relates solubility with CO2
density directly and has a high accuracy in prediction (4% error), it is used
widely in the supercritical-CO2 fluid extraction field (Gómez-Prieto et al.,
2002). Some modification of this model was made by Del Valle and Aguilera
(1988) (Equation 2.23), where C is the concentration of the solute, but it did not
improve the accuracy. However, it did successfully predict the solubility of
vegetable oil (Equation 2.24) in the temperature range of 293–353 K and the
pressure range of 20–80 MPa. It also showed that the solubility behavior of
some bioactive components such as lycopene was very similar to vegetable oil
(Roy et al., 1996) and that their solubilities can be predicted by this equation.
a1 a
ln C2 = k ln ρ1 + a0 + + 22 (2.23)
T T
 18, 708 2, 186, 840  10.724

C = exp  40.361 − +  × ρ ± 2.7 (2.24)
 T T2

Adachi and Lu (1983) developed a five-adjustable-parameter equation (ki

and ai are all adjustable parameters), however the accuracy did not improve
much. The determinant coefficient of regression for the solubility for hazel-
nut oil increased to 0.995 from 0.985 based on the prediction from the Chrastil
equation (Ozkal et al., 2005):
a1
ln C2 = (k1 + k 2ρ1 + k 3ρ12 )ln ρ1 + a0 + (2.25)
T
As for the solubility of the cosolvent, Walsh et al. (1987) gave an associated-
perturbed-anisotropic-chain theory and obtained a complicated phase equi-
librium calculation method. Usually, researchers choose suitable mixing
rules for EOS to simplify calculation.
2.5 Equation of State

EOSs are used to describe the thermodynamic states through the relation-
ship of state parameters such as temperature, pressure, and volume. In
supercritical-CO2 fluid extraction processing, EOS describes the behavior of
solute being dissolved in supercritical fluids. The Peng–Robinson equation
(Peng and Robinson, 1976) is usually used to describe a compressed gas. A
general form of Peng–Robinson equation is

RT aα
P= − (2.26)
V − b V (V + b) + b(V − b)
where
(RTc )2
a = 0.45724
Pc
α = [1 + m(1 − Tr )]2
m = 0.37464 + 1.54226ω − 0.26992ω 2
RTc
b = 0.07780
Pc
 Ps 
ω = −1.000 − log  
 Pc  Tr = 0.7

For mixtures, the following mixing rule is used to calculate the fugacity
coefficient:
n n
a= ∑∑ y y a
i=1 j=1
i j ij
aij = ai a j [1 − kij + (kij − k ji )yi ]

Redlich–Kwong (R–K) (Redlich and Kwong, 1949) is simplified to the fol-

lowing forms:
RT aα
P= − (2.27)
V − b V (V + b)
(RTc )2
a = 0.42747
Pc
α = [1 + m(1 − Tr )]2
m = 0.48 + 1.57 ω − 0.176ω 2
RT
b = 0.08664 c
Pc
 Ps 
ω = −1.000 − log  
 Pc  Tr = 0.7

Both P–R and R–K predict the state well in the compressed gas region, but
P–R shows better prediction in the saturated liquid field. Although P–R and

R–K are widely accepted by researchers, Schmitt and Reid (1986) pointed out
that these kinds of EOSs are not appropriate for solid solute system, as the
essential assumptions are applicable for liquid vapor system, which is con-
sidered the main reason, that the prediction of P–R is not perfect over a wide
pressure range even with optimized parameters. They gave a modified P–R
equation accounting for the solid solute and obtained better prediction over
a wide pressure range (from 5 to 45 MPa). Hartono et al. (2001) pointed out
that a two-parameter equation Mohsen–Nia–Moddaress–Mansoori (MMM)
was as accurate as the modified P–R equation for a biocomponent in the
supercritical region. An alternative choice is the virial EOS. The virial EOS
(Joslin et al., 1996) directly describes the multicomponent fluid mixture with
molecular interactions. There is no adjustable parameter, and the solubility
is obtained directly by the intermolecular potentials of the interaction of the
solvent, solute, and cosolvent. This method performs under supercritical-
CO2 fluid extraction operation conditions with four-body interaction level.
Although the above EOSs have good prediction performance, they require
a lot of critical physical properties of solutes, which are not easily available
and only few are available in the published literatures. Hence some empiri-
cal expressions are still widely used by researchers. For example, Saeki (1995)
expressed an empirical equation based on the power law and Maxwell ther-
modynamic relation. It has good agreement with some fluids such as neon,
hydrogen, deuterium, and carbon monoxide in the supercritical state.
EOSs have a central role in supercritical fluids, because they not only pre-
dict the solubility, but also give the qualitative phase behavior. Hence, more
complicated theories and more multiparameter empirical methods continue
to be developed to formulate the state more accurately. Although the present
model accuracy is limited by their character, they can be used to build most
of the known binary phase diagrams, which are the fundamentals of super-
critical fluid extraction.
2.6 Verification of Solubility of Lycopene

in the Supercritical-CO2 Fluid
To predict the solubility of lycopene in the supercritical-CO2 fluid under vari-
ous conditions, the compressed gas model is based on the phase equilibrium
model, a modified Peng–Robinson method in this study. This equation has
been derived from the equifugacity condition between the solute (lycopene)
and the solvent (CO2), where it is used to predict the phase performance of
solutes with an extensive range of volatility (Li et al., 2003). It is assumed that
the system pressure is significantly greater than the vapor pressure of the
solute, the saturated vapor of the pure solute at sublimation behaves like an
ideal gas, the solute is incompressible, and it is also vital to assume that the

solubility of the solvent in the solute is negligible. Then lycopene solubility

in mole fraction is calculated according to Equation 2.28 (Schmitt and Reid,
1986).
 P vp   1   Vs 
y 2 =  2    exp  2  (2.28)
 P   φ2   RT 

where y2 is the mole fraction of the solute (lycopene) at equilibrium in the

supercritical phase, P is the pressure, ϕ2 is the fugacity coefficient of the spe-
cies in the supercritical phase, P2vp is the vapor pressure of pure solute, V2s is
the molar volume of the solute (assumed to be independent of pressure), R is
the gas constant, and T is the given temperature. P2vp and V2s were acquired
from experimental and literature data. The molar volume of solid lycopene
is 604.2 cm3 mol−1.
To determine the fugacity coefficient, the simplified version of Schmitt and
Reid (1986) is used as Equation 2.29. They confirmed that certain variables
could be neglected because they were found to be sufficiently small:
  b2  
0.5
 b2   P(V1 + b1 )   a1  a2 
ln φ 2 ≈   (Z1 − 1) − ln  −  
  2.828RTb1   2 ×   −  b  
 b1   RT   a1  1 

 (V1 + 2.414b1 ) 
× ln   (2.29)
 (V1 − 0.414b1 ) 

where T is the critical temperature, and the relation of T and Tr and Tc is as
T
Tr = (2.30)
Tc
where Tr is the reduced temperature that is equivalent to the fraction of

the absolute temperature, Tc where the subscript c denotes critical param-
eters, Z1 is the compressibility factor that could be determined by using the
Nelson–Obert generalized compressibility charts (Bejan, 1988) where Pr = P/
Pcr, V1 is the molar volume of CO2 and is determined by the compressibility
Equation 2.31 (Balzhiser and Samuels, 1977):
Z1RT
V1 = (2.31)
P
Variable a is the parameter that describes attractive interactions between

molecules, and variable b is the parameter for volume exclusion and repul-
sion interactions. Therefore, the pure component parameters could either be

of subscript 1 or 2, where subscript 1 represents the solvent and subscript 2

represents the solute:
R2Tc2
a1 = 0.457 [1 + (0.3746 + 1.5423ω − 0.2699ω 2 )(1 − Tr0.5 )]2
Pc
−b ± b 2 − 4 ac (2.32)
2a
RTc
b1 = 0.07780 (2.33)
Pc
Parameters a2 and b2 are determined by nonlinear regression models of the

experimental data. The supercritical temperature and pressure of CO2 are
304.21 K and 73.825 bars, respectively, and were taken from thermodynamic
tables-hydrocarbon (Frenkel et al., 1997).
The acentric factor, ω , of a substance is defined as (Balasubramanian, 2003):
ω = − log Pr at Tr = 0.7 (2.34)

To obtain the values of ω, the reduced vapor pressure Pr at Tr = 0.7 is

required. The values of ω and some other literature data are tabulated in
many thermodynamic tables.
The lycopene solubility was measured under different pressure and tem-
perature conditions as listed in Table 2.7. The increase in lycopene solubility
with isothermal and pressure effects was due to the increase in density. It
was noticed in modeling calculations that the vapor pressure of lycopene
showed significant signs of increase as the temperature rose from 50°C to
70°C, which correlates well with other published data (De la Fuente et al.,
2006). According to the modified Peng–Robinson equation, model lines for
the solubility of lycopene in supercritical-CO2 are presented in Figure 2.13.
The determination of solubility through the modified Peng–Robinson equa-
tion was in reasonably close agreement with the experimental data albeit
with some obvious scattering. The scattering of data was more prominent
under low-density conditions. The plot portrays that the experimental and
the calculated plotted curves follow the same trend, where it is more appar-
ent that solubility increases with both the system pressure and the tem-
perature. Both curves differ slightly and do not follow each other closely at
50°C. This discrepancy could be caused by the very low solubility of lyco-
pene at 50°C, where accurate results are extremely sensitive to the method
of analysis for low-solubility conditions. In addition, Subra et al. (1997) also
indicated that the ambiguity of solubility measurements becomes higher at
lower pressures, and achieving solubility at the lowest temperatures also
amplifies the uncertainty. The 60°C curve for the experimental values fits

TABLE 2.7
Density and Solubility of Lycopene in Supercritical CO2 at 50°C, 60°C,
70°C, and 80°C, and for Pressures 200, 300, and 400 bar
Temperature
(°C) Pressure (bar) CO2 Density (kg/m3) Solubility (y 2 × 10 −6)
50 200 785.0 ± 27.3 0.651 ± 0.17

250 834.8 ± 21.6 0.776 ± 0.12
300 871.0 ± 32.9 0.802 ± 0.09
350 897.4 ± 41.2 0.821 ± 0.13
400 923.8 ± 39.2 0.845 ± 0.09
60 200 724.5 ± 28.4 0.902 ± 0.11
250 787.2 ± 19.6 0.989 ± 0.09
300 830.3 ± 30.2 1.014 ± 0.12
350 860.5 ± 33.5 1.057 ± 0.09
400 890.6 ± 29.3 1.056 ± 0.13
70 200 660.0 ± 19.8 1.150 ± 0.11
250 737.6 ± 21.6 1.231 ± 0.09
300 788.6 ± 23.5 1.290 ± 0.13
350 822.9 ± 33.1 1.304 ± 0.11
400 857.2 ± 29.7 1.340 ± 0.12
80 200 595.4 ± 16.7 1.790 ± 0.09
250 687.1 ± 18.9 1.872 ± 0.13
300 746.3 ± 21.3 1.847 ± 0.15
350 785.0 ± 22.7 1.799 ± 0.09
400 823.7 ± 31.2 1.712 ± 0.19
Source: Modified from Shi, J. et al. 2009. Separation and Purification Technology, 66:
322–328.
very closely to the calculated values. Therefore, for this particular experi-
mental procedure, it can be concluded that the most favorable conditions
for lycopene solubility are at 60°C and 70°C. The solubilities of lycopene at
50°C, 60°C, 70°C, and 80°C were determined in supercritical-CO2 for pres-
sures ranging from 200 to 400 bar. The low solubility was determined by a
single-pass flow apparatus, where off-line solute analysis was performed.
For this reason, it was necessary to ensure that all the solute was collected.
Though the solubilities were very low, the reproducibility of results was
reasonably good. Three replications were conducted for each condition,
and the deviation between measurements ranged from 7% to 10%, except
at some lower conditions where it ranged from 13% to 15%. The solubil-
ity values show the usual trend for solubility increase with both pressure
and temperature. An isothermal increase in solubility with pressure was
observed. Increasing the density of CO2 showed a distinct increase in solu-
bility. The measurement of lycopene solubility is affected possibly by factors

1.6
Mole fraction of lycopene (y2 × 10–6) 1.4
1.2
0.8
0.6
0.4
0.2
0
100 200 300 400 500
Pressure (bars)
FIGURE 2.13
Plot of mole fraction versus pressure for the solubility in supercritical CO2—a comparison
between experimental and calculated values: (♦) 50°C; (▪) 60°C; (▴) 70°C; (—) calculated results
from modeling. (Modified from Shi, J. et al. 2009. Separation and Purification Technology, 66:
322–328.)
such as the experimental method, sampling method, method of quantita-

tion, method of measurement of solubility, degradation of lycopene, and
the degree of purity of the starting material. The presented solubility data
were correlated using the modified Peng–Robinson equation. The proposed
model fits reasonably close to the experimental curve and provides reliable
solubility estimates. It is concluded that the most favorable conditions for
lycopene solubility are at 60°C and 70°C. At 80°C, noticeable signs of ther-
mal degradation were observed.
2.7 Summary
The solubility determines the capacity of supercritical-CO2 fluid extraction,
and measuring the solubility of targeted components in supercritical-CO2
fluid extraction will build a solid base for prediction and further process
designs. There are many factors that influence the solubility of biomole-
cules in supercritical fluids. The density, pressure, and temperature of the
solvent have been noted to have great impacts on the results of the extrac-
tion process. Other factors include the particle size, molecular structure,
and polarity of the targeted carotenoid. The state and composition of the
raw material has an effect due to matrix complexities including lipid, carot-
enoid, and carbohydrate fractions, as well as moisture content and storage

conditions. By understanding the effect of the parameters that influence

the extraction process, conditions may be set to optimize yield and cost
efficiency.
The measurement and prediction of solubility includes not only solubility
in supercritical-CO2 fluid extraction, but also the phase equilibrium data in
multisolvent systems, as the modification of supercritical-CO2 fluid extraction
will play a leading role in the future development which is based on the real-
ity that a lot of bioactive components have low solubility in supercritical-CO2
fluid extraction, and small quantity of a cosolvent can change the solubil-
ity dramatically. Cosolvents will decrease the pressures applied and further
decrease the operation cost, but how much cosolvent is suitable and how
much will the solubility change need more attention. So far, many research-
ers studying cosolvents are at the qualitative stage and require many more
experiments to determine the optimal conditions. The difficulty of prediction
is that cosolvents will change the polarity and phase state of the supercritical-
CO2 fluid extraction system. When determining the parameters that should
be used to maximize the carotenoid yield and solubility, many researchers
attempted to use conditions that may be applicable in large-scale applications.
For example, nontoxic cosolvents and modifiers would be acceptable for food
processing; therefore, a number of researchers opted to use canola oil and
ethanol. Although a high temperature in the extraction process generally
increases the solubility of components in supercritical fluids, the conditions
under which thermally labile-targeted compounds are negatively affected
should be considered. The intensity and the length of heat processing affect
the degree of isomerization, therefore ideally the extraction time and tem-
perature should be minimized. Minimizing such conditions also leads to a
more economically viable process.
Excessively high flow rates may reduce the contact time between the sol-
ute and the solvent and restrict the fluid flow in the sample if it becomes
compacted. The optimal flow rate appears to vary with the targeted mole-
cule; relatively high flow rates having a negative effect on some components,
yet no effect on others. The pressure increases carotenoid yields, however
high carotenoid contents obtained vary with the molecule. Few phase equi-
librium data in or near the critical region are available, which makes the
theory difficult to validate. So developing an accurate and convenient EOS
which can deal with multicomponents will be important. Through EOSs, the
solubility and the phase diagram can be determined, which is a design base
of supercritical fluid extraction. Furthermore, a lot of bioactive components
are extracted together with by-products. In order to improve the purity or
decrease the untargeted component, the selectivity of the supercritical-CO2
fluid extraction system should be improved. Crossover pressure operation is
a choice, which in fact is similar to distillation at the supercritical region. Its
limitation is that usually there is not enough phase equilibrium data in the
supercritical region, and temperature control is not as convenient as pressure
control. Complex cosolvents can be an alternative choice. Some researchers

have observed that at different operational stages, the extraction speeds of

various components are different, so that the components in the extracted
mixture will change. If the batch operation can be changed to a semibatch
operation, the cosolvent can be added to the system at a suitable time to keep
the selectivity at a high level. The nature of the material used as a source
of carotenoids governs the availability of the compounds for the extraction
process. The presence of other components such as lipids may impede the
process or elevate costs due to an elongated extraction time. The manner in
which the material is stored also affects the results, as carotenoids degrade
over time, the rate of which depending on the storage temperature, particle
size, and the presence of additional antioxidants or stabilizers. Optimizing
the water content is also important. There are opportunities to decrease the
pretreatment cost without change or even to increase the efficiency of super-
critical fluid. To decrease the operation cost, pretreating tissues, breaking
down the cell wall and releasing the oil all need to be improved. Flaking
and grinding are widely used methods because the cell breakage percent-
age is high. However, those are laboratory-scale methods. If applied to
industry, decreasing the operation costs and increasing the percentage of
cell breakage should be considered. In other words, to apply the supercriti-
cal fluid t echnology, the solubility prediction and the phase diagrams must
be accurate to give a solid base for design and the operation cost have to be
decreased further.
References
Abbott, A.P., Corr, S., Durling, N.E., Hope, E.G. 2002. Solubility of substituted aro-
matic hydrocarbons in supercritical difluoromethane. Journal of Chemical &
Engineering Data, 47(4): 900–905.
Abbott, A.P., Eardley, C.A., Scheirer, J.E. 2000. CO2/HFC 134a mixtures: Alternatives
for supercritical fluid extraction. Green Chemistry, April: 63–65.
Adachi, Y., Lu, B.C.Y. 1983. Supercritical fluid extraction with carbon dioxide and
ethylene. Fluid Phase Equilibria, 14: 147, 156.
Ambrogi, A., Cardarelli, D.A., Eggers, R. 2002. Fractional extraction of paprika using
supercritical carbon dioxide and on-line determination of carotenoids. Journal of
Food Science, 67(9): 3263–3241.
Ambrogi, A., Cardarelli, D.A., Eggers, R. 2003. Separation of natural colorants using
a combined high pressure extraction-absorption process. Latin American Applied
Research, 33: 323–326.
Ambrosino, P., Galvano, F., Fogliano, V., Logrieco, A., Fresa, R., Ritieni, A. 2004.
Supercritical fluid extraction of Beauvericin from maize. Talanta, 62(3):
523–530.
Balasubramanian, R. 2003. Acentric factor of alkali metals. International Journal of
Thermophysics 24(1): 201–206.

Balzhiser, R.E., Samuels, M.R. 1977. Engineering Thermodynamics. Prentice-Hall, New

Jersey, USA.
Barth, M.M., Zhou, C., Kute, K.M., Rosenthal, G.A. 1995. Determination of optimum
conditions for supercritical fluid extraction of carotenoids from carrot (Daucus
carota L.) tissue. Journal of Agricultural Food Chemistry, 43: 2876–2878.
Bartle, K.D. 1991. Solubilities of compounds in supercritical carbon dioxide. Journal of
Physical and Chemical Reference Data, 20(4): 756.
Baysal, T., Ersus, S., Starmans, D.A.J. 2000. Supercritical CO2 extraction of β-carotene
and lycopene from tomato paste waste. Journal of Agricultural and Food Chemistry,
48: 5507–5511.
Bejan, A. 1988. Advanced Engineering Thermodynamics. John Wiley & Sons, USA.
Berna, A., Cháfer, A., Montón, J.B. 2000. Solubilities of essential oil components of
orange in supercritical carbon dioxide. Journal of Chemical and Engineering Data,
45(5): 724–727.
Bharath, R., Yamane, S., Inomata, H., Adschiri, T., Arai, K. 1993. Phase equilibria of
supercritical CO2—Fatty oil component binary systems. Fluid Phase Equilibria,
83(2): 183–192.
Botch-Jensen, C., Mollerup, J. 1997. Phase equilibria of fish oil in sub- and supercriti-
cal carbon dioxide. Fluid Phase Equilibria, 138(1–2): 179–211.
Brunner, G. 1983. Selectivity of supercritical compounds and entrainers with respect
to model substances. Separation Science and Technology, 10: 289–298.
Brunner, G. 1987. Stofftrennung mit ueberkritischen gasen (gasextraktion). Chemie-
Ingenieur-Technik, 59: 12–22.
Burgos-Solorzano, G.I., Brennecke, J.F., Stadtherr, M.A. 2004. Solubility measure-
ments and modeling of molecules of biological and pharmaceutical interest
with supercritical CO2. Fluid Phase Equilibria, 220: 57–69.
Cadoni, E., De Giorgi, M.R., Medda, E., Poma, G. 2000. Supercritical CO2 extraction
of lycopene and β-carotene from ripe tomatoes. Dyes and Pigments, 44: 27–32.
Castellan, G.W. 1964. Physical Chemistry. 2nd edition. Addison-Wesley, Reading, MA.
Chafer, A., Berna, A., Monton, J.B., Mulet, A. 2001. High pressure solubility data of
the system limonene + linalool + CO2. Journal of Chemical & Engineering Data, 46:
1145–1148.
Chafer, A., Fornari, T., Berna, A., Stateva, R.P. 2004. Solubility of quercetin in super-
critical CO2 + ethanol as a modifier: Measurements and thermodynamic model-
ling. Journal of Supercritical Fluids, 32: 89–96.
Chandra, A., Nair, M.G. 1997. Supercritical fluid carbon dioxide extraction of α- and
β-carotene from carrot (Daucus carota L.). Phytochemical Analysis, 8: 244–246.
Chang, C.J., Randolph, A.D. 1989. Precipitation of microsize organic particles from
supercritical fluids, AIChE Journal 35: 1876.
Chimowitz, E.H., Pennisi, K.J. 1986. Process synthesis concepts for supercritical gas
extraction in the crossover region. AIChE Journal, 32: 1665–1676.
Chrastil, J. 1982. Solubility of solids and liquids in supercritical gases. The Journal of
Physical Chemistry, 86: 3016–3021.
Cocero, M.J., Calvo, L. 1996. Supercritical fluid extraction of sunflower seed oil
with CO2–ethanol mixtures. Journal of the American Oil Chemists’ Society, 73:
1573–1578.
Cocero, M.J., González, S., Pérez, S., Alonso, E. 2000. Supercritical extraction of unsat-
urated products. Degradation of β-carotene in supercritical extraction processes.
The Journal of Supercritical Fluids, 19: 39–44.

Cygnarowicz, M.L., Maxwell, R.J., Seider, W.D. 1990. Equilibrium solubilities of

β-carotene in supercritical carbon dioxide. Fluid Phase Equilibria, 59: 57–71.
de França, L.F., Reber, G., Meireles, M.A.A., Machado, N.T., Brunner, G. 1999.
Supercritical extraction of carotenoids and lipids from buriti (Maura flexuosa),
a fruit from the Amazon region. The Journal of Supercritical Fluids, 14: 247–256.
de la Fuente, J.C., Oyarzún, B., Quezada, N., del Valle, J.M. 2006. Solubility of carot-
enoid pigments (lycopene and astaxanthin) in supercritical carbon dioxide.
Fluid Phase Equilibria, 247: 90–95.
Del Valle, J.M., Aguilera, J.M. 1988. Improved equation for predicting the solubility of
vegetable oils in supercritical CO2. Industrial & Engineering Chemistry Research,
27: 1551–1553.
Dobbs, J.M., Johnston, K.P. 1987. Selectivities in pure and mixed supercritical fluid
solvents. Industrial & Engineering Chemistry Research, 26: 1476–1482.
Dobbs, J.M., Wong, J.M., Johnston, K.P. 1986. Nonpolar co-solvents for solubil-
ity enhancement in supercritical fluid carbon dioxide. Journal of Chemical and
Engineering Data, 31: 303–308.
Dobbs, J.M., Wong, J.M., Lahiere, R.J., Johnston, K.P. 1987. Modification of super-
critical fluid phase behavior using polar cosolvents. Industrial & Engineering
Chemistry Research, 26: 56–65.
Dohrn, R., Bunz, A.P., Devlieghere, F., Thelen, D. 1993. Experimental measurements
of phase equilibria for ternary and quaternary systems of glucose, water, CO2
and ethanol with a novel apparatus. Fluid Phase Equilibria, 83: 149–158.
Dunford, N.T., Goto, M., Temelli, F. 1998. Modeling of oil extraction with supercritical
CO2 from Atlantic mackerel (Scomber scombrus) at different moisture contents.
Journal of Supercritical Fluids, 13: 303–309.
Dunford, N.T., Temelli, F., Leblanc, E. 1997. Supercritical CO2 extraction of oil and
residual proteins from Atlantic mackerel (Scomber scombrus) as affected by mois-
ture content. Journal of Food Science, 62: 289–294.
Fedors, R.F.A. 1974. Method for estimating both the solubility parameters and molar
volume of liquids. Polymer Engineering and Science, 14: 147–154.
Ferreira, S.R.S., Meireles, M.A.A., Cabral, F.A. 1993. Extraction of essential oil of black
pepper with liquid carbon dioxide. Journal of Food Engineering, 20: 121–133.
Frenkel, M., Gadalla, N.M., Hall, K.R., Hong, X., Marsh, K.N. 1997. TRC Thermodynamic
Tables-Hydrocarbon. Thermodynamic Research Center, Texas A&M University
System, USA.
Foster, N.R., Gurdial, G.S., Yun, J.S.L., Liong, K.K., Tilly, K.D., Ting, S.S.T., Singh, H.,
Lee, J.H. 1991. Significance of the crossover pressure in solid-supercritical fluid
phase equilibria. Industrial & Engineering Chemistry Research, 30: 1955–1964.
Foster, N.R., Singh, H., Yun, S.L.J., Tomasko, D.L., Macnaughton, S.J. 1993. Polar and
nonpolar cosolvent effects on the solubility of cholesterol in supercritical fluids.
Industrial & Engineering Chemistry Research, 32(11): 2849–2853.
Gaspar, F., Lu, T., Marriott, R., Mellor, S., Watkinson, C., Al-Duri, B., Santos, R., Seville,
J. 2003. Solubility of Echium, Borage, and Lunaria seed oils in compressed CO2.
Journal of Chemical & Engineering Data, 48: 107–109.
Ghaziaskar, H.S., Eskandari, H., Daneshfar, A. 2003. Solubility of 2-ethyl-1-hexanol,
2-ethylhexanoic acid, and their mixtures in supercritical carbon dioxide. Journal
of Chemical & Engineering Data, 48: 236–240.
Giddings, J.C., Myers, M.N., McLaren, L., Keller, R.A. 1968. High pressure gas chro-
matography of non-volatile species. Science, 162: 67–73.

Gnayfeed, M.H., Daood, H.G., Illés, V., Biacs, P.A. 2001. Supercritical CO2 and subcrit-
ical propane extraction of pungent paprika and quantification of carotenoids,
tocopherols and capsaicinoids. Journal of Agricultural and Food Chemistry, 49:
2761–2766.
Goldman, S, Gray, C.G., Li, W., Tomberli, B., Joslin, C.G. 1996. Predicting solubilities
in supercritical fluids. The Journal of Physical Chemistry, 100: 7246–7249.
Gómez-Prieto, M.S., Caja, M.M., Santa-María, G. 2002. Solubility in supercritical car-
bon dioxide of the predominant carotenes of tomato skin. Journal of the American
Oil Chemists’ Society, 79: 897–902.
Gordon, H.T., Bauernfeind, J.C. 1982. Carotenoids as food colorants. Critical Reviews
in Food Science and Nutrition, 18: 59–97.
Gouveia, L., Nobre, B.P., Marcelo, F.M., Mrejen, S., Cardoso, M.T., Palavra, A.F.,
Mendes, R.L. 2007. Functional food oil coloured by pigments extracted from
microalgae with supercritical CO2. Food Chemistry, 101: 717–723.
Graham, B.F., Lagalante, A.F., Bruno, T.J., Harrowfield, J.M., Trengove, R.D. 1998.
Solubility of p-t-butylcalixarenes in supercritical carbon dioxide: A comparison of
static and dynamic measurement techniques. Fluid Phase Equilibria, 150: 829–838.
Güçlü-Üstündağ, Ö., Temelli, F. 2000. Correlating the solubility behavior of fatty
acids, mono-, di-, and triglycerides, and fatty acid esters in supercritical carbon
dioxide. Industrial & Engineering Chemistry Research, 39: 4756.
Gurdial, G.S., Macnaughton, S.J., Tomasko, D.L., Foster, N.R. 1993. Influence of chem-
ical modifiers on the solubility of o- and m-hydroxybenzoic acid in supercritical
CO2. Industrial & Engineering Chemistry Research, 32: 1488–1497.
Hammam, H. 1992. Solubilities of pure lipids in supercritical carbon dioxide. The
Hansen, B.N., Harvey, A.H., Coelho, J.A.P., Palavra, A.M.F., Bruno, T.J. 2001. Solubility
of capsaicin and β-carotene in supercritical carbon dioxide and in halocarbons.
Journal of Chemical and Engineering Data, 46: 1054–1058.
Hartono, R., Mansoori, G.A., Suwono, A. 2001. Prediction of solubility of biomol-
ecules in supercritical solvents. Chemical Engineering Science, 56: 6949–6958.
Hassan, M.N., Rahman, N.N.A., Ibrahim, M.H., Omar, A.K.M. 2000. Simple fraction-
ation through the supercritical carbon dioxide extraction of palm kernel oil.
Separation and Purification Technology, 19(1–2): 113–120.
Hopper, M.L., King, J.W. 1991. Enhanced supercritical fluid carbon dioxide extrac-
tion of pesticides from foods using palletized diatomaceous earth. Association of
Official Analytical Chemists, 74(4): 661–666.
Hyatt, J.A. 1984. Liquid and supercritical carbon dioxide as organic solvents. The
Journal of Organic Chemistry, 49(26): 5097–5101.
Jay, A.J., Steytler, D.C. 1992. Near critical fluids as solvents for β-carotene. The Journal
of Supercritical Fluids, 5: 274–282.
Johannsen, M., Brunner, G. 1997. Solubilities of the fat-soluble vitamins A, D, E, and K in
supercritical carbon dioxide. Journal of Chemical and Engineering Data, 42: 106–111.
Johnston, K.P., Peck, D.G., Kim, S. 1987. Multicomponent polar mixtures at supercriti-
cal fluid conditions. Industrial & Engineering Chemistry Research, 28(8): 1115–1125.
Joslin, C.G., Gray, C.G., Goldman, S., Tomberli, B., Li, W. 1996. Solubilities in super-
critical fluids from the virial equation of state. Molecular Physics, 89(2): 489–503.
Khosravi-Darani, K., Vasheghani-Farahani, E., Yamini, Y., Bahramifar, N. 2003.
Solubility of poly (α-hydroxybutyrate) in supercritical carbon dioxide. Journal
of Chemical and Engineering Data, 48: 860–863.

King, J.W., Friedrich, J.P. 1990. Quantitative correlations between solute molecular
structure and solubility in supercritical fluids. Journal of Chromatography, 517:
449–458.
King, M.B., Mubarak, A., Kim, J.D., Bott, T.R. 1992. The mutual solubilities of water
with supercritical and liquid carbon dioxide. The Journal of Supercritical Fluids,
5: 296–302.
Koga, Y., Iwai, Y., Hata, Y., Yamamoto, M., Arai, Y. 1996. Influence of cosolvent on
solubilities of fatty acids and higher alcohols in supercritical carbon dioxide.
Fluid Phase Equilibria, 125(1–2): 115–128.
Kraska, T., Leonhard, K.O., Tuma, D., Schneider, G.M. 2002. Correlation and solubil-
ity of low-volatile organic compounds in near- and supercritical fluids. Part I.
Applications to adamantine and β-carotene. The Journal of Supercritical Fluids,
23: 209–224.
Krukonis, V.J., McHugh, M.A., Seckner, A.J. 1984. Xenon as a supercritical solvent. The
Journal of Physical Chemistry, 88: 2687–2689.
Kuk, M.S., Hron, R.J. 1994. Supercritical carbon dioxide extraction of cottonseed with
co-solvents. Journal of the American Oil Chemists’ Society, 71(12): 1353–1356.
Lewin-Kretzschmar, U., Blasberg, L., Harting, P. 2002. Solubility of nitroaromatics
and adamantane in pure and surfactant-modified compressed gases. Tenside
Surfactants Detergents, 39(1): 29–35.
Li, Q., Zhang, Z., Zhong, C., Liu, Y., Zhou, Q. 2003. Solubility of solid solutes in super-
critical carbon dioxide with and without cosolvents. Fluid Phase Equilibria, 207:
183–192.
Li, S., Hartland, S. 1996. New industrial process for extracting cocoa butter and xan-
thines with supercritical carbon dioxide. Journal of the American Oil Chemists’
Society, 73(4): 423–429.
Liong, K.K., Wells, P.A., Foster, N.R. 1991. Diffusion in supercritical fluids. The
Journal of Supercritical Fluids 4: 91–108.
Macías-Sánchez, M.D., Mantell, C., Rodríguez, M., Martínez de la Ossa, E., Lubián,
L.M., Montero, O. 2005. Supercritical fluid extraction of carotenoids and
chlorophyll a from Nannochloropsis gaditana. Journal of Food Engineering, 66:
245–251.
Maheshwari, P., Nikolov, Z.L., White, T.M., Hartel, R. 1992. Solubility of fatty acids in
supercritical carbon dioxide. Journal of the American Oil Chemists’ Society, 69(11):
1069–1076.
Marentis, R.T. 1998. Steps to developing a commercial supercritical carbon dioxide
processing plant. In Supercritical Fluid Extraction and Chromatography, ed. B.A.
Charpentier and M.R. Sevenants. American Chemical Society, Symposium
Series, pp. 127–143.
Mendes, R.L., Nobre, B.P., Coelho, J.P., Palavra, A.F. 1999. Solubility of β-carotene in
supercritical carbon dioxide and ethane. The Journal of Supercritical Fluids, 16:
99–106.
Mitra, S., Wilson, N.K. 1991. Empirical method to predict solubility in supercritical
fluids. Journal of Chromatographic Science, 29(7): 305–309.
Montero, O., Macías-Sánchez, M.D., Lama, C.M., Lubían, L.M., Mantell, C., Rodríguez,
M., de la Ossa, E. 2005. Supercritical CO2 extraction of β-carotene from a marine
strain of the cyanobacterium Synechoccus species. Journal of Agricultural and Food
Chemistry, 53: 9701–9707.

Murga, R., Sanz, M.T., Beltran, S., Cabezas, J.L. 2004. Solubility of syringic and vanil-
lic acids in supercritical carbon dioxide. Journal of Chemical & Engineering Data,
49(4): 779–782.
Norulaini, N.A., Zaidul, I.S., Anuar, O., Azlan, A., Omar, A.K. 2004a. Supercritical
enhancement for separation of lauric acid and oleic acid in palm kernel oil
(PKO). Separation and Purification Technology, 35(1): 55–60.
Norulaini, N.A., Zaidul, I.S., Anuar, O., Azlan, A., Omar, A.K. 2004b. Supercritical
reduction of lauric acid in palm kernel oil (PKO) to produce cocoa butter equiv-
alent (CBE) fat. Journal of Chemical Engineering of Japan, 37(2): 194–203.
Ollanketo, M., Hartonen, K., Riekkola, M.L., Holm, Y., Hiltunen, R. 2001. Supercritical
carbon dioxide extraction of lycopene in tomato skins. European Food Research
and Technology, 212: 561–565.
Ozkal, S.G., Salgin, U., Yener, M.E. 2005. Supercritical carbon dioxide extraction of
hazelnut oil. Journal of Food Engineering, 69: 217–223.
Patton, C.L., Luks, K.D. 1995. Determination of partial molar volumes at critical end
points. Fluid Phase Equilibria, 113: 21–26.
Peng, D.Y., Robinson, D.A.A. 1976. New two-constant equation of state. Industrial &
Engineering Chemistry Fundamentals, 15: 59–64.
Poling, B.E., Prausnitz, J.M., O’Connell, J.P. 2000. The Properties of Gases and Liquids,
5th edition. McGraw-Hill, New York.
Politzer, P., Murray, J.S., Lane, P., Brinck, T. 1993. Relationship between solute
molecular properties and solubility in supercritical CO2. The Journal of Physical
Chemistry, 97: 729–732.
Prausnitz, J.M. 1969. Molecular Thermodynamics of Fluid Phase Equilibria. Prentice-Hall,
Englewood Cliffs, NJ.
Prausnitz, J.M., Lichtenthaler, R.N., Azevedo, E.G. 1986. Molecular Thermodynamics of
Fluid Phase Equilibria, Chapter 9, Prentice-Hall, Englewood Cliffs, NJ.
Raveendran, P., Wallen, S.L. 2003. Exploring CO2-philicity: Effects of stepwise fluori-
nation. Journal of Physical Chemistry B, 107: 1473–1477.
Redlich, O., Kwong, J.N.S. 1949. The thermodynamics of solutions. V. An equation of
state. Fugacities of gaseous solutions. Chemical Reviews, 44: 233–244.
Rizvi, S.S.H., Benado, A.L., Zollweg, J.A., Daniels, J.A. 1986. Supercritical fluid extrac-
tion: Fundamental principles and modeling methods. Food Technology, 40(6): 55–65.
Roy, B.C., Goto, M., Hirose, T. 1996. Temperature and pressure effects on supercriti-
cal CO2 extraction of tomato seed oil. International Journal of Food Science and
Technology, 31(2): 137–141.
Rozzi, N.L., Singh, R.K., Vierling, R.A., Watkins, B.A. 2002. Supercritical fluid extrac-
tion of lycopene from tomato processing byproducts. Journal of Agricultural and
Sabio, E., Lozano, M., Montero de Espinosa, V., Mendes, R.L., Pereira, A.P., Palavra,
A.F., Coelho, J.A. 2003. Lycopene and β-carotene extraction from tomato pro-
cessing waste using supercritical CO2. Industrial and Engineering Chemistry
Research, 42: 6641–6646.
Saeki, S. 1995. Empirical equation of state for supercritical fluids. Journal of Supercritical
Fluids, 8(1): 30–45.
Sakaki, K. 1992. Solubility of β-carotene in dense carbon dioxide and nitrous oxide
from 308 to 323 K and from 9.6 to 20 MPa. Journal of Chemical and Engineering
Data 37: 249–251.

Saldaña, M.D.A., Sun, L., Guigard, S.E., Temelli, F. 2006. Comparison of the solubil-
ity of β-carotene in supercritical CO2 based on a binary and a multicomponent
complex system. The Journal of Supercritical Fluids, 37: 342–349.
Schmitt, W.J., Reid, R.C. 1986. Solubility of monofunctional organic solids in chemi-
cally diverse supercritical fluids. Journal of Chemical & Engineering Data, 31:
204–212.
Shi, J., Khatri, M., Xue, J., Mittal, G., Ma, Y., Li, D. 2009. Solubility of lycopene in
supercritical CO2 fluid affected by temperature and pressure. Separation and
Purification Technology, 66: 322–328.
Singh, H., Yun, S.L.J., Macnaughton, S.J., Tomasko, D.L., Foster, N.R. 1993. Solubility
of cholesterol in supercritical ethane and binary gas mixtures containing ethane.
Industrial & Engineering Chemistry Research, 32(11): 2841–2848.
Škerget, M., Knez, Ž., Habulin, M. 1995. Solubility of β-carotene and oleic acid in
dense CO2 and data correlation by a density based model. Fluid Phase Equilibria,
109: 131–138.
Smith, R.D., Udseth, H.R., Wright, B.W. 1985. Micro-scale methods for characteriza-
tion of supercritical fluid extraction and fraction processes. In Supercritical Fluid
Technology, ed. J.M.L. Penninger, M. Radosz, M.A. McHugh and V.J. Krukonis.
Elsevier Science Publishers B.V, Amsterdam.
Soave, G. 2000. A simple model for the supercritical extraction of solids. Journal of
Sovová, H., Stateva, R.P., Babushka, A.A. 2001. Solubility of β-carotene in supercritical
CO2 and the effect of entrainers. The Journal of Supercritical Fluids, 21: 195–203.
Spanos, G.A., Chen, H., Schwartz, S.J. 1993. Supercritical CO2 extraction of β-carotene
from sweet potatoes. Journal of Food Science, 58(4): 817–820.
Staby, A., Forskov, T., Mollerup, J. 1993. Phase equilibria of fish oil fatty acid ethyl
esters and sub- and supercritical CO2. Fluid Phase Equilibria, 87(2): 309–340.
Staby, A., Mollerup, J. 1993. Separation of constituents of fish oil using supercriti-
cal fluids: A review of experimental solubility, extraction, and chromatographic
data. Fluid Phase Equilibria, 91: 349–386.
Subra, P., Castellani, S., Ksibi, H., Garrabos, Y. 1997. Contribution to the determina-
tion of the solubility of β-carotene in supercritical carbon dioxide and nitrous
oxide, experimental data and modelling. Fluid Phase Equilibria, 131: 269–286.
Sun, M., Temelli, F. 2006. Supercritical carbon dioxide extraction of carotenoids from
carrot using canola oil as a continuous co-solvent. The Journal of Supercritical
Fluids, 37: 397–408.
Sunol, A.K., Hagh, B., Chen, S. 1985. Entrainer selection in supercritical extraction. In
Process Technology Proceedings, ed. J.M.L. Penninger et al., Elsevier, Amsterdam,
pp. 451–458.
Snyder, J.M., Rossell J.M., Friedrich J.P., Christianson, D.D. 1984. Effect of moisture
and particle size on the extractability of oils from seeds with supercritical CO2.
Journal of the American Oil Chemists’ Society 61: 1851–1856.
Teberikler, L., Koseoglu, S., Akgerman, A. 2001. Selective extraction of phosphatidyl-
choline from lecithin by supercritical carbon dioxide/ethanol mixture. Journal of
the American Oil Chemists’ Society, 78(2): 115–119.
Uquiche, E., del Valle, J.M., Ortiz, J. 2004. Supercritical carbon dioxide extraction of
red pepper (Capsicum annum L.) oleoresin. Journal of Food Engineering, 65: 55–66.
Walsh, J.M., Ikonomou, G.D., Donohue, M.D. 1987. Supercritical phase behavior: The
entrainer effect. Fluid Phase Equilibria, 33: 295–314.

Weathers, R.M., Beckholt, D.A., Lavella, A.L., Danielson, N.D. 1999. Comparison of
acetals as in situ modifiers for the supercritical fluid extraction of β-carotene
from paprika with carbon dioxide. Journal of Liquid Chromatography and Related
Technologies, 22(2): 241–252.
Wiebe, R., Gaddy, V.L. 1941. Vapor phase composition of carbon dioxide-water mix-
tures at various temperatures and at pressures to 700 atmospheres. Journal of the
American Chemical Society, 63: 475–477.
Wong, J.M., Johnston, K.P. 1986. Solubilization of biomolecules in carbon dioxide
based supercritical fluids. Biotechnology Progress, 21: 29–39.
Wong, V., Grant, W.S., Cornwell, C.P., Tronson, D. 2001. Supercritical fluid extrac-
tion (SFE) of monoterpenes from the leaves of Melaleuca alternifolia (tea tree).
Molecules, 6: 92–103.
Xing, H.B., Yang, Y.W., Su, B.G., Huang, M., Ren, Q.L. 2003. Solubility of artemis-
inin in supercritical carbon dioxide. Journal of Chemical & Engineering Data, 48:
330–332.
Yin, J.Z., Liu, R.J., Ding, X., Yang, B. 2002. Experimental study on solubility of Hippophae
rhamnoides L. seed oils in supercritical carbon dioxide. Huagong Xiandai/Modern
Chemical Industry, 22(Suppl): 111–113.
Yuan, H., Gao, G.T., Zeng, X.C. 1997. Effects of the co-solvent energy parameter and
dipolar strength on solute residual chemical potential. Fluid Phase Equilibria,
138: 61–68.

3
Mathematical Modeling of Supercritical
Fluid Extraction
Helena Sovová
CONTENTS
3.1 Introduction................................................................................................. 105
3.2 Mass Balance Equations............................................................................. 108
3.2.1 Flow Patterns................................................................................... 109
3.2.2 Thermodynamic and Apparent Solubility.................................. 110
3.3 Mass Transfer Resistances......................................................................... 113
3.3.1 External Mass Transfer.................................................................. 113
3.3.1.1 Internal Mass Transfer.................................................... 114
3.3.2 Linear Driving Force...................................................................... 114
3.3.3 Porous Particles............................................................................... 115
3.4 SFE of Vegetable Oils from Seed.............................................................. 115
3.5 SFE of Volatile Oils..................................................................................... 117
3.6 SFE of Mixtures........................................................................................... 119
Nomenclature....................................................................................................... 120
Greek Letters......................................................................................................... 121
References.............................................................................................................. 121
3.1 Introduction
Medicinal and aromatic plants and other natural products are impor-
tant sources of valuable substances. These substances are often labile and
therefore mild methods should be applied to isolate them from the plants.
Supercritical fluid extraction (SFE) is particularly suited for such isolation
as it does not expose the substances to high temperatures and is relatively
fast due to outstanding transport properties of supercritical fluids. The most
frequent supercritical solvent is carbon dioxide, which is as a “green” solvent
much more acceptable than toxic conventional organic solvents.
The pressurized solvent circulates in extraction equipment between an
extractor containing a fixed bed of plant particles, where the extract dissolves at
supercritical conditions, and a separator, where the solvent is expanded to gas-
eous state and the extract precipitates at reduced solvent power and is collected.
105
Thus, it is a continuous process with respect to the solvent and a batch process
with respect to the plant material. Use of two or three extractors enables a con-
tinuous operation, whereas the exhausted plant material in one of the extrac-
tors is substituted by a fresh one, and two or three separators operated in series
at individually adjusted pressures and temperatures enable a partial fraction-
ation of extract on the basis of different solubility of its components.
A correct adjustment of pressures and temperatures in the extractor and
separators, solvent flow rate, and extraction time are crucial for the efficiency
of the process. It is therefore important to understand the processes in both
parts of the equipment. Especially the mechanisms of extraction, on which
this chapter is focused, are variable in accordance with variable properties
of plant materials.
As carbon dioxide is by far the most frequently applied supercritical sol-
vent in the extraction from plants, all examples of extraction and values of
model parameters in this chapter concern the extraction with carbon diox-
ide, whereas mathematical relations are of general validity.
The easily measurable variables of mathematical models for the extraction
are indicated in Figure 3.1. It is the amount of material fed into the extractor,
the solvent flow rate, which is maintained constant during the whole extrac-
tion run, and the concentration of the extract in the solution flowing out of
the extractor; its concentration in the solvent flowing into the extractor is
assumed to be zero. To describe local mass-transfer rates inside the cylindri-
cal extraction vessel, axial coordinate identical with the main direction of
solvent flow is used in the models.
The extraction kinetics is characterized by the extraction curves, showing
the dependence of extraction yield either on extraction time, t, or solvent-
to-feed ratio (the mass of solvent passed through the extractor divided by
the mass of the feed), q. The slope of the extraction curve e = e(q) is equal to
y = y(H, t)
h=H
Axial
Feed N coordinate
h=0
y=0 Flow rate Q′
FIGURE 3.1
Scheme of cylindrical extractor with a fixed bed of particles and a continuous solvent flow.

Mathematical Modeling of Supercritical Fluid Extraction 107
the instantaneous extract concentration y(H,t), expressed in terms of mass of

the extract per mass of the solvent, and the slope of the extraction curve e = e(t)
is equal to the product of outlet concentration and specific mass flow rate:
de
= q′ y ( H , t ) (3.1)
dt
Thus, modeling the extraction kinetics means modeling the outlet concen-
tration y(H,t) developed as a result of mass transfer from the solid phase to the
fluid phase inside the extractor. Probably the first attempt to review and clas-
sify mathematical models for SFE was made by Reverchon (1997) in his paper
on SFE and fractionation of essential oils. Besides the models based on differ-
ential mass balance discussed in this chapter, he recognizes empirical mod-
els, which contain no information on extraction mechanisms, and the models
based on heat transfer analogy, which could be derived from the mass bal-
ance equations as a special case. A later paper (Reverchon and De Marco, 2006)
gives references to the models applied for SFE of different extracts from differ-
ent raw materials and emphasizes the importance of the plant material shape
and structure, location of extracted substances, and the type of phase equilib-
rium for correct mathematical modeling. The paper on the SFE of oilseeds (del
Valle and de la Fuente, 2006) is the most comprehensive review on equations
from which the models and correlations for the model parameters are built.
Use of the Peng–Robinson equation and semiempirical correlations for solubil-
ity as well as adsorption isotherms are discussed together with mass-transfer
characteristics. Another survey of mathematical models for SFE includes also
a chapter on thermodynamic modeling of high-pressure equilibria (Sovova
and Stateva, 2011). The equilibrium, internal and external mass-transfer resis-
tance, and flow pattern were taken into account in this paper to character-
ize the reviewed models. In parallel, Oliveira et al. (2011) published a review
of mathematical models for both SFE from plants and SFE in countercurrent
supercritical fluid–liquid columns. In the SFE from plants, they distinguished
models with linear driving force, shrinking core models, models with broken
and intact cells (BICs), and a combination of shrinking core model with BICs.
The most frequently applied models for SFE were reviewed recently (Shilpi
et al., 2013). Also, Huang et al. (2012) published an extensive review of SFE
models and their applications. Besides the models based on differential mass
balance as the BIC model and shrinking core model, the authors pay attention
to simplified models that neglect concentration differences in the extractor.
These models either lack any characteristic of solubility, or, on the opposite,
they are based on the assumption that equilibrium is established very fast and
do not contain any kinetic parameter. Both types of simplified models have
formally equal solution for the time dependence of the extraction yield. It is
thus evident that the choice of the model used to fit experimental data cannot
be arbitrary, otherwise incorrect kinetic parameters would be evaluated for a
process controlled by extract solubility in the solvent, and vice versa.

3.2 Mass Balance Equations

The extractor is for mathematical treatment divided into finite-difference
volume elements of height Δh. Mass balances of the extract are written and
rearranged so that differential mass balance equations result when making
Δh → 0 and Δt → 0. The mass balance for the fluid phase flowing through the
extraction bed with interstitial velocity u is then
 ∂y ∂y 
ερf  + u  = j , y( h , t = 0) = y 0 , y( h = 0 , t ) = 0 (3.2)
 ∂t ∂h 

where t = 0 at the moment when the solvent just begins flowing out of the
extractor. The mass balance for the solid phase is
∂x
(1 − ε)ρs = − j , x( h, t = 0) = x0 (3.3)
∂t
The amount of the extract that flows from the plant particles to the solvent
phase in unit volume of extraction bed and in unit time is directly propor-
tional to the fluid-phase mass-transfer coefficient, specific surface area, and
the difference between the extract concentrations at particle surface and in
bulk fluid (driving force):
j = kf a0ρf ( y + − y ) (3.4)

The scheme valid for the fluid phase can be analogously applied to the
solid phase:
j = ks a0ρs ( x − x + ) (3.5)

A more precise description of extract diffusion through the particle to

its surface is based on Fick’s law, characterized by effective diffusivity, De,
requires a definition of particle shape, and uses a spatially variable concen-
tration inside the particle, cs. The respective equations for a sphere of radius
R are
∂cs 3(1 − ε) ∂cs D ∂  2 ∂cs 

j = − a0De , a0 = , = 2e r  (3.6)
∂r r=R R ∂t r ∂r  ∂r 

Equations 3.1 through 3.9, together with a relationship between concen-

trations x+, y+ at particle surface and with equilibrium relationship, con-
stitute mathematical model for SFE. Generally, the equations have to be

solved numerically to give the extraction curves, which are compared with
the experimental curves e(t) or e(q). After adjustment of model parameters
to fit the data measured on small-scale equipment, the model should be
able to predict the extractor performance for any size of extraction equip-
ment. However, as a complete model involves characteristics of external and
internal mass transfers, phase equilibrium, and flow pattern, the number of
parameters is too high to adjust all of them according to the experimental
extraction curves. As many parameters as possible should be fixed using
additional information, for example, the correlations published for external
mass-transfer coefficients and the number of adjusted parameters should be
minimized.
3.2.1 Flow Patterns

Equation 3.2 is written for the plug flow pattern, where the solvent flows in
axial direction and its velocity is of the same value in the whole extractor.
In reality, axial dispersion exists in the flow through a packed bed due to
molecular diffusion and flow irregularities, and the mass balance equation
should therefore include an axial dispersion term:
 ∂y ∂y ∂2 y 
ερf  +u − Dl 2  = j
 ∂t ∂h ∂h 

u ∂y
y − Dl = 0 for h = 0 (3.7)
ε ∂h
∂y
= 0 for h = H
∂h
with Dl being the axial dispersion coefficient. The values of Dl in SFE models
are usually estimated from correlations of Peclet number, for example, the
correlation published by Tan and Liou (1989a). Many researchers observed,
however, that the term for axial dispersion with Dl values estimated this way
had negligible or very small effect on the calculated extraction yield (del Valle
et al., 2000, 2004; Reverchon and Marrone, 2001) and therefore Equation 3.2 is
used frequently in the models instead of Equation 3.7.
For a short extraction bed, a lumped parameter model (model of differen-
tial extractor) is applied (Peker et al., 1992; Goto et al., 1993). The fluid-phase
mass balance equation is then an ordinary differential equation identical
with that for the ideal mixer:
 dy y 
ρf ε  + = j , y(t = 0) = y0 (3.8)
 dt tr 


Comparing the rate of SFE with the solvent flow from the extractor bottom
upward and with the flow in the opposite direction, the extraction with the
flow to the bottom was sometimes found faster (Beutler et al., 1988; Barton
et al., 1992), and this effect was more pronounced at low interstitial veloci-
ties (Dams, 1989). The effect is connected with natural convection that eas-
ily develops in supercritical fluids due to their low kinematic viscosity. The
loaded solvent is usually heavier than pure one and flows faster downward.
It is not yet clear whether the natural convection takes place on microscale,
increasing the external mass-transfer coefficient in gravity-assisted flow and
decreasing it in gravity-opposed flow, as suggested by Recasens and cowork-
ers (Stüber et al., 1996; Guardo et al., 2007) or whether it acts on a larger scale,
changing the flow pattern in the extractor. In any case, the inhomogeneity of
solvent flow resulting from the inhomogeneity of extraction bed void frac-
tion is more pronounced at low velocities when the flow direction is upward
(Dams, 1989; Sovová et al., 1994). The SFE retarded by natural convection was
simulated dividing the flow in the extractor into parallel flows of different
velocities and thus different outlet concentrations (Sovova et al., 1994).
3.2.2 Thermodynamic and Apparent Solubility

The initial distribution of the extract between the fluid and solid phases ful-
fills the mass balance constraint:
ερf y0 = (1 − ε)ρs ( xu − x0 ) (3.9)

When the solution starts flowing out from the extractor immediately after
its pressurization, y0 = 0 can be assumed. Generally, any value of y0 between
0 and equilibrium concentration y+(x0) can be applied in the models. When a
period of static extraction precedes the dynamic extraction with solvent flow,
phase equilibrium y0 = y+(x0) is usually assumed to be established at t = 0.
As at least a part of solute is easily accessible on particle surface after the
mechanical pretreatment and as the diffusion in supercritical fluids is fast,
the assumption of saturated (or almost saturated) solution at t = 0 is justified
and the initial part of the extraction curve contains information on the type
of equilibrium between the solid and fluid phases (Shilpi et al., 2013).
Different scientific disciplines use different units to express the content of
the solute in the mixture. Thus, mole fractions are used in thermodynamic
calculations, for diffusion and mass transfer generally the solute content
is expressed as volumetric concentration, whereas mass fractions or mass
ratios are used with advantage when the density of solution strongly varies
like between the extractor and the separator in the equipment for SFE. The
conversion from the volume-related units, c, cs, and 〈cs〉, in which the equi-
librium relationships were originally formulated, to the mass-related units,
x and y, is based on the assumption of constant density of the fluid phase

(which is regarded equal to the density of a pure solvent, ρf, due to usually
low solubility of the extract in the solvent) and on the assumption of constant
volume of the plant particles with initial density, ρs, regardless of diminish-
ing extract in them:
cs c
x= , y= (3.10)
ρs ρf
The density of supercritical-CO2 can be read from the tables or calculated

using Bender equation of state with 21 coefficients (Brunner, 1994a) or the
equation of state derived by Altunin and Gadetskii (Angus et al., 1976); they
are also available on the Internet.
The equilibrium concentration of the extracted solute in a supercritical sol-
vent is either equal to its solubility or, when the extract is bound to matrix,
lower than its solubility. Del Valle and Urrego (2012) distinguish in this
respect between thermodynamic solubility (extract solubility in the solvent)
and apparent solubility (concentration of the saturated solution when the sol-
ute is extracted from plant matrix). Both solubilities strongly depend on the
extraction pressure and temperature.
A correlation for CO2 thermodynamic solubility of common vegetable oils,
composed of fatty acid esters where fatty acids with 18 carbons in molecule
prevail, was published by del Valle and Aguilera (1988). The correlation, which
is valid in a relatively wide range of pressures and temperatures, was recently
further refined using a more comprehensive database (del Valle et al., 2012):
[ 9.59 − 8.45((ρf /910 ) − 1) − 23.0((ρf /910 ) − 1)2 ]

 ρ 
ys = 0.00807  f  θ
 910 
(3.11)
  1 1   1 1  
exp −4182 1 − 259  −    − 
   T 313  T 313  

As the oil content in seeds is relatively high, at least a part of it has no

interactions with the matrix and thus the apparent solubility read from the
initial slope of their extraction curve is equal to their thermodynamic solu-
bility in CO2.
Most of other substances extracted from plants occur in concentration of
a few percent or even less and usually are bound to matrix. This becomes
evident from their apparent solubility: as the solid-phase concentration
diminishes during the extraction, the equilibrium fluid-phase concentration
is decreasing, too. The extract may be adsorbed on the solid matrix (Goto
et al., 1993) or absorbed in a liquid contained in the plant (Sovova et al., 2001).
Description of the extract–matrix interaction by linear equilibrium relation-
ship with partition coefficient K is mathematically simple and most frequent

in the literature on SFE (Peker et al., 1992; Goto et al., 1993; Reverchon, 1996;
del Valle et al., 2000):
ρs
y + = Kx + , c + = K vcs+ , K = K v (3.12)
ρf
Other relationships used in the SFE models for interaction of the extract
with the plant matrix are Langmuir adsorption isotherm (Silva et al., 2008),
Freundlich adsorption isotherm (Brunner, 1994b), and other adsorption iso-
therms (Salimi et al., 2008). The SFE models can be classified according to the
description of equilibrium fluid-phase concentration into three categories as
shown in Table 3.1.
It seems, however, that the apparent solubility can be described by one func-
tion of solid-phase concentration for vegetable oils and other types of solutes.
A decrease in fluid-phase equilibrium concentration was observed by Perrut
et al. (1997) when the solid-phase concentration dropped to a certain value in
the course of sunflower oil extraction. Therefore, they used in the SFE model
a composed equilibrium relationship with a switch from the thermodynamic
solubility to the linear equilibrium at critical solid-phase concentration xt:
y + = ys for x + ≥ xt , y + = Kx + for x + < xt , Kxt < ys (3.13)

TABLE 3.1
Models for SFE from Plants according to the Equilibrium Relationship
No Effect of Equilibrium Thermodynamic Solubility Adsorption Isotherm
Model with desorption rate Shrinking core model (Goto Equilibrium model (Kubátová
constant (Tan and Liou, et al., 1996) et al., 2002)
1989b)
Kinetic model with 2 rate Simple BIC models Model with internal resistance
constants (Kandiah and (Goodrum et al., 1996; Yoo (Reverchon et al., 1994;
Spiro, 1990; Kubátová and Hong, 1996) Reverchon, 1996)
et al., 2002)
Hot ball model (Bartle Model with external mass Model with internal and
et al., 1990) transfer resistance (Perrut external resistance (Goto
et al., 1997)a et al., 1993; Goodarznia and
Eikani, 1998; Araus et al.,
2009; Melreles et al., 2009)
Internal and external mass BIC model with Model with external resistance
transfer resistance approximate solution (Melreles et al., 2009)
(Reverchon et al., 1993) (Sovova, 1994)
BIC (Gaspar et al., 2003) BIC models (Sovová, 2005; BIC models (Sovová et al.,
Marrone et al., 1998)a 1994a; Reis-Vasco et al., 2000)
BIC with shrinking core
(Fiori et al., 2009)
a Either thermodynamic solubility or adsorption isotherm is applied in the second stage.

y+
Solubility ys
Slope K
Critical concentration xt
x+
FIGURE 3.2
Apparent solubility in CO2 as a function of extract concentration in plant.
The relationship, depicted in Figure 3.2 as a broken line, has proved to be

an efficient tool in SFE (Sovová, 2005), though the discontinuity may cause
problems in the numerical integration of mass balance equations.
An S-shaped dependence, also depicted in Figure 3.2, was proposed later
(del Valle and Urrego, 2012):
ys − Kx +
y + = Kx + + (3.14)
1 + ( xt /x + )b
This dependence is in good agreement with the results of direct oil solu-
bility measurement in the system oil + plant matrix + CO2 (Bulley et al., 1984;
King et al., 1987). To conclude, at least a part of any solute is expected to
interact with the plant matrix because the matrix is a good sorbent and the
supercritical-CO2 is a week solvent.
3.3 Mass Transfer Resistances

3.3.1 External Mass Transfer
The external mass-transfer resistance has little effect on the SFE kinetics as
long as its characteristic time tf
ε y − y+
tf = , j = ερf (3.15)
kf a0 tf
is substantially smaller than the residence time, tr, and the characteristic
time of internal mass-transfer resistance, ti (Sovová, 2012). The external mass-
transfer coefficient increases with increasing solvent velocity, and its value
can be estimated from the published correlations of Sherwood number,

Reynolds number, and Schmidt number, and in the case of natural convec-
tion, also Grashof number (Puiggené, 1997; del Valle and de la Fuente, 2006).
3.3.1.1 Internal Mass Transfer

The internal mass-transfer resistance depends on many factors: what plant
and what part of the plant (leaves, flowers, fruit, seed, root, etc.) is extracted,
where the extract is located in the material, and what was the pretreatment
(drying, soaking with a liquid, different procedures of mechanical disinte-
gration, opening the cavities containing the extract by a sudden decrease in
pressure after pressurization, and others). After the initial fast extraction of
the solute from the particle surface is finished, the internal mass-transfer
resistance usually controls the later extraction stages. Its characteristic time
(Reverchon, 1996; Sovová, 2012) is expressed as
1− ε R2 L2 x − x+
ti = , ti = , ti = , j = (1 − ε)ρs (3.16)
ks a0 15De 3De ti
where R is the radius of a spherical particle and L is half of the thickness of

a slab-shaped particle.
When nonpolar and low-polar substances are extracted, the plant is
usually dried, as larger amounts of water would hinder the access of
supercritical-CO2 into particles. The moisture of dry plants is usually slightly
below 10%, and during the SFE further decreases as water is coextracted:
water solubility in supercritical-CO2 is approximately 1.5 mg/g at 25°C,
3 mg/g at 50°C and 5 mg/g at 75°C (Wiebe and Gaddy, 1941). The perme-
ability of cell membranes changes with water content; they become imper-
meable when there is not enough water in the plant (Brunner, 1994c). The
extremely low values of effective diffusion coefficient, observed frequently
in SFE, can be explained by the low content of water in over-dried plants.
On the other hand, the particles soaked with water, prepared this way for
the extraction of polar compounds such as alkaloids with wet supercritical-
CO2, are well permeable.
3.3.2 Linear Driving Force

When Equations 3.15 and 3.16 are combined with linear phase equilibrium,
Equation 3.12, the concentration at the particle surface can be eliminated and
the mass-transfer rate per unit extractor volume is expressed as a function of
the difference of the concentrations in bulk phases:
Kx − y
j = ερf (3.17)
tf + γKti

3.3.3 Porous Particles

Plant particles are often described as porous bodies with the pores filled
with the solution of extract in the solvent. The interface is then on the pore
surface, and the solute diffuses in the solution filling the pores to the par-
ticle surface. The internal and external volumetric concentrations at the par-
ticle surface are equal and the mass-transfer rate per unit extractor volume
approximated by linear driving force is for spherical particles (del Valle and
de la Fuente, 2006):
ρs x − ρf y
j = kf a0 (3.18)
1 + (kf R/5De )
The particle porosity is measured independently from SFE experiments,

at ambient pressure, and the effective diffusion coefficient is estimated from
the binary diffusion coefficient (del Valle and de la Fuente, 2006; del Valle
et al., 2006) as
β
De = D12 (3.19)
τ
or even simpler, when the tortuosity, τ, is estimated as the inverse value of

particle porosity (Goto et al., 1993), as
De = D12β 2 (3.20)

3.4 SFE of Vegetable Oils from Seed

As the apparent oil solubility in supercritical-CO2, measured from the initial
slope of the extraction curve, is usually equal to its thermodynamic solubil-
ity, the models from the second column of Table 3.1 should be applied. A part
of oil in mechanically pretreated seeds is initially on the particle surface.
Assuming a quasi-steady mass transfer into the plug flow, the yield in the
first extraction stage is (Brunner, 1984):
  t 
e = ys 1 − exp  − r   (3.21)
  tf  

The SFE can be applied as a mild method to obtain thermolabile oil that
remains in the seed after pressing. As the mass-transfer resistance in pressed

or flaked seeds is low, internal diffusion is fast and oil recovery is complete
in a relatively short time. Shrinking core model was applied successfully to
simulate this process. The model distinguishes within a porous spherical
particle a central core where the pores are filled with the solute and a region
between the core and the particle surface where the solute diffuses through
the solvent in the pores. Initially, the core reaches the particle surface, and as
the solute in the particle diminishes, the core shrinks and the internal mass-
transfer resistance increases with increasing length of the path from the core
surface to the particle surface. The model was successfully applied to the SFE
of the prepressed rapeseed (Goto et al., 1996; Germain et al., 2005; del Valle
et al., 2006) and other vegetable oil-rich substrates as flaked rosehip seeds
and olive husks (del Valle et al., 2006).
When, however, less intensive pretreatment methods are used as grind-
ing and milling, a certain region exists inside the particles where the matrix
remains intact and oil diffusion from this region is much slower than from
the regions closer to the particle surface where the walls of the “cells” con-
taining the oil have been broken during the pretreatment. Particularly, the
cells on the particle surface are open, as was shown by the scanning electron
microscope (Reverchon and Marrone, 1997). The BIC models distinguish
between the two regions. The solid-phase mass balance can be written, for
example, as (Sovová, 2005):
∂x + ∂x
G(1 − ε)ρs = − j + js , (1 − G)(1 − ε)ρs = − js (3.22)
∂t ∂t
where G is the initial fraction of the oil in the broken cells, 1 − G is the frac-
tion of the oil in the intact cells, x+ is the concentration in the region with
open cells, and x is the average concentration in the region with the intact
cells, j is given by Equation 3.4, and js is given by Equation 3.5. As the mass-
transfer resistance in the region with open cells is assumed to be zero, x+ and
y+ fulfill the equilibrium condition. Equation 3.22 can be combined with fluid-
phase mass balance equations for any type of flow patterns. In the case of
oilseeds, the equilibrium solubility is the thermodynamic solubility of the
oil in the supercritical solvent, as long as the oil content in particles does not
fall below a critical value. As apparent from Figure 3.3 where experimental
data (Roy et al., 1994) for oil extraction from tomato seeds at extraction condi-
tions of 24.5 MPa and 40°C are modeled, the BIC model simulates also the
slow increase in extraction yield in the second extraction stage, whereas the
shrinking core could match the data in the second extraction stage only with a
horizontal line of asymptotic yield, adjusted separately for each particle size.
Using several simplifying assumptions, an approximate analytical solu-
tion was derived (Sovová, 1994) for the SFE with the plug flow pattern, low
thermodynamic solubility of the extract, no solute–matrix interaction, and
the mass-transfer rate from the intact cells by several orders of magnitude

0.3
0.25
0.2 dp = 0.25 mm
dp = 0.46 mm
e, g/g
0.15 dp = 0.65 mm
dp = 1.02 mm
0.1
0.05
0
0 20 40 60 80
t, min
FIGURE 3.3
BIC model for SFE from particles of size dp (Roy et al., 1994). Model parameters: xu = 0.285 g/g,
tf = 0.1–0.5 min, decreasing with increasing dp, G = 0.21–0.93, decreasing with increasing
dp, ti = 700 min, ys = 8 mg/g CO2, xt = 0.04 g/g, K = 0.05 g CO2/g seed. (Adapted from Roy, B.C.,
Goto, M., Hirose, T. 1996. International Journal of Food Science and Technology, 31(2): 137–141.)
lower than the initial mass-transfer rate from the open cells. The simplified
model closely simulates the extraction of oil from seed particles with low
permeable cell walls (Jokic et al., 2012), however, it should not be applied in
other cases, for example, when a mixture of substances with different solu-
bility is extracted or when the solute interacts with matrix.
With respect to SEM images of seed particles showing their surface cov-
ered with open cells, Fiori et al. (2009) proposed a model that combines the
properties of the shrinking core model and BIC model. The seed particle is
represented by the concentric spherical shells containing oil, which is first
extracted from the outer shell with broken cells, then from the shell below it
where it must surpass one cell wall—a semipermeable woody husk, when this
oil is exhausted, the extraction from the third shell through two walls begins,
etc. A comparison of calculated extraction curves with experimental ones has
shown that the mechanical opening of the cells is not limited to the outer shell;
approximately two shells of cells with broken walls should be considered.
3.5 SFE of Volatile Oils

Essential oils are volatile secondary metabolites produced by plants in
glands, which are of various shape, size, and location. They are obtained
by steam distillation, hydrodistillation, or, in some cases, by pressing. The

extraction with supercritical-CO2 is an alternative, mild method used to

extract the volatile substances from plants. The extent of coextraction of non-
volatile substances soluble in CO2 is adjusted by pressure and temperature.
As the volatile content in plant material is usually low, a few percent or less,
it is in most cases completely adsorbed on the plant matrix and many models
for its extraction include adsorption isotherm, usually linear equilibrium.
The mass-transfer resistance depends not only on the kind of plant, but also
on its drying and mechanical pretreatment. For example, Goto et al. (1993)
found the extraction of peppermint oil to be controlled by linear equilibrium,
the effect of mass-transfer resistance on the extraction rate was negligible.
The model with diffusion in porous spherical particles and the fluid-phase
mass balance equation according to Equation 3.8 was applied. On the other
hand, Reverchon et al. (1993) assumed that the extraction of volatiles from
basil, rosemary, and marjoram leaves was controlled by mass-transfer resis-
tance. The extraction from sage leaves with liquid CO2 was c ontrolled by
mass-transfer resistance (Catchpole et al., 1996) and in other case, with a dif-
ferent sample of plant and with supercritical CO2, both linear equilibrium
and mass-transfer resistance affected the extraction rate (Reverchon, 1996).
Araus et al. (2009) used a model that includes flow with axial dispersion, dif-
fusion in slab particles, and linear equilibrium at particle surface to evaluate
the kinetics of volatile oil extraction from five aromatic plants.
Gaspar et al. (2003) observed in the extraction of volatile oil from oregano
bracts that a part of oil was extracted fast, in fact it was washed out from the
extractor in the solution formed during the static extraction, and the rest
was extracted very slowly. They assumed that a part of the solute had been
liberated from the essential oil glands during the mechanical pretreatment
and formulated a simple type of BIC model where all easily available solute
is dissolved at t = 0 and the solute from intact cells diffuses from slab-like
particles, as described by a modification of hot ball model. The initial wash-
ing out of a part of free essential oil, followed at extraction time t = tr by a
decrease in the extraction rate, is observed also in SFE from other aromatic
plants rich in essential oil, like orange peel (Berna et al., 2000). It is therefore
possible that the difference between the extraction rates in the first and sec-
ond stages is not related to the free and closed solute but to the switch from
the solute that is not bound to matrix to the extraction of adsorbed solute.
Important research on the relationship between the type of glands and
the extraction kinetics is reported in a series of papers (Zizovic et al., 2005,
2007, 2007a). As summarized by Stamenic et al. (2008), the essential oil is
located in secretory cells distributed in the whole volume of plant particle
only in certain plant families, where the above-mentioned diffusion mod-
els are appropriate. In other plant families, the volatile oil is either in frag-
ile ducts or cavities opened by mechanical pretreatment, and the internal
mass-transfer coefficient is low. The herbs of Lamiaceae family, where many
aromatic plants belong, contain essential oil in glandular trichomes located
on the surface of the leaves and flowers. Three fractions of the solute are

distinguished: the first one in the trichomes broken during pretreatment, the
second fraction in the trichomes broken after a certain time of exposition to
supercritical CO2, and the third fraction in the trichomes that remain intact.
A special type of the BIC model was therefore proposed and validated for
Lamiaceae (Zizovic et al., 2005; Stamenic et al., 2008).
3.6 SFE of Mixtures

The extracts from botanic materials are usually complex mixtures, whereas
the above-mentioned SFE models are written for the extraction of one sub-
stance of a given apparent solubility. Their application to seed oils, com-
posed mainly of triacylglycerols, is justified because of similar solubility of
oil components (unless free fatty acids are present in larger percentage). The
content of other components such as sterols, tocopherols, and carotenoids
is small and they practically do not affect the overall extraction curve. The
volatile oils from plants consist of mono- and sesquiterpene hydrocarbons,
oxygenated mono- and sesquiterpenes in variable proportions, and other
volatile substances. The solubility of these compounds in CO2 decreases with
increasing molecular size and polarity, but the difference is relatively low
even at the recommended mild extraction conditions (Della Porta et al., 1999).
In other cases, the extracted oleoresin contains comparable amounts of sub-
stances of different solubility and its kinetics should be simulated by multi-
component models. Otherwise, when a BIC model is applied, the extraction
of more soluble substances, typically volatiles, is related to the extraction
from broken cells and the extraction of easily accessible, less soluble sub-
stances to the extraction from intact cells, in contrast to the reality.
The coextraction of substances of different solubility is modeled in two
ways. First, by simple addition of extraction curves calculated for individ-
ual components. The SFE kinetics of the oil from pressed palm oil fibers
was composed this way from extraction curves for oleic acid, triolein, and
carotenoids (de Franca and Meireles, 2000). The second approach is based
on simultaneous integration of mass balance equations written for indi-
vidual components, which are interconnected by the relationship for mul-
ticomponent equilibrium and/or mutual affecting the diffusion in particles.
Tezel et al. (2000) proposed two multicomponent models. The first one is
a shrinking core model for two extracted substances which cores shrink
in a different rate: the faster extracted substance diffuses first through the
shell of the larger core of the slower extracted compound. The second model
describes the extraction of several substances adsorbed in porous particles:
the Langmuir-like adsorption isotherm was written to represent either a
single compound behavior or a multicomponent behavior. Different extrac-
tion rates of sea buckthorn oil as a whole and its β-sitosterol were simulated,

including the separation factor into the equilibrium relationship (Sovova

et al., 2010).
To sum up, multicomponent models for SFE should be used when the
extract composition is substantially changing with extraction time. Further
research in this field would improve prediction of both overall yield and
extract composition in dependence on extraction conditions.
Nomenclature
a0 Specific surface area, m2/m3
b Exponent in Equation 3.14
c Fluid-phase concentration, kg/m3
cs Solid-phase concentration, kg/m3
〈cs〉 Average concentration in particle, kg/m3
D12 Binary effective diffusion coefficient of extract in the solvent, m2/s
De Effective diffusion coefficient, m2/s
Dl Axial dispersion coefficient, m2/s
e (= E/N) extraction yield, kg/kg
G Initial fraction of oil in broken cells, –
h Axial coordinate, m
H Height of extraction bed, m
j Mass-transfer rate in unit extractor volume, kg/m3 s
js Mass-transfer rate from intact to broken cells, kg/m3 s
L Half of the slab thickness, m
kf External mass-transfer coefficient, m/s
ks Internal mass-transfer coefficient, m/s
K (= KVρs/ρf) partition coefficient, (kg/kg)/(kg/kg)
KV Partition coefficient, (kg/m3)/(kg/m3)
N Feed of plant material, kg
Pe (= Hu/Dl) Peclet number
q (= q′t) solvent-to-feed, kg/kg
q′ (= Q′/N) specific flow rate, kg/kg s
Q′ Flow rate, kg/s
r Radial coordinate, m
R Particle radius, m
t Extraction time, s
tf External mass-transfer characteristic time, s
ti Internal diffusion characteristic time, s
tr (= u/H = γ/q′) residence time, s
T Absolute temperature, K
u Interstitial velocity, m/s
x (= cs/ρs) extract content in solid phase, kg/kg

x+ Solid-phase concentration at particle surface/equilibrium concentra-

tion, kg/kg
x0 Initial extract content in solid phase, kg/kg
xt Transition concentration in Equation 3.13, kg/kg
xu Extract in untreated solid phase, kg/kg
y (= c/ρf) extract content in fluid phase, kg/(kg solvent)
y+ Fluid-phase concentration at particle surface/equilibrium concentra-
tion, kg/(kg solvent)
y0 Initial extract content in fluid phase, kg/(kg solvent)
ys Extract solubility, kg/(kg solvent)
Greek Letters
β Particle porosity, –
γ (= ερf/(1 − ε)ρs) solvent-to-solid mass ratio in extraction bed, kg/kg
ε Void fraction of extraction bed, –
ρf Solvent density, kg/m3
ρs Initial solid density, kg/m3
References
Angus, S., Armstrong, B., de Reuck, K.M. 1976. International Thermodynamic Tables of
Fluid State, Carbon Dioxide. Pergamon Press, Oxford, UK, pp. 37: 45–47.
Araus, K., Uquiche, E., del Valle, J.M. 2009. Matrix effects in supercritical CO2 extrac-
tion of essential oils from plant material. Journal of Food Engineering, 92: 438–447.
Bartle, K.D, Clifford, A.A., Hawthorne, S.B., Langenfeld, J.J., Miller, D.J., Robinson,
R. 1990. A model for dynamic extraction using a supercritical fluid. Journal of
Barton, P., Hughes, R.E., Hussein, M.M. 1992. Supercritical carbon dioxide extraction
of peppermint and spearmint. Journal of Supercritical Fluids, 5: 157–162.
Berna, A., Tarrega, A., Blasco, M., Subirats, S. 2000. Supercritical CO2 extraction
of essential oil from orange peel: Effect of the height of the bed. Journal of
Beutler, H.J., Gährs, H.J., Lenhard, U., Lürken, F. 1988. Einfluß der Lösungsmittelführung
auf den Hochdruck-Extraktions-Prozeß. Chemie Ingenieur Technik, 60: 773–776.
Brunner, G. 1984. Mass transfer from solid material in gas extraction. Berichte der
Bunsengesellschaft für Physikalische Chemie, 88: 887–891.
Brunner, G. 1994a. Gas Extraction. An Introduction to Fundamentals of Supercritical Fluids
and the Application to Separation Processes. Springer, New York, USA, pp. 5–16.
Brunner, G. 1994b. Gas Extraction. An Introduction to Fundamentals of Supercritical Fluids

Brunner, G. 1994c. Gas Extraction. An Introduction to Fundamentals of Supercritical Fluids

Bulley, N.R., Fattori, M., Meisen, A., Moyls, L. 1984. Supercritical fluid extraction of
vegetable oil seeds. Journal of the American Oil Chemists’ Society, 61: 1362–1365.
Catchpole, O.J., Grey, J.B., Smallfield, B.M. 1996. Near-critical extraction of sage, cel-
ery, and coriander seed. Journal of Supercritical Fluids, 9: 273–279.
Dams, A. 1989. Stoffübergang bei der überkritischen Extraktion im Festbett. Chemie
Ingenieur Technik, 61: 712–715.
de Franca, L.F., Meireles, M.A.A. 2000. Modeling the extraction of carotene and lipids
from pressed palm oil (Elaes guineensis) fibers using supercritical CO2. Journal of
Della Porta, G., Porcedda, S., Marongiu, B., Reverchon, E. 1999. Isolation of eucalyp-
tus oil by supercritical fluid extraction. Flavour Fragrance Journal, 14: 214–218.
del Valle, J.M., Aguilera, J.M. 1988. An improved equation for predicting the solu-
bility of vegetable oils in supercritical CO2. Industrial & Engineering Chemistry
Research, 27: 1551–1553.
del Valle, J.M., de la Fuente, J.C. 2006. Supercritical CO2 extraction of oilseeds: Review
of kinetic and equilibrium models. Critical Reviews in Food Science and Nutrition,
46: 131–160.
del Valle, J.M., de la Fuente, J.C., Uquiche, E. 2012. A refined equation for predict-
ing the solubility of vegetable oils in high-pressure CO2. Journal of Supercritical
Fluids, 67: 60–70.
del Valle, J.M., Germain, J.C., Uquiche, E., Zetzl, C., Brunner, G. 2006. Microstructural
effects on internal mass transfer of lipids in prepressed and flaked vegetable
substrates. Journal of Supercritical Fluids, 37: 178–190.
del Valle, J.M., Napolitano, P., Fuentes, N. 2000. Estimation of relevant mass transfer
parameters for the extraction of packed substrate beds using supercritical flu-
ids. Industrial & Engineering Chemistry Research, 39: 4720–4728.
del Valle, J.M., Rivera, O., Mattea, M., Ruetsch, L., Daghero, J., Flores, A. 2004.
Supercritical CO2 processing of pretreated rosehip seeds: Effect of process scale
on oil extraction kinetics. Journal of Supercritical Fluids, 31: 159–174.
del Valle, J.M., Urrego, F.S. 2012. Free solute content and solute-matrix interactions
affect apparent solubility and apparent solute content in supercritical CO2
extractions. A hypothesis paper. Journal of Supercritical Fluids, 66: 157–175.
Fiori, L., Basso, D., Costa, P. 2009. Supercritical extraction kinetics of seed oil: A new
model bridging the ‘broken and intact cells’ and the ‘shrinking-core’ models.
Gaspar, F., Lu, T., Santos, R., Al-Duri, B. 2003. Modelling the extraction of essential
oils with compressed carbon dioxide. Journal of Supercritical Fluids, 25: 247–260.
Germain, J.C., del Valle, J.M., de la Fuente, J.C. 2005. Natural convection retards
supercritical CO2 extraction of essential oils and lipids from vegetable sub-
strates. Industrial & Engineering Chemistry Research, 44: 2879–2886.
Goodarznia, I., Eikani, M.H. 1998. Supercritical carbon dioxide extraction of essential
oils: Modeling and simulation. Chemical Engineering Science, 53: 1387–1395.
Goodrum, J.W., Kilgo, M.K., Santerre, C.R. 1996. Oilseed solubility and extraction
modelling. In Supercritical Fluid Technology in Oil and Lipid Chemistry, ed. J.W.
King and G.R. List. AOCS Press, Champaign, IL, USA, pp. 101–131.
Goto, M., Roy, B.C., Hirose, T. 1996. Shrinking-core leaching model for supercritical-
fluid extraction. Journal of Supercritical Fluids, 9: 128–133.

Goto, M., Sato, M., Hirose, T. 1993. Extraction of peppermint oil by supercritical car-
bon dioxide. Journal of Chemical Engineering Japan, 26: 401–407.
Guardo, A., Coussirat, M., Recasens, F., Larrayoz, M.A., Escaler, X. 2007. CFD stud-
ies on particle-to-fluid mass and heat transfer in packed beds: Free convection
effects in supercritical fluids. Chemical Engineering Science, 62: 5503–5511.
Huang, Z., Shi, X.-H., Jiang, W.-J. 2012. Theoretical models for supercritical fluid
extraction. Journal of Chromatography A, 1250: 2–26.
Jokic, S., Nagy, B., Zekovic, Z. et al. 2012. Effects of supercritical CO2 extraction param-
eters on soybean oil yield. Food and Bioproducts Processing, 90: 693–699.
Kandiah, M., Spiro, M. 1990. Extraction of ginger rhizome: Kinetic studies with super-
critical carbon dioxide. International Journal of Food Science and Technology, 25:
328–338.
King, M.B., Bott, T.R., Barr, M.J., Mahmud, R.S., Sanders, N. 1987. Equilibrium and
rate data for the extraction of lipids using compressed carbon dioxide. Separation
Science and Technology, 22: 1103–1120.
Kubátová, A., Jansen, B., Vaudoisot, J.-F., Hawthorne, S.B. 2002. Thermodynamic and
kinetic models for the extraction of essential oil from savory and polycyclic aro-
matic hydrocarbons from soil with hot (subcritical) water and supercritical CO2.
Journal of Chromatography A, 975: 175–188.
Marrone, C., Poletto, M., Reverchon, E., Stassi, A. 1998. Almond oil extraction by
supercritical CO2: Experiments and modelling. Chemical Engineering Science, 53:
3711–3718.
Melreles, M.A.A., Zahedi, G., Hatami, T. 2009. Mathematical modeling of supercritical
fluid extraction for obtaining extracts from vetiver root. Journal of Supercritical
Fluids, 49: 23–31.
Oliveira, E.L.G., Silvestre, A.J.D., Silva, C.M. 2011. Review of kinetic models for super-
critical fluid extraction. Chemical Engineering Research and Design, 59: 1104–1117.
Peker, H., Srinivasan, M.P., Smith, J.M., McCoy, B.J. 1992. Caffeine extraction rates
from coffee beans with supercritical carbon dioxide. AIChE Journal, 38: 761–770.
Perrut, M., Clavier, J.Y., Poletto, M., Reverchon, E. 1997. Mathematical modeling
of sunflower seed extraction by supercritical CO2. Industrial and Engineering
Chemistry Research, 36: 430–435.
Puiggené, J., Larrayoz, M.A., Recasens, F. 1997. Free liquid-to-supercritical fluid mass
transfer in packed beds. Chemical Engineering Science, 52: 195–212.
Reis-Vasco, E.M.C., Coelho, J.A.P., Palavra, A.M.F., Marrone, C., Reverchon, E. 2000.
Mathematical modelling and simulation of pennyroyal essential oil supercriti-
cal extraction. Chemical Engineering Science, 55: 2917–2922.
Reverchon, E. 1996. Mathematical modeling of supercritical extraction of sage oil.
AIChE Journal, 42: 1765–1771.
Reverchon, E. 1997. Supercritical fluid extraction and fractionation of essential oils
and related products. Review. Journal of Supercritical Fluids, 10: 1–37.
Reverchon, E., De Marco, I. 2006. Supercritical fluid extraction and fractionation of
natural matter. Journal of Supercritical Fluids, 38: 146–166.
Reverchon, E., Donsi, G., Sesti Osseo, L. 1993. Modeling of supercritical fluid extrac-
tion from herbaceous matrices. Industrial & Engineering Chemistry Research, 32:
2721–2726.
Reverchon, E., Marrone, C. 1997. Chemical Engineering Science, 52: 3421.
Reverchon, E., Marrone, C. 2001. Modeling and simulation of the supercritical CO2
extraction of vegetable oils. Journal of Supercritical Fluids, 19: 161–175.

Reverchon, E., Sesti Osseo, L. 1994. Modeling the supercritical extraction of basil oil.
In Proceedings of the 3rd International Symposium on Supercritical Fluids, Strasbourg,
France, ed. G. Brunner and M. Perrut, Institut national polytechnique de
Lorraine, Nancy, France, Tome 2, pp. 189–196.
Roy, B.C., Goto, M., Hirose, T., Navaro, O., Hortacsu, O. 1994. Extraction rates of
oil from tomato seeds with supercritical carbon dioxide. Journal of Chemical
Engineering Japan, 27: 768–772.
Salimi, A., Fatemi, S., Zakizadeh Nei Nei, H., Safaralie, A. 2008. Mathematical mod-
eling of supercritical extraction of valerenic acid from Valeriana officinalis L.
Chemical Engineering & Technology, 31: 1470–1480.
Shilpi, A., Shivhare, U.S., Basu, S. 2013. Supercritical CO2 extraction of compounds
with antioxidant activity from fruits and vegetables waste—A review. Focusing
on Modern Food Industry (FMFI), 2: 43–62.
Silva, C.F., Mendes, M.F., Pessoa, F.L.P., Queiroz, E.M. 2008. Supercritical carbon diox-
ide extraction of macadamia (Macadamia integrifolia) nut oil: Experiments and
modelling. Brazilian Journal of Chemical Engineering, 25: 175–181.
Sovová, H. 1994. Rate of the vegetable oil extraction with supercritical CO2—I.
Modelling of extraction curves. Chemical Engineering Science, 49: 409–414.
Sovová, H. 2005. Mathematical model for supercritical fluid extraction of natural
products and extraction curve evaluation. Journal of Supercritical Fluids, 33:
35–52.
Sovová, H. 2012. Steps of supercritical fluid extraction of natural products and their
characteristic times. Journal of Supercritical Fluids, 66: 73–79.
Sovova, H., Galushko, A.A., Stateva, R.P., K. Rochova, K., M.Sajfrtova, M., M. Bartlova,
M. 2010. Supercritical fluid extraction of minor components of vegetable oils:
β-Sitosterol. Journal of Food Engineering, 101: 201–209.
Sovová, H., Komers, R., Kučera, J., Jež, J. 1994a. Supercritical carbon dioxide extrac-
tion of caraway essential oil. Chemical Engineering Science, 49: 2499–2505.
Sovová, H., Kučera, J., Jež, J. 1994. Rate of the vegetable oil extraction with supercriti-
cal CO2—II. Extraction of grape oil. Chemical Engineering Science, 49: 415–420.
Sovova, H., Stateva, R. 2011. Supercritical fluid extraction from vegetable materials.
Reviews in Chemical Engineering, 27: 79–156.
Sovova, H., Stateva, R.P., Galushko, A.A. 2001. Essential oils from seeds: Solubility
of limonene in supercritical CO2 and how it is affected by fatty oil. Journal of
Stamenic, M., Zizovic, I., Orlovic, A., Skala, D. 2008. Mathematical modelling of
essential oil SFE on the micro-scale—Classification of plant material. Journal of
Stüber, F., Vázquez, A.M., Larrayoz, M.A., Recasens, F. 1996. Supercritical fluid extrac-
tion of packed beds: External mass transfer in upflow and downflow. Industrial &
Engineering Chemistry Research, 35: 3618–3628.
Tan, C.S., Liou, D.C. 1989a. Axial dispersion of supercritical carbon dioxide in packed
beds. Industrial & Engineering Chemistry Research, 28: 1246–1250.
Tan, C. S., Liou, D.C. 1989b. Modeling of desorption at supercritical conditions. AIChE
Journal, 35: 1029–1031.
Tezel, A., Hortacsu, A., Hortacsu, O. 2000. Multi-component models for seed and
essential oil extractions. Journal of Supercritical Fluids, 19: 3–17.

Wiebe, R., Gaddy, V.L. 1941. Vapor phase composition of carbon dioxide water–water
mixtures at various temperatures and at pressures to 700 atmospheres. Journal
of the American Chemical Society, 63: 475–477.
Yoo, K.P., Hong, I.K. 1996. Modeling of the supercritical fluid extraction of oilseeds.
In Supercritical Fluid Technology in Oil and Lipid Chemistry, ed. J.W. King and G.R.
List, Chapter 6. AOCS Press, Champaign, IL, USA.
Zizovic, I., Stamenic, M., Ivanovic, J. et al. 2007a. Supercritical carbon dioxide extrac-
tion of sesquiterpenes from valerian root. Journal of Supercritical Fluids, 43:
249–258.
Zizovic, I., Stamenic, M., Orlovic, A., Skala, D. 2005. Supercritical carbon dioxide
essential oil extraction of Lamiaceae family species: Mathematical modelling
on the micro-scale and process optimization. Chemical Engineering Science, 60:
6747–6756.
Zizovic, I., Stamenic, M., Orlovic, A., Skala, D. 2007. Supercritical carbon dioxide
extraction of essential oils from plants with secretory ducts: Mathematical mod-
elling on the micro-scale. Journal of Supercritical Fluids, 39: 338–346.

4
Biochemical Reactions in Supercritical Fluids
Željko Knez, Maja Leitgeb, and Mateja Primožič
CONTENTS
4.1 Introduction................................................................................................. 127
4.2 Enzyme Catalysis in Nonconventional Media....................................... 131
4.3 Enzyme Catalysis in SCFs......................................................................... 134
4.3.1 Enzyme Stability in SCFs.............................................................. 137
4.3.2 Effect of Pressure............................................................................ 139
4.3.3 Number of Pressurization–Depressurization Steps.................. 141
4.3.4 Effect of Temperature..................................................................... 143
4.3.5 Effect of Water Activity.................................................................. 144
4.4 Enzyme Reactors......................................................................................... 146
4.4.1 Process Schemes and Downstream Processing Schemes......... 146
4.4.1.1 Batch-Stirred Tank Reactor............................................. 146
4.4.1.2 Continuous Packed-Bed Reactor.................................... 146
4.4.1.3 Continuous High-Pressure Enzyme Membrane
Reactor............................................................................... 148
4.4.2 Processing Costs............................................................................. 149
4.5 Conclusion................................................................................................... 149
Acknowledgment................................................................................................. 150
References.............................................................................................................. 150
4.1 Introduction
Enzymatic catalysis has gained considerable attention in recent years as an
efficient tool for the synthesis of natural products, pharmaceuticals, fine
chemicals, and food ingredients.
The production of fine chemicals results in the generation of consider-
able volumes of waste, as the syntheses generally include a number of steps.
The yield of each of these steps is usually 60%–90%, but 10% is also not
unusual. On the basis of these data, we can conclude that typically 1 kg of
end-product leads to the generation of 15 kg of wastes or more. Most of the
generated wastes are solvents and by-products from solvents and intermedi-
ates. Therefore, ideally several reactions should be performed in water or in
supercritical fluids (SCFs).
127
The high selectivity and mild reaction conditions of enzymatic transfor-

mations make them an alternative to the synthesis of complex bioactive
compounds, which are often difficult to obtain by standard chemical routes.
However, the majority of organic compounds are not highly soluble in water,
which was traditionally perceived as the only suitable reaction medium
for the application of biocatalysts. The realization that most enzymes can
function perfectly well under near-anhydrous conditions and, in addition,
display a number of useful properties, for example, highly enhanced sta-
bility and different selectivity, has dramatically widened the scope of their
application to organic synthesis. Another great attraction of using organic
solvents rather than water as a reaction solvent is the ability to perform
synthetic transformations with relatively inexpensive hydrolytic enzymes.
Generally, in vivo, the synthetic and hydrolytic pathways are catalyzed by
different enzymes. However, elimination of water from the reaction mix-
ture enables the “reversal” of hydrolytic enzymes and thus avoids the use
of the expensive cofactors or activated substrates that are required for their
synthetic counterparts. Water is the most common solvent for biochemical
reactions but in a biotechnological perspective, there are a lot of advantages
of conducting enzymatic conversions in monophasic organic solvents as
opposed to water (Dordick, 1989), listed as follows:
• High solubility of most organic (nonpolar) compounds in nonaque-

ous media
• Ability to carry out new reactions impossible in water because of
kinetic or thermodynamic restrictions
• Reduction of water-dependent side reactions
• Insolubility of enzymes in organic media, which allows their easy
recovery and reuse
However, the use of solvents can be problematic because of their toxicity

and flammability, and also due to the increasing environmental concerns. As
a result, SFCs have attracted much attention in recent years as an alternative
to organic solvents for enzymatic reactions.
The present research activity is focused on the development of selective
methods for the production of polyfunctional molecules by enzymatic reac-
tion SCFs especially in supercritical carbon dioxide (SC-CO2).
Among all the possible SCFs, CO2 is largely used. The use of SC-CO2 instead
of organic solvents in biocatalysis presents several additional advantages. It
performs mainly as a lipophilic solvent, nontoxic, nonflammable, and cheap.
SC-CO2 has been most frequently used as a medium for biotransformations:
its critical pressure (7.38 MPa) is “acceptable” and temperature (31.1°C) is con-
sistent with the use of enzymes and/or labile solutes. It has been generally
regarded as safe status. In addition, its “naturalness” is greatly appreciated
by the food- and health-care-related industries. The use of SCFs as solvents

Biochemical Reactions in Supercritical Fluids 129
for enzymatic transformations is a relatively new area of research, which is

expected to expand in the future.
Near the critical point, even small changes in temperature or pressure can
produce large changes in density and solvation ability of SCFs. This attribute
of SCFs can be fruitfully exploited and integrated in biotransformation and
downstream processing steps in a single bioreactor.
Above the critical point, both phases are indistinguishable and the fluid
is monophasic and occupies all the vessel volume. It can be described as a
dense gas or an expanded liquid. Generally, SCFs exhibit liquid-like density
and therefore have a good solvating power, but they retain gas-like com-
pressibility. Consequently, it is possible to control their solvating power by
changing the pressure or temperature, with a continuous transition from a
good to poor solvent. Moreover, low viscosity and high diffusion coefficients
of these fluids enhance mass transport and reaction kinetics. These unique
properties of SCFs enable one to design efficient integrated processes by
coupling an enzymatic reaction with subsequent fractionation and product
recovery steps. Molecules in the SC phase are not uniformly distributed in
space, but the solvent molecules aggregate around the solute through sol-
vent–solute intermolecular interactions forming clusters, where the aggre-
gated molecules are in dynamic equilibrium with the molecules of the free
solvent. Thus, the solvation depends strongly on the density of the SCFs and
differs from that in the liquid solution or gaseous phase (Kajimoto, 1999).
When catalytic reactions are performed in SCFs, the outcome of the reactions
can be affected in a number of ways. In general, replacement of the conven-
tional liquid solvents by SCFs can increase the rate and tune the selectivity of
reactions for the following reasons (Ikariya and Kayaki, 2000):
• Rapid diffusion of solutes or weakening of the solvation around

reacting species facilitates the reactions and sometimes changes the
reaction pathway.
• Local clustering of solutes or solvents resulting in an appreciable
increase in the local concentration of the substrate (and catalyst)
causes acceleration of the reaction.
• Reduction and/or increase in the cage effect often affects the reac-
tion performance of rapid chemical transformations such as radical
reactions.
In addition to the benefits of SCF chemistry, as a reaction media, it pos-

sesses economical, technical, environmental, and health advantages. The
high volatility of CO2 allows it to be completely and easily removed from
the product, resulting in an overall “solvent-free” reaction. By using SC-CO2,
an integrated production process can be performed, because it can act as a
solvent for the reaction and also as a separation medium. The variable solvat-
ing power of SC-CO2 and other SCFs facilitates the integration of biocatalytic
and downstream processing steps in a single robust bioreactor. The main

drawback of SC-CO2 is that it has limited solvating power with respect to

polar compounds. This is a serious limitation for biotechnological applica-
tions where most natural molecules of interest (e.g., alkaloids, carotenoids,
phenols, proteins, and sugars) are only sparingly soluble in SC-CO2. In this
case, a polar cosolvent or the so-called “entrainer,” such as acetone, etha-
nol, methanol, or water, is added in order to increase the polarity of the
medium and to solubilize the target solute via the formation of hydrogen
bonds. Typically, cosolvents are added to the SCF at moderate concentrations
of <10 mol% (Wong and Johnston, 1986). In a batch reactor, the cosolvent can
be added directly into the reactor prior to pressurization, whereas in a con-
tinuous process, the addition should be made to the CO2 inflow via a liquid
pump to deliver a constant flow rate at the operating pressure. However, the
use of another component in the system further increases the complexity
and may also complicate downstream processing. Moreover, the solubility
enhancement effect of the cosolvent is usually limited in the case of very
polar compounds. Two alternative methods have been developed for some
specific cases. To solubilize polyols (e.g., glycerol and sugars), formation of
hydrophobic complex between the polyol and phenylboronic acid (PBAC)
was proposed, due to higher solubility of complex in the SC phase (Castillo
et al., 1994). This method was used to perform the esterification of glycerol
and sugar with oleic acid in SC-CO2. Another method involves the adsorp-
tion of polar substrates onto a solid hydrophilic support such as silica gel.
Compared with the former, this approach is more general because it is not
necessary to have two vicinal hydroxyl groups in the substrate molecule
(Castillo et al., 1994). In addition, recent advances in the understanding of
the chemical properties of materials that are soluble in CO2 have permit-
ted the development of novel surfactants that allow the dissolution of both
hydrophilic and hydrophobic materials in CO2.
The use of SCFs decreases the mass-transfer limitations because of the
high diffusivity of reactants in the SC medium, the low surface tension,
and also the relatively low viscosity of the mixture. The Schmidt number,
Sc = η/ρ ⋅ D (where η is the dynamic viscosity, D the diffusivity, and ρ the
density), for CO2 at 20 MPa is 45 times lower than that for water at 0.1 MPa
and 20°C. High diffusivity and low surface tension of SCFs lead to reduced
internal mass-transfer limitations for heterogeneous chemical or biochemi-
cal catalysis. One of the main advantages of the use of dense gases as a sol-
vent for enzyme-catalyzed reactions is the simple downstream processing.
The physicochemical properties of dense gases are determined by their pres-
sure and temperature and are especially sensitive near their critical point.
By reducing the solvent power of a dense gas in several stages, fractionation
of the product and unreacted reactants is possible. Fractionation is also pos-
sible by extracting the mixture, usually with the same dense gas as used in
the reaction, but under different process conditions. In all downstream pro-
cessing schemes, various particle-formation techniques or chromatographic
techniques can be integrated.

4.2 Enzyme Catalysis in Nonconventional Media

The breakthrough of biocatalysis in nonaqueous media started in the early
1980s (Antonini et al., 1981; Martinek et al., 1981, 1982; Zaks and Klibanov,
1984, 1985). Nowadays, it is well established that many enzymes, such as
lipases, can remain active and stable in pure organic solvents. Changing
the hydrophobicity of predominantly aqueous media through addition of
organic solvents causes the hydrophobic effects, which keep the hydropho-
bic residues buried in the core of the proteins and folded in an aqueous envi-
ronment where enzymes are kinetically trapped in their native structure in
organic solvents (Wescott and Klibanov, 1994; Griebenow and Klibanov, 1996;
Partridge et al., 1999; Klibanov, 2001). In addition, in the media of low water
content, enzyme inactivation, caused by hydrolysis of peptide bonds and
deamidation of asparagine and glutamine residues, is reduced (Klibanov,
2001). Indeed, many times, enzymes are more stable in organic solvents than
in water. For instance, increased thermal stability in dry organic solvents
with substantial increase in enzyme half-life at 100°C compared with water
has been observed (Zaks and Klibanov, 1984, 1988a). The advantages of using
enzymes in organic solvents are listed in Table 4.1.
Many substrates that are insoluble in water can be dissolved by using
organic solvents. Enzymes are often insoluble, an advantage that simplifies
their recovery and reuse many times, which is economically very important.
A change from an aqueous environment can favor a shift in the equilib-
rium, enabling synthetic reactions to be achieved with hydrolytic enzymes.
Moreover, the unique selectivity and activity of enzymatic reactions is
achieved under mild reaction conditions. The disadvantage of using enzymes
is their instability under harsher reaction conditions, which is common in
industrial process. The search for enzymes from various extremophilic organ-
isms may however result in biocatalysts that can withstand more extreme
conditions (Adams and Kelly, 1995; Persidis, 1998). Furthermore, enzymes are
environmentally benign and, unlike metal catalysts, are completely degraded
in nature. Although practically some enzymes do display perfect selectivity,
TABLE 4.1
Advantages of Biocatalysis in Nonaqueous Media
Increased substrate solubility
Simplified recovery of biocatalyst
Shift to synthetic reactions
Mild reaction conditions and minimization of side reactions
Environmentally benign catalyst
High selectivity
Simplified work-up of products
Avoids microbial contaminations

many others can receive a broad range of unnatural substrates still with high
chemo-, regio- or enantioselectivity. Additionally, the use of an organic sol-
vent often simplifies work-up procedures and avoids microbial contamina-
tion of the reaction medium (Faber, 2000; Klibanov, 2001).
The conventional notion that enzymes are only active in aqueous media
has long been discarded, thanks to the numerous studies documenting
enzyme activities in nonaqueous media, including pure organic solvents and
SCFs. Enzymatic reactions in nonaqueous solvents offer new possibilities for
producing useful chemicals (emulsifiers, surfactants, wax esters, chiral drug
molecules, biopolymers, peptides and proteins, modified fats and oils, struc-
tured lipids, and flavor esters). According to conventional notion, enzymes
are active only in water. Historically, enzymatic catalysis has been carried
out primarily in aqueous systems. Although water is a poor solvent for pre-
parative organic chemistry, it is the unique specificity of enzymes that drew
the interest of chemists who were seeking highly selective catalytic agents.
Experiments to place enzymes in systems other than aqueous media date
back to the end of the nineteenth century (Hill, 1898; Kastle and Loevenhart,
1900; Bourquelot and Bridel, 1912, 1913; Dastoli and Price, 1967). Initial stud-
ies considered the addition of small quantities of water-miscible organic sol-
vents like ethanol or acetone to aqueous enzyme solutions ensuring high
availability of water to retain the catalytic activity of enzymes. Then, the
biphasic mixtures (aqueous enzyme solution emulsified in a water-immisci-
ble solvent such as iso-octane or heptane) were used, in which the substrates
from the organic phase diffuse into the aqueous phase that undergoes enzy-
matic reaction and the products diffuse back. The size of the water drop-
lets may be reduced to facilitate mass transfer, resulting in the formation of
microemulsions or reverse micelles, whose stabilization is achieved by add-
ing surfactants (Martinek et al., 1986; Krishna et al., 2002).
Developments in using enzymes in nearly nonaqueous solvents contain-
ing traces of water have stimulated research in achieving various kinds
of enzymatic transformations (Klibanov, 1986, 1989; Schoffers et al., 1996;
Bornscheuer and Kazlauskas, 1999; Gandhi et al., 2000; Giri et al., 2001;
Krishna and Karanth, 2002a; Panke and Wubbolts, 2002; Thomas et al., 2002).
Enzymatic reactions in nonaqueous solvents offer numerous possibilities
for the biotechnological production of useful chemicals using reactions that
are not feasible in aqueous media. These reactions include chiral synthesis
or resolution (Klibanov, 1990; Collins et al., 1992; Stinson, 2000; Zaks, 2001),
production of high-value pharmaceutical substances (Zaks and Dodds, 1997;
Schulze et al., 1998; McCoy, 1999; Patel, 2001; Rasor and Voss, 2001), modifica-
tion of fats and oils (Bornscheuer, 2000a; Lee et al., 2009), synthesis of flavor
esters and food additives (Krishna and Karanth, 2001, 2002b; Krishna et al.,
1999, 2000a, 2000b, 2001a, 2001b), and production of biodegradable polymers
(Kobayashi, 1999), peptides, proteins, and sugar-based polymers (Vulfson,
1998). In nonaqueous solvents, hydrolytic enzymes could undergo synthetic
reactions, while they also exhibit altered selectivities (Klibanov, 2001), pH

memory (Zaks and Klibanov, 1985, 1988a; Klibanov, 1995), increased activity
and stability at elevated temperatures (Zaks and Klibanov, 1984; Ahern and
Klibanov, 1985), regio-, enantio- and stereoselectivity (Bornscheuer, 2000a,
2000b), and may also be affected by their water activity (Halling, 2000).
Currently, there is a considerable interest in the use of enzymes (particularly
lipases, esterases, and proteinases) as catalysts in organic synthesis (Schmid
and Verger, 1998; Bornscheuer, 2000a, 2000b; Carrea and Riva, 2000; Faber,
2000; Liese et al., 2000; Patel, 2000; Koeller and Wong, 2001; Dhake et al., 2013;
Li et al., 2014).
Five major technological advances are believed to have significantly influ-
enced the industry for adopting enzymatic biotransformations (Lilly and
Eighth, 1994): (1) the development of large-scale downstream processing
techniques for the release of intracellular enzymes from micro-organisms;
(2) improved screening methods for novel biocatalysts (Kieslich et al., 1998;
Demirjan et al., 1999; Wahler and Reymond, 2001; Asano, 2002; Ornstein,
2002); (3) the development of immobilized enzymes; (4) biocatalysis in organic
media; and most recently (5) recombinant-DNA technology to produce
enzymes at a reasonable cost. There seems to be no agreement as to why the
biocatalysis in organic media could not take off earlier (Halling and Kvittingen,
1999; Klibanov, 2000; Kvittingen, 2000). Perhaps, the traditional belief that
most enzymes are incompatible with most organic syntheses in nonaque-
ous media also poses a psychological hurdle. Also, until recently, there was
no demand for enantiopure compounds, and hence, no enzymes need to be
used. The establishment of industrial processes (Coleman and Macrae, 1977;
Matsuo et al., 1981) and the realization that most enzymes can function well
in organic solvents (Zaks and Klibanov, 1984, 1985, 1986, 1988b) have height-
ened interest in the use of enzymes. Also, the need for enantiomerically pure
drugs is driving the demand for enzymatic processes. This combined with
the discovery of strikingly new properties of enzymes in organic solvents has
led to the establishment of organic-phase enzyme processes in the industry
(Bornscheuer, 2000b; Liese et al., 2000; Ramsey et al., 2009).
Enzymes occupy a unique position in synthetic chemistry because of their
high selectivities and rapid catalysis under ambient reaction conditions.
Nevertheless, synthetic chemists have been reluctant to employ enzymes
as catalysts, because most organic compounds are water-insoluble and the
removal of water is tedious and expensive. The fact that enzymes are sta-
ble, and in some cases, improve their high specificity in near-anhydrous
media, has dramatically changed the prospects of employing enzymes in
synthetic organic chemistry. The problems that arise for most biotransfor-
mations are low solubility of reactants and products and limited stability of
biocatalysts. Carrying out reactions in an aqueous–organic two-phase sys-
tem would be a solution to overcome the first problem. This is not always
possible due to the limited stability of enzymes at liquid–liquid interface
or in organic solvents. Hence, other approaches are also necessary. These
include addition of complexing agents such as dimethylated cyclodextrins

or adsorbing materials such as XAD-7 resins (Eli Lilly, Indianapolis, USA),

use of membrane-stabilized interface (Sepracor, Marlborough, MA, USA),
and continuous extraction of reaction products (Forschungszentrum-Julich,
Julich, Germany). The catalyst’s stability can be increased using a variety of
methods including the addition of antioxidants (e.g., dithiothreitol), immo-
bilization, cross-linking, separation from deactivating reagents, variation of
reaction conditions, and by genetic engineering (Burton et al., 2002).
Solvent systems used as the reaction media for enzymatic catalysis may be
categorized as: (1) aqueous; (2) water: water-miscible (monophasic aqueous–
organic system); (3) water: water-immiscible (biphasic aqueous–organic sys-
tem); (4) no aqueous (monophasic organic system); (5) anhydrous; (6) SCFs;
(7) reversed micelles; (8) solvent-free systems; (9) gas phase; and (10) ionic
liquids (ILs).
4.3 Enzyme Catalysis in SCFs

During the last decade, the tremendous potential of enzymes as a practi-
cal catalyst for chemical processes in nonaqueous environments has been
well recognized. The use of biocatalysts in solvents other than water offers
many advantages over using pure water. Among these media, SCFs, such
as SC-CO2, exhibit properties similar to organic solvents, but with the addi-
tional capacity of enhancing transport phenomena (due to their high diffu-
sivities) and facilitate reaction products separation by tuning solvent power,
which makes them more attractive to be used as “green designer” solvents
(Jarzebski and Malinowski, 1995; Lozano et al., 2002). The interest of using
biocatalysts in SC-CO2 as well as in other SCFs has been growing rapidly in
recent years, mainly in industrial and pilot plant applications (Perrut, 2000;
Habulin et al., 2007; Knez, 2009; Perez et al., 2010; Varma and Madras, 2010a;
Matsuda, 2013).
The growing interest in SCF technology results from the attractive pos-
sibilities offered by this technique: processing at moderate, usually ambient
temperatures, use of nontoxic, nonflammable, and environmentally accept-
able solvents (usually pressurized CO2), and easy-to-change solvent power
(not possible with conventional organics). For example, SCFs at temperatures
and pressures slightly above the critical points (e.g., 31.1°C and 7.38 MPa for
CO2) exhibit unique combined properties: liquid-like density (and hence sol-
vent power) and high compressibility, very low viscosity, and high diffusiv-
ity. The first two properties make the solvent power controllable by changing
the pressure and/or temperature, whereas low viscosity and high diffusivity
markedly enhance mass transport phenomena and hence kinetics of a pro-
cess. However, enzymes are not soluble in SCFs, and therefore enzymatic
catalysis in SCFs would always be heterogeneous.

The use of enzymes as catalysts in nonaqueous media has been described

in scientific literature since the middle of the 1980s (Zaks and Klibanov, 1985).
Organic media offer certain advantages over aqueous media: stabilization of
enzymes, dissolution of hydrophobic compounds, and the feasibility of shift-
ing thermodynamic equilibrium toward the synthesis of esters and amides
(e.g., in the case of hydrolytic enzymes). Certain SCFs, such as CO2, may pro-
vide an interesting alternative to organic solvents since they exhibit similar
properties and their solvent power, which is dependent on the specific den-
sity (and hence easily controllable by regulation of pressure and tempera-
ture), may be advantageously used in the recovery of products. This opens
the way for an integrated production of product recovery process.
The use of SCFs as media for enzymatic reaction was first proposed, in
the middle of 1980s, by Randolph et al. (1985), Hammond et al. (1985) and
Nakamura et al. (1986). The aim of the initial studies was to demonstrate
that enzymes are active in SC-CO2. Randolph et al. (1985) showed that alka-
line phosphatase and cholesterol oxidase are active in SC-CO2; Hammond
et al. (1985) demonstrated the same for polyphenyl oxidase and Nakamura
et al. (Nakamura et al., 1986) carried out successful transesterification reac-
tions using lipase. Successively, Randolph et al. (1988) examined the effect of
aggregation of cholesterol on cholesterol oxidase activity and found that the
addition of cosolvents (entrainers) promotes aggregation and thus resulted
in an increase in the reaction rate proportional to the degree of aggregation.
Recently, the benefit of using SCFs for enzymatic reactions has been dem-
onstrated by Mori and Okahata (1998), Kamat et al. (1992, 1993), Chaudhary
et al. (1996) and Paiva et al. (2011), for example, improved reaction rates, con-
trol of selectivities by pressure, etc. Some examples of enzymatic reactions
are shown in Figures 4.1 and 4.2. Varma and Madras (2010) demonstrated
that different SCFs used for transesterification of methyl butyrate, ethyl
butyrate, and butyl butyrate influenced the initial reaction rate, for example,
the initial rate of transesterification of butyl butyrate in different SCFs fol-
lowed the order: SC-CO2 < SC-C2H6 < SC-C2H4 < SC-CH4.
Shang et al. (2014) reported the formulation of a CO2-based micelle sta-
bilized by nontoxic 2,6,8-trimethyl-4-nonanol (TMN) series surfactants.
Enantioselection of racemic ibuprofen catalyzed by Candida antarctica lipase
B (CALB) was used as a model reaction and they found out that better resolu-
tion efficiency in high-pressure CO2-based micelles could be achieved within
a relatively short period of time compared with other traditional reactive
systems. The improvement in the conversion of palmitic acid (81%) and yield
OH O OH O OH O
Pseudomonas SP
* OEt OEt + OH
SC-CO2
FIGURE 4.1
Reaction scheme of the enantioselective hydrolysis of HPAE in SC-CO2. (From Hartmann, T.,
Meyer, H.H., Scheper, T. 2001. Enzyme and Microbial Technology, 28: 653.)

R OH O O O
Pseudomonas SP R OH R OH
C + CH3 C O C CH3 +
C C CH3 C OH
R1 H SC-CO2
R1 H R1 H
FIGURE 4.2
Reaction scheme of enzymatic esterification of secondary alcohols in SC-CO2. (From Cernia, E.,
Palocci, C., Soro, S. 1998. Chemistry and Physics of Lipids, 93: 157.)
of dipalmitin (69.44%) were obtained in SC-CO2, whereas in organic solvent

(tert-butanol), lower conversion and yield were detected (Tao et al., 2013).
Another improvement in production of n-octyl oleate catalyzed by immo-
bilized lipase from Rhizomucor miehei (Lipozyme RM IM) was detected in
SC-CO2 when compared with those attained over n-hexane under identical
reaction conditions (Laudani et al., 2007).
Similar results were obtained by Guthalugu et al. (2006) that the maximum
degree of enzymatic hydrolysis of triglycerides of soy-deodorized distillate
was achieved with short incubation time of 1.5 h under SC-CO2, whereas
the conventional method of hydrolysis in n-hexane under similar reaction
conditions of temperature, moisture, and enzyme concentration needed
5 h to achieve 88% of triglyceride hydrolysis. An integrated process for the
enzymatic alcoholysis of lipids and the separation of the products by SCF
extraction has been designed by Weber et al. (2007). Ciftci and Saldana (2012)
demonstrated that SC-CO2 is a promising green solvent for the enzymatic
synthesis of phenolic lipids.
Shin et al. (2012) performed enzymatic ethanolysis of menhaden oil by
lipozyme TL-IM in the SC-CO2 system and solvent-free system. The reaction
rate in the SC-CO2 system was slightly lower than that in the solvent-free sys-
tem, but the reaction products, especially ethyl ester, can easily be separated
from the mixtures.
The production of biodegradable and biocompatible poly-l-lactide has
also been successfully achieved by enzymatic ring-opening polymerization
(e-ROP) of l-lactide in subcritical and supercritical 1,1,1,2-tetrafluoroethane
(R134a). Best results were obtained at 105°C and 5 MPa using the thermo-
stable lipase from Burkholderia cepacia with a maximum polylactic acid yield
above 50% (Guzman-Lagunes et al., 2012). Subcritical R134a was also used as
reaction media for enzyme-mediated syntheses of polyester structures (e.g.,
poly(delta-valerolactone)) (Lopez-Luna et al., 2010).
The most of the research published to date dealt with two problems:
(1) conformation and stability in the SC environment (mainly CO2) and
the effect of pressure on the reaction rate; (2) the effect of water/moisture
content on the activity of the enzyme. Housaindokht and Monhemi (2013)
showed for the first time the possibility of interfacial activation of lipases in
a compressed gas. The analysis showed that in compressed propane the lid
of the lipase was opened and so the active conformation of the enzyme was
resulted. Moreover, it was found that in the compressed propane, similar to

the aqueous solution, the enzyme has native conformation (Housaindokht

and Monhemi, 2013).
In recent years, among nonconventional bioreaction media, biphasic sys-
tem SCF/IL has increasingly attracted attention as emerging green and high-
tech alternative to classical organic solvents to carry out enzymatic reactions
for the preparation of valuable and active materials (Zarevucka and Wimmer,
2008; Lozano et al., 2009; Sureshkumar and Lee, 2009). By combining biocata-
lysts with biphasic systems based on neoteric solvents, for example, ILs and
SC-CO2, interesting alternatives to organic solvents for designing continuous
clean (bio) transformation methods are growing which directly provide pure
products (Lozano et al., 2011a). Besides, the ability of CO2 to force two immis-
cible phases to form one homogeneous phase for the reaction performance
is of great advantage. After completion of the reaction, the homogeneous
fluid phase could be split into two or three phases upon pressure decrease in
order to facilitate the product recovery (Bermejo et al., 2008).
4.3.1 Enzyme Stability in SCFs

The use of enzymes in SCFs presents a number of problems, and the param-
eters that influence the stability of the enzymes increase dramatically. This
is the reason why, up to now, no prediction was made on whether enzymes
are stable under supercritical conditions or whether the equivalent of even
higher activity and selectivity is available compared with the reactions in
organic solvents. In the following sections, the influence of parameters on
the enzyme stability will be discussed.
Early investigations (Hammond et al., 1985; Randolph et al., 1985;
Nakamura et al., 1986) demonstrated that certain enzymes are active in
SC-CO2. Randolph et al. (1988) first studied the conformation of several spin-
labeled variants of cholesterol oxidase in SC-CO2 and concluded that these
proteins were not influenced by the SC-CO2 environment; a similar result
has recently been obtained for lipase (Miller et al., 1991). In contrast, Kasche
et al. (1988) reported that α-chymotrypsin, trypsin, and penicillin amidase
were partially denaturated by SC-CO2 and suggested that the decompres-
sion process led to their denaturation, but no in situ measurements were
conducted to substantiate this suggestion. Most recently, Zagrobelny and
Bright (1992) carried out a more detailed examination of the same problem.
The conformation of trypsin in SC-CO2 was studied at the pressure range of
5–25 MPa and monitored the conformational changes in trypsin in situ as
a function of pressure. Their results clearly demonstrate that (1) significant
changes in protein conformation can be induced by SC solvents, (2) most of
the conformational changes occur during compression, and (3) the native
trypsin conformation is only slightly more stable than the unfolded form.
Performance characteristics, specific rates of conversion, and yield factors
are essential for rating any technological processes. On the whole, enzymatic
reactions in SC-CO2 proceed at rates similar to those of organic solvents such

as n-hexane (Marty et al., 1990, 1992) and cyclohexane (Miller et al., 1991),
ensuring similar rates of processing and enzyme stability. The SC technology
offers important advantages over organic solvent technology, such as ecolog-
ical friendliness and product fractionation, which can easily be linked with
direct micronization and crystallization from SC-CO2 by fluid expansion. In
addition, CO2 does not usually oxidize substrates and products, allowing
the process to be operated at only a temperature of 40°C. Although many
enzymes are stable in SCFs, one should pay considerable attention to find-
ing the correct reaction conditions for each substrate/enzyme/SCF system.
Although successful reactions have been reported with Subtilisin Carlsberg
protease and Candida lipases in SC-CO2, there is also evidence for their insta-
bility (Kamat et al., 1992, 1995; De Carvalho et al., 1994) or the existence of a
narrow pressure range of activity (Ikushima et al., 1995, 1996). These enzymes
are fairly stable in other SCFs such as fluoroform, ethylene, ethane, propane,
and sulfur hexafluoride (Kamat et al., 1992). Manera et al. demonstrated
that different compressed fluids (SC-CO2, propane, n-butane) had different
influence on the enzymatic synthesis of galactooligosaccharides (GOSs) in a
batch mode reactor catalyzed by beta-galactosidase from permeabilized cell
of Kluyveromyces marxianus (Manera et al., 2011). Comparing the GOS produc-
tions in the three reaction systems, the SC-CO2 led to the best results, where
the maximum production was 83 g/L, whereas, for propane and n-butane,
values of 63 and 75 g/L were verified, respectively (Manera et al., 2012).
Immobilized Mucor miehei lipase appears to be very stable in SC-CO2, as it
is a monomeric enzyme with three stabilizing disulfide bonds (Jensen et al.,
1987), which may play a role in maintaining its activity in SC-CO2.
The evaluation of the lipase stability from newly isolated Bacillus sp.
ITP-001 after different times with propane at 45°C and 60°C and pressures
of 5 and 20 MPa, considering the free form of lipase and also the enzyme
immobilized by the sol–gel technique, was studied by Carvalho et al. (2014).
The hydrolytic activity of the free lipase remained constant up to 360 min of
incubation at 60°C and 20 MPa. For the immobilized enzyme, a loss in the
hydrolytic activity was observed after the high-pressure treatment, but the
esterification activity was improved up to 50% at 60°C and 20 MPa.
Various approaches have been applied in order to enhance enzyme sta-
bility and activity. The capability of SC-CO2 to alter the activity of alpha-
amylase after consecutive enzymatic reactions was studied by Senyay-Oncel
and Yesil-Celiktas (2011, 2013). Therefore, alpha-amylase from Aspergillus
oryzae initially treated with SC-CO2 was immobilized in Ca-alginate beads
and NaY zeolite, subsequently assayed several times for the hydrolysis of
soluble starch and when a lower activity value was recorded compared with
the initial activity of the untreated enzyme, the immobilized samples were
retreated with SC-CO2. These consecutive reactions and treatment loops were
repeated till the activity could not be increased with SC-CO2 retreatment in
comparison to the initial activity of the untreated enzyme. The best results
were achieved with NaY zeolite immobilized samples where four successful

loops and 17 reactions were realized till the activity could not be enhanced
to a value higher than the activity of the untreated enzyme (Senyay-Oncel
and Yesil-Celiktas, 2013).
Alternative fuels are becoming important due to diminishing fossil-fuel
reserves and the environmental hazards. Biodiesel is an attractive alternative
fuel and could be synthesized in SC-CO2 using enzyme as catalyst (Varma
and Madras, 2007a; Varma et al., 2010). Because the products and the enzyme
do not dissolve in CO2 at room conditions, separation can easily be achieved
by reducing the pressure and therefore this process is of commercial interest.
The improvement of operational stability of the immobilized CALB by its
coating with ILs, leading to a two-phase systems with respect to the bio-
diesel product, which showed an excellent catalytic behavior in continuous
operation under SC-CO2 conditions (up to 82% biodiesel yield after 12 cycles
of 4 h), was reported by Lozano et al. (2011b). Further, no deactivation of
immobilized CALB (Novozym 435) treated with subcritical R134a under dif-
ferent operation conditions (pressure of 2–8 MPa, temperature of 30–60°C,
incubation time of 1–12 h, different water content enzyme/water and depres-
surization rate) was observed. Subcritical R134a treatment led to significant
increase in activity of Novozym 435, and a maximum residual activity of
300% was measured at 4 MPa, 30°C after 7 h of incubation (Yu et al., 2007).
Nevertheless, one of the most important advantages by using the SCFs as
reaction media is that upstream processing after reaction can be greatly sim-
plified as the technique is easily combined with other unit operations.
4.3.2 Effect of Pressure

The influence of the system pressure on the stability of enzymes is not so sig-
nificant within the pressure ranges of up to 30 MPa. This is of great advan-
tage because it means that the solvent power of the SCF can be adjusted for
reaction performance. On the one hand, the solubility of substances increases
with higher pressures because of the higher fluid density and this is essen-
tial to bring the initial products in the reactor and remove the end-products
from the reactor. On the other hand, higher pressure normally results in
higher reaction rates. Therefore, a pressure increase is in most cases positive
for enzymatic reactions.
An isothermal change in pressure of SCFs may change the reaction rate by
changing either solubility or the reaction rate constant. A pressure increase
improves solubility, thereby increasing production rates and this effect is
most pronounced in the near-critical region. Certain enzymes show consid-
erable apparent pressure activation (Dufour et al., 1995). Pressure can modify
the catalytic behavior of the enzyme by changing, for example, the rate-
limiting step (Gross et al., 1993) or modulating the selectivity of the enzyme
(Okamoto et al., 1991). If an enzyme is stable in an SCF, its stability is usually
not influenced by pressure ranges up to 30 MPa. Conversely, reaction rates
may be influenced by the pressure. In most cases, a pressure increase acts

positively for enzymatic reactions or there are no changes in the reaction

rates. Pressure-induced deactivation of enzymes takes place mostly at pres-
sure exceeding 150 MPa. Reversible pressure denaturation mostly occurs at
pressures below 300 MPa and higher pressures are required to cause irre-
versible denaturation (Cheftel, 1991).
The effect of pressure on the reaction rate constant has not yet been deter-
mined but the effect on the overall production rate has been examined in
several papers. Erickson et al. (1990) carried out transesterification of triglyc-
erides, using lipase from Rhizopus arrhizius. The used reactants were trilau-
rin and palmitic acid and the pressure ranged from 10 to 30 MPa. A strong
negative effect of pressure increase on the rate of palmitic acid incorporation
into triglyceride was detected, especially in the near-critical region. Another
influence by pressure on the catalytic efficiency of immobilized lipase from
R. miehei (Lipozyme RM IM) used as a biocatalyst for lauryl oleate produc-
tion in SC-CO2 was found. With increasing the pressure up to 10 MPa, the
catalytic efficiency of Lipozyme RM IM improved, rising up to a maximum
and decreased at higher pressure values (Knez et al., 2007). The interestifi-
cation of trilaurin and myristic acid, catalyzed by lipase, was investigated
by Miller et al. (1991) in the pressure range 6–11 MPa. The interesterification
rate and the overall rate (based on total trilaurin conversion) increased with
increase in pressure; however, the interesterification rate increased much
more rapidly than the overall rate, indicating that the selectivity of the reac-
tion for interesterification over hydrolysis improved at higher pressures. The
operational stability of enzymes in SC-CO2 is of crucial importance from the
point of view of application. Miller et al. (1991) measured the interesterifica-
tion rate over 80 h of continuous operation and observed no loss of activity
of lipase. Cholesterol oxidase is stable at 10 MPa and 35°C for at least 50 h
(Randolph et al., 1988). Pressure has also been found to have little effect on
the stability of lipase from M. miehei in the range 13–18 MPa, causing only
10% loss of activity (Marty et al., 1990) after 6 days at 40°C, unlike tempera-
ture, effect which contributed to a 20% loss at 60°C. Additionally, in some
cases, a negative effect of pressure on the catalytic activity on biocatalysts
in compressed gases may be observed. The catalytic efficiency of subtilisin
Carlsberg suspended in compressed propane, near-critical ethane, near-
critical CO2, and tert-amyl alcohol at constant temperature and pressure up
to 30 MPa and fixed enzyme hydration was lowered (Fontes et al., 1998). In
near-critical fluids, an increase in pressure of only 20 MPa caused a sixfold
decrease in the catalytic efficiency of subtilisin in CO2.
In SC-CO2, the formation of carbamates is essential for lower enzymatic
activity in this medium. Carbamates are the product of the reaction between
basic free amino groups in enzymes and acidic SC-CO2 (Kamat et al., 1995).
On the other hand, lysozyme lipase unfolded and partially oligomerized in
moist SC-CO2 at 80°C and its denaturation was not caused by interaction
with SC-CO2 but by heating the protein in the presence of water, as found by
Weder (1984).

One of the advantages of using SCFs as enzymatic reaction media is sep-

aration of products from the reaction mixture with changing the pressure
of the SCF. With the respect to the facts mentioned previously, the solvent
power of the SCF can be adjusted for running reactions, and products can
easily be removed from the reactor.
When the lipid-coated lipase was employed in SC-CHF3, the enzyme activ-
ity (Mori et al., 2001) could switch on and off by adjusting the pressure or
temperature of SC-CHF3. The influence of pressure in the operative pressure
range from 7.5 to 30 MPa on the conversion of lactic acid in SC-CHF3 cata-
lyzed by CALB (Novozym 435) was studied by Kavčič et al. (2014). The high-
est conversion of lactic acid after 26 h of reaction performance was obtained
at 30 MPa and 55°C.
The effect of pressure on the extent of conversion and the product composi-
tion in the enzyme-catalyzed hydrolysis of canola oil in SC-CO2 was investi-
gated using lipase from M. miehei immobilized on macroporous anionic resin
(Rezaei and Temelli, 2000). A conversion of 63%–67% (triglyceride disappear-
ance) was obtained at 24–38 MPa. Monoglyceride production was favored at
24 MPa. The amount of product obtained was higher at 24–38 MPa due to
enhanced solubility of SC-CO2. Oliveira et al. (2009) reported, by increasing
the pressure, the inhibition of decyl acetate synthesis catalyzed by immobi-
lized CALB (Novozym 435) in SC-CO2.
The e-ROP of poly(epsilon-caprolactone) was performed in SC-CO2 solvent
medium at pressure range from 12 to 28 MPa. This study also evaluates the
enzyme reuse in order to reduce the impact of the enzyme cost on the pro-
cess. Results for analysis of variance statistical analysis for the first set of
experiments show that the pressure or the solvent density has no significant
influence over the parameters evaluated (Rosso et al., 2013). Response sur-
face methodology was used to predict the effects of pressure (20–30 MPa),
fat:water ratio (1:5–1:30 mol/mol), and their interaction in the free fatty acid
(FFA) production by enzymatic hydrolysis of conjugated linoleic acid (CLA)-
enriched anhydrous milk fat using SC-CO2 and three different biocatalysts
(Lipozyme RM IM, Novozyme 435, and immobilized enzyme from Candida
rugosa) (Prado et al., 2012). The maximum CLA content in FFA form was
obtained using Lipozyme TL IM at 30 MPa, 1:30 fat:water ratio (mol/mol),
and at 55°C.
4.3.3 Number of Pressurization–Depressurization Steps

The influence of pressurization–depressurization steps in batch reactors
on the enzyme activity is of importance to many researchers (Knez et al.,
2001). Pressurizing an enzyme does not play an important role, but depres-
surization is usually the step that influences residual enzyme activity. The
SCF permeates through the enzymes by diffusion, a process relatively slow.
After a certain time, the enzymes are saturated with the SCFs. When the
expansion is too fast and the fluid diffuses out of the enzyme slowly, this

causes yet a higher fluid pressure in the enzyme than in the system. The
pressure difference results in cell cracking, where the cell membranes are
broken by the resulting pressure inside the cells. This causes the unfolding
of the enzymes and therefore destroys the structure which is of importance
for the activity and selectivity. Experiments have shown that depressuriza-
tion from SC-CO2 conditions is much smoother than entering the two-phase
region and expanding the gas of the fluid (Habulin et al., 2005). This can be
explained in combination with a change in density which is continuous in
supercritical conditions. The expanding liquid CO2 causes evaporation of the
fluid accompanied by a large change in density and this volume expansion
causes the unfolding of the enzyme.
Depressurization is of importance when using the benefit of SCFs for
simple downstream processing. In this case, by operating a cascade of
depressurizations (with a possible change in temperature), product frac-
tionation can be achieved (Romero et al., 2005). Due to successful indus-
trial applications, enzymes as biocatalysts should retain their activity for
a considerable period of time. The activity of the lipase from C. antarctica
for the production of isoamyl acetate in SC-CO2 was studied by Romero
et al. (2005) in a tubular reactor, measuring the esterification extent at the
stationary state. The yield of isoamyl acetate was 100% for 30 days and then
slowly decreased. Habulin et al. (1996) found similar results with immobi-
lized Rhizomucor miehie lipase, reporting a 4% decrease in conversion after
1 month of treatment.
Cholesterol oxidase from different sources can exhibit different stabilities
in SC-CO2 (Randolph et al., 1988). By cholesterol oxidation, the cholesterol
oxidase from Gloecysticum retained its activity for 3 days and the one from
Streptomyces sp. for only 1 h. Commercial horseradish peroxidase (HRP)
treated in compressed CO2 promotes a significant decrease in the enzyme
activity, whereas HRP showed good stability in compressed propane, indi-
cating that this enzyme is more stable in propane than in CO2 (Fricks et al.,
2009).
In some cases, half-life of the biocatalysts under pressure could be increased,
as it was the case with Lozano et al. (1996). The half-life of α-chymotrypsin
increased with increasing pressure from 8 to 15 MPa. The influence of pres-
surization–depressurisation cycles of up to 12 times on the Lipozyme RM IM
and TL IM resultant activity was studied by Jenab et al. (2014). The amount
of reaction intermediates decreased by 50%–60% in the product obtained
by using SC-CO2-treated enzymes after 12 pressurization–depressurization
cycles compared with untreated enzymes, whereas there were no significant
changes in the conformational and morphological structures of the treated
enzymes. Kuhn et al. (2011) demonstrated that the stability of the inulinases
from K. marxianus NRRL Y-7571 immobilized in natural montmorillonite
after high-pressure pretreatment in propane was higher than the nontreated
one. Another study on the effect of treatment with compressed propane on
lipases hydrolytic stability (Amano PS, Amano AY30, and a noncommercial

lipase from Yarrowia lipolytica) in free, resuspended, and in immobilized

form showed that the residual enzyme activity after exposure to compressed
propane changes significantly depending on the enzyme presentation form
(Franken et al., 2010).
4.3.4 Effect of Temperature

The effect of temperature is much more significant than that of the pressure.
For enzyme stability, a temperature increase above certain levels, depending
on the enzyme sources, results in the deactivation of the enzymes.
This limitation causes the temperature limits for the solubility of the ini-
tial and end products of the reaction. Normally, at higher pressure levels,
an increase in temperature also results in higher solubilities of substances
in SCFs because the increase in the vapor pressure of the compounds to be
dissolved overcomes the reduction in density. On the other hand, within
the ranges of pressure (10–40 MPa) and temperature (35–60°C) that typi-
cally characterize the supercritical region, an increase in pressure and/or
a decrease in temperature may lead to a decrease in the enzyme turnover
because the diffusion coefficients of the substrates migrating to the active
sites of enzymes are affected (Rezaei et al., 2007a).
The reaction rate also increases at higher temperatures, because enzyme
deactivation may occur at higher temperatures and the optimal temperature
for the enzymatic activities and the one that causes reaction are not necessar-
ily the same. At the moment, no correlation between the temperature and the
stability of the different types of enzymes is available.
Lipase from Aspergillus niger was incubated in SC-CO2 at 30 MPa and dif-
ferent temperatures (Knez et al., 2003). Its residual activity was optimal at
50°C. At higher temperatures, a rapid decrease in activity was observed. This
thermal deactivation was connected to changes in the water distribution in
the system.
In microaqueous media, including SCFs, thermal stability of biocatalysts
could also be improved. The reaction rate for subtilisin protease-catalyzed
reactions increased up to 80% in SC-CO2 (Pasta et al., 1989). Optimal tem-
perature for esterification between n-butyric acid and isoamyl alcohol, cata-
lyzed by porcine pancreas lipase, moved from 40°C at atmospheric pressure
to 50°C in near-critical propane (Habulin and Knez, 2001). The better stabil-
ity of the lipase in the low-water-content environment is a consequence of a
well-known fact that reactions, which may be responsible for the denatur-
ation of enzymes, are hydrolytic and therefore require water (Mattiasson and
Aldercreutz, 1991). The optimal operation temperature for the synthesis of
isoamyl laurate and isoamyl stearate in SC-CO2 with three lipases, Novozym
435, Lipolase 100T, and C. rugosa was observed to be 40–45°C (Varma and
Madras, 2007b). Oliveira et al. (2006) reported that within the range of activ-
ity study of three commercial immobilized lipases submitted to compressed
CO2, propane, and n-butane (temperature from 35°C to 75°C, in the pressure

range of 1–28 MPa, exposure times from 1 to 6 h), temperature and exposure

times affected positively immobilized lipases (Novozym 435, Lipozyme IM)
activity, whereas the decompression rates did not demonstrate to be a rel-
evant variable (Oliveira et al., 2006). Opposite, decompression rate affected
positively enzyme activity, whereas the exposure time and temperature
presented the negative effect on the noncommercial immobilized lipase
from Y. lipolytica activity (De Oliveira et al., 2006). Additional, immobilized
lipase from Y. lipolytica has shown to be more suitable than Lipozyme IM
for enzyme-catalyzed reactions using compressed propane and n-butane as
solvents, but with inferior performance when compared with Novozym 435
treated in these solvents (De Oliveira et al., 2006).
Thermal activation and deactivation (energy of activation and deactivation
enthalpies, respectively) may be determined from the Arrhenius diagram.
The ratio between the amounts of inactive and active forms of the enzyme
at a temperature, where the greatest initial reaction rate was observed, is
expressed with the deactivation constant. If the activation and deactivation
enthalpy values are high, then enzyme activity is very much influenced by
the temperature.
4.3.5 Effect of Water Activity

Water concentration in the reaction system is one of the most important fac-
tors that influence activity of an enzyme. Water is crucial to enzymes and
affects enzyme action in various ways: by influencing enzyme structure via
noncovalent binding and disruption of the hydrogen bonds, by facilitating
reagent diffusion, and by influencing the reaction equilibrium (Krishna,
2002).
Furthermore, as a modifier of the solvent, up to a certain level, water can
modify the solvent properties such as polarity/polarizability as well as the
solubility of the reactants and the products. In addition, depending on the
type of the reaction, water can be a substrate (e.g., in hydrolysis) or a product
(e.g., in esterolysis) of the enzymatic reaction, influencing the enzyme turn-
over in different ways. Controlling water content of ingoing CO2 and sub-
strates as well as precise management of enzyme support and salt hydrates
are important strategies to adjust water level in the reaction media, espe-
cially in supercritical environments (Rezaei et al., 2007b).
The subtilisin Carlsberg-catalyzed transesterification of N-acetyl pheny
lalanine methyl ester (Aaltonen, 1999), N-acetyl phenylalanine ethyl ester
(Turner et al., 2001), N-trifluoroacetyl phenylalanine methyl ester (Rezaei
and Temelli, 2001), and N-trifluoroacetyl phenylalanine ethyl ester (Schmitt-
Rozieres et al., 2000) was studied in SC-CO2. The water content of the reaction
affected the reactivity of the system; for the transesterification of the methyl
esters with ethanol, the optimum concentration of water was determined to
be about 0.74 M, whereas during the transesterification of the ethyl esters
(Smallridge et al., 2002) with methanol, it was about 1.3 M.

Use of an enzyme in pure SC-CO2 may lead to removal of the water, which
is included or bonded to the enzyme. The quantity of the removed water is
temperature- and pressure-dependent and if it is too high, this may lead to
enzyme denaturation and lost its activity.
The solubility of water in SC-CO2 can be calculated by Chrastil equation
(Chrastil, 1982):
a 
c = ρk exp  + b
T 

where c is the solubility (g/L), ρ the CO2 density (g/L), and T the temperature
(K). The calculated water parameters are k = 1.549, a = −2826.4, and b = −0.807.
Some amount of water is necessary in the SCF because water-saturated CO2
causes the inhibition of enzymes and consequent loss of activity. The opti-
mal water concentration has to be determined for each enzyme separately.
Enzymes require some specific amount of water to maintain their active
conformation. Enzyme stability generally decreases with increasing water
concentration, whereas their activities require some water to be present.
Therefore, the water content has to be optimized to find the best balance
between enzyme life and activity.
If water acts as a substrate for enzymatic reaction, the optimal parame-
ters for continuous reaction require among other enough moisture to com-
pensate for complete reaction and sufficient enzyme moisture for losses
due to water solubility in SC-CO2 (Hampson and Foglia, 1999; Martinez
et al., 2002).
However, if the water concentration in the supercritical medium is too
high or if it is a product in reaction increases the humidity, it may cause
enzyme deactivation.
Not surprisingly, all studies published so far pointed out the strong influ-
ence of moisture on enzymatic activity and reaction rates. Optimum water
content in the support was estimated at 10 wt%, irrespective of the operating
conditions (Marty et al., 1992), but this value may be contested in view of
other reports (Leitgeb and Knez, 1990). To prevent dehydration of the enzyme,
the fluid in contact with the protein must contain water. The most hydro-
philic hydrocarbons (e.g., hexane) dissolve 0.01% water but SC-CO2 may dis-
solve as much as 0.3%–0.35% water. However, it is not the solubility of water
itself, but the partition of water between enzymatic support and the solvent
(SC-CO2) that matters. Marty et al. (1992) carried out an extensive analysis of
the partition of water between the enzymatic support and SC-CO2 as a func-
tion of pressure and temperature. They found that increasing temperature
had a negative effect on the adsorption of water to the support but increas-
ing pressure also had a similar effect. This is opposite of the results obtained
by gas adsorption, which suggests that the solvation effect predominates
over the vapor pressure effect. The same authors also extensively studied

the influence of ethanol (entrainer) content in SC-CO2 and found that ethanol
has a strong “drying” effect on the enzyme support: indeed, the more hydro-
philic the fluid, the more pronounced is the dehydration of the enzymes.
Increasing water content, above the optimum level, adversely affects the
overall performance. This appears to be related to hydrophilic hindrance of
the hydrophobic substrate on its way to the active sites on the enzyme and
eventually makes the thermodynamic equilibrium less favorable (Basheer
et al., 1995). Chulalaksananukul et al. (1993) measured the residual activ-
ity of lipase from M. miehei after a day in SC-CO2 at a temperature range
of 40–100°C at various water concentrations. As the temperature rises, the
enzyme molecule at first unfolds reversible and then undergoes one or more
reactions as following: formation of incorrect or scrambled structures, cleav-
age of disulfide bonds, deamination of trypsin residues, and hydrolysis of
peptide bonds. Each process requires water and is therefore accelerated with
increasing water concentration.
4.4 Enzyme Reactors

4.4.1 Process Schemes and Downstream Processing Schemes
Batch, stirred-tank, extractive semibatch, recirculating batch, semicontinu-
ous flow, continuous packed-bed, and continuous-membrane reactors have
been used with dense gases used as solvents.
4.4.1.1 Batch-Stirred Tank Reactor

Batch-stirred tank reactors are usually used for screening enzymatic reac-
tions in dense gases (Knez et al., 1998, 2007; Knez and Habulin, 2002; Laudani
et al., 2007; Knez, 2009; Ciftci and Temelli, 2013). The design of the system is
shown in Figure 4.3.
Initially, the substrates are pumped into the reactor and then the enzyme
preparation is added. Finally, dry gas is pumped into the reactor, up to the
desired pressure. The initial concentration of the reactant must never exceed
its solubility limit in the gas.
4.4.1.2 Continuous Packed-Bed Reactor

A continuous packed-bed reactor is presented in Figure 4.4. The pump deliv-
ers a high-pressure gas into the system. The substrates are pumped into the
system, using a liquid pump. The initial concentration of the reactant must
never exceed its solubility limit in the gas. SC-CO2 is depressurized through
the expansion valves into separator column 4, where the product and the

T = const. PI
2
Gas
FIGURE 4.3
Design of experimental batch-stirred tank apparatus for synthesis under high pressure. 1:
magnetic stirrer and heater; 2: reactor; P: high-pressure pump; PI: pressure indicator.
PI PI PI
HPP TIR
TIR
H 3 FI
Gas HPP
HPP
4
1 2
FIGURE 4.4
Design of continuous experimental apparatus for synthesis under supercritical conditions. 1, 2:
substrates; 3: reactor; 4: separation column; HPP: high-pressure pump; PI: pressure indicator;
TIR: temperature indicator and regulator; H: heat exchanger; FI: flow indicator.
unreacted substrates are recovered. The gas phase is finally vented into the
atmosphere after flow-rate measurement through a rotameter. The gas can be
condensed and recycled on a pilot or industrial scale.
Dalla Rosa et al. (2009) successfully performed continuous lipase-catalyzed
synthesis of fatty acid ethyl esters from soybean oil, and Ciftci and Temelli

(2011) synthetized fatty acid methyl esters from corn oil in the presence of lipase
(Novozym 435) in continuous packed-bed bioreactor using different com-
pressed fluids as reaction media. Also the enzymatic degradation of chemically
synthesized biodegradable plastics was successfully performed in a packed-
bed reactor with immobilized lipase from C. antarctica (Novozym 435) at 40°C
under the continuous flow of SC-CO2 with toluene (Osanai et al., 2006).
4.4.1.3 Continuous High-Pressure Enzyme Membrane Reactor

In a continuous high-pressure enzyme membrane reactor (Figure 4.5), the
membrane with 35-mm diameter is placed between two sintered plates and
fitted in the reactor. A certain amount of the catalyst (hydrated enzyme
preparation) is put in the reactor which is electrically heated, with a heating
jacket, to a constant temperature. The substrates and the gas are pumped
into the membrane reactor with the high-pressure pump. The products and
the unreacted reactants are collected in the separator. The catalyst remains
in the reactor (behind the membrane). Continuous flat-shape and continuous
tubular membrane enzymatic reactors were successfully used for different
enzymatic systems at atmospheric pressure and at supercritical conditions
(Primožič et al., 2009). Rios et al. (2007) performed the synthesis of butyl pro-
pionate in a recirculating tubular membrane bioreactor. The reaction was
catalyzed by CALB immobilized onto alpha-alumina microporous mem-
branes in different IL/SC CO2 biphasic systems at 50°C and 8 MPa. In room
temperature, IL/SC CO2 biphasic systems, the immobilized enzyme-coated
TIR
PI
P PI
4 FI
H
TIR
Gas
P P 3
2
1 1
FIGURE 4.5
High-pressure continuously stirred tank membrane reactor; 1: substrates; 2: magnetic stirrer
and heater; 3: reactor with membrane; 4: separation column; P: high-pressure pump; TIR: tem-
perature regulator and indicator; PI: pressure indicator; FI: flow indicator.

with three different ILs based on dialkylimidazolium cations, showed an

increase in the selectivity of the process (up to 99.5%) compared with SC-CO2
assayed in the absence of room-temperature ILs. Besides, the effect of the
IL media on the enzyme activity and the diffusional limitations across the
IL-layer around the biocatalyst was observed (Hernandez et al., 2006).
Continuous processes, in particular, are favored in the industry because
they are cost-efficient and the reactors can be kept relatively smaller in size
(Tundo, 1991; Hitzler et al., 1998). This reduction in size reduces both costs
and safety problems of the high-pressure equipment needed for SC reactions.
4.4.2 Processing Costs

Economic evaluations were made for the enzyme-catalyzed production of
oleyl oleate in a high-pressure batch-stirred tank reactor (HP BSTR) and in a
high-pressure continuously operated reactor (HP COR). It was assumed that
the reaction mixture is completely precipitated from CO2. The production
costs are strongly dependent on the solubility of the substrates in dense gas,
the enzyme-lifetime, and the productivity of the biocatalyst.
The study shows that the return on investment should be less than 1 year
(Knez et al., 2001).
4.5 Conclusion
The application of SCFs as reaction media for enzymatic synthesis has sev-
eral advantages, such as the higher initial reaction rates, higher conversion,
possible separation of products from unreacted substrates, over solvent-free,
or solvent systems (where either water or organic solvents are used). Owing
to the lower mass-transfer limitations and mild (temperature) reaction con-
ditions, at first the reactions which were performed in nonaqueous systems
will be transposed to supercritical media. An additional benefit of using
SCFs as reaction media is that they give simple and ecologically safe (no heat
and solvent pollution) recovery of products. However, for some specific reac-
tions, solvent-free systems are preferred because of their higher yields. The
main area of development should be related to the hydrolysis of glycerides,
transesterification, esterification, and inter-esterification reactions. As lipases
have high and stable activity in SC-CO2 (even at the high temperature), an
intensive development is expected. Only a little research on the separation
and synthesis of chiral compounds have been published so far. Because
enzymes have extremely high selectivity, and owing to the great importance
of enantioselective synthesis or enantiomeric resolution in the pharmaceuti-
cal industry, the most intense research in this area can be expected, along
with minimizing the use of substances and maximizing their effect.

Acknowledgment
We are sincerely and heartily grateful to Dr. Chiara G. Laudani for her sup-
port and assistance in the preparation of the first version of this manuscript.
References
Aaltonen, O. 1999. Enzymatic catalysis. In Chemical Synthesis Using Supercritical Fluids,
ed. P.G. Jessop, W. Leiner. Wiley-VCH, Weinheim.
Adams, M.W.W., Kelly, R.M. 1995. Enzymes from microorganisms in extreme envi-
ronments. Chem Eng News, 73: 32.
Ahern, T.J., Klibanov, A.M. 1985. The mechanism of irreversible enzyme inactivation
at 100-degrees-C. Science, 228: 1280.
Antonini, E., Carrea, G., Cremonesi, P. 1981. Enzyme catalyzed reactions in water–
organic solvent two-phase systems. Enzyme Microb Technol, 3: 291.
Asano, Y. 2002. Overview of screening for new microbial catalysts and their uses in
organic synthesis—Selection and optimization of biocatalysts. J Biotechnol, 94: 65.
Basheer, S., Mogi, K., Nakajima, M. 1995. Surfactant-modified lipase for the cataly-
sis of the interesterification of triglycerides and fatty acids. Biotechnol Bioeng,
45: 187.
Bermejo, M.D. et al. 2008. Influence of the enzyme concentration on the phase behav-
iour for developing a homogeneous enzymatic reaction in ionic liquid-CO2
media. Green Chem, 10: 1049.
Bornscheuer, U.T. 2000a. Enzymes in Lipid Modification. Wiley-VCH, Weinheim.
Bornscheuer, U.T. 2000b. Biotechnology. Wiley-VCH, Weinheim, 277.
Bornscheuer, U.T., Kazlauskas, R.J. 1999. Hydrolases in Organic Synthesis—Regio- and
Stereoselective Biotransformations. Wiley-VCH, Weinheim.
Bourquelot, E., Bridel, M.J. 1912. Travaux originaux sur une. Reaction synthetisante
de l’emulsine. J Pharm Chim, 7: 569.
Bourquelot, E., Bridel, M. 1913. Synthtse des glucosides d’alcools. B. l’aide de
l’tmulsine et reversibilitt des actions fermentaires. Ann Chim Phys, 29: 145.
Burton S.G., Cowan, D.A., Woodley, J.M. 2002. The search for the ideal biocatalyst.
Nat Biotechnol, 20: 37.
Carrea, G., Riva, S. 2000. Properties and synthetic applications of enzymes in organic
solvents. Angew Chem Int Ed Engl, 39: 2226.
Carvalho, N.B. et al. 2014. Evaluation of activity of Bacillus lipase (free and immobi-
lized) treated with compressed propane. J Mol Catal B, 99: 130.
Castillo, E. et al. 1994. Polar substrates for enzymatic-reactions in supercritical CO2—
How to overcome the solubility limitation. Biotechnol Lett, 16: 167.
Cernia, E., Palocci, C., Soro, S. 1998. The role of the reaction medium in lipase-
catalyzed esterifications and transesterifications. Chem Phys Lipids, 93: 157.
Chaudhary, A.K. et al. 1996. Polyester synthesis and in situ processing using super-
critical fluids. Abstr Pap Am Chem S, 211: 240.

Cheftel, J.C. 1991. Applications des hautes pressions en technologie alimentarie. Ind
Alim Agric, 108: 141.
Chrastil, J. 1982. Solubility of solids and liquids in supercritical gases. J Phys Chem,
86: 3016.
Chulalaksananukul, W., Condoret, J.S., Combes, D. 1993. Geranyl acetate synthesis
by lipase-catalyzed transesterification in supercritical carbon-dioxide. Enzyme
Microb Technol, 15: 691.
Ciftci, D., Saldana, M.D.A. 2012. Enzymatic synthesis of phenolic lipids using flax-
seed oil and ferulic acid in supercritical carbon dioxide media. J Supercrit Fluid,
72: 255.
Ciftci, O.N., Temelli, F. 2011. Continuous production of fatty acid methyl esters from
corn oil in a supercritical carbon dioxide bioreactor. J Supercrit Fluid, 58: 79.
Ciftci, O.N., Temelli, F. 2013. Enzymatic conversion of corn oil into biodiesel in a batch
supercritical carbon dioxide reactor and kinetic modeling. J Supercrit Fluid, 75:
172.
Coleman, M.H., Macrae, A.R. 1977. German Patent DE 27,05,608 [Unilever].
Collins, A.N., Sheldrake, G.N., Crosby, J. 1992. Chirality in Industry. Wiley, Chichester,
NY.
Dalla Rosa, C. et al. 2009. Continuous lipase-catalyzed production of fatty acid ethyl
esters from soybean oil in compressed fluids. Bioresour Technol, 100: 5818.
Dastoli, F.R., Price, S. 1967. Further studies on xanthine oxidase in nonpolar media.
Arch Biochem Biophys, 122: 289.
De Carvalho, I.B., De Sampaio, T.C., Barreiros, S. 1994. Subtilisin hydration and
activity in supercritical and near-critical fluids. In Presented at Proc. 3rd Symp.
Supercrit. Fluids, Strasbourg, October 17–19, p. 155.
Demirjan, D.C., Shah, P.C., Moris-Varas, F. 1999. Screening for novel enzymes. Topics
Curr Chem, 200: 1.
De Oliveira, D. et al. 2006. Influence of compressed fluids treatment on the activity of
Yarrowia lipolytica lipase. J Mol Catal B, 39: 117.
Dhake, K.P., Thakare, D.D., Bhanage, B.M. 2013. Lipase: A potential biocatalyst for
the synthesis of valuable flavour and fragrance ester compounds. Flavour Frag
J, 28: 71.
Dordick, J.S. 1989. Enzymatic catalysis in monophasic organic-solvents. Enzyme
Dufour, E., Hervé, G., Halrtle, T. 1995. Hydrolysis of beta-lactoglobulin by t hermolysin
and pepsin under high hydrostatic-pressure. Biopolymers, 35: 475.
Erickson, J.C., Schyns, P., Cooney, C.L. 1990. Effect of pressure on an enzymatic-
reaction in a supercritical fluid. AIChE J, 36: 299.
Faber, K. 2000. Biotransformations in Organic Chemistry, 4th ed. Springer, Berlin.
Fontes, N. et al. 1998. Effect of pressure on the catalytic activity of subtilisin Carlsberg
suspended in compressed gases. Biochim Biophy Acta, 1383: 165.
Franken, L.P.G. et al. 2010. Effect of treatment with compressed propane on lipases
hydrolytic activity. Food Bioprocess Tech, 3: 511.
Fricks, A.T. et al. 2009. Effects of compressed fluids on the activity and structure of
horseradish peroxidase. J. Supercrit Fluid, 50: 162.
Gandhi, N.N. et al. 2000. Lipase-catalyzed esterification. Catal Rev, 42: 439.
Giri, A. et al. 2001. Biotransformations using plant cells, organ cultures and enzyme
systems: Current trends and future prospects. Biotechnol Adv, 19: 175.

Griebenow, K., Klibanov, A.M. 1996. On protein denaturation in aqueous-organic but

not in pure organic solvents. J Am Chem Soc, 118: 11695.
Gross, M., Auerbach, G., Jeanicke, R. 1993. The catalytic activities of monomeric
enzymes show complex pressure-dependence. FEBS Lett, 321: 256.
Guthalugu, N.K., Balaraman, M., Kadimi, U.S. 2006. Optimization of enzymatic
hydrolysis of triglycerides in soy deodorized distillate with supercritical carbon
dioxide. Biochem Eng J, 29: 220.
Guzman-Lagunes, F. et al. 2012. Enzymatic synthesis of poly-l-lactide in supercritical
R134a. J Supercrit Fluid, 72: 186.
Habulin, M., Knez, Ž. 2001. Activity and stability of lipases from different sources in
supercritical carbon dioxide and near-critical propane. J Chem Technol Biotechnol
76: 1260.
Habulin, M., Krmelj, V., Knez, Ž. 1996. Supercritical carbon dioxide as a medium for
enzymatically catalyzed reaction. In: Proc. High Pressure Chemical Engineering,
ed. R. von Rohr and Ch. Trepp. Elsevier, Amsterdam, p. 85.
Habulin, M., Primožič, M., Knez, Ž. 2005. Stability of proteinase form Carica papaya
latex in dense gases. J. Supercrit Fluid, 33, 27.
Habulin, M. et al. 2007. Supercritical fluids as solvents for enzymatic reactions. Acta
Chim Slov, 54: 667.
Halling, P.J. 2000. Biocatalysis in low-water media: Understanding effects of reaction
conditions. Curr Opin Chem Biol, 4: 74.
Halling, P.J., Kvittingen, L. 1999. Why did biocatalysis in organic media not take off in
the 1930s? Trends Biotechnol, 17: 343.
Hammond, D.A. et al. 1985. Enzymatic-reactions in supercritical gases. Appl Biochem
Biotech, 11: 393.
Hampson, J.W. and Foglia, T.A. 1999. Effect of moisture content on immobilized
lipase-catalyzed triacylglycerol hydrolysis under supercritical carbon dioxide
flow in a tubular fixed-bed reactor. J Am Oil Chem Soc, 76: 777.
Hartmann, T., Meyer, H.H., Scheper, T. 2001. The enantioselective hydrolysis of
3-hydroxy-5-phenyl-4-pentenoicacidethylester in supercritical carbon dioxide
using lipases. Enzyme Microb Tech, 28: 653.
Hernandez, F.J. et al. 2006. A new recirculating enzymatic membrane reactor for ester
synthesis in ionic liquid/supercritical carbon dioxide biphasic systems. Appl
Catal B, 67: 121.
Hill, A.C. 1898. Reversible zymohydrolysis. J Chem Soc, 73: 634.
Hitzler, M.G. et al. 1998. Selective catalytic hydrogenation of organic compounds in
supercritical fluids as a continuous process. Org Process Res Dev, 2: 137.
Housaindokht, M.R., Monhemi, H. 2013 The open lid conformation of the lipase is
explored in the compressed gas: New insights from molecular dynamic simula-
tion. J Mol Catal B, 87: 135.
Ikariya, T., Kayaki, Y. 2000. Supercritical fluids as reaction media for molecular cataly-
sis. Catal Surv Jpn, 4: 39.
Ikushima, Y. et al. 1995. Activation of a lipase triggered by interactions with super-
critical carbon-dioxide in the near-critical region. J Phys Chem, 99: 8941–8944.
Ikushima, Y. et al. 1996. Promotion of a lipase-catalyzed esterification in supercritical
carbon dioxide in near-critical region. Chem Eng Sci, 51: 2817.
Jarzebski, A.B., Malinowski, J.J. 1995. Potentials and prospects for application of
supercritical fluid technology in bioprocessing. Process Biochem, 30: 343.

Jenab, E. et al. 2014. Performance of two immobilized lipases for interesterification

between canola oil and fully-hydrogenated canola oil under supercritical car-
bon dioxide. LWT-Food Sci Technol, 58: 263.
Jensen, B.H., Galluzzo, D.R., Jensen, R.G. 1987. Partial-purification and characteriza-
tion of free and immobilized lipases from Mucor miehei. Lipids, 22: 559.
Kajimoto, O. 1999. Solvation in supercritical fluids: Its effects on energy transfer and
chemical reactions. Chem Rev, 99: 355.
Kamat, S. et al. 1992. Biocatalytic synthesis of acrylates in organic-solvents and
supercritical fluids. 1. Optimization of enzyme environment. Biotechnol Bioeng,
40: 158.
Kamat, S.V. et al. 1993. Biocatalytic synthesis of acrylates in supercritical fluids—
Tuning enzyme-activity by changing pressure. Proc Natl Acad Sci USA, 90: 2940.
Kamat, S. et al. 1995. Biocatalytic synthesis of acrylates in organic-solvents and super-
critical fluids. 3. Does carbon-dioxide covalently modify enzymes. Biotechnol
Bioeng, 46: 610.
Kasche, V., Sch1othauer, R., Brunner, G. 1988. Enzyme denaturation in supercritical
CO2—Stabilizing effect of s–s bonds during the depressurization step. Biotech
Lett, 10: 569.
Kastle, J.H., Loevenhart, A.S. 1900. Concerning lipase, the fat-splitting enzyme, and
the reversibility of its action. J Am Chem Soc, 24: 491.
Kavčič, S., Knez, Z., Leitgeb, M. 2014. Antimicrobial activity of n-butyl lactate obtained
via enzymatic esterification of lactic acid with n-butanol in supercritical trifluo-
romethane. J Supercrit Fluid, 85: 143.
Kieslich, K. et al. 1998. New Frontiers in Screening for Microbial Biocatalysts. Elsevier,
Amsterdam.
Klibanov, A.M. 1986. Enzymatic reactions in liquid and solid paraffins: Application
for enzyme-based temperature abuse sensors. Chemtech, 16: 354.
Klibanov, A.M. 1989. Enzymatic catalysis in anhydrous organic solvents. Trends
Biochem Sci, 14: 141.
Klibanov, A.M. 1990. Asymmetric transformations catalyzed by enzymes in organic-
solvents. Acc Chem Res, 23: 114.
Klibanov, A.M. 1995. Enzyme memory: What is remembered and why? Nature, 374: 596.
Klibanov, A.M. 2000. Answering the question: ‘Why did biocatalysis in organic media
not take off in 1930s?’ Trends Biotechnol, 18: 85.
Klibanov, A.M. 2001. Improving enzymes by using them in organic solvents. Nature,
409: 241.
Knez, Ž. 2009. Enzymatic reactions in dense gases. J Supercrit Fluid, 47: 357.
Knez, Ž., Habulin, M., Krmelj, V. 1998. Enzyme catalyzed reactions in dense gases.
J Supercrit Fluid, 14: 17.
Knez, Ž., Gamse, T., Marr, R. 2001. High pressure process technology: Fundamentals
and applications. In Enzymatic Reactions (Industrial Chemistry Library, Vol. 9), ed.
A. Bertucco, G. Vetter. Elsevier, Amsterdam.
Knez, Ž., Habulin, M. 2002. Compressed gases as alternative enzymatic-reaction
solvents: A short review. J. Supercrit Fluid, 23: 29.
Knez, Ž., Habulin, M., Primožič, M. 2003. Hydrolases in supercritical CO2 and their
use in a high-pressure membrane reactor. Bioprocess Biosyst Eng, 25: 279.
Knez, Ž. et al. 2007. Exploiting the pressure effect on lipase-catalyzed wax ester syn-
thesis in dense carbon dioxide. Biotechnol Bioeng, 97: 1366.

Kobayashi, S. 1999. Enzymatic polymerization: A new method of polymer synthesis.

J Polym Sci A Polym Chem, 37: 3041.
Koeller, K.M., Wong, C.H. 2001. Enzymes for chemical synthesis. Nature, 409: 232.
Krishna, H.S. 2002. Developments and trends in enzyme catalysis in nonconventional
media. Biotechnol Adv, 20: 239.
Krishna, H.S., Karanth, N.G. 2001. Lipase-catalyzed synthesis of isoamyl butyrate. A
kinetic study. Biochim Biophys Acta, 1547: 262.
Krishna, H.S., Karanth, N.G. 2002a. Lipases and lipase-catalyzed esterification reac-
tions in nonaqueous media. Catal Rev, 44: 499.
Krishna, H.S., Karanth, N.G. 2002b. Response surface modelling of lipase-catalyzed
isoamyl propionate synthesis. J Food Sci, 67: 32.
Krishna, H.S., Prapulla, S.G., Karanth, N.G. 2000b. Enzymatic synthesis of isoamyl
butyrate using immobilized Rhizomucor miehei lipase in non-aqueous media.
J Ind Microbiol Biotechnol, 25: 147.
Krishna, H.S., Sattur, A.P., Karanth, N.G. 2001b. Lipase-catalyzed synthesis of iso-
amyl isobutyrate—Optimization using a central composite rotatable design.
Process Biochem, 37: 9.
Krishna, H.S. et al. 1999. Lipase-catalyzed synthesis of isoamyl butyrate: Optimization
by response surface methodology. J Am Oil Chem Soc, 76: 1483.
Krishna, H.S. et al. 2000a. Optimization of isoamyl acetate production by using immo-
bilized lipase from Mucor miehei by response surface methodology. Enzyme
Krishna, H.S. et al. 2001a. Enzymatic synthesis of isoamyl acetate using immobilized
lipase from Rhizomucor miehei. J Biotechnol, 87, 193.
Krishna, H.S. et al. 2002. Reverse micellar extraction for downstream processing of
proteins/enzymes. Adv Biochem Eng Biotechnol, 75: 119.
Kuhn, G.D. et al. 2011. Effect of compressed fluids treatment on the activity of inu-
linase from Kluyveromyces marxianus NRRL Y-7571 immobilized in montmoril-
lonite. Process Biochem, 46: 2286.
Kvittingen, L. 2000. Response from Kvittingen, refers to Answering the question:
‘Why did biocatalysis in organic media not take off in 1930s?’ Trends Biotechnol,
18: 86.
Laudani, C.G. et al. 2007. Lipase-catalyzed long chain fatty ester synthesis in dense
carbon dioxide: Kinetics and thermodynamics. J Supercrit Fluid, 41: 92.
Lee, J.H. et al. 2009. Biodiesel production from various oils under supercritical fluid
conditions by Candida antartica lipase B using a stepwise reaction method. Appl
Biochem Biotechnol, 156: 454.
Leitgeb, M., Knez, Ž. 1990. The influence of water on the synthesis of n-butyl oleate by
immobilised Mucor miehei lipase. J Am Oil Chem Soc, 67: 775.
Li, Y.Y. et al. 2014. Recent advances in engineering of media for enzymatic catalysis
with lipase. Asian J Chem, 26: 3755.
Liese, A., Seelbach, K., Wandrey, C. 2000. Industrial Biotransformations. Wiley-VCH,
Weinheim.
Lilly, M.D., Eighth P.V. 1994. Danckwerts memorial lecture presented at Glaziers’
Hall, London, U.K. 13 May 1993—Advances in biotransformation processes.
Chem Eng Sci, 49: 151.
Lopez-Luna, A. et al. 2010. Lipase-catalyzed syntheses of linear and hyperbranched
polyesters using compressed fluids as solvent media. J Mol Catal B, 67: 143.

Lozano, P., Bernal, J.M., Vaultier, M. 2011b. Towards continuous sustainable processes
for enzymatic synthesis of biodiesel in hydrophobic ionic liquids/supercritical
carbon dioxide biphasic systems. Fuel, 90: 3461.
Lozano, P. et al. 1996. Stability of immobilized alpha-chymotrypsin in supercritical
carbon dioxide. Biotechnol Lett, 18: 1345.
Lozano, P. et al. 2002. Continuous green biocatalytic processes using ionic liquids and
supercritical carbon dioxide. Chem Commun, 7: 692.
Lozano, P. et al. 2009. Dynamic kinetic resolution of sec-alcohols in ionic liquids/
supercritical carbon dioxide biphasic systems. Int J Chem React Eng, 7: A79.
Lozano, P. et al. 2011a. (Bio)catalytic continuous flow processes in scCO2 and/or ILs:
Towards sustainable (bio)catalytic synthetic platforms. Curr Org Chem, 8: 810.
Manera, A.P. et al. 2011. Effect of compressed fluids treatment on the activity, stability
and enzymatic reaction performance of beta-galactosidase. Food Chem, 125: 1235.
Manera, A.P. et al. 2012. Enzymatic synthesis of galactooligosaccharides using pres-
surised fluids as reaction medium. Food Chem, 133: 1408.
Martinek, K., Semenov, A.N., Berezin, I.V. 1981. Enzymatic synthesis in biphasic
aqueous-organic systems. I. Chemical equilibrium shift. Biochim Biophys Acta,
658(1): 76.
Martinek, K. et al. 1982. Colloidal solution of water in organic solvents: A microhet-
erogeneous medium for enzymatic reactions. Science, 218: 889.
Martinek, K. et al. 1986. Micellar enzymology. Eur J Biochem, 155: 453.
Marty, A. et al. 1990. Comparison of lipase-catalyzed esterification in supercritical
carbon-dioxide and in normal-hexane. Biotechnol Lett, 12: 11.
Marty A. et al. 1992. Kinetics of lipase-catalyzed esterification in supercritical CO2.
Biotechnol Bioeng, 39: 273.
Martinez, J.L., Rezaei, K., Temelli, F. 2002. Effect of water on canola oil hydrolysis in
an online extraction-reaction system using supercritical CO2. Ind Eng Chem Res,
41: 6475.
Matsuda, T. 2013. Recent progress in biocatalysis using supercritical carbon dioxide.
J Biosci Bioeng, 115: 233.
Matsuo, T. et al. 1981. European Patent EP 00 35 883 [Fuji Oil].
Mattiasson, B., Aldercreutz, P. 1991. Tailoring the microenvironment of enzymes in
water-poor systems. Trends Biotechnol, 9: 394.
McCoy, M. 1999. Biocatalysis grows for drug synthesis. Chem Eng News, 77: 10.
Miller, D.A., Blanch, H.W., Prausnitz, J.M. 1991. Enzyme-catalyzed interesterification
of triglycerides in supercritical carbon-dioxide. Ind Eng Chem Res, 30: 939.
Mori, T., Okahata, Y. 1998. Effective biocatalytic transgalactosylation in a supercritical
fluid using a lipid-coated enzyme. Chem Commun, 20: 2215.
Mori, T. et al. 2001. Reversible activity control of enzymatic reactions in supercriti-
cal fluid—Enantioselective esterification catalyzed by a lipid-coated lipase in
supercritical fluoroform. Kobunshi Ronbunshu, 58: 564.
Nakamura, K. et al. 1986. Lipase activity in supercritical carbon-dioxide. Chem. Eng.
Commun, 45, 207.
Okamoto, M. et al. 1991. High-pressure proteolytic digestion of food proteins—
Selective elimination of beta-lactoglobulin in bovine-milk whey concentrate.
Agric Biol Chem, 55: 1253.
Oliveira, D. et al. 2006. Assessment of two immobilized lipases activity treated in
compressed fluids. J Supercrit Fluid, 38: 373.

Oliveira, M.V. et al. 2009. Kinetic modelling of decyl acetate synthesis by immobilized
lipase-catalysed transesterification of vinyl acetate with decanol in supercritical
carbon dioxide. J Supercrit Fluid, 50: 138.
Ornstein, R.L. 2002. Improving Enzyme Catalysis: Screening, Evolution and Rational
Design. Marcel Dekker, New York.
Osanai, Y., Toshima, K., Matsumura, S. 2006. Enzymatic transformation of aliphatic
polyesters into cyclic oligomers using enzyme packed column under continu-
ous flow of supercritical carbon dioxide with toluene. Sci Technol Adv Mater, 7:
202.
Paiva, A. et al. 2011. Biocatalytic separation of (R,S)-1-phenylethanol enantiomers and
fractionation of reaction products with supercritical carbon dioxide. J Supercrit
Fluid, 55: 963.
Panke, S., Wubbolts, M.G. 2002. Enzyme technology and bioprocess engineering.
Curr Opin Biotechnol, 13: 111.
Partridge, J., Moore, B.D., Hailing, P.J. 1999. Alpha-chymotrypsin stability in aqueous-
acetonitrile mixtures: Is the native enzyme thermodynamically or kinetically
stable under low water conditions? J Mol Catal B, 6: 11.
Pasta, P. et al. 1989. Subtilisin-catalyzed trans-esterification in supercritical carbon-
dioxide. Biotechnol Lett, l1: 643.
Patel, R.N. 2000. Stereoselective Biocatalysis. Marcel Dekker, New York.
Patel, R.N. 2001. Biocatalytic synthesis of intermediates for the synthesis of chiral
drug substances. Curr Opin Biotechnol, 12: 587.
Perez, M., Sinisterra, J.V., Hernaiz, M.J. 2010. Hydrolases in green solvents. Curr Org
Chem, 14: 2366.
Perrut, M. 2000. Supercritical fluid applications: Industrial developments and eco-
nomic issues. Ind Eng Chem Res, 39: 4531.
Persidis, A. 1998. Extremophiles. Nat Biotechnol, 16: 593.
Prado, G.H.C. et al. 2012. Enzymatic hydrolysis of conjugated linoleic acid-enriched
anhydrous milk fat in supercritical carbon dioxide. J Supercrit Fluid, 66: 198.
Primožič, M. et al. 2009. Hydrolase-catalyzed reactions in membrane reactors at
atmospheric and high pressure. Desalination, 241: 14.
Ramsey, E. et al. 2009. Mini-review: Green sustainable processes using supercritical
fluid carbon dioxide. J Environ Sci (China), 21: 720.
Randolph, T.W., Blanch, H.W., Prausnitz, J.M. 1988. Enzyme-catalyzed oxidation of
cholesterol in supercritical carbon-dioxide. AIChE J, 34: 1354.
Randolph, T.W. et al. 1985. Enzymatic catalysis in a supercritical fluid. Biotechnol Lett,
7: 325.
Rasor, J.P., Voss, E. 2001. Enzyme-catalyzed processes in pharmaceutical industry.
Appl Catal A, 221: 145.
Rezaei, K., Jenab, E., Temelli, F. 2007b. Effects of water on enzyme performance
with an emphasis on the reactions in supercritical fluids. Crit Rev Biotechnol,
27: 183.
Rezaei, K., Temelli, F. 2000. Lipase-catalyzed hydrolysis of canola oil in supercritical
carbon dioxide. J Am Oil Chem Soc, 77: 903.
Rezaei, K., Temelli, F. 2001. On-line extraction-reaction of canola oil using immobi-
lized lipase in supercritical CO2. J Supercrit Fluid, 19: 263.
Rezaei, K., Temelli, F., Jenab, E. 2007a. Effects of pressure and temperature on enzy-
matic reactions in supercritical fluids. Biotech Adv, 25: 272.

Rios, A.P.D.L. et al. 2007. Understanding the chemical reaction and mass-transfer phe-
nomena in a recirculating enzymatic membrane reactor for green ester synthesis
in ionic liquid/supercritical carbon dioxide biphasic systems. J Supercrit Fluid,
43: 303.
Romero, M.D. et al. 2005. Enzymatic synthesis of isoamyl acetate with immobilized
Candida antarctica lipase in supercritical carbon dioxide. J Supercrit Fluid, 33: 77.
Rosso, S.R.C. et al. 2013. Enzymatic synthesis of poly(epsilon-caprolactone) in super-
critical carbon dioxide medium by means of a variable-volume view reactor.
J Supercrit Fluid, 79: 133.
Schmid, R.D., Verger, R. 1998. Lipases: Interfacial enzymes with attractive applica-
tions. Angew Chem Int Ed Engl, 37: 1609.
Schmitt-Rozieres, M., Deyris, V., Comeau, L.C. 2000. Enrichment of polyunsaturated
fatty acids from sardine cannery effluents by enzymatic selective esterification.
J Am Oil Chem Soc, 77: 329.
Schoffers, E., Golebiowski, A., Johnson, C.R. 1996. Enantioselective synthesis through
enzymatic asymmetrization. Tetrahedron, 52: 3769.
Schulze, B. et al. 1998. Review of biocatalysis in the production of chiral fine chemi-
cals. Spec Chem, 18: 244.
Senyay-Oncel, D., Yesil-Celiktas, O. 2011. Activity and stability enhancement of alpha-
amylase treated with sub- and supercritical carbon dioxide. J Biosci Bioeng, 112: 435.
Senyay-Oncel, D., Yesil-Celiktas, O. 2013. Treatment of immobilized alpha-amylase
under supercritical CO2 conditions: Can activity be enhanced after consecutive
enzymatic reactions? J Mol Catal B, 91: 72.
Shang, W.T. et al. 2014. High pressure CO2-controlled reactors: Enzymatic chiral reso-
lution in emulsions. RSC Adv, 4: 24083.
Shin, S.K. et al. 2012. Characteristics of menhaden oil ethanolysis by immobilized
lipase in supercritical carbon dioxide. J Ind Eng Chem, 18: 546.
Smallridge, A.J., Trewhella, M.A., Wang, Z. 2002. The enzyme-catalysed stereose-
lective transesterification of phenylalanine derivatives in supercritical carbon
dioxide. Aust J Chem, 55: 259.
Stinson, S.C. 2000. Chiral drugs. Chem Eng News, 78: 55.
Sureshkumar, M., Lee, C.K. 2009. Biocatalytic reactions in hydrophobic ionic liquids.
J Mol Catal B, 60: 1.
Tao, M.L. et al. 2013. Enzymatic synthesis of dipalmitin in supercritical carbon diox-
ide and mechanism study. Ind Eng Chem Res, 52: 13528.
Thomas, S.M., Di Cosimo, R., Nagarajan, A. 2002. Biocatalysis: Applications and
potentials for the chemical industry. Trends Biotechnol, 20: 238.
Tundo, P. 1991. Continuous Flow Methods in Organic Synthesis. Ellis Horwood ltd.,
Chichester, UK, p. 378.
Turner, C. et al. 2001. Lipase-catalyzed reactions in organic and supercritical solvents:
Application to fat-soluble vitamin determination in milk powder and infant for-
mula. Enzyme Microb Tech, 29: 111.
Varma, M.N., Deshpande, P.A., Madras, G. 2010. Synthesis of biodiesel in supercriti-
cal alcohols and supercritical carbon dioxide. Fuel, 89: 1641.
Varma, M.N., Madras, G. 2007a. Synthesis of biodiesel from castor oil and linseed oil
in supercritical fluids. Ind Eng Chem Res, 46: 1.
Varma, M.N., Madras, G. 2007b. Synthesis of isoamyl laurate and isoamyl stearate in
supercritical carbon dioxide. Appl Biochem Biotechnol, 141: 139.

Varma, M.N., Madras, G. 2010a. Effect of chain length of alcohol on the lipase-
catalyzed esterification of propionic acid in supercritical carbon dioxide. Appl
Biochem Biotechnol, 160: 2342.
Varma, M.N., Madras, G. 2010b. Kinetics of enzymatic synthesis of geranyl butyrate
by transesterification in various supercritical fluids. Biochem Eng J, 49: 250.
Vulfson, E.N. 1998. Novel surfactants: Preparation, applications and biodegradabil-
ity. Surfactant Science Series. Marcel Dekker, New York, p. 279.
Wahler, D., Reymond, J.L. 2001. Novel methods for biocatalyst screening. Curr Opin
Chem Biol, 5: 152.
Weber, A. et al. 2007. A supercritical fluid-assisted, integrated process for by-products
from fat and lipid production. Chem Eng Technol, 30: 732.
Weder, J.K. 1984. Studies on proteins and amino-acids exposed to supercritical
carbon-dioxide extraction conditions. Food Chem, 15: 175.
Wescott, C.R., Klibanov, A.M. 1994. The solvent dependence of enzyme specificity.
Biochim Biophys Acta, 1206: 1.
Wong, J.M., Johnston, K.P. 1986. Solubilization of biomolecules in carbon dioxide-
based supercritical solutions. Biotechnol Prog, 2: 29.
Yu, G. et al. 2007. Stability and activity of lipase in subcritical 1,1,1,2-tetrafluoroethane
(R134a). J Ind Microbiol Biotechnol, 34: 793.
Zagrobelny, J., Bright, F.V. 1992. In situ studies of protein conformation in supercriti-
cal fluids-trypsin in carbon-dioxide. Biotechnol Progr, 8: 421.
Zaks, A. 2001. Industrial biocatalysis. Curr Opin Chem Biol, 5: 130.
Zaks, A., Dodds, D.R. 1997. Application of biocatalysis and biotransformations to the
synthesis of pharmaceuticals. Drug Discov Today, 2: 513.
Zaks, A., Klibanov, A.M. 1984. Enzymic catalysis in organic media at 100°C. Science,
224: 1249.
Zaks, A., Klibanov, A.M. 1985. Enzyme-catalyzed processes in organic solvents. Proc
Natl Acad Sci USA, 82: 3192
Zaks, A., Klibanov, A.M. 1986. Substrate-specificity of enzymes in organic-solvents vs
water is reversed. J. Am. Chem Soc, 108: 2767.
Zaks, A., Klibanov, A.M. 1988a. The effect of water on enzyme action in organic
media. J Biol Chem, 263: 8017.
Zaks, A., Klibanov, A.M. 1988b. Enzymatic catalysis in nonaqueous solvents. J Biol
Chem, 263: 3194.
Zarevucka, M., Wimmer, Z. 2008. Plant products for pharmacology: Application of
enzymes in their transformations. Int J Mol Sci, 9: 2447.

5
Mass Transfer Coefficient of Plant Oil
in Supercritical-CO2 Fluid Extraction
John Shi and Sophia Jun Xue
CONTENTS
5.1 Introduction................................................................................................. 159
5.2 Model Development................................................................................... 162
5.3 Estimation Methods................................................................................... 164
5.3.1 Weighted Linear Least-Squares.................................................... 164
5.3.2 Error-in-Variable Model................................................................. 164
5.4 Experimental Verification.......................................................................... 166
5.5 Summary...................................................................................................... 175
References.............................................................................................................. 175
5.1 Introduction
Separation of one or more components from a complex mixture is a require-
ment for many operations and extensively used in various applications in
the food and biotechnology industries. Application of separation technol-
ogies is used either to recover high-value components from agricultural
commodities, being an important operation for the production of food prod-
ucts such as oil and proteins, or especially for the development of health-
promoting food ingredients and high value-added food products such as
antioxidants and flavors. Separation processes are also used to remove
contaminants, impurities, or toxins such as pesticide residue from food
materials. Separation, often known as “downstream processing,” is totally
devoted to the science and engineering principles of unit operation of sepa-
ration and purification. Over the last 20 years, separation technologies in
the food processing area have undergone an explosive growth. The competi-
tive nature of the biotechnologies applied to the pharmaceutical and food
industries for cost-effective manufacturing has provided much impetus for
the development and use of new separation techniques on a large scale, but
at a lower cost. Aside from the conventional separation techniques such as
solvent and water extraction (solid–liquid contacting extraction or leaching),
159
crystallization, precipitation, distillation, and liquid–liquid extraction, etc.,

have already been incorporated in basic food processing and well estab-
lished and commercialized. A number of newer separation techniques as
promising alternative methods for improved application in food engineering
have been implemented on the commercial scale, including supercritical-CO2
fluid extraction, membrane-based separation, molecular distillation, pres-
sured low-polarity water extraction procedures, etc. These newer techniques
have made impressive advances in obtaining adequate segregations of com-
ponents of interest with maximum speed, minimum effort, and minimum
cost at as large a capacity as possible in production-scale processes. These
techniques have been implemented for the purification of proteins, charac-
terization of aromas, whey protein removal from dairy products, extraction
of health-benefiting fish oil, and clarification of beverages including beer,
fruit juices, and wine. The separation processes and technologies for high
value-added products are based on their polarity and molecular size. Many
potential high-value products can be developed from natural resources by
different separation technologies and processes. For example, carotenoids
including lycopene, β-carotene, astaxanthins, and lutein make up a world
market nearing $1 billion with a growth rate of about 3%. Therefore, efforts
to utilize natural agricultural materials for the production of high value-
added products, especially health-promoting foods and ingredients, are of
great interest to the food and biotechnology industries.
Supercritical-CO2 fluid extraction as emerging “green” processes has
gained increased importance in the food industry in recent years. The
supercritical-CO2 fluid process has many advantages over some conven-
tional separation techniques such as distillation and organic solvent extrac-
tion, especially used to extract bioactive components from plant sources
(Williams, 1981; Rozzi and Singh, 2002). The most significant implication in
the use of supercritical-CO2 fluid is no toxic-chemical involved in the pro-
cess, so there is no toxic-chemical residue in the end products that consider
it safe for use in food and pharmaceutical products. The supercritical-CO2
fluid extraction process has been successfully used to extract high-value
essential oils commonly used as food additives in the industrial scale
(Ferreira et al., 1999).
The extraction process requires pumping supercritical-CO2 fluid through
a packed-bed formed by solid stratum (ground sample) to extract a target
biocomponent (Brunner, 1984; Lee et al., 1986; Sovová, 1994). The flow pat-
tern in a fixed bed during supercritical-CO2 fluid extraction is complex
because of the nature of the intrinsic solid matrix, which experiences some
physical changes during drying (Lewicki and Pawlak, 2003), thus it is dif-
ficult to obtain mass-transfer data empirically. As a result, the most prac-
tical approach is to develop correlation equations based on dimensionless
numbers to model the mass-transfer kinetics. In the past years, although
the supercritical-CO2 fluid extraction process and technologies have been

Mass Transfer Coefficient of Plant Oil in Supercritical-CO2 Fluid Extraction 161
applied extensively, the mass-transfer kinetics of the process is not given

much attention. Therefore, characterization of the supercritical-CO2 fluid
extraction process coupled with an understanding of its thermodynamic
and kinetic behavior is crucial, especially for scale-up of the design of a pilot
plant and of commercial CO2 fluid extraction systems.
One of the important parameters to describe the extraction kinetics is the
mass-transfer coefficient in the supercritical-CO2 fluid phase. The kinetics of
the mass-transfer coefficients are influenced by variables such as diffusivity,
viscosity, density, porosity of the fixed bed, particles/pore size distributions,
and solvent flow rate. The characteristic behavior of these variables can be
expressed in terms of dimensionless numbers, that is, the Reynolds number
(Re), Sherwood number (Sh), Schmidt number (Sc), and Grashof number (Gr).
Puiggene et al. (1997), Wakao and Kaguei (1982), and Tan et al. (1988) have
studied the mass transfer in supercritical-CO2 fluid extraction systems using
dimensionless numbers in correlation models.
An accurate method for predicting the mass-transfer coefficient is required
to develop process modeling and quality control on line. Correlation equa-
tions using characteristic dimensionless numbers are often used for this
purpose. It means that the pure chemical system can be used to simulate the
mixed food material system and obtain more accurate physical properties.
Ferreira (1996) proposed correlation equation in terms of Sherwood number
(Sh), Rayleigh number (Ra = GrSc), Reynolds number (Re), and Grashof num-
ber (Gr), and combined the effects of natural and forced convection together
as follows:
0.525
 Re 
Sh = 1.451Ra1/4  1/2  (5.1)
 Gr 

where Sh is the Sherwood number, Sc is the Schmidt number, Re is the

Reynolds number, and Gr is the Grashof number.
The parameters of Equation 5.1 are estimated for pepper oil system in the
presence of mixed convection. If Ra is expanded as GrSc, the exponent of Gr
is much smaller than Re and Sc, especially in Equation 5.1. That reveals a
weak correlation between Sh and Gr. Both of these two correlations combine
the natural and force convection together.
Based on an assumption that the natural and forced convection are inde-
pendent, their effects can be combined in a linear manner. Lee and Holder
(1995) proposed the following correlation equation:
1/0.6439
Sh (Re1/2Sc1/3 )1.6808  Re 2Sc1/3  0.6439 
= 0 .5265 + 2 .48    − 0 . 8768 
(ScGr )1/4 (ScGr )1/4  Gr  

(5.2)

TABLE 5.1
Mass-Transfer Correlation Parameters for Essential Oils in Supercritical-CO2 Fluid
Extraction
Parameters of Mass-
Transfer Correlation
Sources m n1 n2 Range
Catchpole and King (1994) 0.821 0.66 1/3 1 < Re < 70, 3 < Sc < 11
Tan et al. (1988) 0.381 0.83 1/3 2 < Re < 40, 2 < Sc < 20
Puiggene et al. (1997) 0.206 0.80 1/3 10 < Re < 100, 3 < Sc < 20
King et al. (1997) 0.255 0.52 1/3 Re < 1, 70 < Sc < 100
for 0.3 < Re < 135. The parameters are obtained from the extraction of naph-
thalene and toluene from the porous silica gel.
Taking into account that the natural convection is very weak, the correla-
tion can be simplified as the first item of the right-hand side of Equation 5.2:
Sh = m(Re1/2Sc1/3 )n (5.3)

or
Sh = m(Re n1 Sc n2 ) (5.4)

This assumption is reasonable for essential oil extraction, because usually

the solubility of essential oil in the supercritical-CO2 fluid is not high. For
many dynamic extractions from the solid particles, the extracted material is
below the solubility limit (Bartle et al., 1990). Hence, after the extraction, the
density of the solution of supercritical-CO2 will also not significantly change.
Some mass-transfer correlations are published for supercritical-CO2 fluid
extraction for essential oils as shown in Table 5.1. In all of the above correlations,
the parameters are usually obtained from a least-squares analysis, which is
developed based on the assumptions that the independent variables are accu-
rate. However, all of the dimensionless numbers contain similar experimental
errors. Therefore, the classical least-squares method is not statistically valid to
establish correlation equations among those dimensionless numbers. This is
also a possible reason why published estimation correlations are diverse.
5.2 Model Development

Correlation models are developed to predict the mass-transfer coeffi-
cient using dimensionless grouped numbers during a supercritical-CO2

extraction process. Tan et al. (1988) modeled supercritical-CO2 fluid extrac-

tion of β-naphthol by using Equation 5.5 with superficial velocities ranging
from 4.4 × 10−5 to 3.1 × 10−4 m/s, and reported the m and n values of 0.38 and
0.83, respectively. Similarly, Puiggene et al. (1997) reported 0.206 and 0.80 for
m and n, respectively, for 1,2-dichlorobenzene modeled with Re values rang-
ing from 10 to 100 and Sc from 3 to 20:
Sh = mRe nSc1/3 (5.5)
To improve the capacity of Equation 5.5, Wakao and Kaguei (1982) sug-
gested Equation 5.6 which covers a wider range of Re (3–3000) and Sc (0.5–
10,000) values.
Sh = 2 + 1.1Re 0.6Sc1/3 (5.6)
Most supercritical-CO2 fluid extraction processes of essential oils are con-

ducted with ground biological materials with small particle sizes (diameters:
dp ≤ 0.1 mm) and with a minimal superficial velocity and an Re of not less
than 1, thus using the above equations may be erroneous, given that the vari-
ables fall below the functionality limits of the equations.
Most essential oils are insoluble to some degree in CO2 as shown by the
results of dynamic extraction from solid particles (Press et al., 1997). The
reported solubility of the essential oil extract is below critical limits (Press
et al., 1997), and no significant change in the density is observed, hence the
contribution of natural convection to mass transfer can be negligible. As forced
convection played a principal role, the correlation of dimensionless number
can simply be expressed by the relationship between the Sh, Re, and Sc. The
apparent Sherwood number Sh′ is established as Equation 5.7 to account for
the local mass-transfer coefficient around a single sphere (Schluender, 1981):
Sh′ = Sh ⋅ ϕ (5.7)

where φ = 1 + 1.5(1 − ε) and ε is the percent porosity. Equation 5.8 is proposed

as Model I in this study:
Sh′ = m(Re1/2Sc1/3 )n (5.8)

Equation 5.9 is the generic form and is defined as Model II:
Sh′ = m(Re n1 Sc n2 ) (5.9)

A multivariate optimization technique is used to estimate the model

parameters and for simplicity, homogeneity was assumed for the oil compo-
nents extracted by the supercritical-CO2 fluid.

5.3 Estimation Methods

5.3.1 Weighted Linear Least-Squares
Linear least-squares method is widely used by researchers and is easy to
understand. The objective function of the least-squares method is
ϕ = [y − f(θ)]′ [y − f(θ)] (5.10)

where y represents the response vector of the experiment, θ is the parameter

vector, and f(θ) is the model predictions. This objective function is the sum of
squared residuals. The least-squares method needs an essential assumption
that no error exists in the independent variables. In reality, if the errors in the
independent variables are much less than that of the dependent variable, this
assumption is still considered valid.
The dimensionless numbers are composed of various measured variables.
Therefore, no matter whether the variables are dependent or independent,
they all contain similar measurement errors. If the linear least-squares anal-
ysis is directly applied, the resultant correlations might be misleading. For
the univariate linear models, weighted least-squares can be used to compen-
sate for this defect (Press et al., 1997). The error of the independent variable
is taken into account of the weight. The objective function of linear model
y(x) = a + bx becomes
N
( yi − a − bxi )2
ϕ= ∑ σ 2yi + b 2σ 2xi
(5.11)
i=1
where σxi and σyi are the standard deviations of variables x and y at experi-
ment i, respectively. The weight wi is
1
wi = (5.12)
σ 2yi + b 2σ 2xi

The standard deviation can be calculated based on the error propagation

method (Press et al., 1997). However, when the error structure is complicated,
the expression of the standard deviation is not easy to be derived, and the
replicating experiment is normally a more practical way of obtaining the
standard deviation.
5.3.2 Error-in-Variable Model

Error-in-variable model (EVM) is a better method to solve this problem.
It accounts for all the errors in the variables. It is applicable on linear and

nonlinear multivariate problems. The algorithm developed by Reilly et al.

(1993) is used for estimation. The objective function of EVM is
∑ (x − ξ )′V
1
ϕ= i i
−1
(x i − ξ i ) (5.13)
2 i=1

where xi is the experimental data of the ith trial, ξ is the estimates of

the true value of the variables, and V is the error covariance matrix of
the measurement. Variable metric (or quasi-Newton) method is used to
search the minimum of the objective function ϕ. The Jacobian matrix, J, is
expressed as
 ∂ϕ 
J= (5.14)
 ∂θi 

where J is calculated by the following equation:
J= ∑ Z′(B VB′) i i i
−1
Bi (x i − ξ i ) (5.15)
i=1
where Z is the partial derivatives of the function f versus the parameters:
 ∂f j (ξ i , θ)  (5.16)
Zi = 
 ∂θ k 

and B is the partial derivatives of the function f versus the variables:
 ∂f ( ξ , θ) 
Bi =  s i  (5.17)
 ∂(ξ i )t  ξi = ξi( k )

The estimation of the true values of the variables is obtained by the itera-
tion of the following equation:
ξ(i k + 1) = x i − VB′i (Bi VB′i )−1[f(ξ(i k ) , θ lim) + Bi (x i − ξ i( k ) )] (5.18)

δx → 0
It is the inner iteration at given parameters θ. This method only requires an

adequate model, independent and normally distributed error structure. It is
obviously more flexible than the weighted least-squares method.

5.4 Experimental Verification

Experimental data obtained during tomato seed oil extraction by
supercritical-CO2 fluid and within the defined ranges of 1–10 for Re and less
than 10 for the Sc (Reverchon and Marrone, 2001) is used to validate the pro-
posed correlation functions.
The diffusion coefficient was approximated by the correlation proposed
by Wilke and Chang (1955) and later modified by Sassiat et al. (1987) for
supercritical-CO2 fluid extraction. The diffusion coefficient ranged from
2.1 × 10−8 to 4.28 × 10−8 m2/s at 313.15°K and 24 MPa. The viscosity of CO2 is
determined by the method of Vesovic et al. (1990).
King et al. (1997) published a completed set of data for supercritical-CO2
fluid extraction of primrose oil, where Re < 1.0 and Sc ranging from 70 to 100.
Based on Model I, the weighted least-squares model is used to fit their data.
The objective function, f(θ) (Equation 5.19),
 f = m(Re Sc )
1/2 1/3 n
 (5.19)
 y = Sh′
is linearized as follows:
 f = ln m + n ln(Re1/2Sc1/3 )
 (5.20)
 y = ln Sh′
Model II is complex. Both Sc and Re not only have similar errors with Sh,
but also have covariance.
The weighted least-squares have adjusted variance of independent vari-
ables to some extent. However, it does not work for the variables having
covariance. In other words, the classical weighted least-squares is not valid
here. An alternative is by rewriting the correlation as follows:
n1 n2
kf dp  dpρfU   µ f 
= m (5.21)
D12  µ f   ρf D12 

or
kf = mdp n1 −1ρf n1 − n2 U n1 µ f n2 − n1 D121− n2 (5.22)

and linearizing the form as
ln(kf ) = ln m + (n1 − 1)ln dp + (n1 − n2 )ln ρf

+ n1 ln U + (n2 − n1 )ln µ f + (1 − n2 ) ln D12 (5.23)


Particle size, flow rate, fluid density, mutual diffusion coefficient, and
viscosity are independent variables. Their measurement errors usually
have the same order of error of kf, because the mutual diffusivity D12,
fluid viscosity, and density μf and ρf, respectively, are not easy to be mea-
sured accurately. It also needs to be adjusted for least-squares regression
analysis.
Although kf vs. dp, ρf, U, μf, and D12 can be discussed directly, dimension-
less number can be used to draw more general conclusions. At this point, the
least-squares method is not suitable to be used for analysis, EVM is a better
alternative, regardless of which variable is independent, therefore more flex-
ible to modify the equation to different forms.
According to the experimental data of King et al. (1997), the variance of
y is obtained. There are four groups of replicated experimental data. After
pooling their experimental variance, the standard deviation of Sh is 0.0015.
The standard deviation of independent variable σx is estimated during the
estimation process. Model I is linearized as follows:
ln Sh′ = ln m + n ln(Re1/3 Sc1/3 ) (5.24)

According to the error propagation, the standard deviation of y (ln Sh) is

calculated as follows:
σ Sh
σy = (5.25)
Sh
The estimated parameters are n (2.584 ± 0.35), m (0.0015), and the standard

deviation of Re1/2Sc1/3 (0.28). The standard deviation, σx, is greater than that of
the variable Sh′. If it is assumed that Re1/2Sc1/3 had no error, and the estimated
results via the linear least-squares method are n (0.803 ± 0.16) and m (0.0124).
Figure 5.1 shows the predictions of the models from two kinds of estimations
and their results exhibit different trends. A good agreement between the
Model I and the experimental data is observed. The linear model (the dashed
line in Figure 5.1) shows an apparently poor fit, as revealed by the coefficient
of determination (0.58).
Model II (Equation 5.9) is linearized by the following equation:
ln Sh′ = ln m + n1 ln Re + n2 ln Sc (5.26)

and
f = ln Sh ’− (ln m + n1 ln Re + n2 ln Sc) (5.27)

0.07
Weighted least squares
0.06 Linear least squares
Experimental data
0.05
0.04
Sh′
0.03
0.02
y = 0.0089x
0.01 R2 = 0.5785
0.00
0 1 2 3 4 5
Re1/2Sc1/3
FIGURE 5.1
Experimental data and prediction of Model I. The dashed line is linear trend; Sh′ = Re1/2Sc1/3,
R 2 is the coefficient of determination.
The partial derivatives with respect to the parameters are
∂f
= −1 (5.28)
∂ ln m

∂f
= − ln(Re) (5.29)
∂n1
∂f
= − ln(Sc) (5.30)
∂n2
The partial derivatives with respect to variables are
∂f
=1 (5.31)
∂ ln Sh′

∂f
= − n1 (5.32)
∂ ln Re

∂f
= − n2 (5.33)
∂ ln(Sc)

The experimental and predicted data of both Models I and II are plot-
ted in Figure 5.2a,b. From the experimental data of King et al. (1997), n1
and n2 are estimated as 0.4299 and 0.8783, respectively, and the parameter

(a) 0.06
Model I
0.05
Model II
0.04
Predicted Sh
0.03
0.02
0.01
0
0 0.01 0.02 0.03 0.04 0.05 0.06
Experiment Sh
(b) 0.05
0.04
Sh′ (prediction)
0.03
0.02
Model II
Model I
0.01
0.00
0.00 0.01 0.02 0.03 0.04 0.05
Sh′ (experiment)
FIGURE 5.2
Experimental data and predictions from Model I and Model II. (a) The relationship between
the experimental data and prediction of Sh. (b) The relationship between the experimental
data and prediction of Sh’. (Verification of the experimental data obtained from Reverchon,
E., Marrone, C. 2001. Journal of Supercritical Fluids, 19(2): 161–175, and prediction data generated
from Models I and II.)
m, 0.00084, is very small, compared with other correlations (Equation 5.3),

where 0.1 < m < 1.0. Model II fits the experimental data well (Figure 5.2a). It
is observed that the order of the exponent of the Sc in Model II is similar to
that of Model I. The exponent of Re in Model II is less than that of Model I.
The reason is attributed to the measurement error of the dimensionless num-
bers Re and Sc which cannot be obtained accurately in Model II. Therefore, it
requires more data on various Re with the same Sc to improve the estimation.

The results show the predicted values for Model I are much larger than the
experimental values and that the predicted values also increase when Re is
greater than unity. Model II gives a good fit, thus indicating better predic-
tive capabilities because of its versatility. However, it must be recognized
that as the mass transfer improves and as Sh increases, an increase in devia-
tion could be generated. The probable reason is the stagnation of flow in the
laminar region where Re is <10. The molecular diffusion is insignificant at
this stage when compared with eddy diffusion, thus resulting in poor mass
transfer. This situation is significant usually when the extraction process is
conducted at high pressures on very small particle sizes.
Puiggene et al. (1997) used pure chemicals to mimic the essential oil extrac-
tion system. 1,2-Dichlorobenzene (DCB)/CO2 is absorbed in nonporous glass
beads and extracted by the supercritical-CO2 fluid, which is analogous to the
initial stage of oil extraction. Pure chemical substance is used because it is
easy to characterize and more accurate to measure. In Puiggene et al.’s (1997)
experiment, 10 data points which are located on the range of Re 10–30, and
Sc < 20, is used for estimation. In order to show the effect of Re, five extra
points at fixed Sc and Re ranging from 10 to 100 are obtained (Table 5.2).
As shown in Table 5.3, with the increase in experimental data points, the
estimated values from the nonlinear least-squares (NLLS) method change
more than that of the EVM method, because of the disturbance of the large
error of the independent variables. Considering the error on each variable,
with fewer data points, EVM obtains better and stable estimation results.
TABLE 5.2
The Estimated Values of Sherwood Number Sh’, Schmidt
Number Sc, Reynolds Number Re from the Nonlinear
Least-Squares (NLLS) Method
Run Re Sc Sh′
1 25.3 3.24 3.86
2 27.0 3.01 4.24
3 25.8 4.20 4.26
4 25.8 6.67 4.94
5 24.3 6.42 4.68
6 21.4 7.46 4.62
7 24.9 9.90 5.48
8 21.8 6.90 4.57
9 16.0 18.30 5.27
10 14.8 19.30 5.01
11 15.0 6.90 3.67
12 35.3 6.90 6.01
13 50.7 6.90 7.45
14 66.7 6.90 8.75
15 81.0 6.90 9.79
Source: Data from the paper of Puiggene, J., Larrayoz, M.A., Recasens,
F. 1997. Chemical Engineering Science, 52(2): 195–212.

TABLE 5.3
Estimation Results Based on the Data of Puiggene et al. (1997)
Mass Transfer Parameters
Estimate Method m n1 n2
10 points (1–10) NLLSa 0.517 0.525 0.291
EVM 0.380 0.609 0.316
15 points (1–15) NLLS 0.425 0.578 0.307
EVM 0.422 0.580 0.307
Puiggene et al. (1997) 0.206 0.821 0.333
a Nonlinear least-squares.
14
Prediction (Puiggene et al., 1997)
12 Prediction (15 points)
Prediction (10 points)
10
Sh (prediction)
0
0 2 4 6 8 10 12 14
Sh (experiment)
FIGURE 5.3
Errors for Sherwood number prediction.
Figure 5.3 shows the relationship between the experimental data and pre-
diction. When Sh < 6, the corresponding Re < 30, the prediction of Puiggene
equation (Puiggene et al., 1997) is as good as that of EVM result. This range of
Re is located on the data points for estimation. But when Re approaches 100
(Sh is >6 in Figure 5.3), the deviation emerged and increased as Re increased.
In compared with the parameter estimation of EVM method where Re < 30
(10 points) with Re < 100 (15 points), there prediction matched very well. It
indicates that the kinetics behavior of the supercritical-CO2 fluid extraction
system is consistent in the range of Re 10–100, whereas the estimation by
NLLS is misleading. In the joint confidence region plot (Figure 5.4), it is more
clearly shown that there is no significant difference between the estimated
results when Re < 30 and Re < 100. There is also no significant difference of
m and n1 between this work and that of Puiggene et al. (1997). However, the
relationships between n2 and n1, and n2 and m are significantly different. If
the order of Sc is fixed as 1/3, the EVM estimation results are n1 = 0.58 and

(a) 1
0.9
0.8
0.7 95% JCR contour
Estimation in this work
0.6
Puiggene et al. (1997)
0.5 Catchpole and King (1994)
n1
0.4 Tan et al. (1988)

0.3 Order of Sc:1/3
0.2 King et al. (1997)
This work based on king
0.1 This work (10 points)
0
0.00 0.50 1.00 1.50
n1
(b) 1
95% JCR
0.9 Estimation in this work
0.8 Puiggene et al. (1997)
Catchpole and King (1994)
0.7 Tan et al. (1988)
0.6 Order of Sc:1/3
King et al. (1997)
0.5
n2
This work based on king

0.4 This work (10 points)
0.3
0.2
0.1
0
0.00 0.20 0.40 0.60 0.80 1.00
n2
(c) 1.2
95% JCR contour
1 Estimation in this work
Puiggene et al. (1997)
0.8 Catchpole and King (1994)
Tan et al. (1988)
0.6 Order of Sc:1/3
m
King et al. (1997)

0.4 This work based on king
This work (10 points)
0.2
0
0 0.5 1 1.5 2
n1
FIGURE 5.4
Ninety-five percent joint confidence regions (JCR) of every two parameters. (a) The experi-
mental data n1 estimated from the 95% joint confidence region. (b) The experimental data n2
estimated from the 95% joint confidence region. (c) The experimental data m estimated from
the 95% joint confidence region.

m = 0.42. This is not significantly different from the previous estimation in

this work. That means fixing the exponent of Sc as 1/3 is reasonable. It also
implies that the order of Re is overestimated in other studies (Tan et al. 1988;
Catchpole and King, 1994; Puiggene et al., 1997). The rosemary oil data are
published by Coelho et al. (1997), and the pepper oil data by Ferreira (1996).
Rosemary oil is extracted at pressure ranging from 10 to 16 MPa and at a
temperature ranging from 310 to 320°K. Pepper oil is extracted at pressures
range of 20–30 MPa and temperatures of 300–320°K. Both of these systems
have low Re (Re < 1) and low Sc (Sc almost at the same order). The predic-
tion results show no big deviation for most experimental data as shown in
Figure 5.5. Model II can be extrapolated to low Re to some extent.
The data obtained from Puiggene et al. (1997) is used to predict mass-
transfer coefficient during supercritical-CO2 fluid extraction of essential oil
using Model II. There is an assumption that the oil is considered as a homog-
enous mixture, and therefore regarded as one component. Although the data
of King et al. (1997) also located in low Re region, Sh is much lower than
the model prediction of Puiggene et al. (1997). Comparing the mass-transfer
behavior of the model from Puiggene et al. (1997) with King et al. (1997), it
shows slight significant difference between m–n1 relationship, whereas n2 is
significantly different between the two models. The effect of Sc increases in
low-Re and high-Sc region. When the viscosity increases by one order and
the molecular diffusion decreases a lot, the contribution of eddy diffusion
will become important. As shown in Figure 5.6 at the region of low-Re and
high-Sc, the model from data of Puiggene et al. (1997), Figure 5.6a shows a
better prediction trend. Figure 5.6b overpredicts Sherwood number.
1.40
Pepper oil
1.20 Rosemary oil
1.00
Sh prediction
0.80
0.60
0.40
0.20
0.00
0.00 0.50 1.00 1.50
Sh experiment
FIGURE 5.5
Prediction of Sh, based on pepper oil extraction data (adapted from Ferreira, S.R.S. 1996. Mass
transfer kinetics in the supercritical fluid extraction of pepper oil. PhD thesis, Campinas
University) and rosemary oil extraction data (adapted from Coelho, L.A.F., Oliveira, J.V.,
d’Ávila, S.G. 1997. Journal of High Resolution Chromatography, 20(August): 431–436).

(a)
0.08
0.06
Sh´
0.04
0.02 1.0
0.8
0.6
0.00 0.4
Re
60 0.2
80
100 0.0
Sc
120
(b)
14
12
10
8
Sh
4
100
2 80
60
0
Re
4 40
6 8 10 12
14 20
Sc 16 18 20
FIGURE 5.6
Three-dimensional view of model prediction (a) based on the data of King et al. (1997) and
(b) based on the data of Puiggene et al. (1997).

5.5 Summary
The mass transfer during supercritical fluid extraction of essential oils in
fixed bed under forced convection can be described by the kinetic Models I
and II. The forced convection model describes the kinetics well. Data from
Puiggene et al. (1997) obtained consistent estimation from Re 10 to 100 and
can extrapolate to lower Re to some extent. The exponent of Sc set as 1/3 is
acceptable and the exponent of Re is more than 1/2. That means the flow
pattern contributes more on mass transfer than expected. The parameter
estimation should consider the errors in all the variables for dimensionless
number correlation, which makes the estimates more reliable. The errors in
the independent variables were found to be much larger than those of the
dependent variable for the Sh′ of the data set, and hence, the least-squares
and EIV statistical methods were used to minimize these deviations. The
best estimation was obtained when the Re was <1 and when Sc values were
between 70 and 100. A correlation between Models I and II was shown by
the unique exponent of their Sc’s, whereas the exponent of Re was smaller
than that of Model II. In general, Model II gave better predictions when its Re
was greater than unity. It indicated that the exponents of Re and Sc may not
strictly have a ratio of 3:2. It is found that the errors from Re1/2Sc1/3 are much
larger than that from the apparent Sherwood number Sh′. It also confirmed
that a specific kinetic behavior occurred in the laminar region, where the
mass transfer is poor as a result of poor molecular diffusion. This phenom-
enon usually occurred at high extraction pressures (>20 MPa) and with small
particle sizes of about <300 µm.
References
Bartle, K.D., Clifford, A.A., Hawthorne, S.B., Langenfeld, J.J., Miller, D.J., Robinson,
R. 1990. A model for dynamic extraction using a supercritical fluid. The Journal
of Supercritical Fluids, 3: 143–149.
Brunner, G. 1984. Mass transfer from solid material in gas extraction. Berichte der
Bunsengesellschaft für physikalische Chemie, 88: 887–891.
Catchpole, O.J., King, M.B. 1994. Measurement and correlation of binary diffusion
coefficients in near critical fluids. Industrial & Engineering Chemistry Research,
33(7): 1828–1837.
Coelho, L.A.F., Oliveira, J.V., d’Ávila, S.G. 1997. SFE of rosemary oil: Assessment of
the influence of process variables and extract characterization. Journal of High
Resolution Chromatography, 20(August): 431–436.
Ferreira, S.R.S. 1996. Mass transfer kinetics in the supercritical fluid extraction of pep-
per oil. PhD thesis, UNICAMP, Campinas, 218 pp.

Ferreira, S.R.S., Nikolov, Z.L., Doraiswamy, L.K., Meireles, M.A.A., Petenate, A.J.
1999. Supercritical fluid extraction of black pepper (Piper nigrum L.) essential
oil. Journal of Supercritical Fluids, 14(3): 235–245.
King, J.W., Cygnarowicz-Provost, M., Favati, F. 1997. Supercritical fluid extraction
of evening primrose oil kinetic and mass transfer effects. Italian Journal of Food
Science, 9(3): 193–204.
Lee, A.K.K., Bulley, N.R., Fattori, M., Meisen, A. 1986. Modeling of supercritical
carbon dioxide extraction of canola oilseed in fixed beds. JAOCS, 63(7): 921–925.
Lee, C.H., Holder, G.D. 1995. Use of supercritical fluid chromatography for obtain-
ing mass transfer coefficients in fluid-solid systems at supercritical conditions.
Industrial & Engineering Chemistry Research, 34: 906.
Lewicki, P.P., Pawlak, G. 2003. Effect of drying on microstructure of plant tissue.
Drying Technology, 21(4): 657–683.
Press, W.H., Teukolsky, S.A., Vetterling, W.T., Flannery, B.P. 1997. Numerical Recipes in
Fortran 90. Cambridge University Press, New York.
Puiggene, J., Larrayoz, M.A., Recasens, F. 1997. Free liquid-to-supercritical fluid mass
transfer in packed beds. Chemical Engineering Science, 52(2): 195–212.
Reilly, P.M., Reilly, H.V., Keeler, S.E. 1993. Parameter estimation in the error-in-
variables model. Applied Statistics, 42(4): 693–701.
Reverchon, E., Marrone, C. 2001. Modeling and simulation of the supercritical CO2
extraction of vegetable oils. Journal of Supercritical Fluids, 19(2): 161–175.
Rozzi, N.L., Singh, R.K. 2002. Supercritical fluids and the food industry. Comprehensive
Reviews in Food Science and Food Safety, 1(1): 33–44.
Sassiat, P.R., Mourier, P., Caude, M.H., Rosset, R.H. 1987. Measurement of diffusion
coefficients in supercritical carbon dioxide and correlation with the equation of
Wilke and Chang. Analytical Chemistry, 59(8): 1164–1170.
Schluender, E.U. 1981. Heat transfer between packed, agitated and fluidized beds
and submerged surfaces. Chemical Engineering Communications, 9(1–6): 273–302.
Sovová, H. 1994. Rate of the vegetable oil extraction with supercritical CO2.
I. Modelling of extraction curves. Chemical Engineering Science, 49(3): 409–414.
Tan, C.-S., Liang, S.-K., Liou, D.-C. 1988. Fluid–solid mass transfer in a supercritical
fluid extractor. The Chemical Engineering Journal, 38: 17–22.
Vesovic, V., Wakeham, W.A., Olchowy, G.A., Sengers, J.V., Watson, J.T.R., Millat, J.
1990. The transport properties of carbon dioxide. Journal of Physical and Chemical
Reference Data, 19(3): 763–808.
Wakao, N., Kaguei, S. 1982. Heat and Mass Transfer in Packed Beds. Gordon and Breach
Science Publishers, New York. pp. 138–160; 264–295.
Wilke, C.R., Chang, P. 1955. Correlation of diffusion coefficients in dilute solutions.
AIChE Journal, 8: 264–270.
Williams, D.F. 1981. Extraction with supercritical gases. Chemical Engineering Science,
36(11): 1769–1788.

6
Pressurized Low-Polarity Water
Extraction of Biologically Active
Compounds from Plant Products
Juan Eduardo Cacace and Giuseppe (Joe) Mazza
CONTENTS
6.1 Introduction................................................................................................. 177
6.2 PLPW Extraction Process........................................................................... 178
6.2.1 Basic Concepts................................................................................. 178
6.2.2 Equipment........................................................................................ 180
6.3 Applications of PLPW Extraction............................................................. 184
6.3.1 Effect of the Extraction Temperature and Pressure................... 187
6.3.2 Fractionation of Compounds of Different Polarity.................... 190
6.4 Modeling of PLPW Extraction of Bioactives from Plant Materials.......193
6.5 Conclusions.................................................................................................. 195
References.............................................................................................................. 196
6.1 Introduction
Plants synthesize many classes of organic chemical compounds rang-
ing from simple structures to complex molecules as part of their normal
metabolic processes. These compounds are broadly characterized as (a)

primary metabolites which encompass substances such as nucleic acids,
proteins, lipids, and polysaccharides that are the fundamental, biologically
active chemical units of living plant cells, and (b) secondary metabolites
which typically have larger, more complex chemical architectures that incor-
porate one or more primary metabolites into their structures. Various types
of secondary metabolites synthesized by plants are commonly referred to
as phytochemicals and include carotenoids, phenolics, alkaloids, terpenes,
sterols, saponins, nitrogen-containing compounds, and organosulfur com-
pounds (Liu, 2004). The most studied of the phytochemicals are the phenolics
and carotenoids. Phenolics are compounds possessing one or more aromatic
rings with one or more hydroxyl groups and generally are categorized as
phenolic acids, flavonoids, stilbenes, coumarins, tannins, isoflavones, and
177
lignans. The flavonoids are further classified as flavonols, flavones, flavanols

(catechins), flavanones, and anthocyanins.
It is known that many phytochemicals can significantly affect human
metabolism and health, and therefore, there is considerable interest in the
extraction of these compounds for their incorporation into functional foods,
nutraceuticals, and in pharmaceutical products. Also, certain classes of phy-
tochemicals (terpenes, pyrazines) are useful for the production of flavor and
fragrances and for incorporation into topical preparations.
Phytochemicals typically are not soluble in water under ambient conditions
because of their organic nature and the preponderance of nonionic bonds in
their architectures. However, they are readily soluble in various organic sol-
vents such as aliphatic alcohols, hexanes, dioxanes, acids, ethers, methylene
chloride, trichloroethylene, and acetonitrile. Numerous methods are known
for extracting phytochemicals from plant materials, and most of them are
based on sequential extraction processes incorporating one or more organic
solvents in combination with washing steps (Garcia Viguera et al., 1998; Luque
de Castro et al., 1999; Kahkonen et al., 2001; Gertenbach, 2002; Zhang et al.,
2003; Mazza et al., 2004). Some methods teach us the use of alkali or alkaline
solvents in combination with organic solvents for increased extraction effi-
ciency (Dix and Marutzky, 1984; Amen Chen et al., 1997; Eliasson et al., 2003).
Starting plant materials are usually physically disrupted by means of grind-
ing, shredding, chopping, pulverizing, compressing, or macerating in order
to improve extraction efficiency. Phytochemical extracts must be further
processed to remove all traces of the organic s olvents and to separate and
purify individual phytochemicals. More complex techniques such as hydro-
distillation, pressurized solvent extraction, microwave-assisted extraction,
supercritical-CO2 extraction, and pressurized low-polarity water (PLPW)
extraction have been used in an attempt to reduce the use of organic solvents
and/or increase the efficiency of the extraction process (Jimenez Carmona
et al., 1999; Luque de Castro et al., 1999; King, 2000; Kaufmann and Christen,
2002; Zhang et al., 2003).
6.2 PLPW Extraction Process

6.2.1 Basic Concepts
PLPW extraction, also known as subcritical water extraction, is a technology
that modifies the properties of water by increasing the temperature up to
374°C and keeping the pressure high enough to maintain the water in the
liquid state to improve its extraction ability. It is known that the physical and
chemical properties of water within sealed systems can be manipulated by
concurrently controlling the temperature and pressure, whereby the water
remains in the liquid state even though its temperature may be significantly

Pressurized Low-Polarity Water Extraction 179
increased above its atmospheric boiling point of 100°C. At this condition,

PLPW can be maintained in the liquid form up to a temperature of 374°C and
a pressure of 22.1 MPa (221 bar) after which, it becomes supercritical water.
The low-polarity water can be considered in a region from normal atmo-
spheric pressure and room temperature to 22 MPa and 374°C (Figure 6.1). The
polarity, viscosity, surface tension, and disassociation constant of subcritical
10,000 0 1000 2000
°C
400
20 C
°C
0°
°C
°C
10 °C
=
0°C
40
60
80
T
300°C
°C
500
3
200
m
g/
°C
3 k 3
1000 /m 8 00 /m
0 kg kg
0
90 70
3
4.0
g/m
4.5
3.5 g/m
3.0
/m
kg 0k
2.5
0k
m3
0 40
50
95
2.0
g/
0k
60
1.5
200 360°C
Density = 1000 kg/m3
340°C
320°C
100 300°C
1.0
280°C
Pressure (bar)
Entropy = 0.5 kj/kg K
50 260°C
240°C
220°C
200°C
180°C
10.0
160°C
140°C
120°C
40%
30%
50%
%
20%
60%
0%
80%
70%
= 10
ty = 9
T = 100°C
1.0
lity
Quali
Q ua
80°C
60°C
0.1
0 1000 2000
Enthalpy (kj/kg)
FIGURE 6.1
Pressure–enthalpy chart of water. (Adapted from Haar, L., Gallagher, J.S., Kell, G.S. 1984. NBC/
NRC Steam Tables: Thermodynamic and Transport Properties and Computer Programs for Vapor and
Liquid States of Water in SI Units. Hemisphere Publishing Corporation, Washington, DC.)

water are significantly lower compared to water at ambient temperature

and pressure conditions, thereby significantly altering its chemical proper-
ties to approximate those of the organic solvents. Thus, an increase of water
temperature from 25°C to 200°C, for instance, would decrease its dielectric
constant from 79 to 35, reaching values similar to those for ethanol (24) or
methanol (33). A pressure of 5 MPa (50 bar) would be high enough to keep
the water in the liquid phase during this increase in temperature (Figure 6.1).
A higher extraction pressure would be detrimental for the process because
of a slight increase in the dielectric constant and a considerable increase in
the cost of the equipment. At 25°C, the dielectric constant of water increases
from about 79 to 93 when the pressure increases from 10 to 600 MPa (100–
6000 bar) (Haar et al., 1984). The polarity of pure water would be compa-
rable to water–methanol or water–acetonitrile mixtures at 25°C (Yang et al.,
1998). Consequently, PLPW can easily solubilize organic compounds such as
phytochemicals, which are normally insoluble in ambient water (Miller and
Hawthorne, 2000).
PLPW extraction has been compared with and reported to be superior
to conventional extraction techniques including hydrodistillation (Jimenez
Carmona and Luque de Castro, 1999; Fernandez Perez et al., 2000; Hawthorne
et al., 2000; Soto Ayala and Luque de Castro, 2001), solid–liquid extraction
(Jimenez Carmona et al., 1999; Fernandez Perez et al., 2000; Gamiz Gracia
and Luque de Castro, 2000), and supercritical-CO2 extraction (Clifford et al.,
1999; Kubátová et al., 2001). Some of the benefits that have been mentioned
include higher selectivity, cleanliness, speed, and cost savings of both raw
material and energy. The heat advantage of PLPW extraction over hydrodis-
tillation has been reported at 20 times per kilogram of water, although 10 kg
more of water would be used (Basile et al., 1998). While methods based on
sequential extraction processes with organic solvents are useful for extrac-
tion and purification of small quantities of phytochemicals for research
purposes, they are difficult to scale up to commercial volumes because of
problems associated with cost and safe removal and recovery of the organic
solvent from the extracts and spent plant materials. Furthermore, the types
and concentrations of organic solvents must be carefully selected in order to
avoid structural changes to the target phytochemicals during extraction that
may adversely affect one or more of their desirable physical, chemical, and/
or biological properties.
6.2.2 Equipment
The most commonly used equipment in PLPW systems (Figure 6.2) con-
sists of storage tank (T) for pure water connected to a high-pressure pump
(P), which is connected to a valve (V1) and to a heating coil (HC) housed
within a temperature-controlled chamber (O). The pressure in the line is
displayed by a pressure gage installed outside the oven. The heating coil
is connected to an extraction vessel (EV), which is also mounted inside the

V1
TC
P
CC
BPR
HC O EV
CV
FIGURE 6.2
Diagram of PLPW extractor. T, water tank; P, water pump; HC, heating coil; TC, temperature
controller; E, extraction chamber; O, oven; CC, cooling coil; BPR, back-pressure regulator; CV,
collection vial.
temperature-controlled chamber. The chamber is equipped with a program-

mable temperature controller and recorder (TC). The extraction vessel is
connected to a cooling coil (CC), which in turn is connected to the inlet of
a pressure regulation valve or back-pressure regulator (BPR). The pressure
regulation valve is connected to a collection vessel (CV).
The collection vessel can be changed periodically to provide a plurality of
collection volumes, thereby separating and individually collecting multiple
eluant fractions. A source plant material is loaded into the extraction vessel
and it is maintained inside the vessel during extraction by fitting adequate
frits in the inlet and outlet of the vessel. Water is pumped from the storage
tank into the extraction vessel until a desired in-line pressure is achieved,
usually in the range of about 4–7 MPa. LC pumps and GC ovens have been
used most frequently for pumping and heating the water (Table 6.1). The
pressurized water within the extraction vessel is then heated by raising
the temperature within the extraction chamber, whereas in-line pressure
is maintained at a desired level by the pressure regulation valve. Stainless
steel tubing coils of various lengths have been used as heating and cool-
ing elements. Varied pressure regulators have been used including very
simple home tubing restrictors to automatic backpressure regulators (Table
6.1). Self-contained equipment has also been used for pressurized liquid
or accelerated liquid extraction. Flow-control devices taking action on the
pump achieve precise flow rates of subcritical water through the extrac-
tion vessel. The temperature within the chamber is set and maintained by
automatic controller during an extraction procedure. Components of the

182
TABLE 6.1
Components of PLPW Extraction Systems Used for Extraction of Plant Bioactives
Targeted
Phytochemicals Cell Pump Heater Cooler BPRa Reference
Essential oils 80 × 3 ID mm frit LC pump Coil/oven Coil/cooling Home variable Jimenez Carmona et al.
2 µm (0.5–3.0 mL/ recirculation restrictor (1999)
min) bath (25°C) (2–20 MPa)
Essential oils 150 × 11 ID mm LC pump Coil/oven 1 m coil/cooling Soto Ayala and Luque de
(14 mL) frit 2 µm (2.0 mL/min) recirculation Castro (2001); Fernandez
bath (25°C) Perez et al. (2000); Gamiz
Gracia and Luque de
Castro (2000); Luque de

Castro et al. (1999)
Essential oils (10.4 mL) frit LC pump 3 m × 0.76 mm 1 m coil/ Pressure control Ozel and Clifford (2004);
0.5 µm (1–3 mL/min) ID coil/oven ice-water bath valve (2–9 MPa) Ozel et al. (2003)
Bioactive 100 × 5 ID mm LC pump Coil/oven Coil Manual adjustable Ollanketo et al. (2002)
antioxidant (1.0 mL/min) restrictor
Oxygenates with 50 × 9.4 ID mm Isco 100 D 3 m coil/oven BP regulator Ibañez et al. (2003);
AO activity (3.47 mL) 0.5 µm (1 mL/min) (6–7 MPa) Kubátová et al. (2001)
and 2 µm frits
Fragrance and (10.4 mL) LC pump 1 m coil/oven 40 cm coil/ 11 cm–100 µm Clifford et al. (1999); Basile
flavor water bath restrictor et al. (1998)
compounds (25°C)
Anthocyanins 14.8 ID mm (24 mL/min) Coil/oven Coil/cooling Micro-metering King et al. (2003)
(55 mL) water bath valve (4 MPa)
Phenolics from 100 × 7.0 ID mm LC pump 3 m × 0.76 mm 1 m × 0.76 mm BP Regulator Ho et al. (2007); Cacace and
whole flaxseed 100 × 9.4 ID mm (0.3–4 mL/ ID coil/oven ID coil/water (5.2 MPa Mazza (2006)
and flaxseed 100 × 19.3 ID mm min) bath (room cartridge)
meal 200 × 7.7 ID mm temperature)
a Back-pressure regulator.
system between the pump and the pressure regulation valve must be con-
nected by tubing resistant to high pressure (8–10 MPa) required for PLPW
extraction.
PLPW extraction of bioactives has been usually performed using pure
water. Water may be further processed by distillation or filtration, and
optionally, could be purged with nitrogen to remove all dissolved oxygen
prior to its use. Purified water typically has a pH in the range of 5.9–6.2.
However, if so desired, the pH of the purified water can be adjusted into a
range of 3.5–9.5 with acids or bases prior to its use, to enhance solubilization
and extraction of various phytochemicals.
Extraction of bioactives from flax meal was optimized by modifying the
pH of subcritical water. Thus, buffered water at either high or low pH was
used to improve extraction of bioactives (Ho et al., 2007). Yields of proteins,
lignans, and secoisolariciresinol diglucoside (SDG) were maximized at alka-
line pH 9. The pH was the factor that defined the equilibrium yields of SDG
irrespective of the temperature. At pH 4 and 9, extraction of SDG from flax
meal at 160°C and 190°C reached the same equilibrium yield (Figure 6.3). It
is known that alkaline pH helps to solubilize protein (Oomah et al., 1994;
Wanasundara and Shahidi, 1997). At the same time, pH could have helped
to extract lignans by breaking phenolics–protein interaction and thus releas-
ing phenolics from the plant matrix. Alkaline pH may also have hydrolyzed
complex polymeric phenolics, reducing them to more available and easily
extracted compounds. Whatever mechanisms pH employed, high pH raised
SDG and protein yields. Thus, pH effect overcame the increase in solubility
and yield obtained by temperature.
Carbohydrate extraction from flax meal was improved using PLPW at
pH 4 (Ho et al., 2007). Also, high-temperature pressurized liquid extraction
25 119
20 95
SDG (mg/g meal)
15 71
Yield (%)
10 48
160°C pH 4
5 160°C pH 9 24
190°C pH 4
190°C pH 9
0 0
0 100 200 300 400 500
Time or volume (min or mL)
FIGURE 6.3
PLPW extraction of SDG from flax meal at 160°C and 190°C and pH 4 and 9.

(80–100°C) using acidified water was as effective as acidified 60% methanol

in extracting anthocyanins from grape skins (Ju and Howard, 2003).
Another modification of water to improve its extraction efficiency has
been the addition of sulfur dioxide. Sulfured PLPW (containing 1400 g/mL
sodium metabisulfite) extractions of grape skin phenolics over a temperature
range of 100–160°C were compared with conventional hot water or aqueous
60% (v/v) methanol extractions (50°C, 1 h). PLPW extracts from modified sul-
fured water had higher levels of total anthocyanins and total phenolics than
extracts that used pure water. Furthermore, subcritical water and subcriti-
cal sulfured water extracts had comparable or higher levels of anthocyanins
than extracts obtained using conventional hot water or 60% methanol (Ju
and Howard, 2005).
A modification of the heating system in the subcritical water extraction
could be the replacement of the oven with a water heater interconnected
between the pump and the extraction vessel. In such a system, the extraction
vessel would be provided with a jacket or a surface heater, thereby main-
taining the temperature of PLPW flowing through the extraction vessel. The
jacket may be integral to the extraction vessel, or alternatively, be mountable
onto the exterior surface of the vessel (Mazza and Cacace, 2005).
6.3 Applications of PLPW Extraction

PLPW has been applied to extract a variety of biochemicals from a wide
range of plant species (Table 6.2). It has been used to extract antioxidants
from rosemary (Ibañez et al., 2003) and Taiwan yams (Chen et al., 2004),
anthocyanins from red grape skin (Ju and Howard, 2003, 2005), ginsenosides
from American ginseng (Choi et al., 2003), catechins and epicatechin from tea
leaves and grape seeds (Piñeiro et al., 2004), oil from cedarwood (Eller and
Taylor, 2004), essential oil from oregano (Soto Ayala and Luque de Castro,
2001), anthraquinones (antibacterial, antiviral, and anticancer compounds)
from roots of Morinda citrifolia (Shotipruk et al., 2004), tanshinone I and IIA
from Salvia miltiorrhiza used in Chinese medicine (Ong and Len, 2004), fla-
vones, anilines, and phenols from orange peels (Lamm and Yang, 2003), kava
lactones from kava roots (Kubátová et al., 2001), lignans from whole flaxseed
(Cacace and Mazza, 2006), and flaxseed meal (Ho et al., 2007) and saponins
from cow cockle (Saponaria vaccaria) seed (Guclu-Ustundag and Mazza, 2006).
Plant material for the extraction can include homogenous samples, or mix-
tures of whole plant parts such as seeds, flowers, leaves, stems, and roots,
and also, with source plant materials disrupted and processed by methods
including one or more of grinding, shredding, chopping, pulverizing, com-
pressing, and macerating. The biomass can be fresh hydrated plant materi-
als or plant materials may be dehydrated prior to extraction or alternatively,

TABLE 6.2
Pressurized Low-Polarity Water Extraction

Applications of PLPW Technology to the Extraction of Plant Bioactives
Target
Phytochemicals Plant Material Temperature/Pressure Major Compounds Reference
Essential oils Marjoram 100–175°C (150°C) / a Eucalyptol, linalool, terpinen-4-ol, Jimenez Carmona et al.
2–20 MPa α-terpineol, geraniol, ETMC b (1999)
Essential oils Laurel 50–200 (150°C)/5MPa 1,8-Cineole, 2 unidentified peaks Fernandez (2000); Fernandez
Perez et al. (2000)
Essential oils Clove 125, 250°C/2.4, Eugenol, eugenyl acetate Clifford et al. (1999); Rovio
5, 10, 17 MPa et al. (1999)
Essential oils Thyme 100, 125, 150, 175°C α-Pinene, p-cymene, γ-terpinene, Ozel et al. (2003)
(150°C)/2, 6, 9 MPa limonene, E-3-caren-2-ol, thymol,
carvacrol, caryophyllene
Essential oils Oregano 100–175°C (150°C) Thymol Soto Ayala and Luque de
Castro (2001)
Essential oils A. monocephala 100, 125, 150, 175°C/6 MPa 1,8-Cineole, camphor, α-campholenal, Gogus et al. (2006)
borneol, terpinen-4-ol and many more
Essential oils O. micranthum 100, 125, 150, 175°C/4 to α-Terpineol, linalool, borneol, terpinen- Gogus et al. (2005)
8 MPa 4-ol, and many more
Essential oils Fennel 50–200 (150°C)/2 MPa α-Pinene, mircene, limonene, camphor, Gamiz Gracia and Luque
phelandrene, anethol de Castro (2000)
Phenolic Sage 70, 100, or 150°C/10 MPa Rosmarinic acid, carnosol, carnosic acid, Ollanketo et al. (2002)
antioxidants methyl carnosate
Antioxidant Rosemary leaves 25, 100, 150, 200°C and 100, Scutellarein, rosmanol, genkwanin, Ibañez et al. (2003)
compounds 150, 200°Cc/6 MPa carnosol, carnosic acid, NI 1
Fragrance and Peppermint 150, 175°C and 50, 100, 125, Carvone, pulegone, eucalyptol, Kubátová et al. (2001)
flavor compounds 150, 175, 200°Cc/6 MPa menthone, neomenthol, menthol,
menthyl acetate
(Continued)
185
186
Applications of PLPW Technology to the Extraction of Plant Bioactives
Target
Phytochemicals Plant Material Temperature/Pressure Major Compounds Reference
Fragrance and Savory 100, 150, 175°C and 50, 100, p-Cymene, thymol, carvacrol, linalool, Kubátová et al. (2001)
flavor compounds 125, 150, 175, 200°Cc/ borneol, thymoquinone
6 MPa
Fragrance and Rosemary 125, 150, 175°C α-Pinene, limonene, camphene, camphor, Basile et al. (1998)
flavors 1,8-cineole, borneol
Fragrance and R. canina 50, 100, 150°C/2.5, 5, Benzaldehyde, benzyl alcohol, Ozel and Clifford (2004)
flavor compounds 7.5 MPa phenylethyl alcohol, eicosane, and more
Anthocyanins Eldelberry, 110–160°C (100–120°C) Phenolic acids, ellagic acid, catechin, King et al. (2003)

raspberry, bilberry, epicatechin, resveratrol, quercetin,
chokeberry kaempferol, anthocyanins
Anthocyanins and Grape skin 100–160 by 10°C Monoglucoside and acylated Ju and Howard (2003)
phenolics anthocyanins
Phenolics/lignans Whole flaxseed 100, 120, 140, 160°C/5 MPa SDGd, p-coumaric acid glucoside, ferulic Cacace and Mazza (2006)
acid glucoside
Lignans, proteins Flaxseed meal 130, 160, 190°C/5 MPa SDGd-proteins, carbohydrates Ho et al. (2007)
Saponins Cow cockle 125, 175°C/5 MPa Saponins, cyclopeptides Guclu-Ustundag and
Mazza (2006)
Kava lactones Kava root 25, 50, 100 to 200 by Dihydrokavain, kavain, Kubátová et al. (2001)
25°C/6–7 MPa desmethoxyyangonin, yangonin,
dihydromethysticin
a Selected temperature in parentheses.
b Ethenyl-α,α-4trimethyl-3-(1-methylethenyl)-cyclohexanemethanol.
c Sequential equal time extractions at listed temperatures of one sample load.
d SDG, secoisolariciresinol diglucoside.
processing by one or more of the methods described above prior to extrac-

tion. The plant materials may be packed into an extraction vessel either alone
or in combination with inert physical substrates such as glass wool, glass
beads, resin beads, silica sand, stainless steel wire cloth, and other like sub-
strates, whereby the inert substrates maintain spacing and distribution of the
source plant materials throughout the vessel during the course of the extrac-
tion procedure, thereby facilitating mass transfer while preventing migra-
tion and packing of the plant material against the outlet frits (Mazza and
Cacace, 2005). Recently, Ho et al. (2007) showed that extraction of lignans,
proteins, and carbohydrates with PLPW was positively affected by the addi-
tion of glass beads to flaxseed meal for packing the cell. The overall lignan
yield increased from 10% to 50% at all temperatures tested with co-packing
the meal with glass beads.
6.3.1 Effect of the Extraction Temperature and Pressure

The extraction of bioactives from plant material with the PLPW process is
clearly affected by the temperature. Thus, SDG, the major lignan in flaxseeds,
along with two other phenolic compounds, p-coumaric acid glucoside and
ferulic acid glucoside were extracted with varied success at different tem-
peratures in a PLPW system. Extraction yields of lignans and other phenolic
compounds from flaxseed increased from 10% at 100°C up to approximately
90% at 140–160°C (Cacace and Mazza, 2006). Temperature also affected PLPW
extraction of lignan SDG from flax meal. Yield of flax meal SDG increased
from 30% at 130°C to a maximum of almost 100% at 190°C (Ho et al., 2007). In
PLPW extraction of phenolic compounds from grape seeds at 150°C, catechin
increased by 32% and epicatechin increased by 99% over the recovery at 50°C
extraction (Palma et al., 2001). Saponin yields of PLPW extracts increased
with temperature and time. Although only 33.2% (w/w) of total saponins
was extracted at 125°C in 3 h, 60.2% (w/w) was recovered at 175°C in the
first 15 min extraction from cow cockle ground seeds (Guclu-Ustundag and
Mazza, 2006). The temperature has such a remarkable effect on the extraction
and composition of the final extract that a study of temperature effect is a pre-
requisite in any new PLPW extraction research. The effect of temperature on
extraction of bioactives using PLPW has also been reported in extractions of
lactones from kava root (Kubátová et al., 2001), phenolics from grape skin (Ju
and Howard, 2003), and essential oils from peppermint and savory (Kubátová
et al., 2001), fennel (Gamiz Gracia and Luque de Castro, 2000), rosemary
(Ibañez et al., 2003; Basile et al., 2006), thyme (Ozel et al., 2003), clove (Rovio
et al., 1999), Achillea monocephala (Gogus et al., 2005), Rosa canina (Ozel and
Clifford, 2004), Origanum micranthum (Gogus et al., 2005), and Origanum onites
(Ozel and Kaymaz, 2004). It is clear from these and others studies (Table 6.2)
that temperature has to reach a minimum value for the water to gain the
properties required to extract a given bioactive. Below the required tempera-
ture, the extraction does not occur or results in a very low yield.

100
80
60
Yield (%)
40 Carvone
Menthol
Menthyl acetate
20 β-Caryophyllene
0
50 100 150 200
Temperature (°C)
FIGURE 6.4
Effect of temperature on PLPW extraction of volatile compounds from peppermint. (Adapted
from Kubátová, A. et al. 2001. Flavour and Fragrance Journal, 16(1): 64–73.)
A very good example of the effect of temperature on compound yield is

the extraction of volatile compounds from peppermint (Figure 6.4). Four
group of compounds were extracted, the first included carvone, pulegone,
and piperitone, the second included eucalyptol, menthone, neomenthol,
and menthol, the third menthyl acetate, and the fourth, β-caryophyllene
(Kubátová et al., 2001). Only one line per group has been plotted in Figure 6.4
to simplify the plot. As can be observed, extraction yields increased with
the temperature from values as low as zero to as high as 100%, depending
on the group of compounds evaluated. At temperatures below 50°C, only
compounds of the first two groups were extracted and yields varied depend-
ing on the extraction time. Menthyl acetate and β-caryophyllene were not
extracted or their yields were very low because their solubility in water
at those temperatures was likely very low. In order to begin extraction of
menthyl acetate and β-caryophyllene, temperatures above 50°C and 150°C
had to be used. Increasing the extraction time did not considerably increase
the yield of these compounds when the extraction temperature was kept
below 50°C and 150°C, respectively. Yield points represented in Figure 6.4
are yields of 15 min extraction at each temperature. Thus, 80% yield at 175°C
was calculated by adding yields of five extractions of 15 min. However, at
50°C, methyl acetate yield after 15 min extraction was 1% or 2%, if the extrac-
tion time would be increased five times, the yield would be at most 5%–10%.
Effect of temperature has been attributed to an increase in the solubility of
the compounds (Basile et al., 1998). It is also possible that the temperature
affects the extraction by breaking interactions between the analytes and the
plant matrix (Palma et al., 2001). Because the extraction yields increase with

the temperature and also temperature has to reach a minimum value for the
bioactives to be extracted with water, the extraction temperature must be
selected for the targeted compounds.
There is an optimal temperature at which the yield is maximized for
every bioactive. Ideally this temperature would be related to the modified
properties of the water required for the extraction in the PLPW system,
which in turn are linked to the properties of the bioactive that has to be
extracted. Thus, the most efficient extraction of the lignan SDG and other
phenolics from flaxseed occurred in the temperature range from 140°C to
160°C (Cacace and Mazza, 2006) and from flax meal at 190°C (Ho et al., 2007).
Recommended temperatures for PLPW extraction of other phytochemicals
are: 150°C for phenolic compounds from grape seeds (Palma et al., 2001),
120°C for anthocyanins from berries (King et al., 2003), 100°C for volatile fla-
vor and fragrance compounds from R. canina (Ozel and Clifford, 2004), and
150°C for essential oils from A. monocephala (Gogus et al., 2006).
Most of these extraction temperatures are relatively high and may be detri-
mental when heat-sensitive bioactives are present in the raw material. Thus,
at 160°C, the total SDG measured in flaxseed extracts, solvent wash, and
extracted seed residues was lower than the amount achieved at 140°C, and
there was a lower SDG percentage in the seed residue at 160°C, which may be
attributed to degradation of phenolics. At temperatures higher than 160°C,
degradation of SDG also occurred in PLPW extractions of bioactives from
flax meal. However, the detrimental effect of temperature could be alleviated
when a high rate of extraction removes most of the bioactives in a relatively
short time. Thus, SDG yields of 96%–98% were reached at temperature as
high as 190°C when the rate of the extraction was increased by the increase
in pH to 9 (Ho et al., 2007). Equilibrium time and maximum recovery were
reached in <100 min in extractions avoiding degradation of SDG.
Elevated temperature increases the extraction rate, which in turn reduces
the volume of water required for the extraction and the time to reach maxi-
mum recovery. The increase in the extraction temperature increased the
extraction rate of lignans from flax meal, thus the volume of water required
for the extraction and the time to reach maximum recovery were reduced.
A raise in temperature from 160°C to 190°C reduced the extraction time by
half (Ho et al., 2007). Increased extraction rates produced by temperature
raises have been reported for the extraction of kava lactones from kava roots
(Kubátová et al., 2001) and flavor and fragrance compounds from savory
and peppermint (Kubátová et al., 2001), rosemary (Basile et al., 1998), and
clove (Rovio et al., 1999). However, for some plant material and targeted com-
pounds, the raise in temperature cannot be unlimited.
The combination of high temperatures and long time of extraction must
be avoided when temperature-sensitive bioactives are being extracted. Ju
and Howard (2003) reported that extraction temperatures higher than 100°C
resulted in anthocyanin degradation in pressurized liquid extraction, which
was especially marked at 140°C. Reductions from 24% to 40% of the total

anthocyanin content have been reported with pure and modified PLPW in
the range of temperatures from 110°C to 160°C (Ju and Howard, 2005). It has
been demonstrated that most phenolic compounds react easily at high tem-
perature (65°C) when they are in contact with air. However, at higher tem-
peratures under nitrogen atmosphere, there are no degradations, since the
degradation process for phenolic compounds is an oxidative process requir-
ing the presence of oxygen (Palma et al., 2001). At high temperatures, dark
brownish PLPW extracts with strong burning smell and increased viscos-
ity have been found in extractions from parsley (Mazza and Cacace, 2005),
flax meal (Ho et al., 2007), O. micranthum (Gogus et al., 2005), oregano (Soto
Ayala and Luque de Castro, 2001), and leaves and flowers of A. monocephala
(Gogus et al., 2006). This has been attributed to the presence of browning
reaction products such as furfural, acetylfuran, and 5-methylfurfural in the
extracts of flowers of A. monocephala at 175°C (Gogus et al., 2006). However,
the degradation in PLPW extractions is lower than the effects measured at
atmospheric pressure extractions even at lower temperatures. Thus, PLPW
allows for the use of temperatures higher than those used in conventional
extraction techniques, probably because PLPW requires shorter extraction
times and an oxygen-reduced environment.
The effect of pressure in PLPW extraction of bioactives from diverse plant
material has also been reported. Rovio et al. (1999) studied the effect of four
pressures at two temperatures in PLPW extraction of flavor and fragrance
from clove. No significant differences were found for the recoveries of
eugenol and eugenyl acetate from clove at 25, 50, 100, and 175 kg/cm2 (2.4, 4.9,
9.8, 17.2 MPa). Similar responses have been reported on PLPW extractions
from R. canina (Ozel and Clifford, 2004), oregano (Soto Ayala and Luque de
Castro, 2001), O. micranthum (Gogus et al., 2005), and marjoram (Jimenez
Carmona et al., 1999). These results are in agreement with the change in
water dielectric constant with an increase in pressure. As it has been men-
tioned above, an increase in pressure of 590 MPa (from 10 to 600 MPa at 25°C)
results in an increase in dielectric constant from 79 to 93 (Haar et al., 1984).
6.3.2 Fractionation of Compounds of Different Polarity

The extraction performance of a compound in a PLPW system is related to
the polarity of the compound. Extraction yields of major phenolics present
in flaxseed were not markedly different, indicating that they have a similar
polarity. This suggests that fractionation of the major phenolic compounds in
flaxseed using a PLPW system may not be viable. However, fractionation of
phenolic compounds has been achieved in black currants and parsley (Mazza
and Cacace, 2005). A sequential-temperature extraction of frozen black cur-
rant particles was performed in a PLPW system at temperatures from 80°C
to 240°C. The total phenolic concentration of the extracts decreased from
80°C to 120°C and the yield of the extraction increased until it reached a
plateau. However, when the temperature was further increased up to 240°C,

1000 5000
Concentration (mg/L)
800 4000
TPhen
Concentration (mg/L)
Anthoc
Yield (mg/100 g)
Yield (mg/100 g)
600 TPhen 3000
Anthoc
400 2000
200 1000
0 0
50 100 150 200 250 300
Temperature (ºC)
FIGURE 6.5
Total phenolic yield and extract concentration in PLPW sequential-temperature extractions
from frozen black currant at 80–240°C.
the concentration and the yield increased continuously with the temperature
(Figure 6.5). The concentration of anthocyanins decreased continuously to
zero and the yield increased to reach equilibrium and remained at those val-
ues even after further temperature increase up to 240°C. High-performance
liquid chromatographic (HPLC) chromatograms of samples collected at
80°C, 120°C, and 200°C (Figure 6.6) indicate that high-polarity compounds
were extracted at the initial lower temperatures, and their content in the
extraction cell decreased with further extraction. Although the identification
of the compounds being extracted was not pursued, the increase in yield
at the highest temperatures could be attributed to the extraction of newly
generated high-polarity compounds by hydrolysis of low-polarity poly-
meric compounds or to the extraction of different molecules that otherwise
would not be extracted. Extractions of those compounds are indicated by the
appearance of new peaks at the beginning of the 280 nm chromatograms
of the extracts collected at 200°C and 240°C (Figure 6.6). On the other hand,
anthocyanins were extracted continuously and completely with extractions
at 80°C, 120°C, and 200°C and no new compounds that absorb at 525 nm
were extracted with further increase in temperature. These observations
clearly suggest that by suitably controlling the extraction temperature and
the collection of the extracted fractions, PLPW extraction can be used for the
recovery or fractionation of compounds of different polarity.
PLPW extractions of phenolics from grape seeds (Palma et al., 2001) and
antioxidant compounds from rosemary produced similar results (Ibañez
et al., 2003). In these studies, depending upon the temperature used, there
were large differences in both the identity and recovery rate of the phenolic

(c)
200°C
(b)
120°C
(a)
80°C
0 10 20 30 40 50 60 70 80
Retention time (min)
FIGURE 6.6
HPLC chromatograms of frozen black currant extracts collected in PLPW sequential-temperature
extractions at 80°C (a), 120°C (b), and 200°C (c).
compounds extracted. In grape seed extraction at 150°, catechin increased

by 32% and epicatechin increased by 99% over the recovery from the 50°C
extraction. There were also some compounds that were not detected in
the extracts produced at 50°C and 100°C but which were detected in the
extracts obtained at 150°C. It was considered that the use of higher extrac-
tion temperature helped in the breaking of bonds between the analytes and
the matrix, increasing yields of some compounds or extracting compounds
that otherwise would not be extracted. In rosemary extractions, when water
was heated to 200°C, the dielectric constant of water was reduced and the
solubilities of less polar compounds increased by several orders of magni-
tude, changing the composition of the extracts (Ibañez et al., 2003). Similarly,
Palma et al. (2001) reported that the behavior of the analytes depended on
the matrix, and the analytes in grape seeds were more strongly bonded to
the matrix than in grape skin. Therefore, for grape skins shorter extraction
times and lower temperatures would result in significant increase in yield
and fractionation of phenolics (Palma et al., 2001). The temperature of the
water affects the extraction in two ways, first by changing the dielectric con-
stant of the water and thus the solubility of targeted compounds, and second
by breaking the interactions between the analytes and the matrix.
Production of essential oils richer in oxygenated fragrance compounds
of more value and with less contaminant mono- and sesquiterpenes from
peppermint can be produced by adequate selection of extraction tempera-
ture (Figure 6.4). Oxygenated compounds extract at substantially lower

water temperatures than nonoxygenated compounds (Kubátová et al., 2001).

Extraction at 50°C to 100°C will result in extracts richer on valuable oxy-
genated compounds with a low fraction (<5%) of terpenes such as p-cymene
or β-caryophyllene. Similarly, desirable carnosol-rich rosemary extracts can
be produced by PLPW extraction of rosemary leaves at 100°C. Furthermore,
by using such a procedure, it is possible to obtain enriched extracts with
very high antioxidant activity (Ibañez et al., 2003). Also, continuous PLPW
extraction of oil from marjoram resulted in extracts richer in odoriferous and
hence more valuable oxygenated compounds than hydrodistilled oils, which
contained 11–22 times larger amounts of monoterpenes (Jimenez Carmona
et al., 1999). Therefore, extracts produced by PLPW extractions can give a
higher quality and higher value product with more intense characteristic
natural aroma.
6.4 Modeling of PLPW Extraction of Bioactives

from Plant Materials
Two simple models have been applied to describe the extraction profiles
obtained with PLPW extraction (Kubátová et al., 2002). The first of these
models is based on the thermodynamic distribution coefficient (K D), which
assumes that analyte release from the matrix is rapid compared to elu-
tion, and the second model is a two-site kinetic model which assumes that
the extraction rate is limited by the analyte release rate from the matrix
and is not limited by the thermodynamic (K D) partition that occurs during
elution.
The two models are defined by the following equations (Kubátová et al.,
2002).
Thermodynamic model:
Sb (1 − Sa /S0 ) S
= + a (6.1)

S0 [ KD m/(Vb − Va )d + 1] S0
Kinetic model:
ST
= 1 − [F e− k1t ] − [(1 − F )e− k2t ] (6.2)
S0
Sa: Cumulative mass of the analyte extracted after volume Va (mL)

Sb: Cumulative mass of the analyte extracted after volume Vb
S0: Initial total mass of the analyte in the matrix

Sb/S0 and Sa/S0: Cumulative fraction of the analyte extracted by the

fluid of the volume Vb and Va
ST: Mass of the analyte removed by the extraction fluid after time t
KD: Distribution coefficient; concentration in matrix/concentration in
fluid
F: Fraction of the analyte released quickly
k1 and k2: First-order rate constant (mL−1) for the quickly and slowly
released fractions
d: Density of the extraction fluid at a given condition (g/mL)
m: Mass of the extracted sample (g)
The kinetic model does not include solvent volume, but relies solely on the
extraction time. Therefore, doubling the extractant flow rate should have little
effect on the extraction efficiency per unit time if the extraction efficiency is
controlled by the kinetics of the initial desorption step (assuming the other
extraction parameters remain constant). On the contrary, the thermody-
namic model is only dependent of volume of extractant used. Therefore, the
mechanism of thermodynamic elution and desorption kinetic can be com-
pared simply by changing the flow rate in PLPW extraction. If the concentra-
tion of bioactive compounds increases proportionally with increase in flow
rate at certain extraction time, the extraction mechanism can be explained by
the thermodynamic model. However, if the increase in flow rate has no signifi-
cant effect on the extraction of the bioactive compounds, with the other extrac-
tion parameters kept constant, the extraction mechanism can be modeled by
kinetic diffusion (Kubátová et al., 2002; Cacace and Mazza, 2006). In this case,
plots for several flow rates of the extracted amount or yield of the analyte as a
function of the extraction time must lie on the same line, indicating no effect
of the flow rate. The mechanism of control and therefore the model valid for
PLPW extraction may be different depending on the raw material, the targeted
analyte, and extraction conditions. Thus, phenolic extractions from flaxseed
performed at flow rates from 1 to 4 mL/min were affected by the flow rate,
which indicates that the mass transfer of the solute from the surface of the
solid into the bulk of the water regulated most of the extraction process in a
similar way to the thermodynamic model. However, there was no difference in
the extraction rate among phenolic extractions performed at 1, 0.5, and 0.3 mL/
min, which were not affected by the flow rate. Thus, extractions at low flow
rates would have been controlled by the diffusion in the seeds, as the two-site
kinetic model establishes above. It has been suggested that at low flow rates
(<1 m/min), Biot number may have increased to values higher than 50–100;
in this condition, the effect of the boundary layer is negligible, the extraction
would be controlled by the diffusion inside the seeds, and not show any effect
of the flow rate. No effect of the flow rate has also been found in extractions
of SDG, proteins, and carbohydrates from flaxmeal (Cacace and Mazza, 2006).

100
80
Yield (%)
60
40
0.25 mL/min
0.5 mL/min
20 1 mL/min
2 mL/min
4 mL/min
0
0 10 20 30 40 50 60
Time (min)
FIGURE 6.7
Effect of flow rate on thymol yields in PLPW extractions from savory at 100°C. (Adapted from
Kubátová, A. et al. 2002. Journal of Chromatography A, 975(1): 175–188.)
The data from LPWE extraction for savory, presented in Figure 6.7, clearly
show that the extraction rates of essential oils had an increase proportional to
the water flow rate, and thus for PLW extraction, the thermodynamic elution of
analytes from the matrix is the prevailing mechanism as evidenced by the fact
that extraction rates increased proportionally with the water flow rate. This is
also confirmed by the fact that simple removal calculations based on a single
KD (for each essential oil compound) give good fits to experimental data for
flow rates from 0.25 to 4 mL/min (Kubátová et al., 2002). The increased recov-
ery rates directly proportional to extraction flow rates show that high water
flow rates can be used to shorten PLPW extraction times without increasing
the amounts of fluid needed to achieve a high recovery (Kubátová et al., 2002).
6.5 Conclusions
PLPW extraction, also known as subcritical water extraction, is a highly
promising “green” technology for the extraction and fractionation of biologi-
cally active compounds for use as functional food ingredients and nutraceu-
ticals. PLPW has been applied to extract a variety of phytochemicals from a
wide range of plant species including antioxidants from rosemary and yams,
anthocyanins from berries and red grape skin, ginsenosides from American
ginseng, catechins and epicatechin from tea leaves and grape seeds, essen-
tial oil from oregano, kava lactones from kava roots, lignans from flaxseed
and flaxseed meal, and saponins from cow cockle (S. vaccaria) seed. PLPW

extraction has been compared and reported to be superior to conventional

extraction techniques including solid–liquid extraction (Fernandez Perez
et al., 2000; Gamiz Gracia and Luque de Castro, 2000), hydrodistillation, and
supercritical-CO2 extraction. Key benefits include higher selectivity, cleanli-
ness, speed, and cost savings of both raw material and energy.
References
Amen Chen, C., Pakdel, H., Roy, C. 1997. Separation of phenols from Eucalyptus wood
tar. Biomass Bioenergy, 13(1–2): 25–37.
Basile, A., Jimenez Carmona, M.M., Clifford, A.A. 1998. Extraction of rosemary by
superheated water. J. Agric. Food Chem., 46(12): 5204–5209.
Cacace, J.E., Mazza, G. 2006. Pressurized low polarity water extraction of lignans
from whole flaxseed. J. Food Eng., 77: 1087–1095.
Chen, P.Y., Tu, Y.X., Wu, C.T., Jong, T.T., Chang, C.M.J. 2004. Continuous hot pressurized
solvent extraction of 1,1-diphenyl-2-picrylhydrazyl free radical scavenging com-
pounds from Taiwan yams (Dioscorea alata). J. Agric. Food Chem., 52: 1945–1949.
Choi, M.P.K., Chan, K.K.C., Leung, H.W., Huie, C.W. 2003. Pressurized liquid extrac-
tion of active ingredients (ginsenosides) from medicinal plants using non-ionic
surfactant solutions. J. Chromatogr. A, 983: 153–162.
Clifford, A.A., Basile, A., Al Saidi, S.H.R. 1999. A comparison of the extraction of clove
buds with supercritical carbon dioxide and superheated water. Fresenius’ J. Anal.
Chem., 364(7): 635–637.
Dix, B., Marutzky, R. 1984. Tannin extracts from spruce and pine barks. J. Appl. Polym.
Sci., 1984 (Applied Polymer Symposium 40): 91–100.
Eliasson, C., Kamal Eldin, A., Andersson, R., Aman, P. 2003. High performance liq-
uid chromatographic analysis of secoisolariciresinol diglucoside and hydroxy-
cinnamic acid glucosides in flaxseed by alkaline extraction. J. Chromatogr. A,
1012(2): 151–159.
Eller, F.J., Taylor, S.L. 2004. Pressurized fluids for extraction of cedarwood oil from
Juniperus virginianna. J. Agric. Food Chem., 52: 2335–2338.
Fernandez Perez, V., Jimenez Carmona, M.M., Luque de Castro, M.D. 2000. An
approach to the static–dynamic subcritical water extraction of laurel essential
oil: Comparison with conventional techniques. Analyst, 125(3): 481–485.
Gamiz Gracia, L., Luque de Castro, M.D. 2000. Continuous subcritical water extrac-
tion of medicinal plant essential oil: Comparison with conventional techniques.
Talanta, 51(6): 1179–1185.
Garcia Viguera, C., Zafrilla, P., Tomas Barberan, F.A. 1998. The use of acetone as an
extraction solvent for anthocyanins from strawberry fruit. Phytochem. Anal.,
9(6): 274–277.
Gertenbach, D.D. 2002. Solid–liquid extraction technologies for manufacturing nutra-
ceuticals. In: Functional Foods: Biochemical and Processing Aspects. Volume 2. (Eds.
John Shi, G. Mazza, and Marc Le Maguer), pp. 331–366. Boca Raton, FL: CRC Press.
Gogus, F., Ozel, M.Z., Lewis, A.C. 2006. Extraction of essential oils of leaves and flow-
ers of Achillea monocephala by superheated water. Flavour Frag. J., 21(1): 122–128.

Gogus, F., Ozel, M.Z., Lewis, A.C. 2005. Superheated water extraction of essential oils
of Origanum micranthum. J. Chromatogr. Sci., 43(2): 87–91.
Guclu-Ustundag, O., Mazza, G. 2006. Pressurized low polarity water extraction of
saponins from cow cockle (Saponaria vaccaria) seed. AAFC-PARC Internal
Report. Summerland, BC, Canada.
Haar, L., Gallagher, J.S., Kell, G.S. 1984. National Bureau of Standards/National Research
Council Steam Tables. Hemisphere Publishing Corporation: New York.
Hawthorne, S.B., Grabanski, C.B., Martin, E., Miller, D.J. 2000. Comparisons of
Soxhlet extraction, pressurized liquid extraction, supercritical fluid extraction
and subcritical water extraction for environmental solids: Recovery, selectivity
and effects on sample matrix. J. Chromatogr. A, 892(1–2): 421–433.
Ho, C.H.L., Cacace, J.E., Mazza, G. 2007. Extraction of lignans, proteins and carbohy-
drates from flaxseed meal with pressurized low polarity water. LWT-Food Sci.
Technol. 40: 1637–1647.
Ibañez, E., Kubátová, A., Señoráns, F.J., Cavero, S., Reglero, G., Hawthorne, S.B. 2003
Subcritical water extraction of antioxidant compounds from rosemary plants.
J. Agric. Food Chem., 51(2): 375–382.
Jimenez Carmona, M.M., Luque de Castro, M.D. 1999. Isolation of eucalyptus essen-
tial oil for GC–MS analysis by extraction with subcritical water. Chromatographia,
50(9/10): 578–582.
Jimenez Carmona, M.M., Ubera, J.L., Luque de Castro, M.D. 1999. Comparison of
continuous subcritical water extraction and hydrodistillation of marjoram
essential oil. J. Chromatogr. A, 855(2): 625–632.
Ju, Z.Y., Howard, L.R. 2003. Effects of solvent and temperature on pressurized liq-
uid extraction of anthocyanins and total phenolics from dried red grape skin.
J. Agric. Food Chem., 51(18): 5207–5213.
Ju, Z.Y., Howard, L.R. 2005. Subcritical water and sulfured water extraction of
anthocyanins and other phenolics from dried red grape skin. J. Food Sci., 70:
270–276.
Kahkonen, M.P., Hopia, A.I., Heinonen, M. 2001. Berry phenolics and their antioxi-
dant activity. J. Agric. Food Chem., 49(8): 4076–4082.
Kaufmann, B., Christen, P. 2002. Recent extraction techniques for natural products:
Microwave-assisted extraction and pressurised solvent extraction. Phytochem.
Anal., 13(2): 105–113.
King, J.W. 2000. Advances in critical fluid technology for food processing. Food Sci.
Technol. Today, 14(4): 186–191.
King, J.W., Grabiel, R.D., Wightman, J.D. 2003. Subcritical water extraction of antho-
cyanins from fruit berry substrates. Proceedings of the 6th International Symposium
on Supercritical Fluids. tome 1, pp. 409–418.
Kubátová, A., Jansen, B., Vaudoisot, J.F. Hawthorne, S.B. 2002. Thermodynamic and
kinetic models for the extraction of essential oil from savory and polycyclic aro-
matic hydrocarbons from soil with hot (subcritical) water and supercritical CO2.
J. Chromatogr. A, 975(1): 175–188.
Kubátová, A., Lagadec, A.J.M., Miller, D.J., Hawthorne, S.B. 2001. Selective extrac-
tion of oxygenates from savory and peppermint using subcritical water. Flavour
Frag. J., 16(1): 64–73.
Kubátová, A., Miller, D.J., Hawthorne, S.B. 2001. Comparison of subcritical water and
organic solvents for extracting kava lactones from kava root. J. Chromatogr. A,
923: 187–194.

Lamm, L.J., Yang, Y. 2003. Off-Line coupling of subcritical water extraction with sub-
critical water chromatography via a sorbent trap and thermal desorption. Anal.
Chem., 75(10): 2237–2242.
Liu, R.H. 2004. Potential synergy of phytochemicals in cancer prevention: Mechanism
of action. J. Nutr., 134: 3479S–3485S.
Luque de Castro, M.D., Jimenez Carmona, M.M., Fernandez Perez, V. 1999. Towards
more rational techniques for the isolation of valuable essential oils from plants.
Trends Anal. Chem., 18(11): 708–716.
Mazza, G., Cacace, J.E. 2005. Extraction of phytochemicals. US Patent application.
Mazza, G., Cacace, J.E., Kay, C.D. 2004. Methods of analysis for anthocyanins in plants
and biological fluids. J. AOAC Int., 87(1): 129–145.
Miller, D.J., Hawthorne, S.B. 2000. Solubility of liquid organic flavor and fragrance
compounds in subcritical (hot/liquid) water from 298 K to 473 K. J. Chem. Eng.
Data, 45: 315–318.
Ollanketo, M., Peltoketo, A., Hartonen, K., Hiltunen, R., Riekkola, M.L. 2002.
Extraction of sage (Salvia officinalis L.) by pressurized hot water and conven-
tional methods: Antioxidant activity of the extracts. Eur. Food Res. Technol.,
215(2): 158–163.
Ong, E.S., Len, S.M. 2004. Evaluation of pressurized liquid extraction and pressurized
hot water extraction for Tanshinone I and IIA in Salvia miltiorrhiza using LC and
LC-ESI-MS. J. Chromatogr. Sci., 42: 211–216.
Oomah, B.D., Mazza, G., Cui, W. 1994. Optimization of protein extraction from flax-
seed meal. Food Res. Int., 27(4): 355–361.
Ozel, M.Z., Clifford, A.A. 2004. Superheated water extraction of fragrance compounds
from Rosa canina. Flavour Frag. J., 19(4): 354–359.
Ozel, M.Z., Gogus, F., Lewis, A.C. 2003. Subcritical water extraction of essential oils
from Thymbra spicata. Food Chem., 82(3): 381–386.
Ozel, M.Z., Kaymaz, H. 2004. Superheated water extraction, steam distillation and
Soxhlet extraction of essential oils of Origanum onites. Anal. Bioanal. Chem.,
379(7–8): 1127–1133.
Palma, M., Piñeiro, Z., Barroso, C.G. 2001. Stability of phenolic compounds during
extraction with superheated solvents. J. Chromatogr. A, 921: 169–174.
Piñeiro, Z., Palma, M., Barroso, C.G. 2004. Determination of catechins by means of
extraction with pressurized liquids. J. Chromatogr. A, 1026: 19–23.
Rovio, S., Hartonen, K., Holm, Y., Hiltunen, R., Riekkola, M.L. 1999. Extraction of
clove using pressurized hot water. Flavour Frag. J., 14(6): 399–404.
Shotipruk, A., Kiatsongserm, J., Pavassnt, P., Goto, M., Sasaki, M. 2004. Pressurized
hot water extraction of anthraquinones from the roots of Morinda citrifolia
Biotechnol. Prog., 20: 1872–1875.
Soto Ayala, R., Luque de Castro, M.D. 2001. Continuous subcritical water extrac-
tion as a useful tool for isolation of edible essential oils. Food Chemistry 75:
109–113.
Wanasundara, P.K.J.P.D., Shahidi, F. 1997. Removal of flaxseed mucilage by chemical
and enzymatic treatments. Food Chem., 59: 47–55.
Yang, Y., Belghazi, M., Lagadec, A., Miller, D.J., Hawthorne, S.B. 1998. Elution of
organic solutes from different polarity sorbents using subcritical water. Journal
of Chromatography A 810(1–2): 149–159.
Zhang, R., Xu, Y.Q., Shi, Y. 2003. The extracting technology of flavonoids compounds.
Food and Machinery 1: 21–22.

7
Purification of Orange Peel Oil
and Oil Phase as Functional Foods
by Vacuum Distillation
Mércia de Fátima M. Bettini
CONTENTS
7.1 Introduction................................................................................................. 199
7.1.1 Steam Distillation........................................................................... 200
7.1.2 Simple Distillation and Fractional Distillation........................... 201
7.1.3 Vacuum Fractional Distillation..................................................... 201
7.1.3.1 Significant Reduction of Thermal Hazard................... 202
7.1.3.2 Greater Fractionation Efficiency.................................... 202
7.1.3.3 Enhanced Purity of Distillate......................................... 202
7.2 Industrial Scale Purification of Orange Peel Oil.................................... 203
7.2.1 Citrus Industry................................................................................ 203
7.2.2 Sweet Orange Oil............................................................................ 203
7.2.2.1 Purification of Orange Peel Oil...................................... 203
7.2.2.2 Method of Deterpenation—Vacuum
Fractional Distillation...................................................... 204
7.2.2.3 Folded Oils........................................................................ 204
7.2.2.4 Uses and Applications..................................................... 205
7.2.2.5 Experimental Results....................................................... 206
7.3 Conclusions.................................................................................................. 213
References.............................................................................................................. 215
7.1 Introduction
Distillation is a process of heating a substance until the most volatile constit-
uents change into the vapor phase, and then cooling the vapors to recover the
constituents in liquid form by condensation. The main purpose of distillation
is to separate a mixture into individual components by taking advantage of
their different level of volatilities. Distillation is one of the main methods of
extracting essential oils from plants. The percentage of each constituent in
199
the vapor phase usually depends on its vapor pressure at a certain tempera-
ture. The principle of vacuum distillation may be applied to substances such
as oils that would be damaged by overheating by the conventional method
(Ahmad, 2004). There are different methods of distillation, depending on the
desired product; some of these processes are described below.
7.1.1 Steam Distillation

Steam distillation is one of the common methods of distillation. Although
steam distillation appears to be the best method for extracting essential oils,
supercritical-CO2 extraction is becoming quite popular. Most aromathera-
pists believe that essential oils are only effective when extracted by steam
distillation and all other essential oils should be labeled with their extraction
methods, that is, cold-pressed oils, concretes, absolutes, etc. These prepara-
tions should not be confused with “true” essential oils.
In the steam distillation process, the material to be extracted is carefully
gathered and placed into a copper or stainless steel vat, known as the dis-
tillation chamber. Steam is then generated by heating (preferably low heat)
to build pressure within the distillation chamber causing the glands of the
plant to rupture and release its essence which is then carried by the steam
vapor. The steamy vapor is cooled and condensed in a coil, which is usually
submerged in refrigerated cold water. The water and essential oil separate
and the essential oils usually stay on top of the water due to their lower
densities. The water and essential oil are sent to a collection chamber where
the oil is easily skimmed off the top of the distillated water. The distillated
water may contain many low water-soluble components of the plant. These
by-products of the distillation process are known as “hydrosol” and can be
used for skin care, usually for children and the elderly.
When subjecting a plant material to heat or steam as in the distillation
process, the physicochemical properties of the material may be altered. The
thermolabile components are degraded and here the information given by
some authors with regard to the essential oils may be flawed. To achieve
effective aromatherapeutic benefits, it is important to recognize that the
extracted essential oils are not always in the same form when they were in
the plant. Therefore, when subjecting plant materials to heat or steam as
in the distillation process, the chemical structure within the essential oil
after distillation is different from that found in the original plant material.
For instance, chamomile is converted into the chemical azulene, which
is not found in the plant itself but is produced during distillation (Steam
Distillation, 2004).
When designing equipment for high vacuum process, parameters such
as pressure, pipe sizes and vessel sizes must be adequate to avoid exces-
sive vapor speed and pressure drops. The entire system must be free of
leaks (Hendrix et al., 1992) and compensation for the excess pressure drop

Purification of Orange Peel Oil and Oil Phase as Functional Foods 201
(2 or 3 mmHg) across the entire system must be incorporated into the

design process.
7.1.2 Simple Distillation and Fractional Distillation

If one desires to get a pure compound, the distillation process has to be
repeated many times and this will make it a cumbersome process. In prac-
tice, the vapors are condensed and revaporized in fractionating columns
connected sequentially in series. Equilibrium between liquid and vapor pre-
sumably occurs on each plate. However, it is not likely that equilibrium is
reached on each plate; therefore, it is imperative to calculate the plate effi-
ciency, an important design factor. The column is packed with an inert mate-
rial to promote contact between the liquid and the vapor. One of the most
important design factors in packed columns is to prevent channeling—a
condition where the vapors and liquid flow in opposite directions without
coming into contact.
The number of theoretical plates is determined under a specific set of
operating conditions, during the construction of the column. The effi-
ciency of design is referred to as the height equivalent per theoretical plate
(HETP).
When all the vapors that go up through the column condensed and flow
back down, the returning liquid is called the reflux. The total reflux consists
of the total product received from the outlet when no distillate is produced.
As soon as the product is drawn from the condensed vapors, the amount of
liquid returning through the column is reduced and produces changes to the
operating conditions of the column. If all the condensed vapors are drawn
off, then there will be no down flow to wash the upcoming vapors and in
effect the column would stop functioning all together and become just a long
pipe. However, most columns would retain some amount of reflux, and for
practical considerations, there would be some heat losses through radiation
and convection along the column (Hendrix et al., 1992).
7.1.3 Vacuum Fractional Distillation

Vacuum distillation is a method of distilling substances at temperatures
below their normal boiling point (standard atmospheric pressure). By reduc-
ing the pressure, a much lower distillation temperature will be obtained.
High vacuum distillation may be used for certain classes of chemical com-
pounds that decompose, polymerize, react, or are destroyed by conven-
tional distillation methods. Low cost per pound and high throughput may
be obtained on certain groups of compounds such as vitamins, epoxy res-
ins, highly concentrated pure fatty acids, plasticizers, fatty acid nitrogen
compounds, and a host of other heat-sensitive materials which may require
only deodorizing and decolorizing. The pharmaceutical industry, in their

continuing search for a new product with high and intermediate molecular
weights, finds that it is still an invaluable commercial practice to separate
products by molecular weight from excess reactants and catalysts.
7.1.3.1 Significant Reduction of Thermal Hazard

High vacuum distillation is a safe process to separate mixtures of organic
or silicon compounds, most of which cannot withstand prolonged heat-
ing above 250°C without excessive structural change or decomposition. As
opposed to conventional atmospheric and pressure-packed distillation tow-
ers, the high vacuum process utilizes the heat of condensation as a source of
radiant heat emission to heat the surface film on the evaporator. With short
residence time and lower distilling temperatures, thermal hazard to the
organic material is greatly reduced.
7.1.3.2 Greater Fractionation Efficiency

The best approach to a mechanical method of creating a uniform film thick-
ness with a fast-moving liquid is to feed the liquid to the center of a heated
spinning disc. By centrifugal action, the material spreads as a film across the
heated disc which intimately faces a large-condensing surface. The lighter
compound evaporates and condenses in a fraction of a second. The heavier
residues that are not evaporated slide off the outer edge of the disc into a
concentric residue collector and are discharged. The degree of separation is
a function of the molecular weight differences of the distilled mixture. The
greater the difference in the molecular weights, the purer the distillate. The
closer the molecular weights of the mixture are, the less efficient is the frac-
tionation process, resulting in the need for successive runs of the distillate.
7.1.3.3 Enhanced Purity of Distillate

Purity of the distillate also depends on the film thickness. Controlling
positive pressure and supply to the heated evaporator surface will usually
provide a uniform film throughout the distillation. The absence of air mol-
ecules in the high vacuum distillation column permits most of the distilling
molecules to reach the condenser with relatively few molecules returning
to the liquid film surface in the evaporator. Experimental results show a
relationship between the molecular weight and distillation temperature for
a broad range of different materials. There is no predictable temperature
at which distillation may occur; condensation begins whenever a suffi-
cient temperature difference occurs between the evaporator and condenser.
There is an infinite number of sets of operating conditions for every mate-
rial that is fed through the system. Since pressure is constant, the only vari-
ables are flow rate (governing film thickness) and evaporator temperature
(Myers Vacuum, 2004).

7.2 Industrial Scale Purification of Orange Peel Oil

The orange peel oil and essences are produced by the orange juice industry,
as will be explained in Sections 7.2.1 and 7.2.2.1. With the objective of prepar-
ing products rich in aromatic compounds, compounds that are very impor-
tant for flavor, the deterpenation process on those oils and essences will be
discussed in Sections 7.2.2.2 and 7.2.2.3.
7.2.1 Citrus Industry

Citrus juice processing generates large amount of wastes with almost 50% of
the fruit weight ending up as waste in the form of peel, segment, membranes,
rags, and seeds. Besides that, during juice concentration, volatile constituents
contained in the vapor phase are condensed by the aroma recovery systems.
The depletion of these flavoring components results in decreased juice qual-
ity, unless these compounds are reincorporated back into the juice, the nutri-
tional value will be diminished. The aroma recovery system consists of two
separate phases: essence oil (oil phase) and aqueous essences (water phase).
The average yield of these products is very low. All these products are rich in
aldehydes, esters, and other special volatile compounds. As the storage and
transportation of these products are expensive, the evaluation of the concen-
tration processes is of great interest (Bettini, 2004). It is very important that the
special light fractions and the concentration of flavoring volatile compounds
from the oil phase and peel oil be obtained by vacuum fractional distillation.
7.2.2 Sweet Orange Oil

Expressed sweet orange oil is primarily used for flavoring beverages, soft
drinks, ice cream, sweets, pharmaceutical preparations, and also perfumes.
It contains limonene, minor amounts of other monoterpene hydrocarbons,
linalool, terpineol, aliphatic alcohols, aldehydes, such as octanal, nonanal,
decanal, traces of the sesquiterpenic aldehydes, and sinensal, and methyl
anthranilate, coumarins, and waxes. For flavoring foods and beverages, the
so-called concentrated terpeneless oils or folded oils are preferred and are
commercially available.
7.2.2.1 Purification of Orange Peel Oil

Sweet orange oil is expressed from the fresh peels of ripe citrus fruits auran-
tium L. var. dulcis (Citrus sinensis Os beck) (fam. Rutaceae). The tree probably
originated from the far East between the Himalayas and south-west China.
It is now cultivated in the United States (California, Texas, and Florida),
throughout the Mediterranean region (southern Italy, Sicily, Spain, Algeria,
Tunisia, Morocco, Israel, Cyprus), South Africa, Brazil, and the West Indies.

The main producers of the equipment that expresses oil are the United
States and Cyprus, whereas the hand-pressed equipment is produced in
Guinea (Bradock, 1971). Many investigators have pointed out that the quality
of citrus oils is dependent upon many factors such as soil type, weather, the
method of extraction, and maturity of the fruit.
Citrus oils are located in the oval, balloon-shaped oil sacs or vesicles
located in the outer ring or flavedo of the fruit. Winton and Winton (Ahmad,
2004) described the exact location of these oil sacs in their discussion of the
microscopic structure of the flavedo of the orange.
To extract the oil from the peel of citrus fruits, the oil sacs must be ruptured
by either pressure or rasping. The methods used in Florida were investigated
by von Loesecke and Pulley (Wiley, 1982) and they revealed that extraction
method had an effect on the quality of the oil (Heath, 1986).
Cold-pressed citrus oils may be further processed to remove all or part
of the terpenes and sesquiterpenes. The resulting products are known as
terpenefree, terpeneless, or sesquiterpeneless oils. For products with low ses-
quiterpene concentrations, the term terpeneless is generally used. Citrus
oils from which terpenes have been removed are also called folded oils as
the remaining flavorful oxygenated compounds are more concentrated.
The degree of concentration is often calculated from the ratio of the princi-
pal constituent in the concentrated oil to that in the prime oil from which
it was made.
7.2.2.2 Method of Deterpenation—Vacuum Fractional Distillation

The bulk of terpeneless oils are produced by fractional distillation under
high vacuum (1–2 mmHg) so that the oil boils at a relatively low tempera-
ture to ensure minimum damage to the heat-sensitive constituents during
the long distillation period. At these pressures, the boiling points of the
constituents may be too low for efficient separation, but as the terpenes are
rarely separated individually, this is usually a design problem and reflux
balance can be readily solved if necessary. The aromatic vapors rise through
a fractionating column which enables fractions at defined boiling ranges to
be separated and collected. Depending on the nature of the starting mate-
rial and the desired character of the end product, the various fractions may
be reblended as necessary and any unwanted fractions rejected (Florida
Chemical Company Inc., 2004).
7.2.2.3 Folded Oils

Folded orange oil is the concentrated product obtained from the winter-
ized oil of fresh oranges. The process used will remove unneeded materi-
als while retaining the highly aromatic components. Folded oils are several
times more potent than the original oil and maintain the best fragrance. The
two primary commercial varieties of folded orange oil are 5-fold and 10-fold.

The higher the fold (distillation), the more concentrated is the finish product.
Stable emulsions are more easily formed with folded oils since they required
less oil to produce the desired flavor and fragrance levels (Florida Chemical
Company Inc.). The oil is primarily used as a flavoring agent for both foods
and beverages and pharmaceutical products and is 10 times stronger than
the usual orange oil.
Concentrated fractions with high contents of fragrant compounds such as
ethyl butyrate, valencene, aldehyde C 10, and others can be obtained. Their
smell and taste that come from the original esters and aldehydes of the raw
materials are very good. Cold-pressed orange and grapefruit oils contain
about 90% d-limonene. d-Limonene is a hydrocarbon and has the following
structure:
CH3
H2C CH3
d-Limonene boils at 178°C and is generally thought that d-limonene con-

tributes very little to citrus flavor. However, the oil flavor and taste can be
varied by adjusting the d-limonene concentration. In general, the term 5-fold
oil is used when starting with 5 lb of cold-pressed orange oil and distilling
4 lb; the remaining 1 lb is referred to as the fivefold oil. The folding of oils is
surrounded by secrecy and mysticism and various flavor houses have devel-
oped their own methods. Some use solvent extraction, usually about 70%
ethanol followed by chilling to separate the extracted oil.
Since d-limonene boils at about 27°C and 2 mmHg, and reliable mechani-
cal vacuum pumps are available, it is possible to fractionate d-limonene at
5–10 mmHg absolute pressure. This is probably the most common method
used today. There are some low-boiling point esters and aldehydes that are
distilled with d-limonene, and their separation and recovery are presently
the subject of research.
7.2.2.4 Uses and Applications

Folded orange oils are used as a food ingredient and also as a flavor enhancer
in beverages. They can be incorporated into new and existing formulations
to enhance fragrance and color. A wide array of industrial applications from

general purpose cleaning to air fresheners have been enhanced by the addi-
tion of folded orange oils (Florida Chemical Company Inc.).
Original citrus oils are characterized by the presence of large percentages
of terpenes (C10/H16) and smaller amounts of sesquiterpenes (C15/H24). Both
of them, original and folded oils, carry oxygenated compounds compris-
ing alcohols, aldehydes, ketones, acids, and esters, which are responsible
for the characteristic odor and flavor profiles. The terpenoid composition of
various original citrus oils is similar, and their principle component being
d-limonene. The terpenes possess little intrinsic odor or flavor value but
it would be incorrect to say that they have no flavoring effect. An original
citrus oil from which the terpenes have been removed is significantly flat-
ter and lacks the characteristic freshness associated with a complete peel
oil (Florida Chemical Company Inc., 2004). When part of their terpenes is
removed, folded oils will be obtained.
7.2.2.5 Experimental Results

Figure 7.1 illustrates the industrial production of essences, as oil and water
phases. In one of the stages of the evaporator, there is a column, where the
volatile compounds, the oil, and water phase are evaporated and recovered
in a tank.
7.2.2.5.1 General Description of the System

The Essence Recovery System is connected to TASTE evaporators (Thermally
Short Time Evaporator), which have the first juice stage in the first evapora-
tor effect (i.e., the first stage is the hottest effect or boiler steam effect). The
essence-bearing vapors are “boiled-off” with the water vapors in the first
stage. The vapors are condensed in the shell side of the second stage (sec-
ond effect). The essence-bearing vapors rise to the top of the shell side of
the second stage and through a series of baffles in the tube nest to remove
as much condensable water vapor as possible. The noncondensable portion
of these vapors is removed through the vent line at the top of tube, which is
connected to the barometric condenser with a bypass for the essence system.
This is also repeated in the next tube.
When the essence system is “on,” the noncondensable vapors are routed
to the essence system through remote-controlled air-operated valves, and
the vent line to the barometric condenser is shut off. The essence vapors
enter the essence system at the vent condensers. The vent condenser con-
denses approximately 90%–95% of the essence product. Cold water is used
as a condensing medium. The essence product (condensed) flows downward
through a single line to a dirt trap.
There are two lines running up from the trap, one line is the essence prod-
uct line running to the product chillers, the second line is reflux line, which
is used to control the strength of the essence. The amount of reflux flow is
dependent on the desired strength of the water phase essence. The reflux rate

Back press regulator
and strainer
Product and Ammonia surge
noncondensible cooler drum
Noncondensible
Water out
Dual pressure Sporian UMC-DA-5
Overhead Relief valve
condenser liquid level control
Reflux Metering
chamber valve
Essence recovery
column assay

Stripping col.
and reboiler
Condenser
Barometric
2 T/N
Scrubber
condenser
NH3 liquid out
NH3 liquid in
No 2 T/N vacuum
Essence vacuum Flow meter
500 gal
gauge Water out cold wall
valve tank
Column vaccum Ammonia surge
Atm. steam
gauge
Hot water to hot well

drum
Barometric isolation valve
Bottom dump
On Essence storage tank
Off Water
To hot well
Essence valve Bottom seal
tank
Water to
Purification of Orange Peel Oil and Oil Phase as Functional Foods
baro. cond.
Hot well Steam header

To essence vaccum gauge
FIGURE 7.1
207
Recovery system of water phase + oil phase in the orange juice industry.

is controlled only by the amount of product taken from the system. There is
a constant amount of essence product generated by the overhead condenser,
and the essence product that does not continue through the system flows
back to the tube nest as reflux.
The essence product flows from the dirt trap to the double circuit chiller-
condenser. This unit uses flooded NH3 refrigeration on the shell side. One
circuit of the chiller-condenser is used to chill the essence product, and the
other circuits are used to further condense the noncondensable vapors that
rise from the vent condenser.
The second circuit (noncondensable) of the chiller-condenser is connected
to the bottom of the refrigerated condenser. This condenser is a vertical shell
and tube heat exchanger using flooded NH3 on the shell side. The noncon-
densable vapors flow through the tubes in a final condensing stage. The
chilled product flows to the decanter assembly. The remaining noncondens-
able vapors flow upward and into the evaporator barometric condenser.
The product lines from the bottom of the scrubber and the final condenser
run to the decanter assembly located in the cold wall storage tank. The prod-
uct line from the bottom of the scrubber to the decanter assembly has a flow-
meter located in the line. This flow-meter measures the product flow into the
decanter assembly.
The essence system product flow is created by vacuum generated by the
evaporator barometric condenser. There is a continuous vacuum path from
the evaporator tube to the evaporator barometric condenser, through each
unit. The vacuum in the essence system is controlled by remote controlled
valves (Hendrix et al., 1992).
7.2.2.5.2 Purification Process of Orange Peel Oil and Orange Essence Oil

at Flavor Tec (Figure 7.2)
Peel oil, oil phase, and water phase are the by-products of the citrus-
processing plants. These products can be concentrated to obtain folded oils,
folded essences (in the case of the oil phase and water phase), besides the spe-
cial light fractions and terpenes. Figure 7.2 shows the flow diagram for the
processing of raw materials at Flavor Tec Ltd. As shown, the light fractions
and folded oils or essences are processed by vacuum fractional distillation.
As shown in the first step of Figure 7.2, the volatile compounds that will be
the raw materials for the FTNF (from the named fruit) aromas are obtained
from the natural fruit in the citrus-processing plant, as follows: the peel oil
comes from the peel in a special process of washing, cleaning, centrifuga-
tion, and dewaxing. The oil phase and water phase are also recovered by the
citrus juice process, as shown in Figure 7.1.
In the Flavor Tec process, these raw materials will feed the distillation
equipment to produce the folded oils or folded essences, plus terpenes and
light fractions by high vacuum fractional distillation (second and third steps
in Figure 7.2). By blending the different percentages of the folded oils, folded
essences, and light fractions, the FTNF aromas are produced at Flavor Tec.

Raw material (fruit)
Volatile compounds-peel oil, oil phase,

water phase
Distillation/fractional process
Folded oils/folded Terpenes Light fractions

essences
Selling
FTNF aromas
Analytical evaluations
Shelflife study Juice additon
Shelflife study
FTNF = From the named fruit
FIGURE 7.2
Citrus oils concentration at Flavor Tec., by distillation.
The percentage of each of the compounds will change according to the needs
of the final flavor, for example, freshness, sweetness, ripeness, etc. All the
distilled products are evaluated by physicochemical and sensory analytical
methods. After the analytical evaluations of all the obtained products are
completed, their shelflife at room temperature and cold store will be studied.
The shelflife of the FTNF aromas used in citrus juices will also be evaluated.
Based on these evaluations, it will be possible to guarantee the quality of
the aromas, as well their addition to the final feed. A typical industrial frac-
tional vacuum distillation plant is shown in Figure 7.3. In order to increase
the yield and quality of the final products at Flavor Tec., the distillator is
continuously fed with raw materials for 24 h a day.
Figure 7.4 shows the chromatographic profile of a 10-fold orange oil.
Figure 7.5 shows the profile of the orange terpene, obtained during the dis-
tillation process. The average yield of the terpenes on the process of a 10-fold
oil is about 90% and about 80% on the process of a 5-fold. The purity grade
of d-limonene is a minimum of 96% by high resolution gas chromatography
(HRGC). The different concentrations of each component of single, 5-fold,
10-fold oils, and terpene can be seen in Table 7.1.

FIGURE 7.3
Vacuum fractional distillation at Flavor Tec.
The values of the components as percentage area (% area) were analyzed

by HRGC, using the following analytical conditions:
Gas Chromatograph Shimadzu, 14BPFSC, 115/220 V AC (50 Hz/60 Hz)
and 2 kV A max capacity, detector: flame ionization; T = 220°C, injector:
split/splitless (split = 1:100); T = 200°C, Column: capillary—Shimadzu,
Type-Bonded phase, phase: CBP1 (nonpolar), size: 30 m × 0.25 mm, film
thickness: 0.25 μm, material: fused silica, temperature program = 40–190°C
(4°C/min). The % area (HRGC) of each identified component of the orange
essence oils: single-, 5-fold, and 10-fold products are listed in Table 7.2.
There are more aromatic or fragrant components in the essence oil as com-
pared to peel oil, as can be seen in Tables 7.1 and 7.2. It is therefore advisable
to remember that the peel oil is obtained from the orange peel while the
essence oil from the concentration of the orange juice, the later has more
fresh compounds than the former. Special attention might be given to the
valencene content in the folded oil phases. This component is very impor-
tant for the fruit taste characteristic. During the concentration process of
the essence oil, special light fractions can be obtained, if processed under

7
9 11
5
10
12
2
1
6
4
1 α-Pinene 4 Phelandrene 7 Linalol 10 Neral

2 Sabinene 5 -Limonene 8 Citronelal 11 Geranial
3 Mircene + octanal 6 Nonanal 9 Decanal 12 Valencene
FIGURE 7.4
Chromatographic profile of 10-fold orange oil (HRGC).
ideal conditions. Table 7.3 shows the concentration of one of these fractions,
obtained from the essence oil (oil phase). It is a very light fraction, because
it is rich with top note components, for example, ethyl butyrate and octanal,
as can be seen in Figure 7.6.
Special attention might also be given to the high level of the light compo-
nents, for example, hexanal and ethyl-butyrate, which are very important for
the top notes of a flavor. The FTNF aromas are produced by blending the
folded oils and oil phases plus the special light fractions, as show in Figure 7.2.
At Flavor Tec., this blending is done with different fractions, as show in
Figure 7.2. The FTNF aromas are produced by blending the folded oils and
folded oil phases. It depends on the desired characteristics of the final fla-
vor, for example, fruity, fresh, green, etc. As an example, Table 7.4 shows the
concentration of some components of two different FTNF aromas, called A
and B. They are products of Flavor Tec., after concentration of the raw mate-
rials and blending with light fractions. The main differences between the
aromas A and B are: A is fresher than B, as demonstrated by its ethyl butyr-
ate content, and B has more juice note, as shown in its valencene content.
These special characteristics of each component will give unique flavor notes
when added to the final food product.

3
5
1
2
4 7
6
8
1 α-Pinene 3 Mircene + octanal 5 -Limonene 7 Linalol

2 Sabinene 4 Phelandrene 6 Nonanal 8 Citronelal
FIGURE 7.5
Chromatographic profile of orange terpene (d-limonene) (HRGC).
TABLE 7.1
Area % by HRGC of the Identified Components on the Orange Peel Oils
in Several Concentrations
Evaluated Products (Area %)
Pick No. Components Single Oil 5-Fold Oil 10-Fold Oil Terpene
1 α-Pinene 0.49 0.31 0.13 0.53
2 Sabinene 0.32 0.20 0.13 0.43
3 Mircene + octanal 2.19 1.54 1.08 2.35
4 Phelandrene 0.15 0.10 0.05 0.16
5 d-Limonene 95.36 91.92 81.71 96.30
6 Nonanal 0.03 0.11 0.10 0.02
7 Linalol 0.38 1.01 2.08 0.13
8 Citronelal 0.04 0.13 0.35 0.01
9 Decanal 0.24 1.15 3.07 –
10 Neral 0.06 0.29 0.92 –
11 Geranial 0.10 0.50 1.51 –
12 Valencene 0.04 0.14 0.58 –

TABLE 7.2
% Area by HRGC of Some Components on the Orange Essences or Orange
Oil Phases in Several Concentrations
Evaluated Products (Area %)
Single 5-Fold Oil 10-Fold
Pick No. Components Oil Phase Phase Oil Phase
1 Hexanal 0.01 0.29 0.71
2 Ethyl butyrate 0.03 0.14 0.33
3 T-2-hexanal 0.01 0.02 0.06
4 α-Pinene 0.38 0.93 2.10
5 Sabinene 0.34 0.34 0.67
6 Mircene + octanal 2.17 1.46 2.30
7 Phelandrene 0.08 0.09 0.12
8 d-Limonene 94.57 85.75 68.20
9 Nonanal 0.10 0.09 0.14
10 Linalol 0.70 2.13 2.78
11 Citronelal 0.01 0.13 0.57
12 Decanal 0.30 1.21 3.70
13 Neral 0.11 0.54 2.04
14 Geranial 0.10 0.49 1.00
15 Valencene 0.23 2.31 4.08
7.3 Conclusions
Fractional distillation has been shown to be an efficient process. At high vac-
uum, it is a safer process from an operational point of view, but also much
gentler on the raw materials, because of the short residence time and low
distillation temperature. This results in better fragrant characteristics in the
final products. To obtain top-quality products, the raw materials, orange
TABLE 7.3
Area % by HRGC of the Main Components on the Light
Fraction of the Orange Oil Phase
Oil Phase Light
Pick No. Components Fraction (Area %)
1 Hexanal 5.38
2 Ethyl butyrate 2.54
3 T-2-Hexanal 0.41
4 α-Pinene 13.84
5 Sabinene 4.34
6 Mircene + octanal 12.76
7 Phelandrene 0.64
8 d-Limonene 57.51

4 6
1
2
5
3
8
1 Hexanal 3 T-2-hexanal 5 Sabinene 7 Phelandrene

2 Ethyl butyrate 4 α-Pinene 6 Mircene + octanal 8 -Limonene
FIGURE 7.6
Chromatographic profile of special light fraction of orange oil phase (HRGC).
TABLE 7.4
FTNF Orange Aromas (A and B): HRGC Data
Sample A Sample B
Pick No. Components (Area %) (Area %)
1 Acetaldehyde TR TR
2 Hexanal 0.27 0.10
3 Ethyl butyrate 0.18 0.05
4 T-2-hexanal 0.10 0.05
5 α-Pinene 1.23 0.74
6 Sabinene 0.60 0.28
7 Mircene + octanal 2.98 1.91
8 Phelandrene 0.17 0.09
9 d-Limonene 87.36 86.08
10 Nonanal 0.05 0.10
11 Linalol 1.02 1.70
12 Citronelal 0.12 0.17
13 Decanal 1.38 1.80
14 Neral 0.40 0.54
15 Geranial 0.52 0.80
16 Valencene 0.53 1.17

peel oil and oil phase, need to be well preserved from harvest until their use
in the process.
It is well known that single citrus oils and essences can come in large vol-
umes from the industry. The storage and transportation costs of these prod-
ucts are high, which is another reason why more interest is being shown
in the concentrated products. Research results have shown limonene to be
a good candidate for human clinical chemoprevention trials. Figures 7.4
through 7.6 with chromatograms and Tables 7.1 through 7.3 of the main
components that were obtained from this work at Flavor Tec. show that the
folded oils, essences, and the aromas besides their fragrant components also
maintain good levels of d-limonene concentration when added to juice and
other beverages. Although considerable differences have been observed with
these preparations, wide acceptance by sensory panels has been achieved.
It is very important to note that these folded oils and aromas are useful in
both aspects: (1) as functional foods, because of the limonene content; (2) its
taste and fragrance, because of their fragrant compounds such as esters,
aldehydes, etc. It is very important to bear in mind that the aroma is not a
curative but will, in general, enhance food products.
References
Ahmad, W. PE Chemical Engineering Review: Binary Distillation Weight and Mole
Fractions. Retrieved October 5, 2004. www.reviewpe.com/pe/samples/b6_7htm
Baser, K.H.C., Buchbauer, G. Essential Oils: Science, Technology and Applications, CRC
Press, 2010.
Bettini, M.F.M. Orange concentrate essences by fractional distillation. In: IFU
Symposium, 25, 2004. Stuttgart, Germany.
Bradock, R.J., Hendrickson, R., Kesterson, J.W. Florida Citrus Oils. Agricultural
Experiment Stations, Bulletin 749, 179 p., Gainesville, FL, 1971.
Florida Chemical Company Inc. Folded Orange Oil. Retrieved October, 2003. http://
www.floridachemical.com/msds/329700_Folded_Orange_Oils_MSDS.pdf
Heath, H.B., Reineccius, G. Flavor Chemistry and Technology. AVI Publishing Co.,
Westport, CT, 1986.
Hendrix, C.M. Jr., Hendrix, D.L., Redd, J.B. Process Procedures: Quality Control Manual
for Citrus Processing Plants. Florida, v. 2, Section 1, division B, 1992.
Kubeczka, K.H. Orange Oil Sweet: Essential Oils Analysis by Capillary Chromatography
and Carbon-13 NMR Spectroscopy. John Wiley, Canada, 1982.
Myers Vacuum, Molecular Distillation: The Unique Principle behind a Unique Process.
Retrieved November 3, 2004. www.myers-vacuum.com/mv1.html

8
Membrane Technology for
Production of Nutraceuticals
Marie-Pierre Belleville and Fabrice Vaillant
CONTENTS
8.1 Introduction................................................................................................. 217
8.2 Membrane Processes.................................................................................. 218
8.3 Implementation of Membrane-Based Separation Processes................ 221
8.3.1 Process Analysis.............................................................................223
8.3.2 Experimental Investigation...........................................................223
8.3.3 Modeling and Simulation..............................................................223
8.4 Application to Nutraceutical Production................................................ 224
8.4.1 Peptides............................................................................................ 224
8.4.2 Lipid-Soluble Compounds............................................................. 226
8.4.2.1 Carotenoids....................................................................... 226
8.4.2.2 Tocopherols....................................................................... 227
8.4.2.3 Phenolic Compounds...................................................... 227
8.4.2.4 Terpenoids......................................................................... 229
8.4.2.5 Other Compounds........................................................... 229
8.5 Conclusions.................................................................................................. 230
References.............................................................................................................. 230
8.1 Introduction
During the last decades, membrane technologies have emerged successfully
in a wide range of application areas (water desalination and treatment, food
processing industries, chemical industries, etc.) because they have shown huge
potential to replace conventional separation techniques. Membrane operations
can be used to achieve the different objectives of separation (i.e., concentra-
tion/clarification, purification, fractionation, extraction) but the coupling of a
reactor and a membrane can also lead to a more sustainable reaction-separa-
tion process. In addition, membrane techniques can be operated at low tem-
peratures (i.e., ambient or temperatures less than 50–80°C) and in continuous
mode which limits start up and shut down procedures and leads to consistent
product quality. Working at low temperatures permits to reduce the energy
217
consumption which can represent more than 50% of the separation cost and
preserves the qualities of heat-sensitive compounds. Therefore, membrane pro-
cesses are expected to be one of the most promising candidates for separation
and purification applications (downstream separation processes) involving
biomolecules such as nutraceutical compounds. Indeed, due to their favorable
impact on human health and prevention of certain diseases, the interest for
bioactive compounds has increased dramatically in recent years and achiev-
ing more sustainable production processes is one of the current challenges
of the industry. This chapter will thus focus on refining and purifying bio-
active compounds from natural products using m embrane-based separation
schemes. It is divided into three main sections: first a short presentation of the
most relevant membrane processes is given, then a theoretical approach for
membrane process implementation is developed and finally different applica-
tions to nutraceuticals production are reported.
8.2 Membrane Processes

During the 1960s–1970s, a large number of membrane processes based on
different separation principles have been developed. In all of them, the
membrane which can be considered as the heart of the process acts as a thin
permselective barrier or interface between two phases and the mass transfer
through the membrane occurs thanks to a driving force such as pressure,
concentration, electrical potential, or temperature gradient. A summary of
the characteristics of the most relevant processes for production of nutraceu-
ticals is given in Table 8.1.
Pressure-driven membrane processes (microfiltration [MF], ultrafiltration
[UF], nanofiltration [NF], and reverse osmosis [RO]) are now well estab-
lished for concentration, fractionation, and purification of liquid solutions
and water treatment (Mulder, 1996; Strathmann, 2011). MF is generally used
for fine particles or bacteria removal or turbidity reduction processes (van
Reis and Zydney, 2007; Cissé et al., 2011; Machado et al., 2012), whereas UF
is used for macromolecule concentration and fractionation (Díaz-Reinoso
et al., 2011). The main applications of NF are water treatment (Mondal and
Wickramasinghe, 2008), whey demineralization (Román et al., 2009), and
small organic molecules concentration (Tylkowski et al., 2011; Langevin
et al., 2012; Murakami et al., 2013). Regarding RO, this technique is mainly
used for water desalination (Peñate and García-Rodríguez, 2012) but it is
also widely used for the concentration of thermosensitive aqueous solutions
(Bogianchini et al., 2011; Gunathilake et al., 2014). Nevertheless, the combina-
tion of at least two pressure-driven membranes (MFT/RO, UF/NF, UF/RO,
etc.) permits to achieve better separation (Saidi et al., 2013; Torun et al., 2014;
Kamada et al., 2002).

Membrane Technology for Production of Nutraceuticals 219
TABLE 8.1
Membrane Separation Processes: Characteristics and Applications
Mass Transfer
Membrane Process Driving Force Mechanism Applications
Microfiltration Pressure gradient Convection Clarification and
cold-sterilization
Ultrafiltration Pressure gradient Convection Concentration, fractionation
of macromolecular solutions
Nanofiltration Pressure gradient Diffusion/ Concentration, purification of
convection small organic compounds,
separation of selected salts
Reverse osmosis Pressure gradient Solubilization/ Concentration/desalination
diffusion
Electrodialysis Electrical potential Ion exchange Separation of ions from water
gradient (Donnan and non-ionic solutes
exclusion)
Pervaporation Concentration Absorption/ Separation of mixtures of
gradient diffusion/ volatile liquids
desorption
Membrane distillation/ Partial pressure Evaporation/ Desalination, concentration
osmotic membrane gradient diffusion/
distillation condensation
Except RO, pressure-driven membrane process involves porous inorganic

or polymeric membranes which show different ranges of pore sizes—from
1 nm for NF membranes to 1.4 µm for MF membranes. In the case of UF,
the membranes are generally characterized by their molecular weight cut-
off which refers to the molecular weight of the lowest standard solute which
is 90% retained by the membrane. In such processes, the rejection is mainly
achieved by means of sieving mechanisms, although interactions between
the membrane surface and the solution can influence the separation, espe-
cially in NF where differences in diffusivity and solubility of solutes and
electrostatic interactions between the membrane surface groups and the ions
also play an important role in the separation efficiency (Nagy et al., 2011).
Regarding RO, the membranes used are nonporous and the mechanisms of
separation of species are based on the “solution-diffusion” model. In this
process, the ionic species are generally totally rejected by the membrane.
Small nonionic organic compounds which can be taken up by the mem-
brane are also rejected because they show a much lower mobility than water
molecules.
Among electrically driven membrane processes, electrodialysis is the
most widely used. It permits the selective separation of ionic species of a
solution thanks to an electrical potential gradient (Strathmann, 2010). In
practice, an electrodialysis unit consists of many flat ion exchange mem-
branes (cross-linked polymer matrices which have fixed charge groups to
which mobile ions with opposite charge [counter ions] are attached) forming

distinct channels where the solution to be treated and an extracting solu-

tion (a saline solution) flow, respectively. This membrane stack is placed
between electrodes and when an electrical current is applied, cations will
try to move toward the cathode whereas anions will try to move toward
the anode. However, only ions with the same charge of the counter ions are
thus free to move through the membrane, whereas the ions with the oppo-
site charge are almost totally rejected. At the outlet of the ED unit, the salt
concentration of the solution to be treated decreases while in the other com-
partment, the salt concentration increases. ED is mainly used to concentrate
electrolyte solutions or to dilute or deionized solutions (i.e., demineraliza-
tion of whey or grape must, etc.) (Strathmann, 2010). Nevertheless, the use
of porous membranes (micro or UF membrane) in the replacement of ion-
exchange ones inside the membrane stack allows extending the ED appli-
cation field to the separation of molecules of molecular weight exceeding
about 500 Da (Galier et al., 2005). Combining the separation mechanisms
of conventional membrane filtration (size) and electrophoresis (electrical
charge), such devices are thus suitable for the fractionation of valuable mol-
ecules from complex feed stocks (Firdaous et al., 2009; Langevin et al., 2012;
Husson et al., 2013).
Concentration-driven membrane processes are divided into two major
types of technologies: pervaporation (PV) and membrane contactors (MC).
In PV, species are separated from a liquid mixture by partial vaporization
through a nonporous membrane. The mass transport across the membrane
involves three successive steps: (i) selective absorption into the membrane
at the feed side of the membrane, (ii) selective diffusion through the mem-
brane, and (iii) desorption into the permeate side kept under vacuum.
The selectivity of the PV membrane is thus the key factor of the process.
Hydrophilic membranes are used for dehydration applications, whereas
hydrophobic ones allow organic compounds to be separated. The main
applications of PV are solvent dehydration (Chapman et al., 2008) but PV
can also be used for aroma recovery in food (Pereira et al., 2006), or when
PV is coupled to a reactor, it permits the continuous elimination of one of
the products, thus shifting the reaction equilibrium toward higher conver-
sion (Findrik et al., 2012).
In contrast to PV, MC involves porous membranes generally made not
only of hydrophobic materials that is, classical hydrophobic polymers
[polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE),
polyvinylidene fluoride (PVDF)], but also of modified ceramic membranes
(Brodart et al., 2003; Romero et al., 2006) and modified metallic membranes
(Hengl et al., 2007). The membrane acts as a passive barrier and as a medium
to bring a fluid into contact with another one without dispersion of these
phases into each other. Generally, each fluid flows on the opposite side of
the membrane and as only one or neither of them can penetrate inside the
porosity, one or two fluid/fluid interfaces are thus created at the entrance of
each pore. This family of processes includes the membrane distillation (MD),

the osmotic membrane distillation (OD) [also called osmotic evaporation

(OE)], the liquid–liquid extraction (LLE), and the supercritical fluid–liquid
extraction (SCLE). In MD which is the most studied process, the membrane
porosity is kept empty of liquid and when a driving force (i.e., a thermal
gradient) is applied, evaporation occurs and the volatile compounds dif-
fuse across the membrane and condense either in an aqueous phase: direct
contact membrane distillation (DCMD) or on a cold wall (air gap membrane
distillation), or are removed by a gas flow (vacuum membrane distillation
and sweep gas membrane distillation) (Wang and Chung, 2015). In OD, the
mass transfer also occurs through the immobile gas phase which fills the
membrane pores but the driving force is the water vapor pressure gradi-
ent induced by the difference in the water activity of the aqueous solutions
which flow along the membrane. The main application of MD is the pro-
duction of pure water but MD can also be used for the concentration of
nonvolatile components in aqueous solutions as far as the compounds to be
concentrated are not heat-sensitive (Ding et al., 2010). If it is not the case, OD
which can be operated at low temperatures is more suitable (Cassano et al.,
2011; Cissé et al., 2011); however, problems of corrosion and high production
costs due to the use of concentrated brine can reduce the interest of this
process (Kujawski et al., 2013).
8.3 Implementation of Membrane-Based Separation Processes

Despite the undoubted interest of membrane technologies for separation and
purification of biomolecules, implementation of these processes is not obvi-
ous. The performances are sometimes lower than expected, and the selectiv-
ity and stability for long-term application could not be achieved. These poor
performances mainly result in polarization (i.e., a mass-transfer boundary
layer formed in the vicinity of the membrane due to the accumulation or
depletion of solutes) and fouling (i.e., solutes or particles deposit onto the
membrane surface or into its pores), the major drawbacks of membrane pro-
cesses which have been widely studied over the past decades (Mulder, 1996;
Strathmann, 2010; Guo et al., 2012; Tijing et al., 2015). Although the issue
on these phenomena is still not well understood for all membrane process
types, the occurrences and control of polarization and fouling depend on the
membrane process, the solution to be treated (composition, concentration,
etc.), and of course the membrane properties (cut-off, permeability, chemical
and thermal stability, permselectivity, electrical resistance, etc.). To enhance
the efficiency of the membrane process, it is deemed necessary to deeply
characterize the solution to be treated, to know and understand the process
fundamentals in view to define the right operating parameter taking into
account the fouling propensity of membranes. In other words, it is necessary

Membrane
Optimized
process
Solution Operating
parameters
FIGURE 8.1
Key factors for the optimized membrane process.
to optimize the tryptic “solution-membrane-process,” which is the key factor

for the success of membrane implementation (see Figure 8.1).
The objective of the following section is thus to propose a theoretical
approach for design of membrane-assisted separation processes. As seen in
Figure 8.2, the global route involves three main steps: (i) process analysis,
(ii) experimental investigation at laboratory scale, (iii) modeling and simula-
tion with validation on a pilot scale.
Feed solution
Objective definition
characterization
Process analysis
Process and membrane
identification
Experimental investigation (lab scale)

Experimental (membrane selection, study of Technical
operating parameters effects) feasibility
Modeling and simulation Economic

Modeling and
Experimental investigation (pilote scale) viability
simulation
Model fitting
Dimensioning and investment
FIGURE 8.2
Methodology for the implementation of a membrane process.

8.3.1 Process Analysis

This step is certainly the most important one. It permits to select the suitable
separation process. Firstly, the objective of the separation should be clearly
defined since the choice of the membrane technology depends on it. It is also
important to know the characteristics and properties (nature, size, concen-
tration) of the species that should be separated as well as the global compo-
sition of the feed solution. The information is necessary to select the more
suitable membrane process or the best membrane coupling or membrane
hybrid processes. This choice based on the literature survey should also take
into account the main use of the final product, the disposal of the feed stock,
and the annual production target. The end use of the product will help to
define the expected added value of the product and the process selectivity
(i.e., rejection rate) that should be achieved, whereas the production target
is important to define the productivity threshold (i.e., permeation flux) that
guarantees the viability of the process.
8.3.2 Experimental Investigation

The objective of this step is firstly to verify the technical feasibility of the
selected membrane process. Different suitable membranes are character-
ized in a laboratory-scale plant. Their performances are then compared in
view to choose the best one in terms of membrane flux and selectivity. This
choice also takes into account their chemical, thermal, and mechanical resis-
tances which defined their lifetime and of course their price. At this stage if
the objectives of separation defined previously are achieved, it is possible to
conclude the feasibility of the process. In this case, further experiments are
carried out with the selected membrane varying the operating conditions.
These experiments thus permit to determine the necessary information for
mass-transfer modeling and process optimization.
8.3.3 Modeling and Simulation

The objective of this last step is to propose a first design of the separation
unit and to conclude the process viability. Starting from the few experimen-
tal set of data obtained on the laboratory scale, suitable models are identified
from literature survey, parameterized, and implemented. The obtained data
are then used for simulation calculation of a preindustrial unit. However,
before concluding about the viability of the process, calculated data and
experimental data are required to be compared. For this purpose, experi-
mental investigations should be done on the pilot scale. If significant differ-
ences are observed, the parameters should be adjusted and new calculation
data should be done until to obtain a good agreement between calculated
data and experimental data. Finally, if the economic viability of the process
is checked, investments can be developed.

8.4 Application to Nutraceutical Production

Membrane technologies are generally soft process that allows separation at
lower temperatures and in most cases without phase changes. Therefore,
they are of very high interest for application in which improved taste, flavor,
and color are required but also for increasing absorption and bioavailability
of functional foods, nutraceuticals, and health supplements. The size range
and some properties of the main bioactive compounds found in food sources
are presented in Table 8.2. According to their physical properties, these com-
pounds can be separated selectively using the appropriate membrane pro-
cess. A review of membrane systems that have been used for each family of
bioactive compounds is presented below.
8.4.1 Peptides
Bioactive peptides are most often obtained from hydrolysate of protein-rich
by-products of food industries (marine, dairy, etc.). Therefore, food-grade
bioactive peptides come often in complex matrices of hydrolyzed protein
fractions and require further isolation and purification. Even though, as a pre-
liminary step, classical UF allows to separate larger peptides from the smaller
ones, the main problem is the similarity in size among the numerous peptide
species. Additionally, the high fouling capacity of protein hydrolysate makes
TABLE 8.2
Size Range of Some Bioactive Compounds from Food Products
Structures Size Range (Da) Main Properties
Peptides 700–20,000 Charged
Carotenoids (lycopene, lutein, and other carotenoids) 550–600 Apolar
Fatty acids (polyunsaturated fatty acids …) 280 Apolar
Phytosterol and sterol esters (tocotrienols, tocopherol) 400–450 Apolar
Phenolic compounds
Condensed tannins 1500–4000
Flavonoids and other simple phenolics 200–1000
Stilbenzenes (resveratrol) 200–400
Phenolic acids 180–400 Charged
Organosulfur compounds 200–400
Aliin, allicin, etc. 150–200
Isothiocyanates (cruciferous vegetables) 70–200
Terpenoids monoterpenes in citrus 130–150 Volatiles
Phytoestrogen (daidzein, genestein, etc.) 250–600
Oligosaccharides 5000–10,000

standard pressure-driven membrane processes less efficient. The combina-

tion of membrane separation with enzyme treatment can enhance separation
by diminishing the influence of larger molecules on fouling. Additionally,
coupling the enzymatic hydrolysis with membrane system has several advan-
tages over batch enzymatic treatment, as enzyme is partially reused and sub-
strate is retained, while the product of the reaction permeates more easily
through the membrane. Cross-flow UF have been widely used to obtain food
ingredient or extracts with enhanced b iological and functional properties
from milk protein hydrolysates (Claudia et al., 2013), marine resources, and
plant sources such as rapeseed (He et al., 2013), soybean (Moure et al., 2006),
alfalfa leaf (Xie et al., 2008), pea whey (Gao et al., 2001), and wheat gluten
(Wang et al., 2006).
But when more purified extracts are needed, a more efficient fractionation
of peptides requires the setting up of technological alternatives based on
other properties than their molecular weight such as their charge and their
electrostatic interactions between themselves and the membrane.
Charged UF membranes have been used implementing various succes-
sive filtration steps at different pH corresponding to different charged
states of the membrane and of the peptides to improve separation. But NF
appears to be a better alternative to separate charged peptides through a
combination of size exclusion and charge mechanism (Futselaar et al.,
2002; Butylina et al., 2006). Because of the dimension of pores, of only one
order of magnitude larger than the size of ions, NF membranes have natu-
rally a slightly charged surface, enhancing selectivity based on both size
exclusion and charge interactions with membrane surface according to the
Donan theory. Therefore, they are particularly efficient for the fractionation
of peptides of low-molecular weight (<1000 Da) from protein hydrolysates.
During NF process, the selectivity of the separation depends not only on
charge characteristics and size of solutes, but also on the characteristics
of the initial solution (solute concentration, shape of biomolecules, pH of
solution, presence of competitive ions, salt concentration, and type of salt),
on membrane characteristics (membrane pore size, membrane charges in
term of sign and density, surface roughness, porosity, hydrophobicity, or
hydrophilicity of layer material), and finally on hydrodynamic conditions
(transmembrane pressure, cross-flow velocity). Therefore, the complexity of
NF systems requires numerous test trials using different materials and pro-
cess conditions. Nonetheless, if selectivity can be modulate, concentration
polarization and fouling still remain the main limitation of pressure-driven
process. Alternatively, the application of external electrical field acting as a
second driving force could improve considerably the efficiency of separa-
tion. Electro-filtration or combined process of NF and even UF with elec-
trodialysis represents a technological breakthrough to reduce fouling and
polarization and enhance separation of charged bioactive peptides (Bazinet
and Firdaous, 2009; Bazinet and Firdaous, 2013).

8.4.2 Lipid-Soluble Compounds

8.4.2.1 Carotenoids
Carotenoids can be found soluble in crude oils but also in the form of emul-
sion in aqueous solution such as fruit juices. In each case, the processes to be
implemented must be different as the properties of the initial solution are also
different. For the treatment of crude oil, hydrophobic or slightly h ydrophobic
membranes are usually used and most often with preconversion of fatty
acids into methyl esters or use of organic solvent. Concentration of carot-
enoids and tocophereol from esterified crude oil has been achieved using
flat sheet NF membranes of polyethersulfone (PES) with MCWO 200–400 Da
(Chiu et al., 2009). In these assays, the oil methyl esters flux was between
7.5 and 10 L m−2 h−1 with a retention of β-carotene of 60%–80% at 40°C and
2.5–2.75 MPa which led to conclude that the process may be practical at an
industrial level. Esterification allows reducing oil viscosity and transforming
triglycerides into smaller and more soluble fatty acids methyl ester yield-
ing more efficient separation of carotenoids and tocopherol. Nonetheless, the
conversion is irreversible and further purification step is required to remove
residual methyl esters which are not edible (Othman et al., 2010).
The use of solvent (commonly hexane) increases also considerably the effi-
ciency of the separation due to the higher permeability of lipids through
the membrane (Darnoko and Cheryan, 2006). The main problem is the use
of solvent stable membranes and also the resulting health and environmen-
tal hazards. Ethanol extraction is more safe and ecofriendly and has been
used mainly to obtain an enriched extract of lutein and zeaxanthin from
corn residues by coupling UF with MWCO of 1000 Da to remove residual
proteins and NF with 300 Da to concentrate UF permeate up to 7.6 times
(Tsui and Cheryan, 2007). A more safe, ecofriendly, and efficient purifica-
tion of carotenoids can be achieved by coupling supercritical fluid extraction
with membrane separation. Once, lipids are solubilized into supercritical
carbon dioxide, separation of compounds of low-molecular weight such as
carotenoids can be achieved, although it requires the use of membranes that
can resist the physicochemical properties of the critical point of liquid car-
bon dioxides (18–31 MPa and 40–43°C) (Sarrade et al., 1998). Nonetheless, the
coupling of supercritical extraction with cross-flow membrane is showing
great promise for the selective separation of lipids, although different chal-
lenges still need to be overcome (Sarrade et al., 2002; de Moura et al., 2007;
Ruivo et al., 2008).
In the case of the highly bioactive carotenoids compounds that can be
found in fruit juice, for instance, separation can be much easier although
initial concentration is much lower than in oils. In juices, carotenoids are
found in heterogeneous systems consisting of immiscible liquid completely
dispersed in the aqueous phases. This is the case in some fruit juices where
carotenoids are embedded in small droplets of 0.1 μm diameter or higher sus-
pended in water producing an oil-in-water emulsion. In this case, an effective

separation of droplets can be achieved through MF with hydrophilic mem-

branes showing pore diameter inferior to droplets diameter. The pretreat-
ment and hydrodynamic conditions must preserve the integrity of emulsion
and low pressures and temperature will favor the separation (Kocherginsky
et al., 2003). For example, carotenoids can be concentrated up to 10-fold in
the retentate after simple MF of water melon juice or cashew apple juice, on
ceramic membranes with 0.2 μm pore size at 0.15 MPa (Vaillant et al., 2005;
de Abreu et al., 2013). Carotenoids extract can be further purified following a
sieving or centrifugation step to separate insoluble solids.
8.4.2.2 Tocopherols
Tocopherols (Vitamin E), the natural antioxidants present in crude veg-
etable oils, can also be separated from triacylglycerol and fatty acids simi-
lar to carotenoids. Some authors have succeeded in enriching permeate in
tocopherols after UF of different vegetable oils (peanuts, soybean, sunflower)
on dense and composite polymeric membranes with silicone as the active
layer at high transmembrane pressure (3–5 MPa) and temperature (20–40°C)
(Subramanian et al., 1998). Because the polarity of tocopherols differs slightly
from fatty acids which are more polar, these compounds are selectively more
rejected by the membrane and tocopherols permeate selectively. Nonetheless,
enrichment was poor (15%–30%) and higher purification appeared to require
the use of solvent combined with membrane filtration. Preferential perme-
ation of tocopherols was obtained in relation with fatty acids and their esters
after dilution with hexane and using a dense polymeric membrane with
polyamide active layer (Nagesha et al., 2003). Nonetheless, this experiment
has been performed only on a laboratory scale and the presence of residual
hexane should limit the posterior use of extracts in the food industry.
8.4.2.3 Phenolic Compounds

The recovery of biologically active compounds from the by-product from fruit
industries has attracted considerable economical interest in recent years. In
this sense, the most emblematic sector is the wine industry which has suc-
ceeded in producing a large variety of functional food and nutraceuticals
using all the by-products generated by the activity. The recovery of natural
grape extracts has been implemented successfully, mostly thanks to membrane
processes. Membrane separation processes have been utilized in the concen-
tration and separation of phenolic compounds due to greater separation effi-
ciency, higher purity, mild operation parameters, avoidance or reduced use of
solvents, and easy scaling-up. Inclusively, all pressure-driven processes have
been used such as MF to remove insoluble solids (Nawaz et al., 2006), UF to
remove larger molecules (proteins, polymers, and other impurities) (Kalbasi
et al., 2007), NF often with diafiltration to purify further and concentrate, and
RO to remove selectively water and minerals (Díaz-Reinoso et al., 2009). In all

cases, the main limitation is the fouling of membrane which compromises

process performance in terms of permeate flux, rejection rate, and concentra-
tion factor. A large number of examples of the applications of pressure-driven
membrane processes to the fractionation and concentration of phenolics com-
pounds have been published in recent years; some examples are presented in
Table 8.3. As always in membrane processes, there is no common results and
optimum hydrodynamic conditions varied greatly according to the character-
istics of the feed. Basically, phenolic compounds can be fractionated and con-
centrated according to their molecular weight on membranes accordingly to
their MWCO but filtration performances and final purity will always depend
on the characteristics of the impurities and the other undesirable molecules
that accompany the target molecules. The fouling of membrane is most often
due to polysaccharides, polypeptides, or larger molecules. Phenolic com-
pounds, except condensed tannins, have lower molecular weight and thus
permeate more easily through the UF/NF membranes. Although it is often
neglected, the implementation of simple pretreatment such as enzyme treat-
ment, centrifugation, or prefiltration can drastically improve the membrane
separation process. Like in the case of other bioactive compounds, the use
of solvents can enhance separation, but except for ethanol and supercritical-
CO2, the health and environmental problems inherent to the use of solvent
TABLE 8.3
Examples of Pressure-Driven Process Applied to the Treatment of Fruit Residues
Membrane
Product Process Conditions Permeate Retentate Reference
Almond skin UF 50 KDa Proanthocy- Proanthocy- Prodanov
anidines anidines et al. (2008)
pentamers oligomer (up
to decamer)
Grape UF/NF 250–100 Da High- Diaz-Renoso
pomace molecular et al. (2009)
phenolic
compounds
Grape seed MF 0.22 and 0.45 μm Seed Nawaz et al.
solvent/EtOH polyphenols (2006)
(50%–50%)
Grape juice UF PVDF flat sheet Monomeroc Polymeric acy Kalbasi
10–100 KDa acy et al. (2007)
Coffee brew UF-diafiltration 1 KDa Low- Rufian-
molecular Henares
melanoidins et al. (2007)
Olive mill UF/NF PVDF 1 KDa Tyrosol Cassano
wastewaters et al. (2013)
Orange press UF/NF/OD PES 1KDa Flavonoids/ Cassano
liquor acy et al. (2014)

remain. The supercritical-CO2 extraction has been applied, for instance, to

cocoa seeds with ethanol as the cosolvent. The final liquid extract was nano-
filtered on the polymeric membrane at 40°C and 8 MPa (Sarmento et al., 2008).
In this case, the rejection of polyphenols reached 80%–95% with a significant
permeate flux.
8.4.2.4 Terpenoids
Terpenoids are abundant in some by-products from fruit industries such as
citrus essential oils. Oxygenated terpenes can be collected preferentially using
MCs. Separation is based on the difference in polarity between very apolar
terpenes hydrocarbons and slightly more polar oxygenated terpenoids using
an appropriate solvent, such as hydro-alcoholic mixture preferentially etha-
nol. In this LLE, the solvent and the product are separated by a hydrophobic
porous membrane that allows stabilizing an interface between both solutions,
enabling the transfer of compounds by diffusion. The main limitation is to
avoid phase mixing, emulsification, and consequently the destabilization of
the interface. The process is therefore performed at low pressure, always below
breakthrough pressure (often <50 kPa), and at relatively low cross-flow veloc-
ity (68 < Re < 649) and requires the use of highly hydrophobic membranes
like PP, PTFE, or modified hydrophobic alumina membrane, all with rela-
tively small pores (below 0.4 μm). Actually, the higher the breakthrough pres-
sure which depends on hydrophobicity and pore size, the more stable is the
interface during the process and it allows better hydrodynamic conditions to
enhance mass-transfer coefficients. With existing membranes, mass-transfer
coefficients up to 1.08 × 10−6 m s−1 have been reached (Dupuy et al., 2011), but
there is no doubt that the development of more appropriated membrane could
improve this value, making this process highly efficient in terms of time and
energy savings with respect to the conventional extraction process.
8.4.2.5 Other Compounds

Numerous other compounds with bioactive potential can be separated
through membrane processes. This is the case for specific carbohydrate such
as oligosaccharides like fructooligosaccharides or xylosaccharides that are
mainly used as soluble dietary fiber with important prebiotic effects. To
obtain different fractions of oligosaccharides, in general two stages of UF
and NF system are implemented allowing the fractionation of low-molecular-
weight saccharide mixtures (Minhalma et al., 2006; Akin et al., 2011). The NF
is also often combined with diafiltration to improve purity of the extracts
and RO to concentrate thermally the UF permeate stream. In general, all the
results show a high potential of NF for the separation of low-molecular-weight
oligosaccharides (Goulas et al., 2002; Akin et al., 2011). Phytoestrogenes have
been also selectively separated by NF membranes and concentrated by RO
as shown previously by Dudziak and Bodzek (2010). Examples of membrane

separation of organosulfur compounds from garlic, onion, or cruciferous

vegetable have not been published so far.
8.5 Conclusions
Since the last decades, membrane science and technology offer new options
and great opportunities for the production of nutraceuticals from various
food products or by-products. Indeed membrane operations are good can-
didates for thermo-sensitive compounds processing since they can be oper-
ated at low temperatures without phase change or the use of nonfriendly
environmental solvent. In addition, these processes are often more efficient
than the conventional ones in terms of energy saving, separation capacity
and selectivity, environmental impact, and capital investments in particular
when two or more membrane operations are associated or integrated into
existing industrial processes. Nevertheless, sometimes performances are not
as high as expected mainly due to membrane fouling or in some cases, to
membrane selectivity or membrane resistance. Improvements in material
science could help overcome some of these problems but the more important
is to design and develop innovative membrane-based integrated processes
in view to develop more sustainable and competitive industries. This is the
main challenge of membrane engineering for the future.
References
Akin, O., Temelli, F., Köseoğlu, S. 2001. Membrane applications in functional foods
and nutraceuticals. Critical Reviews in Food Science, 52: 347–371.
Akin, O., Temelli, F., Köseoǧlu, S. 2011. Membrane Applications in Functional Foods
and Nutraceuticals. Critical Reviews in Food Science and Nutrition, 52: 347–371.
Bazinet, L., Firdaous, L. 2009. Membrane processes and devices for separation of bio-
active peptides. Recent Patents on Biotechnology, 3: 61–72.
Bazinet, L., Firdaous L. 2013. Separation of bioactive peptides by membrane pro-
cesses: Technologies and devices. Recent Patents on Biotechnology, 7: 9–27.
Bogianchini, M., Cerezo, A.B., Gomis, A., López, F., García-Parrilla, M.C. 2011.
Stability, antioxidant activity and phenolic composition of commercial
and reverse osmosis obtained dealcoholised wines. LWT—Food Science and
Technology, 44: 1369–1375.
Brodart, F., Romero, J., Belleville, M.P. et al. 2003. New hydrophobic membranes for
osmotic evaporation process. Separation and Purification Technology, 32: 3–7.
Butylina, S., Luque, S., Nyström, M. 2006. Fractionation of whey-derived peptides
using a combination of ultrafiltration and nanofiltration. Journal of Membrane
Science 280: 418–426.

Cassano, A., Conidi, C., Drioli, E. 2011. Clarification and concentration of pome-
granate juice (Punica granatum L.) using membrane processes. Journal of Food
Engineering, 107: 366–373.
Cassano, A., Conidi, C., Giorno L., Drioli, E. 2013. Fractionation of olive mill waste-
waters by membrane separation techniques. Journal of Hazardous Materials,
248–249: 185–193.
Cassano, A., Conidi, C., Ruby-Figueroa, R. 2014. Recovery of flavonoids from orange
press liquor by an integrated membrane process. Membranes, 4: 509–524.
Chapman, P.D., Oliveira, T., Livingston, A.G., Li, K. 2008. Membranes for the dehy-
dration of solvents by pervaporation. Journal of Membrane Science, 318: 5–37.
Chiu, M.C., de Morais Coutinho, C., Gonçalves, L.A.G. 2009. Carotenoids concentra-
tion of palm oil using membrane technology. Desalination, 245: 783–786.
Cissé, M., Vaillant, F., Bouquet, S. et al. 2011. Athermal concentration by osmotic
evaporation of roselle extract, apple and grape juices and impact on quality.
Innovative Food Science and Emerging, 12: 352–360.
Cissé, M., Vaillant, F., Soro, D., Reynes, M., Dornier, M. 2011. Crossflow microfiltration
for the cold stabilization of roselle (Hibiscus sabdariffa L.) extract. Journal of Food
Claudia, M., Francisco, R., Ayoa, F. 2013. Advancements in the fractionation of milk
biopeptides by means of membrane processes. In Bioactive Food Peptides in
Health and Disease, ed. B. Hernández-Ledesma and C.-C. Hsieh, pp. 241–266.
Rijeka, Croatia: In Tech.
Darnoko, D., Cheryan, M. 2006. Carotenoids from red palm methyl esters by nanofil-
tration. Journal of the American Oil Chemists’ Society, 83, 365–370.
de Abreu, F.P., Dornier, M., Dionisio, A.P., Carail, M., Bolzan, A. 2013. Cashew apple
(Anacardium occidentale L.) extract from by-product of juice processing: A focus
on carotenoids. Food Chemistry, 138: 25–31.
de Moura, J.M.L.N., Gonçalves, L.A.G., Sarmento, L.A.V., Petrus, J.C.C. 2007.
Purification of structured lipids using SC-CO2 and membrane process. Journal
of Membrane Science, 299: 138–145.
Díaz-Reinoso, B., Moure, A., Domínguez, H., Parajó, J.C. 2009. Ultra- and nanofiltra-
tion of aqueous extracts from distilled fermented grape pomace. Journal of Food
Díaz-Reinoso, B., Moure, A., Domínguez, H., Parajó, J.C. 2011. Membrane concen-
tration of antioxidants from Castanea sativa leaves aqueous extracts. Chemical
Engineering Journal, 175: 95–102.
Ding, Z., Liu, L., Liu, Z., Ma, R. 2010. Fouling resistance in concentrating TCM extract
by direct contact membrane distillation. Journal of Membrane Science, 362:
317–325.
Dudziak, M., Bodzek, M. 2010. A study of selected phytoestrogens retention by
reverse osmosis and nanofiltration membranes—The role of fouling and scal-
ing. Chemical Papers, 64: 139–146.
Dupuy, A., Athes, V., Schenk, J., Jenelten, U., Souchon, I. 2011. Solvent extraction of
highly valuable oxygenated terpenes from lemon essential oil using a poly-
propylene membrane contactor: Potential and limitations. Flavour and Fragrance
Journal, 26: 192–203.
Findrik, Z., Németh, G., Vasić-Raćki, D., Bélafi-Bakó, K., Csanádi, Z., Gubicz, L. 2012.
Pervaporation-aided enzymatic esterifications in non-conventional media.
Process Biochemistry, 47: 1715–1722.

Firdaous, L., Dhulster, P., Amiot, J. et al. 2009. Concentration and selective separation
of bioactive peptides from an alfalfa white protein hydrolysate by electrodialy-
sis with ultrafiltration membranes. Journal of Membrane Science, 329: 60–67.
Futselaar, H., Schonewille, H., van der Meer, W. 2002. Direct capillary nanofiltration—
A new high-grade purification concept. Desalination, 145: 75–80.
Galier, S., Roux de Balmann, H. 2005. Electrophoretic membrane contactors. Chemical
Engineering Research and Design, 83: 268–275.
Gao, L., Nguyen, K.D., Utioh, A.C. 2001. Pilot scale recovery of proteins from a
pea whey discharge by ultrafiltration. LWT—Food Science and Technology, 34:
149–158.
Goulas, A.K., Kapasakalidis, P.G., Sinclair, H.R., Rastall, R.A., Grandison, A.S. 2002.
Purification of oligosaccharides by nanofiltration. Journal of Membrane Science,
209: 321–335.
Gunathilake, K.D.P.P., Yu, L.J., Rupasinghe, H.P.V. 2014. Reverse osmosis as a potential
technique to improve antioxidant properties of fruit juices used for functional
beverages. Food Chemistry, 148: 335–341.
Guo, W., Ngo, H-H., Li, J. 2012. A mini-review on membrane fouling. Bioresource
Technology, 122: 27–34.
He, R., Girgih, A.T., Malomo, S.A., Ju, X. et al. 2013. Antioxidant activities of enzy-
matic rapeseed protein hydrolysates and the membrane ultrafiltration frac-
tions. Journal of Functional Foods, 5: 219–227.
Hengl, N., Mourgues, A., Pomier, E. et al. 2007. Study of a new membrane evaporator
for the concentration of heat sensitive aqueous solutions. Journal of Membrane
Science, 289: 169–177.
Husson, E., Araya-Farias, M., Gagné, A., Bazinet, L. 2013. Selective anthocyanins enrich-
ment of cranberry juice by electrodialysis with filtration membrane: Influence of
membranes characteristics. Journal of Membrane Science, 448: 114–124.
Kalbasi, A., Cisneros-Zevallos, L. 2007. Fractionation of monomeric and polymeric
anthocyanins from Concord grape (Vitis labrusca L.) juice by membrane ultra-
filtration. Journal of Agricultural and Food Chemistry, 55: 7036–7042.
Kamada, T., Nakajima, M., Nabetani, H., Saglam, N., Iwamoto, S. 2002. Availability
of membrane technology for purifying and concentrating oligosaccharides.
European Food Research and Technology, 214: 435–440.
Kocherginsky, N.M., Tan, C.L., Lu, W.F. 2003. Demulsification of water-in-oil emul-
sions via filtration through a hydrophilic polymer membrane. Journal of
Membrane Science, 220: 117–128.
Kujawski, W., Sobolewska, A., Jarzynka, K., Güell, C., Ferrando, M., Warczok, J. 2013.
Application of osmotic membrane distillation process in red grape juice con-
centration. Journal of Food Engineering, 116: 801–808.
Langevin, M.-E., Roblet, C., Moresoli, C., Ramassamy, C., Bazinet, L. 2012. Comparative
application of pressure- and electrically-driven membrane processes for isola-
tion of bioactive peptides from soy protein hydrolysate. Journal of Membrane
Science, 403–404: 15–24.
Machado, R.M.D., Haneda, R.N., Trevisan, B.P., Fontes, S.R. 2012. Effect of enzymatic
treatment on the cross-flow microfiltration of açaí pulp: Analysis of the fouling
and recovery of phytochemicals. Journal of Food Engineering, 113: 442–452.
Minhalma, M., Beal, L.L., Catarino, I., Mateus, M., de Pinho, M.N. 2006. Optimization
of saccharide fractionation using nanofiltration/ultrafiltration. Desalination,
199: 337–339.

Mondal, S., Wickramasinghe, S.R. 2008. Produced water treatment by nanofiltration

and reverse osmosis membranes. Journal of Membrane Science, 322: 162–170.
Moure, A., Domínguez, H., Parajó, J.C. 2006. Antioxidant properties of ultrafiltration-
recovered soy protein fractions from industrial effluents and their hydroly-
sates. Process Biochemistry, 41: 447–456.
Mulder, M. 1996. Basic Principles of Membrane Technology, 2nd edition. Kluwer
Academic Publishers, Dordrecht, 554 pp.
Murakami, A.N.N., Amboni, R.D.M.C., Prudêncio, E.S. et al. 2013. Concentration
of biologically active compounds extracted from Ilex paraguariensis St. Hil. by
nanofiltration. Food Chemistry, 141: 60–65.
Nagesha, G.K., Subramanian, R., Sankar, K.U. 2003. Processing of tocopherol and FA
systems using a nonporous denser polymeric membrane. Journal of the American
Oil Chemists’ Society, 80: 397–402.
Nagy, E., Kulcsár, E., Nagy, A. 2011. Membrane mass transport by nanofiltration:
Coupled effect of the polarization and membrane layers. Journal of Membrane
Science, 368: 215–222.
Nawaz, H., Shi, J., Mittal, G.S., Kakuda, Y. 2006. Extraction of polyphenols from grape
seeds and concentration by ultrafiltration. Separation and Purification Technology,
48: 176–181.
Othman, N., Manan, A., Alwi, S.R.W., Sarmidi, M.R. 2010. A review of extraction
technology for carotenoids and vitamin E recovery from palm oil. Journal of
Applied Science, 10, 1187–1191.
Peñate, B., García-Rodríguez, L. 2012. Current trends and future prospects in the
design of seawater reverse osmosis desalination technology. Desalination, 284:
1–8.
Pereira, C.C., Ribeiro Jr., C.P., Nobrega, R., Borges, C.P. 2006. Pervaporative recovery
of volatile aroma compounds from fruit juices. Journal of Membrane Science, 274:
1–23.
Prodanov, M., Garrido, I., Vacas, V. et al. 2008. Ultrafiltration as alternative purifica-
tion procedure for the characterization of low and high molecular-mass pheno-
lics from almond skins. Analytica Chimica Acta, 609: 241–251.
Román, A., Wang, J., Csanádi, J., Hodúr, C., Vatai, G. 2009. Partial demineralization
and concentration of acid whey by nanofiltration combined with diafiltration.
Desalination, 241: 288–295.
Romero, J., Draga, H., Belleville, M.P. et al. 2006. New hydrophobic membranes for
contactor processes—Applications to isothermal concentration of solutions.
Desalination, 193: 280–285.
Rufian-Henares, J.A., Morales, F.J. 2007. Effect of in vitro enzymatic digestion on anti-
oxidant activity of coffee melanoidins and fractions. Journal of Agricultural and
Ruivo, R., Couto, R., Simões, P.C. 2008. Supercritical carbon dioxide fractionation of
the model mixture squalene/oleic acid in a membrane contactor. Separation and
Saidi, S., Deratani, A., Ben Amar, R., Belleville, M.-P. 2013. Fractionation of a tuna dark
muscle hydrolysate by a two-step membrane process. Separation and Purification
Sarmento, L.A.V., Machado, R.A.F., Petrus, J.C.C., Tamanini, T.R., Bolzan, A. 2008.
Extraction of polyphenols from cocoa seeds and concentration through
polymeric membranes. Journal of Supercritical Fluids, 45: 64–69.

Sarrade, S., Guizard, C., Rios, G.M. 2002. Membrane technology and supercritical
fluids: Chemical engineering for coupled processes. Desalination, 144: 137–142.
Sarrade, S.J., Rios, G.M., Carlès, M. 1998. Supercritical CO2 extraction coupled with
nanofiltration separation: Applications to natural products. Separation and
Strathmann, H. 2010. Electrodialysis, a mature technology with a multitude of new
applications. Desalination, 264: 268–288.
Strathmann, H. 2011. Introduction to Membrane Science and Technology. Wiley-VCH,
Weinheim, 524 pp.
Subramanian, R., Nakajima, M., Kawakatsu, T. 1998. Processing of vegetable oils
using polymeric composite membranes. Journal of Food Engineering, 38, 41–56.
Tijing, L.D., Woo, Y.C., Choi, J.-S., Lee, S., Kim, S.-H., Shon, H.K. 2015. Fouling and its
control in membrane distillation—A review. Journal of Membrane Science, 475:
215–244.
Torun, M., Rácz, G., Fogarassy, E. et al. 2014. Concentration of sage (Salvia fruticosa
Miller) extract by using integrated membrane process. Separation and Purification
Tsui, E.M., Cheryan, M. 2007. Membrane processing of xanthophylls in ethanol
extracts of corn. Journal of Food Engineering, 83: 590–595.
Tylkowski, B., Tsibranska, I., Kochanova, R., Peeva, G., Giamberini, M. 2011.
Concentration of biologically active compounds extracted from Sideritis ssp. L.
by nanofiltration. Food and Bioproducts Processing, 89: 307–314.
Vaillant, F., Cisse, M., Chaverri, M. et al. 2005. Clarification and concentration of
melon juice using membrane processes. Innovative Food Science & Emerging
Technologies, 6: 213–220.
van Reis, R., Zydney, A. 2007. Bioprocess membrane technology. Journal of Membrane
Science, 297: 16–50.
Wang, P., Chung, T.-S. 2015. Recent advances in membrane distillation processes:
Membrane development, configuration design and application exploring.
Journal of Membrane Science, 474: 39–56.
Wang, J., Zhao, M., Yang, X., Jiang, Y. 2006. Improvement on functional properties
of wheat gluten by enzymatic hydrolysis and ultrafiltration. Journal of Cereal
Science, 44: 93–100.
Xie, Z., Huang, J., Xu, X., Jin, Z. 2008 Antioxidant activity of peptides isolated from
alfalfa leaf protein hydrolysate. Food Chemistry, 111: 370–376.

9
Extraction of Functional Food Ingredients
and Nutraceuticals from Dairy
Geneviève Gésan-Guiziou
CONTENTS
9.1 Introduction................................................................................................. 235
9.2 Extraction/Fractionation of Milk Fat Components................................ 237
9.2.1 Characteristics of Milk Fat............................................................. 237
9.2.2 Processes of Enrichment and Extraction of Milk Fat
Membrane Globule, MFGM Fragments....................................... 240
9.2.3 Isolation of MFGM from Milk at Laboratory Scale.................... 241
9.2.4 Isolation of MFGM from Industrial Sources............................... 241
9.3 Extraction of Milk Proteins as Functional Ingredients
and Nutraceuticals...................................................................................... 243
9.3.1 Milk Proteins................................................................................... 243
9.3.2 Fractionation of Dairy Proteins.................................................... 245
9.3.2.1 Pretreatment of Milk....................................................... 245
9.3.2.2 Isolation of the Whole Casein........................................ 247
9.3.2.3 Concentration of Serum Proteins.................................. 250
9.3.2.4 Fractionation of Individual Serum Proteins................ 253
9.3.2.5 Fractionation of Whole Casein into
Individual Casein............................................................. 259
9.4 Conclusion................................................................................................... 260
References.............................................................................................................. 260
9.1 Introduction
Among the food products, milk is undoubtedly a high source of compo-
nents with functional and physiological properties (Mattila-Sandholm and
Saarela, 2003). It is naturally secreted by mammal females as a whole food
to cover the nutritional needs for newborns’ growth and health and con-
tains many components that provide critical nutritive elements, immuno-
logical protection, and biologically active substances to both neonates and
adults. Severin and Xia (2005) have reviewed the nutraceutical properties of
milk components. Even though most of the claimed physiological properties
235
of milk bioactive components have been carried out in vitro or in animal

model systems (resulting in further research work to prove the hypothesized
properties in humans), the demand of nutraceuticals extracted from milk is
expected to rapidly grow. These functional components and ingredients are
expected to play a major role in complementation, rather than in substitution
to the synthetic pharmacological drugs.
Milk contains many components including fat, proteins, carbohydrate,
vitamins, and minerals. Some of these components are nutraceuticals in
their native form and then require a direct extraction/purification step from
the dairy matrix to produce functional ingredients. This is, for example, the
case for the polar lipids constituting the milk fat globule membrane (MFGM),
which can beneficially affect lipid metabolism, particularly through the
ability of sphingolipids to lower cholesterol absorption. Milk also contains
biologically active proteins such as lactoperoxidase (enzyme), immuno-
globulins, or lactoferrin, as well as various growth factors, which require
specific extraction procedures to be valorized. However, a large range of
nutraceuticals and functional ingredients produced from milk or wheys is
obtained after chemical modifications or hydrolysis of native milk compo-
nents. Some derivatives of lactose (e.g., lactulose) (Gänzle, 2011) or bioactive
peptides issued from the enzymatic hydrolysis of milk proteins, which are
currently the main source of biologically active peptides, even though other
animal and plant proteins contain potential bioactive sequences, offer large
potential for creating new value-added ingredients with applications in
functional food and nutraceuticals. The bioactive peptides released by enzy-
matic hydrolysis of milk proteins can be associated to a variety of activities,
such as opioid antagonistic or angiotensin-converting enzyme inhibitory
ones (Haque et al., 2009). These groups of molecules, obtained after modifi-
cations or hydrolysis of native milk components, are not directly extracted
or purified from milk or whey and this is the reason why they will not be
considered in this chapter.
In order to use the desired milk components as functional ingredients or
nutraceuticals, their fractionation and purification from the complex dairy
matrix is essential. This separation is challenging. The low concentration
of the molecules of interest, the high number of components in the complex
matrix to be treated (more than 2000 molecules referenced in milk), which,
for some of them, exhibit characteristics similar to those of the targeted mol-
ecule, and the strong physicochemical interactions between molecules led
to intricate extraction processes. In recent years, the increased knowledge of
researchers and manufacturers on both the separation technologies and the
characteristics and biological functions of dairy constituents has boosted the
demand and thereby the development of processes adapted for the produc-
tion of high-value dairy ingredients for functional or health-promoting foods.
This chapter describes the main fractionation/purification processes
developed for the preparation of functional ingredients from milk and
derivatives, with a focus on the technologies applied for the extraction of

Extraction of Functional Food Ingredients and Nutraceuticals from Dairy 237
protein, which represent an increasingly used class of nutraceutical prod-

ucts. The contemporary industrial processes available for the fractionation of
milk compounds comprise three main approaches alone or in combination,
namely precipitation, chromatography, and membrane separations. Since the
appropriate selection of a given process depends, to a large extent, on the
nature of the starting material, the concentration of the desired molecules
therein, and the physicochemical characteristics of the molecule to be sepa-
rated from the complex mixtures, we have organized the sections of this
chapter by categories of components. We have then described examples of
technologies available for the separation/extraction of each of them.
9.2 Extraction/Fractionation of Milk Fat Components

9.2.1 Characteristics of Milk Fat
Bovine milk is a complex fluid, with a constant pH around 6.7 at ambient
temperature. It is mainly composed of fat, lactose, proteins (casein micelles
and serum proteins), and minerals, but many other small species are present
in the soluble phase of milk (urea, vitamins, etc.). The average composition of
cow milk is given in Table 9.1 and Figure 9.1.
Bovine milk contains about 40 g/L of lipids. More than 95% of the mass
of lipids in milk is present in the form of spherical milk fat globules, with a
diameter ranging from 0.15 to 15 µm and a volume mean diameter around
4 µm (Walstra et al., 2006). In their native state, the milk fat globules have a
core-shelf structure: the fat globules are composed of a core of triacylglycer-
ols and a surrounding membrane (MFGM). MFGM contains a rich variety
of components, consisting mainly in polar lipids, which comprise phospho-
and sphingolipids, membrane-specific proteins, mainly glycoproteins (e.g.,
butyrophilin, mucins), cholesterol, and enzymes (e.g., xanthine oxidase/
dehydrogenase) (Fong et al., 2007; Lopez, 2011) (Table 9.2). The main milk
polar lipids of the MFGM are the glycerophospholipids (phosphatidyletha-
nolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylino-
sitol) and the sphingolipids (mainly sphingomyelin). When compared with
other sources of polar lipids (soya, colza, sunflower lecithins, and egg leci-
thin) which represent the main origins of polar lipids used in food industry,
MFGM is a rich source of sphingolipids [sphingolipids account for up to one-
third of the MFGM polar lipid fraction (Burling and Graverholt, 2008) with
highly bioactive functions].
For the last 15 years, a great deal of knowledge has been accumulated on
health beneficial factors, protein, and polar lipids of bovine MFGM, and
although controversies exist, several components of the MFGM (in particu-
lar, phospholipids and sphingolipids) have been shown to have an impressive

TABLE 9.1
Average Composition of Bovine Milk and Characteristics (Concentration, Isoelectric
Point, Molecular Weight) of Main Components
Size (µm) or
Molecular Weight
Iso- (Order of
Concentration Electric Magnitude) g/mol
Component g/L Point (or Dalton) Biological Activitiesa
Fat 34–44 0.15–15 µm (fat
globules)
Proteins 32–35
Caseins 25–28 ≈ 4.6 50–500 nm (casein Iron carrier,
micelles) immunomodulator,
bioactive peptides
precursor
Molecule carrier
Serum proteins 5–7 14,200–150,000 Da
β-lactoglobulin 3.2 5.4 18.4 Da (dimer in Retinol carrier, potential
milk 36.8) antioxidant, precursor
for bioactive peptides,
binds fatty acids
α-lactalbumin 1.2 4.8 14.2 Da Lactose synthesis in
mammary gland,
calcium carrier,
immunomodulator,
precursor for bioactive
peptide
Immunoglobulins 0.5–1.0 5–8 150–1000 Da Specific immune
protection (antibodies
and complement
system), potential
precursor for bioactive
peptides
Bovine serum 0.4 5.1 66.3 Da Precursors of bioactive
albumin peptides
Lactoferrin 0.2 8.5 80 Da Antimicrobial,
antioxidative,
anticarcinogenic,
antiinflammotory, iron
transport, call growth,
regulation, precursor
for bioactive peptides,
immunomodulator
Lactoperoxydase 0.03 9.6 78 Da Antimicrobial,
synergetic effect with
immunoglobulins and
lactoferrin
Lactose 48–50 342 Da
Ashes (minerals) 8–9
Water 870–875
a Adapted from Korhonen, H., Pihlanto, A. 2007. Current Pharmaceutical Design, 13: 829–843.

25
Ions
20 Serum
proteins
Casein
micelles
15
Number (log)
Fat
10
Micro-
organisms
5 Somatic
cells
0
–2 –1 0 1 2 3 4 5 6
Size: diameter in nm (log)
FIGURE 9.1
Approximate particle sizes of milk constituents for which separation by means of membrane
filtration can be applied.
array of functionalities. Among the health-beneficial functionalities of the

MFGM components are cholesterolemia-lowering factors, inhibitors of can-
cer cell growth, vitamin binders, inhibitors of Helicobacter pylori, inhibitors of
β-glucuronidase of the intestinal Escherichia coli, xanthine oxidase as a bacte-
ricidal agent, and butyrophilin as a possible suppressor of multiple sclerosis.
TABLE 9.2
Average Composition of the Milk Fat Globule Membrane, MFGM
Concentration
Component (in mg/100 g Fat Globules) (in g/100 g MFGM Dry Matter)
Protein 1800 70
Phospholipids 650 25
Cerebrosides 80 3
Cholesterol 40 2
Monoglycerides Presence (quantity unknown) ?
Carotenoids + Vit. A 0.04 0.0
Fe 0.3 0.0
Cu 0.01 0.0
Total >2570 100
Source: From Walstra, P., Wouters, J.T.M., Geurts, T.J. 2006. Dairy Science and Technology. CRC
Press, Boca Raton, FL, USA.

Milk polar lipids certainly offer the main functionalities of the MFGM: they
have antioxidative activities, as well as antimicrobial and antiviral properties
(Ward et al., 2006); they act against colon cancer, inflammation and gastroin-
testinal infections, cardiovascular diseases, stress, nervous system myelina-
tion, and neurological development (Spitsberg, 2005; Dewettinck et al., 2008;
Küllenberg et al., 2012; Contarini and Povolo, 2013). Besides their nutritional
benefits, milk polar lipids-enriched ingredients have also very interesting
techno-functional properties such as emulsifying and whipping properties
(Ward et al., 2006; Dewettinck et al., 2008; Vanderghem et al., 2010). They can
also be used to prepare liposomes for the encapsulation of bioactive mol-
ecules like tea polyphenols, ascorbic acid, and omega-3 oils (Singh, 2006;
Bezelgues et al., 2009; Farhang et al., 2012).
Even though the MFGM components and milk polar lipids are present at
low levels in dairy products (Table 9.2), resulting in their very challenging
economic fractionation, special attention paid to all of the above properties
has highlighted the potential to use MFGM fragments in manufacturing
nutraceutical and functional foods.
9.2.2 Processes of Enrichment and Extraction of Milk

Fat Membrane Globule, MFGM Fragments
For the last 20 years, different patents and scientific papers have been pub-
lished regarding the isolation and concentration of MFGM components and
the production of the milk polar lipid-rich fractions. The yield and composi-
tion of the final fractions depend on the techniques utilized for their isolation
and on the nature of the starting product (buttermilk/butter serum, cream,
whole milk, or whey cream) (Sodini et al., 2006; Vanderghem et al., 2010). In
a general manner and as seen in various publications, dairy products rich in
polar lipids are enriched in MFGM proteins: the close association of polar
lipids and proteins within the MFGM explaining their comigration during
dairy processing. Among the various dairy products (Table 9.3), buttermilk,
the liquid released after churning butter out of cream, and butter serum, the
liquid obtained after the melting of butter to produce anhydrous milk fat, are
suitable as sources for the isolation of MFGM material. They contain about
2% and 11% of polar lipids, respectively, the latter being the richest source
of MFGM material on dry matter basis. Despite this, buttermilk and butter
serum are still considered to date as low-value products in human nutrition
and are mainly spray-dried to be used in animal feed. Cheese wheys and
whey buttermilk, the aqueous fraction obtained by churning of whey cream
which is separated from cheese whey, and acid buttermilk whey, the aque-
ous fraction obtained by acidification of sweet-cream buttermilk, can also be
considered favorable for MFGM isolation by filtration because of the absence
of casein micelles (Table 9.3). Casein micelles, being colloidally similar in size
to the MFGM fragments, are difficult to separate from the MFGM using size-
based separations such as membrane operation.

TABLE 9.3
Polar Lipid Content of Various Dairy Products during Processing
Concentration
Dairy Product g/100 g on Product g/100 g on Dry Matter
Raw milk 0.03–0.04 0.23–0.32
Skimmed milk 0.02 0.28
Cream 0.19 0.40
Butter 0.14–0.23 0.17–0.26
Buttermilk 0.16 2.03
Butter serum 1.25 11.54
Acid buttermilk whey 0.10 1.84
Cheddar cheese whey 0.02 0.26
Source: From Dewettinck, K. et al. 2008. International Dairy Journal, 18: 436–457.
9.2.3 Isolation of MFGM from Milk at Laboratory Scale

From a methodological point of view, enriched MFGM fractions can be
obtained using a four-step procedure previously reviewed by Dewettinck
et al. (2008) (Figure 9.2). This procedure describes the required steps for the
isolation of MFGM from untreated milk on a laboratory scale (Figure 9.2a).
The main challenges for these different technological steps are to separate
MFGM fragments and milk polar lipids from the other components mainly,
triacylglycerols, proteins, lactose, and minerals.
The first step consists in the separation of fat globules from the milk by
centrifugation (Figure 9.2). Next, the fat globules are washed to remove
undesired components, with specific water, saline, pH buffered, or phos-
phate solutions. A milk salt buffer or sucrose can be added to increase the
density of the serum and minimize MFGM loss (Singh, 2006). Then the glob-
ules are disrupted to release the membrane material, by either churning at
reduced temperatures or applying cycles of freezing and thawing, treatment
with detergents, or by suspension in polar and aprotic solvents (Keenan and
Mather, 2006). This step may lead to loss of proteins due to their dispersion in
the salt/detergent phase. Once the membrane is disrupted, it can be collected
by ultracentrifugation (with high centrifugal force, i.e., 100,000 g), microfiltra-
tion, protein precipitation at low pH, or induction of membrane aggregation
with ammonium sulfate followed by centrifugation (Keenan and Mather,
2006). As previously mentioned, the processing history of the material used
for the isolation will profoundly affect the final results.
9.2.4 Isolation of MFGM from Industrial Sources

Isolation of MFMG materials is more suitable from buttermilk or butter
serum, which contains a high content of polar lipids (Figure 9.2b). Many
attempts have been performed, mainly on a laboratory scale, to isolate

(a) Whole milk (b) Whole milk

Steps 1 and 2 : Separation and
washing of the fat globules
Separation of fat globules Skimmed milk Centrifugation Skimmed milk

Centrifugation using Discharged
Cream
specific butters and washing
solutions Washing of fat globules solutions
Washed cream Cream
Churning
Freezing + thawing cycles Release/extraction of milk fat Pasteurization
Treatment with globule membrane, MFGM
detergents or specific Continuous churning
solvents
Step 3 : Disruption of
Separation/centrifugation Butter
Buttermilk
globules
Butter Melting
Buttermilk
Melting
Centrifugation
Centrifugation
MFGM Butter fat/oil Butter fat/oil

Buttermilk Butter serum
suspensions
Membrane filtration
Step 4 : Collection/purifications
Microfiltration Separation casein Proteins,

Concentration/purification
using membrane technologies
Ultracentrifugation micelles/polar lipids

Precipitation os minerals, lactose
precipitation/aggregation +
casein micelles +
centrifugation Freeze drying
ultrafiltration
Defatting Conc. + diafiltration

Solvent extraction
Dialysis + freeze drying Conc. + spray drying
Pure MFGM proteins Butter serum or buttermilk powders
FIGURE 9.2
Four-step procedure for the milk fat globule membrane (MFGM) isolation in a laboratory (a)
and pilot (b) plant setting. (Adapted from Dewettinck, K. et al., 2008. International Dairy Journal,
18: 436–457.)
MFGM from buttermilk. The authors used membrane techniques such as

microfiltration to selectively separate the components according to their
size; MFGM fragments (one to several micrometers long) recovered in the
retentate and proteins (casein micelles: 200 nm in diameter) removed in
the permeate. By using membrane technologies (mainly ultrafiltration and
microfiltration), the isolation was based on the selective concentration of lip-
ids and removal of casein, whey proteins, lactose, and minerals from the
concentrate. However, the similarity in size of casein micelles and small
MFGM fragments or polar lipid vesicules was reported to be a major obstacle
during isolation. Some authors (Sachdeva and Buchheim, 1997) used ren-
neting and acid coagulation to remove casein micelles (protein in the curd
and phospholipids in the serum phase) prior to the concentration of MFGM
using membrane technologies. Other authors (Corredig et al., 2003) dissoci-
ate casein micelles structure using citrate after microfiltration and diafiltra-
tion; the casein contamination decreases from 30% to 6% of total proteins.
With microfiltration it was possible to obtain an MFGM concentrate contain-
ing approximately 60% (w/w) protein and 35% (w/w) lipid, unfortunately
with a loss of MFGM material especially during the diafiltration phase.

The authors also used chemical treatments such as calcium to precipitate

MFGM fragments (thermocalcic aggregation) and enzymatic treatments
aiming at hydrolyzing proteins. Ultrafiltration, sometimes in combination
with diafiltration, was used to remove lactose and minerals and to concen-
trate milk polar lipids in the final ingredient.
In order to enrich the polar lipid fraction and reach high purity, the pres-
ence of neutral lipids is often problematic, as polar lipids in dairy products
are present mainly as MFGM fragments of lipoprotein particles and the
neutral lipids are intimately associated with them. The separation of a polar
lipids fraction of high purity is therefore challenging. The use of organic
solvent to extract milk polar lipids is the only means today to produce ingre-
dients with high amount of polar lipids and devoid of proteins (polar lipids
>50%, e.g., PL700 product produced by Fonterra, Lacprodan PL-75 product
produced by Arla Foods), but the use of ingredients from organic solvent-
free processes is preferred for food applications.
Supercritical fluid extraction has been reported as a possibility to increase
the milk lipid polars/triacylglycerols ratio by the selective removal of neutral
lipids (Astaire et al., 2003). Supercritical fluid extraction was demonstrated
to significantly reduce the total fat in retentate fractions obtained after
microfiltration 0.8 µm of buttermilk (by 38%) and that the removed fraction
was composed exclusively of non-polar lipids yielding to a final fraction of
≈20 mg polar lipids/g dry matter. Several dairy products enriched in polar
lipids are available on the market. Their composition in polar lipids ranges
between ≈ 15% and 80% depending on the extraction process performed.
9.3 Extraction of Milk Proteins as Functional

Ingredients and Nutraceuticals
9.3.1 Milk Proteins
A thorough knowledge of the physicochemical properties of milk proteins is
strictly necessary for the development of the process of extraction of individ-
ual proteins. Proteins (32–35 g/L expressed as nitrogen, N × 6.38) correspond
to about 95% of the total nitrogen of the milk. They are classically divided
into two primary main groups (caseins and whey (or serum) proteins) which
differ greatly with regard to their physicochemical and biological properties.
About 80% of bovine milk proteins (≈ 25–28 g/kg milk, Table 9.1) consists of
caseins (individual casein ~20 kg/mol) that are insoluble at their isoelectric
point of pH 4.6 at temperature >8°C. Approximately 92% of the caseins are
associated into large globular aggregates, called casein micelles. The struc-
ture of the casein micelles is still not totally elucidated (Bouchoux et al., 2010;
Dalgleish, 2011), but casein micelles can be described as roughly spherical

particles, with outer diameters ranging from 50 to 500 nm (mean diameter

~100–150 nm) (Table 9.1). They are made of four distinct caseins, αs1, αs2, β,
and κ in proportion of 3:1:3:1, and 8% in mass of calcium and phosphate,
often called the colloidal calcium phosphate. The stability of the casein
micelles depends on various factors. First, it is directly linked to the calcium
phosphate content of the micelle. Indeed the minerals contained in casein
micelles are in equilibrium with the aqueous phase. Thus, changing external
conditions of milk such as pH, temperature, or addition of a calcium che-
latant cause alterations in the mineral equilibriums, induce modifications in
the structure and stability of casein micelles, and then cause changes in frac-
tionation processes performances. For instance, the casein micelles dissoci-
ate on removing colloidal calcium phosphate either by the addition of citrate
or EDTA (Ca-chelating agents) or by acidification. The stability of the casein
micelles results also from the zeta potential of casein micelles (≈ −20 mV at
ambient temperature and native pH) and from steric hindrance caused by
the protruding (“hairy”) C-terminal parts of κ-casein, which prevent the
approach of micelles. Removal of this protruding segments using chymosin,
the main enzyme present in the neonate calf stomach, results in the desta-
bilization of the native casein micelle structure and then in the coagulation
of the damaged micelles. The integrity of micelles is also affected by cool-
ing. At temperatures lower than 4°C, β-casein, mainly linked by hydropho-
bic bonds to the micelle, and calcium phosphate are released into the serum
phase, leading to a process of extraction of β-casein (see Section 9.3.2.5). Just
for information, by comparison with bovine milk, human caseins consist of
primarily β- and κ-caseins.
The second group of proteins is constituted by the serum proteins, also
called whey proteins or soluble proteins, because they do not precipitate in
the ionic environment of milk when rennet is added or acidification occurs
down to pH 4.6. The main serum proteins are β-lactoglobulin, β-Lg (~3.2 g/L),
α-lactalbumin, α-La (~1.2 g/L), bovine serum albumin, bovine serum albumin
(~0.4 g/L), immunoglobulins (~0.5–1.0 g/L), minor proteins like lactoferrin
or lactoperoxidase, enzymes (e.g., lysozyme), growth factors, and hormones
(Table 9.1). The whey protein fraction of human milk is very different from
that of bovine milk in that it contains no β-Lg and is very rich in α-La, lac-
toferrin, lysozyme, and stimulatory factors. In a general manner, whey pro-
teins have globular three-dimensional structures and are either basic or acid
proteins. Their main characteristics and specific biological activities of serum
proteins are given in Table 9.1. On the whole, these proteins are excellent
sources of branched-chain amino acids (BCAAs). l-Isoleucine, l-leucine, and
l-valine, all BCAAs, are metabolized directly into the muscle and help protect
against muscle breakdown during heavy exercise. Therefore, whey protein is
a popular protein choice for use in sports nutrition products because it is easy
to digest and is efficiently absorbed into the body.
Each of the extracted proteins or group of proteins has been proven or
implied to have unique functional, nutritional, or nutraceutical properties.

The basic proteins (lactoferrin, lactoperoxidase, lysozyme) have known anti-

microbial properties, whereas the main acidic serum proteins have func-
tions that include lactose synthesis, calcium transport, immunomodulation,
and anticarcinogeniticy (α-La) and retinol transport, fatty acid binding, and
antioxidant activity (β-Lg). Lactoferrin, in particular, has multiple activities,
including antioxidant activity, iron binding, anticarcinogenicity, and immu-
nomodulation. The growing interest of lactoferrin is related to its antibac-
terial properties, by forming an iron complex and inhibiting the growth
of microorganisms by depriving bacteria of iron that is essential for their
growth, and to its interesting nutritional activities due to the transport of
iron in organism. β-Lactoglobulin plays a role in the regulation of mammary
gland phosphorus metabolism, as a transporter of vitamin D, cholesterol, and
retinol, the transfer of passive immunity to the newborn, and the enhance-
ment of pregastric esterase activity. Madureira et al. (2007) summarized a
number of biological functions of this protein. Immunoglobulins, unsurpris-
ingly, impart various immunoprotective functions in a range of functional
foods (Gapper et al., 2007). Table 9.1 gives an idea of the wide range of nutri-
tional, functional, and biological activities properties of the serum proteins.
9.3.2 Fractionation of Dairy Proteins

Most of the dairy proteins ingredients, used as either nutritional or func-
tional ingredients, are the results of a succession of different processing steps
(Figure 9.3). Each stage of the extraction process requires a thorough analy-
sis of the preservation of the desirable qualities (e.g., functional, biological
properties). In particular, special attention must be paid to the successive
heat treatments, the effect of which is cumulative. Like most liquid foods,
milk and its derivatives are very favorable media for spoilage microorgan-
isms. Consequently, pretreatments and temperature–time parameters and
residence time temperature must be chosen in order to control microbial
growth.
9.3.2.1 Pretreatment of Milk
Skimmed milk is generally used as the starting fluid for most extraction proce-
dures for milk proteins (Figure 9.3). Separation of cream from skimmed milk
is a common practice over 100 years ago. Whole milk is usually centrifuged,
at around 50°C, in a cream separator equipped with conical discs running
at 5000 g. As milk fat has a lower density than plasma, the fat globules rise
under the influence of gravity, and the rate of rising is increased when the
centrifugal field is applied. Despite its large application in the dairy industry,
this separation is not totally perfect and the skimmed milk, which generally
represents 90% of the volume of the entering whole milk, still contains a very
low fat content, 0.05 (0.5 g/L) to 0.08%, which strongly influences the efficiency
of downstream processes and the resulting properties of protein products.

Whole milk
Cream Cream separation
Standardization Skimmed milk
Standardized milk
Separation casein micelle/whey proteins
Coagulation Using microfiltration
Wheys
Clarification
Microfiltration, physico- Micellar casein
Caseins chemical treatment Whey/microfiltrate concentrates/isolates
Addition of alkali Concentration/purification Demineralization

Ultrafiltration + diafiltration Nanofiltration; Ion exchange; elctrodialysis
Concentrates/isolates of whey proteins

(WPI, WPC)
Fractionation Fractionation
Caseinates Chromatography; precipitation; filtration Chromatography; percipitation;
membrance separation
Individual serum proteins

Individual caseins
FIGURE 9.3
General overview of the protein fractionation of milk.
The good bacteriological quality of the starting skimmed milk is also a

guarantee of the acceptable quality of the final protein concentrates or
isolates (low content in bacteria, low degradation of the final fractions).
The bacteriological quality of milk is the most variable factors with which
the manufacturer has to content. In developed countries, milk is normally
contaminated by common mesophilic and psychotrophic microflora and
rarely by pathogenic microorganisms. Several operations can be carried out
in order to minimize health hazard and control the bacterial growth during
milk processing. Heat treatment with various combinations of time and tem-
perature (such as pasteurization 72°C for 12 s) is frequently used on an indus-
trial scale, but it usually affects the product. The heat treatment decreases
the pH, shifts the protein–calcium phosphate equilibrium, causes changes
in the micellar structure of casein, and initiates the Maillard reaction, which
permanently modifies the functional and nutritional properties of the whey
proteins. Centrifugation and crossflow microfiltration, MF, are nonthermal
preservation technologies that have been widely applied for the removal
of bacteria (Gésan-Guiziou, 2010). Centrifugation (often referred to as bac-
tofugation) is used for the removal of bacteria and spores from minimally
pasteurized milk because it efficiently removes spores as their density is
higher than that of bacteria. However, the decimal reduction is generally low
(~1 log) and significant protein loss is observed, reaching 2.5%–12% accord-
ing to the types of machines (Gésan-Guiziou, 2010). In that context, MF has
been more and more applicable for the removal of bacteria and becomes an

interesting process to remove bacteria prior to further protein fractionation

steps. MF is classically and specifically applied to skimmed milk, because the
size ranges of fat globules and bacteria overlap. This separation is performed
at a temperature between 35°C and 55°C, with a ceramic membrane with a
pore diameter of 1.4 µm in order to retain bacteria and let the proteins pass
through the membrane. In order to overcome membrane fouling phenom-
ena, most MF plants in the dairies operate using either the hydraulic concept
of Uniform Transmembrane Pressure (UTP) or specific ceramic membrane
with linear hydraulic resistance (Sandblöm, 1974; Gésan-Guiziou, 2010). Total
solids and proteins of skimmed milk are then largely transmitted (99.5 and
99 of transmission, respectively; Maubois and Ollivier, 1997), residual fat is
removed by the membrane, and the decimal reduction of bacteria reaches
3–4 log (Gésan-Guiziou, 2010). The microfiltered 1.4 µm skimmed milk is
then a perfect starting fluid for further extraction of dairy proteins.
9.3.2.2 Isolation of the Whole Casein

The extraction procedures developed for the extraction of each individual
protein require first the separation of the two types of proteins (casein and
serum proteins) from each other. The characteristics of the two groups of
milk proteins differ significantly permitting their ready separation by at
least two principal ways: precipitation/coagulation of caseins as during the
cheese manufacture, and microfiltration using a membrane with a 0.1 µm
mean pore diameter for the removal of casein micelle from the aqueous
phase of milk (Figure 9.3).
9.3.2.2.1 Precipitation/Coagulation of Caseins
The first and most classical way used to separate caseins from serum pro-
teins is based on the destabilization of the casein micelles by precipitation/
aggregation of casein (Figure 9.4). Regardless of the mode used to destabilize
the micelles (isoelectric precipitation or rennet coagulation), the isolated frac-
tion of casein micelles is denatured. The coproduct of the casein destabiliza-
tion is the whey, which can roughly be considered as milk destitute of casein
micelles and fat. Whey contains approximately 65 g/L dry matter with lac-
tose (~50 g/L), nitrogen matter (mainly serum proteins ~6 g/L), ashes (min-
erals ~6 g/L), and fat (0.3 g/L). Its exact composition varies according to the
process from which it comes from (Table 9.4).
In isoelectric precipitation, the destabilization of the casein micelles is
accomplished at the isoelectric point of the caseins around 4.6, either from
chemical acidification or from lactic acid fermentation (as for fresh cheese).
When milk is acidified, the net charge of the micelles decreases, the calcium
and phosphate are removed and the micelles become less and less stable
until caseins precipitate. Acid casein can be produced by sufficient reduc-
tion of the pH of the milk (to 4.6) by the use of hydrochloric or sulfuric acid.
The mix is diluted with four parts water with thorough mixing. After a

Microfiltration Isoelectric Rennet

diafiltration precipitation coagulation
Chemical acidification κ casein hydrolysis

Ions exchanger with chymosin
Biological acidification aggregation of calcium
paracaseinate
Native phosphocaseinate
(93%)
+ Acid casein Rennet casein

Microfiltrate + +
= Acid whey Sweet whey
〈〈Defatted〉〉 sweet whey
Rotatry sieve
centrifugation
FIGURE 9.4
Separation of the two types of proteins (casein/whey proteins) from milk.
short holding period in a vat, the whey is drained off. The curd is washed
twice with cold water, pressed, and milled. Acid casein can also be produced
by inoculating skim milk with acid-producing bacteria and incubating it
at 20–30°C until the acidity reaches 0.64%. The curd is stirred and heated
to 50–65°C. The whey is then drained and the curd washed twice with
cold water and pressed for 10–15 h. It is then milled and ready for further
TABLE 9.4
Approximate Composition of Sweet and Acid Wheys and Permeate of
Skimmed Milk Microfiltration using a 0.1 µm Mean Pore Membrane
(“Ideal” Whey)
Sweet Whey Acid Whey “Ideal” Whey
g/L g/L g/L
pH 6.4 4.6 6.6
Dry matter 66 64 61
Nitrogen matter 6.2 5.8 7.5
Non nitrogen matter 0.37 0.40 1.8
Lactose 52.3 44.3 48
Ashes 5.0 7.5 —
Fat 0.2 0.3 0.0
Calcium 0.5 1.6 0.32
Magnesium 0.07 0.10 0.07
Sodium 0.53 0.51 0.40
Potassium 1.45 1.40 1.60
Chloride 1.02 0.90 —

processing (e.g., drying). In such processes, the collected “acid” whey con-

tains high mineral content because of the release of minerals from the casein
micelles (mainly calcium and phosphate) into the serum phase under acid
conditions. Its final pH is also very acid, close to 4.6 (Table 9.4).
In the case of rennet coagulation, the destabilization of casein micelle is
caused by the proteolysis of the micelle-stabilizing κ-casein. The chymosin,
the principal enzyme present in the calf rennet, releases the glycomacrope-
ptide (C-terminal segments of glycosylated κ-casein) and renders the casein
micelles susceptible to precipitate by Ca2+ at the natural concentration in
milk. Rennet casein is made by adding sufficient rennet and calcium chlo-
ride to skim milk to cause it to clot in 20–30 min. Stirring is commenced
2–5 min after coagulation has started. The temperature is raised to 55–70°C,
and the curd is cooked for 30 min. The “sweet” whey is then drained off. The
final pH of sweet whey is close to the initial milk pH that is to say around
~6.0–6.6; its mineral content is similar to that of milk, and it contains the sol-
uble glycomacropeptide portion of the κ-casein that is released in the serum
phase. Industrially, the different combinations of coagulation operating con-
ditions (temperature, heating time, nature of starters, etc.) give rise to various
types of wheys, and then to various composition of wheys.
9.3.2.2.2 Preparation of Casein Fraction Using Microfiltration

Contrary to these two ways of manufacturing casein, the MF using a 0.1 µm
mean pore diameter is undoubtedly the most promising technology for
the selective separation of casein micelles (Figure 9.4). This operation, first
developed on an industrial scale in the mid-1990s, makes it possible in one
single operation, the separation of milk into a retentate enriched specifically
in native casein micelles and a permeate containing native soluble proteins
(Fauquant et al., 1988; Le Berre and Daufin, 1996; Gésan-Guiziou et al., 1999)
(Figure 9.3). Despite the relative complexity of milk, milk components are
relatively well separated according to their size. Figure 9.1 shows that it
is possible to separate casein micelles from serum proteins provided that
milk is not heat treated because heat denaturation of serum proteins leads
to change in size by aggregation of soluble proteins between each other and
association between them and casein micelles.
Microfiltration leads to two fractions:
• A retentate enriched specifically in native casein micelles.

Diafiltration against water of this retentate allows its purification
into micellar casein isolate, and it is easily concentrated again by
microfiltration and then spray-dried (Pierre et al., 1992; Schuck et al.,
1994) to become a good starting material for further fractionation of
caseins. Taking advantage of the native casein protein self-assembly,
casein micelles have also been proposed as a novel delivery vehicle
for nutraceutical compounds. Casein micelles are in effect nano-
capsules created by nature to deliver nutrients, such as calcium,

phosphate, and protein, to the neonate and can be used also for the
nanoencapsulation and stabilization of hydrophobic nutraceutical
substances for the enrichment of food products.
• A permeate corresponding to the aqueous phase of milk and contain-
ing the native serum proteins (size 2–10 nm). The permeate is some-
times called “ideal whey” because its composition is close to that
of a sweet whey but free of rennet-by-product-glycomacropeptide,
residual fat, and micro-organisms (phage, cellular debris, etc.). It is
a crystal clear fluid that can be sterile if the downstream equipment
prevents its recontamination. The pH of the permeate is similar to
that of milk, and then higher than all pH of wheys that are more
acidic (Table 9.4). The microfiltrate obtained is an excellent starting
substrate both for the production of whey protein isolates (WPI)
with high nutritional and functional properties and for further frac-
tionation and isolation of serum proteins.
From an engineering point of view, this separation is classically conducted

using ceramic membrane either with the UTP system or membranes with lin-
ear hydraulic resistance, as it is done for the removal of bacteria. Separation
is usually performed at 50–55°C, at a crossflow velocity of 7 m/s, a trans-
membrane pressure of ~50 kPa, and a volume reduction ratio, VRF, of 2–4.
At VRF = 3, serum proteins transmission ranges from 65% to 80% depending
on the milk heat treatment (pasteurization, thermalization, or raw skimmed
milk). Permeation flux is ≈ 75 L/h/m2 for 10 h according to the critical sta-
bility criterion, which allows the industry to perform long runs with very
moderate fouling and high selectivity (Gésan-Guiziou et al., 1999). Due to
the high running costs and investments imposed by the tubular ceramic
equipment, the industry starts to operate MF with spiral wound polymer
membranes. The process using organic spiral-wound membranes is not con-
ducted with the UTP system, it is classically performed at low temperatures
(<10°C) to avoid bacteria growth. Permeation flux is reported to be very low
<10 L/h/m2 with a low protein transmission ranging from 20% to 30%. This
high serum protein rejection requires diafiltration against ultrafiltrate (perme-
ate of milk ultrafiltration) to decrease the soluble protein level in the enriched
casein fraction. According to the manufacturers, about 25% of MF plants are
assumed to be equipped with organic membranes today and this proportion
is likely to increase in the next years mainly for cheese applications.
9.3.2.3 Concentration of Serum Proteins

Among the milk proteins, it is mainly the whey proteins that have nutraceu-
tical applications. Nutritional and functional properties of serum proteins
can be exploited either in mixtures (concentrates/isolates of serum proteins)
or in enriched/isolated form of individual serum proteins. In the latter case,
the extraction procedures of individual proteins generally start from either

a concentrate [whey proteins concentrates (WPCs)] or an isolate [whey pro-

tein isolates (WPIs) with proteins/dry matter >90%]. The concentration step
is classically carried out either from wheys or from microfiltrate of milk MF
0.1 µm (Figure 9.3). There are several industrial methods suitable for the
production of WPCs and WPIs. In addition to traditional operations such as
evaporation, modern methods used in industrial whey processing include
membrane operations, ultrafiltration (UF), microfiltration, reverse osmosis,
and demineralization step requiring nanofiltration, electrodialysis, and/or
ion exchange (Gernigon et al., 2011) or ion exchange chromatography.
9.3.2.3.1 Processes Using Membrane Operations

The development of UF membrane processes in the 1970s has offered the
possibilities to fully exploit the nutritional, biological, and functional prop-
erties of the whey proteins. Before UF, the interesting properties of whey
proteins could not be fully exploited because of the damaging effect of heat/
acid treatments used for their separations from whey. It has actually been a
long practice to obtain fully serum proteins, in a denatured form, by heating
acidified whey (temperature > 90°C and pH < 6) according to the technology
used for making whey cheeses (e.g., Ricotta). Today most serum protein con-
centrates are produced using UF. During ultrafiltration of wheys or micro-
filtrate, classically carried out with a cut-off membrane ranging from 10 to
20 kg/mol, low-molecular-weight compounds, such as lactose, minerals,
non-protein nitrogen, and vitamins, are separated in the permeate, whereas
proteins are concentrated in the retentate. Ultrafiltration of wheys permits
WPCs to be obtained with 20%–60% protein (expressed in nitrogen N × 6.38)
in total solids and low quantities of lactose and minerals. By a combina-
tion of UF and diafiltration (addition of water to the concentrate to remove
impurities) and starting from wheys or microfiltrate, it is possible to pro-
duce whey protein concentrates and isolates with 80% and up to 95%–98%
of protein. Most WPCs on the market contain either 35% or ~80% protein
and are important sources of protein for a large variety of food products,
ranging from processed meat and sausages, over health foods, to beverages
and confectionery. The upper limit of 80% obtained from wheys is usually
dictated by the residual fat content of the starting whey-fat that is preferen-
tially concentrated along with proteins. In some cases, pretreatment of whey
is proposed to remove the residual lipids. Among them, some use membrane
operations such as microfiltration that was included as a first step for the
removal of bacteria, casein fines, and fat from whey, thus contributing to an
improved quality of the final product. More recently an optimized pretreat-
ment, based on a “thermocalcic aggregation process” of the residual fat ini-
tially suggested by Pearce et al. (1992) and Maubois’s group (Fauquant et al.,
1985). An optimized pretreatment was proposed. It consists of several steps:
whey is first concentrated by UF until a concentration of 4–5, then the pH of
the retentate is increased and adjusted to 7.5, the temperature is maintained
at 55°C for 8 min (in order to favor the aggregation of the lipoproteins-Ca)

and finally the formed aggregates as well as the small fat globules and bac-
teria are separated using a 0.1 µm membrane MF. The absence of fat in the
“clarified” whey (permeate) and high pH strongly reduce the fouling of sub-
sequent UF resulting in longer running time higher permeation flux. Such a
pre-treatment allows us to obtain WPI with 90% protein in the total solids.
After ultrafiltration, the concentrate of serum proteins is pasteurized, evapo-
rated (or not, depending on the viscosity, protein content, and level of protein
denaturation required), and spray-dried.
Although high performances are obtained by this way, better purity and
functionalities of proteins of WPI are observed from the microfiltrate of
skimmed milk MF (0.1 µm). Due to its composition and because of the native
state of its proteins, the UF of the milk microfiltrate leads to the production of
WPI with high level of purity up to 95%–98% protein in the total solids and
very high functional and nutritional properties. This WPI has recently been
incorporated in products devoted to athletes for aiding the muscle recovery:
this is the case of the WPI produced by Lactalis (Prolacta® ingredients) used
in the Apurna® products.
From a technological point of view, whey UF is performed mainly in multi-
stage spiral wound systems with polyethersulfone membranes. Processes
are currently operated at temperature either around 50°C or 10°C. Operating
at a temperature of 50°C requires a pretreatment in order to avoid severe
fouling during operation, mainly due to calcium phosphate in these condi-
tions. Flux is about twice as high as flux at 10°C, which is a major incen-
tive for operation at such high temperatures, and thermal denaturation of
proteins is minimized. However, due to the relatively low membrane prices,
most of the manufacturers of protein concentrates prefer to operate at a lower
temperature (10–12°C) in spite of a lower flux. In these conditions, a much
lower growth of thermoduric bacteria in the spiral-wound filtration equip-
ment is observed which results in a better microbiological quality of the end
product. In addition, membrane fouling was reduced due to the increase of
solubility of calcium phosphate at low temperature.
9.3.2.3.2 Processes Using Ion-Exchange Chromatography

Because serum proteins are amphoteric molecules, highly purified whey
protein isolates are also produced using ion exchange chromatography
which provides an additional level of selectivity above membrane process-
ing. To date, ion exchange is the main adsorption technique employed, but
with the advent of new technologies, there are potential applications of affin-
ity binding and other techniques that have been historically and economi-
cally limiting.
Ion-exchange separations take advantage of electrostatic interaction
between surface charges on biomolecules, such as proteins, and clusters of
charged groups on the resin phase. At pH lower than their isoelectric point
(pH ~5.0–5.5), proteins become positive and they can be adsorbed on cation
exchangers, and at pH above, they can be adsorbed on anion exchangers.

After the adsorption of the proteins on the resins or columns (generally

performed at pH ~3.2), lactose and other non-protein whey components are
rinsed with water from the ion exchanger. Then alkali is added to a pH
of about 8 to desorb proteins, and the desorbed proteins are eluted from
the ion exchanger, concentrated by UF, evaporated, and spray-dried. There
are several ways of producing protein isolates, using in particular specific
conditions of pH, ionic strength, and ions nature, which give the processors
the opportunity to adjust the composition and functionality of the obtained
fractions. Among them, two major ion exchangers are commercially avail-
able for this application: the Vistec and Spherosil processes. The Vistec pro-
cess uses a cellulose-based cation exchanger, in a stirred tank reactor. WPI
are then produced by a single-stage batch capture of proteins, whereas the
Spherosil process uses a fixed-bed ion-exchanger with porous silica beads
coated with a polymer material having either cationic exchange poten-
tial (─SO3H groups, Spherosil S) or anionic exchange reactivity (─N(CH3)3
groups, Spherosil QMA). Acidified whey (pH <4.6) is generally applied to
Spherosil S and sweet whey (pH 5.5) to Spherosil QMA. Using chromatog-
raphy, protein concentrates are always devoid of lipids and of glycomacro-
peptides. However, UF operation is, for economic reasons, the privileged
way of processing.
9.3.2.4 Fractionation of Individual Serum Proteins

To exploit the particular properties of individual serum proteins, fraction-
ation of whey protein mixture for the isolation of one or a group of proteins
is useful. There is a considerable commercial interest in the production of
individual (purified) serum proteins with well-characterized functional and
biological properties. Considerable progress has been made over the last
20 years in technologies aimed at separation, fractionation, and isolation
in a purified form of many interesting proteins occurring in both bovine
milk and colostrum. To date, the main extraction techniques employed at
an industrial level have been membrane separations and chromatography
(mainly ion-exchange). Several other methods have already been published,
but a lot of them, because they are economically limiting, are only avail-
able on a laboratory scale (Bonnaillie and Tomasula, 2008; Fee et al., 2010;
El-Sayed and Chase, 2011).
Some procedures have led to the industrial manufacture of enriched pro-
tein fractions or separate proteins such as α-lactalbumin and β-lactoglobulin
and minor proteins like lactoferrin, lactoperoxidase, and immunoglobulins.
The cost of extracting these proteins is high but often justified when recog-
nizing the great value-added benefits when incorporated in hygiene prod-
ucts, functional foods, and nutraceutical products. The net result in terms
of purity and functionality depends on the starting materials and process
involved. Serum protein ingredients can contain the same amount of protein
but vary widely in functionality because the amount of various no-protein

compounds and their interactions with proteins differ. In many cases, it is

the interactions among molecules that provide the functional properties
rather than simply the amount of one or several components.
Because of their high protein purity and solution clarity, WPIs, either
obtained from defatted whey or from microfiltrate of skimmed milk MF, are
excellent starting materials for industrial production for further fraction-
ation of concentrates into individual proteins used for nutritional and health
purposes.
9.3.2.4.1 α-Lactalbumin and β-Lactoglobulin

Among the commercially interesting proteins, the two main serum proteins,
α-lactalbumin, α-La, and β-lactoglobulin, β-Lg (Table 9.1), can be produced
in enriched fractions using numerous technologies from WPC, WPI, liquid
whey, or even milk (Kamau et al., 2010). α-Lactalbumin has a great potential
market because of its high content in tryptophan (four residues per mole)
and in infant milk formula. α-La is the main soluble protein of the human
milk, and is therefore required in a concentrated form for the manufacture of
adapted formula for the infant uses. The main utilizations of β-lactoglobulin
appear to be in gel and foam-type products and in the manufacture of protein
hydrolysates for food ingredients. Kamau et al. (2010) give a summary of the
available methods used for the isolation and manufacture of α-lactalbumin
from milk and whey.
9.3.2.4.1.1 Chromatography Chromatography is mostly used industrially

to produce α-La and β-Lg enriched or purified fractions (Outinen et al.,
1996; Etzel, 1999; Etzel et al., 2006; El-Sayed and Chase, 2011). There are
many examples of affinity, hydrophobic, or size exclusion chromatography
for the isolation of whey proteins; however, ion-exchange chromatography
has been investigated by many researchers. Several methods have been
reported and today the ion-exchange chromatography (anion- or mainly
combination of anion and cation exchanges) is one of the most widely used
technologies to separate the two major serum proteins (Gerberding and
Byers, 1998; El-Sayed and Chase, 2011).
The chromatography process features the use of a resin to isolate fraction
of protein from the rest of the whey. With a careful choice of the resin sys-
tem and the eluants α-La and β-Lg can be separated from each other very
precisely. The purification of α-La and β-Lg is mainly based on selective elu-
tion rather than selective adsorption. For example, Thuran and Etzel (2004)
modified a cation-exchange chromatography method previously designed
to fractionate sweet whey, to efficiently fractionate acid whey. They used
different inexpensive food-grade buffers and extraction is performed in two
steps leading to the release of α-La fraction with a high recovery rate (96%)
and purity (93%) on the one hand and WPI containing mainly β-Lg on the
other hand. The high cost of purified fractions obtained by chromatogra-
phy often prevents them from being used in targeted food applications, but

rather in more valuable products for their high nutritional and biological
properties.
9.3.2.4.1.2 Membrane Separations in Combination Several attempts have been

done to separate the two major whey proteins using membrane filtration
only. However, their close characteristics (mainly in sizes and zeta potentials)
lead to difficulties in reaching simultaneously high purity and recovery of
both proteins. During ultrafiltration, β-Lg is normally recovered in the reten-
tate together with bovine serum albumin and immunoglobulins. To increase
the purity of β-Lg, primary removal of impurities (bovine serum albumin,
immunoglobulins) is then necessary. Simultaneously, α-La is recovered in
the permeate with high purity but often with low recovery yield. Almecija
et al. (2007), for instance, investigated the potential of membrane ultrafiltra-
tion for the fractionation of clarified whey. A 300 kDa tubular ceramic mem-
brane was used in a continuous diafiltration mode, but the highest permeate
yields for α-La and β-Lg were 56% and 33%, respectively, at a pH of 9. Muller
et al. (1999) also investigated the separation of α-La using different ultra-
filtration modes with a 300 kDa membrane. They show that performances
(purity, yield) depend on the operation mode and that it was possible, from
a clarified whey concentrates, to get a high purity of α-La (>95%) with sat-
isfactory yield (90%) provided a combined continuous concentration up to a
volume reduction ratio of 15 and diafiltration was carried out.
Membrane separations are then classically used in combination with other
unit operations. Membrane separations in association with a selective enzy-
matic digestion have been attempted for the isolation of α-La and β-Lg from
whey. Recently, isolation of α-La from sweet whey was achieved through an
approach involving membrane fractionation and tryptic treatment (Konrad
and Kleinschmidt, 2008). In the process, the serum proteins are first con-
centrated and fractionated using ultrafiltration membranes, followed by
tryptic hydrolysis of the permeate. In the conditions chosen (45°C, pH of 6.7
and enzyme/substrate ratio of 5 mAU/g), the α-La is slowly hydrolyzed by
trypsin, whereas the β-Lg is totally digested. After stopping the hydrolysis,
the concentration of the final hydrolysate in which bovine serum albumin
remains as the only impurity, it is possible to recover α-La in its native form
with a high purity of about 93% on the basis of total protein. In this process,
the main critical point remains in the correct termination of the tryptic deg-
radation: if the degradation of β-Lg is incomplete, β-Lg leaves as an impurity
and conversely, if the degradation is maintained, the trypsin attacks the α-La
immediately after the completion of β-Lg.
Another isolation technique is based on coupling precipitation and separa-
tion of the aggregates either using membrane separation or centrifugation.
The precipitation on either β-Lg or α-La can be performed under variation of
different environmental or process parameters.
The precipitation of β-Lg can be carried out by use of the limited solubility
of the protein at low ionic strength, pH 4.65, and high protein concentration

using ultrafiltration (Tolkach et al., 2005), by addition of chitosan (Casal et al.,

2006) or by addition of ammonium sulfate (Lozano et al., 2008). β-Lg interacts
reversibly with chitosan by electrostatic interaction, forming a precipitate at
pH 6.2. The β-Lg is then recovered by dissolving the precipitate in sodium
acetate at pH 9 to give a recovery of 90%, with a purity of 95% (Montilla et al.,
2007). The isolated β-Lg maintains its native structure and may be of inter-
est in industrial applications due to the non-toxic chitosan. β-Lg can also be
separated from others whey proteins by two successive precipitations with
50% and 70% ammonium sulfate (Lozano et al., 2008). After dissolution of
the final precipitate, the supernatant enriched in β-Lg is purified by weak
cation-exchange chromatography resulting in a purity up to 95%.
Better results can however be achieved using the selective and reversible
precipitation of α-La. As initially proposed by Pearce (1983, 1992), a heat
treatment combined with pH adjustment process has been developed to
selectively precipitate α-La and then fractionate α-La and β-Lg from whey.
α-La is a strong calcium-binding protein that contains one mole of calcium
per mole of protein. This protein loses its bounded calcium and its stability
when at ~55°C (30 min) pH is lowered to 3.8. At this pH, calcium is released
in the solution and α-La unfolds and precipitates at temperature between
50°C and 65°C while β-Lg remains in solution. Such physicochemical condi-
tions involve the precipitation of α-La together with immunoglobulins and
bovine serum albumin (Bramaud et al., 1997). The separation of the resulting
aggregates consisting of α-La and whey proteins other than β-Lg can be per-
formed by microfiltration or centrifugation. The precipitate enriched in α-La
is resolubilized by the addition of solvent (water) and pH adjustment and
the two forms of α-La [apo-(calcium-free protein) or holo (native with cal-
cium) forms] can be recovered depending of the solvent used: the apo-α-La
is obtained with a calcium-free solvent whereas the native form is obtained
with calcium solvent. The permeate or supernatant contains the β-Lg frac-
tion that can be then processed by UF in combination with diafiltration to
yield purified β-lactoglobulin (95% purity) prior to drying. Starting from the
permeate of milk microfiltration (0.1 µm) performed at temperature lower
than 45°C, this principle can be used to produce high purity nonlactosylated
β-Lg (Maubois et al., 2001). If highly purified β-Lg is obtained through this
process, there are still problems, to our knowledge, related to the purity of
the industrially recovered α-La, that can be recovered from the retentate/
sediment after solubilization at neutral pH, followed by UF. The purity
of α-La does not exceed 60%–70% due to the presence of some denatured
immunoglobulins and contamination by β-Lg and bovine serum albumin.
9.3.2.4.1.3 Other Techniques Recently, new technologies have also been

shown to produce enriched fractions of α-La and β-Lg (Bonnaillie and
Tomasula, 2008). Supercritical CO2 fractionation technology is one of
the methods proposed for the recovery of large-scale serum protein frac-
tions enriched with both α-La and β-Lg from either WPC or WPI. When

supercritical CO2 is injected into solutions containing soluble protein isolate

or serum protein concentrate, fractions containing 70% (w/w) of α-La in solid
form and 95% (w/w) of β-Lg in a soluble liquid form uncontaminated with
chemical additives have been obtained.
9.3.2.4.2 Lactoferrin and Lactoperoxidase

Lactoferrin (LF) and lactoperoxidase (LP) are the two proteins of the serum
having a basic isoelectric point, pI (Table 9.1) (Bonnaillie and Tomasula, 2008).
LF has been extensively investigated in both laboratory and industrial-scale
extraction technologies, but in most cases, LP has been co-eluted as a second-
ary product because of the similarity in their pI values.
Numerous chromatographic technologies are described to obtain enriched
or pure LF and LP fractions from both milk and whey (Fee et al., 2010). But
among them, cation-exchange chromatography of whey is the main one used
on an industrial scale. Owing to the complex nature of milk, the production
of high-value LF and LP requires extensive pre-treatments of milk to remove
fat and caseins by centrifugation, precipitation, and/or filtration, thus lead-
ing to a more commonly capture from whey.
Cation-exchange matrices might be routinely used in basic protein separa-
tions: at the pH of whey (or milk) around the neutrality, LF and LP are spe-
cifically adsorbed on cation exchanger, the other proteins being negatively
charged. Their elution is generally realized through the use of an increasing
ionic strength gradient.
Among the cationic resins (that include phosphocellulose and carboxymethyl
(CM) or sulfopropyl (SP) substituted cellulose, Sephadex and Sepharose),
CM-Sepharose (a weak cation exchanger) and SP-Sepharose (a strong cation
exchanger) resins (GE Healthcare, Sweden) are very well characterized for
high thorough isolation of basic proteins from milk and whey. During the
early development, CM-Sepharose resin was the most widely used matrix for
the isolation of LF from acid and rennet wheys at pH around 7.7, but in recent
times most LF purification methods investigated have used SP-Sepharose
strong cation-exchange resins (SP-Sepharose Fast-Flow™ or Big Beads™ and
Streamline™-SP resins). Several works have been done to optimize the yield
and purity of target protein fractions depending on the nature of the resin,
the starting fluids (whole milk, whey, bovine colostrum), and technology.
Etzel et al. (2000), for instance, optimized a chromatographic process using
SP-Sepharose Big Beads for the isolation of LF from skimmed milk with >90%
purity and 80% recovery in a single-step packed-bed system. Wu and Xu (2009)
reported a purification process for the isolation of LF and Immunoglobulin
G from bovine colostrum using a serial cation-anion exchange chromatog-
raphy system with 95% and 97% final purities, respectively. Anderson and
Matthiasson (2006) extracted pure lactoferrin from WPC using a simulated
moving bed chromatographic technology, with several advantages compared
to non-moving bed columns: increase in productivity by 48%; increase in LF
concentration by 6.5 times; reduction in buffer consumption by 4.3 times.

9.3.2.4.3 Immunoglobulins
Milk contains natural immunoglobulins, Igs, which can be isolated and con-
centrated, either from normal milk or from colostrum. Cow’s colostrum con-
tains substantially higher concentrations of immunoglobulins than mature
milk (20– 200 g/L against 0.15–0.8 g/L in milk) and then can be used as an
appropriate starting fluid for an immunoglobulins extraction procedure.
Immunoglobulin G is the most abundant class of immunoglobulins. It is an
important component of bovine milk, but also comprises the major protein
in bovine colostrum. Immunoglobulins from bovine milk have applications
as supplements in infant formulae, hyperimmune foods, functional foods,
nutraceutical, and pharmaceutical products. Commercial immunoglobulins
products are mostly used in veterinary medicine or neonatal ruminants and
pigs. Because ruminants are born without blood antibodies, they are very
susceptible to infection, and it is highly desirable that they receive protection
either by suckling colostrum for at least one week or by ingesting an immu-
noglobulin concentrate.
There are many processing methods available for separation of immuno-
globulins from various sources, including colostrum and milk; however,
appropriate technologies for large-scale productions are lacking. Traditionally,
isolation of Igs from colostrum has been achieved by precipitation with either
ammonium sulfate or ethanol, followed by chromatography, but this method
is feasible only in small-scale applications (El-Loly, 2007). The development in
membrane separation techniques made industrial-scale isolation of Igs from
various streams using either a single-step process or a combination of several
individual steps. Korhonen et al. (1998), for instance, used various filtration
techniques (reverse osmosis, UF, MF) and cation-exchange resins as a molec-
ular sieve, to concentrate Igs from colostral whey. The Igs level of the final
freeze-dried concentrates varied from 45% to 75%. Ion-exchange chromatog-
raphy can be used in the extraction process of immunoglobulins from dairy
feeds, either as an intermediate or as a final purification step in combination
with other chromatographic or membrane-processing methods.
UF membrane with a cut-off of about 100 kg/mol or more can be used to
isolate Igs from whey, but whey is a poor source of immunoglobulins com-
pared to colostrum or milk produced by hyperimmunized cows. Piot et al.
(2004) obtained enriched Igs fraction using MF and UF techniques: the colos-
trum was first microfiltered using a 0.1 µm pore membrane so as to obtain a
permeate (named “serocolostrum”) which is crystal clear, free of blood, and
somatic cells as well as fat globules and casein micelles. The permeate that
contained 80% of the initial immunoglobulins could then be further concen-
trated using ultrafiltration (100 kg/mol).
9.3.2.4.4 Caseinomacropeptide
The caseinomacropeptide, the C-terminal part of the κ-casein release in
cheese whey by the action of chymosin, has been demonstrated to have a

great variety of biological activities, including the modulation of gastroin-

testinal motility and regulation of the release of gastrointestinal hormones.
It has been shown also to stimulate secretions by the pancreas (Fosset and
Tomé, 2002). This peptide, in particular, contains no Phe, which makes it suit-
able for use as a nutritional protein supplement for patients suffering from
phenylketonuria, who did not digest protein with phenylalanine due to their
lack in the appropriate degrading enzyme.
Isolation and recovery of CMP were mainly shown to be separated from
sodium caseinate. Addition of chymosin to caseinate (obtained by alkalina-
tion of acid casein, Figure 9.3) makes it possible to release the CMP that can
be recovered by chromatography, in the permeate of ultrafiltration or in the
supernatant of centrifugation (Nakano and Ozimek, 2000). Using a two-step
chromatography (gel chromatography on Sephacryl S-200 at pH 7.0 to obtain
a crude CMP fraction; addition of acidic solution, pH 3.5, to the crude glyco-
macropeptide to precipitate contaminating protein and/or peptide; and re-
chromatography of the material soluble in the acidic solution on Sephacryl
S-200 at pH 3.5), Nakano and Ozimek (2000) obtained pure CMP. Their prepa-
ration was of considerably high purity with its amino-acid composition show-
ing only traces (each <1 residue/peptide) of arginine, histidine, phenylalanine,
and tyrosine, the amino acids that do not occur in CMP. More recently, Rojas
and Torres (2013) studied the opportunity to recover the CMP from cheese
whey using thermal treatments (90°C). The first results were attractive: at 90°C
treated sweet whey, 14, 20, and 41 kDa bands were observed, corresponding to
oligomers of the peptide. Its recovery showed an average of 1.5 g/L (34.08%).
These results indicate that industrial-scale CMP production is feasible; how-
ever, further research must be carried out for the biological and nutritional
evaluation of CMP’s incorporation to foodstuff as a supplement.
9.3.2.5 Fractionation of Whole Casein into Individual Casein

There is a considerable interest in fractioning whole casein into individual
caseins. Potential uses include bovine milk-based infant formulas and prepa-
ration of biologically active peptides and specific additives. The native casein
micelles retentate obtained from skimmed milk 0.1 µm MF constitutes an
excellent raw material for preparing individual caseins.
Most studies on the fractionation of the whole casein are related to the
isolation of β-casein, the main component of human casein, which contains
numerous peptide sequences with high physiological properties. The isola-
tion method is based on the preferential solubilization of the very hydro-
phobic β-casein at low temperatures. At ~4°C, β-casein dissociates from the
casein micelle and can be isolated from the rest of the caseins (caseinate,
renneted skimmed milk, etc.) using separation techniques such as UF and
MF or centrifugation. The yield of β-casein is enhanced at low pH (4.2–4.6)
and the β-casein purity is announced to reach 90% (Le Magnen and Maugas,

1991). The co-product (retentate or sediment fractions) is enriched in αs and

κ-caseins. Based on the same principle, a promising process to separate
β-casein directly from whole milk has recently been proposed (Lucey and
Smith, 2009). It operates in two successive MF steps both using polymeric
spiral wound membranes. The first one, operating at a low temperature,
separates β-casein from the retentate-containing casein micelles and fat.
β-Casein is then separated from the rest of the materials present in permeate
by warming the permeate to room temperature resulting in aggregation of
β-casein. The second MF retain the aggregated β-casein while native soluble
proteins passed into the membrane.
9.4 Conclusion
Milk is a unique source of numerous bioactive and functional molecules,
and it clearly appears that in the recent years, the dairy industry has moved
from a commodity food-based industry to one that has increasingly sought
added value products. Nutraceutical products extracted from milk with
implicit claims of health benefits are now common place. The exploitation
of their properties will continue to growth. Several minor milk components
with useful bioactivities (such as growth factors, oligosaccharides, minor
proteins, etc.) still need to be extracted to meet the consumer requirements.
Different techniques have classically been used for the purification of native
functional components, namely chromatography, membrane techniques, and
selective precipitation principles. Chromatography and membrane tech-
niques remain the most commonly used techniques for component extrac-
tion. Using a single technique shows limitations and specially results in a
trade-off between purity and yield. Therefore, there is now a growing interest
in coupling techniques, or coupling one of them with physicochemical treat-
ment. Large studies have also been performed on a laboratory scale and some
effort should be done to further investigate the suitability of the processes for
scale-up. Then the technical and economic feasibility of the coupled systems/
process will probably be one of the major challenges facing the fractionation
industry. The challenge is also to develop processes with both respect of the
native properties of components and total respect of the environment.
References
Almecija, M.C., Ibanez, R., Guadix, A., Guadix, E.M. 2007. Effect of pH on the frac-
tionation of whey proteins with a ceramic ultrafiltration membrane. Journal of
Membrane Science, 288: 28–35.

Anderson, J., Matthiasson, B. 2006. Simulated moving bed technology with a simpli-
fied approach for protein purification: Separation of lactoperoxidase and lacto-
ferrin from whey protein concentrate. Journal of Chromatography A, 1107(1–2):
88–95.
Astaire, J.C., Ward, R., German, J.B., Jimenez-Flores, R. 2003. Concentration of polar
MFMG lipids from buttermilk by microfiltration and supercritical fluid extrac-
tion. Journal of Dairy Science, 86: 2297–2307.
Bezelgues, J.B., Morgan, F., Palomo, G., Crosset-Perrotin, L., Ducret, P. 2009. Milk
fat globule membrane as a potential delivery system for liposoluble nutrients.
Journal of Dairy Science, 92, 2524–2528.
Bonnaillie, L., Tomasula, P.M. 2008. Whey protein fractionation. In Whey Processing,
Functionality and Health Benefits, ed. C.I. Onwulata and P.J. Huth. Wiley
Blackwell, Singapore, pp. 15–39.
Bouchoux, A., Gésan-Guiziou, G., Javier, P., Cabane, B. 2010. How to squeeze a
sponge casein micelles under osmotic stress, a SAXS study. Biophysical Journal,
99(11): 3754–3764.
Bramaud, C., Aimar, P., Daufin, G. 1997. Whey protein fractionation: Isoelectric
precipitation of α-lactalbumin under gentle heat treatment. Biotechnology
Bioengineering, 56(4): 391–397.
Burling, H., Graverholt, G. 2008. Milk—A new source for bioactive phospholipids for
use in food formulations. Lipid Technology, 20: 229–231.
Casal, E., Montilla, A., Moreno, F.J., Olano, A., Corzo, N. 2006. Use of chitosan for
selective removal of β-lactoglobulin from whey. Journal of Dairy Science, 89(5):
1384–1389.
Contarini, G., Povolo, M. 2013. Phospholipids in milk fat: composition, biological
and technological significance, and analytical strategies. International Journal of
Molecular Sciences, 14: 2808–2831.
Corredig, M., Roesch, R.R., Dalgleish, D.G. 2003. Production of a novel ingredient
from buttermilk. Journal of Dairy Science, 86: 2744–2750.
Dalgleish, D. 2011. On the structural models of bovine micelles—Review and possible
improvements. Soft Matter, 7(6): 2265–2272.
Dewettinck, K., Rombaut, R., Thienpont, N., Le, T.T., Messens, K., Van Camp, J. 2008.
Nutritional and technological aspects of milk fat globule membrane material.
International Dairy Journal, 18: 436–457.
El-Loly, M.M. 2007 Bovine milk immunoglobulins in relation to human health.
International Journal of Dairy Science, 2(3): 183–195.
El-Sayed, M.M.H., Chase, H.A. 2011. Trends in whey protein fractionation.
Biotechnology Letters, 33: 1501–1511.
Etzel, M.R. 1999. Isolating β-lactoglobulin and α-lactalbumin by eluting from cation
exchanger without sodium chloride. US Patent 5, 986,063.
Etzel, M.R., Helm, T.R., Vyas, H.K. 2006. Methods and compositions involving whey
protein isolates. US Patent 60, 569,078.
Etzel, M.R., Liten, A.D., Moore, P. 2000. Chromatographic capture of proteins from milk.
In 2nd International Conference on Expanded Bed Adsorption 1998, Whistler, Canada.
Farhang, B., Kakuda, Y., Corredig, M. 2012. Encapsulation of ascorbic acid in lipo-
somes prepared with milk fat globule membrane-derived phospholipids. Dairy
Fauquant, J., Maubois, J.L., Pierre, A. 1988. Microfiltration du lait sur membrane
minérale. Techniques laitières, 1028: 21–23.

Fauquant, J., Vieco, S. Brulé, G., Maubois, J.L. 1985. Clarification des lactosérums doux
par agrégation thermocalcique de la matière grasse résiduelle. Lait, 65: 1–20.
Fee, C.J., Billakanti, J.M., Saufi, S.M. 2010. Methods for purification of dairy nutraceu-
ticals. In Separation, Extraction and Concentration Processes in the Food Beverages
and Nutraceuticals Industries, ed. S.S.H. Rizvi. Woodhead Publishing Ltd.,
Oxford, pp. 450–482.
Fong, B.Y., Norris, C.S., MacGibbon, A.K.H. 2007. Protein and lipid composition of
bovine milk-fat-globule membrane. International Dairy Journal, 17: 275–288.
Fosset, S., Tomé, D. 2002. Nutraceuticals from milk. In Encyclopedia of Dairy Sciences,
1st edition. J.W. Fuquay, P.F. Fox and P.L.H. McSweeney. Elsevier, Academic
Press, Amsterdam, vol. 4, pp. 2108–2002.
Gänzle, M.G. 2011. Lactose: Derivatives. In Encyclopedia of Dairy Sciences, 2nd edi-
tion, ed. J.W. Fuquay, P.F. Fox and P.L.H. McSweeney. Elsevier, Academic Press,
Amsterdam, vol. 3, pp. 202–208.
Gapper, L.W., Copestake, D.E.J., Otter, D.E., Indyk, H.E. 2007. Analysis of bovine
immunoglobulin G in milk, colostrum and dietary supplements: A review.
Analytical and Bioanalytical Chemistry, 389(1): 93–109.
Gerberding, S.J., Byers, C.H. 1998. Preparative ion-exchange chromatography of pro-
teins from dairy whey. Journal of Chromatography A, 8008: 141–151.
Gernigon, G., Schuck, P., Jeantet, R. 2011. Demineralization. In Encyclopedia of Dairy
Science, 2nd edition, ed. J.W. Fuquay, P.F. Fox and P.L.H. McSweeney. Elsevier,
London, UK, 2011, vol. 4, pp. 738–743.
Gésan-Guiziou, G. 2010. Removal of bacteria, spores and somatic cells by centrifu-
gation and microfiltration techniques. In Improving the Safety and Quality of
Milk—Volume 1: Milk Production and Processing, ed. M.W. Griffiths, Woodhead
Publishing Ltd., Oxford, pp. 349–372.
Gésan-Guiziou, G., Boyaval, E., Daufin, G. 1999. Critical stability conditions in cross-
flow microfiltration of skimmed milk: Transition to irreversible deposition.
Journal of Membrane Science, 158: 211–222.
Haque, E., Chand, R., Kapila, S. 2009. Biofunctional properties of bioactive peptides
of milk origin. Food Reviews International, 25(1): 28–43.
Kamau, S.M., Cheison, S.C., Chen, W., Liu, X.-M., Lu, R.-R. 2010. Alpha-lactalbumin:
Its production technologies and bioactive peptides. Comprehensive Reviews in
Food Science and Food Safety, 9: 197–212.
Keenan, T.W., Mather, I.H. 2006. Intracellular origin of milk fat globules and the
nature of the milk fat globule membrane. In Advanced Dairy Chemistry: Lipids,
ed. P.F. Fox and P.L.H. McSweeney. Springer, New York, pp. 137–171.
Konrad, G., Kleinschmidt, T. 2008. A new method for isolation of native α-lactalbumin
from sweet whey. International Dairy Journal, 18: 47–54.
Korhonen, H., Pihlanto, A. 2007. Technological options for the production of health-
promoting proteins and peptides derived from milk and colostrum. Current
Pharmaceutical Design, 13: 829–843.
Korhonen, H., Syväoja, E.L., Vasara, E., Kosunen, T., Marnila, P. 1998. Pharmaceutical
composition, comprising complement proteins, for the treatment of Helicobacter
infections and a method for the preparation of the composition. PCT Patent
Application WO98/00150.
Küllenberg, D., Taylor, L.A., Scheinder, M., Massing, U. 2012. Health effects of dietary
phospholipids. Lipids in Health and Disease, 11: 1–16.

Le Berre, O., Daufin, G. 1996. Skimmilk crossflow microfiltration performance versus

permeation flux to wall shear stress ratio. Journal of Membrane Science, 117: 261–270.
Le Magnen, C., Maugas, J.J. (EURIAL) 1991. Method and device for obtaining beta
casein. Patent PCT/FR 91/00506.
Lopez, C. 2011. Milk fat globules enveloped by their biological membrane: Unique
colloidal assemblies with a specific composition and structure. Current Opinion
in Colloids and Interface Science, 16: 391–404.
Lozano, J.M., Giraldo, G.I., Romero, C.M. 2008. An improved method for isolation of
β-lactoglobulin. International Dairy Journal, 18(1): 55–63.
Lucey, J., Smith, K. 2009. An integrated processing system to produce beta-casein,
native serum protein and casein concentrates from whole milk. Journal of Dairy
Science, 92(E-Suppl. 1): 164.
Madureira, A.R., Pereira, C.I., Gomes, A.M.P., Pintado, M.E., Malcata, F.X. 2007.
Bovine whey proteins—Overview on their main biological properties. Food
Research International, 40: 1197–1211.
Mattila-Sandholm, T., Saarela, M. 2003. Functional Dairy Products. Woodhead
Publishing Limited, Boca Raton, FL: CRC Press.
Maubois, J.L., Fauquant, J., Famelart, M.H., Caussin, F. 2001. Milk microfiltrate, a con-
venient starting material for fractionation of whey proteins and derivatives. In
Proceedings of the 3rd International Whey Conference, Munich September 12–14,
Behr’s Verlag, Hamburg.
Maubois, J.L., Ollivier, G. 1997. Extraction of milk proteins. In Foods Proteins and
Their Applications, ed. S. Damodaran and A. Paraf. Marcel Dekker, New York,
pp. 579–595.
Montilla, A., Casal, E., Javier Moreno, F., Belloque, J., Olano, A., Corzo, N. 2007.
Isolation of bovine β-lactoglobulin from complexes with chitosan. International
Dairy Journal, 17(5): 459–464.
Muller, A., Daufin, G., Chaufer, B. 1999. Ultrafiltration modes of operation for the separa-
tion of α-lactalbumin from acid casein whey. Journal of Membrane Science, 153: 9–21.
Nakano, T., Ozimek, L. 2000. Purification of glycomacropeptide from caseinate
hydrolysate by gel chromatography and treatment with acidic solution. Journal
of Food Science, 65(4): 588–590.
Outinen, M., Tossavainen, O., Syväoja, E.L. 1996. Chromatographic fractionation
of α-lactalbumin and β-lactoglobulin with polystyrenic strongly basic anion
exchange resins. Lebens Wissenschaft Techn, 29(4): 340–343.
Pearce, R.J. 1983. Thermal separation of beta-lactoglobulin and alpha-lactalbumin
in bovine Cheddar cheese whey. Australian Journal of Dairy Technology, 38:
144–148.
Pearce, R.J. 1992. Protein recovery and whey protein fractionation. In Whey and Lactose
Processing, ed. J.G. Zadow. Elsevier Applied Science: London, pp. 271–316.
Pearce, R.J., Marshall, S.C., Dunkerley, J.A. 1992. Reduction of lipids in whey pro-
tein concentrates by microfiltration. International Dairy Federation, Special issue,
9201: 118–129.
Pierre, A., Fauquant, J., Le Graet, Y., Piot, M., Maubois, J.-L. 1992. Native micellar
casein separation through crossflow membrane microfiltration. Lait, 72(5):
461–474.
Piot, M., Fauquant, J., Madec, M.N., Maubois, J.-L. 2004. Preparation of “serocolos-
trum” by membrane microfiltration. Lait, 84: 333–342.

Rojas, E., Torres, G. 2013. Isolation and recovery of glycomacropeptide from milk
whey by means of thermal treatment. Food Science and Technology, 33(1): 14–20.
Sachdeva, S., Buchheim, W. 1997. Recovery of phospholipids from buttermilk using
membrane processing. Kieler Milchwirtschaftliche Forschungsberichte, 49: 47–68.
Sandblöm, R.M. (Alfa-Laval). 1974. Filtering process Swedish Patent No. 7,416,257.
Schuck, P., Piot, M., Méjean, S., Le Graet, Y., Fauquant, J., Brulé, G., Maubois, J.L. 1994.
Spray drying of native phosphocaseinate obtained by membrane microfiltra-
tion. Lait, 74(5): 375–388.
Severin, S., Xia, W.S. 2005. Milk biologically active components as nutraceuticals:
Review. Critical Reviews in Food Science and Nutrition, 45(7–8): 645–656.
Singh, H. 2006. The milk fat globule membrane—A biophysical system for food appli-
cations. Current Opinion in Colloid & Interface Science, 11(2–3): 154–163.
Sodini, I., Morin, P., Olabi, A., Jiménez-Flores, R. 2006. Compositional and functional
properties of buttermilk: A comparison between sweet, sour, and whey butter-
milk. Journal of Dairy Science, 89: 525–536.
Spitsberg, V.L. 2005. Bovine milk fat globule membrane as a potential nutraceutical.
Journal Dairy Science, 88: 2289–2294.
Tolkach, A., Steinle, S., Kulozik, U. 2005. Optimization of thermal pretreatment condi-
tions for the separation of native α-lactalbumin from whey protein concentrates
by means of selective denaturation of β-lactoglobulin. Journal of Food Science, 70:
E557–E566.
Turhan, K.N., Etzel, M.R. 2004. Whey protein isolate and alpha-lactalbumin recovery
from lactic acid whey using cation-exchange chromatography. Journal of Food
Science, 69(2): E66–E70.
Vanderghem, C., Bodson, P., Danthine, S., Paquot, M., Deroanne, C., Blecker, C. 2010.
Milk fat globule membrane and buttermilks: From composition to valorization.
Biotechnologie, Agronomie, Société et Environnement, 14: 485–500.
Walstra, P., Wouters, J.T.M., Geurts, T.J. 2006. Dairy Science and Technology. CRC Press,
Boca Raton, FL, USA.
Ward, R.E., German, J.B., Corredig, M. 2006. Composition, applications, fraction-
ation, technological and nutritional significance of milk fat globule membrane
material. In Advances Dairy Chemistry. Lipids, 3rd edition, ed. P.F. Fox and P.L.H.
McSweeney. Springer, New York, pp. 213–244.
Wu, M.B., Xu, Y.J. 2009. Isolation and purification of lactoferrin and immunoglobulin
G from bovine colostrum with serial cation-anion exchange chromatography.
Biotechnology Bioprocess Engineering, 14(2): 155–116.

Section II
Nano-Microencapsulation,
Delivery System, and
Packaging Technologies

10
Microencapsulation of Food
Ingredients for Functional Foods
Siew Young Quek, Qiong Chen, and John Shi
CONTENTS
10.1 Introduction................................................................................................. 267
10.2 Microencapsulation.................................................................................... 268
10.2.1 Technology....................................................................................... 268
10.2.2 Wall Materials................................................................................. 269
10.2.2.1 Selection of Wall Materials............................................. 269
10.2.2.2 Common Wall Materials................................................. 270
10.2.3 Methods............................................................................................ 270
10.3 Microencapsulation of Lipophilic Bioactive Components.................... 272
10.3.1 Emulsion Types for Delivering Lipophilic Components.......... 278
10.3.1.1 Emulsion Characteristics................................................ 281
10.3.1.2 Emulsion Stability............................................................ 283
10.3.1.3 Emulsifiers and Their Role in Bioactive
Delivery Systems..............................................................284
10.3.1.4 Emulsification Techniques and Conditions................. 290
10.3.2 Drying.............................................................................................. 291
10.3.2.1 Drying Techniques.......................................................... 291
10.3.2.2 Factors Influencing the Performance of
Spray Drying..................................................................... 294
10.4 Current Challenges for Delivering LBCs................................................ 296
10.5 Co-Encapsulation: Merits and Implication............................................. 298
10.6 Concluding Remark....................................................................................300
References.............................................................................................................. 301
10.1 Introduction
Public awareness of high-quality, nutritious foods and their effects on human
health has risen sharply over the years. Well embroiled within the evolution
of consumer food preferences are functional foods and their growing role as
healthy foods for general well-being and disease prevention. The develop-
ment of functional foods has been driven by mounting evidence of health
267
and food relationships as documented in scientific studies. The increasing

pressure on the food industry to produce healthy food products has, as a
result, unveiled the challenge of processing functional ingredients in a way
that meets the requirements of the modern-day consumer.
The major issues limiting the use of functional ingredients without prior
processing include vulnerability to oxidative breakdown, limited water or
lipid solubility, poor thermal stability, and poor taste. Unprocessed, these
ingredients, such as bioactive compounds, are essentially unsuitable for use
as food ingredients due to any of the aforementioned reasons. This leads to
the application of microencapsulation technology as one of the methods in
the production of food ingredients for functional food application.
By utilizing microencapsulation technology, it is possible to produce func-
tional ingredients with desirable properties. Such properties include increas-
ing the shelf-life of an ingredient by protecting from oxidation or masking
undesirable odors, color, and taste of the original ingredient, altogether cre-
ating a product that is viable for commercial use. Other important reasons
include controlling the release of core materials at specific locations, protec-
tion of core materials during processing stages, introducing new colors, fla-
vors, or textures to ingredients, improving or maximizing dispersibility and
solubility of the bioactive in powder form, and improving powder properties
for ease of handling during processing.
The goal of this chapter is to provide readers with an understanding of the
application of microencapsulation processes to produce functional ingredi-
ents for foods. The prevalence of good review articles on microencapsulation
makes it unnecessary to commit heavily to explaining basic principles and
components of microencapsulation processes. Instead, the focus is placed on
the microencapsulation of lipophilic bioactive components (LBCs) and the
development in the area.
10.2 Microencapsulation
10.2.1 Technology
Microencapsulation can be defined as the process of surrounding or envel-
oping small solid particles, liquid droplets, or gas in small capsules ranging
from 1 to 1000 μm (Champagne and Fustier, 2007; Nazzaro et al., 2012). This
technology allows sensitive ingredients to be enveloped as a “core” material
with a polymer matrix or “wall material.” The development of microencap-
sulation products started in the 1950s by National Cash Register Company
(USA) in the research into pressure-sensitive coatings for the manufacture
of carbonless copying paper (Green and Scheicher, 1955). This technology
is now well developed and widely used within different areas, including

Microencapsulation of Food Ingredients for Functional Foods 269
pharmaceutical, chemical, cosmetic, food, and printing industries (Heinzen,

2002). In foods, microencapsulation is currently widely used in the process of
delivering health-promoting functional ingredients aiming to protect ingre-
dients from the environment during both processing and storage, to enhance
the ingredient bioavailability and efficacy (Hogan et al., 2003; Kagami et al.,
2003; Drusch et al., 2006), and to extend flavor perception and mouthfeel over
a longer period of time as well (Anandaraman and Reineccius, 1986; Bangs
and Reineccius, 1988; Kim and Morr, 1996; Shefer and Shefer, 2003). Over
the last few decades, microencapsulation has been extensively studied for
delivery of bioactive components into foods, both water-soluble and lipid-
soluble, including probiotics, dietary lipid and fatty acids, flavors, colorants,
polyphenols, enzymes, vitamins, and minerals.
10.2.2 Wall Materials

The surrounding coating of a microcapsule is often chosen for its protective
abilities when encasing a bioactive compound while in other cases, the deci-
sion is primarily based on making the food ingredient palatable or commer-
cially viable. The choice of wall material ultimately determines the stability
of the microcapsule and thus careful consideration must be given toward the
final coating choices for successful microcapsule formation.
10.2.2.1 Selection of Wall Materials

There are many factors to consider when choosing a suitable wall material
for a specific microencapsulation system. Invariably, within the food indus-
try, microencapsulation is used to protect the food ingredient in addition to
other requirements. After all, a wall that is unstable and cannot protect the
core is useless. As such, the stability of the wall material at different process-
ing environments (e.g., pH and heat) is a critically important requirement.
In addition to stability, a wide range of requirements beyond just stable film
formation must be met in order to satisfy the rigors of the food industry and
the expectations of the consumer.
One of the considerations is the solubility of core and wall materials.
For example, microencapsulated beverage powders containing a bioactive
must have water-soluble wall materials to facilitate the dissolution process
in water. However, a bioactive compound that is intended to be released at
the intestinal tract should preferably have water-insoluble wall material and
enteric coating. The emulsification ability of the wall materials is a crucial
consideration for microencapsulation of LBCs to improve water solubility of
ingredients for application in food products.
Suitable wall materials should also have good film formation properties
to protect the core materials and give high encapsulation efficiency. Good
barrier properties such as oxygen nonpermeability are important to ensure
the stability of the core material during storage. The rheological properties

of wall materials, including viscosity and shear strength, are also impor-
tant considerations. For example, low viscosity is beneficial for spray drying/
chilling processes.
Furthermore, one should also consider if there will be any interaction
between the wall and core materials. For cases like coacervation or inclu-
sion complex formation with cyclodextrins, the interaction between the wall
and core materials is important for the stability of products. Other important
factors to consider include the taste profile of the wall material. Preferably,
wall materials should have a neutral taste so they do not influence the fla-
vor profile of the food products. Cost and availability of the wall materials
are also important considerations. Unlike the pharmaceutical industry, the
profit margin in the food industry is not very high, so the materials should
preferably be readily available and inexpensive. Finally, but very crucially,
the wall materials must be of food grade.
10.2.2.2 Common Wall Materials

When deciding on a suitable compound to be used as the wall material for
microencapsulation, care must be taken to ensure that the design require-
ments of the final product are met and the wall material is suitable for the
core material in question. This is particularly true in the food industry due
to strict regulations preventing the usage of many compounds common with
the pharmaceutical industry, which is also a heavy user of microencapsu-
lation techniques (Wandrey et al., 2010). This sometimes results in a chal-
lenging situation for food processors aiming to apply microencapsulation for
protecting bioactive ingredients. Despite the many requirements that must
be met for a successful wall material, there are a limited number of legally
permissible choices among wall materials to meet these requirements.
Commonly available for use within the food industry are carbohydrates,
proteins, and lipids, and derivatives/complexes of these compounds. These
food-grade materials are able to be used singly or in conjunction with other
materials to produce a suitable microcapsule encasement. A brief overview
of each wider family of wall materials is provided in Table 10.1, along with a
brief description of their common properties.
10.2.3 Methods
There are many ways to prepare microcapsules, including physical, chemical,
and physicochemical techniques (Uhlemann et al., 2002). Physical methods
include spray drying, spray chilling/cooling, fluidized bed coating, extrusion,
freeze drying, and co-crystallization (Gibbs et al., 1999; Gouin, 2004; Desai
and Park, 2005). Interfacial polymerization and molecular inclusion are chem-
ical methods for encapsulation (Desai and Park, 2005). Physicochemical meth-
ods mainly include coacervation, liposome entrapment, and organic phase
separation (Shahidi and Han, 1993; Gibbs et al., 1999; Desai and Park, 2005).

TABLE 10.1
Common Types of Wall Materials and Their Properties
Wall Material Common Properties Common Examples
Carbohydrates Gums (good emulsifiers) Gums
Alginates—Varying water Alginates: Chávarri et al. (2010);
solubility Homayouni et al. (2008)
Agar—Strong gelling agent, Agar: Champagne (2013)
water soluble
Gum arabic—Strong film former, Gum arabic: Chranioti and Tzia
low viscosity in aqueous (2014); Samborska and Czelejewska
solutions, water soluble (2014)
Starches (widely available) Starches
Dextrins—Emulsifier, strong film Dextrins: Ahn et al. (2008)
former, water soluble
Maltodextrin—Nonsweet, good Maltodextrin: Thirundas et al. (2014);
oxidation protection, water Wang et al. (2014)
soluble
Cyclodextrins—Forms stable Cyclodextrins: da Rosa et al. (2014);
inclusion complexes, poor water Szente and Szejtli (2004); Tao et al.
solubility (2014)
Celluloses—Widely available, Celluloses (including methyl/ethyl/
water insoluble hydroxypropylcellulose):
Koupantsis et al. (2014); Setchell
et al. (2005)
Others
Chitosan—Gelling agent, Chitosan: Bastos et al. (2012);
emulsifier, strong film former, Donhowe and Kong (2014); No et al.
water insoluble (2007)
Inulin—Prebiotic, moderately Inulin: Fernandes et al. (2014); Franck
water soluble (2002)
Proteins Gelatine—Gelling agent, water Gelatine: Soukoulis et al. (2014);
soluble, inexpensive Wang et al. (2014)
Whey proteins—Gelling agent, Whey proteins: Chen et al. (2013)
emulsifier, water soluble
Soy proteins Soy proteins: Gao et al. (2014);
Nesterenko et al. (2014)
Caseinates—Emulsifier, strong Caseinates: Hogan et al. (2001);
film former, water soluble Srinivasan et al. (1999)
Lipids Plant oils—Low boiling Plant oils: Alvim et al. (2013); Wilson
temperature, water insoluble and Shah (2007); Zoet et al. (2011)
Waxes—Water impermeable; Waxes (including beeswax and
water insoluble carnauba wax): Blanco-Pascual et al.
(2014); Drusch and Mannino (2009);
Kuang et al. (2010)
Phospholipids—Liposomal Phospholipids: Alvim et al. (2013);
formation; water insoluble Moraes et al. (2013)

Simple Multicore Matrix Multiwall Irregular
FIGURE 10.1
Structures of different types of microcapsules. (Modified from Gharsallaoui, A. et al. 2007. Food
Research International, 40: 1107–1121.)
The selection of the method depends on the economics of processing, core

sensitivity, requirements in particle size of microcapsules, physicochemi-
cal properties of core and wall materials, application, and release properties
(Shahidi and Han, 1993). Depending on the physicochemical properties of the
core, the wall composition, and the used microencapsulation technique, dif-
ferent types of particles can be obtained as shown in Figure 10.1.
Some of the encapsulated food ingredients prepared by preferred microen-
capsulation techniques are summarized in Table 10.2. The two major indus-
trial processes are spray drying and extrusion (Madene et al., 2006). However,
extrusion normally forms particles of rather large size (200–2000 µm)
(Uhlemann et al., 2002), which limits the application of extruded ingredients
where mouthfeel is an important factor (Gharsallaoui et al., 2007). Another
common method, freeze drying, has been reported to have advantages in the
encapsulation of heat-sensitive components (Schwegman, 2009).
10.3 Microencapsulation of Lipophilic Bioactive Components

Currently, there is a high level of interest in LBCs, such as omega-3 fatty acids,
phytosterols, limonene, carotenoids, etc., because of their potential health
benefits. Among these, fish oil is known to be a rich source of omega-3 long-
chain polyunsaturated fatty acids (PUFAs), containing both d ocosahexaenoic
acid (DHA) and eicosapentaenoic acid (EPA). Several health benefits, such as
ameliorative effects on brain and nervous systems, and prevention of cer-
tain diseases, including diabetes, inflammatory and autoimmune disorders,
coronary artery disease, thrombosis, cardiac arrhythmias, and hyperten-
sion, have been associated with the regular consumption of omega-3 fatty
acids (Gurr, 1999; Newton, 2001). Research also shows that the consumption
of fish oil was associated with a reduced risk from all-cause, ischemic heart
disease, and stroke mortality across 36 countries (Zhang et al., 1999). One
important LBC, phytosterol, has been shown to decrease the serum choles-
terol level and the ratio of the low-density lipoprotein (LDL) to high-density

TABLE 10.2
Microencapsulation of Food Ingredients for Functional Foods

Microencapsulation of Bioactive Food Ingredients by Frequently Used Techniques in Food Industry
Core
Frequently Used Technique
Category Examples Wall for Microencapsulation References
Acidulants Ascorbic acid Gelatin, ethylcellulose, Fluidized-bed coating, extrusion Knezevic et al. (1998); Chang et al.
maltodextrin/soy lecithin (2010); Xie et al. (2010)
Sorbic acid Triglyceride
Calcium propionate Glycerol monostearate Nie et al. (2010)
Sodium chloride Vegetable oils, HPMC Li et al. (2010)
Flavoring agents Citrus oil Gum arabic, gum arabic/ Inclusion complexation, Kim and Morr (1996); Mahajan et al.
maltodextrin/modified starch extrusion, centrifugal (2007); Badee et al. (2011)
extrusion, spray drying
Mint oils Modified starch, hydrolyzed Baranauskiene et al. (2007)
starch
Onion oils β-Cyclodextrin Chen and Liu (2011)
Garlic oils β-Cyclodextrin Ayala-Zavala et al. (2008)
Spice oleoresins Maltodextrin/pea glucose Krishnan et al. (2005); Boursier (2014)
syrup, skim milk, gum arabic/
maltodextrin/modified starch
Sweeteners Sugars Stevia glycoside Cocrystallization, fluidized-bed Bipin et al. (2011)
coating
Nutritive sugars Sucrose syrup, gum arabic, Blonde (1969); Goodacre et al. (1989);
(aspartame) monoglycerides/triglycerides Atchley (2010)
Colorants, Annatto Gelatin/carbohydrates, PHBV Extrusion, emulsion Winning (1995); Teixeira et al. (2008)
carotenoid
β-Carotene Corn starch, chitosan/SSPS Berset and Marty (1992); Hou et al.
(2012)
Turmeric Gelatin/carbohydrates, soy Winning (1995); Tapal and Tiku (2012)
protein isolate
(Continued)
273
274
Core
Astaxanthin Whey protein isolate, soluble Emulsification and spray drying Shen and Quek (2014)
corn fiber
Lipids Fish oil NaCA/lactose/maltodextrin, Spray drying, freeze drying, Heinzelmann and Franke (1999);
barley protein vacuum drying Wang et al. (2011)
Linolenic acid Maltodextrin/gum arabic, zein Watanabe et al. (2004); Quispe-
Condori et al. (2011)

Rice bran oil CMC/whey protein Slater (2007)
Phytosterols Slater (2007); Hendrickson et al.
(2009)
Olive oil Sodium caseinate and gelatin; Emulsification and spray drying Calvo et al. (2010)
gum arabic; starch, lactose,
and maltodextrin
Fish oil Gelatine Coavervasion Barrow et al. (2009)
Fish oil Chitosan, maltodextrin, and Emulsification, ultrasonic Klaypradit and Huang (2008)
whey protein isolate atomization and freeze drying
Sunflower oil Dextrin and milk protein isolates Emulsion and spray drying Ahn et al. (2008)
Avocado oil Whey protein isolates and Emulsification and spray drying Bae and Lee (2008)
maltodextrin
Fish oil Cellulose and maltodextrin Emulsification and spray drying Kolanowski et al. (2006)
Fish oil Starch, glucose, and trehalose Emulsification and spray drying Drusch et al. (2006)
Fish oil Cellulose Emulsification and spray drying Kolanowski et al. (2004)
(Continued)

Core
Linoleic acid Whey protein isolates and Emulsification and spray drying Jimenez et al. (2004)
maltodextrin
Olive oil Sodium caseinate and gelatin; Emulsification and spray drying Calvo et al. (2010)
gum arabic; starch, lactose
and maltodextrin
Fish oil Gelatine Coavervasion Barrow et al. (2009)
Fish oil Chitosan, maltodextrin, and Emulsification, ultrasonic Klaypradit and Huang (2008)
whey protein isolate atomization and freeze drying
Polyphenols Cocoa polyphenols Starch Spray drying Vitaglione et al. (2013)
Gallic acid Chitosan, cyclodextrin, and Lyophilization Nunes et al. (2013)
xanthan
Sour cherry Maltodextrin, gum arabic Emulsification, freeze drying Cilek et al. (2012)
polyphenols
Bayberry Cellulose Emulsification, spray drying Zheng et al. (2011)
polyphenols
Black currant Maltodextrin and inulin Spray drying Bakowska-Barczak and Kolodziejczyk
polyphenols (2011)
Raspberry leaf, Sodium alginate and chitosan Extrusion Belščak-Cvitanović et al. (2011)
hawthorn, ground
ivy, yarrow, nettle,
and olive leaf
extracts
(Continued)
275
276
Core
Pomegranate Maltodextrin and soybean Spray drying Robert et al. (2010)
polyphenols protein isolates
Cactus pear pulp Maltodextrin and inulin Spray drying Saenz et al. (2009)
and ethanolic
exracts
Vitamins and Fat soluble (A, D, E, Gelatin/acacia, β-cyclodextrin/ Coacervation, Banville et al. (2000); Junyaprasert
minerals K) HP β-cyclodextrin, PG/PS80/ inclusion complexation, spray et al. (2001); Munoz-Botella et al.
proliposome, CMC/chitosan drying, liposome entrapment (2002); Alencastre et al. (2006); Zhao

et al. (2011)
Water soluble (C, PIB/EC, gum arabic/ Koida et al. (1984); Taxi et al. (2003);
B1, B2, B6, B12, maltodextrin, ZBP, PEG/ Gabizon et al. (2004); Onal and
niacin, folic acid) phospholipids, Langdon (2005)
Enzymes and Lipase, invertase, Egg PC, alginate, Coacervation, spray method, Fugman et al. (1984); Dale et al.
microorganisms amylase, protease polyelectrolyte, sucrose/ liposome entrapment (1997); Kokufuta (1998); Stefan and
starch/hydrolyzed PVA Bahrim (2007)
Probiotics Lactobacillus Maltodextrin Spray drying Anekella and Orsat (2013)
acidophilus, L.
rhamnosus
L. reuteri Whey protein isolates Spray drying Jantzen et al. (2013)
L. reuteri Alginate and chitosan Spray drying Malmo et al. (2013)
(Continued)

Core
L. gasseri, Sodium alginate coated with Extrusion Chávarri et al. (2010)
Bifidobacterium chitosan
bifidum
L. plantarum Calcium alginate coated with Extrusion and freeze drying Gbassi et al. (2009)
whey proteins
L. paracasei, Milk proteins Emulsification and Heidebach et al. (2009)
B. lactis centrifugation
L. casei, Calcium alginate Emulsification Homayouni et al. (2008)
B. lactis
L. acidophilus Sodium alginate Emulsification and freeze drying Kim et al. (2008)
L. acidophilus Calcium alginate Extrusion Chandramouli et al. (2004)
Abbreviation: PHBV = poly(3-hydroxybutyrate-co-3-hydroxyvalerate); HPMC = hydroxypropyl methylcellulose; SSPS = soybean soluble polysaccha-
rides; CMC = carboxymethyl cellulose; HP = hydroxypropyl; PG = propylene glycol; PS80 = polysorbate80; PIB = polyisobutylene; EC = Et
cellulose; PEG = polyethylene glycol; ZBP = zein-bound particle; PC = phosphatidylcholine; PVA = polyvinyl alcohol.
277
lipoprotein (HDL)-bound cholesterol in serum (Mattson and Grundy, 1982).

Other LBCs, such as limonene, have various health benefits, including che-
mopreventive activity against cancers, gastric-acid-neutralizing effect, sup-
porting normal peristalsis, and dissolving cholesterol-containing gallstones
(Sun, 2007). Nevertheless, because of their highly unsaturated nature, these
LBCs are susceptible to oxidants, light, and heat, resulting in poor-quality
products with reduced health-promoting properties. The highly lipophilic
nature of these compounds also results in poor water solubility and absorp-
tion, and these factors limit their use in functional foods.
A wide range of different types of delivery system can be applied to micro-
encapsulate the lipophilic components, and emulsion technology is found
to be a suitable one. The reason is that emulsion-based delivery systems
could meet the requirements to incorporate LBCs into food. The delivery
systems, first, should comprise entirely of permitted and inexpensive food
ingredients; second, they should be able to protect the encapsulated nutri-
ents against chemical degradation; third, they should allow for precise con-
trol over the release of encapsulated components during mastication and
digestion to maximize adsorption; fourth, they should enhance or at least
not adversely affect the bioactivity of the encapsulated components; and
finally, they should not adversely affect the properties of the surrounding
matrix, such as appearance, texture, flavor, and stability of the final product
(McClements et al., 2007). To achieve a suitable formulation, a sound under-
standing of the physicochemical properties of emulsion systems and interac-
tions between emulsifier components is required.
Emulsions, which are in liquid form, have a limitation to their application
in food, since some food systems are in dry bases, such as breakfast cereals,
chocolate, etc. Moreover, LBCs encapsulated in emulsion forms are not as sta-
ble as in dry powders. Drying emulsions into powders is a method to make the
microencapsulated LBCs more stable during storage and extend their appli-
cation in food systems. Existing drying methods, such as spray drying, are
widely used to form microencapules from emulsion due to its advantages and
this technology is suitable to be adopted in the food industry. On the other
hand, it was hypothesized that freeze drying may have advantages over spray
drying for sensitive components because of its low temperature and vacuum
circumstances; thus, freeze drying is also considered to be a suitable method.
The process of microencapsulation of LBCs by spray and freeze drying
consists of two steps: the first is often emulsification of a core material, with
a dense solution of wall materials. The second is drying or cooling of the
emulsions. Thus, not only is the drying process important but the emulsion
technique is also quite crucial.
10.3.1 Emulsion Types for Delivering Lipophilic Components

An emulsion is a suspension of one phase in another in which it is immis-
cible, with one of the phases existing as discrete droplets suspended in the

second phase, and it contains an interfacial layer between the two phases,
including substances known as surfactant material (Dalgleish, 2004). The
substance making up the droplets is called dispersed phase whereas the
substance making up the surrounding liquid is referred to as the continu-
ous phase. Generally, the mean diameter of the droplets in emulsified food
products is somewhere in the range of 0.1–100 μm (McClements, 2007). It is
common practice to describe an emulsion as being oil-in-water (O/W) or
water-in-oil (W/O), where the first phase mentioned is the dispersed phase
and the second phase mentioned is the continuous phase. To deliver LBC oil
into an aqueous food system, an O/W emulsion system is considered.
According to the spatial organization of the oil and water phases, O/W
emulsion can be classified into several types, including single-layer oil-
in-water (O/W), water-in-oil-in-water (W/O/W), multilayer oil-in-water
(M-O/W), and solid lipid particle (SLP) emulsions (Figure 10.2). Single-layer
O/W emulsion system is the basic type of emulsion that is important and
potentially used in foods. The oil droplets are dispersed in an aqueous con-
tinuous phase and only one layer of emulsifiers around the droplet surface
(Dalgleish, 2004). The delivery systems contain some potential advantages:
first, emulsions can be made completely from food-grade ingredients and
can be created by using simple processing operations; second, it could be
possible to deliver the components that are polar, nonpolar, and amphiphilic
within the same system (McClements, 2005b); third, emulsions can be used
either directly in wet or in dried powders, which may facilitate the efficiency
of their transport and utilization (Soottitantawat et al., 2003; Desai and Park,
2005). Because of its ease of preparation and low cost, this technology has
been widely used in food industries although it still contains certain limi-
tations, for example, physical instability when exposed to environmental
stresses, for example, change of pH, modification of temperature, and high
mineral concentrations; a limited type of emulsifier that can be used to form
the interfacial layer that limits the protective ability of the system to a small
range (Dalgleish, 2004). In recent studies, more complex emulsifier systems
Single layer Water-in-oil-in-water Multilayer-oil-in-water Solid lipid particle

Oil-in-water (O/W) (W/O/W) (M-O/W) (SLP) emulsions
FIGURE 10.2
Different types of structured O/W emulsion systems. (Modified from McClements, D.J. 2010.
Annual Review of Food Science and Technology, 1: 241–269.)

(combination of different components by either physical blend or chemical

reaction), for example, Maillard reaction products (MRPs), have been utilized
to improve the properties of O/W emulsion system to protect the delivered
ingredients (Chung et al., 2008). They are also applied to control the release
rate of active agents in response to specific environmental triggers (e.g., pH,
salt concentration, and temperature) due to the ability to manipulate the
thickness and properties of the interfacial layer (Chung et al., 2008).
Apart from the simple O/W emulsions, there is an increase in the application
of various types of emulsions in the food industry for LBCs in recent years,
which are composed of more sophisticated configuration. The first type of
emulsion is called multiple emulsions or double emulsions, for example, water-
in-oil-in-water (W/O/W) emulsions, which are emulsions within emulsions.
It is described as oil droplets entrapping microsized water droplets, which
is dispersed in a bulk aqueous phase, and requires at least two stabilizing
surfactants (Garti and Bisperink, 1998; McClements et al., 2007; Morais et al.,
2008). Another complex emulsion delivery system is multilayer oil-in-water
emulsions (M-O/W), which consists of oil droplets surrounded by multilayer
interfacial membranes, comprising an ionic inner emulsifier layer to produce
a “primary” emulsion and one or more oppositely charged biopolymer layers
to produce a “second” or “third” emulsion (Güzey and McClements, 2006a;
Shi et al., 2015). SLP emulsions are similar to simple O/W emulsions consist-
ing of oil droplets coated with emulsifier suspended in an aqueous continuous
phase. However, the lipid phase is fully solid or partially contains solid lipid
with mean diameters ranging between 50 and 1000 nm (Battaglia et al., 2007).
Some research have stated that complex emulsion systems have some
potential merits. For example, W/O/W emulsion has a highly protective abil-
ity to chemical degradation of functional components caused by interaction
with other water-soluble ingredients (Cournarie et al., 2004). M-O/W emul-
sion could have better resistance to the environmental stresses, for example,
chilling, freezing, dehydration, thermal processing, or mechanical agitation
by controlling the composition and properties of the interfacial layer (Ogawa
et al., 2003; Gu et al., 2005; Harnsilawat et al., 2006; Shi et al., 2015). SLP emul-
sion has the ability to improve the physical stability of the emulsion-based
delivery system by trapping solid LBCs within a solid lipid matrix (Shah
et al., 2007). All these systems have an excellent and exciting opportunity for
slow or controlled release of active entrapped compound (Cournarie et al.,
2004; McClements et al., 2007; Shah et al., 2007). Nevertheless, they have
various drawbacks, which limit their application in industry processing.
Multiple emulsions are relatively complex to formulate, bulky, and prone to
various routes of physical degradation. Moreover, double emulsions consist
of large and polydispersed droplets, which are thermodynamically unstable
with a strong tendency for aggregation (Garti and Benichou, 2004). The main
limitation of multilayer emulsions is that only relatively dilute emulsions
(<5 wt%) could be possible to prepare due to the tendency for aggregation to
occur (McClements et al., 2007). The major limitation of SLP emulsions is that

at high temperatures, which is used to avoid crystallization of the lipid phase

during the homogenization process, chemical degradation of heat-sensitive
lipophilic components may occur (Ribeiro et al., 2003; McClements et al.,
2007). Lamba et al. (2015) provide a comprehensive review on the research of
double emulsion for food application.
10.3.1.1 Emulsion Characteristics

The efficient production of high-quality emulsion-based food products depends
on understanding the relationship between their bulk physicochemical
characteristics and their colloidal properties, as well as on the successful
implementation of this knowledge in practice. The main characteristics of
emulsions are droplet size and size distribution, droplet charge, interfacial
properties, etc.
10.3.1.1.1 Droplet Size

Many of the most important properties of an emulsion are determined by the
size of the droplets that they contain, such as the stability (McClements and
Dungan, 1993), the visual properties (Chantrapornchai et al., 1998), and the
rheological properties (Pal, 1996) of an emulsion. Consequently, it is particu-
larly important for food scientists to reliably control, measure, and report the
size of the droplets within the emulsion. If all the droplets are of the same size,
the emulsion is then referred to as “monodisperse,” which is shown in a single
number with a unit, radius, or diameter. However, in practice, droplets in food
emulsions are normally in a wide range of sizes. This is defined as “polydis-
perse” emulsion, which is characterized by droplet size distribution (DSD)
(Güzey and McClements, 2006a; McClements, 2007). DSD is defined as the num-
ber (or volume) of droplets that fall into specified size categories (McClements,
2007). It is more convenient to represent the full DSD by a measure of the cen-
tral tendency (mean values) (McClements, 2007). Some ways could be applied
to control the DSD, for example, varying homogenization conditions or sys-
tem composition. Smaller droplets can be produced by increasing the inten-
sity or duration of homogenization, or by increasing the concentration of the
emulsifier used in the emulsion (Walstra, 1993; McClements, 2007). Numerous
analytical techniques can be applied to analyze the droplet size and size dis-
tribution, including microscopy (Kalab et al., 1995; Kirby et al., 1995), scatter-
ing (light, x-ray, neutron) (Shukla et al., 2002; O’Hagan et al., 2005), electrical
pulse counting (McClements, 2005a), sedimentation techniques (McClements,
2005a), ultrasonic spectrometer (Coupland and McClements, 2004), and elec-
troacoustics (McClements, 2008). The technique selection is based on the drop-
let size of the sample and the detection range of the instrument.
10.3.1.1.2 Droplet Charge

The formation of droplet charge is due to the absorption of molecules to
their surface that are ionized or ionizable. The electrical characteristics of

the droplet depend on the type and concentration of the surface-active mol-
ecules present as well as on the physical properties of the surrounding aque-
ous phase (e.g., the charge of proteins depends on the pH of the solution
compared to their isoelectric point) (Tadros, 1994; McClements et al., 2007).
The droplet charge is quite important in an emulsion because it determines
the nature of its interactions with other charged species, for example, small
ions, or its behavior in the electric field (McClements, 2005c; Güzey and
McClements, 2006a). Aggregation of droplets in an emulsion could be pre-
vented when the droplets are coated by the same type of emulsifier, which
present the same electric charge, and the charge of the droplets is sufficiently
large, which gives an electrostatic repulsive force (Gupta and Muralidhara,
2001; McClements, 2005d). Different emulsifiers have different electrical
characteristics, for example, Tween has a smaller negative electric charge
(Silletti et al., 2007) and lecithin has a larger negative charge (El-Shaboouri,
2002). Therefore, the electrical characteristics can be controlled by carefully
selecting the emulsifier type.
The electrical characteristics of an emulsion droplet are often represented
by the zeta-potential, which is considered as the electrical potential that
exists at the “shear plane” of a particle, which is some small distance from
its surface. Zeta-potential is derived from measuring the mobility distribu-
tion of a dispersion of charged particles as they are subjected to an electric
field (McClements, 2007). As this electric potential approaches zero, particles
tend to aggregate. Particle electrophoresis and electroacoustics are the main
techniques to measure the droplet charge in emulsion systems (McClements,
2005a, 2008). Many of the commercial instruments available for measuring
the zeta-potential of emulsion droplets combine particle electrophoresis/
electroacoustics measurements with dynamic light-scattering measurements
so that both the droplet charge and the droplet size distribution can be deter-
mined using the same instrument.
10.3.1.1.3 Interfacial Properties

The interfacial layers of O/W food emulsions contain adsorbed materials, for
example, emulsifier molecules, in order to prevent them from aggregation.
The interfacial characteristics is described as the nature of the interfacial
region (≈ 1–50 nm thick) (McClements, 2007) that separates the oil and aque-
ous phases, including its thickness, composition, interfacial tension, electri-
cal charge, and rheological properties (McClements and Demetriades, 1998).
The electric charge of the interface influences the interaction with other mol-
ecules, and therefore the emulsion stability. The thickness and rheology of
the interfacial layer influences the stability of the emulsion, and also deter-
mines the rate at which molecules adsorb or desorb from the surface of the
droplets (McClements, 2007). The properties of the interfacial layers depend
not only on the type, concentration, and interactions of materials adsorbed
but also on the events that occur before, during, and after emulsion forma-
tion, for example, complexation, competitive adsorption, and layer-by-layer

formation (Rousseau, 2000; Dickinson, 2003). Therefore, the interfacial prop-

erties of an emulsion could be controlled by the selection of specific emul-
sifier types. The interfacial information, such as thickness, concentration
profile at an interface, adsorption kinetics, and changes in molecular con-
formation, can be obtained by using some analytical techniques, including
microscopy, reflection, spectroscopy, and small-angle scattering (Malmsten
et al., 1995; McClements, 2005a).
10.3.1.2 Emulsion Stability

The stability of emulsions normally refers to the ability to resist changes
in its physicochemical properties during the processing and even the stor-
age (McClements et al., 2007). In general, emulsions are thermodynami-
cally unstable systems because contact between oil and water molecules
is energetically unfavorable due to the different densities of water and oil.
Hence, they tend to break down and minimize the contact area over time
(McClements and Demetriades, 1998). The primary physical mechanisms
of instability are creaming, flocculation, coalescence, and Ostwald ripening
(Sæther et al., 2004) as shown in Figure 10.3.
Creaming is the process in which droplets move upward due to their
lower density compared to the surrounding liquid. The creaming rate can be
reduced by lowering the droplet radius, by increasing the continuous-phase
viscosity or by decreasing the difference in density between the two phases
(Beydoun et al., 1998). Coalescence refers to the increase in droplet size by
accretion and gradually leads to the separation of the oil and the aqueous
phase (Sæther et al., 2004). This process is always irreversible. Ostwald ripen-
ing is the process by which larger particles grow at the expense of smaller
ones due to the higher solubility of the smaller particles (Gibbs–Thomson
or Kelvin effect) and to molecular diffusion through the continuous phase
Kinetically stable emulsion
Creaming Flocculation Coalescence Ostwald ripening
FIGURE 10.3
Illustration of primary physical mechanisms of instability.

(Capek, 2004; Tadros et al., 2004; McClements, 2005d). Ostwald ripening
occurs in O/W emulsions containing a large amount of water-soluble lipids,
such as essential oils, since slightly water-soluble oil can transfer between
droplets at significant rates (Taylor, 1998; Kabalnov, 2001; Capek, 2004).
Flocculation is the process whereby two or more droplets stick together
but maintain their individual integrity (McClements, 2005d; McClements
et al., 2007). The interaction energy is a combination of attractive force, which
is dependent on London–van der Waals forces, and repulsive force due to
surfactants present at the interface (Weiss et al., 2008). Generally, two types
of droplet–droplet interactions are distinguished, that is, bridging floccu-
lation and depletion flocculation (McClements, 2000). Bridging flocculation
normally occurs when a high-molecular-weight polymer at a significantly
low concentration adsorbs onto two or more emulsion droplets, forming
bridges. Depletion flocculation occurs due to the presence of a nonadsorb-
ing biopolymer in the emulsion continuous phase, which can promote asso-
ciation of oil droplets by inducing an osmotic pressure gradient within the
continuous phase surrounding the droplets (McClements, 2000). If the added
biopolymer is either unadsorbed or poorly adsorbed, it is squeezed out of the
area between two approaching emulsion droplets. The bulk phase concen-
tration between the emulsion droplets becomes less than the overall solution
concentration, resulting in osmotic imbalance. The net effect is that the drop-
lets are attracted toward each other, resulting in flocculation. The attraction
energy is determined by the concentration of the polymer, and the range of
interaction depends on the radius of gyration of the biopolymer molecule
(McClements, 2000). The bonds formed during depletion flocculation are
generally weak, flexible, and reversible. The extent and nature of the floccu-
lation can strongly influence the properties of the emulsion, such as appear-
ance, rheology, and sensory perception (McClements, 2005d).
Emulsion stability has been reported to affect the quality and functionality
of dried microcapsules (Kim and Morr, 1996). Thus, it is necessary to pro-
duce a stable emulsion before drying. The stability of emulsions during pro-
cessing is affected by many factors, including emulsifiers, temperature, pH,
ionic strength, and high pressure either individually or collectively (Hunt
and Dalgleish, 1995; Keogh and O’Kennedy, 1999; Je and Rosenberg, 2000;
Djordjevic et al., 2004; Comas et al., 2006; Güzey and McClements, 2006b;
Choi et al., 2007; Wooster and Augustin, 2007; Jafari et al., 2008b; Yuan et al.,
2008).
10.3.1.3 Emulsifiers and Their Role in Bioactive Delivery Systems

The wall materials need to have emulsifying characteristics since it is cru-
cial to form a stable emulsion of fine droplets of the core material mixed
with the wall solution for spray drying to produce microcapsules (Risch
and Reineccius, 1988). Thus, an effective wall material must be an effective
emulsifier in the first place, which is defined as surface-active molecules

with hydrophilic (water soluble) and lipophilic (oil soluble) parts, that can
spontaneously migrate from solution and adsorb onto the dispersed lipid
droplet interfaces (Reineccius, 1988). Once adsorbed, they facilitate further
droplet disruption by lowering the interfacial tension, thereby reducing the
size of the droplets produced during homogenization (Reineccius, 1988).
Emulsifiers also reduce the tendency for droplets to aggregate by form-
ing protective membranes and/or by generating repulsive forces between
the droplets (McClements, 2008). A good emulsifier should rapidly adsorb
to the surface of the lipid droplets formed during homogenization, rapidly
lower the interfacial tension by a significant amount, and protect the drop-
lets against aggregation during emulsion processing, storage, and utilization
(Walstra, 1993; McClements, 1999). The large group of emulsifiers, including
natural, synthetic, and semisynthetic emulsifiers, can be easily divided into
two groups based on their molecular weights, namely, the small-molecule
or low-molecular-weight (LMW) surfactants and polymers (McClements
et al., 2007). Despite the successful elaboration of many synthetic polymers
as delivery systems (Chen et al., 2006), these cannot be used in food applica-
tions that require compounds generally recognized as safe.
10.3.1.3.1 Low-Molecular-Weight Surfactants

Food-grade surfactants with low-molecular weight include glycerol (mono-
glycerides and diglycerides), sorbitan esters of fatty acids, polyoxyethylene
sorbitan esters of fatty acids, phospholipids, lecithins, and sucrose esters
(Bos and Vliet, 2001). They contain long-chain fatty acid residues, which pro-
vide the hydrophobic group binding to the lipid phase of the O/W inter-
face and causing adsorption (Krog and Sparsø, 2004). Head groups of these
surfactants are quite different, which could be glycerol, substituted phos-
phoglyceryl moieties (in phospholipids), or sorbitan highly substituted with
polyoxyethylene chains (Garti, 2002) (Figure 10.4). These substances provide
various hydrophilic–lipophilic balance values, which determine their usage
in different types of emulsions.
LMW surfactants generate low interfacial tensions because these mol-
ecules adsorb strongly to the O/W interface and have few steric constraints
to prevent them from packing closely (Lee et al., 1996; Bos and Vliet, 2001).
However, generally, adsorbed layers of these small molecules may be quite
easily disrupted because they generally do not give highly cohesive or vis-
cous surface layers (Bos and Vliet, 2001).
10.3.1.3.2 Biopolymers
Proteins and polysaccharides are the two most important biopolymers in
food emulsions. Proteins act as emulsifiers but they behave differently from
small molecules because of their individual molecular structures. Specific
proteins, generally, give many food emulsions their characteristic properties.
When a protein adsorbs onto an O/W interface, the hydrophobic regions of
their structures, which are created by clusters of appropriate amino acid side

(a)
O HO
O HO
OH
P
O O OH
O
(b)
O
O
w
O OH
O
x
HO OH
O O
z y
FIGURE 10.4
Structures of glycerophospholipid (a) and polysorbate (b).
chain, lie on or partially dissolve in the oil phase. Some structures may be
considered as especially important, for example, a hydrophobic side of an
α-helical portion of a protein, created by the hydrophobic side chains that lie
outside the peptide core of the helix (Krochta, 2002).
Compared to LWM surfactants, proteins behave in a different way as sur-
factants. Emulsions formed by proteins are more stable against coalescence
because steric repulsion between the protein-covered oil droplets is very
effective in opposing aggregation (Palazolo et al., 2005). It is less sensitive to
high salt concentrations than LWM surfactants (Gu et al., 2005). Moreover,
proteins can form a viscoelastic film around the oil droplets or air cells via
noncovalent intermolecular interactions and via covalent intermolecular
disulfide cross-linking, but not in the LMW surfactants (Dickinson, 1997).
Protein adsorption may often be considered as being irreversible because
adsorption and desorption of protein tends to be considerably slower due to
the higher molecular weight and the cooperative nature of adsorption com-
pared to LMW surfactants. However, many proteins are not very suitable
for making fine emulsions; in other words, it takes more energy to obtain
small droplets than with small molecule surfactants (Bos and Vliet, 2001).
This is primarily due to their large molar mass and the structure of the
protein itself (the polypeptide backbone) will prevent close packing at the
points of contact with the interface (the side chains), and as a result, a thicker
O/W interface can be produced (Bos and Vliet, 2001; Dalgleish, 2004). Also,
molar concentration of proteins is small at a given mass concentration, caus-
ing Gibbs elasticity to be relatively small, which means that prevention of
recoalescence is less efficient (Krog and Sparsø 2004; Palazolo et al., 2005).

Further, the adsorption layer of proteins on the droplets obtained by emul-

sification is not an equilibrium layer, different to that of the LWM surfac-
tants (Krog and Sparsø, 2004). Some researchers have been interested in
determining the mechanism of protein adsorption on the oil droplet sur-
faces. Literature shows that most of the proteins adsorbed to interfaces are
capable of changing conformation at the time of adsorption or afterward
(Douillard et al., 1993; Rodriguez Patino et al., 2007). When a protein absorbs
onto the interface, it is affected by spreading pressure induced by external
force (e.g., stirring), which pulls apart the native structure to maximize the
amount of hydrophobic contact with the oil interface (Douillard et al., 1993).
Consequently, surface denaturation occurs.
Proteins with well-known specific emulsifying properties are from milk,
soy, meat, fish, egg, and plant origins (Nieuwenhuyzen and Szuhaj, 1998).
Milk proteins consist of two main classes: caseins and whey proteins. These
have been well studied as emulsifiers (Hunt and Dalgleish, 1994; Euston
et al., 1995, 2001; Hogan et al., 2001; Srinivasan et al., 2002; Djordjevic et al.,
2004; Harnsilawat et al., 2006; Vega and Roos, 2006).
Bovine casein consists of four types of protein fractions with substantially
different properties: αs1-, αs2-, β-, κ-casein, approximately 44%, 11%, 32%, and
11% of the whole casein, respectively (Robson and Dalgleish, 1987; Fox, 2001).
Generally, all caseins have relatively random and flexible structures, and
contain high level of prolines, which prevents the formation of secondary
structures (α-helices, β-sheets, and β-turns) and tertiary structures, making
the caseins stable to denaturation and contributing to high surface activity
(Fox, 2001).
Commercially, caseins are also converted into a number of other vari-
ants such as sodium caseinate, calcium caseinate, potassium caseinate, a
combination of these, and rennet casein (McClements, 2005c). Caseins and
caseinates have good interfacial properties and amphiphilic character, in
particular sodium caseinate (NaCA), which offers the physical and func-
tional characteristics required to encapsulate lipid core materials (Vega
et al., 2005; Vega and Roos, 2006). For example, NaCA is relatively soluble and
can be dispersed rapidly in an aqueous phase and is easily absorbed at the
oil–water interface (Kinsella, 1984), which is due to the removal of calcium
phosphate that is usually present in milk caseins. Emulsions stabilized by
NaCA are relatively heat stable, especially at pH 6.5 (Guo et al., 1989; Hunt
and Dalgleish, 1995), although NaCA molecules in emulsions are more sus-
ceptible to degradation than those in solution during heating (Srinivasan
et al., 2002). However, at extremely high temperature, for example, over
132°C, the emulsifying capacity of NaCA reduces significantly (Guo et al.,
1996). The drawback of NaCA is sensitive to pH, limiting its application in
food systems (Chen, 2002). However, some researchers (Lieske and Konrad,
1994) found that although it has poor solubility near its isoelectric point,
the encapsulation properties of the soluble fraction increased dramatically
(Lieske and Konrad, 1994).

The surfactant properties, described as above, give sodium caseinate the

encapsulation capacity as a primary emulsifying agent for various applica-
tions, such as spray drying (Pedersen et al., 1998; Hogan et al., 2001; Sliwinski
et al., 2003). When NaCA is used as an encapsulating wall material, the pro-
tein concentrations should be higher and oil phase volumes are better to be
lower, rather than those normally used in emulsion studies, where dilute
protein solution and high oil/protein ratios are applied (Tornberg, 1978;
Britten and Giroux, 1993). A thicker interfacial layer around the surface of the
droplets helps to protect the encapsulated materials during the drying pro-
cess. Research on factors influencing the protein load on the surface of the
droplets has been conducted and they found that increasing the ratio of oil to
protein decreases the protein load on the surface of oil droplets to some cer-
tain values (Hunt and Dalgleish, 1994; Hogan et al., 2001). Hogan et al. (2001)
reported that protein load values decreased from 3.1 to 2.0 mg/m2 when oil-
to-protein ratios increased from 0.21 to 0.50, but it did not decrease further
when the ratio increased to 3.0.
The other common protein emulsifier, whey protein, is also a complex
mixture of different protein fractions: β-lactoglobulin (55%), α-lactalbumin
(24%), serum albumin (5%), and immunoglobulins (15%) (Morr and Ha,
1993; Fox, 2001). Because of their compact, globular conformation, whey
proteins remain highly soluble at all pH values and even at their isoelec-
tric points (around 5) and tend to be denatured above the thermal denatur-
ation temperature (90°C, 10 min for native whey proteins) (Monahan et al.,
1996; Fox, 2001). There are two commonly used whey proteins in encapsu-
lation, namely, whey protein isolate (WPI) and whey protein concentrate.
Compared to NaCA, whey proteins have higher nutritional values, with
whey protein concentrate containing 35%–75% or more protein and WPI
at least 90% protein (Keogh and O’Kennedy, 1999). The emulsifying abil-
ity of whey proteins is poorer than that of NaCA since they are naturally
globular and need to be unfolded before adsorbing to oil/water interface
(Je and Rosenberg, 2000). However, whey proteins have been successfully
used as a wall system to encapsulate milk fat via spray drying (Keogh and
O’Kennedy, 1999). This is supported by another study done by Young et al.
(1993a), showing that using whey proteins as a wall system to encapsulate
anhydrous milk fat by spray drying obtained encapsulation yield higher
than 90%. In another study, Caraway essential oil has also been encapsu-
lated in a wall system consisting of whey proteins, which should provide
it an effective protection against oxidation (Bylaite et al., 2001). Shi et al.
(2015) also reported that double layers of lycopene-microemulsion by WPI
and HMP (high-methylester-pectin) exhibited high stability when they sub-
jected it to environmental stress (pH, ions strength, and heat) compared to
nonemulsified lycopene-in-oil droplets.
A small number of naturally occurring polysaccharides having hydro-
phobic character, for example, gum arabic (GA), have been chemically
modified to introduce nonpolar groups (McClements, 2008). GA is the most

commonly used biopolymer emulsifier in flavor beverage emulsions (Tan,

1997). It is composed of molecules with highly branched arabinogalactan
blocks attached to a polypeptide backbone (Randall et al., 1989). The hydro-
phobic polypeptide chain anchors the molecules to the oil droplet surface,
while the hydrophilic arabinogalactan blocks extend into the solution,
providing stability against droplet aggregation through steric and electro-
static repulsion (Philips and Williams, 1995; Islam et al., 1997; Jayme et al.,
1999). GA has the potential to form a stable emulsion by itself because it
can act both as an emulsifier that has the ability to form a protective film
around emulsion droplets and as a stabilizer that can prevent or retard the
dynamic instability of emulsion system. However, the surface-active por-
tion in GA is relatively small, and the concentration of GA needed to obtain
long-term emulsion stability is rather high (12% for a 20% orange oil emul-
sion) (Williams and Phillips, 2000). McNamee et al. (1998) also showed that
a stable emulsion with droplet size <1 µm could only be produced by oil-to-
GA ratio <2.0. This result further indicates that the formation of a fine and
stable emulsion requires large quantity of GA. GA was also found to be the
least effective material for orange oil emulsions when compared with WPI,
NaCA, and soy protein isolate (Kim and Morr, 1996).
10.3.1.3.3 Complex Emulsifier Systems

Since emulsifiers vary considerably in their ability to form and stabilize
emulsions, as well as in their cost, ease of utilization, ingredient compat-
ibility, and environmental sensitivity, there is not a single emulsifier that
is ideal for use in every food product. At present, there are few natural
emulsifiers that can be used in foods that are capable of providing good
emulsion stability to freeze–thaw cycling, thermal processing, and high
mineral contents (Ogawa et al., 2003). For this reason, extensive research
on more complex emulsifier systems, for example, combination of emulsi-
fiers or emulsifier with non-surface-active carbohydrates, have been carried
out to improve emulsion stability (Dickinson et al., 1990; Dickinson and
Yamamoto, 1996; Agboola et al., 1998; Bos and Vliet, 2001; Bylaite et al., 2001;
Comas et al., 2006; Güzey and McClements, 2006b; Gu et al., 2007; Wooster
and Augustin, 2007; Mun et al., 2010).
In many food systems, for example, ice cream and whipped creams, LMW
surfactants are used together with proteins. This is not because they are nec-
essary to produce an emulsion but their primary role is to control stability
(Dickinson and Yamamoto, 1996; Agboola et al., 1998; McCare, 1999; Euston
et al., 2001; Scuriatti et al., 2003; Gu et al., 2005; Jiménez-Flores et al., 2005;
Comas et al., 2006). For example, in the whipped cream processing, a stable
product is only achieved by destabilization of the initial O/W emulsion,
which means fat droplets are able to absorb to and stabilize air bubbles dur-
ing the whipping process. The destabilization of emulsion cannot occur in
the absence of LMW surfactant, such as phospholipids, as the emulsions are
stable to coalescence (van Aken, 2003). LMW surfactants are able to displace

or interact with proteins from the fat globule surface and lower emulsion
stability.
The combination of proteins with carbohydrates having non-surface activ-
ity has been investigated in order to improve the stability of emulsions
(Hogan et al., 2001; Güzey and McClements, 2006b; Wooster and Augustin,
2007; Mun et al., 2010). Research found that addition of carbohydrates to
emulsion systems increases the emulsion stability to freeze–thaw cycling.
Gu et al. (2007) studied the influence of sucrose to β-lactoglobulin-stabilized
emulsions. They found that the addition of sucrose to the emulsion prior to
freezing greatly improved its freeze–thaw stability. Mun et al. (2008, 2010)
concluded that maltodextrin could improve the stability of emulsion to
endure freeze–thaw cycling and freeze drying process. They also found that
maltodextrin with high dextrose equivalent (DE) value had better perfor-
mance for stabilizing. However, Klinkesorn et al. (2004) demonstrated that
in the presence of a high concentration of maltodextrin, rapid creaming of
emulsion would be promoted. On the other hand, this combination has been
extensively studied in microencapsulation (Rosenberg and Young, 1993;
Young et al., 1993a,b; Heinzelmann and Franke, 1999; Heinzelmann et al.,
2000; Chung et al., 2008).
10.3.1.4 Emulsification Techniques and Conditions

A stable emulsion with minimum droplet size can increase the microencap-
sulation efficiency and the shelf-life of encapsulated core materials (Ishido
et al., 2002; Minemoto et al., 2002; Soottitantawat et al., 2003, 2005). To achieve
this, selection of emulsifiers, emulsification techniques, and conditions are
crucial. Generally, there are two types of emulsification techniques: hydro-
static emulsification, for example, ultrasound and Ultra-Turrax, and dynamic
high-pressure emulsification, for example, two-stage high-pressure homog-
enizer and microfluidizer (Floury et al., 2003). Mongenot et al. (2000) stated
that their results clearly showed that the use of ultrasound increased aroma
retention and limited the diffusion of the most volatile and polar compounds
during drying when the wall material has low emulsifying ability and a low
viscosity compared to the use of Ultra-Turrax. However, no significant dif-
ference in emulsion size was observed between these two techniques. Jafari
et al. (2008b) compared the efficiency of microfluidizer and ultrasonifier (or
ultrasonic dismembrator). Of the two device methods, microfluidization pro-
duced the most stable emulsion with a small droplet size. The emulsion was
then converted into powders showing the least amount of unencapsulated
oil. They also demonstrated that the different performance of hydrostatic
and dynamic high-pressure emulsification techniques on the emulsion prop-
erties is mainly due to their different effects on the biopolymers.
Besides emulsification techniques, homogenization conditions, including
pressure and number of passes, can significantly influence the properties of
emulsions. Je and Rosenberg (2000) demonstrated that the surface coverage of

the globules by protein is affected by the number of homogenization passes.

For instance, a higher number of homogenization passes will give rise to a
relatively greater surface coverage, and then emulsion stability will be depen-
dent on steric forces and emulsion becomes less sensitive to the salt concentra-
tion. Yuan et al. (2008) studied β-carotene emulsions by varying the first-stage
and second-stage pressures from 60 to 140 MPa and 6 to 14 MPa, respectively.
Their results showed that increasing the homogenization pressure could
largely decrease the droplet size because the shear forces and turbulence are
pressure dependent. They also found that increasing the number of cycles
through the homogenizer resulted in a significant decrease in droplet size
and size distribution of emulsions. These results are in line with the find-
ings of other authors (Jimenez et al., 2004; Tan and Nakajima, 2005). However,
“overprocessing” may occur when homogenization pressure is relatively
high. Yuan et al. (2008) found an optimum pressure of about 100 MPa in which
droplet size and distribution reached a minimum, but after pressure over that
point, the stability of emulsions was reduced. Floury et al. (2003) worked on
methylcellulose as emulsifier and observed that emulsification at a high pres-
sure >150 MPa could lead to degradation of long-chain molecules and forma-
tion of polymers with significantly lower molecular weight. This degradation
of methylcellulose reduced its emulsifying ability and consequently increased
droplet collision and aggregation in association with adjacent droplets. They
concluded overall that a pressure less than 150 MPa was optimum to produce
real submicron emulsions without any “overprocessing.”
10.3.2 Drying
10.3.2.1 Drying Techniques
Drying is one of the most widely used methods for large-scale production
of encapsulated flavors and volatiles (Deis, 1997). During drying processes,
water in an encapsulated system is reduced to a level to inhibit or slow down
the growth of spoilage microorganisms as well as the occurrence of chemical
reactions. In addition to this preservation aspect, it also reduces the costs
and/or difficulties of product packaging, handling, storage, and distribution
due to the reduced weight and bulk volume of dried products and their lon-
ger shelf stability (Gharsallaoui et al., 2007; Toledo, 2007). There are numer-
ous drying techniques commonly used for food products, among which
spray drying and freeze drying processes are most suitable for drying O/W
emulsions closely related to encapsulated products as powders.
Spray drying food emulsions has been used in the food industry since the
late 1950s and is a well-established process that can produce large amounts of
material (Gouin, 2004). It is defined as the transformation of emulsion into a
dried particulate form (e.g., powders, agglomerates, or granules). The emulsion
system is atomized, which is broken up into a spray of fine droplets, and is
ejected into a drying chamber. The spray is then contacted with and suspended

by a hot drying medium (generally air or more rarely an inert gas as nitrogen),
allowing the moisture to evaporate and the droplets to be transformed into
dry particles of almost the same size and shape (Barbosa-Cánovas et al., 2005;
Gharsallaoui et al., 2007). A typical drying process is shown in Figure 10.5.
Spray driers differ in size, shape, and atomizer. Two types of atomizers are
widely used: high-pressure and centrifugal wheel (Barbosa-Cánovas et al.,
2005), although other types of atomizers exist, for example, ultrasonic, two-
fluid, disk/cup rotating, and pneumatic (Huang et al., 2006; Xiao et al., 2008;
Stepanov et al., 2011). The main advantage of pressure atomizer is control-
lability to the finished powder size, while centrifugal atomizer is suitable for
viscous or corrosive materials. In pressure atomizer, 1-fluid pressure nozzle
is the most common one, while 2-fluid pressure nozzle can be used in some
special cases, for example, granulation (Legako and Dunford, 2010).
There are several drawbacks when applying spray drying to dehydrate
O/W emulsions. For example, some low-boiling-point bioactive components
can be lost during the drying process and the core substance may also be
on the surface of the capsule, which may encourage oxidation and possible
flavor changes of the encapsulated products (Dziezak, 1988; Desobry et al.,
1997; Madene et al., 2006). This technology also applies elevated temperature,
which is necessary for drying. High temperatures and the presence of oxygen
may lead to increased oxidation of certain components such as PUFAs. Spray
drying also produces very fine powders (typically in the range of 10–100 μm
in diameter), which may need further processing to make the dried particles
more soluble if it is applied in the liquid matrix (Madene et al., 2006).
Nevertheless, the following merits of the spray drying process have ensured
its dominance as a microencapsulation technique (Barbosa-Cánovas et al., 2005;
Feed
Hot air
Atomizer
Drying
chamber
Air Scrubber
Exhaust
Cyclone
Dry product
FIGURE 10.5
Typical spray drying (open cycle, co-current) layout. (Modified from Barbosa-Cánovas, G.V.
et al. 2005. In Food Powers: Physical Properties, Processing, and Functionality, ed. G.V. Barbosa-
Cánovas, E. et al. Kluwer Academic/Plenum Publishers, New York, pp. 271–304.)

Desai and Park, 2005; Madene et al., 2006; Gharsallaoui et al., 2007): (1) it is rela-
tively inexpensive to operate compared with many other techniques; (2) it can
be operated easily and can be a continuous drying operation; (3) it is adaptable
to full automatic control; (4) it is an effective technology for large-scale produc-
tion; (5) it is possible to produce powders with constant quality throughout the
dryer when drying conditions are held constant; (6) it can produce free-flowing
particles of small size and spherical shape with a well-defined particle size dis-
tribution, and therefore good physical stability of finished products; and (7)
its relatively short drying time, when compared with other drying processes,
for example, drum drying, makes it relatively suitable for drying certain heat-
sensitive materials, for example, flavor.
Freeze drying (lyophilization) is one of the most useful drying processes
that is used to dry heat-sensitive substances that are unstable in aqueous
solutions (Barbosa-Cánovas et al., 2005). The freeze drying process mainly
consists of three steps (Barbosa-Cánovas et al., 2005): (1) the product is fro-
zen; (2) the product is dried under sublimation of ice under reduced pres-
sure; and (3) the water vapor is removed. A common freeze drying system
is shown in Figure 10.6. Freeze drying is recognized as the best method of
producing dried food products of the highest quality (Schwegman, 2009).
However, relatively few research (Desobry et al., 1997; Heinzelmann et al.,
2000; Madene et al., 2006; Choi et al., 2007) stated that freeze drying of an
O/W emulsion could be an alternative method for the production of dry
emulsion. These research have shown the merits of freeze drying of emul-
sions. The technology can prevent deterioration due to oxidation or chemical
modification of the products since the drying process is carried out under
vacuum conditions, so there is a virtual absence of air. Moreover, the drying
Heated plate
Product tray
Drying chamber
Noncondensibles
exhaust
Vapor flux Condenser Vacuum pump
FIGURE 10.6
A common freeze drying system. (Modified from Barbosa-Cánovas, G.V. et al. 2005. Drying.
In Food Powers: Physical Properties, Processing, and Functionality, ed. G.V. Barbosa-Cánovas, E.
et al. Kluwer Academic/Plenum Publishers, New York, pp. 271–304.)

temperature is lower than the ambient temperature. Therefore, products that

decompose or undergo changes in structure, texture, appearance, and fla-
vor as a consequence of high temperature can be dried under vacuum with
minimal physical and chemical damage (Barbosa-Cánovas et al., 2005).
However, this drying method is less attractive than others for commercial
applicability. First, it is very expensive and the cost is almost 50 times higher
than that of spray drying (Desobry et al., 1997) because of its slow drying rate
and the use of vacuum. Second, it is energy intensive because the product has
to be frozen first and then heated to sublime the ice and bound water. Third,
it requires long processing time due to the resistance to heat and mass trans-
fer (Kim, 1996). Another important reason that prevents the extensive use of
this technique is that a homogeneous ice crystal distribution is hardly formed
because of the difficulty to set a heat-removal system (Geankoplis, 2003).
10.3.2.2 Factors Influencing the Performance of Spray Drying

Apart from the different types of spray driers, many other factors, includ-
ing infeed properties and drying conditions, affect the performance of spray
drying on encapsulation of sensitive bioactive components, and are detailed
below.
10.3.2.2.1 Solid Content of Infeed Emulsion

With the increase of the emulsion total solids, the retention of core materials
improves, which is due to the rapid formation of semipermeable membrane
(allowing evaporation and diffusion of water but effectively retaining flavor
compounds) on the droplet surface (Rosenberg et al., 1990; Rosenberg and
Sheu, 1996). However, the solubility of the wall material limits the increase of
the solid content. The insoluble material does not have the ability to protect
the core but can lead to processing problems, for example, agglomeration
and difficulties to atomization.
10.3.2.2.2 Molecular Weight (Size) and Relative Volatility of the Core Material
The increase of the molecular size of core materials generally results in slow
diffusion rate; subsequently, the molecules will take more time to reach
the atomized droplet surface during drying and retention will increase.
Moreover, the surface of the droplet becomes impermeable to the core sub-
stance more quickly during drying, when diffusions effectively stop at low
moisture content (Goubet et al., 1998). On the other hand, relative volatility of
a compound is calculated with respect to water (Bhandari, 2005). Retention of
aroma compounds is based on their relative volatility in the mixture before
drying. The higher the relative volatility, the lower the retention.
10.3.2.2.3 Types of Wall Materials

Wall materials containing good emulsifying ability and film-forming abil-
ity effectively protect the core materials from loss or oxidation/evaporation

since they are not only forming a stable emulsion but also forming a fine and
dense network during drying, which could prevent lipid separation from the
emulsion during dehydration.
10.3.2.2.4 Ratio of Oil to Wall Materials

The literature has shown that a suitable ratio of oil to wall materials is impor-
tant to ensure optimum encapsulation. Hogan et al. (2001) studied the micro-
encapsulation properties of NaCA using the spray drying method with soy
oil as the dispersed phase. They found that increasing the oil-to-protein ratio
from 0.25 to 3.0 resulted in a progressive decrease in microencapsulation
efficiency from 89.2% to 18.8%. They also concluded that the oil-to-protein
ratio should be no more than one; otherwise, the formed emulsion could be
destabilized during spray drying.
10.3.2.2.5 Viscosity of Infeed Emulsion

The viscosity of an emulsion is quite important because it strongly influ-
ences the behavior of emulsion on drying, the movement of core materials
to the surface of powders, the size of dried powders, and the thickness of the
encapsulant (Reineccius, 1988; Rosenberg et al., 1990). Rosenberg et al. (1990)
found that the optimum retention of volatiles was a function of wall material
concentration, which corresponded to an emulsion viscosity ranging from
125 to 250 mPa · s for ethyl caproate emulsions containing GA and approxi-
mately 105 mPa · s for allylguaiacol emulsions containing maltodextrin. It
was suggested that emulsions with these optimum viscosities were easy to
atomize to produce ideal spherical particles.
10.3.2.2.6 Emulsion Stability

The microencapsulation efficiency of core materials is reported to be influ-
enced by the stability of the initial emulsion: the better the stability, the
higher the efficiency. For example, Liu et al. (2000) did a series of work on
the effect of emulsion stability on the retention of emulsified hydrophobic
flavors during spray drying. They found that the natural log of mean drop-
let diameters was linearly proportional to the natural log of time. The final
retention correlated negatively with the emulsion stability. This result dem-
onstrated that unstable emulsion was broken inside the droplet, resulting in
appreciable loss of flavor during drying.
10.3.2.2.7 Emulsion Size

Research has reported that emulsion size has a considerable effect on the
microencapsulation efficiency of materials with a hydrophobic core (Risch
and Reineccius, 1988; Sheu and Rosenberg, 1995; Liu et al., 2001; Minemoto
et al., 2002; Soottitantawat et al., 2003; Soottitantawat et al., 2005). All their
results show that the microencapsulation efficiency improved with decreased
droplet size of the initial emulsion. Risch and Reineccius (1988) claimed that

a smaller emulsion size yielded microcapsules with higher total oil content
and lower nonencapsulated oil.
10.3.2.2.8 Flowrate of Dry Air

Generally, high flowrate of dry air causes rapid heat and mass transfer asso-
ciated with the drying process (Coumans et al., 1994). Thus, less fat migration
to the droplet surface takes place before a crust or semipermeable membrane
forms, giving a high retention of the core material.
10.3.2.2.9 Inlet and Outlet Temperatures

Since the outlet temperature is normally subject to the inlet temperature and
the feed rate (Kim et al., 2009), the effect of the outlet temperature on the
encapsulation of food oils is controversial and unclear; however, low outlet
temperature is preferred (Jafari et al., 2008a). Thus, inlet temperature is the
main parameter to influence the drying behavior. In general, at high dry-
ing temperature, the moisture is evaporated quickly and the surface solidi-
fies rapidly, which consequently reduces the surface fat (Jafari et al., 2008a).
However, if the inlet temperature is too high, ballooning occurs when steam
is formed in the interior of the drying droplet, causing the droplet to puff,
thereby producing a thin-walled hollow particle. On the other hand, when the
drying temperature is too low, the semipermeable membrane forms slowly,
resulting in a migration of more dissolved solids to the droplet surface. This
leads to low encapsulation yield and sticky powders (Rosenberg et al., 1990;
Anker and Reineccius, 1998). Inlet air temperature of 150–200°C and outlet
temperature of 70–90°C are commonly used in the commercial production of
spray-dried milk products (Young et al., 1993a; Fäldt and Bergenståhl, 1996;
Keogh and O’Kennedy, 1999; Kim et al., 2009).
10.3.2.2.10 Infeed Temperature

In low-viscosity systems, reduction of infeed temperature may help increase
the microencapsulation efficiency of the core substance due to the increase of
infeed viscosity. On the other hand, infeed temperature should be increased
to the point that higher infeed solids may be used, resulting in better reten-
tion (Thijssen, 1971). One problem with high infeed temperatures could be
microbial growth in these materials and probably oxidation of oils or evapo-
ration of volatile compounds before drying (Reineccius, 2001).
10.4 Current Challenges for Delivering LBCs

Over the last two decades, microencapsulation of various LBCs has been
studied, including essential oils, fish oil, vegetable oils, fatty acids, phytos-
terols, carotenoids, and fat-soluble vitamins (Table 10.2). One of the most

extensively studied LBCs is fish oil, and the most common methods used for
the microencapsulation of fish oil are emulsification and spray drying using
a variety of wall materials (Kaushik et al., 2014). Coacervation has also been
demonstrated as a good method for encapsulating fish oil and was reported
to have equivalent bioavailability as to those obtained from soft gel capsules
(Barrow et al., 2009). However, oxidation of LBCs during processing and
storage due to the inefficiency of encapsulants and high drying temperature
remains a challenge to overcome. Over the years, efforts have been made to
prevent lipid oxidation in microencapsulated fish oil. This includes the dis-
covery of new natural wall material (e.g., sugar beet pectin) (Drusch, 2007),
modification of natural wall materials (e.g., modified cellulose and modified
starch) (Kolanowski et al., 2004; Kolanowski et al., 2006; Drusch et al., 2006;
Drusch and Schwarz, 2006), physical blends (e.g., emulsifiers with carbo-
hydrates), and MRPs (protein–polysaccharides conjugates) (Augustin et al.,
2006). Other methods, including the addition of antioxidants (Kamal-Eldin
and Yanishlieva, 2002; Medina et al., 2003; Jacobsen et al., 2008), optimiza-
tion of processing conditions, and modification of infeed emulsion com-
positions, have also been tried to improve the properties of dried fish oil
microcapsules (Thijssen, 1971; Coumans et al., 1994; Jafari et al., 2008a; Kim
et al., 2009).
In addition, not many research have shown a successful protection of
microencapsulated fish oil that contains a high level of EPA and DHA during
storage. Kim (1996) tried to apply complex wall materials containing gelatin,
agar, and span 80 (1:3:1) for the encapsulation of fish oil (25% wt. in total solid).
They found 80% retention of EPA and DHA after 3 days of storage at 30°C.
Tan et al. (2009) observed less than 80% of EPA and DHA remained after a
7-day-storage trial at 40°C using alginate/starch blend wall materials. In a
more recent research, Shen et al. (2010) studied the retention of EPA and DHA
in microcapsules containing different ratios of chitosan, glucose, and Hi-Cap
after a 4-week storage at 25°C without the presence of light and o xygen. They
reported the loss of approximately 10% EPA and DHA. However, the fish oil
they used contains lower level of EPA and DHA (less than 30% of total oil)
than the above studies, which may explain lower oxidation level compared
with fish oil containing higher amount of EPA and DHA.
Furthermore, fishy odor has been perceived in microcapsules (Kolanowski
et al., 2007; Lawlor et al., 2010). This is not acceptable by some consumers,
thus limiting their use and impacting on their market value. Few studies
reported strategies to mask the unpleasant fishy odor to improve the sen-
sory profiles of fish oil products. Serfert et al. (2010) studied the impact of
different flavors, for example, apple, orange, lemon, crisped mint, banana,
and vanillin, on odor masking. They observed that apple/vanillin in a ratio
of more than 1.0 was most effective in masking the fishy odor and improv-
ing the taste of the reconstituted fish oil. Chen et al. (2013) successfully used
limonene as a flavor (also has health benefits) to mask the fishy odor of the
microcapsules containing fish oil and phytosterol.

Lycopene microemulsion formed by WPI with HMP exhibited the highest

physical stability. Lycopene was more stable in the two-layer microemulsion
that was prepared with WPC and HMP against environmental stresses, such
as at different pH, NaCl concentrations, and temperatures, compared to the
one-layer lycopene microemulsions. The one-layer lycopene emulsions were
unstable at low pH, but the two-layer WPC with HMP emulsions tended to
be less stable in the neutral pH range of 6.12–7. More than 90 g/100 g of total
lycopenes was lost in the one-layer microemulsion at the lower pH range of
2.01–3.03. The stability of the lycopene-rich microemulsions decreased with
increasing NaCl concentrations for all samples. The stability of the micro-
emulsion was reduced after thermal treatments while the total lycopene
content was reduced by 36.8, 23.2, and 11.1 g/100 g in the corn oil phase, the
one-layer, and two-layer microcapsules after the thermal process, respec-
tively. The overall results suggested the optimized lycopene microemulsion
was more stable to thermal treatment and change of pH, but it was sensitive
to NaCl treatment. The results were positive in characterizing this technol-
ogy as a useful step in the development of lycopene-rich microemulsion to
expend its applications to water-based food systems in commercial produc-
tion such as applications in beverage and bakery products (Shi et al., 2015).
10.5 Co-Encapsulation: Merits and Implication

When more than one compound is encapsulated in the same system, it is
termed co-encapsulation (CE). Some researchers have reported that CE of dif-
ferent core materials can enhance the bioactivity of individual components,
for example, CE of gallium with gentamicin in liposomes enhanced the anti-
microbial activity of gentamicin against Pseudomonas aeruginosa (Halwanil
et al., 2008). Sometimes, more than one LBC is required to be delivered in the
same food matrix. In these cases, the encapsulated LBCs have to be individu-
ally added into the food, which may not be convenient for manufacturers.
For this purpose, CE can be a useful way to deliver more than one LBC into
food matrices.
CE technique has been widely applied in drug delivery systems, either in
liposome form or in capsule form (Gürsoy et al., 2004; Iyer and Kailasapathy,
2005), to provide additional benefits, as the co-encapsulated drug compo-
nents would work synergistically. In food application, CE is not prevalent
and only limited research in this area, including a few on CE of LBCs, has
been done. Chen et al. (2013a,b) studied the CE of fish oil with phytosterol
ester and limonene. It has been reported that the intake of plant sterols and
fish oil together has synergistic effects on cardiovascular disease since they
both reduce the cholesterol level and the ratio of LDL/HDL (Micallef and
Garg, 2008; Khandelwal et al., 2009). Furthermore, intake of both the above

LBCs together could also reduce systemic inflammation in hyperlipidemic

individuals (Micallef and Garg, 2009). In addition, phytosterols have been
found to promote the oxidation stability of other bioactive lipids (Yasukazu
and Etsuo, 2003). Introducing limonene into the system is reported to have
a positive effect on masking the fishy odor while adding extra values for
human health with respect to its bioactivity as mentioned earlier.
Similar to microencapsulation, CE can be achieved by several techniques
such as using emulsion delivery system, freeze drying, and spray drying.
The methods applied would affect the microencapsulation efficiency and
powder properties. Chen et al. (2013a) found significantly higher retention
of volatile fraction (p < 0.05) and lower surface oil in the spray-dried micro-
capsules containing fish oil, phytosterol ester, and limonene than those from
freeze drying even though powders from both dehydration methods showed
good redispersion properties. The oxidation of the encapsulated oils was
comparable for both spray- and freeze-dried samples during a 7-day accel-
erated storage trial but the loss of limonene flavor was significantly higher
in the freeze-dried samples (p < 0.05). Sensory evaluation indicated that
the addition of limonene could mask the unpleasant fishy odor in the co-
encapsulated microcapsules. Overall, the study showed that freeze drying
did not produce powders with much superior properties and did not show
better protection toward the core materials than spray drying. In another
study, Chen et al. (2013b) further showed that the microcapsule properties,
and hence the powder stabilities, were influenced by the inlet and outlet tem-
peratures of the spray drying process.
Apart from the encapsulation methods, other factors such as core materials
interactions and concentrations, and storage stability should be considered for
CE. Since CE involves two or more different bioactive compounds, these com-
pounds may react freely, affecting the synergy aim of CE. Studying the inter-
actions between different core materials in emulsion systems and the storage
stability are therefore crucial. In a study by Calvo et al. (2010), the use of com-
mercial antioxidant butylated hydroxytoluene (BHT) did not provide extra
protection toward microencapsulated extra virgin olive oil during storage.
However, in the studies by Chen et al. (2013a,b), the synergistic effect of the co-
encapsulated LBCs was evident. On the other hand, CE may provide synergis-
tic stability for only a certain storage period. An example is the CE of probiotics
with prebiotic using chitosan as the coating material (Iyer and Kailasapathy,
2005). The finding suggested that CE provided higher probiotic (Lactobacillus
acidophilus CSCC2409) survival rate for up to 2 weeks, before becoming lower
than the control containing only probiotic itself. However, another probiotic
strain, L. acidophilus CSCC2400, which was co-encapsulated with prebiotic
with the same wall material, showed better survival rate up to 6-week storage
time. Thus, storage stability, as dependent on the type of microencapsulated
core materials, is one of the main factors to target when investigating CE.
The ratio among the different bioactive compounds in the formulation
may also play a significant role. This ratio has to be optimized so that the

effectiveness of encapsulation is enhanced and maximized (Chen, 2012). In

addition, wall thickness may enhance or inhibit the release of the encap-
sulated compounds; therefore, the wall-to-core ratio is another important
aspect to consider. Although a higher concentration of core material is often
desirable, it is not always practical. Low wall-to-core ratio will result in thin
wall on the microcapsules, which may break during processing and thus fail
to provide protective effects toward the core material. However, if the wall-
to-core ratio is too high, then the wall will be thicker than expected, and
the co-encapsulated bioactive components may not be delivered effectively.
Additionally, this will lead to the formation of larger particles that will nega-
tively affect the textural properties of the food product. This is reflected in
Chen et al. (2013b) where the ratio of wall to core of 4:1 gave the best micro-
encapsulation efficiency for LBCs compared to the 1:1 and 2:1 ratios. At the
ratio of 1:1, fish oil was found to be overly loaded, leading to larger particle
sizes after reconstitution of microcapsules. These overloaded oil caused the
deposition of fat on the surface of the droplets, reducing the encapsulation
efficiency and storage stability.
The use of different wall materials will affect the microencapsulation effi-
ciency and the stability of the microcapsules. For example, Calvo et al. (2010)
showed that carbohydrate-based wall materials (carboxymethylcellulose and
maltodextrin) were not effective in co-encapsulating olive oil with BHT com-
pared to those containing a combination of protein (sodium caseinate) and
carbohydrate (maltodextrin) in terms of protection toward oxidative stability.
10.6 Concluding Remark

There is an undeniable growing interest among food technologists in the
enormous potential of microencapsulation of functional ingredients for the
food industry, and this has been demonstrated by the exponential increase in
the number of publications (nonscientific and scientific articles, and patents)
published over the years since the mid-1950s. The latest development in the
area of encapsulation of functional ingredients involved the application of
nanotechnology to produce nano-size particles, which is called nanoencap-
sulation. Nanoencapsulation has the same concept as microencapsulation
except that the final products are in the nanoscale. This can require rela-
tively new technology to perform depending on the production method cho-
sen and the intended delivery system. The methods include the use of lipid
nanocarriers (Aditya et al., 2014; Wang et al., 2014), nanoemulsions (Rao and
McClements, 2012; Yu and Huang, 2012), nanoliposomes (Maherani et al.,
2012), and nanoparticles (Rao and McClements, 2013; Paques et al., 2014;
Bagheri et al., 2014; Lv et al., 2014; Pandita et al., 2014; Penalva et al., 2015).
A number of reviews have been made in nanoencapsulation in the recent

years (Mozafari et al., 2008; Quintanilla-Carvajal et al., 2010; Fathi et al., 2012,
2014). However, consumer perception toward the application of nanotechnol-
ogy in food products may raise concern on the acceptability of the ingredi-
ents produced. In addition to new technology, stability, and quality of the
functional ingredients as mentioned above, one aspect of vital importance
is to study the bioavailability of these functional ingredients. Compared to
the pharmaceutical area, this aspect has been relatively less studied for food
ingredients, especially in vivo, and this would be the focus in future research.
References
Aditya, N.P., Macedo, A.S., Doktorovov, S., Souto, E.B., Kim, S., Chang, P.S., Ko, S.
2014. Development and evaluation of lipid nanocarriers for quercetin deliv-
ery: A comparative study of solid lipid nanoparticles (SLN), nanostructured
lipid carriers (NLC), and lipid nanoemulsions (LNE). LWT—Food Science and
Technology, 59(1): 115–121.
Agboola, S.O., Singh, H., Munro, P.A., Dalgleish, D.G., Singh, A.M. 1998. Stability of
emulsions formed using whey protein hydrolysate: Effects of lecithin addition
and retorting. Journal of Agricultural and Food Chemistry, 46: 1814–1819.
Ahn, J.-H., Kim, Y.-P., Lee, Y.-M., Seo, E.-M., Lee, K.-W., Kim, H.-S. 2008. Optimization
of microencapsulation of seed oil by response surface methodology. Food
Chemistry, 107: 98–105.
Alencastre, J.B., Bentley, M.V.L.B., Fabiola, S., de Moragas, M., Viladot, J.L., Marchetti,
J.M. 2006. A study of the characteristics and in vitro permeation properties of
CMC/chitosan microparticles as a skin delivery system for vitamin E. Revista
Brasileira de Ciencias Farmaceuticas, 42(1): 69–76.
Alvim, I.D., de Souza, F.D., Koury, I.P., Jurt, T., Dantas, F.B.H. 2013. Use of the spray
chilling method to deliver hydrophobic components: Physical characterization
of microparticles. Food Science and Technology, 33: 34–39.
Anandaraman, S., Reineccius, G.A. 1986. Stability of encapsulated orange peel oil.
Food Technology, 40(11): 88–93.
Anekella, K., Orsat, V. 2013. Optimization of microencapsulation of probiotics in rasp-
berry juice by spray drying. LWT—Food Science and Technology, 50(1): 17–24.
Anker, M.H., Reineccius, G.A. 1998. Influence of spray-dryer air temperature on
retention and shelf life of encapsulated orange oil. In Flavor Encapsulation,
eds. S.J. Risch, G.A. Reineccius, American Chemical Society, Washington DC,
pp. 78–86.
Atchley, F. 2010. Method and device for flavoring smokeless tobacco. U.S. P.A. Publ.
US 2010282267 (A1): 11.
Augustin, M.A., Sanuansri, L., Bode, O. 2006. Maillard reaction products as encapsu-
lants for fish oil powders. Journal of Food Science, 71(2): E25–E32.
Ayala-Zavala, J.F., Soto-Valdez, H., Gonzalez-Leon, Z., Alvarez-Parrilla, E., Martin-
Belloso, O., Gonzalez-Aguilar, G.A. 2008. Microencapsulation of cinnamon leaf
(Cinnamomum zeylanicum) and garlic (Allium sativum) oils in β-cyclodextrin.
Journal of Inclusion Phenomena and Macrocyclic Chemistry, 60(3–4): 359–368.

Badee, A.Z.M., El-Nawawi, S.A., Aly, H.M., Abd El-Kader, A.E. 2011. Morphology,
properties and safety of microencapsulated orange peel oil. World Applied
Sciences Journal, 13(8): 1821–1826.
Bae, E.K., Lee, S.J. 2008. Microencapsulation of avocado oil by spray drying using
whey protein and maltodextrin. Journal of Microencapsulation, 25(8): 549–60.
Bagheri, L., Madadlou, A., Yarmand, M., Mousavi, M.E. 2014. Spray-dried alginate
microparticles carrying caffeine-loaded and potentially bioactive nanoparticles.
Food Research International, 621: 113–1119. doi: 10.1016/j.foodres.2014.05.040.
Bakowska-Barczak, A.M., Kolodziejczyk, P.P. 2011. Black currant polyphenols: Their
storage stability and microencapsulation. Industrial Crops and Products, 34(2):
1301–1309. doi: 10.1016/j.indcrop.2010.10.002.
Bangs, W.E., Reineccius, G.A. 1988. Corn starch derivatives: Possible wall materials
for spray-dried flavour manufacture. Flavour Encapsulation, eds. G.A. Reineccius
and S.J. Risch. American Chemical Society, Washington D.C., pp. 12–28.
Banville, C., Vuillemard, J.C., Lacroix, C. 2000. Comparison of different methods for
fortifying cheddar cheese with vitamin D. International Dairy Journal, 10(5–6):
375–382.
Baranauskiene, R., Bylaite, E., Zukauskaite, J., Venskutonis, R.P. 2007. Flavor retention
of peppermint (Mentha piperita L.) essential oil spray-dried in modified starches
during encapsulation and storage. Journal of Agricultural and Food Chemistry,
55(8): 3027–3036.
Barbosa-Cánovas, G.V., Ortega-Rivas, E., Juliano, P., Yan, H. 2005. Drying. In Food
Powers: Physical Properties, Processing, and Functionality, ed. G.V. Barbosa-
Cánovas, E. Ortega-Rivas, P. Juliano and H. Yan. Kluwer Academic/Plenum
Publishers, New York, pp. 271–304.
Barrow, C.J., Nolan, C., Holub, B.J. 2009. Bioequivalence of encapsulated and micro-
encapsulated fish-oil supplementation. Journal of Functional Foods, 1(1): 38–43.
Bastos, D.D., Goncalves, M.P., de Andrade, C.T., Araujo, K.G.D., Leao, Mhmd. 2012.
Microencapsulation of cashew apple (Anacardium occidentale, L.) juice using a
new chitosan-commercial bovine whey protein isolate system in spray drying.
Food and Bioproducts Processing, 90, 683–692.
Battaglia, L., Trotta, M., Gallarate, M., Carlotti, M.E., Zara, G.P., Bargoni, A. 2007. Solid
lipid nanoparticles formed by solvent-in-water emulsion-diffusion technique:
Development and influence on insulin stability. Journal of Microencapsulation,
24(7): 672–684.
Belščak-Cvitanović, A., Stojanović, R., Manojlović, V., Komes, D., Cindrić, I.J.,
Nedović, V., Bugarski, B. 2011. Encapsulation of polyphenolic antioxidants from
medicinal plant extracts in alginate–chitosan system enhanced with ascorbic
acid by electrostatic extrusion. Food Research International, 44(4): 1094–1101.
Berset, C., Marty, C. 1992. Formation of nonvolatile compounds by thermal degra-
dation of β-carotene: Protection by antioxidants. Methods in Enzymology, 213:
129–142.
Beydoun, D., Guang, D., Chabra, R.P., Raper, J.A. 1998. Particle settling in oil-in-water
emulsions. Powder Technology, 97(1): 72–76.
Bhandari, B. 2005. Spray drying—An encapsulation technique for food flavors. In
Drying of Products of Biological Origin, ed. A.S. Mujumdar. Science Publisher,
Enfield, USA.
Bipin, R.P., Vijaysinh, V.C., Satyavan, S.D., Kirti, B.M. 2011. Healthy sugar and process
for preparation of the same. Publication number WO2011048616 A2.

Blanco-Pascual, N., Koldeweij, R.B.J., Stevens, R.S.A., Montero, M.P., Gomez-

Guillen, M.C., Ten Cate, A.T. 2014. Peptide microencapsulation by core-shell
printing technology for edible film application. Food and Bioprocess Technology,
7: 2472–2483.
Blonde, P. 1969. Sugar-like Artificial Sweetening Compositions and Their Production.
South African Patent. ZA6804238: 23.
Bos, M.A., Vliet, T.V. 2001. Interfacial rheological properties of absorbed protein layers
and surfactants: A review. Advances in Colloid and Interface Science, 91: 437–471.
Boursier, B. 2014. Encapsulation agents comprising a pea maltodextrin and/or
pea glucose syrup, compositions containing it and its preparation. US Patent
No. US 8,883,243 B2.
Britten, M., Giroux, H.J. 1993. Interfacial properties of milk protein-stabilized emul-
sions as influenced by protein concentration. Journal of Agricultural and Food
Chemistry, 41: 1187–1191.
Bylaite, E., Nylander, T., Venskutonis, R., Jonsson, B. 2001. Emulsification of cara-
way essential oil in water by Lecithin and β-lactoglobulin: Emulsion stabil-
ity and properties of the formed oil aqueous interface. Colloids and Surfaces B:
Biointerfaces, 20: 327–340.
Calvo, P., Hernández, T., Lozano, M., González-Gómez, D. 2010. Microencapsulation
of extra-virgin olive oil by spray-drying: Influence of wall material and olive
quality. European Journal of Lipid Science and Technology, 112(8): 852–858.
Capek, I. 2004. Degradation of kinetically-stable O/W emulsions. Advances in Colloid
and Interface Science, 107: 125–155.
Champagne, C.P. 2013. Algal hydrocolloids for the production and delivery of pro-
biotic bacteria. In Functional Ingredients from Algae for Foods and Nutraceuticals,
ed. H. Dominguez. Woodhead Publishing Ltd., Cambridge.
Champagne, C.P., Fustier, P. 2007. Microencapsulation for the improved delivery
of bioactive compounds into foods. Current Opinion in Biotechnology, 18(2):
184–190.
Chandramouli, V., Kailasapathy, K., Peiris, P., Jones, M. 2004. An improved method of
microencapsulation and its evaluation to protect Lactobacillus spp. in simulated
gastric conditions. Journal of Microbiological Methods, 56(1): 27–35.
Chang, D.W., Abbas, S., Hayat, K., Xia, S.Q., Zhang, X.M., Xie, M.Y., Kim, J.M. 2010.
Encapsulation of ascorbic acid in amorphous maltodextrin employing extrusion
as affected by matrix/core ratio and water content. International Journal of Food
Science and Technology, 45(9): 1895–1901.
Chantrapornchai, W., Clydesdale, F., McClements, D.J. 1998. Influence of droplet size
and concentration on the colour of oil-in-water emulsions. Journal of Agricultural
Chávarri, M., Marañón, I., Ares, R., Ibáñez, F.C., Marzo, F., Villarán, M.D.C. 2010.
Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules
improves survival in simulated gastro-intestinal conditions. International Journal
of Food Microbiology, 142(1–2): 185–189.
Chen, H. 2002. Formation and properties of casein films and coatings. In Protein Films
and Coatings, ed. H. Chen. CRC Press, Boca Raton, FL, Chapter 7.
Chen, L.-Y., Remondetto, G.E., Subirade, M. 2006. Food protein-based materials as
nutraceutical delivery systems. Trends in Food Science & Technology, 17: 272–283.
Chen, Q. 2012. Co-encapsulation of fish oil with phytosterol esters and limonene. PhD
thesis, The University of Auckland, New Zealand.

Chen, Q., McGillivray, D., Wen, J., Zhong, F., Quek, S.Y. 2013a. Co-encapsulation of
fish oil with phytosterol esters and limonene by milk proteins. Journal of Food
Engineering, 117(4): 505–512.
Chen, Q., Zhong, F., Wen, J., McGillivray, D., Quek, S.Y. 2013b. Properties and stabil-
ity of spray-dried and freeze-dried microcapsules co-encapsulated with fish oil,
phytosterol esters, and limonene. Drying Technology, 31(6): 707–716.
Chen, X.F., Liu, N.N. 2011. Study of microcapsules technology of onion oil flavor by
β-cyclodextrin. Zhongguo Tiaoweipin, 36(3): 61–637.
Choi, M.J., Briancon, S., Bazile, D., Royere, A., Min, S.G., Fessi, H. 2007. Effect of cryo-
protectant and freeze-drying process on the stability of W/O/W emulsions.
Drying Technology, 25(5): 809–819.
Chranioti, C., Tzia, C. 2014. Arabic gum mixtures as encapsulating agents of freeze-
dried fennel oleoresin products. Food and Bioprocess Technology, 7(4): 1057–1065.
Chung, C., Sanuansri, L., Augustin, M.A. 2008. Effects of modification of encapsu-
lant materials on the susceptibility of fish oil microcapsules to lipolysis. Food
Biophysics, 3: 140–145.
Cilek, B., Luca, A., Hasirci, V., Sahin, S., Sumnu, G. 2012. Microencapsulation of
phenolic compounds extracted from sour cherry pomace: Effect of formula-
tion, ultrasonication time and core to coating ratio. European Food Research and
Technology, 235(4): 587–596.
Comas, D.I., Wagner, J.R., Tomás, M.C. 2006. Creaming stability of oil in water
(O/W) emulsions: Influence of pH on soybean protein-lecithin interaction. Food
Hydrocolloids, 20: 990–996.
Coumans, W.J., Kerkhof, P.J.A.M., Bruin, S. 1994. Theoretical and practical aspects of
aroma retention in spray drying and freeze drying. Drying Technology, 12(1–2):
99–149.
Coupland, J.N., McClements, D.J. 2004. Analysis of droplet characteristics using low-
intensity ultrasound. Food Emulsions, ed. S.E. Friberg, K. Larsson and J. Sjöblom.
Marcel Dekker, Inc., New York, pp. 573–592.
Cournarie, F., Rosilio, V., Chéron, M., Vauthier, C., Lacour, C.B., Grossiord, J.L.,
Seiller, M. 2004. Improved formulation of W/O/W multiple emulsion for insu-
lin encapsulation; Influence of the chemical structure of insulin. Colloid Polymer
Science, 282: 562–568.
da Rosa, C.G., Borges, C.D., Zambiazi, R.C., Rutz, J.K., da Luz, S.R., Krumreich, F.D.,
Benvenutti, E.V., Nunes, M.R. 2014. Encapsulation of the phenolic compounds
of the blackberry (Rubus fruticosus). LWT-Food Science and Technology, 58(2):
527–533.
Dale, D.A., Gaertner, A.L., Park, G., Becker, N.T. 1997. Enzyme-containing coated
granules. The International Application according to the Patent Cooperation
Treaty. WO 9723606 (A1): 27.
Dalgleish, D.G. 2004. Food emulsions: Their structures and properties. In Food
Emulsions, ed. S.E. Friberg, K. Larsson and J. Sjöblom. Marcel Dekker, Inc., New
York, pp. 1–44.
Deis, R.C. 1997. Spray-drying-innovative use of an old process. Food Product Design
7: 97–113.
Desai, K.G.H., Park, H.J. 2005. Recent developments in microencapsulation of food
ingredients. Drying Technology, 23(7): 1361–1394.
Dickinson, E. 1997. Enzymic crosslinking as a tool for food colloid rheology control
and interfacial stabilization. Trends in Food Science & Technology, 8: 334–339.

Dickinson, E. 2003. Hydrocolloids at interfaces and the influence on the properties of

dispersed systems. Food Hydrocolloids, 17: 25–39.
Dickinson, E., Euston, S.R., Woskett, C.M. 1990. Competitive macromolecules and sur-
factants at the oil/water interface. Progress in Colloid and Polymer Science, 82: 65–72.
Dickinson, E., Yamamoto, Y. 1996. Viscoelastic properties of heat-set whey protein-
stabilized emulsion gels with added lecithin. Journal of Food Science, 61(4): 811–816.
Djordjevic, D., Kim, H.-J., McClements, D.J., Decker, E.A. 2004. Physical stability of
whey protein-stabilized oil-in-water emulsions at pH3: Potential n-3 fatty acid
delivery systems (Part A). Journal of Food Science, 69(5): 351–355.
Donhowe, E.G., Kong, F.B. 2014. Beta-carotene: Digestion, microencapsulation, and
in vitro bioavailability. Food and Bioprocess Technology, 7(2), 338–354.
Douillard, R., Lefebvre, J., Tran, V. 1993. Applicability of Gibbs’ law to protein adsorp-
tion isotherms. Colloid and Surfaces A: Physicochemical and Engineering Aspects,
78(1–3): 109–113.
Drusch, S. 2007. Sugar beet pectin: A novel emulsifying wall component for microen-
capsulation of lipophilic food ingredients by spray-drying. Food Hydrocolloids,
21: 1223–1228.
Drusch, S., Mannino, S. 2009. Patent-based review on industrial approaches for the
microencapsulation of oils rich in polyunsaturated fatty acids. Trends in Food
Science & Technology, 20: 237–244.
Drusch, S., Schwarz, K. 2006. Microencapsulation of two different types of n-
octenylsuccinate-derivatised starch. European Food Research and Technology, 222:
155–164.
Drusch, S., Serfert, Y., Schwarz, K. 2006. Microencapsulation of fish oil with n-
ctenylsuccinate-derivatised starch: Flow properties and oxidative stability.
o
European Journal of Lipid Science and Technology, 108: 501–512.
Drusch, S., Serfert, Y., Van Den Heuvel, A., Schwarz, K. 2006. Physicochemical char-
acterization and oxidative stability of fish oil encapsulated in an amorphous
matrix containing trehalose. Food Research International, 39(7): 807–815.
Dziezak, J. D. 1988. Microencapsulation and encapsulation ingredients. Food
El-Shaboouri, M.H. 2002. Positively charged nanoparticles for improving the oral
bioavailability of cyclosporin-A. International Journal of Pharmaceutics, 249(1–2):
101–108.
Euston, S.E., Munro, P.A., Dalgleish, D.G. 1995. Competitive adsorption between
sodium caseinate and oil-soluble and water-soluble surfactants in oil-in-water
emulsions. Journal of Food Science, 60: 1124–1131.
Euston, S.R., Finnigan, S.R., Hirst, R.L. 2001. Aggregation kinetics of heated whey
protein-stabilised emulsions: Effect of low-molecular weight emulsifiers. Food
Hydrocolloids, 15: 253–262.
Fäldt, P., Bergenståhl, B. 1996. Spray-dried whey protein/lactose/soybean oil emul-
sions. 2. Redispersability, wettability and particle structure. Food Hydrocolloids
10: 431–439.
Fathi, M., Martín, Á., McClements, D.J. 2014. Nanoencapsulation of food ingredients
using carbohydrate based delivery systems. Trends in Food Science & Technology,
39(1): 18–39.
Fathi, M., Mozafari, M.R., Mohebbi, M. 2012. Nanoencapsulation of food ingredients
using lipid based delivery systems. Trends in Food Science & Technology, 23(1):
13–27.

Fernandes, R.V.D., Borges, S.V., Botrel, D.A., de Oliveira, C.R. 2014. Physical and
chemical properties of encapsulated rosemary essential oil by spray drying
using whey protein-inulin blends as carriers. International Journal of Food Science
and Technology, 49(6): 1522–1529.
Floury, J., Desrumaux, A., Axelos, M.A.V., Legrand, J. 2003. Effect of high pres-
sure homogenisation on methylcellulose as food emulsifier. Journal of Food
Engineering, 58(3): 227–238.
Fox, P.F. 2001. Milk proteins as food ingredients. International Journal of Dairy
Franck, A. 2002. Technological functionality of inulin and oligofructose. British Journal
of Nutrition, 87(SupplementS2), S287–S291.
Fugman, D.A., Shirai, K., Jackson, R.L., Johnson, J.D. 1984. Lipoprotein lipase- and
phospholipase a2-catalyzed hydrolysis of phospholipid vesicles with an encap-
sulated fluorescent dye. Effects of apolipoproteins. Biochimica et Biophysica Acta,
Lipids and Lipid Metabolism, 795(2): 191–195.
Gabizon, A., Shmeeda, H., Horowitz, A.T., Zalipsky, S. 2004. Tumor cell targeting of
liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conju-
gates. Advanced Drug Delivery Reviews, 56(8): 1177–1192.
Gao, Z.M., Zhu, L.P., Yang, X.Q., He, X.T., Wang, J.M., Guo, J., Qi, J.R., Wang L.J., Yin,
S.W. 2014. Soy lipophilic protein nanoparticles as a novel delivery vehicle for
conjugated linoleic acid. Food & Function, 5(6): 1286–1293.
Garti, N. 2002. Food emulsifiers: Structure–reactivity relationships, design, and appli-
cations. In Physical Properties of Lipids, eds. A.G. Marangoni and S.S. Narine.
Marcel Dekker, New York, pp. 265–386.
Garti, N., Benichou, A. 2004. Recent developments in double emulsions for food
applications. In Food Emulsions, eds. S.E. Friberg, K. Larsson and J. Sjöblom.
Marcel Dekker, Inc., New York, pp. 353–412.
Garti, N., Bisperink, C. 1998. Double emulsions: Progress and applications. Current
Opinion in Colloid & Interface Science, 3(6): 657–667.
Gbassi, G.K., Vandamme, T., Ennahar, S., Marchioni, E. 2009. Microencapsulation of
Lactobacillus plantarum spp. in an alginate matrix coated with whey proteins.
International Journal of Food Microbiology, 129(1): 103–105.
Geankoplis, C.J. 2003. Transport Processes and Separation Process Principles (Includes
Unit Operations). Prentice Hall PTR, Upper Saddle River, NJ.
Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., Saurel, R. 2007. Applications
of spray-drying in microencapsulation of food ingredients: An overview. Food
Gibbs, B.F., Kermasha, S., Alli, I., Mulligan, C.N. 1999. Encapsulation in the food
industry: A review. International Journal of Food Science and Nutrition, 50: 213–224.
Goodacre, B.C., Pembroke, A.G., Shukla, D.P. 1989. Preparation of Low-Calorie, High-
Intensity Sweetener Compositions. European Patent Application. EP 0334617
(A2): 11.
Goubet, I., Le Quere, J.-L., Voilley, A.J. 1998. Retention of aroma compounds by carbo-
hydrates: Influence of their physicochemical characteristics and of their physi-
cal state. A review. Journal of Agricultural and Food Chemistry, 46: 1981–1990.
Gouin, S. 2004. Micro-encapsulation: Industrial appraisal of existing technologies and
trends. Trends in Food Science & Technology, 15: 330–347.
Green, B.K., Scheicher, L. 1955. Pressure Sensitive Record Materials. U. Patent. NO. 2,
217, 507, Ncr C.

Gu, Y.S., Decker, A.E., McClements, D.J. 2005. Production and characterization of oil-
in-water emulsions containing droplets stabilized by multilayer membranes
consisting of beta-lactoglobulin, iota-carrageenan and gelatin. Langmuir, 21(13):
5752–5760.
Gu, Y.S., Decker, E.A., McClements, D.J. 2007. Application of multi-component bio-
polymer layers to improve the freeze–thaw stability of oil-in-water emulsions:
β-lactoglobulin-ι-carrageenan-gelatin. Journal of Food Engineering, 80(4): 1246–1254.
Guo, M., Fox, P.F., Flynn, A., Kindstedt, P.S. 1996. Heat-induced modification of the
functional properteis of sodium caseinate. International Dairy Journal, 6: 473–485.
Guo, M., Fox, P.F., Flynn, A., Mohammad, K.S. 1989. Heat-induced changes in sodium
caseinate. Journal of Dairy Research, 56: 503–512.
Gupta, R., Muralidhara, H.S. 2001. Interfacial challenges in the food industry:
A review. Trends in Food Science & Technology, 12: 382–391.
Gurr, M.I. 1999. The nutritional and biological properties of the polyunsaturated fatty
acids. In Lipids in Nutrition and Health: A Reappraisal, eds. M.I. Gurr. The Oily
Press, England, pp. 119–158.
Gürsoy, A., Kut, E., Özkırımlı, S. 2004. Co-encapsulation of isoniazid and rifampi-
cin in liposomes and characterization of liposomes by derivative spectroscopy.
International Journal of Pharmaceutics, 271(1–2): 115–123.
Güzey, D., McClements, D.J. 2006a. Formation, stability and properties of multilayer
emulsions for application in the food industry. Advances in Colloid and Interface
Science, 128–130: 227–248.
Güzey, D., McClements, D.J. 2006b. Influence of environmental stresses on O/W
emulsions stabilized by β-lactoglobulin-pectin and β-lactoglobulin-pectin-
chitosan membranes produced by the electrostatic layer-by-layer deposition
technique. FOBI, 1: 30–40.
Halwanil, M., Yebiol, B., Suntres, Z.E., Alipour, M., Azghani, A.O., Omril, A. 2008.
Co-encapsulation of gallium with gentamicin in liposomes enhances antimicro-
bial activity of gentamicin against Pseudomonas aeruginosa. Journal of Antimicrobial
Chemotherapy, 62(6): 1291–1297.
Harnsilawat, T., Pongsawatmanit, R., McClements, D.J. 2006. Influence of pH and
ionic strength on formation and stability of emulsions containing oil droplets
coated by beta-lactoglobulin-alginate interfaces. Biomacromolecules, 7: 2052–2058.
Heidebach, T., Först, P., Kulozik, U. 2009. Microencapsulation of probiotic cells by
means of rennet-gelation of milk proteins. Food Hydrocolloids, 23(7): 1670–1677.
Heinzelmann, K., Franke, K. 1999. Using freezing and drying techniques of emulsions
for the microencapsulation of fish oil to improve oxidation stability. Colloids and
Surfaces B: Biointerfaces, 12: 223–229.
Heinzelmann, K., Franke, K., Jensen, B., Haahr, A.-M. 2000. Protection of fish oil from
oxidation by microencapsulation using freeze-drying techniques. European
Journal of Lipid Science and Technology, 120: 114–121.
Heinzen, C. 2002. Microencapsulation solve time dependent problems for foodmak-
ers. European Food and Drink Review, 3: 27–30.
Hendrickson, W.A., Finney, J.M., Moberg, O.C., Rueb, C.J., Bowman, R.G., Rao, C.S.,
Bentley, N.M. 2009. Encapsulation of oxidatively unstable compounds. P.I. Appl.
WO 2009089115 (A1) 51.
Hogan, S.A., McNamee, B.F., O’Riordan, E.D., O’Sullivan, M. 2001. Emulsification
and microencapsulation properties of sodium caseinate/carbohydrate blends.
International Dairy Journal, 11: 137–144.

Hogan, S.A., O’Riordan, E.D., O’Sullivan, M. 2003. Microencapsulation and oxidation

stability of spray-dried fish oil emulsions. Journal of Microencapsulation, 20(5):
675–688.
Homayouni, A., Azizi, A., Ehsani, M.R., Yarmand, M.S., Razavi, S.H. 2008. Effect of
microencapsulation and resistant starch on the probiotic survival and sensory
properties of synbiotic ice cream. Food Chemistry, 111(1): 50–55.
Hou, Z.Q., Zhang, M., Liu, B., Yan, Q.L., Yuan, F., Xu, D.X., Gao, Y.X. 2012. Effect of chi-
tosan molecular weight on the stability and rheological properties of β-carotene
emulsions stabilized by soybean soluble polysaccharides. Food Hydrocolloids,
26(1): 205–211.
Huang, L.X., Kumar, K., Mujumdar, A.S. 2006. A comparative study of a spary dryer
with rotary disc atomizer and pressure nozzle using computational fluid
dynamic simulations. Chemical Engineering and Processing, 45(6): 461–470.
Hunt, J.A., Dalgleish, D.G. 1994. Adsorption behaviour of whey protein isolate and
caseinate in soya oil-in-water emulsions. Food Hydrocolloids, 8(2): 175–187.
Hunt, J.A., Dalgleish, D.G. 1995. Heat stability of oil-in-water emulsions contain-
ing milk proteins: Effect of ionic strength and pH. Journal of Food Science, 60(5):
1120–1123: 1131.
Ishido, E., Hakamata, K., Minemoto, Y., Adachi, S., Matsuno, R. 2002. Oxidation pro-
cess of linoleic acid encapsulated with a polysaccharide by spray-drying. Food
Science and Technology Research, 8(1): 85–88.
Islam, A.M., Philipis, G.O., SIjivo, A., Snowden, M.J., Williams, P.A. 1997. A review of
recent developments on the regulatory, structural and functional aspect of gum
arabic. Food Hydrocolloids, 11: 493–505.
Iyer, C., Kailasapathy, K. 2005. Effect of co-encapsulation of probiotics with prebiotics
on increasing the viability of encapsulated bacteria under in vitro acidic and bile
salt conditions and in yogurt. Journal of Food Science, 70(1): 18–23.
Jacobsen, C., Let, M.B., Nielsen, N.S., Meyer, A.S. 2008. Antioxidant strategies for pre-
venting oxidative flavour deterioration of foods enriched with n-3 polyunsatu-
rated lipids: A comparative evaluation. Trends in Food Science & Technology, 19:
76–93.
Jafari, S.M., Assadpoor, E., Bhandari, B., He, Y. 2008b. Nano-particle encapsulation of
fish oil by spray drying. Food Research International, 41: 172–183.
Jafari, S.M., Assadpoor, E., He, Y., Bhandari, B. 2008a. Encapsulation efficiency of
food flavours and oils during spray drying. Drying Technology: An International
Journal, 26: 816–835.
Jantzen, M., Göpel, A., Beermann, C. 2013. Direct spray drying and microencapsula-
tion of probiotic Lactobacillus reuteri from slurry fermentation with whey. Journal
of Applied Microbiology, 115(4): 1029–36.
Jayme, M.L., Dunstan, D.E., Gee, M.L. 1999. Zeta potential of gum arabic stabilized
oil-in-water emulsions. Food Hydrocolloids, 13(6): 459–465.
Je, L.S., Rosenberg, M. 2000. Whey protein-based microcapsules prepared by double
emulsification and heat gelation. Academic Press, 33: 80–88.
Jimenez, M., Garcia, H.S., Beristain, C.I. 2004. Spray-drying microencapsulation
and oxidative stability of conjugated linoleic acid. European Food Research and
Jiménez-Flores, R., Ye, A., Singh, H. 2005. Interactions of whey proteins during heat
treatment of oil-in-water emulsions formed with whey protein isolate and
hydroxylated lecithin. Journal of Agricultural and Food Chemistry, 53: 4213–4219.

Junyaprasert, V.B., Mitrevej, A., Sinchaipanid, N., Boonme, P., Wurster, D.E. 2001. Effect
of process variables on the microencapsulation of vitamin a palmitate by gelatin-
acacia coacervation. Drug Development and Industrial Pharmacy, 27(6): 561–566.
Kabalnov, A. 2001. Ostwald ripening and related phenomena. Journal of Dispersion
Science and Technology, 22(1): 1–12.
Kagami, S., Sugimura, S., Fujishima, N., Matsuda, K., Kometani, T., Matsumura, Y.
2003. Oxidative stability, structure, and physical characteristics of microcap-
sules formed by spraying drying of fish oil with protein and dextrin wall mate-
rials. Journal of Food Science, 68(7): 2248–2255.
Kalab, M., Allan-Wojtas, P., Miller, S.S. 1995. Microscopy and other imaging tech-
niques in food structure analysis. Trends in Food Science & Technology, 6: 177–186.
Kamal-Eldin, A., Yanishlieva, N.V. 2002. n-3 fatty acids for human nutrition: Stability
considerations. European Journal of Lipid Science Technology, 104: 825–836.
Kaushik, P., Dowling, K., Barrow, C.J., Adhikari, B. 2014. Microencapsulation of
omega-3 fatty acids: A review of microencapsulation and characterization meth-
ods. Journal of Functional Foods, 1–14, doi:10.1016/j.jff.2014.06.029.
Keogh, M.K., O’Kennedy, B.T. 1999. Milk fat microencapsulation using whey pro-
teins. International Dairy Journal, 9: 657–663.
Khandelwal, S., Demonty, I., Jeemon, P., Lakshmy, R., Mukherjee, R., Gupta, R., Snehi,
U. et al. 2009. Independent and interactive effects of plant sterols and fish oil
n-3 long-chain polyunsaturated fatty acids on the plasma lipid profile of mildly
hyperlipidaemic Indian adults. British Journal of Nutrition, 102: 722–732.
Kim, C.H. 1996. Studies on the microencapsulation of w-3 polyunsaturated fatty
acids. Korean Journal of Food Science and Technology, 28(4): 743.
Kim, E.H.J., Chen, X.D., Pearce, D. 2009. Surface composition of industrial spray dried
milk powders. 2. Effects of spray drying conditions on the surface composition.
Journal of Food Engineering, 94: 169–181.
Kim, S.-J., Cho, S.Y., Kim, S.H., Song, O.-J., Shin, I.-S., Cha, D.S., Park, H.J. 2008. Effect
of microencapsulation on viability and other characteristics in Lactobacillus aci-
dophilus ATCC 43121. LWT—Food Science and Technology, 41(3): 493–500.
Kim, Y.D., Morr, C.V. 1996. Microencapsulation properties of gum arabic and several
food proteins: Spray-dried orange oil emulsion particles. Journal of Agricultural
Kinsella, J.E. 1984. Milk proteins: Physicochemical and functional properties. Critical
Review of Food Science and Nutrition, 21: 197–262.
Kirby, A.R., Gunning, A.P., Morris, V.J. 1995. Atomic force microscopy in food research:
A new technique comes of age. Trends in Food Science & Technology, 6: 359–369.
Klaypradit, W., Huang, Y.-W. 2008. Fish oil encapsulation with chitosan using ultra-
sonic atomizer. LWT—Food Science and Technology, 41(6): 1133–1139.
Klinkesorn, U., Sophanodora, P., Chinachoti, P., McClements, D.J. 2004. Stability and
rheology of corn oil-in-water emulsions containing maltodextrin. Food Research
International, 37: 851–859.
Knezevic, Z., Gosaki, D., Hraste, M., Jalsenjak, I. 1998. Fluid-bed microencapsulation
of ascorbic acid. Journal of Microencapsulation, 15(2): 237–252.
Koida, Y., Kobayashi, M., Hirata, G., Samejima, M. 1984. Studies on microcapsules. III.
Influence of molecular weight of polyisobutylene in the microencapsulation of
ascorbic acid. Chemical & Pharmaceutical Bulletin, 32(12): 4971–4978.
Kokufuta, E. 1998. Polyelectrolyte-coated microcapsules and their potential applica-
tions to biotechnology. Bioseparation, 7(4/5): 241–252.

Kolanowski, W., Jaworska, D., Weissbrodt, J., Kunz, B. 2007. Sensory assessment of
microencapsulated fish oil powder. Journal of the American Oil Chemists’ Society,
84(1): 37–45.
Kolanowski, W., Laufenberg, G., Kunz, B. 2004. Fish oil stabilisation by microen-
capsulation with modified cellulose. International Journal of Food Science and
Nutrition, 55(4): 333–343.
Kolanowski, W., Ziolkowski, M., Webbrodt, J., Kunz, B., Laufenberg, G. 2006.
Microencapsulation of fish oil by spray drying-impact on oxidative stability.
Part 1. European Food Research and Technology, 222: 336–342.
Koupantsis, T., Pavlidou, E., Paraskevopoulou, A. 2014. Flavour encapsulation in milk
proteins—CMC coacervate-type complexes. Food Hydrocolloids, 37, 134–142.
Krishnan, S., Bhosale, R., Singhal, R.S. 2005. Microencapsulation of cardamom oleo-
resin: Evaluation of blends of gum Arabic, maltodextrin and a modified starch
as wall materials. Carbohydrate Polymers, 61(1): 95–102.
Krochta, J.M. 2002. Proteins as raw materials for films and coatings: Definitions,
current status, and opportunities. In Protein-Based Films and Coatings, ed.
A. Gennadios. CRC Press, Boca Raton, FL, 2002.
Krog, N.J., Sparsø, F.V. 2004. Food emulsifiers: Their chemical and physical prop-
erties. In Food Emulsions, eds. S.E. Friberg, K. Larsson and J. Sjöblom. Marcel
Dekker, Inc., New York, pp. 45–91.
Kuang, S.S., Oliveira, J.C., Crean, A.M. 2010. Microencapsulation as a tool for incor-
porating bioactive ingredients into food. Critical Reviews in Food Science and
Nutrition, 50(10): 951–968.
Lamba, H., Sathish, K., Sabikhi, L. 2015. Double emulsions: Emerging delivery system
for plant bioactives. Food Bioprocess Technology, 87: 09–728.
Lawlor, J.B., Gaudette, N., Dickson, T., House, J.D. 2010. Fatty acid profile and sensory
characteristics of table eggs from laying hens fed diets containing microencap-
sulation fish oil. Animal Feed Science and Technology, 156(3–4): 97–103.
Lee, S.K., Klostermeyer, H., Schrader, K., Buchheim, W. 1996. Rheological properties
and microstructure of model processed cheese containing low molecular weight
emulsifiers. Nahrung, 40: 189–194.
Legako, J., Dunford, N.T. 2010. Effect of spray nozzle design on fish oil-whey protein
microcapsules properties. Journal of Food Science, 75(6): E394–E400.
Li, Y.O., Diosady, L.L., Wesley, A.S. 2010. Iodine stability in iodized salt dual fortified
with microencapsulated ferrous fumarate made by an extrusion-based encapsu-
lation process. Journal of Food Engineering, 99: 232–238.
Lieske, B., Konrad, G. 1994. Thermal Modification of sodium caseinate. 1. Influence
of temperature and pH on selected physicochemical and functional properties.
Milchwissenschaft, 49: 16–20.
Liu, X.D., Atarashi, T., Furuta, T., Aishima, S., Ohkawara, M., Linko, P. 2001.
Microencapsulation of emulsified hydrophobic flavors by spray drying. Drying
Technology, 19(7): 1361–1374.
Liu, X.D., Furuta, T., Yoshii, H., Linko, P. 2000. Retention of emulsified flavor in
a single droplet during drying. Food Science and Technology Research, 6(4):
335–339.
Lv, Y., Yang, F., Li, X.Y., Zhang, X.M., Abbas, S. 2014. Formation of heat-resistant nano-
capsules of jasmine essential oil via gelatin/gum Arabic based complex coacer-
vation. Food Hydrocolloids, 353: 05–314.

Madene, A., Jacquot, M., Scher, J., Desobry, S. 2006. Flavour Encapsulation and con-
trolled release: A review. International Journal of Food Science and Technology, 41:
1–21.
Mahajan, A.S., Tilkari, Y.P., Dubal, S.A., Momin, S.A. 2007. Microencapsulation of
sweet orange oil by spray drying using different wall materials. FAFAI Journal,
9(4): 71–82.
Maherani, B., Arab-Tehrany, E., Kheirolomoom, A., Cleymand, F., Linder, M. 2012.
Influence of lipid composition on physicochemical properties of nanoliposomes
encapsulating natural dipeptide antioxidant l-carnosine. Food Chemistry, 134(2):
632–640.
Malmo, C., La Storia, A., Mauriello, G. 2013. Microencapsulation of Lactobacillus
reuteri DSM 17938 cells coated in alginate beads with chitosan by spray drying
to use as a probiotic cell in a chocolate Soufflé. Food and Bioprocess Technology,
6(3): 795–805.
Malmsten, M., Lindstrom, A.-L., Wärnhem, T. 1995. Ellipsometry studies of interfacial
film formation in emulsion systems. Journal of Colloid and Interface Science, 173:
297–303.
Mattson, F.H., Grundy, S.M. 1982. Optimizing the effect of plant sterols on cholesterol
absorption in man. American Journal of Clinical Nutrition, 35: 697–700.
McCare, C.H. 1999. Heat stability of milk emulsions: Phospholipid-protein interac-
tions. International Dairy Journal, 9: 227–231.
McClements, D.J. 1999. Emulsion rheology. In: Food Emulsions-Principles, Practice and
Techniques. CRC Press, Washington, DC, pp. 235–266.
McClements, D.J. 2000. Comments on viscosity enhancement and depletion floccula-
tion by polysaccharides. Food Hydrocolloids, 14: 173–177.
McClements, D.J. 2005a. Characterization of emulsion properties. In Food Emulsions:
Principle, Practice, and Techniques, ed. D.J. McClements. CRC Press, Boca Raton,
FL, pp. 461–514.
McClements, D.J. 2005b. Context and background. In Food Emulsions: Principles, Practice,
and Techniques, ed. D.J. McClements. CRC Press, Boca Raton, FL, pp. 1–26.
McClements, D.J. 2005c. Emulsion ingredients. In Food Emulsions: Principle, Practices,
and Techniques, ed. D.J. McClements. CRC Press, Boca Raton, FL, pp. 95–174.
McClements, D.J. 2005d. Emulsion stability. In Food Emulsions: Principles, Practice, and
Techniques, ed. D.J. McClements. CRC Press, Boca Raton, FL, pp. 269–339.
McClements, D.J. 2007. Critical review of techniques and methodologies for charac-
terization of emulsion stability. Critical Reviews in Food Science and Nutrition, 47:
611–649.
McClements, D.J. 2008. Lipid-based emulsions and emulsifiers. In Food Lipids
Chemistry, Nutrition and Biotechnology, ed. C.C. Akoh and D.B. Min. CRC Press/
Taylor & Francis Group, Boca Raton, FL.
McClements, D.J. 2010. Emulsion design to improve the delivery of functional
lipophilic components. Annual Review of Food Science and Technology, 1:
241–269.
McClements, D.J., Decker, E.A., Weiss, J. 2007. Emulsion-based delivery systems for
lipophilic bioactive components. Journal of Food Science, 72(8): 109–124.
McClements, D.J., Demetriades, K. 1998. An integrated approach to the development
of reduced-fat food emulsions. Critical Reviews in Food Science and Nutrition,
38(6): 511–536.

McClements, D.J., Dungan, S.R. 1993. Factors that affect the rate of oil exchange
between oil-in-water emulsion droplets stabilized by a nonionic surfactant:
Droplet size, surfactant concentration, and ionic strength. Journal of Physical
Chemistry, 97: 7304–7308.
McNamee, B.F., O’Riordan, E.D., O’Sullivan, M. 1998. Emulsification and microen-
capsulation properties of gum Arabic. Journal of Agricultural and Food Chemistry,
46: 4551–4555.
Medina, I., González, M.J., Pazos, M., Medaglia, D.D., Sacchi, R., Gallardo, J.M. 2003.
Activity of plant extracts for preserving functional food containing n-3-PUFA.
European Food Research and Technology, 217(4): 301–307.
Micallef, M.A., Garg, M.L. 2008. The lipid-lowering effects of phytosterols and (n-3)
polyunsaturated fatty acids are synergistic and complementary in hyperlipid-
emic men and women. The Journal of Nutrition, 6: 1086–1090.
Micallef, M.A., Garg, M.L. 2009. Anti-inflammatory and cardioprotective effects of
n-3 polyunsaturated fatty acids and plant sterols in hyperlipidemic individuals.
Atherosclerosis, 204: 476–482.
Minemoto, Y., Hakamata, K., Adachi, S., Matsuno, R. 2002. Oxidation of linoleic acid
encapsulated with gum Arabic or maltodextrin by spray-drying. Journal of
Microenapsulation, 19(2): 181–189.
Monahan, F.J., McClements, D.J., German, J.B. 1996. Disulfide-mediated polymeri-
sation reactions and physical properties of heated WPI-stabilised emulsions.
Journal of Food Science, 61: 504–509.
Mongenot, N., Charrier, S., Chalier, P. 2000. Effect of ultrasound emulsification on
cheese aroma encapsulation by carbohydrates. Journal of Agricultural and Food
Chemistry, 48(3): 861–867.
Moraes, M., Carvalho, J.M.P., Silva, C.R., Cho, S., Sola, M.R., Pinho, S.C. 2013.
Liposomes encapsulating beta-carotene produced by the proliposomes
method: Characterisation and shelf life of powders and phospholipid vesicles.
International Journal of Food Science and Technology, 48: 274–282.
Morais, J.M., Santos, O.D.H., Nunes, J.R.L., Zanatta, C.F., ERocha-Filho, P.A.R. 2008.
W/O/W multiple emulsions obtained by one-step emulsification method and
evaluation of the involved variables. Journal of Dispersion Science and Technology,
29: 63–69.
Morr, C.V., Ha, E.Y.W. 1993. Whey protein concentrates and isolates: Processing and
functional properties. Critical Review of Food Science, 33: 431–476.
Mozafari, M.R., Khosravi-Darani, K., Borazan, G.G., Cui, J., Pardakhty, A., Yurdugul, S.
2008. Encapsulation of food ingredients using nanoliposome technology.
International Journal of Food Properties, 11(4): 833–844.
Mun, S., Cho, Y., Decker, E.A., McClements, D.J. 2008. Utilization of polysaccharide
coatings to improve freeze–thaw and freeze-drying stability of protein-coated
lipid droplets. Journal of Food Engineering, 86: 508–518.
Mun, S., McClements, D.J., Surh, J. 2010. Influence of maltodextrin type and multi-
layer formation on the freeze–thaw stability of model beverage emulsions stabi-
lized with β-lactoglobulin. Food Science and Biotechnology, 19(1): 7–17.
Munoz-Botella, S., Martin, M.A., del Castillo, B., Lerner, D.A., Menendez, J.C. 2002.
Differentiating geometrical isomers of retinoids and controlling their photo-
isomerization by complexation with cyclodextrins. Analytica Chimica Acta,
468(1): 161–170.

Nazzaro, F., Orlando, P., Fratianni, F., Coppola, R. 2012. Microencapsulation in food
science and biotechnology. Current Opinion in Biotechnology, 23(2): 182–186.
Nesterenko, A., Alric, I., Silvestre, F., Durrieu, V. 2014. Comparative study of encap-
sulation of vitamins with native and modified soy protein. Food Hydrocolloids,
38: 172–179.
Newton, I.S. 2001. Long-chain fatty acids in health and nutrition. In Omega-3 Fatty
Acids: Chemistry, Nutrition, and Health Effects, ed. F. Shahidi and J.W. Finley.
Oxford University Press, The United States of America, pp. 14–27.
Nie, L., Parker, M.D., Bassi, S.D., Maningat, C.C. 2010. Injection apparatus for produc-
ing molded food products. U.S. US20060340781 20060126.
Nieuwenhuyzen, W.V., Szuhaj, B.F. 1998. Effects of lecithins and proteins on the sta-
bility of emulsions. Fett/Lipid, 100: 282–291.
No, H.K., Meyers, S.P., Prinyawiwatkul, W., Xu, Z. 2007. Applications of chitosan for
improvement of quality and shelf life of foods: A review. Journal of Food Science,
72: R87–R100.
O’Hagan, P., Hasapidis, K., Coder, A., Helsing, H., Pokrajac, G. 2005. Particle size
analysis of food powders. In Encapsulated and Powdered Food, ed. C. Onwulata.
CRC Press, Boca Raton, FL, pp. 215–246.
Ogawa, S., Decker, E.A., McClements, D.J. 2003. Influence of environmental condi-
tions on the stability of oil in water emulsions containing droplets stabilized by
lecithin-chitosan membranes. Journal of Agricultural and Food Chemistry, 51(18):
5522–5527.
Onal, U., Langdon, C. 2005. Performance of zein-bound particles for delivery of ribo-
flavin to early fish larvae. Aquaculture Nutrition, 11(5): 351–358.
Pal, R. 1996. Effect of droplet size on the rheology of emulsions. AIChE Journal, 42 (11):
3181–3190.
Palazolo, G.G., Sorgentini, D.A., Wagner, J.R. 2005. Coalescence and flocculation in
o/w emulsions of native and denatured whey soy proteins in comparison with
soy protein isolates. Food Hydrocolloids, 19(3): 595–604.
Pandita, D., Kumar, S., Poonia, N., Lather, V. 2014. Solid lipid nanoparticles
enhance oral bioavailability of resveratrol, a natural polyphenol. Food Research
International, 621: 165–1174. doi: 10.1016/j.foodres.2014.05.059.
Paques, J.P., Sagis, L.M.C., van Rijn, C.J.M., van der Linden, E. 2014. Nanospheres
of alginate prepared through w/o emulsification and internal gelation
with nanoparticles of CaCO3. Food Hydrocolloids, 40: 182–188. doi: 10.1016/j.
foodhyd.2014.02.024.
Pedersen, G.P., Fäldt, P., Bergenståhl, B., Kristensen, H.G. 1998. Solid state character-
ization of a dry emulsion: A potential drug delivery system. International Journal
of Pharmacy, 171: 257–270.
Penalva, R., Esparza, I., Agüeros, M., Gonzalez-Navarro, C.J., Gonzalez-Ferrero, C.,
Irache, J.M. 2015. Casein nanoparticles as carriers for the oral delivery of folic
acid. Food Hydrocolloids, 44: 399–406.
Philips, G.O., Williams, P.A. 1995. Interaction of hyrocolloids in food systems.
In: Ingredient Interactions: Effect on Food Quality, ed. A.G. Gaonkar, Marcel
Dekker, New York, pp. 131–170.
Quintanilla-Carvajal, M., Camacho-Díaz, B., Meraz-Torres, L., Chanona-Pérez, J., Alamilla-
Beltrán, L., Jimenéz-Aparicio, A., Gutiérrez-López, G. 2010. Nanoencapsulation:
A new trend in food engineering processing. Food Engineering Reviews, 2(1): 39–50.

Quispe-Condori, S., Saldana, M.D.A., Temelli, F. 2011. Microencapsulation of flax

oil with zein using spray and freeze drying. LWT—Food Science and Technology,
44(9): 1880–1887.
Randall, R.C., Philipis, G.O., Williams, P.A. 1989. Fractionation and characterization
of gum from Acacia Senegal. Food Hydrocolloids, 3(1): 65–75.
Rao, J., McClements, D.J. 2012. Food-grade microemulsions and nanoemulsions: Role
of oil phase composition on formation and stability. Food Hydrocolloids, 29(2):
326–334. doi: 10.1016/j.foodhyd.2012.04.008.
Rao, J.J., McClements, D.J. 2013. Optimization of lipid nanoparticle formation for
beverage applications: Influence of oil type, cosolvents, and cosurfactants on
nanoemulsion properties. Journal of Food Engineering, 118(2): 198–204. doi:
10.1016/j.jfoodeng.2013.04.010.
Reineccius, G.A. 1988. Spray-drying of Food Flavours. In: Flavor Encapsulation, eds.
S.J. Risch and G.A. Reineccius. Washington, DC, American Chemical Society,
pp. 55–66.
Reineccius, G.A. 2001. Multiple-core encapsulation; The spray drying of food ingredi-
ents. In Microencapsulation of Food Ingredients, ed. P. Vilstrup, Leatherhead Food
RA Publishing, London, pp. 151–185.
Ribeiro, H.S., Ax, K., Schubert, H. 2003. Stability of lycopene emulsions in food sys-
tems. Journal of Food Science, 68(9): 2730–2734.
Risch, S.J., Reineccius, G.A. 1988. Spray dried orange oil: effect on emulsion size
on flavor retention and shelf life stability. In: Flavor Encapsulation, eds. G.A.
Reineccius, S.J. Risch. Washington, D.C., American Chemical Society, pp. 67–77.
Robert, P., Gorena, T., Romero, N., Sepulveda, E., Chavez, J., Saenz, C. 2010.
Encapsulation of polyphenols and anthocyanins from pomegranate (Punica gra-
natum) by spray drying. International Journal of Food Science & Technology, 45(7):
1386–1394.
Robson, E., Dalgleish, D.G. 1987. Interfacial composition of sodium caseinate emul-
sions. Journal of Food Science, 52: 1694–1698.
Rodriguez Patino, J.M., Minones Conde, J., Linares, H.M., Pedroche Jimenez, J.J.,
Carrera Sanchez, C., Pizones, V., Rodriguez, F.M. 2007. Interfacial and foam-
ing properties of enzyme-induced hydrolysis of sunflower protein isolate. Food
Hydrocolloids, 21(5–6): 782–793.
Rosenberg, M., Kopelman, I.J., Talman, Y. 1990. Factors affecting retention in spray-
drying microcapsulation of volatile materials. Journal of Agricultural and Food
Chemistry, 38: 1288–1294.
Rosenberg, M., Sheu, T.Y. 1996. Microencapsulation of volatiles by spray-drying in
whey-based wall systems. International Dairy Journal, 6: 273–284.
Rosenberg, M., Young, S.L. 1993. Whey proteins as microencapsulating agents-micro-
encapsulation of anhydrous milk fat: Structure evaluation. Food Structure, 12:
31–41.
Rousseau, D. 2000. Fat crystals and emulsion stability: A review. Food Research
Saenz, C., Tapia, S., Chavez, J., Robert, P. 2009. Microencapsulation by spray drying
of bioactive compounds from cactus pear (Opuntia ficus-indica). Food Chemistry,
114(2): 616–622.
Sæther, Ø., Sjöblom, J., Dukhin, S.S. 2004. Droplet flocculation and coalescence in
dilute oil-in water emulsion. In Food Emulsions, eds. S.E. Friberg, K. Larsson and
J. Sjöblom. Marcel Dekker, Inc., New York, pp. 190–230.

Samborska, K., Czelejewska, M. 2014. The influence of thermal treatment and spray
drying on the physicochemical properties of Polish honeys. Journal of Food
Processing and Preservation, 38(1): 413–419.
Schwegman, J.J. 2009. Understanding the physical properties of freeze-dried materi-
als. Innovations in Pharmaceutical Technology, 29: 72–74, 76–77.
Scuriatti, M.P., Tomás, M.C., Wagner, J.R. 2003. Influence of soybean protein isolates
phosphatidylcholine interaction on the stability of oil-in-water emulsions.
JAOCS, 80(10): 1093–1100.
Serfert, Y., Drusch, S., Schwarz, K. 2010. Sensory odour profiling and lipid oxidation
status of fish oil and microencapsulated fish oil. Food Chemistry, 123: 968–875.
Setchell, K.D.R., Brzezinski, A., Brown, N.M., Desai, P.B., Melhem, M., Meredith, T.,
Zimmer-Nechimias, L., Wolfe, B., Cohen, Y., Blatt, Y. 2005. Pharmacokinetics of
a slow-release formulation of soybean isoflavones in healthy postmenopausal
women. Journal of Agricultural and Food Chemistry, 53: 1938–1944.
Shah, K.A., Date, A.A., Joshi, M.D., Patravale, V.B. 2007. Solid lipid nanopar-
ticles (SLN) of tretinoin: Potential in topical delivery. International Journal of
Pharmaceutics, 345: 163–171.
Shefer, A., Shefer, S. 2003. Novel encapsulation system provides controlled release of
ingredients. Food Technology, 57(11): 40–42.
Shen, Q., Quek, S.Y. 2014. Microencapsulation of astaxanthin with blends of milk pro-
tein and fiber by spray drying. Journal of Food Engineering, 1231: 65–171.
Shen, Z.P., Augustin, M.A., Sanguansri, L., Cheng, L.J. 2010. Oxidative stability of
microencapsulated fish oil powders stabilized by blends of chitosan, modified
starch, and glucose. Journal of Agricultural and Food Chemistry, 58: 4487–4493.
Sheu, T.Y., Rosenberg, M. 1995. Microencapsulation by spray-drying ethyl caprylate
in whey-protein and carbohydrate wall systems. Journal of Food Science, 60(1):
98–103.
Shi, J., Xue, J., Wang, B., Wang, W., Ye, X., Quek, S.Y. 2015. Optimization of formulation
and influence of environmental stress on stability of lycopene-microemulsion.
LWT—Food Science and Technology, 609: 99–1008.
Shukla, A., Janich, M., Jahn, K., Krause, A., Kiselev, M.A., Neubert, R.H.H. 2002.
Investigation of pharmaceutical oil/water microemulsions by small-angle scat-
tering. Pharmaceutical Research, 19(6): 881–886.
Silletti, E., Vingerhoeds, M.H., Norde, W., van Aken, G.A. 2007. The role of electrostat-
ics in saliva-induced emulsion flocculation. Food Hydrocolloids, 21(4): 596–606.
Slater, N. 2007. Process for encapsulation of edible oil products with whey protein
and encapsulated edible oil products. P.I. Appl., WO 2007034213 (A1): 30.
Sliwinski, E.L., Lavrijsen, B.W.M., Vollenbroek, J.M. 2003. Effects of spray drying on
physicochemical properties of milk protein-stabilised emulsions. Colloids and
Surfaces B: Biointerfaces, 31: 219–229.
Soottitantawat, A., Bigeard, F., Yoshii, H., Furuta, T., Ohkawara, M., Linko, P. 2005.
Influence of emulsion and powder size on the stability of encapsulated d-limonene
by spray drying. Innovative Food Science & Emerging Technologies, 6(1): 107–114.
Soottitantawat, A., Yoshii, H., Furuta, T., Ohkawara, M., Linko, P. 2003.
Microencapsulation by spray drying: Influence of emulsion size on the reten-
tion of volatile compounds. Journal of Food Science, 68(7): 2256–2262.
Soukoulis, C., Yonekura, L., Gan, H.H., Behboudi-Jobbehdar, S., Parmenter, C., Fisk, I.
2014. Probiotic edible films as a new strategy for developing functional bakery
products: The case of pan bread. Food Hydrocolloids, 39: 231–242.

Srinivasan, M., Singh, H., Munro, P.A. 1999. Adsorption behaviour of sodium and cal-
cium caseinates in oil-in-water emulsions. International Dairy Journal, 9: 337–341.
Srinivasan, M., Singh, H., Munro, P.A. 2002. Formation and stability of sodium casein-
ate emulsions: Influence of retorting (121oC for 15 min) before or after emulsifi-
cation. Food Hydrocolloids, 16: 153–160.
Stefan, D., Bahrim, G. 2007. Preparation and use of intelligent invertase micro and
nanocapsules. Romanian Biotechnological Letters, 12(3): 3287–3293.
Stepanov, V., Qiu, H.W., Surapaneni, A. 2011. Production of novel CL-20-based com-
positions by spray drying. International Annual Conference of ICT, Fraunhofer-
Institut fuer Chemische Technologie, Karlsruhe, Germany, 110/111-110/116
(ISSN 0722-4087).
Sun, J. 2007. d-Limonene: Safety and clinical applications. Alternative Medicine Review,
12(3): 259–264.
Szente, L., Szejtli, J. 2004. Cyclodextrins as food ingredients. Trends in Food Science &
Technology, 15(3–4), 137–142.
Tadros, T., Izquierdo, P., Esquena, J., Solans, C. 2004. Formation and stability of nano-
emulsions. Advances in Colloid and Interface Science, 108–109: 303–318.
Tadros, T.F. 1994. Fundamental principles of emulsion rheology and their applica-
tions. Colloid and Surfaces A: Physicochemical and Engineering Aspects, 91: 39–55.
Tan, C.P., Nakajima, M. 2005. [beta]-Carotene nanodispersions: Preparation, charac-
terization and stability evaluation. Food Chemistry, 92: 661–671.
Tan, C.T. 1997. Beverage emulsions. In Food Emulsions, ed. S.E. Friberg. Marcel Dekker,
New York, pp. 491–524.
Tan, L.H., Chan, L.W., Heng, P.W.S. 2009. Alginate/starch composites as wall material
to achieve microencapsulation with high oil loading. Journal of Microenapsulation,
26(3): 263–271.
Tao, F., Hill, L.E., Peng, Y., Comes, C.L. 2014. Synthesis and characterization of beta-
cyclodextrin inclusion complexes of thymol and thyme oil for antimicrobial
delivery applications. LWT-Food Science and Technology, 59: 247–255.
Tapal, A., Tiku, P.K. 2012. Complexation of curcumin with soy protein isolate and its
implications on solubility and stability of curcumin. Food Chemistry, 130(4): 960–965.
Taxi, C.M.A.D., De Menezes, H.C., Santos, A.B., Grosso, C.R.F. 2003. Study of
the microencapsulation of Camu-Camu (Myriaria dubia) juice. Journal of
Microencapsulation, 20(4): 443–448.
Taylor, P. 1998. Ostwald ripening in emulsions. Advances in Colloid and Interface Science,
75: 107–163.
Teixeira, Z., Duran, N., Guterres, S.S. 2008. Annatto polymeric microparticles: Natural
product encapsulation by the emulsion-solvent evaporation method. Journal of
Chemical Education, 85(7): 846–947.
Thijssen, H.A. 1971. Flavour retention in drying preconcentrated food liquids. Journal
of Applied Chemistry and Biotechnology, 21(12): 372.
Thirundas, R., Gadhe, K.S., Syed, I.H. 2014. Optimization of wall material concentra-
tion in preparation of flaxseed oil powder using response surface methodology.
Journal of Food Processing and Preservation, 38: 889–895.
Toledo, R.T. 2007. Dehydration. In Fundamentals of Food Process Engineering, ed. R.T.
Toledo. Springer, Boston, MA, pp. 431–473.
Tornberg, E. 1978. Functional characterisation of protein stabilized emulsions:
Emulsifying behaviour of proteins in a valve homogenizer. Journal of Science and
Food Agriculture, 29: 867–879.

Uhlemann, J., Schleifenbaum, B., Bertram, H.J. 2002. Flavor encapsulation technologies:
An overview including recent developments. Perfumer and Flavorist, 27: 52–61.
van Aken, G.A. 2003. Competitive adsorption of protein and surfactants in highly
concentrated emulsions: Effect on coalescence mechanisms. Colloid and Surfaces
A: Physicochemical and Engineering Aspects, 213(2–3): 209–219.
Vega, C., Kim, E.H.J., Chen, X.D., Roos, Y.H. 2005. Solid-state characterization of
spray-dired ice cream mixes. Colloids and Surfaces B: Biointerfaces, 45: 66–75.
Vega, C., Roos, Y.H. 2006. Invited review: Spray-dried dairy and dairy-like emulsions–
compositional considerations. Journal of Dairy Science, 89: 393–401.
Vitaglione, P., Barone Lumaga, R., Ferracane, R., Sellitto, S., Morelló, J.R., Reguant
Miranda, J. et al. 2013. Human bioavailability of flavanols and phenolic
acids from cocoa-nut creams enriched with free or microencapsulated cocoa
polyphenols. The British Journal of Nutrition, 109(10): 1832–1843.
Walstra, P. 1993. Principles of emulsion formation. Chemical Engineering Science,
48: 333–350.
Wandrey, C., Bartkowiak, A., Harding, S.E. 2010. Materials for Encapsulation. Springer,
New York.
Wang, J.-L., Dong, X.-Y., Wei, F., Zhong, J., Liu, B., Yao, M.-H., Quek, S.Y., Chen, H.
2014. Preparation and characterization of novel lipid carriers containing micro-
algae oil for food applications. Journal of Food Science, 79(2): E169–E177.
Wang, R.X., Tian, Z.G., Chen, L.Y. 2011. A novel process for microencapsulation of fish
oil with barley protein. Food Research International, 44(9): 1735–1741.
Watanabe, Y., Fang, X., Minemoto, Y., Adachi, S., Matsuno, R. 2002. Suppressive effect
of saturated acyl l-ascorbate on the oxidation of linoleic acid encapsulated with
maltodextrin or gum Arabic by spray-drying. Journal of Agricultural and Food
Chemistry, 50(14): 3984–3987.
Weiss, J., Decker, E.A., McClements, D.J., Kristbergsson, K., Helgason, T., Awad, T.
2008. Solid lipid nanoparticles as delivery systems for bioactive food compo-
nents. Food Biophysics, 3(2): 146–154.
Williams, P.A., Phillips, G.O. 2000. Gum Arabic. In Handbook of Hydrocolloids, ed. P.A.
Williams and G.O. Philipis. Woodhead Publishing Ltd., Cambridge, United
Kingdom, pp. 162–168.
Wilson, N., Shah, N. 2007. Microencapsulation of vitamins. ASEAN Food Journal,
14(1): 1–14.
Winning, M. 1995. Micro-encapsulated Colors—Natural colors with improved stabil-
ity. Agro-Food-Industry Hi-Tech, 6(5): 13–15.
Wooster, T.J., Augustin, M.A. 2007. The emulsion flocculation stability of protein-carbo-
hydrate diblock copolymers. Journal of Colloid and Interface Science, 313: 665–675.
Xiao, Z.F., Xie, X.Y., Yuan, Y.J., Liu, X.D. 2008. Influence of atomizing parameters on
droplet properties in a pulse combustion spray dryer. Drying Technology, 26(4):
427–432.
Xie, Z.G., Wang, F.E., Liu, H.Y., Guo, S.D., Zhu, A.X., Niu, H.X. 2010. Gelatin-walled
microencapsulated diet for larval shrimp (Penaeus japnicus Bate) manufactured
using the fluidized bed coating process. Aquaculture Research, 42(1): 65–73.
Yasukazu, Y., Etsuo, N. 2003. Antioxidant effects of phytosterol and its components.
Journal of Nutritional Science and Vitaminology, 49(4): 277–280.
Young, S.L., Sarda, X., Rosenberg, M. 1993a. Microencapsulating properties of whey
proteins. 1. Microencapsulation of anhydrous milk fat. Journal of Dairy Science,
76: 2868–2877.

Young, S.L., Sarda, X., Rosenberg, M. 1993b. Microencapsulating properties of whey

Proteins. 2. Combination of whey proteins with carbohydrates. Journal of Dairy
Science, 76: 2878–2885.
Yu, H.L., Huang, Q.R. 2012. Improving the oral bioavailability of curcumin using
novel organogel-based nanoemulsions. Journal of Agricultural and Food Chemistry,
60(21): 5373–5379. doi: 10.1021/jf300609p.
Yuan, Y., Gao, Y., Zhao, J., Mao, L. 2008. Characterization and stability evaluation
of [beta]-carotene nanoemulsions prepared by high pressure homogenization
under various emulsifying conditions. Food Research International, 41: 61–68.
Zhang, J., Sasaki, S., Amano, K., Kesteloot, H. 1999. Fish consumption and mortal-
ity from all causes, ischemic heart disease, and strole: An ecological study.
Preventive Medicine, 28(5): 520–529.
Zhao, L.P., Xiong, H., Peng, H.L., Wang, Q., Han, D., Bai, C.Q., Liu, Y.Z., Shi, S.H.,
Deng, B. 2011. PEG-coated lyophilized proliposomes: Preparation, characteriza-
tions and in vitro release evaluation of vitamin E. European Food Research and
Technology, 232(4): 647–654.
Zheng, L., Ding, Z., Zhang, M., Sun, J. 2011. Microencapsulation of bayberry poly-
phenols by ethyl cellulose: Preparation and characterization. Journal of Food
Engineering, 104(1): 89–95.
Zoet, F., Grandia, J., Sibeijn, M. 2011. Encapsulated fat soluble vitamin. NL patent,
50668.

11
Nano-Microencapsulation
Technology and Applications in
Fortified and Functional Foods
Yao Olive Li
CONTENTS
11.1 Introduction................................................................................................. 320
11.1.1 Concept and Historical Development
of Microencapsulation................................................................ 320
11.1.2 Overview of Microencapsulation Technology in Food
Applications..................................................................................... 322
11.1.3 Importance of Microencapsulation in Fortified
and Functional Food Development.............................................. 322
11.1.4 Criteria for Developing Microencapsulated Delivery
Systems............................................................................................. 326
11.1.4.1 Bioavailability of Micronutrients
and Nutraceuticals........................................................ 326
11.1.4.2 Encapsulation Efficiency................................................. 327
11.1.4.3 Microcapsule Morphology and Size............................. 328
11.1.4.4 Storage Stability................................................................ 329
11.2 Processing Techniques............................................................................... 329
11.2.1 Physical and Mechanical Methods............................................... 330
11.2.2 Chemical Methods.......................................................................... 332
11.2.3 New Advancements....................................................................... 336
11.2.3.1 Controlled Release Technology...................................... 336
11.2.3.2 Microencapsulation Coupling with Controlled
Release............................................................................... 338
11.2.3.3 Nanolevel Delivery Systems........................................... 339
11.2.3.4 New Advancements in Equipment/Device
Development..................................................................... 341
11.3 Coating or Shell Materials Used...............................................................342
11.3.1 Hydrophilic Coatings.....................................................................344
11.3.2 Hydrophobic Coatings...................................................................345
11.3.3 Enteric and Reverse-Enteric Coatings..........................................346
11.4 Case Studies of Food Applications Based on Different Categories
of Nutraceuticals......................................................................................... 349
319
11.4.1 Microencapsulation and Delivery Systems for Vitamins

and Minerals.................................................................................... 349
11.4.1.1 Microencapsulation of Ferrous Fumarate for Salt
Fortification....................................................................... 350
11.4.1.2 Application of Extrusion-Based
Microencapsulation in Rice Fortification.....................354
11.4.2 Microencapsulation and Delivery Systems for Major
Nutraceuticals.................................................................................. 356
11.4.2.1 Microencapsulation-Based Delivery Systems
for Probiotics..................................................................... 357
for Omega-3 and Omega-6 Oils..................................... 358
for Phytochemicals.......................................................... 359
11.5 Conclusion and Perspectives..................................................................... 361
References.............................................................................................................. 363
11.1 Introduction
11.1.1 Concept and Historical Development of Microencapsulation
Microencapsulation is defined as a technology of enveloping small solid par-
ticles, liquid droplets, or gases in a coating matrix (Benita, 1996). The coated
or entrapped material, also known as the core, fill, internal phase, or payload,
is usually the active ingredient, which needs to be protected from the envi-
ronment or released at a controlled rate. The coating material is called the
capsule, wall, shell, membrane, carrier, encapsulant, or matrix (Benita, 1996).
Microencapsulation was originally developed by Barrett K. Green of the
National Cash Register (NCR) Corp. in the 1950s, with a process called coac-
ervation to create carbonless paper (Benita, 1996). The process involved a
soluble polymer, such as gelatin, induced to come out of solution and form a
shell around dispersed droplets of oil at the interface with a water medium.
The gelatin shell is hardened by the addition of glutaraldehyde, and the
microscopic beads are collected and dried (Clark, 2002). Since then, many
technologies for preparing microparticles have been developed for applica-
tions in pharmaceutical, food, cosmetic, chemical, and printing industries, as
briefly summarized in Table 11.1 (Madene et al., 2006).
As shown in Figure 11.1, the design of a microencapsulated system gen-
erally involves a core material, a wall material, and an appropriate tech-
nique/process required to coat or entrap the core by the wall material. The
core material is the key factor that needs to be protected or released at a
defined rate, while the wall material and process/technique collectively play
an important role in the physical and chemical properties of the formed

Nano-Microencapsulation Technology and Applications 321
TABLE 11.1
Widespread Applications of Microencapsulation in a Number of Industries
Application Microencapsulated Actives References
Pharmaceutical Oral, topical or transdermal, Wang et al. (2003); Lamprecht et al. (2000);
parenteral drugs Carafa et al. (2004); Kshirsagar (2000);
Chen and Lu (1999); Park et al. (2005)
Biological Cells, vaccines, hormones, Sefton et al. (2002); Cleland et al. (1997);
antigens, plasmid DNAs, Lee et al. (1997); Tinsley-Bown et al.
enzymes (2000); Hao et al. (2001); Genta et al.
(2001)
Food Acidulents, flavours, artificial Kirby (1991); Gibbs et al. (1999); Gouin
sweeteners, colourants, (2004); Schrooyen (2001); Desai et al.
enzymes, microorganisms, (2005)
probiotics, leavening agents,
antioxidants, preservatives,
vitamins, minerals, amino
acids, essential oils
Agro-chemical Pesticides, herbicides Tsuji (2001)
Cosmetic and Vitamin E, fragrances, Dingler (1999); Schmitt et al. (1998);
personal care perfumes, plant extracts Benita (1996)
microparticles, such as particle size, permeability, porosity, density (bulk

and particle density), flowability, integrity, reactivity/stability, release prop-
erties, and bioavailability. For each active ingredient, the appropriate choice
of process and wall materials depends on the end use of the microencapsu-
lated particles. For example, microencapsulated flavors for the application
in extrusion-based processed foods should be heat resistant and insoluble
while in the barrel so the flavor is protected against thermal degradation and
flash-off at the exit of the die (Bhandari et al., 2001).
Core ingredient Wall material
Technique/process
Microparticles with desired properties
FIGURE 11.1
Schematic relationship between the core material, the wall material, and the required tech-
nique used in microencapsulation systems.

11.1.2 Overview of Microencapsulation Technology
in Food Applications
Many well-developed microencapsulation techniques and numerous wall
materials have been used in food since the 1980s, which results in many
food ingredients being microencapsulated, as shown in Table 11.2. Different
techniques and shell materials will be reviewed in Sections 11.2 and 11.3,
respectively.
There are a number of reasons for the food industry’s use of microencap-
sulation technologies, including the following:
• Encapsulation or entrapment can protect the active ingredients from

degradation due to environmental conditions such as heat, moisture,
air, and light.
• The physical properties of original core materials can be modified
to make it easy for handling, for example, liquid ingredients can be
converted to free-flowing powders.
• Evaporation or leakage of the core material to the outside environ-
ment can be reduced or controlled, with components such as the
volatile flavoring agents.
• The unpleasant taste or appearance of some food ingredients can be
masked.
• Several active ingredients can be segregated within a food matrix
in separate forms, which prevents undesirable interactions between
them.
• The food ingredients can be tailored to either release slowly or at a
certain point within the process of the digestive system.
11.1.3 Importance of Microencapsulation in Fortified

and Functional Food Development
Micronutrients, namely, vitamins and minerals, are compounds required only
in minute amounts that enable the body to produce enzymes, hormones, and
other substances essential for proper growth and development. As tiny as the
amounts are, however, the consequences of their absence are severe. Their addi-
tion into foods has been practiced by the food industry for years as a cost-effec-
tive means of alleviating micronutrient malnutrition. Some common examples
of micronutrients, which have been added into foods, include iron, iodine, cal-
cium, zinc, vitamins A and D, as well as several B vitamins (Black, 2003).
Nutraceuticals are physiologically active components naturally present in
foods or added to them as functional ingredients, which have a role in health
and well-being of the population. Examples of nutraceuticals, which have
been used to develop functional foods, include polyphenols and other phyto-
antioxidants, omega-3 and omega-6 fatty acids, probiotics, and prebiotics

TABLE 11.2
Nano-Microencapsulation Technology and Applications

Microencapsulated Food Ingredients
Purposes for
Food Ingredients Examples Functions or Properties Microencapsulation Applications
Acidulants Adipic, fumaric, citric, Active agents used as preservation Prevent interactions with other Bread, tea, cured meats,
lactic and ascorbic aids, flavour modifiers components and desserts, baking mixes,
acids self-degradation and pet foods
Flavours Citrus, mint, onion, Volatile, reactive, susceptible to Enhance stability, convert to Microwavable and
garlic oils, spices, heat and moisture free flowing powder, control extruded foods,
menthol, peppermint release chewing-gums, instant
beverages/desserts,
confectionery, toothpaste
Sweeteners Sugar, artificial Susceptible to heat, moisture, and Prevent degradation by Chewing-gum,
sweeteners such as other components temperature and moisture, confectionery, baking
aspartame reduce hygroscopicity, foods, mouthwash, and
improve flowability, control toothpaste
release
Colourants Beta-carotene, turmeric, Less water soluble, less stable to Convert to free flowing form All sorts of processed
annatto, natural colors oxidation for easy-handling, improve foods
solubility and stability
Enzymes and Neutrase, lipase, Taste modification, texture control, Control release, improve Cheese, fermented foods,
microorganisms lysozyme, pepsin, aroma formation, spoilage stability, enhance water treatment
amylases, proteases prevention effectiveness by reducing
amount used or ripening time
Nutritional Vitamins, minerals, Unstable, reactive, causing Improve stability, mask Fortified foods, breakfast
ingredients amino acids, essential off-flavour and discoloration off-flavour, prevent interaction cereals, dairy products,
oils with other components, infant foods
control release
(Continued)
323
324
Microencapsulated Food Ingredients
Purposes for
Food Ingredients Examples Functions or Properties Microencapsulation Applications
Miscellaneous Preservatives, Functional food additives which Improve solubility, enhance Canned foods, package
additives antioxidants, levening are preferred to add in small functionality, control release, foods, baked foods and
agents amount but with uniform reduce amount used baking mixes
distribution
Source: Kirby, C. 1991. Food Science and Technology Today, 5(2): 74–78; Gibbs, B., Kermasha, S., Alli, I., Mulligan, C. 1999. International Journal of Food
Science and Nutrition, 50(3): 213–224; Gouin, S. 2004. Trends in Food Science & Technology, 15(7): 330–347; Schrooyen, P., Van der Meer, R., De
Kruif, C. 2001. Proceedings of the Nutrition Society, 60: 475–479; Desai, K.G.H. 2005. AAPS PharmaSciTech, 6(2): E202–E208.

(Hasler, 1998). However, there are compounds that can be classified as either
a micronutrient or nutraceutical or both. An example is β-carotene, which
acts as a provitamin A precursor and also as a phyto-antioxidant.
As the ultimate goal is effective use, that is, bioavailability, of these com-
ponents by the body, the goal in food fortification is to ensure that they are
delivered and released at proper time and location in the body. The bioavail-
ability of many compounds with poor water solubility or low permeabil-
ity in the small intestine can be improved by appropriate control of food
production and consumption. Therefore, innovative delivery technologies
are always on demand in generating effective delivery systems that can pro-
tect active ingredients, and are able to deliver and release them in a soluble
form at an appropriate site in the body. This chapter focuses on the use of
microencapsulation techniques for effective delivery of selected vitamins
and minerals in fortified staple foods such as salt and rice, and the extension
of these successful technology platforms to the delivery of nutraceuticals in
functional food development will be also discussed.
To take advantage of the benefits from the consumption of essential micro-
nutrients and nutraceuticals, actions are required (i) to ensure stability of
the compounds during processing, handling, and storage, as well as in the
gastrointestinal (GI) tract and (ii) to facilitate controlled release at the site of
the GI tract for proper absorption (Champagne and Fustier, 2007). However,
most of these compounds have limited solubility and stability (Fang and
Bhandari, 2010), or are highly reactive and may interact with many other
food components (Li et al., 2010). Additionally, many have unpleasant flavor
or color that detracts the attractiveness of the food (Kosaraju et al., 2008).
These factors impose several limitations on direct addition of micronutrients
and nutraceuticals to foods; therefore, the development of effective delivery
systems has gained increased urgency in the food industry. Any effective
delivery system must be designed so that it does not adversely impact the
physicochemical and organoleptic properties of the finished food product in
which it is incorporated, while also being able to achieve desirable absorp-
tion of the bioactive components or their bioavailability upon consumptions
of the fortified or functional foods (Lesmes and McClements, 2009). This has
led to numerous attempts to develop food-grade delivery systems to encap-
sulate, protect, and deliver nutraceuticals and micronutrients through novel
food products. Appropriate selection of encapsulation techniques alleviates
many of the problems related to the direct addition of bioactive compounds
to foods. Microcapsules have the potential to protect sensitive compounds,
eliminate or minimize nutritional loss of functional ingredients, incorpo-
rate time-release mechanisms to finished formulations, preserve desirable
flavors and aromas or mask unpleasant taste and appearance, and transform
potent plant or herb extracts into liquid or solid microcapsules for easy han-
dling (Huang et al., 2010). All these functionalities have been exploited in
drug and vaccine delivery in the pharmaceutical sector, and are increasingly
used to add value to novel food products in the food industry.

Microencapsulation has become increasingly important in the develop-

ment of fortified and functional foods in recent years. The latest advances in
drug delivery have their roots in basic microencapsulation, while the tech-
nology has opened up to a broad range of applications in other fields. In
contrast to its applications in drug delivery, its applications in food products
have to meet the additional criterion that the addition of the encapsulated
bioactive ingredients should not adversely affect or alter the sensory proper-
ties of the food vehicle (Champagne and Fustier, 2007).
The use of microcapsules for the protection and controlled release of
nutrients is a growing area of interest to the food science and technology
community, as many of these advantages are applicable to micronutrients
and nutraceuticals used in food fortification. Specifically, nanoparticles for-
mulated in liquid preparations can be incorporated in appropriate matrix
materials and then converted into solid forms either as free-flowing pow-
ders or granular particles. These encapsulated ingredients can be added to
food products easily without segregation and sedimentation, without being
noticed by the consumer, and/or with enhanced bioavailability (Augustin
and Sanguansri, 2008; Augustin and Hemar, 2009). In addition, appropriate
encapsulation techniques will maintain the micronutrients in a stable envi-
ronment, separated from other food components and additives and would
provide efficient delivery into the body without causing major sensory
changes.
Microencapsulation technologies can be used to create agglomerated pow-
der from liquid ingredients that matches the particle size or other physical
properties such as density of the granular food vehicles to prevent segrega-
tion or to prevent visual or textural clues that can be noticed by consumers.
11.1.4 Criteria for Developing Microencapsulated Delivery Systems

11.1.4.1 Bioavailability of Micronutrients and Nutraceuticals
One of the key objectives of microencapsulation of micronutrients and
nutraceuticals is to improve their absorption rates in the body. Methods for
bioavailability determination involve human/animal tests (in vivo) or simu-
lated experiments performed in a laboratory (in vitro). In vivo methods pro-
vide direct data of bioavailability and have been used for a great number
of nutrients and their delivery systems in foods. However, ethical restric-
tions and lengthy approval process of protocols when humans and/or ani-
mals are used in biological or clinical research greatly limit the use of this
type of methods. Another drawback of in vivo tests relates to variations in
physiological state of participating individuals and possible interactions of
the target nutrient with other components in the diet. Thus, as an alternative
to in vivo tests, in vitro methods are extensively used in the food industry
since they are rapid, safe, cost efficient, and do not raise ethical or legal issues
(Parada and Aguilera, 2007).

In a typical in vitro test, the digestion process is simulated under controlled

conditions using a variety of enzymes, for example, lingual lipase, amylase,
protease, pepsin, pancreatin, and so on, while the final transport and absorp-
tion process is commonly assessed using differentiated cell monolayers
obtained from human intestinal epithelium (Caco-2 cell cultures) (Gangloff
et al., 1996). An understanding of the basic physicochemical and physiologi-
cal processes that occur as a delivery system passes through the human GI
tract is required to develop effective in vitro models that accurately simulate
digestion (Artursson et al., 1994; McClements and Li, 2010).
Successful in vitro models have been developed to evaluate the bioavail-
ability of micronutrients in foods. Gangloff et al. (1996) evaluated the in vitro
bioavailability of iron in meat using a simulated digestion system—while
pepsin was used for the gastric phase, the intestinal phase was simulated by
adding pancreatin and bile extract before the uptake of iron by Caco-2 cell
monolayers was measured. The authors observed that iron absorption was
higher in meat than in other media. Zhu et al. (2006) also used this approach
to evaluate the iron uptake by Caco-2 cells when comparing different iron
compounds (NaFeEDTA, FeCl3, and FeSO4) and examining the influence of
other food components such as ascorbic acid (absorption enhancer) and an
iron-chelating agent (absorption inhibitor). The authors observed similar
absorption for all the iron compounds in the absence of ascorbic acid; how-
ever, when adding the enhancer the absorption was significantly improved.
Similarly, when the inhibitor was added, the absorption of the three iron
compounds decreased to a similar extent.
An even simpler test can approximate the iron release rate in the simu-
lated gastric acid (pH 1 HCl solution) based on United States Pharmacopeia
(USP): Chapter <711> Dissolution, USP29 – NF24, p. 2673. This method gives a
good initial indication of iron digestibility in the stomach. The good agree-
ment between the results obtained by this method and by in vivo tests justi-
fies the use of this technique for quick screening (Swain et al., 2003). With an
aid of appropriate in vitro bioavailability procedures, it is possible to design
optimized formulations for an iron microcapsule in order to obtain enhanced
absorption of this important mineral in the small intestine (Li et al., 2009a).
11.1.4.2 Encapsulation Efficiency

Besides bioavailability, another requirement for an effective microencapsu-
lation system for micronutrients or nutraceuticals is acceptable encapsula-
tion efficiency, which is a measure of how well the microcapsules separate
the core from the environment. It is generally defined as the ratio (in per-
centage) between the weight of the core ingredient actually encapsulated
and its total weight at the beginning of encapsulation process. There are
numerous studies in the literature that involve the use of specific meth-
ods to quantify the encapsulation efficiency for various delivery systems
(Gaonkar et al., 2014).

To confirm whether microencapsulation results in an effective protective

layer for micronutrients and nutraceuticals, it is imperative to evaluate the
encapsulation efficiency. Romita et al. (2011) evaluated the encapsulation
efficiency in spray-dried ferrous fumarate microcapsules coated by one of
several selected biopolymers. The microcapsules were immersed in a pH 7
EDTA (ethylenediaminetetraacetic acid) solution for 5 min, during which
exposed iron will be leached while the polymeric portion of the capsules
will remain intact. As the amount of iron leached is directly proportional to
the amount of iron exposed, the authors were able to evaluate the microen-
capsulation efficiency, concluding that microcapsules prepared using HPMC
have the high coverage and protection of ferrous fumarate.
The same authors also analyzed the encapsulation efficiency of spry-dried
ferrous fumarate delivery system using time-of-flight secondary ion mass
spectrometry (TOF-SIMS), which uses a pulsed primary ion beam to desorb
and ionize species from a sample surface. The CH3O ion signal, attributed
to the biopolymer, indicated that the microcapsule surface was mainly cov-
ered by wall material, while the signal for surface iron was small, suggesting
that iron is well enclosed in the system and protected from the environment
(Romita et al., 2011).
11.1.4.3 Microcapsule Morphology and Size

Microencapsulation technologies can allow us to produce micronutrient/
nutraceutical delivery systems for food vehicles with different particle
size ranges. An important goal is that the particles resemble the physical
characteristics of the selected food vehicles in terms of shape, size, color,
and appearance, which ensures the resulting fortified foods have desired
physical, chemical, nutritional, and organoleptic properties, ultimately

meeting the requirements of consumer acceptability and product shelf-life
stability. Pedroza-Islas et al. (1999) reported that the effect of different wall
materials and also their ratios with respect to the core material were critical
for obtaining desirable microcapsules, that is, those have smooth surface and
are spherical in shape and within a narrow size distribution.
Among physical/mechanical microencapsulation technologies, extrusion
followed by surface coating is flexible and able to readily generate microcap-
sules with different particle sizes, ranging from several hundred microns
to several millimeters, which subsequently ensures the microparticles can
match the size of a wide variety of staple foods. For example, for producing
encapsulated ferrous fumarate for its inclusion in refined table salts with
particle sizes ranging from 300 to 1000 μm, particle agglomeration based on
extrusion or fluidized-bed technique followed by surface coating was evalu-
ated (Diosady, 2007; Li et al., 2010). Both size-enlargement techniques were
able to produce microparticles approximating the size of salt grains. Surface
modifications including coverage of whitening agent followed by a film
coating resulted in iron microcapsules with an opaque, white appearance

and smooth surface, which ultimately makes them visually indistinguish-

able when blended into salt. The matching size and density of the iron
microcapsules also reduces the possibility of particle segregation during
salt storage and distribution. The detailed development of this extrusion-
based microencapsulation process for salt double fortification will be dis-
cussed in Section 11.4.
For evaluating the morphology and size of microcapsules to be used in
food fortification, several microscopy techniques, laser particle size distribu-
tion, and infrared spectroscopy can be used. Scanning electron microscopy
(SEM) is most widely used as it allows the analysis of the surface of the par-
ticle as well as its size and shape (Prasertmanakit et al., 2009).
11.1.4.4 Storage Stability

The stability of micronutrients in the microcapsule itself and when added
into a food product is of paramount importance when developing effective
delivery systems. The stability of the microcapsule determines the protec-
tion of the active ingredients from environmental challenges, such as mois-
ture, temperature, or oxygen, and from potential interactions with other food
components. For example, the storage stability of double-fortified salt sam-
ples prepared by adding microencapsulated ferrous fumarate into iodized
salt was studied over a period of 5 months in Nigeria and Kenya (tropical
conditions) (Oshinowo et al., 2004, 2007; Diosady et al., 2006). The study con-
firmed the results obtained in the laboratory storage tests under the simi-
lar controlled conditions, which involved high temperature and humidity.
Microencapsulated ferrous fumarate was prepared by agglomerating fer-
rous fumarate with a binder (HPMC or Opadry®) and finally coated with soy
stearin and titanium dioxide as color-masking agent. The authors observed
the retention of ferrous iron was around 83% as it was protected by the
microcapsule from reacting with iodine. In addition, the polyethylene film
bundle wrapping the packaging material was enough to protect the iodine
compound from the environmental conditions, resulting in iodine retention
in excess of 85% in most cases. The authors concluded that salt double forti-
fied with iodine and microencapsulated iron can protect both iodine and the
ferrous iron during distribution and retail under typical tropical conditions.
11.2 Processing Techniques

Current microencapsulation techniques can be classified based on the
microparticle formation mechanism. These include physical or mechani-
cal processes (such as spray drying, spray chilling/cooling, extrusion,
and fluidized-bed coating) and chemical processes (such as coacervation,

Matrix Simple Multiwall Multicore Irregular
FIGURE 11.2
Microcapsules and microspheres. (Adapted from Gibbs, B. et al. 1999. International Journal of
Food Science and Nutrition, 50(3): 213–224.)
co-crystallization, molecular inclusion, and interfacial or in situ polymer-

ization). In some cases, a combination of processes is used, for example, in
formation of a single or double emulsion followed by spray drying (Madene
et al., 2006).
The formed microparticles are categorized as either microcapsules or
microspheres (Figure 11.2), based on the structure, or more precisely, the
mutual position of the core and the shell. In a microcapsule, the active ingre-
dient is a continuous, concentrated phase and enveloped by a protective
layer of coating material. Usually, a two-step process will be needed to pro-
duce microcapsules, including the formation of the core particles followed
by a coating process. In contrast, in a microsphere, the active substance
is dispersed in the structure and entrapped within the matrix material,
which sometimes only involves a single step of entrapment (Adamiec and
Marciniak, 2004). Microcapsules or microspheres may have diameters rang-
ing from a few microns to a few millimeters.
11.2.1 Physical and Mechanical Methods

Spray drying is a commonly used method of drying a liquid feed through a
hot gas. The liquid feed is pumped through an atomizer that produces fine
droplets into the main drying chamber. Typically, the hot gas is air, but when
sensitive materials are processed or oxygen-free drying is required, nitrogen
gas or supercritical fluid such as CO2 is used instead. It is a well-established
technology involving commercially available equipment. It is extensively
used to produce powdery particles ranging in size from 1 to 150 µm, which
contain value-added ingredients, such as fragrances or flavors. The advan-
tages of this process are relatively low in cost and ease of scale up. The
microparticles prepared by the technique can quickly release the core ingre-
dients without leaving any shell debris, due mainly to the high water solubil-
ity of the shell materials used. However, the suitable shell materials for this
process are limited. Also, the concerns of solvent flammability and toxicity
severely restrict the use of organic solvents for conventional spray drying
operations. Other limitations of this technique include a generally low pay-
load (<40%) and problems with heat-sensitive materials (Benita, 1996; Gouin,
2004; Yuliani et al., 2004; Madene et al., 2006; SwRI website).

Spray chilling/cooling is similar to spray drying, where the core material

is emulsified in a molten wall material then atomized to disperse droplets,
which are immediately mixed with a cooling medium and subsequently
solidified into powder form (Madene et al., 2006). This is probably the least
expensive process, which can be used to convert liquid hydrophilic ingre-
dients into free-flowing powders with improved heat stability and delayed
release in wet environments. However, as a “matrix encapsulation” pro-
cess, rather a “true” core/shell encapsulation, it leads to a significant pro-
portion of unprotected active ingredients on the particle surface or sticking
out of the wall material, which subsequently affects the effectiveness of the
encapsulation.
Freeze drying, also called lyophilization, is one of the most useful processes
for drying thermo-sensitive ingredients in aqueous solutions that are unsta-
ble. It involves the sublimation/removal of water content from dissolved
or dispersed solids. The food industry widely uses the technology to pre-
serve plant or animal products in dehydrated powder forms. In the case of
microencapsulation operation, it can be used to dehydrate and convert food
emulsions into powders. The technique is relatively simple and can provide
better particle properties compared to spray drying and drum drying, such
as resistance to oxidation and intact shape of microcapsules (Madene et al.,
2006). Nonetheless, it normally requires a long processing time for dehydra-
tion, ~20 h depending on the materials and the loads (Desai and Park, 2005a).
Fluidized-bed coating involves suspending a bed or column of solid parti-
cles in a moving gas stream, usually air, and a liquid-coating formulation is
sprayed onto the individual particles. The freshly coated particles are cycled
into a zone where the coating formulation is dried either by solvent evapo-
ration or cooling. Three types of fluidized beds are available such as top
spray, tangential spray, and bottom spray. They vary in the nozzle’s location
or configuration used to apply the coating solution. This technique is gener-
ally an efficient way to apply a uniform layer of shell materials onto solid
particles. Basically, all kinds of shell materials can be used in this process
such as polysaccharides, proteins, lipids, and emulsifiers. In addition, it is
highly thermal efficient due to good gas–solid contact in which optimal heat
and mass transfer rates could be reached. On the other hand, its limitations
are also obvious; it can be only used for encapsulating solid particles, and the
particle size of the end products cannot be less than ~10 µm (Gouin, 2004).
Extrusion was first introduced as an encapsulation process by Swisher in
1957 to coat volatile and unstable flavors (Gibbs et al., 1999; Madene et al.,
2006). Essential oils were dispersed in glassy carbohydrate matrices (such as
corn syrup solids and glycerin) at >100°C, and then extruded into a dehydrat-
ing liquid such as isopropyl alcohol. The solidified material was then sepa-
rated into small pieces and vacuum dried. This process was later modified to
encapsulate microorganisms and enzymes at low temperatures (Gouin, 2004).
The active ingredients were mixed with plasticized composite matrices, such
as starch/fat or starch/polyethylene glycol. The dry mixture was converted

to a wet paste by incorporating ~20% (w/w) water and then extruded. The
exiting rope was cut into pieces between 500 to 1000 µm and air dried. This
technique can provide virtually full protection to the core ingredients by the
surrounding wall materials. Also, the use of glassy polymers can provide an
essentially impermeable barrier against oxygen, which enables prolonged
shelf life of the end product. However, this process can only produce large
particles, typically >500 µm, which greatly limits its applications. In addi-
tion, the suitable shell materials or binders are limited to glassy carbohy-
drates and carbohydrate derivatives (Gibbs et al., 1999).
Coextrusion is a relatively new technique for encapsulation. It creates fibers
consisting of active ingredients within fluid, high-viscosity, glassy shell
materials. These fibers can be chopped to form microcylinders, or when the
viscosity is low and the surface tension of the fluid is high these extrudates
would thermodynamically break up into tiny droplets, forming microcap-
sules. The typical coextrusion systems include stationary nozzle coextru-
sion, centrifugal coextrusion, or slightly altered spinning disk coextrusion.
In the former two processes, concentric nozzles are used to pump the core
material through the inner nozzle and the shell formulation through the
annulus, allowing “true core-shell” morphologies. Spinning disk coextru-
sion involves a suspension of the core material dispersed in the carrier mate-
rial. The mixture is then extruded through the rotating disc in such a way
that the excess coating fluid is atomized and separated from the coated par-
ticles (SwRI website).
This technique can be treated as “true” encapsulation, which gives the
microcapsules unique properties allowing release of the core ingredients at
a defined rate (Gouin, 2004). The high operating cost and specific require-
ments of the equipment greatly limit the application of this technique. In
addition, the core and shell materials must be mutually immiscible liquids,
for example, polar liquids like aqueous solutions in the core require hot melt
shell materials like waxes; whereas with water-immiscible oils as the core, an
aqueous polymer solution that can gel rapidly is required for the shell (SwRI
website).
11.2.2 Chemical Methods

Coacervation is the separation into two liquid phases in colloidal system
(IUPAC, 1997). It starts with an aqueous colloid solution in an appropriate
solvent. According to the nature of the colloid, when the environmental con-
ditions change, such as pH change, the solubility of the colloid is reduced
and a large part of the colloid can be separated out into a new phase. The
original one-phase system becomes two phases, one is rich and the other
is poor in colloid concentration. The colloid-rich phase in a dispersed state
appears as amorphous liquid droplets called coacervate droplets, which can
be deposited to produce the wall material of the resultant capsules. This con-
cept is then borrowed from colloid chemistry to describe the basic process

of capsule wall formation, and initiates the development of microencapsula-

tion technology by the first developer, B.K. Green, in 1957. The first coacer-
vative capsules developed by B.K. Green for carbonless paper were made
using gelatin as a wall in an “oil-in-water” system. Later developments pro-
duced “water-in-oil” systems for highly polar and water-soluble cores. The
process involves three steps: particle or droplet formation, coacervative wall
formation, and capsule isolation. It is considered as a “true” microencapsula-
tion technique, as the core material is completely entrapped by the matrix.
There are two methods of coacervation. Simple coacervation uses a second
more water-soluble polymer or an aqueous non-solvent for the gelatin. This
produces the partial dehydration/desolvation of the gelatin molecules at a
temperature above the gelling point, and subsequently results in the separa-
tion of a liquid gelatin-rich phase in association with an equilibrium liquid
(gelatin-poor). Complex coacervation systems use two oppositely charged
polymers and form the capsules by the ionic interaction, as illustrated in
Figure 11.3. The gelatin–gum arabic (gum acacia) system is mostly studied.
At pH values below the isoelectric point of gelatin, gelatin becomes posi-
tively charged while gum arabic continues to be negatively charged, and
then the gelled coacervate capsules containing the core material will be sepa-
rated out in the system.
Advantages include:
• Very high payloads up to 99% (Gouin, 2004).

• Can be used to achieve sustained release or controlled release based
on mechanical stress or temperature as the activating stimulus.
Gelatin solution Form emulsion of core in gelatin Core material

(40–60°C) solution (40–60°C) in liquid form
Form liquid coacervation by adjusting pH/adding polyanion (such

as gum arabic) and dilution water (40–60°C)
Form gel shell
Crosslink and harden microcapsules
Harvest microcapsules
FIGURE 11.3
Schematic flow diagram of complex coacervation.

Limitations are summarized below:
• Very expensive and complicated, thus it is hard to commercialize.

• The chemical cross-linker involved in the final step, usually glutaral-
dehyde, may not be approved for food use under certain jurisdictions.
Gelation involves the formation of gelled microcapsules or microspheres

using techniques such as cooling, cross-linking, or a chemical reaction. A
well-known system is alginate–calcium cross-linked beads, which were ini-
tially developed for immobilization of live cells and enzymes. It has been
rapidly adapted to many other applications mainly due to its extreme ease
of preparation on a lab-scale and mild-processing conditions. Unfortunately,
the process is difficult to scale up and the operation is costly. In addition, the
obtained microcapsules are very porous and allow fast and easy diffusion
of water and other fluids in and out of the matrix. Such good permeability
is desired for carrying live cells or enzymes but is not suitable for protecting
most active ingredients.
Liposome entrapment was originally used in the pharmaceutical industry,
and in recent years, it has been used in many other applications, such as
food-based delivery systems. The process can be achieved by dispersing a
bilayer-forming polar lipid, such as lecithin, in an aqueous medium con-
taining dissolved active ingredients. The thus formed particles are typically
spherical in shape with a relatively narrow size range from several nano-
meters to several hundred nanometers. Liposomes may contain a single or
multiple layers of amphiphilic polymolecular membranes, which closely
resemble the natural structure of cell membranes.
Generally, liposomes are “kinetically stable,” that is, they are only stable
for a short period of time, similarly to emulsions. Because of this, many prin-
ciples and techniques of emulsion formation can be also applied to the devel-
opment of liposomes (Taylor and Davidson, 2005).
Other technical issues with this technique include that the process is hard
to scale up. Moreover, liposomes are usually in aqueous forms, which impart
great stability of water-soluble materials in high water activity applications,
but limit their usefulness when the coated ingredients need to be in a dry
state.
Molecular inclusion is an advanced technique that is highly specific. It gen-
erally refers to the supra-molecular association of a ligand (the core material)
into a cavity-bearing substrate (the shell material) (Gouin, 2004). Particularly,
β-cyclodextrin, an enzymatically modified starch molecule, is used in this
molecular-level technique. Cyclodextrins are hollow truncated cone-shaped
molecules with an inner diameter of approximately 5–8 A (Figure 11.4), suf-
ficient for inclusion of one or more volatile flavor molecules or essential oil
compounds (Desai and Park, 2005a). The guest molecules, which are apolar,
can be entrapped within the hydrophobic internal cavity by hydrogen bond-
ing, van der Waals forces, or entropy-driven hydrophobic effect. In contrast,

CH2OH 15.3 Å
O 7.8 Å
O O
CH2OH OH HO OH O CH2OH Secondary
HO Apolar
cavity hydroxyl rim
O HO O
OH HO
O
O
OH
CH2OH
CH2OH OH
O 7.8 Å
HO HO Primary
OH O hydroxyl rim
OH O
O O HO
CH2OH O CH2OH
FIGURE 11.4
Molecular structure and microstructure of β-cyclodextrin. (Adapted from Yuliani, S. et al.
2004. Food Reviews International, 20: 163–185.)
its hydrophilic external part requires water as the suspension medium.

When the water molecules in the center of the cyclodextrin are replaced by
less polar molecules, the complex is precipitated (Gibbs et al., 1999).
The microparticles obtained from this technique have unique, sustained
release properties and thermal/chemical stability of the active ingredients
entrapped. However, the relatively high cost of the shell material, cyclo
dextrin, and the low yield of the end product greatly restrict its current com-
mercial applications (Gouin, 2004).
Other chemical techniques include co-crystallization, solvent evapora-
tion/extraction from emulsions, and interfacial or in situ polymerization.
Co-crystallization can be achieved by incorporating aroma compounds into
supersaturated sucrose syrup at the time of spontaneous crystallization at
high temperature (>120°C) and low moisture (95–97°Bx). A small number of
studies have reported that due to its relative simplicity, co-crystallization
can serve as an economical and flexible process (Madene et al., 2006).
Solvent evaporation/extraction is applied to convert droplets formed by
emulsification to solid particles. The mechanism of solvent elimination
from the emulsion droplets is not well known, but considered to have great
influence on the particle morphology and release behavior (Rosca et al.,
2004). These techniques are primarily applied in the development of drug
delivery systems, but attracted some interest by food scientists and tech-
nologists recently. Interfacial or in situ polymerization involves the forma-
tion of capsule shell on the emulsion droplet/particle surface by indirect
or direct polymerization. Interfacial polymerization occurs based on the
classical Schotten–Baumann reaction between an acid chloride and a com-
pound containing an active hydrogen atom, such as an amine or alcohol,
polyesters, polyurea, and polyurethane. In situ polymerization, without
the presence of reactants in the core material, happens when monomers
are directly added to the system. Both polymerization methods are used
for the encapsulation of herbicides and pesticides with rare applications in
food (Benita, 1996; Gibbs et al., 1999).

As discussed above, all microencapsulation techniques have pros and

cons. Generally, physical techniques are less expensive and easy to scale up,
but have the drawbacks of relatively low payloads and imperfect particle
properties. In contrast, chemical processes are costly and involve compli-
cated concepts, but typically can provide well-defined particle structures
and desired release properties. Chemical processes are often reported in for-
mulating drug delivery systems or for making value-added products. For
most applications in the food industry, physical processes are widely used
for the purpose of protection or effective delivery of various ingredients
including flavors, colorants, nutritional ingredients, and other functional
additives (Madene et al., 2006).
11.2.3 New Advancements

11.2.3.1 Controlled Release Technology
Controlled release is defined as technology that can be used to deliver one or
more active ingredients at a desired site and/or time or with a specific rate
(Pothakamury and Barbosa-Canovas, 1995). This technology has its roots in
the drug industry, but has spread quickly to other areas such as agrochemi-
cals (i.e., pesticides), fertilizers, veterinary drugs, plant extracts, and food
products (Das, 1983). A timely and targeted release improves the effective-
ness and functionality of the active ingredients and ensures optimal dosage
(Gouin, 2004).
Traditional methods of achieving controlled release can be categorized as
physical entrapment and chemical binding. Physical methods include dis-
solution, dispersion, adsorption, and encapsulation, while chemical meth-
ods involve covalent or ionic bonding between the active ingredient and the
polymer carrier, such as molecular inclusion, co-crystallization, and copo-
lymerization (Das, 1983). Microencapsulation technology can incorporate
elements of controlled release systems. Many recently developed microen-
capsulation formulations have fine-tuned controlled release properties. Such
applications in food industry will not only provide value-added products,
but also broaden the application range of various food additives and develop
totally new ingredients with novel properties (Gouin, 2004).
The fundamental mechanisms of controlled release have been well stud-
ied. Primarily, there are four mechanisms by which the active ingredient can
be released from a delivery system, namely, diffusion, swelling, biodegrada-
tion, and osmotic pressure (Pothakamury and Barbosa-Canovas, 1995).
As illustrated in Figure 11.5, under appropriate circumstances, the active
ingredient passes through the polymer matrix or the film/membrane by
diffusion into the external environment. When release continues, the rate
may gradually decrease with matrix system, or remains fairly constant with
membrane-based system. Because the active ingredient in the matrix system
has progressively longer distance to travel and thus requires longer diffusion

FIGURE 11.5
Controlled release by matrix-based (a) and membrane-based (b) diffusion. (Adapted from
Medical Device Link at http://www.devicelink.com/mpb/archive/97/11/003.html)
time, while in membrane-diffusion system the membrane has essentially

uniform structure and constant thickness which plays a key role for control-
ling the release rate.
Figure 11.6 shows the mechanism of swelling-controlled release. The
systems are initially in dry form and, when consumed, will absorb water
or other body fluids and swell. During swelling the matrix or membrane
undergoes a transition from a glassy to a gel state. The polymer chains in
the gel state are more mobile than those in the glassy state, which allows the
active ingredient to diffuse out of the swollen network quickly. Most materi-
als used in this system are hydrogels, which swell when placed in aqueous
fluids, absorbing a great deal of water without dissolving. It is worth noting
that this system can be adapted for targeted delivery of oral drugs or nutri-
ents to the intestine by using pH-sensitive polymers. These polymers swell
at high pH but collapse at low pH value, which enables the release of active
ingredients in the upper small intestine rather in the stomach.
The biodegradable system (Figure 11.7) differs from the above two sys-
tems as it involves the change of chemical structures of the carrier poly-
mers. Specifically, in diffusion-controlled systems the barrier or matrix is
not affected during the release process, while in swelling-controlled sys-
tems the matrix undergoes only physical changes. Biodegradable polymers
are used to carry active ingredients and release them at a specific site and
rate when the polymers are broken down by either bulk hydrolysis or sur-
face erosion.
FIGURE 11.6
Controlled release by reservoir-based (a) or matrix-based (b) swelling. (Adapted from Medical
Device Link at http://www.devicelink.com/mpb/archive/97/11/003.html)

FIGURE 11.7
Controlled release by bulk-erosion (a) or surface-erosion (b) biodegradation. (Adapted from
Medical Device Link at http://www.devicelink.com/mpb/archive/97/11/003.html)
In an osmotic system, the water-soluble core material is released at a con-

trolled rate by using osmotic pressure as the driving force. A semi-permeable
membrane with a small orifice is used to enclose the core, where the mem-
brane is selectively permeable to water but not to the core material. In an
aqueous environment, a great osmotic pressure is generated inside the micro-
capsule when water permeates through the membrane and gets absorbed by
the core material. Once the accumulated osmotic pressure exceeds the maxi-
mum force that the walls can hold, the active core will be released through
the orifice (Pothakamury and Barbosa-Canovas, 1995).
The food industry is taking advantage of controlled release technology for
developing novel, value-added food additives to achieve different require-
ments, such as prolonged or sustained release (controlled release rate),
delayed release (at right time), and targeted release (at right site). The com-
mon stimuli employed for the release of the encapsulated ingredient in food
applications include:
• A change in environmental parameters, such as temperature, mois-

ture, or pH, for example, the release of flavor in melting-activated fat
or wax coatings
• The application of osmotic pressure or mechanical shear, such as the
release of sweetener in chewing gum
• The addition of solvents, such as the release of flavor in dry beverage
or cake mixes when water is added
• A combination of two or more stimuli
11.2.3.2 Microencapsulation Coupling with Controlled Release

Microencapsulation coupled with controlled release technology is currently
an active research area. For example, more than 1000 patents were filed in
2002 and over 300 of them were directly related to food ingredient encapsu-
lation (Gouin, 2004). Relative to the direct improvements of either encapsu-
lation techniques or coating materials based on available processes in food

industry, a straightforward approach for developing new microencapsula-

tion systems is to adapt the ones used in pharmaceutical or other chemical
applications and transfer them into food-related processes.
Double or multiple emulsions coupled with an appropriate drying pro-
cess, either physically (spray drying) or chemically (solvent evaporation/
extraction), have been widely used in the pharmaceutical industry for devel-
oping oral drug delivery systems. They have received increasing attention
as potential vehicles for the controlled release of food compounds. Both
hydrophilic and hydrophobic ingredients can be incorporated in double or
multiple emulsions with the primary emulsion being either water-in-oil or
oil-in-water. The emulsions can be stabilized by using appropriate emulsifi-
ers, such as lecithin or other phospholipids, and food-grade surfactants, such
as Span 83 (hydrophilic) and Tween 80 (hydrophobic) (Jiao and Burgess, 2003).
Modifications can be made to the secondary or tertiary emulsions by involv-
ing polymeric complexes, such as proteins and polysaccharides, to form liq-
uid membranes at the outer interface of the primary emulsion droplets. The
combination of milk whey protein and gum arabic (Espinosa-Andrews et al.,
2005), gelatin and carrageenan (Gu et al., 2005), as well as soy protein iso-
late and maltodextrin (Rosenberg et al., 2005) were investigated for this pur-
pose. Recently, this concept is developed to an extreme as “colloidosomes.”
Dinsmore et al. (2002) presented a flexible approach to prepare solid capsules
with precisely controlled size, permeability, mechanical strength, and com-
patibility. The so-called self-assembly process involved, first, the dispersion
of the selected active ingredient in a w/o or o/w emulsion, followed by the
surface tension-driven deposition of solid colloidal particles onto the emul-
sion droplets. The colloidal particles were fused together by physical (sinter-
ing) or chemical (cross-linking) means to form a complete shell around the
active ingredient. Colloidosomes made in such way are promising with a
wide variety of potential mechanism for release, such as by controlled pores,
controlled rupture stress, or using self-swelling core materials. However,
considerable work, such as the investigation of food-grade colloidal poly-
mers and cross-linkers, needs to be done before this process can be adapted
for food ingredients.
11.2.3.3 Nanolevel Delivery Systems

Another technology primarily applied in the pharmaceutical industry, nano-
encapsulation, has shown its relevance for food usage with targeted delivery
of probiotics and nutrients to the intestinal tract (Gouin, 2004). The process
involves the formation of a complex between a polymeric active ingredient
(typically a protein) and a polymeric vector, without forming an actual coat-
ing barrier. The complex often exhibits different solubility and stability char-
acteristics and sometimes has an increased uptake rate by the epithelium, for
example, mucosal cells, which result in an overall increase in the bioavail-
ability of the active ingredients. Chitosan and some biodegradable polymers

such as polyesters and polyanhydride have been used to make nanoparti-

cles for drug delivery, while globular proteins, such as β-lactoglobulin and
bovine serum albumin, are under the study to be used as nanoparticle carri-
ers for controlled delivery of food ingredients (Ko, 2005).
Nanoparticulate delivery systems can also be achieved by microemul-
sions, which have their root in emulsions but present totally different prop-
erties. An emulsion is a dispersion of two immiscible liquids in the form
of droplets with diameters ranging between 0.1 and 3.0 µm. Such systems
are generally optically opaque and possess a finite stability, which may be
enhanced by the addition of amphiphilic molecules or viscosity enhancers.
In contrast, a microemulsion is a single-phase solution of three or four com-
ponents: surfactant or cosurfactant, oil, and water, which is formed spon-
taneously and stabilized by an interfacial film of surfactant molecules. It is
considered to be thermodynamically stable due to the ultralow oil–water
interfacial tension. Microemulsions are intrinsically isotropic, optically clear,
low in viscosity, large in interfacial areas, and high in solubilization capacity
for both hydrophilic and lipophilic compounds (Solans and Kunieda, 1996).
Moreover, microemulsions are dynamic systems with the structure that may
or may not be droplets, with a mean size of less than 200 nm, in general
between 10 and 50 nm. With their unique properties, microemulsions have
attracted more and more attention and stimulated many novel applications
in pharmaceutical, biological, food, cosmetic, and agrochemical industries.
Porter and Charman (2001) reported that microemulsions enhanced absorp-
tion of oral drugs and nutrients, especially hydrophobic ones. Dungan (1997)
reviewed the use of microemulsions in the food industry to enhance the
antioxidant effects of vitamins C and E, and pointed out that microemul-
sions are potentially safe, highly effective, scalable, and low-cost systems for
food use. However, several disadvantages of microemulsion systems, such as
sensitivity to temperature changes, the high amount of surfactants required,
and the use of alcohol as cosurfactants, have limited their applications in
many food products (Gaonkar and Prabhakar, 2003). A recent advance of
introducing biocompatible linker molecules into microemulsion formula-
tions (Acosta et al., 2005) has opened the possibility of using microemulsions
to formulate food-grade delivery systems. Using lecithin as the major surfac-
tant, the microemulsion formulation involves hexyl polyglucoside or sodium
octanoate as the hydrophilic linker and sorbitan monooleate as the lipophilic
linker. This linker-based biocompatible microemulsion is then free of alcohol
and has high solubilization capacity (Acosta et al., 2005).
In spite of the attractive properties of microemulsions, the fact that micro-
emulsion formulations are liquid solutions greatly impedes their applica-
tions in solid drug or food products. Recent advances in the pharmaceutical
industry provide a promising solution with an anhydrous microemulsion,
so-called self-microemulsifying drug delivery system (SMEDDS). SMEDDS
is a mixture of oils, surfactants, and cosurfactants, which are emulsified
in an aqueous medium under conditions of gentle agitation and digestive

motility that would be encountered in the GI tract (Kang et al., 2004). The
solid or semi-solid SMEDDS can be achieved by adding solidifying excipi-
ents (adsorbents and polymers) (Attama et al., 2003; Schwarz, 2003; Patil et al.,
2004) or using one or more fatty substances with a melting point higher than
the room temperature (about 25°C) (Barthelemy et al., 2001). Moreover, many
research studies and patents have also revealed that SMEDDS enhances the
bioavailability of poorly water-soluble drugs (Crison and Amidon, 1997; Kim
et al., 2000; Kang et al., 2004).
11.2.3.4 New Advancements in Equipment/Device Development

Besides the newly invented systems involving combined techniques and wall
materials, some studies were focused more on the process or device used
to make microcapsules, for example, supercritical fluids were used to alter
the organic solvent to assist the process, and a microfluidics-based device
was invented to produce uniform double emulsions in a single step. Porta
et al. (2005) proposed supercritical-assisted atomization (SAA) as an alterna-
tive to conventional jet-milling process. The SAA process was based on the
solubilization of supercritical carbon dioxide in a liquid solution contain-
ing the drug. The ternary mixture was then sprayed through a nozzle and
forming the microparticles. As reported by Utada et al. (2005), a microcapil-
lary device with coaxial jets was developed to generate precisely controlled
double emulsion in a single step. The device consists of three glass capillar-
ies, an outer square tube and two cylindrical inner tubes lying in one axis
(Figure 11.8). Three fluids that make up the double emulsion flow through
the capillaries separately. The inner fluid is pumped through a tapered injec-
tion capillary, and the middle fluid is pumped through the outer region and
forming a coaxial, unidirectional flow with the inner fluid at the capillary
exit. The outer fluid is pumped through the outer region from the opposite
direction. The three fluids are forced to through the exit orifice formed by
the other capillary, rupturing into droplets of inner fluid within droplets of
middle fluid dispersed in the outer fluid. The geometry and relative flow
rates can be used to adjust the size and number of inner droplets. With its
Outer fluid Middle fluid
Inner
fluid
Collection tube Injection tube
FIGURE 11.8
Schematic of the coaxial microcapillary fluidic device. (Adapted from Utada, A.S. et al. 2005.
Science, 308: 537–541.)

advantage of three-dimensional coaxial flow, this general device for encap-

sulation structures opens up a new and promising technique for producing
uniform microscale emulsions.
11.3 Coating or Shell Materials Used

As important as the choice of microencapsulation technique is, the selec-
tion of wall material also plays a crucial role in the development of microen-
capsulated food ingredients. Generally, shell materials are required to have
some of the following characteristics: film forming, pliable, tasteless, nonhy-
groscopic, soluble in an aqueous media or solvent, and/or able to exhibit a
phase transition like melting or gelling. Specifically, for food use the coating
material should also
• Be easily digested by the body

• Have no interaction with the core material
• Be nonsticky
• Be impervious to water
• Be inexpensive
• Should not impart sensory changes
• Comply with food regulations and local customs
(Kirby, 1991; Gibbs et al., 1999, Schrooyen et al., 2001; Gouin, 2004; SwRI
website).
Numerous coating materials have been used in food ingredient micro-
encapsulation. Most of them are natural or are derivatives of plant or ani-
mal food products, which have been approved by FDA (Food and Drug
Administration) as GRAS (generally recognized as safe) materials. Table 11.3
lists some commonly used coating materials for microencapsulation of food
ingredients and their applicable techniques. In most cases, the coating mate-
rials are categorized according to the chemical components from food source
materials (as shown in Table 11.3), such as carbohydrates, proteins, cellu-
lose and derivatives, lipids and waxes, as well as other natural or synthetic
polymers.
Another way to classify the coating or shell materials can be based on the
solubility in aqueous or organic solvents. There is an abundance of water-
soluble polymers, which are primarily extracted or isolated from natu-
ral food source materials, and can be divided into polysaccharides-based
and non-polysaccharides-based materials. Polysaccharide-based poly-
mers include carbohydrates, celluloses, gums, and their derivatives, while

TABLE 11.3
Commonly Used Coating Materials for Microencapsulation of Food Ingredients
Category Coating Materials Applicable Techniques
Carbohydrates Starch, dextran, maltodextrins, Spray drying, freeze drying,

modified starch, cyclodextrins, corn fluidized-bed coating,
syrup, sucrose, and sugar derivatives extrusion, coextrusion,
coacervation, cocrystallization,
molecular inclusion
Cellulose and Methyl-, ethyl-, and Fluidizedbed coating, spray
derivatives carboxymethylcellulose (CMC), drying, coacervation
hydroxypropylmethylcellulose
(HPMC), and other derivatives
Gums Gum acacia, gum arabic, agar/agrose, Spray drying, coacervation,
pectin/polypectate, algin/alginate, gellation
carrageenan, and other gums
Lipids and Fats, fatty acids, vegetable oils, mono-, Spray chilling/cooling, fluidized-
derivatives di-, or triglycerides, hydrophilic or bed coating, emulsion plus
lipophilic waxes such as shellac, solvent evaporation/extraction,
beeswax, PEG (polyethylene glycol), liposome entrapment
paraffin
Proteins Gluten, casein, gelatin, zein, albumin, Coacervation, emulsion, spray
peptides, whey or soy protein isolates drying
Source: Gouin, S. 2004. Trends in Food Science & Technology, 15(7): 330–347; Schrooyen, P., Van der
Meer, R., and De Kruif, C. 2001. Proceedings of the Nutrition Society, 60: 475–479; Kirby, C.
1991. Food Science and Technology Today, 5(2): 74–78; Gibbs, B. et al. 1999. International
Journal of Food Science and Nutrition, 50(3): 213–224; SwRI website: Southwest Research
Institute (SwRI®), available at http://www.swri.org/4org/d01/microenc/microen/
default.htm
non-polysaccharide-based materials include proteins and their derivatives.

Some synthetic polymers are also water soluble and have been found in
many pharmaceutical applications as effective encapsulants, such as poly-
vinyl-alcohol (PVA), poly-ethylene-glycol (PEG) and their copolymers. Most
organic soluble polymers are synthetic materials or modified from natural
ingredients, such as PEG esters, poly-lactic acid (PLA), ethyl cellulose, cel-
lulose acetate propionate (CAP), shellac, and zein. They have been found in
some specific applications for encapsulating core materials or as excipients in
drug delivery systems (Avis et al., 1999; Kwon, 2005; Ghosh, 2006).
There are many reviews in the literature that well discuss the properties
and specific applications of these wide-ranging shell materials; specifically, a
recent handbook (2014) under Dr. Anil Gaonkar’s edition, Microencapsulation
in the Food Industry—A Practical Implementation Guide, provides a compre-
hensive review on coating materials used in food applications based on
the classification of food components. Hence, this review will focus on the
encapsulant selection process during the development of microencapsulated

ferrous fumarate for salt fortification (Yadava et al., 2012). Overall, the selec-
tion of appropriate shell materials (or encapsulants) is mainly based on three
criteria, namely, excellent barrier properties, great film forming or wettabil-
ity, and narrow-ranged phase transition point suitable for the end use of the
designed delivery system. Besides these three characteristics, the selection of
encapsulants also needs to consider the compatibility of the coating materi-
als with the encapsulation techniques employed.
In general, when developing a microencapsulation system, the tech-
niques and coating materials need to be considered together, as they usu-
ally influence each other. For example, the glassy carbohydrates, including
cellulose derivatives, were investigated as coating materials for encap-
sulating extruded iron microparticles during the selected fluidized-bed
coating process. As shown in Table 11.3, these materials are suitable for
fluidized-bed operation due to their unique film-forming properties and
phase transition. Also, they are expected to protect the stability of the core
ingredients (i.e., micronutrients) in dried forms while achieving desirable
bioavailability through instant release when the iron particles are released
in the digestive system. More importantly, these materials are relatively
inexpensive and widely available even in developing countries. On the
other hand, certain gums, for example, sodium alginate, can be used in
the internal gelation for making extruded rice analogues. This polymer
has been used extensively in numerous applications due to its well-known
gelling effect. The broad availability and relative low cost of the materials
will enable the marketability of the successful formulations developed in
the laboratory studies.
11.3.1 Hydrophilic Coatings

Water-soluble, or hydrophilic, coatings are commonly used to coat phar-
maceutical drug tablets with an immediate release profile. In the study of
microencapsulated ferrous fumarate for salt fortification, the microencap-
sulation layer should disintegrate or dissolve completely in the stomach to
release the iron in the premix; therefore, hydrophilic coatings are suitable.
However, most water-soluble coatings do not provide adequate protection
against water compared to other coating materials. Nonetheless, a number of
newer formulations have the ability to provide adequate moisture protection
and unmodified drug release (Dow Chemical Company, 2008a).
Many hydrophilic encapsulants used in pharmaceutical film-coating pro-
cesses are based on HPMC (hydroxypropyl methylcellulose). HPMC is a cel-
lulose ether that is manufactured by reacting propylene oxide and methyl
chloride with an alkaline cellulose (Figure 11.9). This cellulose-based poly-
mer is available in different levels of methoxyl and hydroxypropyl substitu-
tion. Depending on the substitution ratios and overall molecular weight, the
polymer exhibits different physicochemical properties such as viscosity, sol-
ubility in nonaqueous systems and gelation characteristics (Roller and Jones,

CH2OH
O
H
O
H
OR H
H
n
H OR
R = –H, –CH3, –CH2CH(OH)CH
FIGURE 11.9
Chemical structure of HPMC. (Adapted from McGinity, J., ed. 1997. Aqueous Polymeric Coatings
for Pharmaceutical Dosage Forms. Marcel Dekker, New York.)
1996). Various grades of HPMC are available and are used in a wide range
of applications such as emulsification, binding, thickening as well as film
formation. HPMC is suitable for this study as it exhibits good film-forming
characteristics, immediately dissolves in gastric fluids, and is recognized as
an acceptable food additive by the FDA (Dow Chemical Company, 2008a).
The HPMC grade used in film-coating applications has a relatively low vis-
cosity, enabling faster and more efficient coating. The viscosity of this poly-
mer and its derivatives could vary from 2.4 to 18 mPa s (2% water solution
at 20°C) (Dow Chemical Company, 2008b). In general, film-coating HPMC
grades contain approximately 28%–30% methyl substitution and 7%–12%
hydroxypropyl substitution, allowing them to be soluble in aqueous and
hydroalcoholic solutions. HPMC can be used alone as a film former or in
conjunction with additives such as plasticizers, anti-tacking agents, and colo-
rants. The HPMC-based encapsulants chosen in the study of microencapsu-
lated iron particles for salt double fortification are summarized in Table 11.4,
along with their compositions.
In addition to HPMC, PVA-based polymers are commonly used in appli-
cations that require immediate release characteristics. The main advantage
of PVA systems is the enhanced moisture protection properties. Kollicoat
IR White from BASF Chemicals and Opadry AMB from Colorcon Ltd. are
PVA-based ready-to-use coating systems that were investigated in this study.
Both coating formulations contain titanium dioxide as a colorant, which can
potentially minimize the amount of titanium dioxide used in the color-
masking step or eliminate the step altogether.
11.3.2 Hydrophobic Coatings

Hydrophobic coatings, such as hydrogenated vegetable oils, are insoluble in
water and can provide good moisture protection. In the study of microen-
capsulated iron particle for salt fortification, the objective of the coating is to

TABLE 11.4
HPMC-Based Encapsulants Chosen in the Study of Double-Fortified Salt Program
Encapsulant and Supplier Composition Comments
Methocel E LV Premium Hydroxypropyl methyl The series consist of polymers

series cellulose (HPMC) with the same substitution
(Dow Chemicals, USA) ratios, but vary in viscosity. E3,
E6, and E15 were investigated
in the study.
Opadry White Hydroxypropyl methyl This coating blend is a one-step
(Colorcon, USA) cellulose (HPMC), system that incorporates the
polyethylene glycol film-forming agent and
(plasticizer), talc (anti- additives all in one dry
tacking agent), and concentrate, making the
titanium dioxide (colorant) coating process faster.
Sepifilm LP770 Hydroxypropyl methyl Sepifilm is a commercial,
(SEPPIC, France) cellulose (HPMC), stearic ready-to-use coating
acid (plasticizer), formulation that is soluble in
microcrystalline cellulose the gastric juice and is widely
(binder), and titanium used to coat moisture-sensitive
dioxide (colorant) particles.
provide protection from moisture and iodine in the salt. Hydrophobic coat-
ings have the ability to achieve the goal due to the relative impermeability
of the coating in an aqueous environment. However, the coating may not
dissolve properly in the stomach, resulting in reduced iron release.
Soy stearin, which is fully hydrogenated soybean oil, has been used in pre-
vious research on double-fortified salt at the University of Toronto and has
shown good results in terms of maintaining the stability of iron and iodine
(Yusufali, 2001). Unfortunately, due to the lipid material’s poor film-forming
capacity, the soy stearin–coated premix exhibited low density, causing it to
float on water. In addition, the premix did not easily dissolve in pH 1 solu-
tion, indicating that it had a reduced iron dissolution profile (Lo, 2006), or
somewhat reduced iron in vitro bioavailability (Li et al., 2009a). Therefore,
alternate coating materials were investigated in the study to develop premix
with a hydrophobic barrier that did not under-perform in terms of iron solu-
bility in gastric fluids.
11.3.3 Enteric and Reverse-Enteric Coatings

Enteric coatings, sometimes also called sustained or delayed release coat-
ings, are comprised of pH-sensitive polymers, which remain intact in the
acidic environment of the stomach and then become deprotonated and dis-
solved in basic media or at nearly neutral pH values such as that present in
the small intestine (Lin and Kao, 1991). Some examples of enteric coatings
include shellac, zein, CAP, and other cellulose ester polymers. An example

of enzymatic response coatings is proteins cross-linked using transgluta-

minase. Cross-linking helps improve heat and moisture resistance of the
proteins. Microcapsules made of these cross-linked proteins can be broken
down by trypsin and chymotrypsin thus releasing the entrapped content into
the small intestine (Yildirim and Hettiarachchy, 1998). Another enzyme-con-
trolled release system relies on the existence of enzyme-producing microor-
ganisms in the colon. The colonic microflora produces a variety of enzymes,
including azoreductase, various glycosidases, and, at lower concentrations,
esterases and amidases, which can be exploited for colon-specific drug deliv-
ery (Liu et al., 2003). By taking advantage of these enzymes, biodegradable
coating materials have been developed for enzyme-controlled drug delivery
that could be extended to food applications (Leopold, 1999; Nunthanid et al.,
2008).
Unlike other food active ingredients, microencapsulated micronutrients
(vitamins and minerals) must meet the additional requirement as of high
bioavailability, while retaining good stability and sensory properties. High
bioavailability can be achieved by controlled release of these nutrients at
the right site in the body, for example, the intestine, where essentially all
nutrients are absorbed. Similarly, many oral drug delivery systems have
been developed for targeted release in the colonic region. These colon-
specific drug delivery systems usually use two kinds of polymeric coatings,
pH-dependent polymers such as various enteric glassy coatings, or colonic-
bacterial degradable polymers such as chitosan and poly(lactic-co-glycolic
acid) (Lorenzo-Lamosa et al., 1998; Rodriguez et al., 1998; Lamprecht et al.,
2000). The enteric polymers are pH sensitive and will experience a dramatic
increase in the solubility due to the change in local pH from around pH 1
in the stomach to above pH 4 in the intestine (Desai, 2005). Thus, a swell-
ing-controlled mechanism releases the active drug contents (Pham and Lee,
1994). It seems reasonable to adapt this concept to food-based delivery of
micronutrients by targeted release at the intestine.
In contrast, reverse-enteric coatings are hydrophobic at above approxi-
mately pH 6.5, and present hydrophilic properties at gastric pH (<3) (Avis
et al., 1999). These coatings can potentially provide a good moisture barrier
to the coated iron premix without hindering the gastric acid dissolution pro-
file. This is because of the fact that most nonheme iron, presented in the diet
as ferrous or ferric form, is mainly absorbed in the duodenum, where low
pH favors solubility of iron. Further down the intestine, iron absorption rate
is significantly reduced, namely, duodenum > jejunum > ileum, due to the
insoluble ferric complexes likely formed in the end of the GI tract pathway
(Miret et al., 2003). Therefore, compared to enteric coating materials, reverse-
enteric coatings can be explored as the encapsulants for iron microparticles
based on their high solubility in acid solution but low solubility in neutral
solutions, in other words, leading to highly bioavailable and stable delivery
systems for iron. However, only a very few of reverse-enteric coatings are
approved for pharmaceutical use. One example is the Eudragit® E series of

polymers from Degussa, which include reverse-enteric coatings containing a

cationic copolymer based on dimethylaminoethyl methacrylate and neutral
methacrylic esters. The series of polymers includes Eudragit® E 100 in pellet
form and Eudragit® E PO in powder form, which are both soluble up to pH
5.0 and swellable above pH 5.0 (Kwon, 2005). The chemical structure of the
polymer is shown in Figure 11.10.
Eudragit E is insoluble in a neutral medium such as saliva and rapidly
disintegrates at low pH due to the protonation of the pendant tertiary amine
group (Kim, 2004). This property enables the polymer to be used in taste
masking and moisture protection applications. Therefore, it is suitable to
protect the iron premix from moisture without adversely affecting the iron
dissolution profile in stomach acids.
In summary, among the three groups of encapsulants studied, hydrophilic
materials, specifically Methocel E6 and Sepifilm LP770, could form strong
films on the surface of color-masked iron particles that protect the ferrous
iron in the premixes from interacting with the salt matrix or iodine. The iron
premix formulations made by these HPMC-based polymers retained ~95%
of the original ferrous iron and ~95% of the original iodine in dry, refined
iodized salt after 3 months storage of double-fortified salt at 35°C and 60%
relative humidity (RH). The premixes had high bulk and particle densities,
matching that of salt grains; high in vitro iron bioavailability; excellent par-
ticle integrity; and improved appearance and color compared to the premix
made by the previous technique of fluidized-bed agglomeration/coating,
thus meeting all study objectives (Yadava et al., 2012). In contrast, the enteric
coating, Aquacoat, and the hydrophobic coating, soy stearin, were not able
to retain as much iodine as the hydrophilic coatings, suggesting that hydro-
philic coatings offer more protection at lower encapsulation levels (e.g., 10%
coating level, w/w). Most hydrophilic coatings are based on HPMC and are
CH3 CH3
CH2 C CH2 C
C O C O
C OR
CH3
H2C N
CH3
R = –CH3, C4H9
FIGURE 11.10
Eudragit E chemical structure. (Adapted from Degussa, Specifications and test methods for
EUDRAGIT® E 100, EUDRAGIT® E PO, and EUDRAGIT® E 12,5. [Online]. Available at http://
eudragit.evonik.com/product/eudragit/en/products-services/eudragit-products/protective-
formulations/e-100/pages/default.aspx. Accessed on April 22, 2008.)

relatively inexpensive compared to reverse-enteric coatings such as Eudragit,

thus making them better options for field study and later commercialization
(Yadava, 2008).
11.4 Case Studies of Food Applications Based

on Different Categories of Nutraceuticals
As stated above, an effective combination of appropriate coating materials
and encapsulation techniques is the key for developing a microencapsulated
system as it plays an important role in the physical and chemical properties of
the resulting microparticles, such as particle size, porosity, density, flowabil-
ity, integrity, reactivity/stability, and release properties (Gharsallaoui et al.,
2007). For each active ingredient, the appropriate choices of process and wall
materials depend greatly on the end use of the microencapsulated particles.
The following subsections will review several specific applications in detail.
11.4.1 Microencapsulation and Delivery Systems

for Vitamins and Minerals
Schrooyen et al. (2001) summarized the available technologies for microen-
capsulation of vitamins and minerals. Water-soluble vitamins, for example,
vitamin C, were dispersed in a molten fat or wax and spray-cooled/chilled.
Another technique involved fluidized-bed coating, where the dry water-sol-
uble vitamins were suspended in an upward-moving stream of air and a mol-
ten fat or wax was sprayed onto the particles. Compared to the solid particles
formed by using the above two techniques, liposomes were used to incorpo-
rate ascorbic acid in liquid food systems, where a dehydration–rehydration
procedure was used instead of organic solvent to form liposomes. Taylor and
Davidson (2005) reviewed the applications of liposomes in the food industry
and indicated the potential function of liposomes to stabilize and protect
vitamins in food systems, for example, liposome-entrapped vitamin A (reti-
nal) demonstrated great stability in terms of decreased light- or heat-induced
degradation rates. Liposomes can be also used to microencapsulate minerals
such as ferrous sulfate and calcium lactate. Lecithin liposomes containing
iron and calcium have been applied in the fortification of fluid foods, such as
milk (Schrooyen et al., 2001).
Besides the general reviews, several research groups published their indi-
vidual study results regarding the microencapsulation of vitamins and min-
erals. For instance, Desai and Park (2005b) reported the microencapsulation
of vitamin C in tripolyphosphate (TPP) cross-linked chitosan by spray dry-
ing. Vitamin C solution (1% w/v) was well mixed with 1% w/v chitosan solu-
tion, followed by homogenization with 1% w/v TPP solution. The prepared

microspheres were spherical in shape and had smooth surface, with a mean
particle size of 6–10 µm. The volume of cross-linking agent solution (TPP)
added in the system had great impact on the particle properties, encapsu-
lation efficiency, and release properties. The authors claimed that vitamin
C was dispersed at the molecular level in the TPP–chitosan matrix and the
release of vitamin C from the microspheres followed Fick’s law of diffusion.
Madziva et al. (2005) reported using a combination of alginate and pectin to
microencapsulate folic acid for food use. Folic acid was dispersed in the gel
polymer matrix combining alginate and pectin, and the mixture was then
pumped through a nozzle with a continuous flow of nitrogen into a gently
agitated aqueous solution of calcium chloride, where microcapsules formed,
which were then air dried or freeze dried. Jimenez-Alvarado (2005) reported
using a double emulsion system (w/o/w type) to encapsulate a ferrous iron
solution in the inner aqueous phase. With the investigation of various oils and
emulsifiers and their individual concentration levels, the author concluded
that the iron double emulsion thus formed could be used for food fortification.
The research group headed by Hurrell and Zimmermann is working
on iron delivery systems based primarily on micronized ferric phosphate.
They have also investigated simultaneous delivery of vitamin A, iron, and
iodine through fortified salt (Windhab et al., 2005). Recently, they proposed
a novel concept, Multi Microcapsule (MULMICAP), for food fortification,
especially in salt. The capsules were produced by continuous microprocess-
ing, including multistep milling, dispersing/emulsification, cold spraying,
and powder mixing operations. Hydrogenated palm oil was used to pack
iodine, micronized ferric pyrophosphate, and retinyl palmitate, and form
subcapsules which were then integrated into spherical Multi Microcapsules
with a d iameter of 20–100 µm. Such microcapsules were applied to African
salt by attachment to the surfaces of 1–2 mm salt crystals. The triple-fortified
salt (TFS) made by this way was reported to have excellent color stability
and relatively high stability of vitamin A and iodine (87% and 84% retained,
respectively) after 6 months’ storage. The clinical trials showed that iron sta-
tus and vitamin A status in the body were significantly improved in goitrous
children in Morocco after consumption of this TFS for 10 months (Windhab
et al., 2005).
11.4.1.1 Microencapsulation of Ferrous Fumarate for Salt Fortification

For the past 15 years, the Food Engineering research group headed by
Dr. Levente L. Diosady at University of Toronto has been active in develop-
ing microencapsulation-based techniques to fortify staple foods, such as salt,
sugar, and rice, with multiple micronutrients. To prevent unwanted interac-
tions between added micronutrients and food carriers, this research group
has focused on microencapsulation for the stabilization and controlled deliv-
ery of micronutrients and nutraceuticals for improved nutritional value of
foods for consumers in both developing and developed countries.

Micronutrient malnutrition is a severe nutritional problem affecting over

one-third of the world’s population especially those living in developing
nations. The effects are dramatic: children’s intellectual and physical devel-
opment are retarded, their resistance to disease is destroyed by the damage
to their immune system. In adults, it leads to reduced work capacity, reduced
immunity, and huge loss of life in maternal and infant death. This results in
adverse economic effects, contributing, if not leading, to the cycle of poverty
and disease. As micronutrients are required in minute quantities, it is now
realized that providing micronutrients through food fortification is by far
the most cost-effective means to improving the health and economic status
of the poorest people on the planet.
To take advantage of the progress in global salt iodization programs, the
Food Engineering group at University of Toronto has developed a process
for the agglomeration and microencapsulation of ferrous fumarate to form
an iron premix, which could be added to iodized salt at a ratio of 1:100 or
1:150, thus preventing the loss of iodine through iron–iodine interaction.
Engineering application of this approach required the manufacture of
encapsulated iron premix particles that were essentially unnoticeable to the
consumer. This involved (1) agglomeration of the premix particles to match
the size and to approximate the density of salt grains, (2) color masking of the
dark red color of ferrous fumarate, and (3) chemical protection from mois-
ture, preventing the leaching out of iron ions or penetration of iodate ions
into the iron premix particles.
Double-fortified salt (DFS) thus produced with encapsulated ferrous
fumarate premix was used in determining the consumer acceptability and
stability of DFS. Small-scale tests examining the acceptability of typical local
foods prepared with DFS were carried out in more than 10 countries, includ-
ing India, Morocco, Ivory Coast, and Kenya, under the supervision of the
Micronutrient Initiative (MI), an NGO based in Ottawa, Canada. The sta-
bility of DFS was demonstrated by introducing samples of DFS, packed in
typical commercial packages, into the local salt distribution system in four
regions of Kenya and Nigeria. The packages were tracked through the distri-
bution system, and the temperature and humidity history was recorded. The
exposed samples were unaffected in terms of organoleptic properties and
stability of ferrous iron and iodine (Oshinowo et al., 2004, 2007).
The encapsulation process was adapted to fluidized-bed agglomerators
and coaters (Wurster-type fluidized-bed processors) and scaled from 5
through 20 kg, 60, and 300 kg batch sizes to full production units of 600 kg/
batch, in the facilities of Glatt Air Techniques in New Jersey, USA (Diosady
et al., 2004). The quality assurance and quality control systems were then
developed and tested to allow the tracking of iodine and iron in salt by
untrained operators, either in the salt plants or in the field (Yuan et al., 2008).
Fluidized-bed agglomeration followed by lipid coating has the advantage
of high throughput and low-operating cost. However, the microcapsules pro-
duced by this process had surface defects, porous texture, low density, and

FIGURE 11.11
Surface defects on encapsulated ferrous fumarate made by fluidized-bed agglomeration
and soy stearin coating (left: SEM image; right: microscopic image under normal light).
(From Li, Y.O. 2009. Development of microencapsulation-based technologies for micronutri-
ent fortification in staple foods for developing countries. PhD thesis, University of Toronto.
Available at https://tspace.library.utoronto.ca/bitstream/1807/26536/1/Li_Yao_O_200906_
PhD_Thesis.pdf)
marginally acceptable color, as shown in Figure 11.11. The low density of the
iron premix made with fluidized-bed technique leads to a problem when the
salt is added to water the iron particles float and could be unintentionally
discarded by consumers as impurities.
Extrusion agglomeration was then explored to produce particles with
higher density, reduced porosity, and smoother surface that allow better film
coating at lower coating loads. Other advantages of extrusion include its flex-
ibility in forming particles with different size scales, ranging from several
hundred microns to several millimeters, which ensures the premix particles
can match the size of a wide variety of staple foods (Li, 2009).
During process development, ferrous fumarate powder was first mixed
with selected binders such as wheat or rice flour, and then water and oil were
added to form an extrudable dough mass. The mixture was then extruded,
cut, and dehydrated to obtain cylindrical particles matching the size of typi-
cal table salt grains (300–700 μm). These particles were then covered with
a thin layer of whitening agent (titanium dioxide) and encapsulated using
hydrophilic film coatings (Li et al., 2011a; Yadava et al., 2012). These stud-
ies found that the best binder for preparing an extrudable dough capable
of carrying high ferrous fumarate content (75%) was durum semolina, and
to a slightly lesser extent, wheat flour (Li et al., 2011a). Water and short-
ening (hydrogenated vegetable oils) in the extrudable dough were added
at 16%–20% and 2.5% level on dry basis, respectively, to provide plastic-
ity and lubrication to the dough. The resulting ferrous fumarate extrudates
had a bulk density of 1.40 g cm−3 and a particle density of 1.70–1.85 g cm−3,
approximating that of iodized salt (1.86 g cm−3), which simplifies uniform
iron distribution in the DFS.

The color masking and hydrophilic polymer coating were further

optimized (Yadava et al., 2012). Several color-masking techniques and
polymer coating materials were investigated. HPMC-based, polyvinyl
alcohol-polyethylene glycol (PVA-PEG) copolymer-based, enteric and
reverse-enteric coatings were tested. Color masking was achieved by blend-
ing ferrous fumarate extrudates with 25% (w/w) of titanium dioxide until
the surface, that is, the brown color of the particles, was fully covered. This
was followed by film coating done in a top spray lab-scale fluidized-bed pro-
cessor (Uni-Glatt model, Glatt Air Techniques, Ramsey, NJ, USA). Among the
coating materials tested, HPMC offered a better protection at a relatively low
encapsulation level (10%, w/w) compared to other coating materials studied
(Yadava et al., 2012).
DFS was then prepared by blending the preprocessed iron premix into
iodized salt at a ratio of between 1:150 and 1:200, resulting in an iron con-
centration of 1000 ppm in DFS. A storage stability test showed that the DFS
retained more than 90% of the original iodine and more than 93% of the
ferrous iron after 3 months of storage at 35°C and 60% RH (Li et al., 2011a;
Yadava et al., 2012). Besides the excellent storage stability, the DFS also exhib-
ited desirable particle density, good in vitro bioavailability and acceptable
organoleptic properties (Li et al., 2010). Figure 11.12 illustrates the production
steps of the ferrous fumarate premix during the preparation process of DFS.
The process has been tested in India on a pilot scale.
The DFS technology has been introduced to pharmaceutical manufactur-
ers in India, and MI initiated production of the iron premix. With the coop-
eration of the Indian government, MI initiated a major feeding trial in South
India. A total of 3.4 million school children received school lunches prepared
with DFS produced locally, using the premix manufactured in India. Initially
85% of the children were anemic, yet after 10 months of the program this
decreased by approximately 35% (Andersson et al., 2008), representing the
cure of some 1 million children by this simple, cost-effective intervention.
Fefum powder Extruded agglomerate Color-masked particles Encapsulated
FIGURE 11.12
Evolution of ferrous fumarate premix during the double-fortified salt preparation process
(digital microscopic images taken under normal room light). (From Li, Y.O. 2009. Development
of microencapsulation-based technologies for micronutrient fortification in staple foods for
developing countries. PhD thesis, University of Toronto. Available at https://tspace.library
.utoronto.ca/bitstream/1807/26536/1/Li_Yao_O_200906_PhD_Thesis.pdf)

11.4.1.2 Application of Extrusion-Based Microencapsulation
in Rice Fortification
Another successful application of encapsulated premix-based fortification
approach was in the development of Ultra Rice® technology. Ultra Rice is a
reconstituted, nutrient-fortified rice premix made by extrusion, resembling
the shape, size, and appearance of common rice kernels. When formulated
with the appropriate micronutrients and blended at a 1:100 or 1:200 ratio with
normal market rice, this staple becomes a simple and powerful fortification
system.
In the 1980s, Dr. James Cox and his son, Robert Cox, invented this idea
and later obtained a series of patents for Ultra Rice technology (Cox 1982,
1991; Cox and Cox, 1989). The Coxes later transferred the patents to PATH
(Program for Appropriate Technology in Health—a Seattle-based NGO)
in 1997. Reconstituted Ultra Rice grains were made by extrusion of a wet
dough mixture containing selected micronutrients, rice flour, and a struc-
tural ingredient—sodium alginate. The extruded rice kernel-shaped parti-
cles were stabilized by a surface modification step where a calcium solution
was sprayed on the rice grain surface and the ensuing cross-linking reaction
between alginate and calcium hardened the particles.
The surface-coating process or the diffusion-driven alginate–calcium
gelation leads to two problems: (1) the post-extrusion, surface coating step
is hard to control for uniform distribution of calcium ions on the grain sur-
face and; (2) the hardened grains tend to crack and disintegrate during cook-
ing due to starch expansion against the rigid shell. These constraints have
greatly hindered the commercialization of the technology. The process was
improved by the development of controlled internal gelation (as illustrated
in Figure 11.13). Specifically, sodium alginate and a calcium salt (with lim-
ited solubility, e.g., CaSO4) were added to the formulation prior to extrusion.
With the aid of appropriate sequestrants, the cross-linking reaction was
delayed until the completion of the extrusion. The experiments included a
preliminary screening for appropriate techniques/materials followed by an
optimization study based on statistical formulation designs. The optimized
formulations not only led to a simplified process by removing the post-
extrusion coating step, but also resulted in improved grain integrity as all
of the alginate could be converted to a completely interconnected structure
throughout the grain, which enclosed and protected the added micronu-
trients within the rice matrix, ultimately improving its commercial accept-
ability. The most effective internal gelation system is comprised of sodium
alginate, calcium sulfate (CaSO4), and sodium tripolyphosphate (STPP) at an
optimized ratio of 3:3:0.6 (w/w) (Li, 2009).
Ultra Rice now contains a robust antioxidant system for stable vitamin A
fortification (Li et al., 2009b), and has been formulated for multi-nutrient for-
tification (iron, zinc, thiamin, and folic acid) (Li et al., 2008a,b, 2011b). Further
processing refinement has resulted in improved organoleptic properties

External gelation External gelation

“fish-net” or shell model “interconnected” matrix model
Extrusion Alginate Extrusion

CaSO4
Cutting Cutting
Coating CaCl2
Drying Drying
Rice starch granule (~5 μm) Rice starch granule (~5 μm)
Extruded rice grain

Simplified process with
controlled gelation rate
Alginate-Ca network Alginate-Ca network
at the surface with pore size ~1 μm throughout the grain
00002420KV X2.00K 15.0μm 00002120KV X2.00K 15.0μm
Rice made by external gelation Rice made by internal gelation
FIGURE 11.13
Schematic process flow for Ultra Rice production using external (left) and internal (right) gela-
tion techniques and microscopic images of rice premix (left: SEM; right: digital microscopic
image under normal room light).
and particle integrity of the reconstituted kernels, ultimately improving its

commercial acceptability. Ultra Rice has been successfully introduced to
Colombia, Brazil, India, and China under the supervision of PATH.
In summary, the extrusion-based microencapsulation technology plat-
form developed for salt and rice fortification enables the production of
microencapsulated particles ranging in size from 300 μm to 10 mm, while
other unit operations, such as spray drying, freeze drying, and fluidized-
bed agglomeration, could form smaller 10–500 μm particles. Chemical
methods can produce even smaller particles, for example, nano- or micro-
emulsions and microfluidic techniques can lead to particles at nano scales.
Appropriate combinations of these physical and chemical methods make
it possible to produce microencapsulated particles covering a broad size
range for effective delivery of bioactive ingredients or nutraceuticals in food
applications.

11.4.2 Microencapsulation and Delivery Systems for Major

Nutraceuticals
Over the past two decades, numerous microencapsulation techniques have
matured as seen in many practical applications for protection, modifica-
tion, and controlled delivery of many food ingredients, as summarized in
Section 11.1.2 and Table 11.1, including acidulants, flavors, sweeteners, col-
orants, enzymes and microorganisms, antioxidants, leavening agents, and
nutritional ingredients such as vitamins and minerals (Kirby 1991; Gibbs,
et al. 1999; Schrooyen et al. 2001; Gouin, 2004). Most successful applications
lie in the encapsulation and delivery of these rather common food ingredient
in conventionally processed foods, for example, the “4Bs”—beverages, break-
fast cereals, bakery goods, and bars (snacks) (Feder, 2006). More recently,
mature processes have been found in extended applications for advanced
delivery of rare bioactive ingredients, for example, various nutraceuticals
such as omega-3 fatty acids, probiotics/prebiotics, natural polyphenols,
phytosterols, and medicinal/herbal extracts (Zuidam and Shimoni, 2010;
Munin and Edwards-Lévy, 2011; Onwulata, 2012).
As estimated in “Food encapsulation: A global strategic business report”
(Global Industry Analysts, Inc. 2010), the world market share involving the
use of encapsulated food ingredients was more than $26 billion in 2009, and
is predicted to rise to nearly $40 billion by 2015. For a long time, microen-
capsulation was regarded as too expensive for the food industry to use,
until recent advances presented more and more cost-effective preparation
methods and scalable, massive production lines that contribute to the afford-
ability of microencapsulation technologies for effective delivery of various
bioactive ingredients in novel, value-added food products (Shelke, 2005).
A recent patent search reveals that there are over 22,000 patents filed glob-
ally (World Intellectual Property Organization [WIPO]) when “encapsula-
tion of nutrients in food applications” was used as the key words. There are
~1500 hits during a search of the U.S. patent database (United States Patent
and Trademark Office [USPTO]) when “encapsulation,” “nutrient,” and
“food” were used together in the description/specification of the patents.
Specifically, main-stream suppliers of nutrients and nutraceuticals, such as
BASF, DSM, Kraft, Cognis, Balchem, and Firmenich, contribute greatly to
this field. Some examples of innovative delivery technologies for various
nutraceuticals, including vitamins and minerals, from rather small, emerg-
ing companies, include the following:
• NutraLease™, Ltd. (Mishor Adumim, Israel) has developed a unique,

patent-pending technology to produce micelles, which are self-
assembled, structured liquid particles with a diameter of 30 nm or
less. These particles are designed to readily penetrate cell membranes
and dramatically increase the bioavailability of the phytonutrients
carried and protected by the micelles (Food Product Design, 2006).

• Aquanova (Darmstadt, Germany) claimed that its patented

NovaSOL® technology is able to deliver a wide variety of active
ingredients in a stable, crystal-clear aqueous solution. The solubiliz-
ing system has a colloidal micelle structure, enabling an ultrafine
distribution (even below the wavelength of light) of carrying ingre-
dients, including fat-soluble vitamins, omega-3 fatty acid, coenzyme,
isoflavones, flavonoids, carotenoids, phyto-extracts, and essential
oils. This system is expected to bring much higher bioavailability
of the carrying ingredients compared to other formulations on the
market (Aquanova, 2013).
• Proprietary Nutritionals Inc. (Kearny, NJ, USA) launched Benexia™
ALA Powder, a new patent-pending, neutral-tasting, water-soluble
omega-3 microencapsulated powder for numerous food applications
(Shelke, 2006).
• Longevinex (San Dimas, CA, USA) introduced a micron-sized trans-
resveratrol delivery system for enhanced absorption, which is fur-
ther microencapsulated in an envelope of all natural plant dextrins
and starches to preserve resveratrol in its trans-resveratrol form
from degradation by light, heat, and oxygen (Shelke, 2006).
• Maxx Performance (Chester, NY, USA) recently introduced a solu-
tion that allows manufacturers to add green tea extract (rich in poly-
phenol antioxidants but with a bitter after-taste) to baked goods and
other formulations without compromising flavor (Shelke, 2006).
• LiveTheSource (Ft. Lauderdale, FL, USA) announced its launch of
“daily source,” the first-ever nanoencapsulated liquid vitamin, min-
eral, and herbal supplement. The company that claims the herbal
compounds in daily source will “provide a substantial increase in
nutritional value, first, due to their synergy, and second, due to their
nano-encapsulation” (Shelke, 2006).
11.4.2.1 Microencapsulation-Based Delivery Systems for Probiotics

As defined by the FAO (Food and Agriculture Organization) and the WHO
(World Health Organization), probiotics are “live microorganisms that
when administered in adequate amounts confer health benefits to the host”
(Jorgen, 2001, p. 9). In this definition, there are three main components that
are critical for any food products to be qualified for the claim. The “live
microorganisms” commonly include lactic acid-producing bacteria, primar-
ily from the Lactobacillus, Bifidobacterium, and Streptococcus genera. The “ade-
quate amounts” typically require that the viable microorganisms are not less
than 10 million CFUs (colony-forming units) per gram in the food that car-
ries them. The “health benefits” that have been identified in the literature
include helping to prevent colon cancer, improving or preventing constipa-
tion, modulating blood lipids and reducing blood pressure, lowering serum

cholesterol concentrations, improving lactose intolerance symptoms, and

increasing the resistance to infectious intestinal diseases (Nagpal et al., 2012).
To develop effective delivery systems for probiotics, the selection of encap-
sulants and the food carriers are of paramount importance. The encapsu-
lant or food matrix needs to be non-antimicrobial so as not to damage the
live sells, and also needs to be able to provide protection from environmen-
tal factors so as to offer extended shelf stability during storage. Ideally, the
delivery system should also impart a degree of controlled or targeted release
across the small and large intestine so as to ensure the efficacy of probiotics
in the body. All of these requirements pose a great challenge for developing
microencapsulation technology or delivery systems for probiotics, with the
ultimate goal of ensuring its stability and bioavailability during food pro-
cessing, storage, distribution, and consumption.
Some successful concepts in probiotics delivery systems include the entrap-
ment of live cells in extruded polymer matrices leading to dry tablets, or the
entrapment in hydrocolloid solutions leading to soft gel capsules, prior to
the end use of probiotics in food products. Also, fat or polymer coatings are
reported in the literature with successful results (Harel and Tong, 2014). The
typical choice of encapsulants includes naturally occurring polysaccharides
and proteins, as they are nontoxic to live cells and have been widely tested
for encapsulation efficiency in many other delivery systems. The production
of probiotics-bearing microcapsules typically follows a two-step process,
where the formation of microparticle can be made by extrusion, atomiza-
tion, or emulsion, which is then followed by dehydration via spray drying,
air drying, fluidized-bed drying, or freeze drying. Cross-linking effects or
gelling of polymer solutions can be also introduced after either spraying or
dropping the particles into cross-linking solution or emulsifying in oil.

for Omega-3 and Omega-6 Oils
With the more awareness of the health benefits from oils or foods rich in
omega-3 and omega-6 fatty acids, the food industry has seen the increased
demand from consumers for novel functional foods fortified with these poly-
unsaturated fatty acids (PUFAs). Specifically, these PUFAs are recognized
for their ability to reduce the risk of cardiovascular disease, to protect aging
populations from cognitive decline and dementia, and to aid in visual and
brain development in infants (Wang et al., 2006). Also, omega-3 and omega-6
PUFAs are precursors to anti-inflammatory mediators, which have shown
beneficial effects toward allergies, diabetes, Alzheimer’s, and other related
neurodegenerative diseases (Kralovec et al., 2012).
Fish oils represent the most widely used source of n-3 and n-6 PUFAs
including the long-chain versions of eicosapentaenoic acid (EPA) and doc-
osapentaenoic acid (DHA) for nutritional supplementation and food for-
tification. Algal oil is the primary source of DHA for infant formulas

(Koletzko et al., 2008). In contrast, α-linolenic acid (ALA; 18:3n-3) is abundant

in widely consumed plant sources such as flaxseed oil, soybeans, canola, and
nuts, and can be broken down in the presence of certain enzymes to form
EPA and DHA (Larsen et al., 2011).
The major challenge in food applications of n-3 and n-6 fatty acids or
oils is primarily related to the extreme instability of these PUFAs at higher
temperatures and in the presence of oxidizing agents in the environment,
as these PUFAs are easily oxidized, resulting in unpleasant off-flavors and
odors, which further compromise the product quality. The common indus-
trial practice to overcome this challenge is to entrap n-3- and n-6-rich oils in
a protective microencapsulation system, often with the addition of various
antioxidants in the finished formulations. Specifically, spray drying has been
used as the most common method in the current industrial practice to form
encapsulated n-3 and n-6 powders due to its ease of processing and low-
operating cost. Other encapsulation methods used for protecting and deliv-
ery of these PUFAs include spray cooling/chilling, fluidized-bed coating,
freeze drying, and extrusion-based microencapsulation process. Complex
coacervation is also investigated as a means to achieving much higher pay
loads (40%–90%) and lower surface oils (~0.2% of the total oil), wherein a
number of proteins such as gelatin, whey proteins, soy proteins, and pea
proteins, and polysaccharides such as low and high methoxy pectins, gellan
gum, and xanthan gum, are explored to achieve a delicate, layer-by-layer
design for the encapsulated system (Dardelle and Normand 2007). Other
emulsion-based delivery technologies are also explored recently, including
nanoemulsions (Tadros et al., 2011; McClements, 2012) and microemulsions
(Cho et al., 2008; Zheng et al., 2011), where the microcapsules formed can be
used directly for food applications in emulsion form and also can be spray
dried for applications in powder form.
11.4.2.3 Microencapsulation-Based Delivery Systems for Phytochemicals

Phytochemicals belong to a group of chemical compounds naturally occur-
ring in plants, which possess various biological activities that are beneficial
to human health. A large number of phytochemicals have been found and
identified in fruits, vegetables, legumes, whole grain cereals, nuts, and seeds.
According to their chemical structures, they can be classified as polyphe-
nols, carotenoids, phytosterols, betalains, organosulfurs, and glucosinolates.
Polyphenols can be further divided into flavonoids such as anthocyanins,
isoflavones, phenolic acids, and non-flavonoid polyphenols such as cur-
cumin and coumarins. Common carotenoids that are found in an abundance
of food sources include carotene, lutein, and lycopene.
As reported in the literature findings, phytochemicals have presented a
wide range of health benefits. Due to their multiple mechanisms of biological
activities such as antioxidant, anti-inflammatory, anticancer, and antibacte-
rial properties, they can stimulate the immune system, modulate enzyme

activities, and regulate cholesterol synthesis and blood pressure (Herber,

2008; Scalbert et al., 2011; Tokuşoğlu and Hall III, 2011).
Lee and Wong (2014) conducted a comprehensive review on micro-
nanoencapsulation techniques that have been developed in recent years for
effective protection and delivery of selected phytochemicals in various food
applications. Spray drying is the most commonly used method for formu-
lating stable and bioavailable powder premixes containing selected phyto-
chemicals, such as polyphenols and carotenoids, with enhanced solubility
and/or improved functionality in finished food products such as bever-
ages and bakery goods. Other encapsulation processes for phytochemicals
include freeze drying, coacervation (Rivera et al., 2010; Burin et al., 2010),
micro- and nanoemulsions (Huang et al., 2010; Shi et al., 2015), molecular
inclusion, and liposome complexes. For example, Huang et al. (2010) reported
the use of nanoemulsions for solubilization, subsequently improved bio-
availability, of non-water-soluble phytochemicals such as curcumin, resvera-
trol, and carotenoids including lycopene, β-carotene, lutein, and zeaxanthin.
Most recently, based on their many years research experience in lycopene
extraction, characterization, and applications in functional food develop-
ment, Shi et al. (2015) reported the use of a two-layer microemulsion system
consisting of whey protein concentrate (WPC) and high-methylester-pectin
(HMP) to stabilize lycopene under various environmental stresses such as
thermal treatment and pH changes. The two-layer lycopene-microemulsion
is optimized to achieve much-improved stability than the original one-layer
microemulsion system (Shi et al., 2015).
Until very recently, a rather novel and advanced encapsulation technique,
namely, supercritical precipitation process, has found its application for
encapsulating highly value-added food ingredients to generate ultrafine
nanoparticles. The use of supercritical fluids such as supercritical carbon
dioxide has been practiced in many years, mainly for extraction of bioactive
ingredients that are thermally labile or noncompatible with organic solvents.
With its root applications in drug delivery, supercritical fluid encapsulation
is progressively explored for protection and formulating refined nanopar-
ticles with controlled release properties for herbal extracts such as rosemary
antioxidants (Visentina et al., 2012), lutein (Jin et al., 2009), and carotenoid
fraction from pink shrimp residue (Mezzomo et al., 2012).
According to the review article from Silva and Meireles (2014), supercritical
encapsulation processes can be classified based on the function of the super-
critical fluid in the process. For example, if the supercritical fluid is used as
solvent, the process is called rapid expansion of supercritical solution (RESS),
which involves the solubilization of active compounds and wall materials
together in the supercritical fluid. The homogeneous mixture is rapidly
expanded at atmospheric pressure using a suitable nozzle; consequently, the
sudden change in pressure causes the vaporization of the supercritical fluid
and the precipitation of the dissolved materials. If the supercritical fluid is
used as cosolvent or solute, the process is called particles from gas saturated

solutions (PGSS). The process does not require the active core or the poly-
mer encapsulant be dissolved in the supercritical fluid, rather the supercriti-
cal fluid is dissolved in a substrate, and the thus-formed substrate solution
or suspension is then carried by a solvent. The ensuing rapid expansion of
the saturated solution system through a nozzle under a moderate pressure
causes the formation of solid or liquid particles due to the intense cooling
effect caused by the release of CO2. If the supercritical fluid is used as anti-
solvent, the encapsulation process is called supercritical antisolvent precipi-
tation (SASP). In this process, the core substance and the wall material are
solubilized in an organic solvent (primary solvent) to form an active solution,
which is then supersaturated by a dense gas or supercritical fluid (called anti-
solvent), eventually leading to the nucleation and precipitation of ultrafine
particles containing the active core and the encapsulant. This technique has
a great advantage, namely, able to micronize and encapsulate polar compo-
nents, such as the components that are not typically soluble in CO2; therefore,
this method has been found in more applications compared to other super-
critical processes. An extension of SASP is to combine the efficiency of using a
supercritical fluid as the antisolvent with the effect of emulsification process,
which leads to a process called supercritical fluid extraction of emulsions
(SFEE). In the process, the supercritical fluid is employed with dual func-
tions as an antisolvent and an extractor for the removal of the organic solvent
from the droplets of oil-in-water (o/w) or water-in-oil (w/o) emulsions, which
ultimately results in nanoparticles with controlled particle size and uniform
particle size distribution. Due to the ease of operation with mild-processing
conditions, high yield/payload, high encapsulation efficiency, and formation
of well-defined nanoparticles with desirable controlled-release properties,
the SASP, and SFEE have been so far used for nanoencapsulation of a number
of value-added phytochemicals including lutein, β-carotene, extracts from
grape seeds, green tea, rosemary, and annatto seeds, as well as quercetin
(Silva and Meireles, 2014).
11.5 Conclusion and Perspectives

Clearly, there are many technical barriers to overcome when developing
effective delivery systems for micronutrients and nutraceuticals. Potential
interaction between the added ingredients and the intrinsic components in
the food vehicle, leading to adverse effects on sensory properties of the food
vehicle and stability and bioavailability of the added nutrients, is foremost
among these barriers. The selection of appropriate food vehicles is of utmost
importance as the success of a fortification program depends on regular con-
sumption of the fortified food by the target populations in uniform amounts.
To prevent the possible interactions so as to avoid degradation of the added

nutrients as well as maintain the desired stability and organoleptic proper-

ties of the fortified foods, microencapsulation techniques and/or effective
delivery systems are critical.
The program at the Food Engineering Group at the University of Toronto
enhanced the understanding in this field with respect to the interactions
between the added components and the food carriers, which will lead to
the development of more stable delivery systems for food fortification. The
technology platform is based on a broad concept of microencapsulation,
and combines a series of unit operations, such as particle enlargement via
extrusion or fluidized-bed agglomeration, surface modification, color/flavor
masking, film over-coating by the use of rotating disc or fluidized-bed coat-
ing method. The technology is adaptable to formulating customized delivery
systems for active ingredients in broader applications. The current research
focuses on extending this technology platform for developing “engineered”
foods and food ingredients containing both essential micronutrients and
desirable nutraceuticals, consequently leading to novel functional food
products with added nutritional value for health promotion and disease
prevention. Clearly, to achieve this research goal, an integrated approach
of choosing several nano-microencapsulation techniques and using them
in a combination appropriate to the selected food carriers is essential, with
respect to meeting the principle fortification criteria: technical and economi-
cal feasibility, clinical effectiveness, and consumer acceptance.
Specifically, investigations are underway on the potential use of plant-
derived biopolymers such as dietary fibers and plant protein isolates as
building blocks or coating materials for microencapsulating micronutri-
ents (vitamins and minerals) or other nutraceuticals such as phytochemi-
cals, antioxidants, or fermentation by-products from probiotic bacteria. As
there are numerous studies in the literature indicating that soluble dietary
fibers and plant proteins can help in weight control/management, these
compounds would be desirable components of functional food products for
overweight or obese consumers. Currently, the literature lacks of informa-
tion on their utilizations as delivery matrices for other minor ingredients,
focusing only on using them for bulking and excipients in food product
development.
The novel use of plant-derived biopolymers, for example, protein isolates
and refined dietary fiber extracts, as both biologically functional compo-
nents (nutraceuticals) and technologically functional components (structural
or delivery matrix) for minor bioactive ingredients, will permit the fabri-
cation of integrated delivery systems that combine the multiple functions
of encapsulation in protection, targeted delivery, controlled release, and/or
enhanced absorption in novel food product development.
While stability and bioavailability are of major concerns when developing
effective delivery systems for micronutrients and nutraceuticals as discussed
in this chapter, the concepts of targeted delivery and controlled release have
become more attractive to the food industry, especially in functional food

development. These concepts originating from oral drug delivery systems,

when adapted to food applications, are still mostly focused on nutrient
absorption in the body, but the potential use of them in protection of the
active components during manufacturing and distribution presents further
opportunities. Modern food production practice involves many components
and multiple processing stages, which require precise control of operation
conditions for preserving active ingredients at all stages. Encapsulated
ingredients are increasingly used to prevent premature or unwanted inter-
actions. Controlled release of various ingredients, at a targeted stage or at a
desired release rate, during food formulating, processing, distribution, even
at the cooking preparation stage, can be critical factors for obtaining desired
physicochemical and sensory properties and the ultimate effectiveness of
the finished food products. Therefore, a systematic and holistic approach
should be followed when developing “smart” delivery systems for nutraceu-
ticals and micronutrients.
References
Acosta, E., Nguyen, T., Witthayapanyanona, A., Harwell, J., Sabatini, D. 2005. Linker-
based bio-compatible microemulsions. Environmental Science & Technology,
39(5): 1275–1282.
Adamiec, J., Marciniak, E. 2004. Microencapsulation of oil/matrix/water system dur-
ing spray drying process. Drying 2004-Proceedings of the 14th International Drying
Symposium, Sao Paulo, Brazil, Vol. C, pp. 2043–2050.
Andersson, M., Thankachan, P., Muthayya, S., Goud, B.R., Kurpad, A.V., Hurrell, R.F.,
Zimmermann, M.B. 2008. Dual fortification of salt with iodine and iron: A ran-
domized, double blind, controlled trial of micronized ferric pyrophosphate and
encapsulated ferrous fumarate in southern India. American Journal of Clinical
Nutrition, 88(5): 1378–1387.
Aquanova. 2013. NovaSOL®—Crystal Clear Solution: Overview. Available at: http://
www.aquanova.de/media/public/pdf_produkte%20unkosher/NovaSOL_
OVERVIEW.pdf. Accessed on August 20, 2013.
Artursson, P., Lindmark, T., Davis, S., Illum, L. 1994. Effect of chitosan on the per-
meability of monolayers of intestinal epithelial cells (Caco-2). Pharmaceutical
Research, 11(9): 1358–1361.
Attama, A.A., Nzekwe, I.T., Nnamani, P.O., Adikwu, M.U., Onugu, C.O. 2003. The
use of solid self-emulsifying systems in the delivery of diclofenac. International
Journal of Pharmaceutics, 262: 23–28.
Augustin, M.A., Hemar, Y. 2009. Nano- and micro-structured assemblies for encapsu-
lation of food ingredients. Chemical Society Reviews, 38(4): 902–912.
Augustin, M.A., Sanguansri, L. 2008. Encapsulation of bioactives. In Food Materials
Science, ed. J. Aguilera and P. Lillford. Springer, New York, pp. 577–601.
Avis, K.E., Shukla, A.J., Chang, R.K. ed. 1999. Pharmaceutical Unit Operations: Coating.
CRC Press, Boca Raton, FL, p. 182.

Barthelemy, P. (Mions, FR), Benameur, H. (Genas-Azieu, FR). 2001. Composition with

sustained release of active principle, capable of forming a microemulsion. US
Patent # 6,309,665.
Benita, S. ed. 1996. Microencapsulation Methods and Industrial Applications. Marcel
Dekker, Inc., New York.
Bhandari, B., D’Arcy, B.R., Young, G. 2001. Flavor retention during high tempera-
ture short time extrusion cooking process: A review. International Journal of Food
Black, R. 2003. Micronutrient deficiency—An underlying cause of morbidity and
mortality. Bulletin—World Health Organization, 81(2): 79–79.
Burin, V.M., Falcão, L.D., Gonzaga, L.V., Fett, R., Rosier, J.P., Bordignon-Luiz, M.T.
2010. Colour, phenolic content and antioxidant activity of grape juice. Food
Science and Technology (Campinas), 30(4): 1027–1032.
Carafa, M., Marianecci, C., Lucania, G., Marchei, E., Santucci, E. 2004. New vesicu-
lar ampicillin-loaded delivery systems for topical application: Characterization,
in vitro permeation experiments and antimicrobial activity. Journal of Controlled
Release, 95: 67–74.
Champagne, C.P., Fustier, P. 2007. Microencapsulation for the improved delivery
of bioactive compounds into foods. Current Opinion in Biotechnology, 18(2):
184–190.
Chen, W., Lu, D.R. 1999. Carboplatin-loaded PLGA microspheres for intracerebral
injection: Formulation and characterization. Journal of Microencapsulation, 16:
551–563.
Cho, Y.H., Kim, S., Bar, E.K., Mok, C.K., Park, J. 2008. Formation of a cosurfactant-
free O/W microemulsion using non-ionic surfactant mixtures. Journal of Food
Science, 73: E115–E121.
Clark, J.P. 2002. Food encapsulation: Capturing one substance by another. Food
Technology, 56(11): 63–64, IFT Institute of Food Technologists.
Cleland, J.L., Lim, A., Barron, L., Duenas, E.T., Powell, M.F. 1997. Development of a
single-shot subunit vaccine for HIV-1: Part 4. Optimizing microencapsulation
and pilsatile release of MN rgp120 from biodegradable microspheres. Journal of
Controlled Release, 47: 135–150.
Cox, J. 1982. Method for forming shaped products for human and/or animal con-
sumption or as marine bait and products produced thereby. U.S. Patent No.
4,362,748. Lynden, WA.
Cox, J. 1991. Synergistic flavor enhancement nutritional compositions and methods.
U.S. Patent No. 5,034,378. Lynden, WA.
Cox, J., Cox, J. 1989. Cohesive vegetable products and process for manufacture. U.S.
Patent No. 4,844,936. Lynden, WA.
Crison, J.R., Amidon, G.L. 1997. Method and formulation for increasing the bioavail-
ability of poorly water-soluble drugs. U.S. Patent # 5,993,858. Ann Arbor, MI.
Dardelle, G., Normand, V. 2007. Method for preparing microcapsules by coacerva-
tion. World Patent, WO 2007/113706A1.
Das, K.G. ed. 1983. Controlled Release Technology—Bioengineering Aspects. A Wiley-
Interscience Publication, John Wiley, New York.
Degussa, Specifications and test methods for EUDRAGIT® E 100, EUDRAGIT® E
PO and EUDRAGIT® E 12,5. [Online]. Available at: http://eudragit.evonik.
com/product/eudragit/en/products-services/eudragit-products/protective-
formulations/e-100/pages/default.aspx. Accessed on April 22, 2008.

Desai, K.G.H. 2005. Preparation and characteristics of high-amylose corn starch/

pectin blend microparticles: A technical note. AAPS PharmaSciTech, 6(2):
E202–E208.
Desai, K.G.H., Park, H.J. 2005a. Recent developments in microencapsulation of food
ingredients. Drying Technology, 23: 1361–1394.
Desai, K.G.H., Park, H.J. 2005b. Encapsulation of vitamin C in tripolyphosphate cross-
linked chitosan microspheres by spray drying. Journal of Microencapsulation, 22:
179–192.
Dinsmore, A.D., Hsu, M.F., Nikolaides, M.G., Marquez, M., Bausch, A.R., Weitz, D.A.
2002. Colloidosomes: Selectively permeable capsules composed of colloidal
particles. Science, 298: 1006–1009.
Diosady, L. 2007. Salt formulation and process for production thereof. Canadian
Patent 2238,925.
Diosady, L., Oshinowo, T., Venkatesh Mannar, M.G. 2004. Scale-up of production of salt
double fortified with iodine and iron. Presentation abstract Th45. The Report of the
2004 International Nutritional Anemia Consultative Group Symposium, Lima, Peru.
Diosady, L., Yusufali, R., Oshinowo, T., Laleye, L. 2006. A study of storage and dis-
tribution of double fortified salts in Kenya. Journal of Food Engineering, 76(4):
547–556.
Dow Chemical Company 2008a. Methocel Cellulose Ethers: Technical Handbook.
[Online]. Available at: http://www.dow.com/PublishedLiterature/dh_004f/
0901b8 038004fa1b.pdf?filepath=methocel/pdfs/noreg/192–01062.pdf&from
Page=GetDoc. Last accessed: May 25, 2008.
Dow Chemical Company 2008b. Methocel Cellulose Ethers in Aqueous Systems for Tablet
Coating. [Online]. Available at: http://www.dow.com/PublishedLiterature/
dh_004a/0901b8038004ab56.pdf?filepath=methocel/pdfs/noreg/198–00755
.pdf&fromPage=GetDoc. Last accessed: May 25, 2008.
Dungan, S.R. 1997. Microemulsions in foods: Properties and applications. In Industrial
Applications of Microemulsions, eds. C. Solans and H. Kunieda, Surfactant Science
Series, Vol. 66, CRC Press, Boca Raton, FL, pp. 147–174.
Espinosa-Andrews, H., Alvarez-Ramirez, J., Vermon-carter, E.J., Perez-Orozco, J.P.
2005. Release kinetics of ascorbic acid from w/o/w multiple emulsions stabi-
lized with biopolymers. 2005 IFT (Institute of Food Technologists) Annual Meeting
Proceedings, New Orleans, LA, USA.
Fang, Z., Bhandari, B. 2010. Encapsulation of polyphenols—A review. Trends in Food
Science & Technology, 21(10): 510–523.
Food Product Design 2006. Nanoencapsulation aids fortification. Available at: http://
www.foodproductdesign.com/articles/2006/05/nanoencapsulation-aids-
fortification.aspx. Accessed on August 16, 2013.
Feder, D. 2006. Well Noted: The Four Bs of Nutraceuticals. Food Processing. Available
at http://www.foodprocessing.com/articles/2006/252.html. Accessed on
August 20, 2013.
Gangloff, M.B., Glahn, R.P., Miller, D.D., Van Campen, D.R. 1996. Assessment of iron
availability using combined in vitro digestion and Caco-2 cell culture. Nutrition
Research, 16(3): 479–487.
Gaonkar, A.G., Prabhakar, B.R. 2003 Microemulsions in foods: Challenges and
applications. In Surfactant Science Series, Vol. 109, Adsorption and Aggregation of
Surfactants in Solution, eds. K.L. Mittal and D.O. Shah, CRC Press, Boca Raton,
FL, pp. 407–430.

Gaonkar, A.G., Vasisht, N., Khare, A.R., Sobel, R. ed. 2014. Microencapsulation in the
Food Industry: A Practical Implementation Guide. Academic Press of Elsevier,
San Diego, USA.
Genta, I., Perugini, P., Pavanetto, F., Maculotti, K., Modena., T., Casado, B., Lupi, A.,
Iadarola, P., Conita, B. 2001. Enzyme loaded biodegradable microspheres in vitro
ex vivo evaluation. Journal of Controlled Release, 77: 287–295.
Research International, 40(9): 1107–1121.
Ghosh, S.K. 2006. Functional coatings and microencapsulation: A general perspective—
Chapter 1. In Functional Coatings, ed. S.K. Ghosh, Wiley-VCH Verlag GmbH &
Co. KGaA. pp. 1–22.
Gibbs, B., Kermasha, S., Alli, I., Mulligan, C. 1999. Encapsulation in the food industry:
A review. International Journal of Food Science and Nutrition, 50(3): 213–224.
Global Industry Analysts, Inc. 2010. Food encapsulation: A global strategic business report.
Accessed online at: http://www.researchandmarkets.com/reports/1382377/
food_encapsulation_global_strategic_business.
Gouin, S. 2004. Microencapsulation: Industrial appraisal of existing technologies and
trends. Trends in Food Science & Technology, 15(7): 330–347.
Gu, Y.S., Decker, E.A., Mcclements, D.J. 2005. Influence of gelatin and/or I-carrageenan
on oil-in-water emulsions containing droplets stabilized by beta-lactoglobulin.
2005 IFT (Institute of Food Technologists) Annual Meeting Proceedings, New
Orleans, LA, USA.
Hao, T., McKeever, U., Hedley, M.L. 2001. Biological potency of microsphere encapsu-
lated plasmid DNA. Journal of Controlled Release, 69: 249–259.
Harel, M., Tong, Q. 2014. Protection and delivery of probiotics for use in foods.
In Microencapsulation in the Food Industry: A Practical Implementation Guide,
ed. A.G. Gaonkar, N. Vasisht, A.R. Khare, R. Sobel, Academic Press of Elsevier,
San Diego, USA, pp. 469–481.
Hasler, C.M. 1998. Functional foods: Their role in disease prevention and health pro-
motion. Food Technology-Champaign Then Chicago, 52(11): 63–147. Institute of
Food Technologists.
Herber, D. 2008. Multitargeted therapy of cancer by ellagitannins. Cancer Letters,
269(2): 262–268.
Huang, Q., Yu, H., Ru, Q. 2010. Bioavailability and delivery of nutraceuticals using
nanotechnology. Journal of Food Science, 75(1): R50–R57.
IUPAC. 1997. International Union of Pure and Applied Chemistry. Available at: http://
www.iupac.org/publications/compendium/. Accessed on June 26, 2007.
Jiao, J., Burgess, D. 2003. Rheology and stability of water-in-oil-in-water multiple
emulsions containing Span 83 and Tween 80. AAPS PharmaSci, 5(1): 1–12.
Jimenez-Alvarado, R. 2005. Iron double emulsion stabilization. 2005 IFT (Institute of
Food Technologists) Annual Meeting Proceedings, New Orleans, LA, USA.
Jin, H., Xia, F., Jiang, C., Zhao, Y., He, L. 2009. Nanoencapsulation of lutein with
hydroxypropylmethyl cellulose phthalate by supercritical antisolvent. Chinese
Journal of Chemical Engineering, 17(4): 672–677.
Jorgen, S. 2001. Health and nutritional properties of probiotics in food including pow-
der milk with live lactic acid bacteria. Report of a joint FAO/WHO expert consulta-
tion on evaluation of health and nutritional properties of probiotics in food including
powder milk with live lactic acid bacteria. FAO/WHO, Córdoba, Argentina.

Kang, B.K., Lee, J.S., Chon, S.K., Jeong, S.Y., Yuk, S.Y., Khang, G., Lee, H.B., Sho, S.H.
2004. Development of self-microemulsifying drug delivery systems (SMEDDS)
for oral bioavailability enhancement of simvastatin in beagle dogs. International
Journal of Pharmaceutics, 274: 65–73.
Kim, C. 2004. Advanced Pharmaceutics: Physicochemical Principle. CRC Press, Boca
Raton, FL, pp. 454.
Kim, H.J., Yoon, K., Hahn, M.Y., Park, E.S., Chi, S.C. 2000. Preparation and in vitro
evaluation of self-microemulsifying drug delivery systems containing ideben-
one. Drug Development & Industrial Pharmacy, 26(5): 523–527.
Kirby, C. 1991. Microencapsulation and controlled delivery of food ingredients. Food
Science and Technology Today, 5(2): 74–78.
Ko, S. 2005. Preparation of beta-lactoglobulin and bovine serum albumin nanopar-
ticles. 2005 IFT (Institute of Food Technologists) Annual Meeting Proceedings,
New Orleans, LA, USA.
Koletzko, B., Lien, E., Agostoni, C., Böhles, H., Campoy, C., Cetin, I., Decsi, T.
et al. 2008. The roles of long-chain polyunsaturated fatty acids in pregnancy,
lactation and infancy: Review of current knowledge and consensus recom-
mendations. Journal of Perinatal Medicine, 36 (1): 5–14. Online doi: 10.1515/
JPM.2008.001.
Kosaraju, S.L., Labbett, D., Emin, M., Konczak, I., Lundin, L. 2008. Delivering poly-
phenols for healthy ageing. Nutrition and Dietetics, 65: S48–S52.
Kralovec, J.A., Zhang, S., Zhang, W., Barrow, C.J. 2012. A review of the progress in
enzymatic concentration and microencapsulation of omega-3 rich oil from fish
and microbial sources. Food Chemistry, 131: 639–644.
Kshirsagar, N.A. 2000. Drug delivery systems. Indian Journal of Pharmacology, 32: S54–S61.
Kwon, G.S. ed. 2005. Polymeric Drug Delivery Systems. Taylor & Francis, Boca Raton,
p. 142.
Lamprecht, A., Torres, H.R., Schafer, U., Lehr, C. 2000. Biodegradable microparticles
as a two-drug controlled release formulation: A potential treatment of inflam-
matory bowel disease. Journal of Controlled Release, 69: 445–454.
Larsen, R., Eilertsen, E.K.E., Elvevoll, E.O. 2011. Health benefits of marine oils and
ingredients. Biotechnology Advances, 29: 508–518.
Lee, H.K., Park, J.H., Kwon, K.C. 1997. Double-walled microparticles for single shot
vaccine. Journal of Controlled Release, 44: 283–293.
Lee, S.J., Wong, M. 2014. Nano- and microencapsulation of phytochemicals. In Nano-
and Microencapsulation for Foods, ed. H-S. Kwak. Wiley-Blackwell, Oxford, UK.
ISBN: 978-1-118-29233-4, pp. 119–152.
Leopold, C.S. 1999. Coated dosage forms for colon-specific drug delivery.
Pharmaceutical Science & Technology Today, 2(5): 197–204.
Lesmes, U., McClements, D.J. 2009. Structure–function relationships to guide ratio-
nal design and fabrication of particulate food delivery systems. Trends in Food
Science & Technology, 20(10): 448–457.
Li, Y.O. 2009. Development of microencapsulation-based technologies for micro-
nutrient fortification in staple foods for developing countries. PhD the-
sis, University of Toronto. Available at: https://tspace.library.utoronto.ca/
bitstream/1807/26536/1/Li_Yao_O_200906_PhD_Thesis.pdf
Li, Y.O., Diosady, L.L., Jankowski, S. 2008a. Effect of iron compounds on the storage
stability of multiple-fortified Ultra Rice®. International Journal of Food Science and
Technology, 43(3): 423–429.

Li, Y.O., Diosady, L.L., Jankowski, S. 2008b. Stability of vitamin B1 in Ultra Rice®
in the presence of encapsulated ferrous fumarate. International Journal of Food
Sciences and Nutrition, 59(1): 24–33.
Li, Y.O., Diosady, L.L., Jankowski, S. 2011b. Folic acid stability in the presence of vari-
ous formulation components including iron compounds in fortified extruded
Ultra Rice® over prolonged storage at 40°C and 60% relative humidity (RH).
International Journal of Food Science and Technology, 46(2): 379–385.
Li, Y.O., Diosady, L.L., Wesley, A. 2009a. Iron in vitro bioavailability and iodine
storage stability in double-fortified salt. Food and Nutrition Bulletin, 30(4):
327–335.
Li, Y.O., Diosady, L.L., Wesley, A. 2010. Iodine stability in iodized salt dual fortified
with microencapsulated ferrous fumarate made by an extrusion-based encapsu-
lation process. Journal of Food Engineering, 99(2): 232–238.
Li, Y.O., Lam, J., Diosady, L.L., Jankowski, S. 2009b. Antioxidant system for the pres-
ervation of vitamin A in Ultra Rice®. Food and Nutrition Bulletin, 30(1): 82–89.
Li, Y.O., Yadava, D., Lo, K., Diosady, L.L., Wesley, A. 2011a. Feasibility and optimi-
zation study of using cold-forming extrusion process for agglomerating and
microencapsulating ferrous fumarate for salt double fortification with iodine
and iron. Journal of Microencapsulation, 28(7): 639–649.
Lin, S.-Y., Kao, Y.-H. 1991. Tablet formulation study of spray-dried sodium diclofenac
enteric-coated microcapsules. Pharmaceutical Research, 8(7): 919–924.
Liu, L., Fishman, M.L., Kost, J., Hicks, K.B. 2003. Pectin-based systems for colon-
specific drug delivery via oral route. Biomaterials, 24(19): 3333–3343.
Lo, K.L. 2006. Development of appropriate technologies for the production of double
fortification of salt with iron and iodine. MASc thesis, University of Toronto.
Lorenzo-Lamosa, M.L., Remunan-Lopez, C., Vila-Jato, J.L., Alonso, M.J. 1998. Design
of microencapsulated chitosan microspheres for colonic drug delivery. Journal of
Controlled Release, 52: 109–118.
Madene, A., Jacquot, M., Scher, J., Desobry, S. 2006. Flavor encapsulation and con-
trolled release—A review. International Journal of Food Science and Technology,
41(1): 1–21.
Madziva, H., Kailasapathy, K., Phillips, M. 2005. Alginate-pectin microcapsules as
a potential for folic acid delivery in foods. Journal of Microencapsulation, 22(4):
343–351.
McClements, D.J. 2012. Nanoemulsion versus microemulsions: Terminology, differ-
ences, and similarities. Soft Matter, 8: 1719–1728.
McClements, D.J., Li, Y. 2010. Review of in vitro digestion models for rapid screening
of emulsion-based systems. Food & Function, 1(1): 32–59.
McGinity, J. ed. 1997. Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms.
Marcel Dekker, New York.
Mezzomoa, N., de Pazb, E., Maraschin, M., Martín, A., Cocero, M.J., Ferreira, S.R.S.
2012. Supercritical anti-solvent precipitation of carotenoid fraction from pink
shrimp residue: Effect of operational conditions on encapsulation efficiency. The
Journal of Supercritical Fluids, 66: 342– 349.
Miret, S., Simpson, R.J., Mckie, A.T. 2003. Physiology and molecular biology of dietary
iron absorption. Annual Review of Nutrition, 23: 283–301.
Munin, A., Edwards-Lévy, F. 2011. Encapsulation of natural polyphenolic compounds:
A review. Pharmaceutics, 3: 793–829. doi: 10.3390/pharmaceutics3040793.

Nagpal, R., Kumar, A., Kumar, M., Behare, P.V., Jain, S., Yadav, H. 2012. Probiotics,
their health benefits and applications for developing healthier foods: A review.
FEMS Microbiology Letters, 334(1): 1–15. Published online May 28, 2012. doi:
10.1111/j.1574–6968.2012.02593.x.
Nunthanid, J., Huanbutta, K., Luangtana-anan, M., Sriamornsak, P., Limmatvapirat, S.,
Puttipipatkhachorn, S. 2008. Development of time-, pH-, and enzyme-controlled
colonic drug delivery using spray-dried chitosan acetate and hydroxypropyl
methylcellulose. European Journal of Pharmaceutics and Biopharmaceutics, 68(2):
253–259.
Onwulata, C.I. 2012. Microencapsulation and functional bioactive foods. Journal of Food
Processing and Preservation. Online early doi: 10.1111/j.1745-4549.2012.00680.x.
Oshinowo, T., Diosady, L., Yusufali, R., Laleye, L. 2004. Stability of salt double-
fortified with ferrous fumarate and potassium iodate or iodide under storage
and distribution conditions in Kenya. Food and Nutrition Bulletin, 25(3): 264–270.
Oshinowo, T., Diosady, L., Yusufali, R., Wesley, A. 2007. An investigation of the
stability of double fortified salt during storage and distribution in Nigeria.
International Journal of Food Engineering, 3(4). Article 13.
Parada, J., Aguilera, J. 2007. Food microstructure affects the bioavailability of several
nutrients. Journal of Food Science, 72(2): R21–R32.
Park, J.H., Ye, M., Park, K. 2005. Biodegradable polymers for microencapsulation of
drugs. Molecules, 10: 146–161.
Patil, P., Joshi, P., Paradkar, A. 2004. Effect of formulation variables on preparation
and evaluation of gelled self-emulsifying drug delivery system (SEDDS) of
Ketoprofen. AAPS PharmSciTech, 5(3) Article 42. Available at: http://www.aap
spharmscitech.org. Last accessed on June 29, 2009.
Pham, A.T., Lee, P.I. 1994. Probing the mechanisms of drug release from hydroxypro-
pylmethyl cellulose matrices. Pharmaceutical Research, 11: 1379–1384.
Pedroza-Islas, R., Vernon-Carter, E.J., Durán-Domı´nguez, C., Trejo-Martı´nez, S.
1999. Using biopolymer blends for shrimp feedstuff microencapsulation—
I. Microcapsule particle size, morphology and microstructure. Food Research
International, 32(5): 367–374.
Porta, G.D., Vittori, C.D., Reverchon, E. 2005. Supercritical assisted atomization:
A novel technology for microparticles preparation of an asthma-controlling
drug. AAPS PharmaSciTech, 6(3): E421–E428.
Porter, C., Charman, W. 2001. In vitro assessment of oral lipid based formulations.
Advanced Drug Delivery Review, 50: S127–S147.
Pothakamury, U.R., Barbosa-Canovas, G.V. 1995. Fundamental aspects of controlled
release in foods. Trends in Food Science & Technology, 6: 397–406.
Prasertmanakit, S., Praphairaksit, N., Chiangthong, W., Muangsin, N. 2009. Ethyl cel-
lulose microcapsules for protecting and controlled release of folic acid. AAPS
PharmSciTech, 10(4): 1104–1112.
Rivera, T., Crouse, J., Given, P.S. 2010. Microencapsulated citrus phytochemicals
comprising citrus limonoids and application to beverages. World Patent # WO
2010/090971 A1.
Rodriguez, M., Vila-Jato, J.L., Torres, D. 1998. Design of a new multiparticulate sys-
tem for potential site-specific and controlled drug delivery to the colonic region.
Journal of Controlled Release, 55: 67–77.
Roller, S., Jones, S.A. ed. 1996. Handbook of Fat Replacers. CRC Press, Boca Raton, FL.

Romita, D., Cheng, Y.-L., Diosady, L.L. 2011. Microencapsulation of ferrous fumarate
for the production of salt double fortified with iron and iodine. International
Journal of Food Engineering, 7(3). Article 5.
Rosca, I.D., Watari, F., Uo, M. 2004. Microparticle formation and its mechanism in
single and double emulsion solvent evaporation. Journal of Controlled Release,
99: 271–280.
Rosenberg, M., Rosenberg, Y., Frenkel, L., Dragich, A.M. 2005. Microencapsulation of
lipids in wall systems consisting of soy proteins and carbohydrates. 2005 IFT
(Institute of Food Technologists) Annual Meeting Proceedings, New Orleans, LA,
USA.
Scalbert, A., Andres-Lacueva, A., Arita, M., Kroon, P., Manach, C., Urpi-Sarda, M.,
Wishart, D. 2011. Databases on food phytochemicals and their health-promoting
effects. Journal of Agricultural and Food Chemistry, 59: 4331–4348.
Schrooyen, P., Van der Meer, R., De Kruif, C. 2001. Microencapsulation: Its application
in nutrition. Proceedings of the Nutrition Society, 60: 475–479.
Schwarz, J. 2003. Solid self-emulsifying dosage form for improved delivery of poorly
soluble hydrophobic compounds and the process of preparation thereof. US
Patent Application # 2003 0 072 789.
Sefton, M.V., Maya, M.H., Lahootia, S., Babenseeb, J.E. 2002. Making microencapsu-
lation work: Conformal coating, immobilization gels and in vivo performance.
Journal of Controlled Release, 65: 173–186.
Shelke, K. 2005. Encapsulation technologies protect key ingredients and deliver them at
just the right moment. Food Processing, June 27, 2005. Available at: http://www
.foodprocessing.com/articles/2005/421.html. Accessed on August 20, 2013.
Shelke, K. 2006 Ingredients and formulation: The future impact of nanoingredients.
Food Processing, October 30, 2006. Available at http://www.foodprocessing
.com/articles/2006/227.html. Accessed on August 18, 2013.
Shi, J., Xue, S.J., Wang, B., Wang, W., Ye, X., Quek, S.Y. 2015. Optimization of for-
mulation and influence of environmental stresses on stability of lycopene-
microemulsion. LWT-Food Science and Technology, 60(2): 999–1008.
Silva, E.K., Meireles, M.A.M. 2014. Encapsulation of food compounds using super-
critical technologies: Applications of supercritical carbon dioxide as an antisol-
vent. Food and Public Health, 4(5): 247–258. doi: 10.5923/j.fph.20140405.06.
Solans, C., Kunieda, H. ed. 1996. Industrial Applications of Microemulsions, Surfactant
Science Series, Vol. 66. CRC Press, Boca Raton, FL, pp.1–20.
SwRI Website: Southwest Research Institute (SwRI®). Available at: http://www.swri
.org/4org/d01/microenc/microen/default.htm
Swain, J.H., Newman, S.M., Hunt, J.R. 2003. Bioavailability of elemental iron powders
to rats is less than bakery-grade ferrous sulfate and predicted by iron solubility
and particle surface area. The Journal of Nutrition, 133(11): 3546–3552.
emulsions. Advances in Colloid and Interface Science, 108–109, 303–318.
Taylor, T.M., Davidson, P.M. 2005. Liposomal nanocapsules in food science and agri-
culture. Critical Reviews in Food Science and Nutrition, 45: 587–605.
Tinsley-Bown, A.M., Fretwell, R., Dowsett, A.B., Davis S.L., Farrar, G.H. 2000.
Formulation of poly (d, l-lactic-co-glycolic acid) microparticles for rapid plas-
mid DNA delivery. Journal of Controlled Release, 66: 229–241.
Tokuşoğlu, Ö., Hall, C.A. (III) ed. 2011. Fruit and Cereal Bioactives: Sources, Chemistry,
and Applications. Taylor & Francis Group, LLC, CRC Press, Boca Raton, FL.

Utada, A.S., Lorenceau, E., Link, D.R., Kaplan, P.D., Stone, H.A., Weitz, D.A. 2005.
Monodisperse double emulsions generated from a microcapillary device.
Science, 308: 537–541.
Visentina, A. Rodríguez-Rojoa, S., Navarretea, A., Maestric, D., Coceroa, M.J. 2012.
Precipitation and encapsulation of rosemary antioxidants by supercritical anti-
solvent process. Journal of Food Engineering, 109(1): 9–15.
Wang, C., Harris, W.S., Chung, M., Lichtenstein, A.H., Balk, E.M., Kupelnick, B. 2006.
n-3 Fatty acids from fish or fish-oil supplements, but not alpha-linolenic acid,
benefit cardiovascular disease outcomes in primary- and secondary-prevention
studies: A systematic review. American Journal of Clinical Nutrition, 84: 5–17.
Wang, J., Ng, C.W., Win, K.Y., Shoemakers, P., Lee, T.K.Y., Feng, S.S., Wang, C.H. 2003.
Release of paclitaxel from polylactide-co-glucolide (PLGA) microparticles and
discs under irradiation. Journal of Microencapsulation, 20: 317–327.
Windhab, E.J., Wagner, T., Zimmermann, M., Hurrell, R.F. 2005. Processing and appli-
cation of multi-fortified food systems. Chemie Ingenieur Technik, 77: 1180.
Yadava, D. 2008. Microencapsulation-based technologies for the double fortification
of salt with iron and iodine. MASc thesis, University of Toronto.
Yadava, D., Li, O., Diosady, L.L., Wesley, A.S. 2012. Optimisation of polymer coat-
ing process for microencapsulating ferrous fumarate for salt double fortification
with iodine and iron. Journal of Microencapsulation, 29(8): 729–738.
Yildirim, M., Hettiarachchy, N. 1998. Properties of films produced by cross-linking
whey proteins and 11S globulin using transglutaminase. Journal of Food Science,
63(2): 248–252.
Yuliani, S., Bhandari, B., Rutgers, R., D’Arcy, B. 2004. Application of microencapsu-
lated flavor to extrusion product. Food Reviews International, 20: 163–185.
Yuan, J., Li, Y., Ue, J., Wesley, A., Diosady, L.L. 2008. Development of field test kits
for determination of microencapsulated iron in double fortified salt. Food and
Nutrition Bulletin of United Nations, 29: 288–297.
Yusufali, R. 2001. Appropriate technologies for double fortification of salt. MASc the-
sis, University of Toronto.
Zheng, M.Y., Liu, G., Wang, Z.W., Baoyindugurong, J.H. 2011. Formation and charac-
terization of self-assembling fish oil microemulsions. Colloid Journal, 73: 319–326.
Zhu, L., Glahn, R.P., Yeung, C.K., Miller, D.D. 2006. Iron uptake by Caco-2 cells from
NaFeEDTA and FeSO4: Effects of ascorbic acid, pH, and a Fe (II) chelating agent.
Journal of Agricultural and Food Chemistry, 54(20): 7924–7928.
Zuidam, N., Shimoni, E. 2010. Overview of microencapsulates for use in food prod-
ucts or processes and methods to make them. In Encapsulation Technologies for
Active Food Ingredients and Food Processing, ed. N.J. Zuidam and V. Nedovic.
Springer, New York, pp. 3–29.

12
Microencapsulation and Delivery
of Omega-3 Fatty Acids
Luz Sanguansri and Mary Ann Augustin
CONTENTS
12.1 Introduction................................................................................................. 374
12.2 Drivers for Delivery of Omega-3 Fatty Acids into Foods..................... 375
12.3 Incorporation of Omega-3 Oils into Foods............................................. 376
12.4 Microencapsulation.................................................................................... 377
12.4.1 Microencapsulation in the Food Industry.................................. 378
12.4.2 Microencapsulation of Omega-3 Oils.......................................... 378
12.5 Manufacture of Microencapsulated Omega-3 Ingredients.................. 378
12.5.1 Elements of Microcapsule Production......................................... 379
12.5.1.1 Omega-3 Oil Core Selection........................................... 379
12.5.1.2 Encapsulant Selection...................................................... 379
12.5.1.3 Formulation Design......................................................... 381
12.5.1.4 Choosing the Process...................................................... 381
12.5.1.5 The Format for Delivery.................................................. 381
12.5.2 Encapsulating Materials................................................................ 382
12.5.2.1 Challenges for Selection of Materials
for Omega-3 Oil Encapsulation...................................... 383
12.5.2.2 Classes of Encapsulant Materials Used
for Omega-3 Oil Encapsulation...................................... 383
12.5.3 Mechanical Processes..................................................................... 385
12.5.3.1 Emulsion Preparation and Stabilization....................... 386
12.5.3.2 Spray-Drying.................................................................... 387
12.5.3.3 Extrusion........................................................................... 389
12.5.3.4 Gelation and Particle Formation.................................... 390
12.5.3.5 Secondary Coating Application..................................... 391
12.5.4 Chemical-Based Processes............................................................. 391
12.5.4.1 Coacervation..................................................................... 391
12.5.4.2 Inclusion Complexation.................................................. 393
12.5.4.3 Liposome Encapsulation................................................. 393
12.5.5 Emerging Technologies.................................................................. 393
12.5.5.1 Supercritical Fluid Spraying........................................... 393
12.5.5.2 Nanoencapsulation.......................................................... 393
373
12.6 Properties of Microencapsulated Omega-3 Ingredients....................... 394

12.7 Food as Delivery Systems for Omega-3 Oils.......................................... 395
12.7.1 Infant Formula................................................................................ 395
12.7.2 Bakery and Cereal Products.......................................................... 396
12.7.3 Dairy Products................................................................................ 396
12.7.4 Meat and Fish Products................................................................. 397
12.7.5 Drinks and Beverages.................................................................... 397
12.7.6 Confectionery.................................................................................. 397
12.8 Considerations for Successful Delivery of Omega-3
Oils into Commercial Food Products...................................................... 397
12.8.1 Compliance with Regulatory Standards..................................... 398
12.8.2 Definition of Final Product Application to Ensure
the Correct Delivery Format......................................................... 398
12.8.3 Understanding of the Protection and Release
Requirements during Processing................................................. 398
12.8.4 Understanding of How and Where to Add
the Microencapsulated Ingredient............................................... 398
12.8.5 Understanding Possible Interactions with Other Ingredients..... 398
12.8.6 Understanding the Shelf-Life, Sensory, and Physical
Properties of the Final Product..................................................... 399
12.8.7 Working in Partnership through Collaboration......................... 399
References.............................................................................................................. 399
12.1 Introduction
The functional food revolution and an increasing consumer awareness of the
link between health and food have resulted in a growing demand for foods
that offer benefits for healthy living (Klont, 1999; Augustin, 2001). Significant
among the growing class of functional foods are foods that are fortified with
omega-3 fatty acids.
The high degree of unsaturation of omega-3 oils makes them very sus-
ceptible to oxidation. Oxidation of omega-3 oils can occur at all stages in
the supply chain, from the handling of raw materials through to process-
ing, storage and handling of the oil during food manufacturing process,
and subsequently in the storage and handling of the final omega-3 enriched
food products. This makes the incorporation of omega-3 fatty acids into
foods a significant challenge as their susceptibility to oxidation and devel-
opment of off-flavors affects the sensory properties of the omega-3 fortified
foods (Michelsen et al., 2001; Trautwein, 2001; Ennen, 2002; Augustin and
Sanguansri, 2003).
To overcome some of the issues relating to stability of omega-3 oils, the use
of microencapsulation technology has been explored. Microencapsulation,
which involves the packaging of a core within a secondary material in

Microencapsulation and Delivery of Omega-3 Fatty Acids 375
small particles, is proved to be a successful strategy for protecting sensi-

tive ingredients and enabling their delivery into foods (Pszczola, 1998).
Microencapsulation technology has proved to be a powerful technique for
tailoring the properties and functionality of sensitive ingredients for use in
specific systems. It has the potential to offer novel solutions for stabilization
and improved delivery of omega-3 fatty acids in food.
This chapter reviews the issues relating to incorporation of omega-3 fatty
acids in food. It includes a discussion of microencapsulation technology and
its application for delivery of omega-3 fatty acids in the food industry, the
reasons for its use, the materials used as encapsulants, the techniques used,
and examples of its use in product applications. Trends and innovations in
new materials, emerging processes and technical challenges associated with
the selection of encapsulants, and appropriate techniques for the produc-
tion of microencapsulated products are discussed. Important considerations
for successful delivery of omega-3 oils into the functional foods market are
highlighted.
12.2 Drivers for Delivery of Omega-3 Fatty Acids into Foods

Omega-3 fatty acids have been associated with their many health benefits
relating to heart health, brain function, eye health, joint health, mood, behav-
ior, cancer, diabetes, skin disorders, pregnancy, and lactation (Rice, 1991,
1992; Connor et al., 1996; Buttriss, 1999; Michelsen et al., 2001; Muggli, 2001;
Olsen and Secher, 2002; O’Shea, 2003).
Interest in delivery of omega-3 fatty acids into foods has primarily been
driven by consumer awareness of the health benefits of omega-3 fatty acids
and an increased consumer demand for foods fortified with omega-3 fatty
acids that have desirable nutritional, sensory, and functional attributes.
There is also a growing recognition that modern diets are deficient in
omega-3 fatty acids. The average intake of omega-3 fatty acids by consum-
ers has been found to be grossly inadequate due to the low consumption
levels of fish, one of the major sources of omega-3 fatty acids in the diet.
Major health and nutrition organizations and scientific authorities recom-
mend that intakes of long-chain polyunsaturated fatty acids (PUFAs) should
be increased by eating two to three fatty fish meals per week or by supple-
mentation. The International Society of the Study of Fatty Acids and Lipids
(ISSFAL), American Heart Association (AHA), Institute of Medicine (IOM),
and British Nutrition Foundation (BNF) all make recommendations on the
daily intake of omega-3 fatty acids. As a general guideline, the minimum
recommended daily intake for adults is approximately 500 mg of combined
docosahexaenoic acid, C22:6n-3 (DHA) and eicosapentaenoic acid, C20:5n-3
(EPA) per day for cardiovascular health, or 900 mg of combined DHA and

EPA if one has coronary heart disease (BNF, 1999; AHA, 2000; IOM, 2002;
ISSFAL, 2004).
In 2000, the Food and Drug Administration (FDA) announced a quali-
fied health claim for dietary supplements containing EPA and DHA and
the reduced risk of coronary heart disease. The FDA recommends that con-
sumers do not exceed more than a total of 3 g/day of EPA and DHA with
no more than 2 g/day from a dietary supplement. In 2004, the FDA allowed
a qualified health claim for omega-3 fatty acids (DHA and EPA) and car-
diovascular health. To qualify for the health claim in the United States,
foods must contain a minimum of 125 mg long-chain omega-3 fatty acids
per serving, and be part of a food that is low in fat, saturated fat, trans fat,
and cholesterol (USFDA, 2004). In Australia and New Zealand, a nutritional
claim has been allowed for omega-3 fatty acids, if the food contains a mini-
mum 30 mg total EPA and DHA, or 200 mg alpha-linolenic acid, C18:3n-3
(ALA) per serving, when part of a food that is low in saturated fat and trans
fatty acids (FSANZ, 2002).
The accumulating evidence about the health benefits of omega-3 fatty acids,
the increasing awareness of consumers about the benefits of omega-3 fatty
acids and advances in processing PUFA ingredients are making omega-3–
enriched foods a reality in the market place (Newton, 1996; Garcia, 1998;
Ennen, 2002; LaBell, 2002a; Palka, 2002). The advent of the nutritional and
health claims has opened up new opportunities for foods rich in omega-3
fatty acids. More products enriched with omega-3 fatty acids are becoming
available in the market and starting to gain the acceptance of consumers.
12.3 Incorporation of Omega-3 Oils into Foods

Omega-3 oils, such as fish oil, are prone to oxidative deterioration and to the
development of objectionable off-flavors and odors even when precautions
are taken such as protection from light and oxygen and low-temperature
storage (Valenzuela et al., 1993). Oxidation also affects the nutritional qual-
ity and safety of the oil to consumers. The fishy taste and odor associated
with omega-3 oils from marine sources is another hurdle for incorporation
of omega-3 fish oils into food products.
The development of oxidative rancidity of omega-3 oils is a factor that has
limited its use in foods. The biggest challenge to the omega-3 oil manufac-
turers and suppliers is overcoming the development of undesirable taste and
odors in the end products and the difficulties in using the oil as an ingredi-
ent in food and beverage applications (Sunley, 1998).
As the oxidation of unsaturated fatty acids is an autocatalytic process,
unsaturated oils used for incorporation into foods must be of the high-
est quality practically achievable in order to minimize the development of

off-flavors. This means that the oils should be extracted from raw materials
of good quality and the bulk oils should be refined, stabilized, packaged,
and stored under appropriate conditions, which minimize exposure to fac-
tors that promote oxidation such as air, oxygen, and light.
Antioxidants may be added to protect the oil from oxidation. Synthetic
antioxidants, such as tertiary butyl hydroquinone, butylated hyroxylanisole,
and butylated hyroxytoluene, singly or as mixtures, have been used in fish
oils. The push toward natural ingredients has resulted in natural antioxi-
dants such as tocopherols, ascorbic acid, carotenoids, and rosemary extracts
becoming a more popular option (Valenzuela et al., 1993; Shahidi and Kim,
2002). Mixture of tocopherol, lecithin, and ascorbyl palmitate was found to
be effective for retarding oxidation in fish oil (Hamilton et al., 1998). Even
with the addition of antioxidants, unprotected oils can still oxidize very rap-
idly once they are exposed to a food environment.
Omega-3 oils may be added directly to foods but this approach has its limi-
tations. The oil is not protected from the food matrix during processing of
the food product, thereby exposing the oil to environments (e.g., air, oxygen,
heat) that promote the oxidation of unprotected fats. This has undesirable
consequences on the shelf stability of the omega-3–fortified food product.
This has in part been addressed by the addition of antioxidants to the bulk
oil and to the food systems.
An alternative approach is to use microencapsulated omega-3 oils
(Andersen, 1998). With the use of microencapsulation, the oil is protected
from the harsh processing environments during processing. A good micro-
encapsulation system also protects the oil from interactions with the food
matrix and environment during storage. This allows the omega-3 fortification
of food products without affecting the taste and shelf-life of the final food.
The principles of designing microcapsules and the use of microencapsula-
tion and delivery technologies for incorporation of omega-3 fatty acids into
foods are discussed in more detail below.
12.4 Microencapsulation
Microencapsulation is the science of packaging components (referred to as
the core or active) within a secondary material (referred to as the encap-
sulant or coat) and delivering them in small particles (i.e., microcapsules).
Microencapsulation is used as a means of isolating an ingredient from the
reactions of the surrounding materials or environment (Karel, 1990). It may
be used for stabilizing a sensitive ingredient, masking flavors and for con-
trolled delivery of active components. An associated benefit is the improved
ease of handling when microencapsulated ingredients are delivered in a
powder format.

12.4.1 Microencapsulation in the Food Industry

Microencapsulation technology has been used for flavor encapsulation
since 1930 (Blenford, 1986). In recent years, the functional food and ingredi-
ent industries have been identified as potentially significant growth areas
for the technology, especially for the encapsulation of unstable new func-
tional ingredients. There are greater demands being made on the integrity
of the microcapsules to provide controlled delivery of the core material
at the desired time and site (Pothakamury and Barbosa-Canovas, 1995;
Reineccius, 1995; Pszczola, 1998; Brazel, 1999; Gibbs et al., 1999; Clarke, 2002).
Microencapsulation is an enabling technology that provides novel solutions
for the fortification of food, while ensuring that the taste, aroma, or texture
of food is not compromised (Pszczola, 1998; Brazel, 1999; Gibbs et al., 1999;
Augustin et al., 2001; Clarke, 2002).
12.4.2 Microencapsulation of Omega-3 Oils

The main advantage of omega-3 oil encapsulation is the protection afforded
to the oil against oxidation during storage and processing. This is because
the microencapsulated oils have an in-built barrier (i.e., the encapsulant) that
isolates the oil from the surrounding environment. Hence, the oxidation of
polyunsaturated oils is inhibited and the shelf-life of these products during
storage and processing may be improved (Andersen, 1998; Uicich et al. 1999).
The level of protection afforded against oxidation depends on the encapsu-
lant used and the integrity of the microcapsule. The oil may be protected
from direct exposure to light and oxygen during storage and from the other
components in the food matrix during food processing and storage, thus
reducing potentially undesirable reactions with other food ingredients in
a complex food system. Microencapsulation can also be used to mask the
undesirable taste and odor that may be imparted by the oil, thus enabling
the production of omega-3 fortified foods with desirable sensory properties
(Bartz, 2002).
12.5 Manufacture of Microencapsulated Omega-3 Ingredients

The production of a microencapsulated omega-3 product involves the selec-
tion of the omega-3 oil (i.e., the core), choosing the ingredients to be used as
encapsulants, developing the formulation comprising the required amount
of omega-3 oil to encapsulant ratio and applying an appropriate process for
production.
To ensure that the microencapsulated product is used effectively for for-
tification of foods, the properties of the microcapsule need to be tailored to

suit the food application. It is important to ensure that the omega-3 oil core
is protected from the harsh processing environments and that the release
of the core is not triggered prematurely. Many different encapsulant mate-
rials combined with different processing technologies have been used for
the encapsulation of omega-3 oils resulting in microcapsules with different
properties (Iwami et al., 1987; LaBell, 1991; Imagi et al., 1992; Taguchi et al.,
1992; Valenzuela et al., 1993; Lauretzen, 1994; Andersen, 1995; Lin et al.,
1995). However, there are still limitations to the use of microencapsulated
ingredients in some applications. An integrated approach to microcapsule
production and its final product application is required for the success of a
microencapsulated ingredient (Table 12.1).
12.5.1 Elements of Microcapsule Production

12.5.1.1 Omega-3 Oil Core Selection
The first step is to choose a source of omega-3 oil. It may be chosen from a
range of polyunsaturated oils that are rich in omega-3 fatty acids. The long-
chain omega-3 fatty acids, DHA and EPA, are found mainly in fish, such as
tuna, salmon, sardines, mackerel, and in marine algae and seaweeds. The
shorter chain omega-3 fatty acid, ALA, can be found in many plant sources
such as linseed, canola, hemp, perilla, and some vegetables such as spinach,
green peas, and beans (Ley-Jacobs, 2000; Michelsen et al., 2001; Trautwein,
2001). The major commercial source of DHA and EPA is presently from fish.
However, there is increasing interest in algal oils (Mukherjee, 1999; Belbarbi
et al., 2000) and transgenic oil seeds (Abbadi et al., 2001) and krill oil (Nichols
et al., 2010) as alternative sources of long-chain omega-3 fatty acids.
It is essential to use high-quality oil with a low degree of oxidation. The
specific refining and deodorizing process applied in the processing of com-
mercial omega-3 oil, the source of the raw material (e.g., fish oil, algal oil,
plant oils, etc.), the fatty acid composition, and the addition of antioxidant
mixtures to the oil will influence the stability and the sensory properties of
the microencapsulated oil product. The oxidative stability of microencap-
sulated omega-3 oils is dependent also on the type of the antioxidant used.
Tocopherol proved to be more effective than ascorbyl palmitate for the pro-
tection of microencapsulated fish oil powder against oxidation (Baik et al.,
2004).
12.5.1.2 Encapsulant Selection

The encapsulant can be selected from a wide variety of natural or syn-
thetic polymers depending on the characteristics desired in the final
microcapsule. The composition as well as the physical and chemical prop-
erties of the encapsulant material can influence the functional properties

TABLE 12.1
Elements of Microencapsulation: An Integrated Approach to the Development
of Microencapsulated Ingredients
Production of
Technological Activities Microencapsules Market-Related Activities
• Identify core CORE • Work with a supplier of the

• Characterize core core and user of the
• Physical and chemical + ingredient
properties • Obtain specifications
• Stability • Consider cost
• Choose material ENCAPSULANT • Establish reliable source of
• Use without modification MATERIAL material
• Modify to alter functionality • Obtain specifications
• Characterize material ↓ • Consider cost
• Physical and chemical
properties
• Stability
• Design formulation FORMULATION OF • Consider what is available in
• Consider MICROCAPSULE the market place
• Interactions between SYSTEM • Range of formulations
components available
• Stability of formulation ↓ • Limitations of existing
• Triggers for release of core formulations
• Market need for
microcapsules with
differentiated functionality
• Choose appropriate process PROCESSING OF • Consider
• Conventional MICROCAPSULE • Need for capital
• New/emerging investment
• Define unit process variables ↓ • Processing cost
and apply process • Process efficiency and
• Assess process reliability
• Consistency • Alternative processes
• Efficiency
• Determine composition MICRO • Develop product
• Gross composition ENCAPSULATED specifications and
• Proportion of encapsulated PRODUCT applications
and free core • Align with end users
• Characterize microcapsule • Assess suitability
• Physical and chemical • Provide technical service
properties
• Stability
• Triggers for release
of the final microcapsule and the choice of processing technologies to be

used for microencapsulation. For encapsulation of omega-3 fatty acids, it
is desirable to choose materials from a natural source, which have good
oxygen barrier properties in order to afford the protection of the core from
oxidation.

12.5.1.3 Formulation Design

The design of formulations for encapsulation of omega-3 fatty acids requires
an understanding of the properties of the oil and the required function of the
encapsulant materials and any other ingredients or additives chosen for the
formulation. The primary aim is to design a formulation that results in high
encapsulation efficiency and a microcapsule that gives high oxidative stabil-
ity to the omega-3 oil core. A high oil load in the microcapsule is also desir-
able provided this does not compromise the quality of the microcapsule.
The development of a successful formulation for a target application
requires knowledge about the stability of the chosen oil; the encapsulating
properties of the ingredients; the likely interactions between the omega-3
oil core, the encapsulant, and other components in the formulation; and the
reactivity of the microcapsule formulation in the final product application. It
has been shown that factors such as the type of encapsulant material, addi-
tion of antioxidant, chelating agent, emulsifier, stabilizers, and salts affect the
stability of microencapsulated oil products (Danviriyakul et al., 2002).
12.5.1.4 Choosing the Process

The selection of unit processes and process conditions for omega-3 oil encap-
sulation requires some consideration. A number of standard unit processes
suitable for production of microencapsulated omega-3 oils are widely avail-
able in the food industry. Standard unit processes (e.g., mixing, homog-
enization, and drying) have traditionally been used for the production of
microencapsulated ingredients such as omega-3 oil powders as it usually
involves the preparation of an emulsion and drying.
There are possibilities for the application of emerging food-processing
operations (e.g., microfluidization, ultrasound, high pressure processing) in
the manufacture of microencapsulated ingredients. The use of these tech-
nologies at various stages of the microencapsulation process, in place of
standard unit processes, may be used to produce microcapsules with unique
properties.
The choice of the processes used for microencapsulation will depend on
the properties required and the cost for the production of microcapsules.
Even with using standard unit processes, the cost of the production of
microcapsules can vary significantly depending on which microencapsula-
tion technology is being used. If nonstandard unit processes are used, there
may be a need for capital investment. It is important to consider the essential
requirements of the microencapsulated ingredient in the final product, and
whether the product can carry the additional cost.
12.5.1.5 The Format for Delivery

For addition into a dry or powdered food product formulation, it is necessary
to convert the liquid microcapsules containing omega-3 oils into powder to

make it commercially feasible for dry blending. This means that a drying
step is needed as an integral part of the microencapsulation process. It is
important to match the physical properties (e.g., particle size and bulk den-
sity) of the microcapsule to that of the powder with which it is to be dry
blended in order to minimize or avoid physical separation or segregation of
the ingredients during transport and storage. For liquid food applications,
a liquid format (e.g., emulsion) is sometimes preferred but a powder format
that can easily be rehydrated into a stable emulsion or dispersion is also suit-
able (Christensen et al., 2001).
12.5.2 Encapsulating Materials

Proteins, carbohydrates, lipids, gums, polysaccharides, and cellulose materi-
als are commonly used for the microencapsulation of omega-3 fatty acids
and oils (Table 12.2). They can be used alone or in combination to achieve cer-
tain functions either during production or in the final product. Some of the
new developments in materials for the microencapsulation of omega-3 fatty
acids involve the application of clever chemistry and processing to modify
the properties of the native food ingredient to achieve new functional prop-
erties not present in their standard form.
A good encapsulant for omega-3 fatty acids protects the oil in the micro-
capsule from degradation during processing and storage and also masks the
undesirable taste and odor associated with the microencapsulated omega-3
oil ingredient when added into a food product. The encapsulant must be
approved for food use if the formulation is to be incorporated into food
products. The encapsulant materials may be used in combination with other
components in the formulation such as oxygen scavengers, antioxidants,
TABLE 12.2
Materials Used as Encapsulants of Omega-3 Oil Encapsulation
Material Class Types of Materials
Proteins Albumin, caseinate, gelatin, gluten, peptides, soy protein, whey

proteins, zein, other vegetable proteins
Sugars and sugar products Fructose, galactose, glucose, maltose, sucrose, oligosaccharide,
corn syrup solids, dried glucose syrup
Starch and starch products Maltodextrins, dextrins, starches, modified starches
Gums Agar, alginates, carrageenan, gum acacia, gum arabic, pectins
Cellulose material Acetylcellulose, carboxymethyl cellulose, cellulose acetate
butylate phthalate, cellulose acetate phthalate, ethyl cellulose,
methyl cellulose, nitrocellulose
Other carbohydrates Chitosan, cyclodextrins
Lipids Acetoglycerides, beeswax, diglycerides, natural fats and oils,
fractionated fats, hardened fats, lecithin, liposomes,
monoglycerides, paraffin, tristearic acid, waxes

chelating agents, and surfactants. Cost is also a consideration for the selec-
tion of the encapsulant.
12.5.2.1 Challenges for Selection of Materials for Omega-3

Oil Encapsulation
It is important to consider the encapsulant that will be used in the formula-
tion and process for the production of the microcapsule. The ability of the
encapsulant to stabilize and protect the oil core during processing and stor-
age of the microencapsulated omega-3 oil ingredients and the mechanisms
required for release of omega-3 fatty acids at the appropriate time and site
need to be taken into account.
Other criteria that need to be considered during encapsulant selection
include the mechanical strength requirement of the microcapsule, the com-
patibility of the encapsulant material with the target food product, its nutri-
tional value, sensory properties, and aesthetic properties (Brazel, 1999; Gibbs
et al., 1999).
The availability of a very limited range of suitable encapsulant materials
allowed for food use still remains the biggest challenge in encapsulant mate-
rial selection, especially when more sophisticated microcapsule properties
are required by food manufacturers and consumers. The requirement for
good powder flow properties, dispersibility, and solubility are now becom-
ing standard prerequisites for omega-3 oil powder microcapsules, with the
requirements for oxygen, moisture, heat, shear, and pH barrier properties
becoming important characteristics of the encapsulant surrounding the core.
Food-grade materials commercially available meet some of these require-
ments. Modification of the existing food-grade materials to achieve differenti-
ated encapsulant functionality may be required to achieve new properties in
microcapsules and improved performance of microencapsulated ingredients.
12.5.2.2 Classes of Encapsulant Materials Used for Omega-3

Oil Encapsulation
12.5.2.2.1 Proteins
Proteins possess many excellent functional properties. They have been
used as encapsulants for omega-3 fatty acids and oils because of their good
film-forming and emulsifying properties (Kim and Moore, 1995; Lee and
Rosenberg, 2000b; Yin and Xu, 2000; Hogan et al., 2001; Rosenberg and Lee,
2004). The most commonly used proteins are gelatin, whey protein, casein,
soy protein, and albumin.
The good water solubility of proteins is desirable during the preparation
of the encapsulant in order to achieve the required encapsulant functionality
when using an all-aqueous-based process. However, the good water solubil-
ity characteristics of the encapsulant generally result in rapid dissolution of
the encapsulant when the microcapsule is in contact with water in the final

application. This may be accompanied by the release of the core to the envi-
ronment, which presents a disadvantage in some applications.
In order to improve the resistance of protein encapsulants to moist envi-
ronments, the solubility of protein wall materials can be modified using a
number of techniques. This may be achieved by coagulating the protein by
heat or by the action of pH (Serpelloni and Boursier, 1995). Lee and Rosenberg
(2000a,b) used a double emulsification and gelation process to prepare whey
protein microcapsules (containing a milkfat core), which had limited water
solubility for controlled core release in food applications. Others have
improved stability of protein-based fish oil capsules by using transglutamin-
ase to cross-link soy proteins in double emulsions (Cho et al., 2003).
The good oxygen barrier properties of some proteins (Iwami et al., 1987,
1988; Fang et al., 2003) make them desirable as a choice of encapsulant for
protecting oxygen-sensitive cores like omega-3 fatty acids and oils against
oxidation.
Proteins have been used in combination with carbohydrates in the formu-
lation of microencapsulated oil ingredients (Skelbaek and Andersen, 1994;
Schrooyen et al., 2001). Lin et al. (1995) reported that wall materials contain-
ing gelatin, caseinate, and maltodextrin provided optimal protection against
oxidation of microencapsulated squid oil. Further, the oil stability was
improved with the addition of emulsifier (lecithin) and a stabilizer (Avicel).
12.5.2.2.2 Carbohydrates
Maltodextrins, corn syrup solids, and sugars have been used in the formula-
tions for omega-3 oil encapsulation, but not on their own because they have
poor emulsifying and film-forming properties. Starches have also been used
for the encapsulation of oil and oil-soluble cores. They have been made more
suitable for encapsulating oils by chemical modification to increase their
lipophilicity and improve their emulsifying properties. Starches, such as
high amylose cornstarch, may also be used for site-specific delivery as dem-
onstrated by the application of high amylose starch as a food-grade enteric
encapsulant (Dimantov et al., 2004).
Gums have traditionally been used in the flavor industry but also have
applications as encapsulating materials for omega-3 oils. The gum most com-
monly used is gum acacia, although it is the protein fraction in acacia gum
preparations that is responsible for its emulsification properties. Modified
celluloses such as methylcellulose and hydroxypropyl methylcellulose in
combination with maltodextrin have been demonstrated to be suitable
encapsulants for the stabilization of fish oil in high oil load powders with
40%–50% oil (Kolanowski et al., 2004).
Cyclodextrins, which are cyclic molecules derived enzymatically from
starch, have some unique encapsulation properties. They have the ability
to form inclusion complexes with flavors and oil-soluble nutrients and can
provide stabilization, during manufacturing and storage of some core mate-
rials that cannot be stabilized by other methods. The use of cyclodextrin is

currently limited because of its high cost. Nevertheless, β-cyclodextrin offers

advantages as a microencapsulant because it has good controlled release
properties, with binding occurring up to 200°C. The release of the complexed
material is influenced by moisture. β-Cyclodextrin has been approved by the
FDA and granted GRAS status.
12.5.2.2.3 Lipid Materials

Fats have been used mainly as a secondary coating to primary microcap-
sules to improve their water barrier properties (Wu et al., 2000).
Liposomes are artificially made microscopic membrane vesicles consist-
ing of one or more concentric layers of lipids (Kirby, 1991; Gibbs et al., 1999).
These versatile materials can be used for the delivery of water- or oil-soluble
core materials and are therefore suitable for microencapsulating a range of
food ingredients, including vitamins, omega-3 oils, minerals, and flavors.
Stable microcapsules, ranging from a few nanometers to microns in diam-
eter, may be made which make them suitable for a range of applications. The
microcapsules produced by liposome entrapment are more versatile and less
fragile than fat-encapsulated ones. One area of application where liposomes
could have the advantage over other methods is the prevention of oxida-
tion of unsaturated fats in food emulsions such as spreads, margarines, or
mayonnaise. However, this needs further development to optimize loading,
protection, and delivery.
12.5.2.2.4 Protein–Carbohydrate Conjugates

An alternative to the use of physical blends of protein and carbohydrates is
to form protein–carbohydrate conjugates. Kato and Kobayashi (1991) demon-
strated that protein–dextran conjugates, developed by coupling proteins to
cyanobromide-activated dextran or by linking proteins with dextran through
the naturally occurring Maillard reaction, have excellent emulsifying proper-
ties. These conjugates were superior to the native protein, commercial emul-
sifiers from sucrose–fatty acid esters and polyglycerine esters, especially
at high salt concentration and at acidic pH. The emulsifying properties of
protein–dextran conjugates were greatly enhanced by preheating at 100°C.
Protein–carbohydrate conjugates, formed as a result of the Maillard reac-
tion, have been shown to be superior to physical blends of protein and car-
bohydrate for encapsulation of omega-3 oils. The conjugates have excellent
film-forming, emulsifying, and oxygen-scavenging properties. Omega-3 oils
encapsulated within Maillard reaction products of film-forming protein and
carbohydrate have longer shelf-life compared to those encapsulated with
proteins and carbohydrate blends (Sanguansri and Augustin, 2001).
12.5.3 Mechanical Processes

The selection of appropriate processing technologies for microencapsula-
tion of omega-3 fatty acids depends on the desired properties of the final

Omega-3 oil + encapsulant material
Emulsification
Stable oil emulsion

(liquid emulsion microcapsule)
Spray drying
Omega-3 oil powder

(primary powder microcapsule)
Secondary coating in fluidized bed

(optional process)
Omega-3 oil powder

with secondary coating
FIGURE 12.1
Generalized process for production of spray-dried microencapsulated omega-3 oil powder
with optional secondary coating.
microcapsules such as oil loading, shelf-life, release properties, microcapsule

format, and the final product application (Matsuno and Adachi, 1993).
Microencapsulated omega-3 oil products are generally produced in pow-
der form for better stability and ease of handling. Liquid emulsions are also
available for more specialized liquid food applications. A generalized pro-
cess used for the production of microencapsulated omega-3 oil ingredients
is shown in Figure 12.1. Stable omega-3 oil-in-water emulsions may be heat
treated in liquid state or converted by spray-drying into free flowing micro-
encapsulated powders in order to improve their shelf stability.
12.5.3.1 Emulsion Preparation and Stabilization

Preparation of an emulsion involves the dispersion of the oil phase into very
fine droplets in which the action of an emulsifying agent at the surface of

the oil droplets provides protection or functionality to form stable oil-in-

water emulsions. High shear processes such as homogenization or micro-
fluidization may be used for dispersing the oil into droplets and forming
the emulsion. Emulsifiers, antioxidants, and other additives may be incorpo-
rated into both the oil phase and the aqueous phase prior to homogenization
(Watanabe et al., 2002). The emulsion formulation and other variables such as
temperature, total solids, pH, and storage conditions influence the emulsion
properties and stability (Djordjevic et al., 2004a,b; Rodriguez-Huezo et al.,
2004). Stable emulsion microcapsules can be dried into a powder for greater
shelf stability.
The selection of appropriate materials that can form a strong film around
the oil droplets, which will remain intact during processing and storage, is
essential for providing protection and stability to the omega-3 oil droplets.
Both the physical stability of the emulsion during processing and the storage
and oxidative stability need to be considered in the development of omega-3
oil emulsions.
A range of proteins, emulsifiers, and stabilizers have been used for the
stabilization of omega-3 oil-in-water emulsions. The oxidative stability of
omega-3 oil-in-water emulsions is dependent on the type of protein and
emulsifiers used in the formulation (Miyashita et al., 1995; Donnelly et al.,
1998; Pin Der Duh and Gow, 1999; Nuchi et al., 2001; Formuso et al., 2002; Hu
et al., 2003). This is because the oxidation of an oil-in-water emulsion droplet
is influenced by the properties of the interfacial membrane surrounding the
lipid core as well as the properties of the bulk phase. Important determi-
nants of oxidative stability of emulsions include the thickness of the inter-
facial membrane of an emulsion droplet (Silvestre et al., 2000), the size of
the surfactant hydrophobic tail group size (Chaiyasit et al., 2000), the type
of antioxidants, the partitioning of the added antioxidants in the emulsions
(Frankel et al., 1996; Hu et al., 2004), and the level of hydroperoxides, which
may be present in both lipids and surfactants (Nuchi et al., 2001).
Advances in emulsion technology provided the possibility of designing
multiple emulsions for the controlled delivery of bioactives. An example is a
method of producing an oil-in-water-in-oil (o/w/o) emulsion where the oil or
oil-soluble core (flavors) is emulsified with a protein to form the o/w emul-
sion and then this emulsion is further emulsified with a liquefied fat that
is solid at room temperature. The resulting o/w/o emulsion is then atom-
ized into a food product as a delivery vehicle for the core (Merchant and
Nicholson, 2000). Benichou et al. (2004) have also developed hybrids of natu-
ral polymers for entrapment and slow release of active matters in double
emulsion systems.
12.5.3.2 Spray-Drying
Spray-drying has been generally referred to as an encapsulation technology
because it is one of the oldest encapsulation methods and has been in use

since the 1930s (Blenford, 1986). It is still the most commonly used microen-
capsulation method in the food industry. The process of spray-drying is effi-
cient and cost-effective, uses equipment that is readily available, and produces
particles of reasonably good quality. Food ingredients microencapsulated by
this method include fats, oils, flavors, and oil-soluble materials. Encapsulant
materials are usually proteins, carbohydrates, or combinations of both.
The general process involves the dispersion of the core material into a poly-
mer solution, forming an emulsion or dispersion, followed by homogeniza-
tion of the liquid and atomization of the mixture into the drying c hamber
(Re, 1998). This leads to the evaporation of the solvent (water) and the for-
mation of matrix-type microcapsules. The advantage of the spray-drying
process is that it can be operated on a continuous basis. The microcapsules
produced are soluble in water. Proper adjustment and control of the process-
ing conditions enables the production of a desired bulk density and particle
size distribution of the powder.
Advances in spray-dried microencapsulated ingredients have been made
possible by new dryer and atomizer designs. New dryer designs allow dry-
ing at lower outlet temperatures (multistage dryers), longer residence time
(tall-form dryer), and therefore provide milder drying conditions for mini-
mizing deterioration of heat-sensitive microcapsules. New atomizer designs
can improve the properties of microcapsules produced in spray-dryers. New
atomizer designs have been developed by Particle Coating Technology, USA,
and Niro A/S, Denmark. Both these atomizer designs can be fitted into a
spray-dryer set-up for the production of particles with a narrower size dis-
tribution than conventional atomizers.
Many spray-dried formulations have involved the dispersion of liquid cores
in a carrier material prior to spray-drying (Witteveen et al., 2001; Minemoto
et al., 2002; Partanen et al., 2002; Subramaniam et al., 2004). For the produc-
tion of microencapsulated omega-3 oils, it is essential to design the formula-
tion and apply the appropriate process to obtain stable liquid microcapsules
before conversion of these into powder by drying. The formulation and pro-
cessing steps that take place prior to spray-drying are important steps in the
production of stable omega-3 oil powder microcapsules. Prior to the spray-
drying step, the omega-3 oils are essentially encapsulated oil droplets in an
aqueous system (e.g., emulsion, dispersion, and coacervate systems) and the
formation of a stable liquid microcapsule is essential for the manufacture of
acceptable microencapsulated oil powders.
Omega-3 fish oils have been encapsulated using a variety of encapsulant
formulations which are subsequently spray-dried into powder microcap-
sules. These formulations include proteins (sodium caseinate and whey
protein isolate) (Fair-Flow Europe, 2001), proteins in combination with
carbohydrates as wall materials (Barrier and Rousseau, 1998; Keogh et al.,
2001; Kagami et al., 2003), and protein–carbohydrate conjugates (Sanguansri
and Augustin, 2001), which improve the stability of the omega-3 oil core
against oxidation.

Spray-drying can easily be substituted by any other drying method for

production of powdered omega-3 oils such as freeze-drying (Heinzelmann
et al., 2000a,b; Grattard et al., 2002), fluid bed-drying (Skelbaek and Andersen,
1994), refractive windows drying, and filtermat drying depending on final
powder properties desired and the cost that the microencapsulated product
can bear in the market.
Although drying is only one of the steps involved in the production of
microcapsules, the control of the processing conditions during drying
has a significant influence on the physical properties of the final powder
microcapsule.
Processing conditions such as temperature, shear, pH of the emulsion,
total solids, drying rate, and order of addition of each of the components
used during the manufacture of omega-3 oil powders influence the micro-
capsule properties and stability. Depending on the formulation, the drying
method (e.g., spray-drying, freeze-drying) used to convert emulsions and
dispersions into powder can also influence the physical structure and stabil-
ity of microcapsules during storage.
12.5.3.3 Extrusion
Extrusion is used to produce high-density encapsulated products via mix-
ing of molten carriers with active ingredients followed by solidification. It
involves projecting a dispersion or an emulsion of the core and the encap-
sulant through a die or nozzle at high pressures. It has been mainly used
in flavor encapsulation, but the process has also been used for omega-3 oil
encapsulation. The cost of extrusion is believed to be twice that of spray-dry-
ing (Tuley, 1996); however, it ranks second behind spray-drying as the most
frequently used process for encapsulating flavors (Reineccius, 1991). The use
of food-grade hydrating liquid or solvent to solidify the extruded product
into pellets or powder is an important factor in considering this method for
food ingredient production.
The formulation and the processing conditions during extrusion affect
the properties of microcapsule. Particle size was reduced with increased
screw speed and melt temperature during processing and increasing the
hydrophilic–lipophilic balance (HLB value) of the emulsifier in the formula-
tion when sunflower oil was encapsulated in starch matrices via extrusion
(Yilmaz et al., 2001).
There are a number of modifications to the extrusion process aimed at
improving the process and the microcapsule release properties. These
include the following:
• High-pressure method: This method, developed by Ottens Flavors

and Delta Food Group, USA, was designed to microencapsulate key
volatile compounds, flavors, and fragrances by using high pressure
during the coating process. This process is said to keep flavors from

evaporating during processing. The core is protected in matrices of

carbohydrates and fats. Release temperatures are selected such that
flavors added to the food before processing are retained unaltered
after cooking. The new capsule forms are said to provide controlled
release, which enables multistage release or break-on-demand tar-
geted delivery.
• Modified extrusion process: This method, developed by Fuisz
Technologies Ltd, utilizes low-level heating to entrap a particulate
dispersion within a thin matrix. The final products are said to have
enhanced flavors, or sustained-release flavor delivery, such as is
required in chewing gums.
Extrusion has been used to encapsulate both particulate and liquid cores
in a protein carrier that has been heated to create a melt and then denatured
by pH adjustment and treated with an enzyme to form the microcapsule
(Janda et al., 1995). Heat sensitive and readily oxidizable cores were encap-
sulated by extrusion using a range of proteins and carbohydrate materials
to achieve discrete solid microcapsules with controlled release properties
(Van Lengerich, 2003). Cores, including lipids, have been encapsulated in a
carbohydrate or protein matrix that is gelatinized or polymerized during
extrusion process. The microcapsule is claimed to release in the digestive
tract (Katzen, 1976). A patent describes the use of extrusion process for the
protection of omega-3 oils (Saleeb and Arola, 1999).
12.5.3.4 Gelation and Particle Formation

A process often used for encapsulation of oil and oil-soluble cores involves
the use of biopolymer gels as entrapment matrices. Examples include oil and
oil-soluble cores encapsulated in alginate beads by extrusion to form the par-
ticles, followed by drying (Truter et al., 2000), and oils that are sensitive to
oxygen (e.g., wheat germ oil, evening primrose oil) encapsulated in alginate
by emulsification (Chan et al., 2000).
A number of methods have been developed for more efficient particle for-
mation. These include the following:
• Centrifugal extrusion: This is one of the more established methods of

microcapsule particle formation. It is generally a liquid coextrusion
process utilizing nozzles consisting of concentric orifices located on
the outer circumference of a rotating cylinder (Schlameus, 1995).
• Microject method: This has been developed by Ganan-Calvo (2003) for
the production of a core and shell-type microcapsule particle. Ripoll
et al. (2004) have also developed an electrified coaxial jet method for
production of micro- and nanometric size microcapsule particles.

All these methods which result in the formation of wet-gelled particles

require a method for segregation and collection of the microcapsules prior to
drying to produce dry free-flowing microcapsules.
12.5.3.5 Secondary Coating Application

Secondary coating application has been widely used in the flavor industry
to produce agglomerated powders or granules subsequently coated with
another material (Schleifenbaum et al., 2004), to improve the shelf-life and
sensory properties of oil-based flavors (Pacifico et al., 2001; So, 2001). This
method has also been used to apply a thin polymeric layer of secondary coat-
ing to spray-dried fish oil microcapsules to increase the degree of protection
and controlled release of the primary fish oil microcapsules (Souza-Neves-
Cardoso et al., 2001).
Fluidized bed coating is the most common method used for coating appli-
cations. It is accomplished by suspending or fluidizing particles of a core
material in an upward stream of air and applying an atomized coating mate-
rial to the fluidized particles. The coating to be applied may be a molten fat
(e.g., hydrogenated fats, stearines, fatty acids, emulsifiers, waxes) or materi-
als from a range of film-forming materials (such as proteins, starches, gums,
or maltodextrin) dissolved in a solvent (e.g., water). When molten fat is used,
cool air is used to harden the coating. When a soluble coating is used, hot air
is used to evaporate the solvent and harden the coating. Fat-coated core ingre-
dients are released by an increase in temperature or by physical fracture,
whereas core ingredients coated with water-soluble materials are released
when water is added to redissolve the coating. This process is limited to
the microencapsulation of powder particles with diameters of >100 µm, as
particles of less than 100 µm tend to either clump to form agglomerates or
get carried away in the exhaust air (Balassa and Fanger, 1971; Jackson and
Lee, 1991). This method is not suitable for primary encapsulation of an oil
or fat-based product such as omega-3 fatty acids; however, it can be used to
apply a secondary coating to the primary powder microcapsule (Figure 12.1).
Fluidized bed coatings can be used to enhance, time, or tune the release
properties of the primary omega-3 oil microcapsules.
Another method used for coating application is the spinning disk system.
It is a fast coating method for controlled release applications, developed by
Particle Coating Technologies (LaBell, 2002b). The coating materials used in
fluid bed coating processes may be applied to this system.
12.5.4 Chemical-Based Processes

12.5.4.1 Coacervation
Coacervation has been defined as the separation of two liquid phases in col-
loidal systems. The more concentrated phase is termed the coacervate phase

and the other phase is called the equilibrium solution. Complex coacerva-
tion occurs when two oppositely charged colloids interact to form a complex
(IUPAC, 1972).
The underlying cause of coacervation is the phase separation of one or
more hydrocolloids from the initial biopolymer solution. Coacervation may
be induced by a change in temperature or by the addition of a second sub-
stance such as a concentrated aqueous ionic salt solution or a nonsolvent.
The coacervate coats the emulsified oil droplets by phasing polymers out of
solution. The coat can then be hardened by using a chemical or enzymatic
cross-linker. It is the hardening of the shell that remains the greatest chal-
lenge, especially for food applications (Courts, 1973; Versic, 1988).
Complex coacervates between proteins and polysaccharides that inter-
act to form an electrostatically bound complex are most commonly used in
the food industry. Protein–polysaccharide complexes have been shown to
exhibit better functional properties than that of the proteins and polysaccha-
ride combination, in a number of applications (Schmitt et al., 1998). The com-
plex generally has superior interfacial properties, an effect that is attributable
to the simultaneous presence of the two biopolymers, and hence it functions
well in an encapsulant capacity.
In the food industry, coacervation has typically been used for encapsula-
tion of flavor oils but it has the potential for application to a range of other
microencapsulated food ingredients (Gouin, 2014). Microcapsules have
been produced using a plant protein (e.g., soy protein) and a polyelectrolyte
with an opposite charge to the protein subjected to complex coacervation
and followed by hardening with crosslinking agent (e.g., dialdehydes and
tannins). Microcapsules produced are suitable for a range of applications
including food (Richard and Morteau, 2004). A complex coacervate with
gelatin-carboxymethyl cellulose crosslinked with glutaraldehyde has been
used for flavor delivery (Bakker et al., 1999). Chitosan-coacervated micro-
capsules have been shown to have good diffusion release characteristics
(Pandya and Knorr, 1991).
This technique may be adapted for the encapsulation of omega-3 fatty
acids (Lamprecht et al., 2001). Soeda et al. (2003) describe a simple coac-
ervation technique for the encapsulation of flavors and oil-soluble cores
including omega-3 fatty acids, using transglutaminase as the crosslinking
agent for hardening the capsule wall formed by salting out. The resulting
product has been shown to be suitable for a wide range of food applica-
tions. Oil-soluble cores (e.g., vitamins and PUFAs) have been encapsulated
by crosslinking a protein, sugar, and a water-soluble salt, by heating the
mixture under specific conditions, to produce a microcapsule that is sub-
stantially insoluble in boiling water for at least 3 min (Chaundy et al., 1992).
A double emulsification and an enzymatic gelation method using transglu-
taminase-crosslinked protein encapsulant has been suggested by Cho et al.
(2003) to improve the storage stability of fish oil and achieve controlled
release.

12.5.4.2 Inclusion Complexation

Inclusion complexation is the term used when cyclodextrins are used for
microencapsulating core materials. Unlike other techniques inclusion, com-
plexation takes place at a molecular level and utilizes cyclodextrin molecules
as the encapsulating medium.
A β-cyclodextrin complex has been shown to enhance the oxidation stability
of onion and garlic oils as well as mask undesirable odor and flavor (Dziezak,
1988). Inclusion complexes using cyclodextrins have been used for the encap-
sulation of omega-3 fatty acids to enhance their stability to temperature, light,
oxidation, polymerization, or double bond migration and mask undesirable
taste and odor (Reichenbach, 1996; Djedaini-Pilard et al., 2000; Kim et al.,
2000). Yoshii et al. (1997) demonstrated that a complex between DHA oil and
cyclodextrin prepared by a twin-screw kneader is able to produce a microen-
capsulated omega-3 powder with very high resistance to oxidation in fish meal
paste application even without an antioxidant in the formulation.
12.5.4.3 Liposome Encapsulation

Liposome encapsulation is a term used to microencapsulate or trap core mate-
rials using liposomes. Liposomes have been used to encapsulate omega-3
fatty acids, by dissolving the oil in phospholipid before water is added and
sonicated to form the encapsulated material. The entrapped oil is protected
from oxidation and the preparation can be added directly to food products
as a dispersion or it can be dried into a free flowing powder (Haynes et al.,
1991). Food-approved pro-liposomes are available, which can be used for the
microencapsulation of food ingredients, and are much simpler to use.
12.5.5 Emerging Technologies

12.5.5.1 Supercritical Fluid Spraying
This is a relatively new technique, primarily developed for the pharmaceuti-
cal industry, in which the core material and the microencapsulating material
(typically a polymer) are dispersed and/or dissolved in a supercritical fluid,
such as carbon dioxide. The supercritical fluid is ejected from a nozzle in the
form of a spray. The carbon dioxide flashes-off very rapidly, leaving residual
particulate material. By careful control of the operational parameters and
choice of materials, successful microencapsulation has been achieved and
the production of very uniform powder particles is possible (Shine and Gelb,
1998; Breitenbach et al., 2000).
12.5.5.2 Nanoencapsulation
Nanoencapsulation uses vesicular systems in which the active ingredi-
ent is confined to a cavity surrounded by a unique polymeric membrane.

The preparation of nanocapsules containing a membrane-forming mol-

ecule, a coemulsifier, and a lipophilic component has been used to encap-
sulate a range of food ingredients, including omega-3 fatty acids. The
microcapsules are 40–80 nm in size and are suitable for the delivery of
components into clear liquid drinks, as well as other beverages and foods
(Weder et al., 2000). Hydrophobic nanospheres encapsulated in pH- or
moisture-sensitive microspheres have been shown to improve the shelf-
life of foods and beverages and prolong the sensation of flavor (Shefer
and Shefer, 2003).
12.6 Properties of Microencapsulated Omega-3 Ingredients

Omega-3 powder microcapsules are small particles, typically containing
25%–60% oil surrounded by a matrix or encapsulant. The physical properties
of the microencapsulated omega-3 oil ingredients and their stability need to
be defined as these are important considerations for suppliers and users of
these ingredients. The final powder properties need to be tailored to meet
the requirements in the final food product.
Most microencapsulated omega-3 ingredients are supplied as powders
that are reconstituted and hence the ability of the powder to form a stable
dispersion or emulsion upon reconstitution is a desirable attribute. Powder
particle size, encapsulation efficiency, solubility, and dispersibility can vary
depending on the properties of the emulsion or dispersion before drying,
process conditions used during drying, and the method of drying. Particle
size is an important consideration in the design of the microcapsule for tar-
geted delivery and application (LaBell, 2002b).
Storage conditions such as temperature, humidity, oxygen availability,
light, and packaging can greatly influence the stability of the microcapsules,
depending on the core and encapsulant properties as well as the structure
and morphology of the powder microcapsule. Most of the microencapsu-
lated omega-3 products developed have had significant limitations in terms
of storage stability and shelf-life. Most encapsulated products have now been
successfully commercialized without requiring low temperature storage
(e.g., 4–10°C).
Understanding lipid oxidation of microencapsulated oil ingredients is
important because oxidation results in the loss of nutritional value and
development of undesirable flavors and odors during storage of the ingre-
dient and when it is used in a wide range of commercial food applications.
The stability of microencapsulated omega-3 oil powders to oxidation can
be improved by appropriate selection of encapsulant materials, processing,
packaging, and storage conditions.

12.7 Food as Delivery Systems for Omega-3 Oils

Omega-3 fatty acids and oils are key ingredients for the development
of functional food products. Higgins et al. (1999) have highlighted the
potential of fortification with omega-3 fatty acids in food as a vehicle for
increasing omega-3 intake by consumers. With the use of microencapsula-
tion technology, new and innovative ways for the protection and delivery
of omega-3 fatty acids into a range of food products are made possible
(O’Shea, 2003). However, there are significant challenges involved dur-
ing production and storage of the final food product containing omega-3
fatty acids, because of the susceptibility of these components to oxida-
tion and development of undesirable flavors and odors. Developments in
microencapsulation technologies and delivery strategies have resulted
in an increasing number of successful omega-3–fortified products in the
marketplace.
Omega-3 oil–enriched products launched worldwide include pet foods,
dietary supplements, dairy products, processed fish, meat and egg prod-
ucts, snacks, meals, infant formula and baby foods, bakery products, and
beverages. At the forefront of Omega-3 oil–enriched foods are infant for-
mula and baby follow-on foods, bread, dairy products, nutrition bars, and
margarines. Additionally niche products fortified with omega-3 oils such as
soups, ice tea drinks, cakes, biscuits, and seafood products where there has
been restoration of omega-3 fatty acids are commercially available (Newton,
1996).
12.7.1 Infant Formula

Infant formula has been a major segment of focus for delivery of omega-3
fatty acids to food because of its benefits to early childhood development
(Andersen, 1998). Fortification of powder infant formula with omega-3 fatty
acids is quite challenging, due to the long shelf-life of the product (usually
3 years from manufacture) and the limited number of ingredients allowed
in infant formula. The major challenge is to be able to add the health ben-
efits of omega-3 oil to infant formula without affecting its odor, flavor, and
shelf-life during storage. The first successful commercial entry into this
market was seen in Australia and New Zealand in 1998, using microencap-
sulated omega-3 oils that had been specially formulated using ingredients
already in use for infant formula manufacture. Omega-3–encapsulated
powder was incorporated without affecting the sensory properties, shelf-
life, and the ingredient listing of the original product (Sanguansri and
Boghossian, 1991). There are now a number of commercial infant formula
products fortified with omega-3 fatty acids using a whole range of incorpo-
ration technologies.

12.7.2 Bakery and Cereal Products

Bread is a very popular food delivery vehicle for a number of functional
ingredients including omega-3 fatty acids. Fortification of bread with omega-3
fatty acids requires that the omega-3 oil ingredient be protected from the liq-
uid environment, shear, and heat during the bread-making process. As fresh
bread is a short shelf-life product (<1 week), long-term oxidative stability of the
omega-3 oil is not required but it is essential that the oil be not released from
the microcapsule during the bread manufacture. The incorporation of encap-
sulated omega-3 oils into bread has been made possible by microencapsula-
tion without affecting sensory properties and shelf-life (Rennie et al., 2003).
A number of other cereal-based products such as breakfast bars and muesli
bars fortified with microencapsulated omega-3 oils are available worldwide.
Omega-3 oils can also be delivered as a coating onto a dry food particle
(soy nuggets and cereals) where a PUFA-coated particle is further coated
with a sugar solution and dried to a moisture content of the same level as the
original dry food particle (Dalziel et al., 2004). Encapsulated sardine oil in
egg white protein has been incorporated into cookies without affecting the
taste and quality of the final product (Taguchi et al., 1992).
12.7.3 Dairy Products

There are opportunities for microencapsulated ingredients for fortifica-
tion of dairy foods (Augustin, 2003). Dairy products have been targeted by
a number of food companies as a delivery vehicle for omega-3 fatty acids.
Delivery into dairy products has been attempted in two ways: by incorpora-
tion of encapsulated omega-3 oil into the cow diet (Lacasse et al., 2002) or by
fortification of the dairy product during its manufacture.
Omega-3 fish oil (refined, deodorized, and hydrogenated) was incorpo-
rated with an all-vegetable fat blend or a dairy blend mixture of butter and
vegetable oils with a view to the manufacture of a longer shelf-life low calo-
rie spread (40% fat) (Young et al., 1993).
The use of yogurt as a delivery vehicle for omega-3 oils is less challeng-
ing. The sensory properties of omega-3 oil fortified yogurt were found to be
superior with the use of microencapsulated tuna oil compared to tuna oil in
the free-form (Sharma et al., 2003). A yogurt drink (Omega-3 laban drink)
has been successfully commercialized in the Middle East under the Live
Well brand. Cheese and cheese-based products have also been successfully
fortified with omega-3 fatty acids without affecting their flavor and shelf-life.
The fortification of fresh milk with omega-3 oils is more difficult because
of the delicate flavor of milk. Milk products fortified with microencapsu-
lated nutrients including omega-3 fatty acids have been developed, where
the nutrients, which comprise both water-soluble and oil-soluble component,
were dispersed in a hydrogenated oil containing lecithin and encapsulated
in gelatin and sorbitol forming the outer layer (Shin et al., 2004).

12.7.4 Meat and Fish Products

Microencapsulated omega-3 fatty acids have been successfully incorpo-
rated into meat and fish products. Examples of meat products fortified with
omega-3 oils are ham, sausages, and salami. Enrichment of white fish fillets
with omega-3 fatty acids has also been possible by adding microencapsu-
lated omega-3 oil powders in the crumbed formulation with optional flavors
being added.
12.7.5 Drinks and Beverages

Some microencapsulated omega-3 oil formulation has been successfully
used in a number of functional juice and beverage formulations. Market
success is mainly in the short shelf life and refrigerated product category.
Careful selection of encapsulant material that do not release the omega-3
oil under low pH juice environment is important. Production of shelf-stable
omega-3 beverages remains a big challenge.
12.7.6 Confectionery
Even with sweet and strong flavored confectionery products, the addi-
tion of omega-3 fatty acids can be difficult. New orange and mint-flavored
pastille-like products have been developed as a vehicle for omega-3 fatty
acids delivery to increase intake of omega-3 fatty acids by consumers (Kolan
owski and Swiderski, 1999). Their most promising formulation had EPA and
DHA concentration of only 0.8%–1.0% even with strong flavorings added to
mask the fishy flavor in the final product.
12.8 Considerations for Successful Delivery of Omega-3

Oils into Commercial Food Products
Ideally it is desirable to design omega-3 oil microcapsules that are stable in
many different applications, but in reality this is not a practical option, as
the microencapsulated ingredient has to be matched to the target food appli-
cation. Food ingredient manufacturers will normally have microcapsule
formulations designed for different food category applications, for exam-
ple, infant formula, bakery, dairy, and liquid beverage application, each of
which has different requirements. There are different challenges in the final
food formulation with regard to food standard regulations, shelf-life, and
release properties of the omega-3 oil microcapsules. Some of the key issues
for successful delivery of omega-3 fatty acids into commercial food products
include the following.

12.8.1 Compliance with Regulatory Standards

Prior to deciding on the microcapsule formulation, it is mandatory to check
the regulatory standards to ensure that ingredients chosen as encapsulants
are approved for food use in each country where the product will be mar-
keted or sold. This step will minimize the time for regulatory approval when
the final product is launched into the market, and also avoids possible refor-
mulation and testing as in the case where a nonfood grade material has been
used during the initial trials.
12.8.2 Definition of Final Product Application to Ensure

the Correct Delivery Format
For dry blending with other food powders, it is necessary for the microen-
capsulated powder to be matched in terms of water activity, bulk density,
and particle size so as to achieve a homogeneous product and avoid possible
physical separation of the different powders during transport and storage.
For liquid product applications, the rehydration and redispersion behavior
of the powder is important if a powder format is chosen. Alternatively, a liq-
uid format for microencapsulated omega-3 ingredients could be used.
12.8.3 Understanding of the Protection and Release

Requirements during Processing
Knowledge and understanding of the processing steps and unit operations
involved in the manufacture of the final food product is important to be able
to design the microcapsule properties in such a way that the core will be
protected during the process and released at the desired time.
12.8.4 Understanding of How and Where to Add

the Microencapsulated Ingredient
Familiarity with the food manufacturing plant set-up and layout is also
required in order to identify the appropriate stage of addition of the micro-
capsule (e.g., how and where the microcapsule can be added) without the
need for any process modification.
12.8.5 Understanding Possible Interactions with Other Ingredients

When the new microencapsulated omega-3 food ingredient is added to a
food product, the final food product properties and its shelf-life may be
affected due to possible ingredient interactions. Sometimes these interac-
tions may be minimized by minor modification to the process and/or the
formulation. However, sometimes even small changes are not possible due
to limitations in the plant layout/set up or further modifications could add
significant capital investment. Therefore, formulating the new omega-3 food

ingredient has to be approached in such a way that possible negative ingre-

dient interactions are minimized.
12.8.6 Understanding the Shelf-Life, Sensory, and Physical

Properties of the Final Product
Addition of omega-3 food ingredients to short shelf-life products with a very
strong flavor is much less of a problem than the addition to food products
with delicate flavors and longer shelf-life. Therefore, the requirements of the
final food product have to be considered when designing the microcapsule
formulation to suit the final application.
12.8.7 Working in Partnership through Collaboration

There is great advantage when the food ingredient supplier/company who
develops the microencapsulation technology, and the food company manu-
facturing the final food product can work in partnership during the develop-
ment of the new food product with added omega-3 oils (Meyers, 1998; Rennie
et al., 2003). The food ingredient supplier or the company which develops
the microencapsulated omega-3 ingredient will understand the perfor-
mance and limitations of the microcapsule when added to the food product.
In addition, the food manufacturer will have a better understanding of the
market and consumer requirements of the final food product.
By choosing an appropriate encapsulant and microencapsulation process,
controlled, sustained, or delayed release of omega-3 oil in the final product
application or for targeted release at specific sites in the gastrointestinal tract
after consumption may be achieved. With carefully fine-tuned controlled
release properties, microencapsulation is no longer just an added-value tech-
nique, but the source of totally new ingredients with desirable properties
(Gouin, 2004).
The food industry expects increasingly complex properties from food
ingredients and such complex properties can often only be provided by
microencapsulation. With the use of appropriate microencapsulation tech-
nologies, the food industry has the ability to develop functional omega-3
ingredients and products and improve the nutritional content of food with-
out affecting the taste, aroma, and texture of food, and without reducing
their bioavailability and functionality (Schrooyen et al., 2001).
References
Abbadi, A. et al. 2001. Transgenic oilseeds as sustainable source of nutritionally rel-
evant C20 and C22 polyunsaturated fatty acids? European Journal of Lipid Science
and Technology, 103:106.

American Heart Association. 2000. Dietary Guidelines, A Statement for Healthcare

Professionals from the Nutrition Committee of the American Heart Association—
Krauss et al. 102(18): 2284—Circulation.htm, Revision, http://circ.ahajournals
.org/cgi/content/full/106/21/2747.
Andersen, S. 1995. Microencapsulated omega-3 fatty acids from marine sources. Lipid
Technology, 7(4):81.
Andersen, S. 1998. Omega-3 fatty acids in food fortification. Leatherhead Food RA Food
Industry Journal, 1(1):33.
Augustin, M.A. 2001. Functional foods: An adventure in food formulation, Food
Australia, 53:428.
Augustin, M.A. 2003. The role of microencapsulation in the development of func-
tional dairy foods, Australian Journal of Dairy Technology, 58:156.
Augustin, M.A. et al. 2001. Microencapsulation of food ingredients, Food Australia,
53:220.
Augustin, M.A., Sanguansri, L. 2003. Polyunsaturated fatty acids: Delivery, innova-
tion and incorporation into foods, Food Australia, 55:294.
Baik, M.Y. et al. 2004. Effects of antioxidants and humidity on the oxidative stability
of microencapsulated fish oil. Journal of the American Oil Chemists’ Society, 81:355.
Bakker, M.A.E., Galema, S.A., Adrianus, V. 1999. Microcapsules of gelatin and car-
boxy methyl cellulose. European Patent Application EP0937496-A2.
Balassa, L.L., Fanger, G.O. 1971. Microencapsulation in the food industry. Critical
Reviews in Food Technology, 2:245.
Barrier, P., Rousseau, J.Y. 1998. Powder comprising microcapsules based on fish oils
rich in polyunsaturated fatty acids. French Patent Application FR2758055-A1.
Bartz, V. 2002. Vitamin-protected fish oil for oral administration to children or non-
compliant patients is contained in gelatin microcapsules coated with an aroma-
sweetener mixture. German Patent DE20105126-U1.
Belbarbi, E.H., Molina, E., Cristi, Y. 2000. A process for high yield and scalable recov-
ery of high purity eicosapentaenoic acid esters from microalgae and fish oil.
Enzyme and Microbial Technology, 26:516.
Benichou, A., Aserin, A., Garti, N. 2004. Double emulsions stabilized with hybrids of
natural polymers for entrapment and slow release of active matters. Advances in
Colloid and Interface Science, 108–109:29.
Blenford, D. 1986. Fully protected. Food Flavouring Ingredients Processing Packaging,
July:245.
Brazel, C.S. 1999. Microencapsulation: Offering solutions for the food industry. Cereal
Foods World, 44:388.
Breitenbach, A., Mohr, D., Kissel, T. 2000. Biodegradable semi-crystalline comb poly-
esters influence the microsphere production by means of a supercritical fluid
extraction technique (ASES). Journal of Controlled Release, 63:53.
British Nutrition Foundation. 1999. Summary of the findings from the British
Nutrition Foundation conference, which was held on 1 December. http://www
.nutrition.org.uk
Buttriss, J. 1999. N-3 Fatty acids and health. BNF Nutrition Bulletin, 24(87):71.
Chaiyasit, W. et al. 2000. Ability of surfactant hydrophobic tail group size to alter lipid
oxidation in oil-in-water emulsions. Journal of Agricultural and Food Chemistry,
48:3077.
Chan, L.W., Lim, L.T., Heng, P.W.S. 2000. Microencapsulation of oils using sodium
alginate. Journal of Microencapsulation, 17:757.

Chaundy, F.K., Bower, D.K., Killbride, T.K. Jr. 1992. Process for incorporating a mate-
rial in a crosslinked gelatin and product therefrom. United States Patent US
5153177.
Cho, Y.H, Shim, H.K., Park, J. 2003. Encapsulation of fish oil by an enzymatic gelation
process using transglutaminase cross-linked proteins. Journal of Food Science,
68:2717.
Christensen, K.L., Pedersen, G.P., Kristensen, H.G. 2001. Preparation of redispersible
dry emulsions by spray drying. International Journal of Pharmacy, 212:187.
Clarke, J.P. 2002. Food encapsulation: Capturing one substance by another. Food
Technology, 56(11):63.
Connor, W.E., Lowensohn, R., Hatcher, L. 1996. Increased docosahexaenoic acid lev-
els in human newborn infants by administration of sardines and fish oil during
pregnancy. Lipids, 31(Suppl.):S183.
Courts, A. 1973. Gelatin emulsions in the formation of microcapsules. Chemistry and
Industry, 19:943.
Dalziel, S.M., Friedmann, T.E., Schurr, G.A. 2004. Process for dry coating a food par-
ticle or encapsulating a frozen liquid particle. United States Patent Application
US 2004/0131730-A1.
Danviriyakul, S. et al. 2002. Physical stability of spray-dried milk fat emulsion as
affected by emulsifiers and processing conditions. Journal of Food Science, 67:6.
Dimantov, A. et al. 2004. Study of high amylase corn starch as food grade enteric
coating in a microcapsules model system. Innovative Food Science and Emerging
Technologies, 5:93.
Djedaini-Pilard F. et al. 2000. Aqueous solution of polyunsaturated fatty acids
or derivatives, solubilized and stabilised by formation of complex with
gamma-cyclodextrin, used in food, cosmetic or pharmaceutical compositions.
International Patent WO200053637-A1.
Djordjevic, D. et al. 2004a. Physical stability of whey protein-stabilised oil-in water
emulsions at pH 3: Potential omega-3 fatty acids delivery systems (part A).
Journal of Food Science, 69:C351.
Djordjevic, D., McClements, D.J., Decker, E.A. 2004b. Oxidative stability of whey
protein stabilised oil-in-water emulsions at pH 3: Potential omega-3 fatty acid
delivery systems (part B). Journal of Food Science, 69:C356.
Donnelly, J. L, Decker, E.A., McClements, D.J. 1998. Iron-catalyzed oxidation of men-
haden oil as affected by emulsifiers. Journal of Food Science, 63:997.
Duh, P-D., Yen, W.J., Yen, G.C. 1999. Oxidative stability of polyunsaturated fatty acids
and soybean oil in an aqueous solution with emulsifiers. Journal of the American
Oil Chemists’ Society, 76:201.
Dziezak, J.D. 1988. Microencapsulation and encapsulated ingredients. Food Technology,
42(4):136.
Ennen, S. 2002. The rising tide of omega-3. Food Processing’s Wellness Foods, September/
October:19.
Fair-Flow Europe. 2001. Fighting heart disease doesn’t have to be a fishy business.
Fair-Flow Reports, FFE 411/01/CG9, p1.
Fang, X. et al. 2003. Microencapsulation of linoleic acid with low- and high-molecular
weight components of soluble soybean polysaccharide and its oxidation pro-
cess. Bioscience, Biotechnology, and Biochemistry, 67:1864.
Food Standards Australia and New Zealand. 2002. FSANZ 1.2.8 www.foodstandards
.gov.au/foodstandardscode

Formuso, L.B., Corredig, M., Akoh, C.C. 2002. Effect of emulsifier on oxidation prop-
erties of fish oil-based structured lipid emulsions. Journal of Agricultural and Food
Chemistry, 50:2957.
Frankel, E.N. et al. 1996. Evaluation of antioxidant activity of rosemary extracts, car-
nosol and carnosic acid in bulk vegetable oils and fish oil and their emulsions.
Journal of the Science of Food and Agriculture, 72:201.
Ganan-Calvo, A.M. 2003. Enhanced food products. United States Patent US6589579-B2.
Garcia, D.J. 1998. Omega-3 long chain PUFA nutraceuticals. Food Technology, 52(6):44.
Gibbs, B.F. et al. 1999. Encapsulation in the food industry: A review. International
Journal of Food Science and Nutrition, 50(3):213.
Gouin, S. 2004. Microencapsulation: Industrial appraisal of existing technologies and
trends. Trends in Food Science and Technology, 15:330.
Grattard, N. et al. 2002. Influence of physical state and molecular mobility of freeze
dried maltodextrin matrices on the oxidation rate of encapsulated lipids. Journal
of Food Science, 67:3002.
Hamilton, R.J. et al. 1998. Effects of tocopherols, ascorbyl palmitate and lecithin on
autoxidation of fish oils. Journal of the American Oil and Chemical Society, 75:813.
Haynes, L.C., Levine, H., Finley, J.W. 1991. Liposome composition for the stabilization
of oxidisable substances. United States Patent US 5015483.
Heinzelmann, K. et al. 2000a. Microencapsulation of fish oil by freeze drying tech-
niques and influence of process parameters on oxidative stability during stor-
age. European Food Research and Technology, 211:234.
Heinzelmann, K. et al. 2000b. Protection of fish oil from oxidation by microencap-
sulation using freeze drying techniques. European Journal of Lipid Science and
Technology, 102:114.
Higgins, S., Carroll, Y.L., O’Brien, N.M., Morrissey, P.A. 1999. Use of microencapsu-
lated fish oil as a means of increasing n-3 polyunsaturated fatty acid intake.
Journal of Human Nutrition and Dietetics, 12:265.
Hogan, S.A. et al. 2001. Microencapsulating properties of whey protein concentrate
75. Journal of Food Science, 66:675.
Hu, M., McClements, D.J., Decker, E.A. 2003. Lipid oxidation in corn oil-in-water
emulsions stabilized by casein, whey protein isolate and soy protein isolate.
Journal of Agriculture and Food Chemistry, 51:1696.
Hu, M., McClements, D.J., Decker, E.A. 2004. Emulsion droplets engineered to improve
the oxidative stability of n-3 fatty acids in functional foods. Lipid Technology, 16:79.
Imagi, J. et al. 1992. Retarded oxidation of liquid lipids entrapped in matrices of sac-
charides or proteins. Bioscience, Biotechnology, and Biochemistry 56:1236.
Institute of Medicine. 2002. Dietary Reference Intakes for Energy, Carbohydrate, Fiber,
Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids. Released on September 5.
National Academy of Science, Washington, DC. www.iom.edu/Object.File/
Master/4/154/0.pdf
International Society of the Study of Fatty Acids and Lipids. 2004. Report on Dietary
intake of essential fatty acids, June. http://www.issfal.org/news-links/
resources/publications/PUFAIntakeReccomdFinalReport.pdf
IUPAC. 1972. Compendium of Chemical Terminology, 2nd edition. Blackwell Scientific
Publications, Oxford, UK. 1997, 31:611.
Iwami, K. et al. 1987. Spray dried gliadin powders inclusive of linoleic acid
(microcapsules): Their preservability, digestibility and application to bread
making. Agricultural and Biological Chemistry, 51:3301.

Iwami, K. et al. 1988. Stability of gliadin encapsulated unsaturated fatty acids against
autoxidation. Journal of Agriculture and Food Chemistry, 36:160.
Jackson, L.S., Lee, K. 1991. Microencapsulation and the food industry. Lebensmittel-
Wissenschaft Technologie, 24:289.
Janda, J., Bernacchi, D., Frieders, S. 1995. Microencapsulation process. United States
Patent US5418010.
Kagami, Y. et al. 2003. Oxidative stability, structure, and physical characteristics of
microcapsules formed by spray drying of fish oil with protein and dextrin wall
materials. Journal of Food Science, 68:2248.
Karel, M. 1990. Encapsulation and controlled release of food components. In
Biotechnology and Food Process Engineering, ed. H.G. Schwartzberg and M.A. Rao.
Marcel Dekker, New York.
Kato, A., Kobayashi, K. 1991. Excellent emulsifying properties of protein–dextran
conjugates. In Microemulsions and Emulsions in Foods, ed. M. El-Nokaly and
D. Cornell. ACS, Washington, p. 213.
Katzen, S. 1976. Preserved nutrients and products. United States Patent US3962416.
Keogh, M.K. et al. 2001. Stability to oxidation of spray dried fish oil powder microen-
capsulated using milk ingredients. Journal of Food Science, 66:217.
Kim, S.J. et al. 2000. Improvement of oxidative stability of conjugated linoleic acid
(CLA) by microencapsulation in cyclodextrins. Journal of Agriculture and Food
Chemistry, 48:3922.
Kim, Y.D., Moore, C.V. 1995. Encapsulating properties of several food proteins. IFT
Annual Meeting (Poster), 193.
Kirby, C. 1991. Microencapsulation and controlled delivery of food ingredients. Food
Science and Technology Today, 5(2):74.
Klont, R. 1999. Healthy ingredients driving innovation. World of Food Ingredients,
March/April:34.
Kolanowski, W., Laufenberg, W., Kunz, B. 2004. Fish oil stabilization by microen-
capsulation with modified cellulose. International Journal of Food Science and
Nutrition, 55:333.
Kolanowski, W., Swiderski, F. 1999. New product containing polyunsaturated fatty
acids omega-3 EPA, DHA—Sensory quality and possibility of diet supplemen-
tation. Roczniki Panstwowego Zakladu Higieny, 50:427.
LaBell, F. 2002a. Heart healthy omega-3s. Prepared Foods, 171(8):63.
Labell, F. 2002b. New, precise methods for encapsulation. www.preparedfoods.com
Labell, F. 1991. Custom encapsulation protects ingredients. Food Processing, 52(12):42.
Lacasse, P. et al. 2002. Addition of protected fish oil to diets for dairy cows. I. Effects
on yield, composition and taste of milk. Journal of Dairy Research, 69:511.
Lamprecht, A., Schafer, U., Lehr, C.M. 2001. Influences of process parameters on
preparation of microparticle used as a carrier system for ω-3 unsaturated fatty
acid ethyl esters used in supplementary nutrition. Journal of Microencapsulation,
18:347.
Lauretzen, D. 1994. Food enrichment with marine omega-3 fatty acids. International
Food Ingredients, 1/2:41.
Lee, S.J., Rosenberg, M. 2000a. Preparation and some properties of water insoluble,
whey protein based microcapsules. Journal of Microencapsulation, 17:29.
Lee, S.J., Rosenberg, M. 2000b. Whey protein based microcapsules prepared by dou-
ble emulsification and heat gelation. Lebensmittel-Wissenschaft und Technologie,
33:80.

Ley-Jacobs, B.M. 2000. EPA and DHA. In DHA: An Essential Building Block of the Brain.
BL Publications, Temecula, CA, p. 14.
Lin, C.C., Lin, S.Y., Hwang, L.S. 1995. Microencapsulation of squid oil with hydro-
phobic macromolecules for oxidative and thermal stabilization. Journal of Food
Science, 60:36.
Matsuno, R., Adachi, S. 1993. Lipid encapsulation technology—Techniques and appli-
cations to food. Trends in Food Science and Technology, 4:256.
Merchant, M.M., Nicholson, V.J. 2000. Microcapsule flavour delivery system. United
States Patent US6136364.
Meyers, M.A. 1998. Capturing solutions. International Food Ingredients, 5:18.
Michelsen, B., Madsen, S.L., Gotfredsen, P. 2001. Polyunsaturated fatty acids. In Guide
to Functional Foods, ed. J. Young. Leatherhead Publishing, Surrey, England,
p. 141.
Minemoto, Y. et al. 2002. Oxidation of linoleic acid encapsulated with soluble soy-
bean polysaccharide by spray drying. Bioscience, Biotechnology, and Biochemistry,
66:1829.
Miyashita, K. et al. 1995. Oxidative stability of triglycerides from orbital fat of tuna
and soybean oil in an emulsion. Fisheries Science, 61:273.
Muggli, R. 2001. Health benefits of long-chain polyunsaturated fatty acids. Food
Technology International, 22:24.
Mukherjee, K.D. 1999. Production and use of microbial oils. INFORM, 10:308.
Newton, I. 1996. Novel odourless marine long chain n-3 PUFA for food enrichment.
IFT Annual Meeting: Book of Abstracts, p. 70.
Nichols, P.D., Petrie, J., Singh, S. 2010. Long-chain omega-3 oils—An update on sus-
tainable sources. Nutrients, 2(6):572–585.
Nuchi, C.D., McClements, D.J., Decker, E.A. 2001. Impact of Tween 20 hydroperox-
ides and iron on the oxidation of methyl linoleate and salmon oil dispersions.
Journal of Agriculture and Food Chemistry, 49:4912.
Olsen, S.F., Secher, N.J. 2002. Low consumption of sea food in early pregnancy as a
risk factor for preterm delivery: Prospective cohort study. British Medical Journal,
324:447.
O’Shea, M. 2003. Health benefits of marine-based omega-3 fatty acids. Cereal Foods
World, 48:236.
Pacifico, C.J., Wu, W-H., Fraley, M. 2001. Sensitive substance encapsulation. United
States Patent US6251,478-B1.
Palka, A. 2002. What’s new with fatty acids? World of Food Ingredients, September:88.
Pandya, Y., Knorr, D. 1991. Diffusion characteristics and properties of chitosan coac-
ervate capsules. Process Biochemistry, 26:75.
Partanen, R. et al. 2002. Encapsulation of sea buckthorn kernel oil in modified starches.
Journal of the American Oil Chemists’ Society, 79:219.
Pothakamury, U.R., Barbosa-Canovas, G.V. 1995. Fundamental aspects of controlled
release in foods. Trends in Food Science Technology, 6:397.
Pszczola, D.E. 1998. Encapsulated ingredients: Providing the right fit. Food Technology,
52(12):70.
Re, M.I. 1998. Microencapsulation by spray drying. Drying Technology, 16:1195.
Reichenbach, A.W. 1996. Kinetics of the oxidative stabilisation of unsaturated fatty
acids encapsulated in cyclodextrins and NMR analysis of encapsulated fatty
acid conformation, PhD Thesis, Ohio State University.
Reineccius, G.A. 1991. Carbohydrates for flavor encapsulation. Food Technology, 45(3):144.

Reineccius, G.A. 1995. Controlled release techniques in the food industry. In

Encapsulation and Controlled Release of Food Ingredients, ed. S.J. Risch and G.A.
Reineccius. ACS, Washington.
Rennie, M. et al. 2003. Tip top up bread with omega-3 DHA: Results of partnership
between Clover Corporation Ltd. and George Weston Foods Ltd. Food Australia,
55:506.
Rice, R. 1991. Fish oil—Food for thought! A challenge for food technology. European
Food & Drink Review, Spring: 27.
Rice, R. 1992. Fish oil—How can the balance be redressed? Lipid Technology, 4(1):14.
Richard, J., Morteau, S. 2004. Plant protein-based microcapsule. U.S. Patent
Application US2004/0151778-A1.
Ripoll, A.B. et al. 2004. Production of capsules and particles for improvement of food
products. U.S. Patent Application US2004/0161498-A1.
Rodriguez-Huezo, M.E. et al. 2004. Microencapsulation by spray drying of multiple
emulsions containing carotenoids. Journal of Food Science, 69:E351.
Rosenberg, M., Lee, S.J. 2004. Water-insoluble, whey protein-based microspheres pre-
pared by an all-aqueous process. Journal of Food Science, 69:50.
Saleeb, F.Z., Arola, V.K. 1999. Method of preparing glass stabilized material. United
States Patent US5972395.
Sanguansri, L., Augustin, M.A. 2001. Encapsulation of food ingredients. International
Patent Publication WO 01/74175 A1.
Sanguansri, L., Boghossian, V. 1991. Stability trial and sensory evaluation of infant
formula with added omega-3 powder microcapsules. Unpublished Report.
Schlameus, W. 1995. Centrifugal extrusion encapsulation. In Encapsulation and
Controlled Release of Food Ingredients, ed. S.J. Risch and G.A. Reineccius. ACS,
Washington, p. 96.
Schleifenbaum, B. et al. 2004. Encapsulated flavour and/or fragrance preparations.
United States Patent Application US2004/0081735-A1.
Schmitt, C. et al. 1998. Structure and technofunctional properties of protein-polysaccharide
complexes: A review. Critical Reviews in Food Science and Nutrition, 38:698.
Schrooyen, P.M.M., van der Meer, R., De Kruif, C.G. 2001. Microencapsulation: Its
application in nutrition. Proceedings of the Nutrition Society, 60:475.
Serpelloni, M., Boursier, B. 1995. Encapsulation carriers for oxidizable substances
useful in the nutritional supplementation. Food Ingredients Europe, Conference
Proceedings, p. 68. Messegelande Frankfurt, Germany.
Shahidi, F., Kim, S.K. 2002. Marine lipids as affected by processing and their quality
preservation by natural antioxidants. In Bioactive Compounds in Foods: Effects of
Processing and Storage, ed. T.C. Lee and C.T. Ho. ACS, Washington.
Sharma, R. et al. 2003. Applications of microencapsulated omega-3 fatty acids in dairy
products. Australian Journal of Dairy Technology, 58:211.
Shefer, A., Shefer, S. 2003. Novel encapsulation system provides controlled release of
ingredient. Food Technology, 57(11):40.
Shin, Y.S., Jeong, G.H., Kim, S.J. 2004. Enriched milk with capsules containing nutri-
ents. United States Patent US6797293-B2.
Shine, A.D., Gelb, J. Jr. 1998. Microencapsulation process using supercritical fluids.
United States Patent US 5,766,637.
Silvestre, M.P.C. et al. 2000. Ability of surfactant headgroup size to alter lipid and
antioxidant oxidation in oil-in-water emulsions. Journal of Agriculture and Food
Chemistry, 48:2057.

Skelbaek, T., Andersen, S. 1994. A microencapsulated oil or fat product. International

Patent Application WO94/01001-A1.
So, R.S. 2001. Encapsulated liquid product. United States Patent US6174554-B1.
Soeda, T., Nakanishi, M., Inoue, T. 2003. Edible microcapsules containing for exam-
ple, vitamin(s), fats and oils for addition to dried food such as soup, chewing
gum and pouch-packed foods comprise edible hydrophobic core and capsule
wall containing transglutaminase as crosslinking agent. United States Patent
US6592916-B2.
Souza-Neves-Cardoso, F. de., Grosso, C., Almeida-Vitali de, A. 2001. Starch microcap-
sules obtained by spray drying and coated using fluid bed. Brazilian Journal of
Food Technology, 4:131.
Subramaniam, A. et al. 2004. Spray-dried compositions and method for their prepara-
tion. United States Patent US6723359-B2.
Sunley, N. 1998. Ingredients challenge. Food Review, 25(12):29.
Taguchi, K. et al. 1992. Oxidative stability of sardine oil embedded in spray dried egg
white powder and its use for n-3 unsaturated fatty acid fortification of cookies.
Bioscience, Biotechnology, and Biochemistry, 56:560.
Trautwein, E.A. 2001. N-3 Fatty acids—Physiological and technical aspects for their
use in food. European Journal of Lipid Science and Technology, 103:45.
Truter, P.A., Xulu, P.M., Igwe, N. 2000. Microparticles and process for making mic-
roparticles. International Patent Application WO 00/33673.
Tuley, L. 1996. Breaking free—Update on controlled flavour release. International Food
Ingredients, 3:14.
Uicich, R. et al. 1999. Bioavailability of microencapsulated ferrous sulphate in fluid
cow’s milk. Studies in human beings. Nutrition Research, 19:893.
US Food and Drug Administration. 2004. FDA announces qualified health claims
for omega-3 fatty acids, September. www.fda.gov/bbs/topics/news/2004/
NEW01115.html
Valenzuela, A., Nieto, S., Uauy, R. 1993. Technological challenges to assess omega-3
polyunsaturated fatty acids from marine oils for nutritional and pharmacologi-
cal use. Grasas y Aceites, 44:39.
Van Lengerich, B.H. 2003. Embedding and encapsulation of controlled release par-
ticles and encapsulated products. European Patent Application EP 1342548-A1.
Versic, R.J. 1988. Coacervation for flavour encapsulation. In Flavour Encapsulation, ed.
S.J. Risch and G.A. Reineccius. ACS, Washington, p. 370.
Watanabe, Y. et al. 2002. Suppressive effect of saturated acyl l-ascorbate on the oxida-
tion of linoleic acid encapsulated with maltodextrin or gum Arabic by spray
drying. Journal of Agriculture and Food Chemistry, 50:3984.
Weder, H.G., Weder, M.A., Andreas, S. 2000. Use of “nanofood” in foodstuff final
products for humans and animals. Canadian Patent CA 02331661.
Witteveen, F. et al. 2001. Stable spray-dried composition in a carbohydrate substrate
and process for obtaining said composition. International Patent Application
WO01/35764-A1.
Wu, W.H. et al. 2000. Low melt encapsulation with high laurate canola. United States
Patent US 6153236.
Yilmaz, G. et al. 2001. Encapsulation of sunflower oil in starch matrices via extrusion:
Effect of the interfacial properties and processing conditions on the formation of
dispersed phase morphologies. Carbohydrate Polymers, 45:403.

Yin, X., Xu, S. 2000. Microencapsulation of EPA and DHA: Wall material selection.
Food & Fermentation Industries, 26(1):33.
Yoshii, H. et al. 1997. Oxidative stability of powdery tridocosahexanoin included in
cyclodextrin and its application to fish meal paste. Bioscience, Biotechnology, and
Biochemistry, 61:1376.
Young, F.V.K., Barlow, S.M., Madsen, J. 1993. Unhydrogenated fish oil in low-calorie
spreads. INFORM, 4:1140.

13
Packaging Functional Foods: From Basic
Requirements to Nano Perspectives
Louise Deschênes
CONTENTS
13.1 Introduction................................................................................................. 409
13.2 Packaging Practices versus Functional Foods Requirements.............. 410
13.2.1 Fruits and Vegetables..................................................................... 410
13.2.2 Probiotics.......................................................................................... 411
13.2.3 Intermediate Moisture Products................................................... 413
13.2.4 Oils and Fats.................................................................................... 413
13.3 Choice of Packaging Materials.................................................................. 414
13.4 Active Packaging......................................................................................... 415
13.5 Nanotechnology.......................................................................................... 416
13.6 Summary...................................................................................................... 419
References.............................................................................................................. 419
13.1 Introduction
A decade ago, the market of functional foods was considered in emergence.
Since then, the trends have been still in favor of a growing market for nutra-
ceuticals which are now well established in important niches of food prod-
ucts. For instance, the integration of bioactive components in milk products
and beverages is now widely spread. This evolution has instigated a seri-
ous challenge to all manufacturers to keep abreast with the use of adequate
packaging materials for its products. The interest in functional foods is
intrinsically related to the potential of their bioactive components to provide
health benefit. Consequently, the preservation of these bioactive components
is a crucial factor in providing these anticipated health benefits. However,
there have been recurring reports of failures in maintaining the quality of
functional foods (Labuza, 2000; Bell, 2006; Destaillats, 2011).
Functional foods represent a wide variety of products including pre- and
probiotics, dried food (fibers, probiotics, tea, herbs, etc.), fermented prod-
ucts (yoghurts, kefir, vegetables), and fresh fruits and vegetables. Their
bioactive components (minerals, vitamins, fibers, peptides, proteins, n-3
409
polyunsaturated fats, antioxidants, enzymes, symbiotics, other micronutri-

ents) range from very simple molecules to complex living organisms like
bacteria. The chemical nature of the bioactive components and the way in
which they are presented to customers in the form of dried extracts, fresh
food, and processed food products drastically influence their shelf-life and,
as such, their packaging requirements. Unfortunately, there are only a lim-
ited number of documents addressing this critical issue and the available
data are fragmented, being dispersed in the literature in studies that are
involved with other fields of research. A comprehensive review would be
useful to summarize the existing knowledge on their preservation require-
ments, and thus to better orchestrate an appropriate packaging approach.
In addition to classical approaches, the potential of nanotechnologies in
improving packaging of functional foods will be discussed.
13.2 Packaging Practices versus Functional Foods

Requirements
The effect of packaging on the variation in food composition during stor-
age depends on the technology used and the storage conditions. As a rule
of thumb, functional foods packaging and preservation practices are based
on the conditions applied to products of similar origin and format. This
approach can give satisfactory results for several products but should not
be taken as a universal rule regarding all functional foods. In some cases,
the usual technologies and packaging materials could be detrimental to the
shelf-life of certain functional ingredients. In general, they are particularly
sensitive to moisture and oxygen (Bell, 2006). Hopefully, in several cases,
minor adaptations of the packaging technologies can greatly improve the
shelf-life of functional foods. Examples are given in the following sections
for selected classes of functional foods and nutraceuticals.
13.2.1 Fruits and Vegetables

The effects of processing and preservation technologies on minerals and
vitamins have been extensively documented for decades (Schroeder, 1971;
Karmas and Harris, 1988; Reddy and Love, 1999; Rickman et al., 2007a,b;
Serrano et al., 2011; Amorim et al., 2012). In addition to vitamins and min-
erals, several other important functional phytochemicals such as polyphe-
nols, flavanoids, prostaglandins, and sulfur compounds are present in fresh
and processed plant products (Rickman et al., 2007a; Serrano et al., 2011;
Çağlarırmak and Zeki Hepçimen, 2013).
Phenolic compounds are closely associated with the nutritional quality of
foods. Their properties of interest are based on their antioxidant capability

Packaging Functional Foods 411
(Shahidi and Naczk, 2004). As with other antioxidant compounds, these

molecules easily oxidize in the presence of oxygen. Phenolic compounds
are present in cereals, legumes, oilseeds, fruits, and plant origin bever-
ages (tea, cocoa, beer, wine, fruit juices, etc.). Processed plant products with
medium-to-high water activity will be the most susceptible to browning
and phenolic degradation (Maltini et al., 2003; Elizaveta, 2012; Venir and
Maltini, 2013). Conditioning and packaging of these products require the
control of oxygen level. Their stability to processing and storage conditions
is also affected and greatly depends on technologies and conditions used
(Heinonen, 2002; Serrano et al., 2011; Giovanelli et al., 2013; Tadapaneni
et al., 2014). In general, minimal processing is less detrimental than high-
temperature treatments and is often a way to preserve the bioactive con-
stituents in functional foods. However, the effect of minimal processing
and packaging technologies on the nutritional quality of functional foods is
not well established (Oms-Oliu et al., 2012; Coskun and Pazır, 2013). Novel
technologies such as high-pressure processing have demonstrated poten-
tial in better preservation of bioactive components in fruit transformation
(Rawson et al., 2011). The results are dependent on processing conditions
(Sampedro and Zhang, 2012; Vigneault et al., 2012) and on the type of fruits
and vegetables (Sánchez-Moreno et al., 2010).
To extend the shelf-life of minimally processed food products, packag-
ing conditions also become an important issue. Modified atmosphere pack-
aging (MAP) is a minimal process well known for its capacity to enhance
the shelf-life of fresh fruits and vegetables (Goswami and Mangaraj, 2011;
Arvanitoyannis, 2012). The shelf-life improvement obtained using MAP is
usually associated with the preservation of normal appearance and firmness.
MAP slows down or matches the metabolism of packaged fresh products
and microflora to avoid fermentation and thus minimizes microbial growth
(Nicola and Fontana, 2014). For fresh fruits and vegetables, the level of anti-
oxidants (vitamins, phenolics, and anthocyans) depends on atmosphere and
storage conditions (Kader, 2009). However, the impact could be multi-fold.
For instance, MAP of spinach was shown to better preserve vitamin C but
was detrimental to flavonoids which were found in higher amounts in the
air-packed samples (Gil et al., 1999). The retardation of ripening was sug-
gested to explain the lower level of anthocyanins (Serrano et al., 2011) and
phenolic compounds (Kader, 2009) in MAP fruits. The lack of data in the
literature does not allow the extension of such conclusions to the entire class
of fresh fruits and vegetables and optimization should be done on a case-by-
case basis.
13.2.2 Probiotics
The viability of microorganisms may be affected by several factors, packag-
ing conditions being one of them. The integration of probiotics into foods
involved the use of dried cultures. Dried probiotic cultures are affected

by processing and storage conditions such as drying (Fu and Chen, 2011;
Peighambardoust et al., 2011; Gong et al., 2014), freezing (Alur and Grecz,
1975; Bozoğlu et al., 1987), moisture (Fu and Chen, 2011; Poddar et al., 2014),
light (Peighambardoust et al., 2011), and air (Bozoğlu et al., 1987). Oxygen and
moisture levels are known for their detrimental influence on the survival of
dried bacteria (Greiff, 1971; Bozoğlu et al., 1987; da Cruz et al., 2007). In addi-
tion to external packaging, encapsulation could also be used as a primary
packaging system able to optimize the preservation of dried probiotic cul-
tures (Chan and Zhang, 2002; Heidebach et al., 2012). As a general guideline,
oxygen and water vapor should be carefully controlled to ensure ideal pack-
aging conditions of dried cultures.
The viability of probiotics in functional foods is also influenced by pro-
cessing and storage conditions. It has been reported that the survival of both
Lactobacillus and Bifidobacterium species may diminish drastically during
storage of the food products, resulting in low numbers of living cells at the
time of consumption (De Vuyst, 2000). Oxygen is often pointed out as being
of major concern for survivals of these cell cultures (da Cruz et al., 2007;
Saarela, 2011). Lactobacillus and Bifidobacterium spp. are sensitive to oxygen
toxicity (Talwalkar et al., 2004). Lactobacillus acidophilus is microaerophilic
(Gomes and Malcata, 1999; De Vuyst, 2000). The other popular probiotics,
Bifidobacteria, are usually considered strictly anaerobic (da Cruz et al., 2007)
with requirements for oxygen barrier packaging (Saarela, 2011), although
some strains of Bifidobacterium lactis have demonstrated a relative tolerance
to oxygen (De Vuyst, 2000). Strains of these bacteria grown in defined oxygen
concentrations (0%, 5%, 10%, 15%, and 21% of oxygen) showed a substantial
decrease in lactate production with increased oxygen uptake (Talwalkar and
Kailasapathy, 2003). Dave and Shah have demonstrated that for living cells
contained in yogurts, oxygen concentration in the product and container
significantly influences the survival of probiotics (Dave and Shah, 1997a,
b). Interestingly, intrinsic and inducible hydrogen peroxide resistance can
contribute to probiotic survival during processing and storage (Talwalkar
and Kailasapathy, 2004; Brioukhanov and Netrusov, 2007; Oberg et al., 2011).
This type of resistance is reported to be strain-dependent (Oberg et al., 2011).
In order to reduce the impact of oxygen on probiotic concentration, strain-
selection for commercial products usually considers bacterial resistance to
oxygen abuse. This criterion could contribute in reducing the variety of pro-
biotic strains present in the products offered on the market.
The response to oxidative/redox stress should be taken into account in the
selection of strains not only for yogurt (Talwalkar et al., 2004) but also for
milk (Bolduc et al., 2006) probiotic formulations. Milk is often seen as an
ideal media for integration of nutraceuticals. Gliguem and Birlouez-Aragon
(2005) have reported that vitamin C in fortified milks is particularly sensitive
to degradation and that high oxygen and light barrier materials are essen-
tial for its preservation. They have also reported that short storage time and
low-temperature limit the impact on protein degradation. Bolduc et al. (2006)

have demonstrated that the oxygen level affects the survival of probiotics
survival in nonfermented milks.
Tripati and Giri (2014) have recently reviewed the impact of processing
and storage conditions on survivals of probiotics. They have also specially
addressed packaging concerns. Their overview of the literature supports the
fact that the survival of probiotics in food products can be improved by opti-
mization of encapsulation and using oxygen barrier materials combined to
smart packaging technologies (e.g., oxygen scavengers).
13.2.3 Intermediate Moisture Products

Numerous food products can be classified as intermediate moisture foods.
Typical examples of intermediate moisture foods (IMF) are cakes, syrups,
toaster pastries, fruit rolls, and soft bars. The intermediate moisture levels in
foods are generally related to a water content of 15%–40% and a water activ-
ity of 0.65–0.85 (Barbarosa-Canovas and Vega-Mercado, 1996). Intermediate
moisture nutraceutical foods, like semi-soft food bars, belong to this cate-
gory. Despite the relative stability at ambient temperature, the rates of some
deterioration processes are very high at an intermediate moisture level (Rao
et al., 2012). Browning reactions are particularly important in IMF (Labuza,
1974). Both enzymatic and nonenzymatic browning can take place and affect
the quality of the product (Maltini et al., 2003). Lipid oxidation is also a prob-
lem in IMF, provoking rancidity and unacceptable sensory defects (Kader,
2009). The rate of lipid oxidation increases as the water activity (aw) decreases
below the monolayer moisture content. This type of products would greatly
benefit from high barrier materials integrating oxygen scavengers.
The survival of probiotics is also of concern in low moisture foods and
IMF. It has been reported that in several cases the lower the water, the better
the survival of Bifidobacterium and Lactobacillus species (Klu et al., 2012). The
importance of water activity on the survival of probiotics in low and inter-
mediate moisture products is supported by more recent findings obtained
from investigating the survival of Lactobacillus rhamnosus GG in oil matrices
under various processing and storage conditions (Endo et al., 2014).
13.2.4 Oils and Fats

Extracted natural oils and fats contain molecules with unsaturated com-
pounds and natural antioxidants like tocopherols. These molecules are eas-
ily oxidized when exposed to oxygen and light. Consequently, light and gas
barrier packaging materials are mandatory to preserve the functionality of
these products.
Conjugated linoleic acid (CLA) is a natural fatty acid recognized for its
anticancer properties (Thompson et al., 1997). It has also been reported to
have other beneficial effects on physiological functions (Dhiman et al., 2006;
Benjamin and Spener, 2009). When CLA is present in its original matrix,

its preservation seems to be relatively good within the usual shelf-life of

fresh products. CLA can be found in dairy products and meat. It was dem-
onstrated by Lavillonnière et al. (1998) that the CLA level in French cheese
depends more on milk origin than on cheese production and maturation.
CLA is also relatively stable in other dairy products such as yogurt and ice
cream (Dhiman et al., 2006).
13.3 Choice of Packaging Materials

The mechanisms of degradation of the bioactive constituents in packaged
functional foods are mainly related to UV sensitivity, water activity, micro-
bial load, and oxidation processes. Based on these considerations, the pack-
aging technologies should be selected for their potential to control light,
humidity level, temperature, and atmosphere conditions. Table 13.1 sum-
marized some factors to be taken into account in the choice of appropriate
packaging conditions for different types of functional foods. This list is not
exhaustive and specific tests are recommended to ensure the preservation of
minimal levels of bioactive constituents.
TABLE 13.1
Functional Foods and Packaging Requirements
Deterioration Packaging
Factor Products Deterioration Effect Requirements
Oxygen Fermented milks Vitamin oxidation Controlled oxygen
Decrease in survival barrier
probiotic bacteria
Oils and fats Lipid oxidation High barrier
Rancidity
Processed fruits General nutritional quality High barrier
and vegetables loss
Degradation of antioxidants
and polyphenols
Moisture Fresh fruits and Vitamin degradation Controlled
vegetables atmosphere
Dried probiotics Decrease in the active load Moisture barrier
Light Oils and fats Oxidation, rancidity Light barrier
Fermented milks Oxidation of vitamins
Protein-based Modification of proteins and
products amino acids
Microorganisms Sprouts Flavor problems Moisture control
Pathogen development
Temperature Dried herbs High barrier

Common packaging technologies could be a simple solution to extend the

shelf-life of functional foods. They include gas flushing, barrier, controlled
atmosphere, modified atmosphere, and vacuum packaging. However, new
advanced packaging approaches could provide additional tools to increase
the shelf-life of bioactive functional food constituents.
13.4 Active Packaging

Active packaging is a class of technologies in which dynamic physical and/
or chemical interactions between the content and the containers induce
modifications of the internal conditions resulting in shelf-life improvement.
Active packaging can positively influence food preservation by integrating
more sophisticated systems of oxygen scavenging, release of synthetic or
bioactive compounds, moisture and atmosphere control, and antimicrobial
surface properties (Imran et al., 2010). It can benefit to both food quality and
safety (de Kruijf et al., 2002). Realini and Marcos (2014) recently published a
review discussing currently available active packaging systems. Table 13.2
presents a list of various active packaging systems.
Several active packaging options can be adapted to MAP of fresh plant
and sensitive products. They include gas absorbers for oxygen, carbon diox-
ide, ethylene, and water vapor scavenging. Polymer films can also be doped
with UV-absorbent agent (Goswami and Mangaraj, 2011). Active agent
release is another mode of active packaging. Antioxidant release was one
of the first active packaging systems to be implemented in the industry.
TABLE 13.2
Active Packaging Systems
Parameter to
Control Mechanism of Action Packaging Active Constituent
Moisture Physical dehydrators Anhydrous salts, starch, clay
Gas atmosphere Oxygen scavengers Ascorbates, ferrous compounds,
oxidizable polymers, enzymes
Ethylene absorbers Zeolites, cristobalites, clays,
alumino-silicates, active carbon,
permanganates
Odors absorbers Active carbon, zeolites, catalyzers
CO2 absorbers Calcium hydroxide
CO2 generators Ascorbic acid/sodium bicarbonate,
iron carbonate
Microbial load Bacteriocins, vegetable extracts, Nisin, lactoferrin, silver, acetic acid,
metals, organic acids, chelators, imazalil, benomyl, EDTA, natural
fungicides, antibiotics antimicrobials

Traditionally, hindered phenolic compounds are added to the food con-

tact layer which act as oxygen scavengers and chelators, the most popu-
lar additives being butylated hydroxyanisole, butylated hydroxytoluene,
and tertiary butyl hydroquinone (Decker et al., 2010). Cereal-, nut-, and oil-
packaging materials often contain such additives to increase their storage
stability (Furia, 1980; Miltz et al., 1988). For functional foods, complex chains
of chemical interactions involving vitamins, polyphenols, and enzymes
are critical (Kikugawa et al., 1990). The release of antioxidants naturally
involved in the biochemical processes would present an interesting strategy.
New active packaging systems are using natural molecules as antioxidants.
These antioxidants have been integrated in polymer films in the form of
pure molecules such as α-tocopherol, carvacol, and thymol or in the form of
natural extracts (Colín-Chávez et al., 2013). Functional natural antioxidants
are themselves considered as interesting additives in active food packaging
systems (Castro-López Mdel et al., 2014). Their potential applications are not
restricted to synthetic-based materials but also include the area of edible
films and coatings (Silva-Weiss et al., 2013). The advantages of using natural
compounds in packaging materials could be particularly beneficial to the
preservation of functional foods in enhancing stability, bioavailability, and
sensory quality of these products.
With the use of active packaging, new materials can be tailored to ensure
efficacy and/or safety of functional foods through incorporation of agents to
control the internal environment. Nanotechnologies present a high potential
in the development of new active and smart packaging innovations.
13.5 Nanotechnology
Nanotechnologies are now offering a new spectrum of opportunities in
improving food packaging technologies, from nano-based packaging mate-
rials to nanosensing of pathogens and toxins. Such new approaches are
considered having potential and advantages in ensuring healthier and safer
foods (Kalpana Sastry et al., 2013). Moreover, it has been pointed out by
Senturk et al. (2013) that “Packaging area is the most useful application in
the practicability perspective about nanotechnology to foods.” This point of
view is also in agreement with a perception of better acceptance of nanotech-
nologies related to packaging as compared to “nano-foods” (Siegrist et al.,
2008, 2011; Bryksa and Yada, 2012). This section is not intended to cover all
aspects of nanotechnologies related to food packaging and safety. Our inten-
tion is to present some examples of nano-based packaging technologies that
could be of interest in preserving quality and shelf-life of nutraceuticals and
functional foods, or allowing new options for bioactive component delivery
from external packaging. The perspectives of implementation should take

toxicological assessment and regulation compliance into consideration in

each particular case (Sonkaria et al., 2012; Kalpana Sastry et al., 2013; Pérez-
Esteve et al., 2013; Reig et al., 2014).
A large variety of nano-based composite packaging (NCP) materials has
been developed primarily to enhance barrier properties of low-cost packag-
ing containers and films. One of the earliest examples of commercial appli-
cation concerns nanocomposites integrating nano-clay for the production of
gas barrier plastic bottles for beverages. Their low cost of manufacturing has
contributed to their success on the market (Duncan, 2011). Several commer-
cial formulations have been developed and are available for beverage and oil
packaging (Momin et al., 2013), most of them being based on poly(ethylene
terephthalate) and Nylon 6 polymer matrices (Duncan, 2011; Rhim and Kim,
2013). Research has also been done regarding NCP materials based on bio-
degradable polymer matrices (Rhim and Kim, 2013). They are expected to
contribute to both shelf-life improvement and waste reduction from pro-
cessing food (García et al., 2010; Rhim and Kim, 2013). However, their opti-
mization is more challenging in terms of reproducibility and compatibility
(Sanchez Garcia and Lagaron, 2012). Higher barrier properties can also be
obtained by coating polymeric materials with inorganic nanoparticles. Such
coatings have been shown to reduce the permeation of oxygen and carbon
dioxide of poly(ethylene terephthalate) and other polymeric materials. They
are considered as an alternative to oxygen scavengers (Rhim and Kim, 2013).
They could be particularly interesting when sachet forms of oxygen scaven-
gers could not be considered. However, the choice of the formulation should
match the conditions required for the nanosystem to be active. For instance,
in the case of films integrating TiO2 (Mills et al., 2006; Ranjan et al., 2014) and
ZnO (Espitia et al., 2012) nanoparticles, the photocatalytic effect is expected
to depend on UVA light exposure. Other inorganic nanoparticles such as
silver (Intawiwat et al., 2012; Kanmani and Rhim, 2014) and cerium diox-
ide (Althues et al., 2007) have been studied for the development of anti-UV
packaging materials. The advantage is that it allows limiting UV penetration
while maintaining the transparency of the material (Intawiwat et al., 2012).
The impact of the use of NCP materials on the preservation of bioactive
components of green tea was investigated by Zhao et al. (2012). In their study,
they used a film prepared by blending polyethylene with a mixture of nano-
Ag, nano-TiO2, and attapulgite which was further combined to an aluminum
layer. Overall, when compared with the packaging without nanopowder,
they report that the NCP packaging material allowed for a better preservation
of amino acids, polyphenols, vitamin C, and chlorophyll over a 1-year period.
Apart from NCP systems increasing resistance and barrier properties,
several other applications of nanotechnologies to food packaging are under
investigation. With regard to MAP systems, nanoperforated (Parker, 2009)
and nanoporous (Goswami and Mangaraj, 2011) films provide low-cost
options to modify internal packaging atmosphere (Parker, 2009). Other inno-
vations are focusing on the controlled delivery of bioactive compounds from

nanostructured materials. Jiang et al. (2005) have mentioned that the release
of bioactive compounds such as enzymes and proteins from electrospun
nanofibers presents several advantages as compared to classical encapsu-
lation: “facile, high loading efficiency/capacity, mild preparation condition
and relatively steady release characteristics.” Edible nanocoatings formu-
lated from functional and bioactive food-grade components are particularly
interesting in the field of functional foods. Edible coatings applied directly
onto the food are already currently used to provide barrier protection and
improve the shelf-life and quality of a large variety of foods, from fresh to pro-
cessed products (Weiss et al., 2006). It is believed that nanoscience can greatly
contribute in optimizing their formulations and efficacy (Weiss et al., 2006;
Fabra et al., 2013). The potential of whey protein aggregates to serve as build-
ing blocks for the production of nanocoatings has been recently reviewed
and discussed by Ramos et al. (2014). Polysaccharides such as starch, car-
rageenan, and chitosan also received a great deal of interest (Kittitheeranun
et al., 2012; Pinheiro et al., 2012; Heydari et al., 2013; Mihindukulasuriya
and Lim, 2014; Zambrano-Zaragoza et al., 2014). A recent patent (Guerrero
et al., 2014) described lipid nanoparticle-based nanocoatings for long-term
preservation of cereals, seeds, and fresh foods. Such formulations are pre-
sented as having higher stability and coating efficiency, with a potential for
nutraceuticals controlled release. It is worth mentioning that regardless of
nano-approaches, it has been stressed out that in the case of edible coatings,
regulatory aspects and acceptance can slow down the implementation of
such technologies (Rojas-Graü et al., 2010).
Nanotechnologies can also be associated to smart packaging under the
form of time–temperature indicators (TTI), gas detectors, and nanosensors
(Mihindukulasuriya and Lim, 2014; Ranjan et al., 2014). TTI systems are
intended to be used as abuse indicators. They are usually based on a color
change when a certain condition is broken. The property of colloidal gold
nanoparticles to irreversibly agglomerate under freezing has been used by
Timestrip® to develop a visual accidental freezing indicator for chilled foods
(Kagan et al., 2007). Timestrip® is already available and has been optimized
to be adapted to a variety of temperature thresholds (Anonymous). Other
smart indicators are developed to detect the changes in gas composition, the
presence of organic molecules, and more (Duncan, 2011).
The most promising and implementable nano-packaging innovations are
related to sensor incorporation for the detection of food deterioration and
monitoring storage conditions, increasing barrier properties and strength
of materials, oxygen scavenging, and prevention of growth of pathogens.
Toxicological studies need to be conducted to ensure the safety of nano-based
innovations for food applications. The lack of data and clear guidelines in this
regard can contribute to slow down the implementation of nano-innovations
in the food sector. Depending of the legislation, case-by-case assessments or
precautionary approaches should be considered (Reig et al., 2014). Despite
these limitations, some technologies are already available on the market.

A list of current nano-based materials available for food packaging is pre-

sented in the review of Reig et al. (2014). Further examples of applications
and discussion on the status of nanotechnologies for food applications can
be found in recent reviews and textbooks (Duncan, 2011; Frewer et al., 2011;
Sonkaria et al., 2012; Durán and Marcato, 2013; Lopes et al., 2013; Momin
et al., 2013; Pérez-Esteve et al., 2013; Rhim and Kim, 2013; Mihindukulasuriya
and Lim, 2014; Reig et al., 2014).
13.6 Summary
Guaranteeing significant levels of beneficial bioactive constituents in func-
tional foods is a great challenge to manufacturers. The shelf-life of functional
foods is critically dependent on their storage and packaging conditions that
are applied throughout the entire distribution chain. These conditions have
to be precisely adapted to the specific needs of each and every functional
food. Unfortunately, research on packaging conditions effects on functional
foods is lacking. To optimize the preservation of these sensitive products,
there is an urgent need for appropriate data. Nevertheless, the establishment
of specific labeling regulations and standards to functional foods will hope-
fully stimulate further research in this area. These investigations should be
undertaken to supply data supporting the determination of shelf-life and
expiration dates ensuring health benefit claims. Active packaging and nano-
based systems constitute promising approaches to improve functional food
preservation.
References
Althues, H., Henle, J., Kaskel, S. 2007. Functional inorganic nanofillers for transparent
polymers. Chemical Society Reviews, 36(9):1454–1465.
Alur, M.D., Grecz, N. 1975. Mechanism of injury of Escherichia coli by freezing and
thawing. Biochemical and Biophysical Research Communications, 62(2):308–312.
Amorim, K., Lage-Yusty, M.A., López-Hernández, J. 2012. Short communication:
Changes in bioactive compounds content and antioxidant activity of seaweed
after cooking processing. CYTA—Journal of Food, 10(4):321–324.
Arvanitoyannis, I.S. 2012. Principles of MAP and definitions of MAP, CA and AP. In
Modified Atmosphere and Active Packaging Technologies, ed. I.S. Arvanitoyannis.
CRC Press, Boca Raton, FL.
Barbarosa-Canovas, G.V., Vega-Mercado, H. 1996. In Dehydration of Foods. Chapman &
Hall, New York.
Bell, L.N. 2006. Nutraceutical stability concerns and shelf life testing. In Handbook of
Nutraceuticals and Functional Foods, ed. R.E.C. Wildman. CRC Press, Boca Raton, FL.

Benjamin, S., Spener, F. 2009. Conjugated linoleic acids as functional food:

An insight into their health benefits. Nutrition and Metabolism, 6(36),
doi:10.1186/1743-7075-6-36.
Bolduc, M.P., Raymond, Y., Fustier, P., Champagne, C.P., Vuillemard, J.C. 2006.
Sensitivity of bifidobacteria to oxygen and redox potential in non-fermented
pasteurized milk. International Dairy Journal, 16(9):1038–1048.
Bozoğlu, T.F., Özilgen, M., Bakir, U. 1987. Survival kinetics of lactic acid starter cul-
tures during and after freeze drying. Enzyme and Microbial Technology, 9:531–537.
Brioukhanov, A.L., Netrusov, A.I. 2007. Aerotolerance of strictly anaerobic micro-
organisms and factors of defense against oxidative stress: A review. Applied
Biochemistry and Microbiology, 43(6):567–582.
Bryksa, B.C., Yada, R.Y. 2012. Challenges in food nanoscale science and technology.
Journal of Food and Drug Analysis, 20(Suppl. 1):418–421.
Çağlarırmak, N., Zeki Hepçimen, A. 2013. An investigation of nutrational values of
dried vegetables. The Journal of Food, 38(6):327–333.
Castro-López Mdel, M., López-Vilariño, J.M., González-Rodríguez, M.V. 2014.
Analytical determination of flavonoids aimed to analysis of natural samples
and active packaging applications. Food Chemistry, 150(1):119–127.
Chan, E.S., Zhang, Z. 2002. Encapsulation of probiotic bacteria Lactobacillus acidoph-
ilus by direct compression. Food and Bioproducts Processing: Transactions of the
Institution of Chemical Engineers, Part C, 80(2):78–82.
Colín-Chávez, C., Soto-Valdez, H., Peralta, E., Lizardi-Mendoza, J., Balandrán-
Quintana, R. 2013. Diffusion of natural astaxanthin from polyethylene active pack-
aging films into a fatty food simulant. Food Research International, 54(1):873–880.
Coskun, F., Pazır, F. 2013. Impact of non-thermal processing technologies on quality of
some fruit juices. Journal of Hygienic Engineering and Design, 5:18–24.
da Cruz, A.G., Faria, J.d.A.F., Van Dender, A.G.F. 2007. Packaging system and probi-
otic dairy foods. Food Research International, 40(8):951–956.
Dave, R.I., Shah, N.P. 1997a. Effectiveness of ascorbic acid as an oxygen scavenger
in improving viability of probiotic bacteria in yoghurts made with commercial
starter cultures. International Dairy Journal, 7(6–7):435–443.
Dave, R.I., Shah, N.P. 1997b. Viability of yoghurt and probiotic bacteria in yoghurts
made from commercial starter cultures. International Dairy Journal, 7(1):31–41.
Decker, E.A., Chen, B., Panya, A., Elias, R.J. 2010. Understanding antioxidant mecha-
nisms in preventing oxidation in foods. In Oxidation in Foods and Beverages and
Antioxidant Applications: Understanding Mechanisms of Oxidation and Antioxidant
Activity, Vol. 1, eds. Decker, E., Elias, R., and McClements, D.J. Woodhead
Publishing, Cambridge, UK.
de Kruijf, N., van Beest, M., Rijk, R., Sipiläinen-Malm, T., Paseiro Losada, P., De
Meulenaer, B. 2002. Active and intelligent packaging: Applications and regula-
tory aspects. Food Additives and Contaminants, 19(Suppl.):144–162.
Destaillats, F. 2011. Formulating functional foods with long-chain polyunsaturated
fatty acids: Challenges and opportunities. European Journal of Lipid Science and
Technology, 113(10):1293–1295.
De Vuyst, L. 2000. Technology aspects related to the application of functional starter
cultures. Food Technology and Biotechnology, 38(2):105–112.
Dhiman, T.R., Ure, A.L., Walters, J.L. 2006. Conjugated linoleic Acid found in milk and
meat. In Omega 3 Fatty Acid Research, ed. M.C. Teale. Nova Science, New York.

Duncan, T.V. 2011. Applications of nanotechnology in food packaging and food safety:
Barrier materials, antimicrobials and sensors. Journal of Colloid and Interface
Science, 363(1):1–24.
Durán, N., Marcato, P.D. 2013. Nanobiotechnology perspectives. Role of nanotech-
nology in the food industry: A review. International Journal of Food Science and
Technology, 48(6):1127–1134.
Elizaveta, S. 2012. Water activity concept and its role in food preservation. Meridian
Ingineresc, 4:40–48.
Endo, A., Teräsjärvi, J., Salminen, S. 2014. Food matrices and cell conditions influence
survival of Lactobacillus rhamnosus GG under heat stresses and during storage.
International Journal of Food Microbiology, 174:110–112.
Espitia, P.J.P., Soares, N.F.F., Coimbra, J.S.R., de Andrade, N.J., Cruz, R.S., Medeiros,
E.A.A. 2012. Zinc oxide nanoparticles: Synthesis, antimicrobial activity and
food packaging applications. Food and Bioprocess Technology, 5(5):1447–1464.
Fabra, M.J., Busolo, M.A., Lopez-Rubio, A., Lagaron, J.M. 2013. Nanostructured bio-
layers in food packaging. Trends in Food Science and Technology, 31(1):79–87.
Frewer, L.J., Norde, W., Fischer, A., Kampers, F. 2011. Nanotechnology in the Agri-Food
Sector: Implications for the Future. Wiley-VCH Verlag GmbH & Co, Weinheim,
Germany.
Fu, N., Chen, X.D. 2011. Towards a maximal cell survival in convective thermal dry-
ing processes. Food Research International, 44(5):1127–1149.
Furia, T.E. 1980. Antioxidants as stabilizers for fats, oils, and lipid-containing foods.
In Handbook of Food Additives, ed. T.E. Furia. CRC Press, Boca Raton, FL.
García, M., Forbe, T., Gonzalez, E. 2010. Potential applications of nanotechnology in
the agro-food sector. Ciencia e Tecnologia de Alimentos, 30(3):573–581.
Gil, M.I., Ferreres, F., Tomás-Barberán, F.A. 1999. Effect of postharvest storage and
processing on the antioxidant constituents (flavonoids and vitamin C) of fresh-
cut spinach. Journal of Agricultural and Food Chemistry, 47(6):2213–2217.
Giovanelli, G., Brambilla, A., Sinelli, N. 2013. Effects of osmo-air dehydration treat-
ments on chemical, antioxidant and morphological characteristics of blueber-
ries. LWT—Food Science and Technology, 54(2):577–584.
Gliguem, H., Birlouez-Aragon, I. 2005. Effects of sterilization, packaging, and stor-
age on vitamin C degradation, protein denaturation, and glycation in fortified
milks. Journal of Diary Science, 88(3):891–899.
Gomes, A.M.P., Malcata, F.X. 1999. Bifidobacterium spp. and Lactobacillus acidophilus:
Biological, biochemical, technological and therapeutical properties relevant for
use as probiotics. Trends in Food Science and Technology, 10(4–5):139–157.
Gong, P., Zhang, L., Han, X. et al. 2014. Injury mechanisms of lactic acid bacteria
starter cultures during spray drying: A review. Drying Technology, 32(7):793–800.
Goswami, T.K., Mangaraj, S. 2011. Advances in polymeric materials for modified
atmosphere packaging (MAP). In Multifunctional and Nanoreinforced Polymers for
Food Packaging. Woodhead Publishing Limited, Cambridge, UK.
Greiff, D. 1971. Protein structure and freeze-drying: The effects of residual moisture
and gases. Cryobiology, 8(2):145–152.
Heidebach, T., Först, P., Kulozik, U. 2012. Microencapsulation of probiotic cells for
food applications. Critical Reviews in Food Science and Nutrition, 52:291–311.
Heinonen, L.M. 2002. Antioxidants in fruits, berries and vegetables. In Fruit and
Vegetable Processing—Improving Quality, ed. W. Jongen. CRC Press, Cambridge.

Heydari, A., Alemzadeh, I., Vossoughi, M. 2013. Functional properties of biodegrad-

able corn starch nanocomposites for food packaging applications. Materials and
Design, 50:954–961.
Imran, M., Revol-Junelles, A.M., Martyn, A. et al. 2010. Active food packaging evo-
lution: Transformation from micro- to nanotechnology. Critical Reviews in Food
Science and Nutrition, 50(9):799–821.
Intawiwat, N., Myhre, E., Øysaæd, H., Jamtvedt, S.H., Pettersen, M.K. 2012. Packaging
materials with tailor made light transmission properties for food protection.
Polymer Engineering and Science, 52(9):2015–2024.
Jiang, H., Hu, Y., Li, Y., Zhao, P., Zhu, K., Chen, W. 2005. A facile technique to prepare
biodegradable coaxial electrospun nanofibers for controlled release of bioactive
agents. Journal of Controlled Release, 108(2–3):237–243.
Kader, A.A. 2009. Effects on nutritional quality. In Modified and Controlled Atmospheres
for the Storage, Transportation, and Packaging of Horticultural Commodities, ed. E.M.
Yahia. CRC Press, Boca Raton, FL.
Kagan, M., Mirsky, V., Wolfbeis, O. 2007. Indicateur de froideur irréversible. Google
Patents EP 2035796 A2.
Kalpana Sastry, R., Anshul, S., Rao, N.H. 2013. Nanotechnology in food processing
sector-An assessment of emerging trends. Journal of Food Science and Technology,
50(5):831–841.
Kanmani, P., Rhim, J.W. 2014. Physical, mechanical and antimicrobial properties of
gelatin based active nanocomposite films containing AgNPs and nanoclay. Food
Hydrocolloids, 35:644–652.
Karmas, E., Harris, R.S. 1988. Nutritional Evaluation of Food Processing, 3rd edition. Van
Nostrand Reinhold, New York.
Kikugawa, K., Kunugi, A., Kurechi, T. 1990. Chemistry and implications of degrada-
tion of phenolic antioxidants. In Food Antioxidants, ed. B.J.F. Hudson. Springer-
Verlag, Essex, UK.
Kittitheeranun, P., Dubas, S.T., Dubas, L. 2012. Layer-by-layer surface modification
of fruits with edible nano-coatings. Applied Mechanics and Materials, ed. M.
Othman, Vols. 229–231, pp. 2745–2748.
Klu, Y.A.K., Williams, J.H., Phillips, R.D., Chen, J. 2012. Survival of Lactobacillus rham-
nosus GG as influenced by storage conditions and product matrixes. Journal of
Food Science, 77(12):M659–M663.
Labuza, T.P. 1974. Oxidative changes in foods at low and intermediate moisture lev-
els. In Water Relations of Foods: Proceedings of an International Symposium, Glasgow,
September 1974, ed. R. Duckworth. Academic Press, London, UK.
Labuza, T.P. 2000. The search for shelf life—An update on continued efforts in under-
standing practical strategies for determining and testing the shelf life of food
products. Food & Testing Analysis, 6(2): 26–36.
Lavillonnière, F., Martin, J.C., Bougnoux, P., Sébédio, J.L. 1998. Analysis of conjugated
linoleic acid isomers and content in French cheeses. Journal of the American Oil
Chemists’ Society, 75(3):343–352.
Lopes, C.M., Fernandes, J.R., Martins-Lopes, P. 2013. Application of nanotechnology
in the agro-food sector. Food Technology and Biotechnology, 51(2):183–197.
Maltini, E., Torreggiani, D., Venir, E., Bertolo, G. 2003. Water activity and the preserva-
tion of plant foods. Food Chemistry, 82(1):79–86.
Mihindukulasuriya, S.D.F., Lim, L.T. 2014. Nanotechnology development in food
packaging: A review. Trends in Food Science and Technology, 40: 149–167.

Mills, A., Doyle, G., Peiro, A.M., Durrant, J. 2006. Demonstration of a novel, flexible,
photocatalytic oxygen-scavenging polymer film. Journal of Photochemistry and
Photobiology A: Chemistry, 177(2–3):328–331.
Miltz, J., Hoojjat, P., Han, J.K., Giacin, J.R., Harte1, B.R., Gray, I.J. 1988. Loss of antioxi-
dants from high-density polyethylene. Its effect on oatmeal cereal oxidation. In
Food and Packaging Interactions, ed. J.H. Hotchkiss. American Chemical Society,
Cornell.
Momin, J.K., Jayakumar, C., Prajapati, J.B. 2013. Potential of nanotechnology in func-
tional foods. Emirates Journal of Food and Agriculture, 25(1):10–19.
Nicola, S., Fontana, E. 2014. Fresh-cut produce quality: Implications for a systems
approach. In Postharvest Handling. A Systems Approach, ed. R.L.S.W.J. Florkowski,
B. Brueckner and S.E. Prussia. Academic Press, San Diego, CA.
Oberg, T.S., Steele, J.L., Ingham, S.C. et al. 2011. Intrinsic and inducible resistance to
hydrogen peroxide in Bifidobacterium species. Journal of Industrial Microbiology
and Biotechnology, 38(12):1947–1953.
Oms-Oliu, G., Odriozola-Serrano, I., Soliva-Fortuny, R., Elez-Martínez, P., Martín-
Belloso, O. 2012. Stability of health-related compounds in plant foods through
the application of nonthermal processes. Trends in Food Science and Technology,
23(2):111–123.
Parker, N.J.B. 2009. An oxygen producing and carbon dioxide absorbing device for
inclusion in food packages. Kingdom Patent 2450860-A, UK.
Peighambardoust, S.H., Golshan Tafti, A., Hesari, J. 2011. Application of spray drying
for preservation of lactic acid starter cultures: A review. Trends in Food Science
and Technology, 22(5):215–224.
Pérez-Esteve, E., Bernardos, A., Martínez-Máñez, R., Barat, J.M. 2013. Nanotechnology
in the development of novel functional foods or their package. An overview based
in patent analysis. Recent Patents on Food, Nutrition and Agriculture, 5(1):35–43.
Pinheiro, A.C., Bourbon, A.I., Medeiros, B.G.D.S. et al. 2012. Interactions between
κ-carrageenan and chitosan in nanolayered coatings—Structural and transport
properties. Carbohydrate Polymers, 87(2):1081–1090.
Poddar, D., Das, S., Jones, G. et al. 2014. Stability of probiotic Lactobacillus paracasei dur-
ing storage as affected by the drying method. International Dairy Journal, 39(1):1–7.
Quintanar, G.D., de la Luz Zambrano, Z.M., Alvarez, C.A., Mercado, S.E. 2014.
Composition of solid lipid nanoparticles for the long-term conservation of
fruits, vegetables, seeds, cereals and/or fresh foodstuffs using a coating. Google
Patents EP 2698066 A2.
Ramos, O.L., Pereira, R.N., Rodrigues, R., Teixeira, J.A., Vicente, A.A., Xavier Malcata,
F. 2014. Physical effects upon whey protein aggregation for nano-coating pro-
duction. Food Research International, 66:344–355.
Ranjan, S., Dasgupta, N., Chakraborty, A.R. et al. 2014. Nanoscience and nano-
technologies in food industries: Opportunities and research trends. Journal of
Nanoparticle Research, 16(6):1–23.
Rao, Q., Rocca-Smith, J.R., Labuza, T.P. 2012. Moisture-induced quality changes of
hen egg white proteins in a protein/water model system. Journal of Agricultural
and Food Chemistry, 60(42):10625–10633.
Rawson, A., Patras, A., Tiwari, B.K., Noci, F., Koutchma, T., Brunton, N. 2011. Effect
of thermal and nonthermal processing technologies on the bioactive content
of exotic fruits and their products: Review of recent advances. Food Research
International, 44(7):1875–1887.

Realini, C.E., Marcos, B. 2014. Active and intelligent packaging systems for a modern
society. Meat Science, 98(3):404–419.
Reddy, M.B., Love, M. 1999. The impact of food processing on the nutritional qual-
ity of vitamins and minerals. Advances in Experimental Medicine and Biology,
459:99–106.
Reig, C.S., Lopez, A.D., Ramos, M.H., Cloquell Ballester, V.A. 2014. Nanomaterials: A
map for their selection in food packaging applications. Packaging Technology and
Science, 27(11):839–866.
Rhim, J.W., Kim, Y.T. 2013. Biopolymer-based composite packaging materials with
nanoparticles. In Innovations in Food Packaging, 2nd edition. Academic Press,
London, UK.
Rickman, J.C., Barrett, D.M., Bruhn, C.M. 2007a. Nutritional comparison of fresh, fro-
zen and canned fruits and vegetables. Part 1. Vitamins C and B and phenolic
compounds. Journal of the Science of Food and Agriculture, 87(6):930–944.
Rickman, J.C., Bruhn, C.M., Barrett, D.M. 2007b. Nutritional comparison of fresh, fro-
zen, and canned fruits and vegetables II. Vitamin A and carotenoids, vitamin E,
minerals and fiber. Journal of the Science of Food and Agriculture, 87(7):1185–1196.
Rojas-Graü, M.A., Soliva-Fortuny, R., Martín-Belloso, O. 2010. Edible coatings: Past,
present and future. Stewart Postharvest Review, 6(3):1–5.
Saarela, M.H. 2011. Probiotic functional foods. In Functional Foods: Concept to Product.
2nd edition. Woodhead Publishing Limited, Cambridge, UK.
Sampedro, F., Zhang, H.Q. 2012. Recent developments in non-thermal processes.
In Food and Industrial Bioproducts and Bioprocessing, ed. N.T. Dunford. Wiley-
Blackwell, Oxford, UK.
Sanchez Garcia, M.D., Lagaron, J.M. 2012. Nanocomposites for food and bever-
age packaging materials. In Food Materials Science and Engineering. Blackwell
Publishing Ltd., Chichester, UK.
Sánchez-Moreno, C., De Ancos, B., Plaza, L., Elez-Martínez, P., Pilar Cano, M. 2010.
Effects of high-pressure processing and pulsed electric fields on nutritional qual-
ity and health-related compounds of fruit and vegetable products. In Nonthermal
Processing Technologies for Food, ed. G.V.B.-C.H.Q. Zhang, V.M. Balasubramaniam,
C.P. Dunne, D.F. Farkas, and J.T.C. Yuan. Wiley-Blackwell, Oxford, UK.
Schroeder, H.A. 1971. Losses of vitamins and trace minerals resulting from processing
and preservation of foods. American Journal of Clinical Nutrition, 24(5):562–573.
Senturk, A., Yalcin, B., Otles, S. 2013. Nanotechnology as a food perspective. Journal of
Nanomaterials & Molecular Nanotechnology, 2:6. doi: http://dx.doi.org/10.4172/
2324-8777.1000125.
Serrano, M., Díaz-Mula, H.D., Valero, D. 2011. Antioxidant compounds in fruits and
vegetables and changes during postharvest storage and processing. Stewart
Postharvest Review, 7(1):1.
Shahidi, F., Naczk, M. 2004. Phenolics in Food and Nutraceuticals. Taylor & Francis
Group, Boca Raton, FL.
Siegrist, M., Nowack, B., Kastenholz, H. 2011. Environmental considerations of and
societal reactions to nanotechnology in the food sector. In Nanotechnology in the
Agri-Food Sector: Implications for the Future. Wiley-VCH Verlag GmbH & Co.,
Weinheim, Germany.
Siegrist, M., Stampfli, N., Kastenholz, H., Keller, C. 2008. Perceived risks and per-
ceived benefits of different nanotechnology foods and nanotechnology food
packaging. Appetite, 51(2):283–290.

Silva-Weiss, A., Ihl, M., Sobral, P.J.A., Gómez-Guillén, M.C., Bifani, V. 2013. Natural
additives in bioactive edible films and coatings: Functionality and applications
in foods. Food Engineering Reviews, 5(4):200–216.
Sonkaria, S., Ahn, S.H., Khare, V. 2012. Nanotechnology and its impact on food and
nutrition: A review. Recent Patents on Food, Nutrition and Agriculture, 4(1):8–18.
Tadapaneni, R.K., Daryaei, H., Krishnamurthy, K., Edirisinghe, I., Burton-Freeman,
B.M. 2014. High-pressure processing of berry and other fruit products:
Implications for bioactive compounds and food safety. Journal of Agricultural
and Food Chemistry, 62(18):3877–3885.
Talwalkar, A., Kailasapathy, K. 2003. Metabolic and biochemical responses of probi-
otic bacteria to oxygen. Journal of Dairy Science, 86(3):2537–2546.
Talwalkar, A., Kailasapathy, K. 2004. A review of oxygen toxicity in probiotic yogurts:
Influence on the survival of probiotic bacteria and protective techniques.
Comprehensive Reviews in Food Science and Food Safety, 3(3):117–124.
Talwalkar, A., Miller, C.W., Kailasapathy, K., Nguyen, M.H. 2004. Effect of packaging
materials and dissolved oxygen on the survival of probiotic bacteria in yoghurt.
International Journal of Food Science and Technology, 39(6):605–611.
Thompson, H., Zhu, Z., Banni, S., Darcy, K., Loftus, T., Ip, C. 1997. Morphological
and biochemical status of the mammary gland as influenced by conjugated lin-
oleic acid: Implication for a reduction in mammary cancer risk. Cancer Research,
57(22):5067–5072.
Tripathi, M.K., Giri, S.K. 2014. Probiotic functional foods: Survival of probiotics dur-
ing processing and storage. Journal of Functional Foods, 9(1):225–241.
Venir, E., Maltini, E. 2013. Relevance of physical properties in the stability of plant-
based food products. Indian Journal of Experimental Biology, 51(11):894–904.
Vigneault, C., Leblanc, D.I., Goyette, B., Jenni, S. 2012. Invited review: Engineering
aspects of physical treatments to increase fruit and vegetable phytochemical
content. Canadian Journal of Plant Science, 92(3):373–397.
Weiss, J., Takhistov, P., McClements, D.J. 2006. Functional materials in food nanotech-
nology. Journal of Food Science, 71(9):R107–R116.
Zambrano-Zaragoza, M.L., Mercado-Silva, E., del Real-López, A., Gutiérrez-Cortez,
E. Cornejo-Villegas, M.A., Quintanar-Guerrero, D. 2014. The effect of nano-
coatings with α-tocopherol and xanthan gum on shelf-life and browning
index of fresh-cut “red Delicious” apples. Innovative Food Science and Emerging
Technologies, 22:188–196.
Zhao, L., Li, F., Chen, G. et al. 2012. Effect of nanocomposite-based packaging on pres-
ervation quality of green tea. International Journal of Food Science and Technology,
47(3):572–578.

14
High-Pressure Processing of Foods
toward Their Industrialization
and Commercialization:
An Up-to-Date Overview
Giovanna Ferrentino, Sara Spilimbergo, and Alberto Bertucco
CONTENTS
14.1 Introduction................................................................................................. 427
14.2 HHP Technology......................................................................................... 428
14.2.1 Process Principles and Equipment............................................... 429
14.2.2 State of the Art of HHP..................................................................430
14.2.2.1 Inactivation Mechanisms................................................430
14.2.2.2 Inactivation of Microorganisms in Foods.................... 431
14.2.2.3 Effects on Food Quality Attributes............................... 432
14.2.3 Commercial Applications.............................................................. 435
14.3 DPCD Technology...................................................................................... 436
14.3.1 Process Principles and Equipment............................................... 437
14.3.2 State-of-the-Art DPCD................................................................... 438
14.3.2.1 Inactivation Mechanisms................................................ 438
14.3.2.2 Inactivation of Microorganisms in Foods.................... 438
14.3.2.3 Effects on Food Quality Attributes............................... 441
14.3.3 Commercial Applications..............................................................442
14.4 Potentials of High-Pressure Technologies and Conclusions................445
References..............................................................................................................446
14.1 Introduction
Increasing consumer demand for minimally processed, additive-free, shelf-
stable products prompted food scientists to explore other physical preser-
vation methods as alternative to traditional treatments such as freezing,
canning, or drying that rely on heating or cooling operations. Although
these technologies help ensure a high level of food safety, the heating and
cooling of foods may contribute to the degradation of various food quality
attributes. The color, flavor, and texture of foods processed solely by heating
427
(thermal pasteurization up to 80°C and sterilization up to 120°C) may be

irreversibly altered. To ameliorate the undesirable thermal effects on foods,
considerable efforts have been made in commercial and academic circles to
develop nonthermal technologies other than heating or cooling operations.
Some of the investigated technologies are ionization radiation, high
hydrostatic pressure (HHP), pulsed electrical fields, high-pressure homog-
enization, UV decontamination, pulsed high-intensity light, high-intensity
laser, pulsed white light, manothermosonication (combined ultrasonic, heat,
and pressure), oscillating magnetic fields, high voltage arc discharge, and
streamer plasma. Among these emerging technologies, the most promising
ones for food application are high-pressure processing, namely HHP and
dense-phase carbon dioxide (DPCD).
This chapter will describe and discuss the current knowledge in the pas-
teurization and sterilization of foodstuff by means of HHP and DPCD tech-
nologies. The most significant achievements will be summarized, including
fundamental concepts, different hypotheses of bacteria inactivation mecha-
nisms, and the main applications in the food industry focusing on the effects
exerted on the microbial and quality aspects. Further, commercial applica-
tions of the technologies will be described and some conclusions about the
two high-pressure processing will be drawn.
14.2 HHP Technology

The application of HHP processing for food technology began when Hite
(1899) demonstrated that the shelf-life of milk and other food products
could be extended by pressure treatment. This technology applies HHP to
materials by compressing the surrounding water and transmitting pressure
throughout the product uniformly and rapidly (Hyashi, 1989). The use of
high pressure in food processing is an extension of a technology that is com-
monly employed in many other industrial processes, notably in the manu-
facturing of ceramics, diamonds, superalloys, simulators, and sheet metal
forming.
The advances achieved in ceramics and metallurgical industries in the use
of HHP techniques during the 1970s and 1980s have led to the possibility
of treating food by this method at an industrial level. The first commercial
HHP-treated products appeared on the market in 1991 in Japan, where HHP
processing is now being used for products such as fruit juices, jams, sauces,
rice, cakes, and desserts. There is an increasing worldwide interest in the
use of HHP because of the advantages of this technology over other meth-
ods of processing and preservation. An important issue in the application
of high-pressure technology in the food preservation/processing indus-
try is the regulatory approval, which focuses upon microbiological and

High-Pressure Processing of Foods 429
toxicological safety of food products. Generally high-pressure preservation

processes reduce the microbial load to the same level achieved by tradi-
tional technologies, while delivering higher-quality products. However,
in order to implement this new technology in the food industry, there is
an increased need to understand the mechanism and kinetics of pressure-
induced degradation/denaturation inactivation of several food compounds
(e.g., nutrients, proteins, microorganisms, enzymes) and the way in which
the degradation/denaturation/inactivation is affected by other parameters
(e.g., temperature, pH).
14.2.1 Process Principles and Equipment

HHP in food processing uses a pressure range of 100–1000 MPa, thus s pecial
equipments are needed to generate and endure such high pressures. A typi-
cal HHP system consists of a high-pressure vessel and its closure, a pressure
generation system, and a temperature control device. The heart of the HHP
system is the pressure vessel and its wall thickness determines the maxi-
mum working pressure. Depending on the internal diameter of the v essel,
the maximum working pressure is in the range of 400–600 MPa. In case
higher pressures are required, prestressed vessel designs as multilayer
vessels or wire-wound vessels are used (Mertens, 1995). HHP can be gen-
erated by either direct compression or indirect compression (Figure 14.1).
In the case of direct, piston-type compression, the pressure medium in the
high-pressure vessel is directly pressurized by a piston, driven at its larger
diameter end by a low-pressure pump. The indirect compression method
uses a high-pressure intensifier which pumps the pressure medium from
(a) (b)
Low pressure pump

Piston
Pressurization chamber
Heat transfer system
Internal reaction volume Pressure
intensifier
Closure
Reservoir
FIGURE 14.1
Example of a direct (a) and indirect (b) system for the generation of HHP. (Adapted from
Norton, T., Sun, D.-W. 2008. Food Bioprocess Technology, 1:2–34.)

the reservoir into the closed and de-aerated high-pressure vessel, until the
desired pressure is reached (Mertens, 1995).
There are three major types of high-pressure processing of food prod-
ucts: the batch, semi-continuous, and continuous systems. Batch systems
can process both liquid and solid products, but these have to be pre-packed.
In-line continuous systems can be applied only to pumpable products (e.g.,
fruit juice). The product is pumped into the pressure vessel and pressur-
ized using a floating piston, which separates the product from the pressure
medium. For batch systems, the overall cycle time is the sum of the number
of single steps: filling, closing, pressure built up, pressure holding, pressure
releasing, opening, and taking out. For liquid products, continuous treat-
ment also is possible using tube reactors or special valve systems (Van den
Berg et al., 2002).
14.2.2 State of the Art of HHP

14.2.2.1 Inactivation Mechanisms
A fundamental understanding of the inactivation of microorganisms and
enzymes by pressure is needed to make this technology a reliable alterna-
tive to conventional processes. This effort needs to take into account also the
effect of food components and environmental parameters (Oxen and Knorr,
1993; Seyderhelm et al., 1996).
HHP brings about a number of changes in the morphology, cell mem-
brane, or biochemical reactions of microorganisms and all these processes
are related to the inactivation of microorganisms. For instance, Hamada
et al. (1992) noticed changes in colony form after pressure treatment of
Saccharomyces cerevisiae. Especially, the cell membrane is considered to be the
major target for the pressure-induced inactivation of microorganisms, and it
is generally accepted that the leakage of intracellular constituents through
the permeabilized cell membrane is the most direct reason of cell death by
HHP. However, if the applied pressure was not severe enough to induce a
total permeabilization of cell, the permeabilization took place only in the
outer membrane; in the case of Gram-negative bacteria, the permeabilized
membrane was rapidly restored after pressure release (Hauben et al., 1996).
The fluidity of cell membrane plays an important role in the susceptibility of
microorganisms to pressure treatments.
Microorganisms with less fluid membranes are more sensitive to HHP
(Macdonald, 1992). Conversely, increased membrane fluidity protected
against pressure inactivation (Steeg et al., 1999). The denaturation of key
enzymes in microorganisms by pressure has been regarded as another
important reason of cell death. Because intracellular enzymes seemed not
to be a determining factor of pressure resistance (Simpson and Gilmour,
1997), membrane-bounded ATPase was considered to be such a key enzyme
(Wouters et al., 1998).

Intracellular fluid compounds have been found in the cell suspending fluid
after pressure treatment demonstrating that leaks occur while cells are held
under pressure (Shimada et al., 1993). Membrane damage has been shown to
occur later than cell death, suggesting that dye exclusion measurements used
to assess this pressure effect may be used for the characterization of microbial
pressure inactivation (Ulmer et al., 2000). Knowledge of cell damage and repair
mechanism could lead to food preservation based on the hurdle concept and
to novel HHP applications (Hauben et al., 1996; Chilton et al., 2001). Studies
have shown that lysis of starter bacteria releasing intracellular proteinases
is important in cheese ripening. To this extent, it is worth to quote the study
of Malone et al. (2002) who reported the inactivation, physical damage, and
lysis of Lactococcus lactis caused by HHP. Treatments performed at 300 MPa
showed intracellular and cell envelope damage of the cheese-making strains
L. lactis subsp. cremoris MG1363 and SK11. Cell suspensions treated at 200 or
300 MPa did not differ significantly from the control, whereas cells treated at
>400 MPa had decreased cell wall hydrolase activity. In addition, cells treated
at 100 MPa released significantly more reducing sugar than all other samples,
indicating that this pressure activates cell wall hydrolase activity or increases
cell wall accessibility to the enzyme.
14.2.2.2 Inactivation of Microorganisms in Foods

The first report on HHP-processed commercial food product was fruit jams by
Horie et al. (1991) from the Meidiya Food Factory Co. in Japan. Strawberry jams
were processed at 294 MPa for 20 min and elimination of yeast was reported
as well as bacteria. The application of HHP to fruit products has been con-
sidered the most effective and realistic, because the inherent low pH of fruits
can inhibit the growth of most spoilage bacteria. Further, the yeast and mold
which survive such low pH range are relatively susceptible to HHP (Aleman
et al., 1996; Garcia-Graells et al., 1998; Linton et al., 1999; Prestamo et al., 1999;
Zook et al., 1999). In this way, the shelf-life extension of fresh-cut pineapple
was achieved by application of 340 MPa/15 min (Aleman et al., 1994). Parish
(1998) applied HHP to orange juice reporting D-values of vegetative cells of
S. cerevisiae between 1 and 38 s for the treated sample at pressures between
500 and 350 MPa. The native flora of the orange juice showed D-values rang-
ing from 3 to 74 s. HHP at 20 MPa for 10 min, pressures of 300 and 350 MPa
reduced the population of Gram-negative bacteria, yeasts, and molds of let-
tuce and tomatoes by at least one log cycle (Arroyo et al., 1997). Other exam-
ples of HHP applications on vegetable products are the increased shelf-life of
vegetable juices by HHP without quality loss (Lee et al., 1996; Sohn and Lee,
1998). However, the limiting parameter of these products processing is often
the presence of browning enzymes that need higher pressure levels to be inac-
tivated (Eshtiaghi and Knorr, 1993; Seyderhelm et al., 1996).
Milk was found to provide the microorganisms protection against HHP.
Only 2 log reductions at 340 MPa were observed when Listeria was treated

in milk, whereas almost 7 log reductions were observed when the same
microorganisms were treated in buffer solution (Styles et al., 1991). The pro-
tective effect of milk against HHP was observed again with other species
of microorganisms (Patterson and Kilpatrick 1998). However, there were no
significant differences between skim and whole milk. HHP of 400 MPa at
7°C reduced aerobic plate counts of whole milk and skim milk equally by
1 log cycle. Some researches to date have concentrated on the application of
HHP to inactivate microorganisms in cheese to increase cheese safety and
shelf-life. Gallot-Lavallee (1998) studied the efficiency of HHP treatment for
the destruction of L. monocytogenes in goat cheese from raw milk finding
that 450 MPa for 10 min or 500 MPa for 5 min treatments achieved more than
5.6 log reductions of this microorganism without significantly affecting the
sensory characteristics of cheese. Prestamo et al. (2000) reported that the
microbial population of tofu pressurized at 400 MPa and 5°C for 5, 30, and
45 min decreased from an initial microorganism count of 5.54 × 104 cfu/g to
0.31, 1.56, or 2.38 log units, respectively.
As concerns HHP application to meat products, the treatment inactivated
Citrobacter, Pseudomonas, and Listeria in minced meats (Carlez et al., 1993).
An increase (50°C) or decrease (4°C) in the temperature enhanced the effects
of pressure treatments. However, partial discoloration of minced beef was
observed above 150 MPa. Processed meat products such as spreadable sau-
sage were more adequate to pressure treatment than fresh meat. Inactivation
kinetics of Escherichia coli and Listeria innocua inoculated in spreadable sau-
sage were investigated and 5 log reductions of microorganisms were estab-
lished (Zenker et al., 2000). Shigehisa et al. (1991) and Patterson et al. (1995)
reported the effectiveness of pressure treatment against important food-
borne pathogens such as Campylobacter jejuni, E. coli O157:H7, Listeria mono-
cytogenes, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus,
and Yersinia enterocolitica in poultry meat and cooked sausages.
As concerns HHP application to fish products, a number of studies dem-
onstrated the extended shelf-life of the treated foods such as cod (Ohshima
et al., 1993), minced mackerel (Fuji et al., 1994), prawns (Lopez-Caballero
et al., 2000), smoked salmon cream (Capri et al., 1995), or surimi (Miyao et al.,
1993). In Table 14.1, the most recent results on microbial inactivation in foods
induced by HHP are listed.
14.2.2.3 Effects on Food Quality Attributes

Several studies were performed to investigate color changes of HHP pro-
cessed fruit and vegetable products such as orange juice, fruit jam, and
tomato juice. Chemical and spectrophotometric analysis showed that HHP
largely preserves fresh color. Also, the effect of high pressure on meat and
meat products has been studied by many authors because high-pressure
processing may offer potential to preserve and restructure meat, provided
the red color can be maintained. For fruit jams (e.g., strawberry jam), HHP

TABLE 14.1
Summary of the Recent Studies on Microbial Inactivation by HHP
Foodstuff Microorganism Treatment Log Reduction Reference
Apple juice E. coli 0.5–7 min, 21°C 7 log Moody et al.
300–600 MPa (2014)
Purple sweet Yeasts and molds 2.5–25 min >4 log Wang et al.
potato Total aerobic 400–600 MPa (2013)
nectar bacteria
Mango pulp Natural microbial 1–20 min Inactivation to Liu et al.
flora 300–600 MPa undetectable level (2013)
Skimmed S. aureus 5 min Inactivation to Syed et al.
milk 700 MPa undetectable level (2014)
Orange juice
Dry cured Enterococcus 9.5 min 4 log Belletti et al.
ham faecalis 750 MPa (2013b)
Tomato pulp Bacillus coagulans 15 min 5.7 log Zimmermann
spore 600 MPa et al. (2013)
60°C
Mango pulp Natural microbial 5 min Inactivation to Kaushik et al.
flora 600 MPa undetectable level (2014)
Dry cured Serratia 8 min Inactivation to Belletti et al.
ham liquefaciens 650 MPa undetectable level (2013a)
Hard clams Vibrio 4 min Inactivation to Mootian et al.
parahaemolyticus 450 MPa undetectable level (2013)
Chinese Natural microbial 40°C Inactivation to Yulin et al.
water-boiled flora 400 MPa undetectable level (2014)
salted duck
Strawberry E. coli O157:H7 2 min Inactivation to Huang et al.
puree Salmonella spp. 450 MPa undetectable level (2013)
21°C
Meat slurry S. aureus 6 min Inactivation to Yao et al.
350 MPa undetectable level (2015)
Lotus root Natural microbial 10 min Inactivation to Dong et al.
flora 400 MPa undetectable level (2013)
Beetroot juice L. innocua 10 min >4 log Sokołowska
E. coli 400 MPa et al. (2014)
Pomegranate Natural microbial 5 min Inactivation to Chen et al.
juice flora 400 MPa undetectable level (2013)
was found to retain the fresh flavor much more than traditional thermal
processing (Watanabe et al., 1991; Kimura et al., 1994; Dervisi et al., 2001).
In the experiments carried out by Zabetakis et al. (2000) on the effects of HHP
on strawberry flavor compounds, the highest flavor stability was observed
when samples were treated with pressures lower than 800 MPa and they
were stored at 4°C and 30°C. In studies conducted by Rodrigo et al. (2007),
no color degradation of tomato appeared under combined thermal and HHP
treatment (300–700 MPa, 60 min, 65 °C). Basak and Ramasawamy (1998) have

carried out an extensive study on the effect of pressure on the texture of

fruits and vegetables. They observed that the change in firmness of treated
samples was dependent on both pressure level and pressurization time.
As concerns the effect of pressure on the flavor of milk and dairy prod-
ucts, HHP at 400–500 MPa for 3–15 min followed by refrigeration were
found to result in a shelf-life comparable to that of thermal pasteurization
(Rademacher and Kessler, 1996). HHP only slightly influenced milk viscos-
ity. Increase in viscosity was explained to be the result of casein micelle dis-
integration. Johnston et al. (1994) have reported the properties of acid-set
gels prepared from HHP-treated milk. Results indicated an improved tex-
ture (rigidity and resistance to breaking) and syneresis resistance of the gels,
measured by drainage or by centrifugation.
Several studies have been performed to evaluate the effect of HHP on
food protein. It has been shown that pressure affects the protein leading to
protein denaturation, aggregation, or gel formation. Ovalbumin, the main
component of egg white, was denatured under high pressures, as confirmed
by the decrease in its α-helical content and DSC endothermic enthalpies
(Hayakawa et al., 1994). Also, in the study conducted on the effect of pressure
at different temperature (10°C, 25°C, and 60°C) and pH (7.6 and 8.8) levels on
selected properties of egg white solutions, it was observed that the pressure
induced an increase in turbidity, surface hydrophobicity, and susceptibility
to enzymatic hydrolysis, whereas it resulted in a decrease in protein solubil-
ity, denaturation enthalpy, and trypsin inhibitory activity. Moreover, it was
reported that the pressure-induced changes in the selected properties were
dependent on the pressure and temperature applied and the pH (Plancken
et al., 2005).
Studies also reported that HHP treatment at different temperatures
induced different effects on meat texture since the weak linkages stabiliz-
ing the secondary, tertiary, and quaternary structures of a protein respond
differently to heat and pressure (Galazka and Ledward, 1998). Bouton et al.
(1977) found that a pressure of about 100 MPa applied for 2.5 min or longer
to post-rigor muscle at 40–60°C improved the tenderness of the meat. Beilken
et al. (1990) reported that pressure treatment at 150 MPa during treatment
at 40–80°C prevents the development of the myofibrillar component of
toughness, but has little or no effect on the connective tissue component of
toughness, other than to raise the temperature at which heat treatment alone
produces a decrease in this component.
As concerns the application of HHP to sea food products, most of the stud-
ies have been carried out to show its effects on fish proteins (Angsupanich and
Ledward, 1998; Angsupanich et al., 1999; Etienne et al., 2001). The breakdown
of ATP-related products is catalyzed by certain dephosphorylases inherent
in fish muscles. Shoji and Saeki (1989) observed a decrease in carp muscle
treated at 350 and 500 MPa and subsequent storage at 5°C. This was due to
protein denaturation and deactivation of enzymes (involved in the degrada-
tion of ATP and related compounds) during HHP treatment. High-pressure

treatment of surimi produced a new gel product with excellent flavor, luster,
density, and elasticity, quite different from those treated by heat (Yoshioka
and Yamada, 2002). Furthermore, the pressure-induced gels retained the nat-
ural qualities (as color and flavor) of the raw material without the formation
of cooked color and flavor (Okamoto et al., 1990). Pressure-induced surimi
gels from marine species like pollack, sardine, skipjack, tuna, and squid have
been reported to be smoother, more elastic, and sensory superior to those
produced by heat (Thakur and Nelson, 1998; Venugopal et al., 2001).
14.2.3 Commercial Applications
Several years ago, a number of constraints in manufacturing HHP equip-
ment were related to the capacity of pumps to reach a given high pressure
and the fatigue of pressurization vessels after a number of cycles. Engineers
worked to improve the strength of HHP vessels, the vessel’s resistance to a
high number of cycles, and the capacity of the pumps. Today, more compa-
nies around the world are specialized in the manufacture of high-pressure
processing equipment. Some of these companies are Avure® (the United
States and Sweden; formerly, Flow International Systems and ABB Pressure
Systems), Hyperbaric® (Spain), Engineering Pressure Systems International
EPSI® (Belgium), Kobe Steel® (Japan), Stansted Fluid Power®, Ltd (the United
Kingdom), Resato International® (The Netherlands), UNIPRESS® (Poland),
UHDE® (Germany), and ACB Pressure System-Alstom Hyperbar® (France).
Today, high-pressure equipment in most research centers and some food
industries have been typically manufactured by one or more of these com-
panies; in some cases, specific designs for processing target food products
are utilized.
Comparing the different types of equipment is a difficult task because each
one offers unique characteristics based on different operation parameters,
such as range of operating pressure, the systems used to heat and cool down
the process, the pressurization systems, the volume of chamber, and the
layout of system. The typical pressure range of HHP equipment is around
600 MPa, but some companies in Europe, such as Stansted Fluid Power®
Ltd, have equipment capable of achieving up to 1400 MPa and a tempera-
ture range of 20–150°C in just seconds. Avure® offers two equipment layouts,
horizontal or vertical, based on the type of product processed and available
space in the facility; they typically make equipment for industrial use with a
vessel capacity of 35–320 L. The horizontal layout in particular offers many
advantages; the traceability of the product during processing, for example, is
totally controlled because the inlet and outlet are located on different sides
of the equipment, making the installation of equipment easier and cheaper
than the vertical layout. In addition, the equipment is not very high and does
not require additional equipment to raise the inlet and outlet, making the
process of loading and unloading products easier; thus, this layout usually
works well in continuous mode (Hernando-Sàinz et al., 2008). In sterilization

trials, some of the high-pressure equipment used require raising the cham-
ber temperature to at least 90°C, with a pressure level of no lower than 600–
800 MPa. The capacity of such equipment depends on the specific use at the
time: for laboratory scale, 0.02–1.5 L; for pilot plant, 2–50 L; for industrial use,
up to 150 L. Improvement in this type of equipment would be to manufac-
ture a more efficient and faster pumping system to reduce come-up times,
which would be highly desirable (Juliano et al., 2009).
14.3 DPCD Technology
DPCD has been increasingly investigated as a technique able to induce the
inactivation of the natural microbial flora but also pathogens occurring in
solid and liquid matrices (Arreola et al., 1991; Zhou et al., 2009; Spilimbergo
and Ciola, 2010; Ferrentino and Spilimbergo, 2011).
In DPCD technique, food is contacted with either subcritical or supercriti-
cal CO2 for a certain amount of time in a batch, semi-batch, or continuous
manner.
CO2 is considered a GRAS (generally recognized as safe) substance, which
means it can be used for food products. The critical temperature (31.1°C) is
compatible with the thermal stability of most materials, and the critical pres-
sure (7.3 MPa) is easily reached in industrial processes (Spilimbergo et al.,
2013). Supercritical CO2 is CO2 at temperature and pressure above its critical
point values and exists as a single phase. It has the unique ability to diffuse
through solids like a gas and dissolve materials like a liquid. Additionally, it
can readily change in density upon minor changes in temperature or pres-
sure. Subcritical (gaseous or liquid) CO2, on the other hand, is CO2 at a tem-
perature or pressure below its thermodynamically critical point values.
The DPCD technique presents some advantages over HHP related to the
milder conditions it employs. Besides the environmentally benign nature
of the DPCD process (CO2 is nontoxic), the CO2 pressures applied for pres-
ervation purposes are much lower (generally <20 MPa) as compared to
the hydrostatic pressures employed in HHP (~300–600 MPa). Hence, this
makes it easier to control and manage pressure in the DPCD technique
(Spilimbergo et al., 2002). The main drawback of DPCD technology is that
direct contact between CO2 and the microorganisms to be killed must be
ensured. Therefore, while the application to liquids has been widely inves-
tigated, applications of DPCD to solid foods have received increased atten-
tion only in the last years due to: (1) the complexity of the matrix which can
make the CO2 bactericidal action more difficult; (2) the structure of the foods
which needs to be resistant to pressurized CO2; (3) the lack of information
about the inactivation mechanism which is almost obscure and scarcely
studied.

14.3.1 Process Principles and Equipment

Several batch, semibatch, as well as continuous systems of the DPCD
asteurization/sterilization process have been tested for various foods
p
(Damar and Balaban, 2006; Garcìa-Gonzalez et al., 2007). In a batch system,
CO2 and the food to be treated are stationary in an autoclave during treat-
ment. In a semicontinuous system, a continuous flow of CO2 through the
autoclave (which contains the food to be treated) is effected, while a continu-
ous system allows flow of both CO2 and the liquid food through the system.
A semi-continuous system is much more efficient than a batch one, because
it takes at least one order of magnitude less time to reach the same CO2 con-
centration in the water phase of the food, as compared to a batch operation
device (Elvassore et al., 2000). Continuous apparatus, however, holds the
most promise with regard to industrial implementation. However, if for liq-
uid foods some systems have been designed and realized, no implementa-
tion has been approached for solid foods. In 1999, Praxair (Chicago, IL, USA)
developed a continuous DPCD system for liquid foods using the concept
shown in Figure 14.2. CO2 and the products are pumped through the system
and mixed before passing through the high-pressure pump, which increases
the pressure to the process levels. The product temperature is controlled in
holding coils. Residence time is adjusted by setting the flow rate of the prod-
uct through the coils. At the end of the process, an expansion valve is used to
release CO2 from the mixture. It is possible to pull out the residual CO2 in the
food into a vacuum tank. This system has been shown to be very effective in
killing pathogens and spoilage bacteria in a short time (Folkes 2004; Lecky
and Balaban 2004; Kincal et al., 2005; Damar and Balaban 2006; Lim et al.,
2006; Parton et al., 2007).
Pump 2
CO2
P Chiller 6 Hold tube 9
3
7
Main pump
8
CO2 tank
1
Vacuum
Heating
system Expansion
valve
Pump
Treated
4 Juice 5
juice
stream
FIGURE 14.2
A continuous DPCD system. (From Ferrentino, G. et al. 2009b. Journal of Food Science,
74:E333–E341.)

14.3.2 State-of-the-Art DPCD

14.3.2.1 Inactivation Mechanisms
The mechanism concerning the inactivation of microorganisms and
enzymes exerted by DPCD is still a matter of study. In 1985, Daniels and co-
workers thoroughly reviewed the major theories to explain the bacteriostatic
action of (gaseous) CO2. Most of these theories have also been used in more
recent years by many different authors (Lin et al., 1993; Hong and Pyun, 1999;
Spilimbergo and Bertucco, 2003; Damar and Balaban, 2006) to explain the
bacteriostatic action of pressurized CO2. Theories explaining the inactiva-
tion mechanism of microorganisms in liquid foods involve the diffusion and
solubility of pressurized CO2 in the liquid; the decrease in the pH due to the
CO2 dissolution in the food water content to form carbonic acid, which disso-
ciates into bicarbonate, carbonate, and hydrogen ionic species; the microor-
ganisms membrane modification with an increase of the membrane fluidity
and permeability; the diffusion of CO2 into the cells; changes in the cellular
environment, such as a decrease in pH, key enzyme inactivation, inhibitory
effect of molecular CO2 and bicarbonate on cellular metabolism, and dis-
ordering of the intracellular electrolyte balance; the cell membrane rupture
caused by the increase in the internal pressure, and the resultant and the
removal by extraction of vital constituents from cells and cell membranes.
Same inactivation mechanism has been hypnotized also for solid products
based on the fact that the DPCD inactivation method is claimed to be appli-
cable to any food product having an aqueous phase, or being surrounded by
a sufficient amount of an aqueous phase to dissolve sufficient CO2 to inacti-
vate the target microorganisms and/or enzymes (Damar and Balaban, 2006;
Garcìa-Gonzalez et al., 2007). Results demonstrated that sterilization kinetics
were strongly affected by the presence of water. The reason why wet micro-
bial cells are more prone to DPCD inactivation is probably the direct result
of an increased CO2 solubility (and hence an increased formation and disso-
ciation of H2CO3), which liberates more H+ ions that subsequently reduce the
pH of the food to lower values. In addition, Lin et al. (1993) also attributed
the synergistic effect of water to swollen cell walls and membranes due to
the presence of water, by which these biological barriers are expanded to
become more penetrable by CO2 (Dillow et al., 1999).
14.3.2.2 Inactivation of Microorganisms in Foods

DPCD was successfully applied for the pasteurization of a wide group of fruit
juices and beverages: orange juice (Arreola et al., 1991), apple juice (Gui et al.,
2006; Ferrentino et al., 2009a; Gasperi et al., 2009), grapefruit juice (Ferrentino
et al., 2009b), mandarin juice (Lim et al., 2006), coconut water (Damar et al.,
2009), hibiscus beverage (Ramirez-Rodrigues et al., 2013), beer (Dagan and
Balaban, 2006), grape juice (Gunes et al., 2005), watermelon juice (Lecky and
Balaban, 2004), and kiwi and peach juice (Spilimbergo and Ciola, 2010).

Most of the studies reported that microbial inactivation is highly depen-

dent on the processing parameters such as time, pressure, and temperature
as well as the composition of the food. Microbial inactivation is also highly
dependent on the type of microorganisms present in the food matrix due
to distinct microbial cell microstructure and the diffusion of CO2 into the
microbial cells (Ballestra et al., 1996).
Ferrentino et al. (2009a) treated apple juice prepared from “Annurca”
apple puree with a DPCD batch system. Microbial inactivation kinetics
showed that 5 log reductions of natural flora in apple juice were observed at
16 MPa, 60°C and 40 min for DPCD treatment. Bae et al. (2009) investigated
the lethal effect of DPCD (temperature: 65°C, 70°C, pressure: 8, 10, 12 MPa,
time: 10–40 min) on Alicyclobacillus acidoterrestris spores (106 –107 spores/mL)
suspended in apple juice. A. acidoterrestris spores were completely inacti-
vated by DPCD to undetectable levels above 65°C, 10 MPa for 40 min and
70°C, 8 MPa for 30 min.
Fresh squeezed red blush grapefruit juice was treated with continuous
DPCD equipment to inactivate yeasts and molds and total aerobic micro-
organisms. Five log reductions for yeasts and molds and total aerobic
microorganisms occurred at 34.5 MPa, 40°C and 7 min of residence time
(Ferrentino et al., 2009b), whereas the treatment of fresh watermelon juice
with the same apparatus showed 6 log reductions of aerobic microorgan-
isms in 5 min.
Very few researches have been addressed applying DPCD to fresh-cut
fruits. Haas et al. carried out the first study in 1989 on fresh strawber-
ries, honeydew melon, and cucumber in order to delay surface molding.
The treatment had a positive effect on molding, especially for strawberries
showing a preservative action. Recently, Ferrentino et al. (2012) applied the
treatment for the pasteurization of fresh-cut coconut showing promising
results in the inactivation of the natural microbial flora (mesophilic micro-
organisms, lactic acid bacteria, coliforms, and yeasts and molds) with a
batch device. DPCD has been also applied to some vegetables. Kuhne and
Knorr (1990) carried out experiments on fresh celery and leafstalks showing
a substantial inactivation effect on total count. Their data indicated that a
treatment carried out for 30 min at 40°C and 62.8 MPa induced a reduction
in the total plate count of approximately 104 cfu/g. They also reported the
inefficiency to inactivate spores on the same substrates and under the same
experimental conditions.
As concerns the application of DPCD to meat products, Sirisee et al. (1998)
tested the tolerance of E. coli and S. aureus in ground beef and observed the
longer treatment time to inactivate both target microbes compared to the
same treatment carried out on the same microorganisms in a liquid phos-
phate buffer solution. One log cycle reduction of E. coli in ground beef took
178 min, but only 1.7 min was needed to achieve the same inactivation level
in liquid phosphate buffer solutions. The increase in microbial resistance to
the treatment was attributed to the nature of the matrix and compounds,

carbohydrates, and fats that exerted a protective effect. However, a reduction

equal to 5 and 1 log cycles was observed for B. thermosphacta inoculated on
skinned and minced meat, respectively.
The first attempt to apply the DPCD treatment for the preservation of sea
food products was carried out by Wei et al. (1991). In this study, shrimp sam-
ples spiked with Listeria were treated by DPCD at 5.85 MPa, 35°C for 2 h.
The results demonstrated that the treatment reduced the bacterial counts by
only 35%–45% but by increasing the pressure to 13.7 MPa, a microbial reduc-
tion equal to 99% was obtained. More recently, DPCD was applied to the
processing of oysters. The authors reported that the level of total bacterial
inactivation achieved with the DPCD treatment (10 MPa for 30 min at 37°C
or 17 MPa for 60 min at 60°C) was comparable to that achieved with several
FDA-approved post-harvest processing for oysters, namely HHP and quick
freezing (Prapaiwong et al., 2009). In Table 14.2, the most recent results on
microbial inactivation in foods induced by DPCD are listed.
TABLE 14.2
Summary of the Recent Studies on Microbial Inactivation by DPCD
Log
Foodstuff Microorganism Treatment Reduction Reference
Fresh-cut carrot E. coli 10 min Inactivation to Ferrentino et al.
12 MPa undetectable (2014)
35°C levels
Shrimp and conch Natural 42 min >3 log Chen et al. (2014)
microbial flora 14 MPa
55°C
Hibiscus Sabdariffa Natural 6.5 min Inactivation to Ramirez-Rodrigues
beverage microbial flora 34.5 MPa undetectable et al. (2013)
40°C levels
Apple juice S. cerevisiae 140 min 6.7 log Ortuño et al. (2013)
Orange juice 35 MPa
36°C
Cooked ham Natural 5 min Inactivation to Ferrentino et al.
microbial flora 12 MPa undetectable (2013c)
50°C levels
Raw bovine milk Natural 70 min 5 log Hongmei et al.
microbial flora 25 MPa (2014)
50°C
Dry cured ham L. monocytogenes 15 min 7 log Ferrentino et al.
12 MPa (2013b)
50°C
Orange juice Natural 20 min 5 log Yuk et al. (2014)
microbial flora 10 MPa
42°C
Fresh-cut Microbial spoilage 30 min Inactivation to Zhang et al. (2013)
pineapple 15 MPa undetectable
40°C levels

14.3.2.3 Effects on Food Quality Attributes

The first study, performed by Haas et al. in 1989, reported the disruption of
the texture of some products, such as strawberries, honeydew melon, and
cucumber, after DPCD. Moreover, some food samples showed a color change
after treatment, especially orange juice and eggs, they became less yellow,
probably due to the removal of carotenoid pigments. In recent years, the
application of this technique to foodstuff has attracted more and more inter-
est. For instance, Folkes (2004) used a continuous DPCD system for pasteuri-
zation of beer and compared its physical and sensory attributes with that of
fresh and heat pasteurized beer. Aroma and flavor of the treated beer were
not significantly different from fresh beer after being stored for 1 month at
1.67°C, while heat-treated beer was significantly different in taste and aroma.
Lim et al. (2006) treated mandarin juice with CO2 using a continuous sys-
tem and measured the pH, °Brix, acidity, cloud, and color after DPCD treat-
ment (13.8–41.4 MPa, 25–45°C, 7–9 min). DPCD enhanced cloud up to 38.4%,
increased lightness and yellowness, and decreased redness of mandarin
juice. The treated samples had higher acidity than the untreated samples,
but, pH and °Brix did not change after treatment.
Some studies clearly demonstrated that DPCD could also improve the
physical quality of liquids. Arreola et al. (1991) and Balaban et al. (2001) mea-
sured an enhanced cloud stability of DPCD-treated juices compared with
untreated samples. Another improvement worthwhile mentioning is the
effect of DPCD treatment on the browning in cloudy apple juice. The treat-
ment resulted in a significant reduction of the browning degree in the juices
during storage at 4°C as compared to untreated sample, indicating that the
process effectively could prevent the browning of apple juice (Gui et al., 2006).
As concerns panel tests on consumer acceptance of liquid food products
processed by DPCD, some studies reported sensory evaluations conducted
by panelists to better understand consumer perspective and, of course,
acceptance of DPCD-treated foods. Sensory evaluation indicated that the
DPCD treated juices were indistinguishable from the untreated sample
with no significant difference in flavor, aroma, and overall acceptability.
The DPCD-treated juices were ranked higher and thus more likeable by
the sensory panel than the heat pasteurized samples (Arreola et al., 1991;
Folkes, 2004; Damar and Balaban, 2005; Lecky and Balaban, 2004; Del Pozo-
Insfran et al., 2006). DPCD was shown to be detrimental when applied for
the preservation of fresh-cut fruits and vegetables with a soft structure due
to the adverse effect of the process on fruit firmness and texture. Several
studies concluded that DPCD process is more adequate for products where
a firm texture is not essential, such as fruit cocktails, creams, juices, or other
types of fruit preserves. Valverde et al. (2010) showed a loss of consistency
when the process was applied on fresh-cut pears manifested with a softer
aspect and a loss of liquid from the samples. Similar results were reported by
Spilimbergo et al. (2013) performing DPCD on fresh-cut carrot: a significant

texture reduction was observed in such extent that the panelists judged the
samples no more carrot like. Different results were achieved when DPCD
was applied to fresh-cut coconut: the texture of the samples was preserved
after the treatment and no significant differences were measured in color,
pH, acidity, and sensory attributes between the DPCD-treated and untreated
samples (Ferrentino et al., 2013a).
As concerns the application of DPCD to meat products, a complete analysis
was recently performed by Choi et al. (2008) who investigated the effect of
DPCD for sterilization purpose on meat quality and protein denaturation
of the porcine longissimus dorsi muscle. It was observed that DPCD pres-
sure and temperature could induce molecular interactions and proteins
conformation, leading to protein denaturation and aggregation in the meat
(Messens et al., 1997). In the same study, the DPCD treatment (7.4 MPa at
31.1°C for 10 min) did not affect the muscle pH, total weight, tenderness, and
water-holding capacity of the treated meat.
Several studies demonstrated the efficiency of DPCD in increasing the
quality of meats inducing the extraction of cholesterol and other lipids. This
potential is becoming more and more attractive due to consumer concern
over dietary intake of fat and cholesterol. It has been reported that CO2 under
supercritical conditions solubilizes a portion of the lipid components and
removes them from the food matrix. Chao et al. (1991) found that up to 36.9%
of the cholesterol and 71.2% of total lipids in the meat could be removed dur-
ing the process.
Studies applied for the preservation of sea food products showed inconsis-
tent results of DPCD impact on their quality traits. Wei et al. (1991) reported
that the outer layer of the shrimps treated at 13.7 MPa, 35°C for 2 h turned
whitish and gave the appearance of being cooked rapidly at low temperature
or soaked in acid. In addition, loss of small amount of liquid from the tissue
was observed giving the idea of some tissue damaging caused by the treat-
ment. On the contrary, results of a sensory analysis, assessed by a panel of
13 people, performed on oysters treated by DPCD revealed that the samples
remained acceptable considering their physical appearance, texture, and
smell after the exposure to DPCD process (Meujo et al., 2010).
14.3.3 Commercial Applications

Despite the huge research and development efforts performed in the last
20 years, at present there is no commercial food product pasteurized by
DPCD that is marketed, even though several companies have actively worked
in this field (e.g., Praxair, PoroCrit LLC, Shimadzu, and Air Liquide) (Damar
and Balaban, 2006; Garcìa-Gonzalez et al., 2007).
In terms of DPCD technology commercialization, to our knowledge the
most notable public commercialization efforts have been performed by
Praxair Inc., which licensed the technology from the University of Florida
(Balaban et al., 1988; Balaban 1998). This system, commercialized under

the trademark “Better Than Fresh” (BTF), was advertised as an adequate

non-thermal alternative to thermal pasteurization to produce fresh tasting
juice (Connery et al., 2005). Praxair constructed different mobile BTF units
for demonstration purposes (Garcìa-Gonzalez et al., 2007), and in 2003 the
company announced that Sun Orchard, Inc. (Tempe, AZ, US) would install
the first BTF non-thermal processing system at its Florida facility to process
juice. Once installed at Sun Orchard’s Florida facility at the second quarter of
2003, it was foreseen to process juice at a rate of 40 gallons/min (about 150 L/
min). Sun Orchard, however, never implemented Praxair’s BTF technology
on a commercial scale and the juice producing company did not pursue it
any further, using its own proprietary ultra-light heat pasteurization process
instead. The provider of industrial gasses itself also seemed to have com-
pletely abandoned the BTF program, as it is not a current product line at
Praxair anymore.
Entrepreneur Marc Sims (Porocrit LLC, Berkeley, CA) designed a hollow
fiber membrane contactor for continuous DPCD pasteurization of pulp-
free juices that improved the current technology due to its CO2 saturation
enhancements. Sims licensed the technology to the Chicago Research Center
of Air Liquide (Countryside, IL) for further development from 2000 until his
untimely death in 2005 (Sims, 2000).
Air Liquide has since abandoned research interests including commer-
cialization efforts, and without Sims, Porocrit has also ceased operations.
The only other DPCD industrial activity involves pilot-scale equipment
for continuous treatment of liquid foods and manufactured by Mitsubishi
Kakoki Co. (Tokyo, Japan) for Shimadzu Co. (Kyoto, Japan). Maximal flow
rates of 3.0 and 20.0 kg/h were capable of being pumped through a 5.8 l
holding treatment vessel, but the commercial development of the technol-
ogy in Japan currently remains at the laboratory research level only (Garcìa-
Gonzalez et al., 2007).
The commercial utility and overall capabilities of the Porocrit LLC DPCD
pilot plant demonstration system were evaluated during the early 2000s at
the Chicago Research Center of Air Liquide in collaboration with originator
Marc Sims (Figure 14.3).
As concerns solid foods, the development of a continuous DPCD process
seems to be still far away. This probably has to do with the fact that, from
an industrial-scale point of view, continuous DPCD process, in which solid
materials have to be fed into and out of the pressure reactor, is much more
difficult to handle as compared to liquid foods. An interesting design in this
respect might be the supercritical CO2 extrusion process developed by the
Agrotechnology and Food Sciences Group of Wageningen University and
Research Center (Langelaan et al., 1999; Bartels et al., 2002) to isolate valuable
or undesired components from solids in a continuous manner (Figure 14.4).
In the last years, during the FP7 PRESERF EU project recently closed,
the research group of Dr. Spilimbergo showed the technical feasibility and
development of a semi-continuous DPCD process for the pasteurization of

2
CO
o d
d fo 2
ui CO
Fl
FIGURE 14.3
The Porocrit LLC scCO2 system (designed by Marc Sims, Berkeley, CA) consists of microporous
polypropylene membranes through which liquid product is pumped. CO2 diffuses across the
membranes to inactivate microorganisms in the food product under supercritical conditions
of 7.3 MPa and 30–45°C. (From Balaban M.O., Ferrentino, G. 2012. Dense Phase Carbon Dioxide:
Applications to Foods and Pharmaceuticals. Wiley-Blackwell, Oxford, UK.)
meat, fruit, and vegetable products in such a way that sustainability benefits
were qualified. Product characteristics were addressed and a pilot plant was
constructed to demonstrate the feasibility of the first market application of
the process in an industrial environment.
Concluding, although it has often been stated that “commercialization
could be a matter of time” or that “it is going to be a reality within a
Substrate
P atm. High pressure extraction zone P atm.

(P > 74 bar)
Plug 1 Plug 2
SC–CO2
+
CO2 (g) SC–CO2 Extrudate
extract
Extract
FIGURE 14.4
Schematic diagram of a supercritical-CO2 extrusion process developed by the Agrotechnology
and Food Science Group of Wageningen University and Research Center. (From Balaban M.O.,
Ferrentino, G. 2012. Dense Phase Carbon Dioxide: Applications to Foods and Pharmaceuticals. Wiley-
Blackwell, Oxford, UK.) Patent number WO 99/26707. (From Langelaan, H.C., Bartels, P.V.,
Hulleman, S.H.D. 1999. Method for the extraction of a substance from a starting material and
extraction apparatus for carrying out the method. WO Patent 99/26707; Bartels, P.V., Hulleman,
S.H.D., Langelaan, H.C. 2002. Method for the extraction of a substance from a starting material.
US Patent 6,491,892 B1.)

short time,” the DPCD pasteurization process is still looking for its first
commercial success. The following factors seem to determine successful
commercialization of the technology (Damar and Balaban, 2006): (1) sig-
nificant differentiation of DPCD products from those existing in the mar-
ket regarding taste, quality, and shelf-life is needed; (2) niche areas where
no other conventional processing is possible need to be found, such as
tropical juices that are very sensitive to heat; and (3) competing non-ther-
mal technologies may be more appropriate for certain products (Balaban
and Ferrentino, 2012).
14.4 Potentials of High-Pressure Technologies
and Conclusions
The successful introduction of a new technology demands competitive
advantages over existing ones. In the case of HPP, a constraint is the large
capital investment, which is overcome by operating HPP plants at full capac-
ity. Processing of seasonal commodities requires identifying a product mix
achieving maximum utilization of the equipment investment.
As concerns DPCD, in the future, it could become one of the most available
emergent preservation technologies. However, to meet this high expectation,
consumers and stakeholders must be convinced about the improvements
this new technology represents over competing technologies.
Both processes clearly seem to represent some distinctive opportunities
when applied to some food products, as they do not change organoleptic and
nutritional attributes to a detrimental level. In addition, they can be used as
a single technology, as a “hurdle” in combined methods, or as a complemen-
tary step with mild thermal processes. As an example, studies have been
recently published showing the advantages of the simultaneous application
of DPCD and high-power ultrasounds (HPU) for microbial inactivation of
liquid (Ortuño et al., 2012; Cappelletti et al., 2014) foodstuffs. They demon-
strated that when DPCD and HPU were coupled, the time needed to reach
8 log reductions of different microbial strains was reduced, on average by
95%, compared to DPCD alone.
It is clear that some technological and some regulatory hurdles still need
to be overcome before the supply chain can receive the benefits of HHP and
DPCD preservation. The major problem that novel, non-thermal preservation
methods face in gaining widespread acceptance is that thermal processes are
so firmly established and are capable of producing foods that are safe and of
a high quality and nutritional value in large volumes at very low processing
costs. We are, however, confident that the scientific researches performed so
far constitute significant results towards the production of foods industrially
processed and preserved by these new technologies.

References
Aleman, G.D., Farkas, D.F., Mcintyre, S., Torres, J.A., Wilhelmsen, E. 1994. Ultra-high
pressure pasteurization of fresh cut pineapple. Journal of Food Protection, 57:931–934.
Aleman, G.D., Ting, E.Y., Mordre, S.C. et al. 1996. Pulsed ultra high pressure treat-
ments for pasteurization of pineapple juice. Journal of Food Science, 61:388–390.
Angsupanich, K., Edde, M., Ledward, D.A. 1999. Effects of high pressure on the
myofibrillar proteins of cod and turkey muscle. Journal of Agricultural and Food
Chemistry, 47:92–99.
Angsupanich, K., Ledward, D.A. 1998. High pressure treatment effects of cod (Gadus
morhua) muscle. Food Chemistry, 63:39–50.
Arreola, A.G., Balaban, M.O., Wei, C.I., Peplow, A., Marshall, M., Cornell, J. 1991. Effect
of supercritical carbon dioxide on microbial populations in single strength
juice. Journal of Food Quality, 14:275–284.
Arroyo, G., Sanz, P.D., Prestamo, G. 1997. Effect of high pressure on the reduction of
microbial populations in vegetables. Journal of Applied Microbiology, 82:735–742.
Bae, Y.Y., Lee, H.J., Kim, S.A., Rhee, M.S. 2009. Inactivation of Alicyclobacillus acidoter-
restris spores in apple juice by supercritical carbon dioxide. International Journal
of Food Microbiology, 136:95–100.
Balaban, M.O. 1998. Method and apparatus for continuous flow reduction of micro-
bial and/or enzymatic activity in a liquid product using carbon dioxide. US
Patent 6,723,365 B2 (April 20, 2004) and U.S. Patent Application 2004/0131739
A1 (July 8, 2004).
Balaban M.O., Ferrentino, G. 2012. Dense Phase Carbon Dioxide: Applications to Foods
and Pharmaceuticals. Wiley-Blackwell, Oxford, UK.
Balaban, M.O., Kincal, D., Hill, S., Marshall, M.R., Wildasin, R. 2001. The synergistic
use of carbon dioxide and pressure in nonthermal processing of juices. Paper
Presented at the Institute of Food Technologists (IFT) Annual Meeting, New Orleans,
LA, June 23–27.
Balaban, M.O., Marshall, M.R., Wicker, L. 1988. Inactivation of enzymes in foods
with pressurized CO2. U.S. Patent 5,393,547 (February 28, 1995) and WO Patent
90/02799 (March 22, 1990).
Ballestra, P., Dasilva, A.A., Cuq, J.L. 1996. Inactivation of Escherichia coli by carbon
dioxide under pressure. Journal of Food Science, 61:829–831.
Bartels, P.V., Hulleman, S.H.D., Langelaan, H.C. 2002. Method for the extraction of a
substance from a starting material. US Patent 6,491,892 B1.
Basak, S., Ramasawamy, H.S. 1998. Effect of high pressure processing on texture of
selected fruit and vegetables. Journal of Texture Studies, 29:587–601.
Beilken, S.L., Macfarlane, J.J., Jones, P.N. 1990. Effect of high pressure during heat
treatment on the Warner-Bratzler shear force values of selected beef muscles.
Journal of Food Science, 55:15–18.
Belletti, N., Garriga, M., Aymerich, T., Bover-Cid, S. 2013a. Inactivation of Serratia
liquefaciens on dry-cured ham by high pressure processing. Food Microbiology,
35:34–37.
Belletti, N., Garriga, M., Aymerich, T., Bover-Cid, S. 2013b. High pressure inactivation
of a virulent Enterococcus faecalis on dry-cured ham: Modeling the effect of pro-
cessing parameters. Innovative Food Science & Emerging Technologies, 18:43–47.

Bouton, P.E., Ford, A.E., Harris, P.V., Macfarlane, J.J., O’shea, J.M. 1977. Pressure-heat
treatment of post-rigor muscle: Objective–subjective measurements. Journal of
Food Science, 42:857–859.
Cappelletti, M., Ferrentino, G., Spilimbergo, S. 2014. Supercritical carbon dioxide
combined with high power ultrasound: An effective method for the pasteuri-
zation of coconut water. The Journal of Supercritical Fluids, 92:257–263.
Capri, G., Gola, S., Maggi, A., Rovere, P., Buzzoni, M. 1995. Microbial and chemical
shelflife of high pressure treated salmon cream at refrigeration temperatures.
Industrial Conserve, 70:386–397.
Carlez, A., Rosec, J.P., Richard, N., Cheftel, J.C. 1993. High pressure inactivation of
Citrobacter freundii, Pseudomonas fluorescens and Listeria innocua in inoculated
minced beef muscle. Lebensmittel-Wissenschaft & Technologie, 26:357–363.
Chao, R.R., Mulvaney, S.J., Bailey, M.E., Fernando, L.N. 1991. Supercritical CO2 condi-
tions affecting extraction of lipid and cholesterol from ground beef. Journal of
Food Science, 56:183–187.
Chen, M., Sui, X., Ma, X., Feng, X., Han, Y. 2014. Application of response surface
methodology to optimise microbial inactivation of shrimp and conch by super-
critical carbon dioxide. Journal Science Food and Agriculture. 95:1016–1023. doi:
10.1002/jsfa.6783.
Chen, D., Xi, H., Guo, X. et al. 2013. Comparative study of quality of cloudy pome-
granate juice treated by high hydrostatic pressure and high temperature short
time. Innovative Food Science & Emerging Technologies, 19:85–94.
Chilton, P., Isaacs, N.S., Manas, P., Mackey, B.M. 2001. Biosynthetic requirements for
the repair of membrane damage in pressure-treated Escherichia coli. International
Journal of Food Microbiology, 71:101–104.
Choi, Y.M., Ryu, Y.C., Lee, S.H. et al. 2008. Effects of supercritical carbon dioxide
treatment for sterilization purpose on meat quality of porcine longissimus
dorsi muscle. Lebensmittel-Wissenschaft & Technologie, 41:317–322.
Connery, K.A., Shah, P., Coleman, L., Hunek, B. 2005. Commercialization of Better Than
FreshTM dense phase carbon dioxide processing for liquid food. In: Proceedings of
the International Symposium on Supercritical Fluids, Orlando, FL, 60:285.
Dagan, G.F., Balaban, M.O. 2006. Pasteurization of beer by a continuous dense-phase
CO2 system. Journal of Food Science, 71:E164–E169.
Damar, S., Balaban, M.O. 2005. Cold pasteurization of coconut water with a continu-
ous dense phase CO2 system. In: IFT Annual meeting Book of Abstract, ed. The
Society of Food Science & Technology, Institute of Food Technology, Chicago,
Illinois, USA, 54F:2.
Damar, S., Balaban, M.O. 2006. Review of dense phase CO2 technology: Microbial
and enzyme inactivation, and effects on food quality. Journal of Food Science,
71:R1–R11.
Damar, S., Balaban, M.O., Sims, C.A. 2009. Dense phase CO2 processing of coconut
water. International Journal of Food Science & Technology, 44:666–673.
Del Pozo-Insfran, D., Balaban, M.O., Talcott, S.T. 2006. Microbial stability, phy-
tochemical retention, and organoleptic attributes of dense phase CO2 pro-
cessed muscadine grape juice. Journal of Agricultural and Food Chemistry,
54:5468–5473.
Dervisi, P., Lamb, J., Zabetakis, I. 2001. High pressure processing in jam manufacture:
Effect on textural and colour properties. Food Chemistry, 73:85–91.

Dillow, A.K., Dehghani, F., Hrkach, J.S., Foster, N.R., Langer, R. 1999. Bacterial inac-
tivation by using near- and supercritical carbon dioxide. Proceedings of the
National Academy of Sciences of the United States of America, 96:10344–10348.
Dong, P., Kong, M., Yao, J. et al. 2013. The effect of high hydrostatic pressure on the
microbiological quality and physicochemical properties of lotus root during
refrigerated storage. Innovative Food Science & Emerging Technologies, 19:79–84.
Elvassore, N., Sartorello, S., Spilimbergo, S., Bertucco, A. 2000. Micro-organisms inac-
tivation by supercritical CO2 in a semi-continuous process. Proceedings of the 7th
Meeting on Supercritical Fluids, Antibes, France.
Eshtiaghi, M.N., Knorr, D. 1993. Potato cube response to water blanching and high
hydrostatic pressure. Journal of Food Science, 58:1371–1374.
Etienne, M., Fleurence, J, Rehbein, H. et al. 2001. Species identification of formed fish-
ery products and high pressure-treated fish by electrophoresis: A collaborative
study. Food Chemistry, 72:105–112.
Ferrentino, G., Balzan, S., Dorigato, A., Pegoretti, A., Spilimbergo, S. 2012. Effect of
supercritical carbon dioxide pasteurization on natural microbiota, texture and
microstructure of fresh-cut coconut. Journal of Food Science, 77:E137–E143.
Ferrentino, G., Balzan, S., Spilimnergo, S. 2013b. Supercritical carbon dioxide pro
cessing of dry cured ham spiked with Listeria monocytogenes: Inactivation
k inetics, color, and sensory evaluations. Food and Bioprocess Technology,

6:1164–1174.
Ferrentino, G., Balzan, S., Spilimbergo, S. 2013c. Optimization of supercritical carbon
dioxide treatment for the inactivation of the natural microbial flora in cubed
cooked ham. International Journal of Food Microbiology, 161:189–196.
Ferrentino, G., Belscak-Cvitanovic, A., Komes, D., Spilimbergo, S. 2013a. Quality
attributes of fresh-cut coconut after supercritical carbon dioxide pasteuriza-
tion. Journal of Chemistry, Article ID 703057, 9 pages.
Ferrentino, G., Bruno, M., Ferrari, G., Poletto, M., Balaban, M.O. 2009a. Microbial inac-
tivation and shelf life of apple juice treated with high pressure carbon dioxide.
Journal of Biological Engineering, 3:3.
Ferrentino, G., Calliari, N., Bertucco, A., Spilimbergo, S. 2014. Validation of a math-
ematical model for predicting high pressure carbon dioxide inactivation kinet-
ics of Escherichia coli spiked on fresh cut carrot. The Journal of Supercritical Fluids,
85:17–23.
Ferrentino, G., Plaza, M.L., Ramirez-Rodrigues, M., Ferrari, G., Balaban, M.O.
2009b. Effects of dense phase carbon dioxide pasteurization on the physi-
cal and quality attributes of a red grapefruit juice. Journal of Food Science,
74:E333–E341.
Ferrentino, G., Spilimbergo, S. 2011. High pressure carbon dioxide pasteurization of
solid foods: Current knowledge and future outlooks. Trends in Food Science &
Technology, 22:427–441.
Folkes, G. 2004. Pasteurization of beer by a continuous dense-phase CO2 system. PhD
dissertation, University of Florida, Gainesville.
Fuji, T., Satomi, M., Nakatsuka, G., Yamaguchi, T., Okuzumi, M. 1994. Changes of
freshness indexes and bacterial flora during storage of pressurized mackerel.
Journal of the Food Hygiene Society of Japan, 35:195–200.
Galazka, V.B., Ledward, D.A. 1998. High pressure effects on biopolymers. In
Functional Properties of Food Macromolecules, ed. S.E. Hill, D.A. Ledward, J.R. and
Mitchell. Springer, US.

Gallot-Lavallee, T. 1998. Effectiveness of high pressure treatment for destruc-

tion of Listeria monocytogenes in raw milk goat cheese. Sciences des Aliments,
18:647–655.
Garcìa-Gonzalez, L., Geeraerd, A.H., Spilimbergo, S. et al. 2007. High pressure carbon
dioxide inactivation of microorganisms in foods: The past, the present and the
future. International Journal of Food Microbiology, 117:1–28.
Garcia-Graells, C., Hauben, K.J.A., Michiels, C.W. 1998. High-pressure inactivation
and sublethal injury of pressure-resistant Escherichia coli mutants in fruit juices.
Applied and Environmental Microbiology, 64:1566–1568.
Gasperi, F., Aprea, E., Biasioli, F. et al. 2009. Effects of supercritical CO2 and N2O pas-
teurization on the quality of fresh apple juice. Food Chemistry, 115:19–136.
Gui, F.Q., Wu, J.H., Chen, F. et al. 2006. Change of polyphenol oxidase activity, color,
and browning degree during storage of cloudy apple juice treated by super-
critical carbon dioxide. European Food Research and Technology, 223:427–432.
Gunes, G., Blum, L.K., Hotchkiss, J.H. 2005. Inactivation of yeasts in grape juice using
a continuous dense phase carbon dioxide processing system. Journal of the
Science of Food and Agriculture, 85:2362–2368.
Haas, G.J., Prescott, H.E., Dudley, E. et al. 1989. Inactivation of microorganisms by
carbon dioxide under pressure. Journal of Food Safety, 9:253–265.
Hamada, K, Nnakatomi, Y., Shimada, S. 1992. Direct induction of tetraploids or
homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydro-
static pressure. Current Genetics, 22:371–376.
Hauben, K.J.A, Wuytack, E.Y., Soontjens, C.C.F., Michiels, C.W. 1996. High-pressure
transient sensitization of Escherichia coli to lysozyme and nisin by disruption of
outer-membrane permeability. Journal of Food Protection, 59:350–355.
Hayakawa, I., Kanno, T., Tomita, M., Figio, Y. 1994. Application of high pressure for
spore inactivation and protein denaturation. Journal of Food Science, 59:159–163.
Hernando-Sàinz, A., Tárrago-Mingo, S., Purroy-Balda, F. et al. 2008. Advances in
design for successful commercial high pressure food processing. Food Australia,
60:154–159.
Hite, B.H. 1899. The effect of pressure in the preservation of milk. West Virginia
Agricultural Experiment Station Bulletin, 58:15–35.
Hong, S.-I., Pyun, Y.R. 1999. Inactivation kinetics of Lactobacillus plantarum by high
pressure carbon dioxide. Journal of Food Science, 64:728–733.
Hongmei, L., Zhong, K., Liao, X., Hu, X. 2014. Inactivation of microorganisms natu-
rally present in raw bovine milk by high-pressure carbon dioxide. International
Journal of Food Science & Technology, 49:696–702.
Horie, Y., Kimura, K., Hori, K. 1991. Development of a new fruit processing method
by high hydrostatic pressure. Journal of the Agricultural Chemical Society of Japan,
65:1469–1474.
Huang, Y., Ye, M., Chen, H. 2013. Inactivation of Escherichia coli O157:H7 and
Salmonella spp. in strawberry puree by high hydrostatic pressure with/
without subsequent frozen storage. International Journal of Food Microbiology,
160:337–343.
Hyashi, R. 1989. Application of high pressure to food processing and preservation:
Philosophy and development. In Engineering and Food Species, ed. W.E.L. Spiess,
H. Schubert. Elsevier Applied Science, England, p. 815.
Johnston, D.E., Murphy, R.J., Birks, A.W. 1994. Stirred-style yoghurt-type product
prepared from pressure treated skim-milk. High Pressure Research, 12:215–219.

Juliano, P., Knoerzer, K., Barbosa-Canovas, G.V. 2009. High-pressure thermal pro-
cesses: Thermal and fluid dynamic modeling principles. In Mathematical
Modeling of Selected Food Processes, ed. R. Simpson. CRC Press, Boca Raton.
Kaushik, N., Kaur, P.B., Rao, P.S., Mishra, H.N. 2014. Effect of high pressure processing
on color, biochemical and microbiological characteristics of mango pulp (Mangi
fera indica cv. Amrapali). Innovative Food Science & Emerging Technologies, 22:40–50.
Kimura, K., Ida, M., Yoshida, Y., Ohki, K., Fukumoto, T., Sakui, N. 1994. Comparison of
keeping quality between pressure-processed and heat-processed jam: Changes
in flavor components, hue and nutritional elements during storage. Bioscience,
Biotechnology and Biochemistry, 58:1386–1391.
Kincal, D., Hill, S., Balaban, M.O., Marshall, M.R., Wei, C. 2005. A continuous high
pressure CO2 system for microbial reduction in orange juice. Journal of Food
Science, 70:M249–M254.
Kuhne, K., Knorr, D. 1990. Effects of high pressure carbon dioxide on the reduction
of microorganisms in fresh celery. Internationale Zeitschrift f€ur Lebensmittel-
Technik, Marketing, Verpackung und Analytik, 41:55–57.
Langelaan, H.C., Bartels, P.V., Hulleman, S.H.D. 1999. Method for the extraction of a
substance from a starting material and extraction apparatus for carrying out
the method. WO Patent 99/26707.
Lecky, M., Balaban, M.O. 2004. Continuous high pressure carbon dioxide pro-
cessing of watermelon juice. Institute of Food Technologists Annual Meeting,
Las Vegas, NV, vol. 49H-3, p. 133.
Lee, D.U., Park, J.Y., Kang, J.I., Yeo, I.H. 1996. Effect of high hydrostatic pressure on
the shelf life and sensory characteristics of Angelica keiskei juice. Korean Journal
of Food Science Technology, 28:105–108.
Lim, S., Yagiz, Y., Balaban, M.O. 2006. Continuous high pressure carbon dioxide pro-
cessing of mandarin juice. Food Science and Biotechnology, 15:13–18.
Lin, H.-M., Yang, Z.Y., Chen, L.-F. 1993. Inactivation of Leuconostoc dextranicum with
carbon dioxide under pressure. Chemical Engineering Journal and the Biochemical
Engineering Journal, 52:B29–B34.
Linton, M., McClements, J.M.J., Patterson, M.F. 1999. Survival of Escherichia coli
O157:H7 during storage in pressure-treated orange juice. Journal of Food
Protection, 62:1038–1040.
Liu, F., Wang, Y., Bi, X., Guo, X., Fu., S., Liao, X. 2013. Comparison of microbial inactiva-
tion and rheological characteristics of mango pulp after high hydrostatic pres-
sure treatment and high temperature short time treatment. Food and Bioprocess
Lopez-Caballero, M.E., Perez-Mateos, M., Borderias, J.A., Montero, P. 2000. Extension
of the shelf life of prawns (Penaeus japonicus) by vacuum packaging and high-
pressure treatment. Journal of Food Protection, 63:1381–1388.
MacDonald, A.G. 1992. Effects of high hydrostatic pressures on natural and artifi-
cial membranes. In High Pressure and Biotechnology, ed. C. Balny, R. Hayashi,
K. Heremans and P. Masson. John Libbey Eurotext Ltd., Montrouge, pp. 67–75.
Malone, A.S., Shellhammer, T.H., Courtney, P.D. 2002. Effects of high pressure on the
viability, morphology, lysis and cell wall hydrolase activity of Lactococcus lactis
subsp. cremoris. Applied Environmental Microbiology, 68:4357–4363.
Mertens, B. 1995. Hydrostatic pressure treatment of food: Equipment and processing.
In New Methods of Food Preservation. Glasgow: Blackie, Academic and Professional,
ed. G.W. Gould. Chapman & Hall, London, pp. 135–158.

Messens, W., Van Camp, J., Huyghebaert, A. 1997. The use of high pressure to mod-
ify the functionality of food proteins. Trends in Food Science and Technology,
8:107–112.
Meujo, D.A.F., Kevin, D.A., Peng, J., Bowling, J.J., Liu, J., Hamann, M.T. 2010. Reducing
oyster-associated bacteria levels using supercritical fluid CO2 as an agent of
warm pasteurization. International Journal of Food Microbiology, 138:63–70.
Miyao, S., Shindoh, T., Miyamori, K., Arita, T. 1993. Effects of high pressurization
on the growth of bacteria derived from surimi (fish paste). Journal of Japanese
Society of Food Science and Technology, 40:478–484.
Moody, A., Marx, G., Swanson, B.G., Bermúdez-Aguirre, D. 2014. A comprehensive
study on the inactivation of Escherichia coli under nonthermal technologies:
High hydrostatic pressure, pulsed electric fields and ultrasound. Food Control,
37:305–314.
Mootian, G.K., Flimlin, G.E., Karwe, M.V., Schaffner, D.W. 2013. Inactivation of Vibrio
parahaemolyticus in Hard Clams (Mercanaria mercanaria) by high hydrostatic
pressure (HHP) and the effect of HHP on the physical characteristics of hard
clam meat. Journal of Food Science, 78:E251–E257.
Norton, T., Sun, D.-W. 2008. Recent advances in the use of high pressure as an effective
processing technique in the food industry. Food Bioprocess Technology, 1:2–34.
Ohshima, T., Ushio, H., Koizumi, C. 1993. High pressure processing of fish and fish
products. Trends in Food Science and Technology, 4:370–375.
Okamoto, M., Kawamura, Y., Hayashi, R. 1990. Application of high pressure to food
processing: Textural comparison of pressure- and heat-induced gels of food
proteins. Agricultural and Biological Chemistry, 54:183–189.
Ortuño, C., Martínez-Pastor, M.T., Mulet, A., Benedito, J. 2012. An ultrasound-enhanced
system for microbial inactivation using supercritical carbon dioxide. Innovative
Food Science and Emerging Technologies, 15:31–37.
Ortuño, C., Martínez-Pastor, M.T., Mulet, A., Benedito, J. 2013. Application of high power
ultrasound in the supercritical carbon dioxide inactivation of Saccharomyces
cerevisiae. Food Research International, 51:474–481.
Oxen, P., Knorr, D. 1993. Baroprotective effects of high solute concentrations against inac-
tivation of Rhodotorula rubra. Lebensmittel-Wissenschaft & Technologie, 26:220–223.
Parish, M.E. 1998. High pressure inactivation of Saccharomyces cerevisiae endogenous
microflora and pectinmethylesterase in orange juice. Journal of Food Protection,
18:57–65.
Parton, T., Bertucco, A., Bertoloni, G. 2007. Pasteurisation of grape must and tomato
paste by dense-phase CO2. Italian Journal of Food Science, 19:425–437.
Patterson, M.F., Kilpatrick, D.J. 1998. The combined effect of high hydrostatic pres-
sure and mild heat on inactivation of pathogens in milk and poultry. Journal of
Food Protection, 61:432–436.
Patterson, M.F., Quinn, M., Simpson, R., Gilmour, A. 1995. Sensitivity of vegetative
pathogens to high hydrostatic pressure treatment in phosphate-buffered saline
and foods. Journal of Food Protection, 58:524–529.
Plancken, I.V., der Loey, A.V., Hendrickx, M.E. 2005. Combined effect of high pressure
and temperature on selected properties of egg white proteins. Innovative Food
Science and Emerging Technologies, 6:11–20.
Prapaiwong, N., Wallace, R.K., Arias, C.R. 2009. Bacterial loads and microbial com-
position in high pressure treated oysters during storage. International Journal of
Food Microbiology, 131:145–150.

Prestamo, G., Lesmes, M., Otero, L., Arroyo, G. 2000. Soybean vegetable protein
(tofu) preserved with high pressure. Journal of Agriculture and Food Chemistry,
48:2943–2947.
Prestamo, G., Sanz, P.D., Fonberg-Broczek, M., Arroyo, G. 1999. High pressure
response of fruit jams contaminated with Listeria monocytogenes. Letters in
Applied Microbiology, 28:313–316.
Rademacher, B., Kessler, H.G. 1996. High pressure inactivation of microorganisms
and enzymes in milk and milk products. In Abstracts of Poster Presentation. High
Pressure Processing of Foods Symposium, Cologne, Flair Flow Europe, p. 10.
Ramirez-Rodrigues, M.M., Plaza, M.L., Ferrentino, G., Balaban, M.O., Reyes De Corcuera,
J.I., Marshall, M.R. 2013. Effect of dense phase carbon processing on microbial
stability, physicochemical attributes of Hibiscus sabdariffa beverage. Journal of
Food process Engineering, 36: 125–133. doi: 10.1111/j.1745–4530.2011.00663.x.
Rodrigo, D., van Loey, A., Hendrickx, M. 2007. Combined thermal and high pres-
sure colour degradation of tomato puree and strawberry juice. Journal of Food
Engineering, 79:553–560.
Seyderhelm, I., Bogulawski, S., Michaelis, G., Knorr, D. 1996. Pressure induced inacti-
vation of selected food enzymes. Journal of Food Science, 60:164–168.
Shigehisa, T., Ohmori, T., Saito, A., Taji, S., Hayashi, R. 1991. Effects of high hydro-
static pressure on characteristics of pork slurries and inactivation of microor-
ganisms associated with meat and meat products. International Journal of Food
Microbiology, 12:207–216.
Shimada, S., Andou, M., Naito, N., Yamada, N., Osumi, M., Hayashi, R. 1993. Effects
of hydrostatic pressure on the ultra-structure of internal substances in the
yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 40:123–131.
Shoji, T., Saeki, H. 1989. Use of high pressure in food. In Progress of High Pressure Use
in Food Industry, ed. R. Ihayashi. San-ei Publications, Tokyo, Japan, pp. 75–87.
Simpson, R.K., Gilmour, A. 1997. The effect of high hydrostatic pressure on the
activity of intracellular enzymes of Listeria monocytogenes. Letters in Applied
Microbiology, 25:48–53.
Sims, M. 2000. Method and membrane system for sterilizing and preserving liquids
using carbon dioxide. U.S. Patent 6,331,272 B1 and WO Patent 00/41805.
Sirisee, U., Hsieh, F., Huff, H.E. 1998. Microbial safety of supercritical carbon dioxide
processes. Journal of Food Processing and Preservation, 22:387–403.
Sohn, K.H., Lee, H.J. 1998. Effect of high pressure treatment on the quality and stor-
age of kimchi. International Journal of Food Science and Technology, 33:359–365.
Sokołowska, B., Ska̧pska, S., Jolanta Niezgoda, J., Rutkowska, M., Dekowska, A.,
Rzoska, J.S. 2014. Inactivation and sublethal injury of Escherichia coli and Listeria
innocua by high hydrostatic pressure in model suspensions and beetroot juice.
High Pressure Research: An International Journal, 34:147–155.
Spilimbergo, S., Bertucco, A. 2003. Non-thermal bacteria inactivation with dense
CO2. Biotechnology and Bioengineering, 84:627–638.
Spilimbergo, S., Ciola, L. 2010. Supercritical CO2 and N2O pasteurization of peach
and kiwi juice. International Journal of Food Science and Technology, 45:1619–1625.
Spilimbergo, S., Elvassore, N., Bertucco, A. 2002. Microbial inactivation by high-
pressure. Journal of Supercritical Fluids, 22:55–63.
Spilimbergo, S., Komes, D., Vojvodic, A., Levaj, B., Ferrentino, G. 2013. High pressure
carbon dioxide pasteurization of fresh-cut carrot. Journal of Supercritical Fluids,
79:92–100.

Steeg, P.T., Hellemons, J.C., Kok, A. 1999. Synergistic actions of nisin, sublethal ultra-
high pressure, and reduced temperature on bacteria and yeast. Applied and
Environmental Microbiology, 65:4148–4154.
Styles, M.F., Hoover, D.G., Farkas, D.F. 1991. Response of Listeria monocytogenes and
Vibrio parahaemolyticus to high hydrostatic pressure. Journal of Food Science,
56:1404–1407.
Syed, O.A., Buffa, M., Guamis, B., Saldo, J. 2014. Effect of compression and decom-
pression rates of high hydrostatic pressure on inactivation of Staphylococcus
aureus in different matrices. Food and Bioprocess Technology, 7:1202–1207.
Thakur, B.R., Nelson, P.E. 1998. High pressure processing and preservation of food.
Food Reviews International, 14:427–447.
Ulmer, H.M., Gaenzle, M.G., Vogel, R.F. 2000. Effects of high pressure on survival and
metabolic activity of Lactobacillus plantarum. Applied Environmental Microbiology,
66:3966–3973.
Valverde, M.T., Marín-Iniesta, F., Calvo, L. 2010. Inactivation of Saccharomyces cerevi-
siae in conference pear with high pressure carbon dioxide and effects on pear
quality. Journal of Food Engineering, 98:421–428.
Van den Berg, R.W., Hoogland, H., Lelieveld, H.L.M., Van Schepdael, L. 2002. High
pressure equipment designs for food processing applications. In Ultra High
Pressure Treatments of Foods, ed. M.E.G. Hendrickx, and D. Knorr. Kluwer
Academic/Plenum Publishers, New York, pp. 23–51.
Venugopal, V., Kamat, A.S., Bongirwar, D.R. 2001. Processing of foods using high
hydrostatic pressure. Indian Food Industry, 20:65–69.
Wang, Y., Yi, J., Yi, J., Dong, P., Hu, X., Liao, X. 2013. Influence of pressurization
rate and mode on inactivation of natural microorganisms in purple sweet
potato nectar by high hydrostatic pressure. Food and Bioprocess Technology,
6:1570–1579.
Watanabe, M., Aria, E., Kumeno, K., Homma, K. 1991. A new method for producing
non-heated jam sample: The use of freeze concentration and high pressure ster-
ilization. Biological Chemistry, 55:2175–2176.
Wei, C.I., Balaban, M.O., Fernando, S.Y., Peplow, A.J. 1991. Bacterial effect of high pres-
sure CO2 treatment on foods spiked with Listeria or Salmonella. Journal of Food
Protection, 54:189–193.
Wouters, P., Glaasker, E., Smelt, J.P.P.M. 1998. Effects of high pressure on inactivation
kinetics and events related to proton efflux in Lactobacillus plantarum. Applied
and Environmental Microbiology, 64:509–514.
Yao, J., Zhou, B., Wang, R. et al. 2015. Inactivation of Staphylococcus aureus by high
hydrostatic pressure in saline solution and meat slurry with different initial
inoculum levels. Food and Bioproducts Processing, 94:592–600. doi: 10.1016/j.
fbp.2014.06.005.
Yoshioka, K., Yamada, A. 2002. Textural properties and sensory evaluation of soft
surimi gel treated by high pressurization. In Trends in High Pressure Bioscience
and Biotechnology, ed. R. Hayashi. Elsevier Science, Amsterdam, pp. 475–480.
Yuk, H.G., Sampedro, F., Fan, X., Geveke, D.J. 2014. Nonthermal processing of orange
juice using a pilot-plant scale supercritical carbon dioxide system with a gas–
liquid metal contactor. Journal of Food Processing and Preservation, 38:630–638.
Yulin, F., Keping, Y., Huhu, W. et al. 2014. Impact of high hydrostatic pressure treat-
ment on microbial communities in Chinese water-boiled salted duck. Journal of
Food Protection, 7:1142–1147.

Zabetakis, I., Koulentianos, A., OrrunÄo, E., Boye, I. 2000. The effect of high hydro-
static pressure on strawberry flavour compounds. Food Chemistry, 71:51–55.
Zenker, M., Heinz, V., Knorr, D. 2000. Hydrostatischer hochdruck zur erhoehungder
mikrobiellen sicherheit streichfaehiger rohwuerste. LVT, 45:89–92.
Zhang, B.-Y., Samapundo, S., Pothakos, V. et al. 2013. Effect of atmospheres combin-
ing high oxygen and carbon dioxide levels on microbial spoilage and sensory
quality of fresh-cut pineapple. Postharvest Biology and Technology, 86:73–84.
Zhou, L., Wang, Y., Hu, X., Wu, J., Liao, X. 2009. Effect of high pressure carbon dioxide
on the quality of carrot juice. Innovative Food Science and Emerging Technologies,
10:321–327.
Zimmermann, M., Schaffner, D.W., Aragão, G.M.F. 2013. Modeling the inactivation
kinetics of Bacillus coagulans spores in tomato pulp from the combined effect
of high pressure and moderate temperature. LWT—Food Science and Technology,
53:107–112.
Zook, C.D., Parish, R.J., Braddock, R.J., Balaban, M.O. 1999. High pressure inactivation
kinetics of Saccharomyces cerevisiae ascopores in orange and apple juices. Journal
of Food Science, 64:533–535.

15
Spray-Drying of Nano- and
Microcapsules of Nutraceuticals
Xiang Li, Nicolas Anton, and Thierry F. Vandamme
CONTENTS
15.1 Introduction................................................................................................. 456
15.2 Nano-Microencapsulation Technology................................................... 457
15.2.1 Structures of Nano- and Microparticles...................................... 457
15.2.2 Technologies for Nano-Microencapsulation............................... 458
15.2.3 Wall Material for Spray-Drying.................................................... 458
15.2.4 Core Materials for Spray-Drying in the Food Industry............ 460
15.2.4.1 Flavors................................................................................ 460
15.2.4.2 Vitamins............................................................................ 461
15.2.4.3 Lipids................................................................................. 462
15.3 Drying Process of Nano- or Microparticles............................................ 462
15.3.1 Atomization (Spray-Drying).........................................................463
15.3.2 Influences on Nano- or Microparticle Properties from
Different Parameters during the Spray-Drying Process...........464
15.4 Application of Büchi Mini Spray-Dryer B-290 and Büchi Nano
Spray-Dryer B-90 on Nano-Microencapsulation of Nano-
Emulsion of Vitamin E Acetate................................................................. 465
15.4.1 Büchi Mini Spray-Dryer B-290...................................................... 466
15.4.2 Büchi Nano Spray-Dryer B-90....................................................... 467
15.5 Nano-Emulsion........................................................................................... 468
15.5.1 High-Energy Emulsification Methods......................................... 469
15.5.2 Low-Energy Emulsification Methods.......................................... 470
15.5.3 Nano-Microencapsulation of Nano-Emulsion
of Vitamin E Acetate....................................................................... 470
15.5.3.1 Choice of Wall Materials................................................. 470
15.5.3.2 Sample Preparation.......................................................... 471
15.5.3.3 Nano-Microencapsulation of Nano-Emulsion
of Vitamin E Acetate by Spray-Drying......................... 472
15.5.4 Redispersion of Nano-Emulsion from the Spray-Dried
Nano- or Microparticles................................................................. 473
15.6 Conclusion................................................................................................... 474
References.............................................................................................................. 475
455
15.1 Introduction
Today’s scientists and engineers are finding a wide variety of ways to delib-
erately make materials at the nano- and microscale to take advantage of their
enhanced properties such as higher strength, higher stability, lighter weight,
increased control of light spectrum, and greater chemical reactivity than
their larger-scale counterparts. In the food industry, microencapsulation is
more commonly used than nanoencapsulation.
For food applications, the substance that is encapsulated may be called
the core material, the active agent, fill, internal phase, or payload phase. The
substance that is encapsulating may be called the coating, membrane, shell,
carrier material, wall material, external phase, or matrix. The carrier material
of encapsulates used in food products or processes should be food grade and
able to form a barrier for the active agent and its surroundings.
Encapsulates might also be defined by their particle size, for example,
nanoparticles, microcapsules, microreservoir, etc.
The possible benefits of microencapsulated ingredients in the food indus-
try could be
• Immobilization of active agents in food-processing systems

• Superior handling of the active agent (e.g., conversion of a liquid
active agent into a powder, which might be dust-free, free flowing,
and might have a more neutral smell)
• Improved stability in the final product and during processing (i.e.,
less evaporation of the volatile active agent and/or no degradation or
reaction with other components in the food product such as oxygen
or water)
• Controlled release (differentiation, release by the right stimulus)
• Improved safety (e.g., reduced flammability of volatiles like aroma,
no concentrated volatile oil handling)
• Adjustable properties of active components (particle size, structure,
oil- or water-soluble, color)
• Off-taste masking
• Creation of visible and textural effects (visual cues)
Microencapsulation is a rapidly expanding technology which is a unique

way to package materials in the form of micro- and nanoparticles and has
been well developed and accepted within the pharmaceutical, chemical,
food, and many other industries. Spray-drying is the most commonly used
encapsulation technique for food products. A successful spray-drying encap-
sulation relies on achieving high retention of the core materials, especially
volatiles and minimum amounts of the surface oil on the powder particles

Spray-Drying of Nano- and Microcapsules of Nutraceuticals 457
for both volatiles and nonvolatiles during the process and storage. The prop-
erties of wall and core materials and the prepared emulsion along with the
drying process conditions will influence the efficiency and retention of core
compounds.
Also, spray-drying technique has been widely used for drying heat-
sensitive foods, pharmaceuticals, and other substances, because of the sol-
vent rapid evaporation from the droplets, although most often considered
a dehydration process, spray-drying can also be used as an encapsulation
method when it entraps “active” material within a protective matrix, which
is essentially inert to the material being encapsulated. Compared to the other
conventional microencapsulation techniques, it offers the attractive advan-
tage of producing microcapsules in a relatively simple continuous processing
operation. This chapter will present a brief overview of the main consider-
ations involved in the application of spray-drying for microencapsulation,
with a special emphasis on the sizes given by the process parameters during
the microencapsulation.
15.2 Nano-Microencapsulation Technology

Nano- and microencapsulation is a technique in which the core compounds
are enclosed and protected by wall materials. Particles produced by encap-
sulation technology present a core/shell structure, which is composed of two
parts, the so-called core material and wall material. Active ingredients are
either embedded in the core materials, or form the core material itself, coated
by the wall material.
Nano- and microencapsulation technology presents numerous advantages
in the food industry (Gharsallaoui et al., 2007; Pérez-Masiá et al., 2014), such
as protection and stabilization of the active ingredients in core materials
from the environment, increasing the shelf-life of nutraceutical ingredients
and insuring their health-promoting properties, easier to handle, having
controlled release of the core material, masking the taste of the core material
and the dilution of the core material when it should be used in a very small
quantity.
15.2.1 Structures of Nano- and Microparticles

Microparticles are generally small spheres with diameter from a few microm-
eters to a few millimeters and nanoparticles are normally submicron-sized
particles. According to the nature of core materials, the physicochemi-
cal properties of wall materials, and the encapsulation techniques, differ-
ent types of particles can be produced, illustrated in Figure 15.1, ranging
from a simple morphology up to more complex morphologies: simple sphere

Simple Irregular Multicore Multiwall Matrix
FIGURE 15.1
Morphologies of different types of particles.
coating by the wall material; particles presenting an irregular shape core;

many core particles entrapped in one matrix of the wall material; several
distinct core particles within the same capsule composed by the wall mate-
rial and multiwall particles.
15.2.2 Technologies for Nano-Microencapsulation

The encapsulation technologies are classified into three major categories
(Li et al. 2011; Murugesan and Orsat, 2012):
1. Chemical methods: polymerization or interface polycondensation,

molecular inclusion, etc.
2. Physicochemical methods: coacervation, emulsion-solvent evapora-
tion, phase separation, rapid expansion of supercritical fluids, etc.
3. Physical methods: spray-drying, freeze-drying, fluidized-bed tech-
nology, pan coating, extrusion, etc.
Suitable encapsulation method ensures the production of high-quality

encapsulated particles. The selection of encapsulation method is according
to the specific application such as the required type and size of particles,
physicochemical properties of the core material and the wall material, and
the mechanism of release. In the food industry, for regulatory reasons, prin-
cipally physical methods can be used. Various food ingredients encapsulated
by different encapsulation methods are listed in Table 15.1. In this chapter,
we focus on the spray-drying technology, which is one of the oldest and very
common methods for the encapsulation of food ingredients and additives in
the nutraceutical industries.
15.2.3 Wall Material for Spray-Drying

Since the wall material presents different encapsulating properties and
release characteristics for core materials, the choice of the wall material
is very important for the encapsulation efficiency and nano- or micropar-
ticle stability during storage. The wall material is generally designed for
protecting the core material from the outside environment, preventing
the interaction of the core compound with other ingredients and having

TABLE 15.1
Food Ingredients Encapsulated by Different Encapsulation Methods
Encapsulated Encapsulation
Ingredients Wall Material Technology Reference
Tuna oil Gelatin/sodium Coacervation Wang et al. (2014)
hexametaphosphate
(SHMP)
Peppermint oil Gelatin/gum arabic Coacervation Dong et al. (2011)
Sesame oil Maltodextrin/modified Fluidized-bed Jeong et al. (2009)
starch/gum arabic/ granulation
gellan gum
Fennel oleoresin Gum arabic/ Freeze-drying Chranioti and
maltodextrin, modified Tzia (2014)
starch/chitosan
Fish oil Barley protein Spray-drying Wang et al. (2011)
Flavor oil Maltodextrin Freeze-drying Kaasgaard and
(r-carvone) emulsion Keller (2010)
Limonene or medium Gum acacia/gelatin Coacervation Leclercq et al.
chain triglycerides or (2009)
menthol
Rosemary essential oil Gum arabic/modified Spray-drying Fernandes et al.
starch/maltodextrin/ (2014)
inulin
Clove extract Maltodextrin Spray-drying, Cortés-Rojas
freeze-drying et al. (2014)
d-Limonene β-Cyclodextrin Spray-drying, Yuliani et al.
extrusion (2006)
β-Carotene Maltodextrin Spray-, freeze-, Desobry et al.
and drum-drying (1997)
Folic acid Whey protein/resistant Electrospraying, Pérez-Masiá et al.
starch nanospray-drying (2014)
a controlled release of the core material at desirable conditions. A suitable

wall material for spray-drying should present a high emulsifying activity,
a good stability, a rapid formation of a fine and dense crust during the
drying process, a good compatibility with other ingredients in the for-
mulation, and should have a good retention and protection of core com-
pounds. In addition, most samples for spray-drying in the food industry
are aqueous formulations, thus the available wall materials must have a
good solubility in water.
According to the desirable properties of the final encapsulated particles
and the nature of the core material, the wall materials used for spray-drying
can be a choice from a variety of natural and synthetic polymers (e.g., modi-
fied starches, pectin, gum arabic, whey protein, gelatin, alginate, polyvi-
nyl alcohol): (1) Carbohydrates such as starches, maltodextrins, are often
used for the encapsulation of food ingredients. These polymers present

good solubility and low viscosity at high concentration; however, they have
generally poor interfacial properties that are important for high encapsula-
tion efficiency. To improve the encapsulating properties, these compounds
are often associated with other wall materials or are chemically modified,
such as lycoat®, a modified starch specifically developed for the aqueous
film coating for immediate release, solid oral dosage forms. Maltodextrin
is a polysaccharide produced from starch by partial hydrolysis. It is widely
used as a food additive and encapsulation carrier, due to its relatively low
cost, nature aroma taste, low sweetness, and low viscosity at high concen-
trations. Researchers in the literature concluded that maltodextrins with
dextrose equivalence from 10 to 20 are suitable to be used as the wall mate-
rial (Gharsallaoui et al. 2007). (2) Gum arabic is one of the most common
wall materials in microencapsulation by spray-drying process due to its
good emulsifying efficiency and film-forming properties. This natural gum
is a complex mixture of arabinogalactan oligosaccharides, polysaccharides,
and glycoproteins. Gum arabic is highly soluble in water and the presence
of proteins in gum arabic ensures its emulsification properties, thus keep-
ing good oil retention. (3) Proteins, such as whey protein and soy protein,
present amphiphilic and film-forming properties that are suitable for the
encapsulation of hydrophobic core materials (Gharsallaoui et al. 2007).
Whey protein is a mixture of globular proteins isolated from whey, includ-
ing alpha-lactalbumin, beta-lactoglobulin, immunoglobulin, and serum
albumin. It is considered as a suitable wall material due to its functional
properties required for the encapsulation process. Compared with animal
proteins that are largely used as wall materials, plant proteins, such as bar-
ley protein, should be also a good candidate for the encapsulation applica-
tions. They also present emulsifying and film-forming properties and they
are less expensive and can reduce the risk of spreading diseases (Wang et al.
2011).
15.2.4 Core Materials for Spray-Drying in the Food Industry

Spray-drying is a common and low-cost technique for the production of
encapsulated food materials. This technology is widely used for the protec-
tion of bioactive compounds or functional foods from outside environment,
such as flavors, lipids, and vitamins. Here we take some examples of food
ingredients, which are mostly encapsulated by spray-drying process.
15.2.4.1 Flavors
Flavors are very important food ingredients and can promote the consump-
tion of some food products. Spray-drying is the most common technology
for the encapsulation of flavors. The encapsulation of flavors can prevent
the loss of volatile flavors and enhance the stability of flavors in the core of
encapsulated particles by reducing the degradative reactions. Bylaitë et al.

(2001) demonstrated that the caraway essential oil was successfully encapsu-
lated by whey protein and the wall system provided an effective protection
of the core material against oxidation. The flavor of d-limonene is encap-
sulated by spray-drying with various wall materials (gum arabic, malto-
dextrin, and modified starch) (Soottitantawat et al. 2005a) and the modified
starch showed higher stability of encapsulated d-limonene than other wall
materials. Rosemary essential oil was encapsulated by various combinations
of wall materials between gum arabic, modified starch, maltodextrin, and
inulin (Fernandes et al. 2014). The results showed that the different mixtures
of wall materials had significant effects on the characteristics of the obtained
microparticles. The spray-drying technology can encapsulate not only liq-
uid flavors, but also solid flavors. l-menthol, a solid flavor, was encapsulated
by spray-drying by using gum arabic and modified starch as wall materi-
als (Soottitantawat et al. 2005b). The results indicated that the flavor-loading
capacity depended on the type of the wall material. Modified starch showed
higher l-menthol retention; however, gum arabic seemed to be a more suit-
able wall material in this study due to the good l-menthol retention and the
lower surface l-menthol contents.
15.2.4.2 Vitamins
Since vitamins are often partially denaturized or damaged during storage
or food preparation, the encapsulation should be considered as a good solu-
tion for the preservation of vitamins. Ascorbic acid (vitamin C) in juices has
been successfully encapsulated and protected by the spray-drying process
(Rodríguez-Hernández et al. 2005; De Oliveira et al. 2009) and the retention
of vitamin C can reach up to 95% (De Oliveira et al. 2009). Folic acid, a water-
soluble vitamin, is encapsulated by whey protein and a commercial resistant
starch by using a Nano Spray-Dryer B-90 (Büchi) (Pérez-Masiá et al. 2014).
Spherical submicron capsules were obtained after nanospray-drying pro-
cess. Whey protein showed higher encapsulation efficiency than the resis-
tant starch. In the folic acid stability study, particles encapsulated by whey
protein showed greater stability than resistant starch ones in both aqueous
solution and dry form situations. The whey protein capsules in the dry form
can keep the bioactive stability of folic acid at 100% in darkness conditions
when 40% of nonencapsulated folic acid was degraded. Vitamin E acetate
formulated in nano-emulsion was encapsulated by different wall materials
by using the spray-drying technology (Li et al. 2011). The results showed
that the nano-emulsion of vitamin E acetate was successfully encapsulated
and the stability of vitamin E acetate in the spray-dried particles was evalu-
ated by the high-performance liquid chromatography (HPLC) method. The
results showed very high encapsulation efficiency of nano-emulsion of vita-
min E acetate, approximately 100% and the spray-drying process did not
induce the degradation of the encapsulated molecule and preserve the integ-
rity of the nano-emulsion system.

15.2.4.3 Lipids
Encapsulation of lipids is very important in the food industry. This tech-
nique transforms the liquid lipids into dry powders, which is easier to han-
dle, improves their dispersion in food products, and reduces their oxidation.
Numerous papers have been published about the encapsulation of lipids by
spray-drying (Kagami et al. 2003; Bae and Lee, 2008; Drusch and Berg, 2008).
The linoleic acid was encapsulated by spray-drying and its oxidation was
studied by Minemoto et al. (2002). Microparticles of linoleic acid encapsu-
lated by gum arabic showed higher stability and better oxidation resistance
than that encapsulated by maltodextrin. Fish oil is usually encapsulated into
nano- or microcapsules. Thus, the unpleasant taste of fish oil can be masked
and the encapsulation form can prevent the oxidation of fish oil. In the
study of Tan et al. (2009), fish oil was encapsulated by spray-drying by using
alginate/starch blends as the wall material (Tan et al. 2009). The addition
of alginate in the wall material demonstrated higher fish oil encapsulation
efficiency. And the microencapsulated fish oil exhibited higher stability than
unencapsulated fish oil. In another work, fish oil was for the first time encap-
sulated by barley protein (Wang et al. 2011). An emulsion was first formed
by mixing barley protein and fish oil. The solid microcapsules were then
obtained through the spray-drying process. These barley protein microcap-
sules exhibited high encapsulation and loading efficiency and a strong abil-
ity to protect fish oil against oxidation.
15.3 Drying Process of Nano- and Microparticles

Nanomaterials (micro materials) require to be dried during processing. Water
molecules are approximately 0.35 nm in size. When talking about nanoma-
terials (micro materials), water certainly cannot be considered a continuum.
This water is also responsible for capillary pressure and drying stresses in
nano (micro) porous solids.
Drying water at a molecular level involves a play of interaction forces
between molecules of gas, molecules of liquid, and nano- or microparticles,
which are only several times larger. The surface tends to assume an equilib-
rium state in which the overall surface energy is the lowest. When molecules
of water are removed from the surface one by one during drying, new sur-
face configurations emerge. Then, understanding of drying at the molecular
level can be very helpful in drying on the nanometric (micrometric) scale
and can be used to obtain the designed product properties.
Drying can be used in a production cycle of nano- or microparticles and
nano- or microcapsules. In all cases, the drying process is essential in obtain-
ing the designed and expected properties of the final product. Moreover, in

many cases, these properties could not be obtained without a specific dry-
ing technique. Different methods can be applied. Principally, we can note
the spray-drying using ultrasonic nebulizers and electrospraying. Also the
method of supercritical-drying and the solvent replacement technique can
be used.
15.3.1 Atomization (Spray-Drying)

By now, when the agroalimentary industrial world refines its technological
processes to bring more controls of the manufacturing costs, the progressive
passage of batch processes to the continuous processes is undoubtedly one of
the strong axes of the technological revolutions of the next years. More par-
ticularly, in the field of drying and obtaining particles of determined sizes,
the food industry can currently use in particular from the technology of
atomization for the manufacturing of ingredients and also for the improve-
ment of the productivity of the presentations in the form of powders.
Today, drying by atomization opens very interesting alternative ways
compared to discontinuous technologies like freeze-drying. The technologi-
cal improvements, original associations of the multistage solutions integrat-
ing one or more successive stages of drying by atomization, make it possible
to optimize the kinetics of desorption of strongly thermosensitive products.
By respecting the paces of drying, the temperatures of the coatings of the
products can remain lower than the maximum limits fixed to avoid any risk
of degradation of the functional and biological properties of the ingredients
to be stabilized.
A method of drying the particles, atomization is carried out in a continu-
ous way in order to process liquid products possibly thermosensitive into
powder intended for food, chemical, or pharmaceutical industries. The prin-
ciple of spray-dryer is illustrated in Figure 15.2. The liquid sample is fed by
a supply pump and dispersed into thin droplets through the fluid nozzles.
Thin droplets are atomized in contact with hot air in the drying chamber.
And finally, dried particles are separated through a separation cyclone and
recovered in the collector.
This operation is fast and is done at a moderate temperature. It consists of
removing the water of the product to be dried by pulverizing it in a large hot
air draught. The product to be dried, which can be a solution, a suspension,
or still a pasty liquid (thixotropic), is sent in the atomizer starting from a tank
of food. One generally uses a positive-displacement pump with a continuous
variable flow for this transfer. The atomizer allows the pulverization of the
product in a fog of fine droplets. Three types of atomizers and drying can be
used in the following:
• Centrifugal atomization. The product is dispersed with a co-current

in the air of drying by the turbine of a centrifugal spray placed at the
top of the room.

4 5
3
1a
10
1b
7 2
FIGURE 15.2
Schematic of a spray-dryer: 1a, Atomization turbine or nozzle; 1b, countercurrent nozzle; 2,
supply pump under supply tank; 3, drying air filter; 4, hot air oven; 5, air distributor; 6, drying
chamber; 7, transport channel; 8, separation cyclone; 9, extraction ventilator; 10, chimney for
humid air evacuation.
• Atomization with countercurrent: a tube with a high pressure is

then used.
• Atomization by a bi-fluid tube: this tube is placed at the co- or
countercurrent of the air in the conical part of the tower. In this case,
the atomization of the liquid is carried out by compressed air. The
product to be dried can be maintained at a given temperature.
15.3.2 Influences on Nano- or Microparticle Properties from

Different Parameters during the Spray-Drying Process
Influence of the inlet temperature of air on the powder: It is not because the inlet
temperature of air is of 250°C that the product will be carried at this tem-
perature. Indeed, the design of the installation is made so that the solution
does not touch the diffuser of air. Very intense evaporation around the
droplet of liquid makes that the heart of the particle is maintained at low
temperature.
The temperature of the particle rises only during the secondary phase of
drying which is carried out during the fall of the particle in the room.

In general, it is admitted that this temperature will be approximately 15°C

lower than the temperature of the exit air, but this rise will be carried out
only during a relatively short time. In addition, if the product is thermosensi-
tive, it has to be cold at exit on a fluidized bed or in any other cooled carrier.
The classical use consists in having an air flow defined with a regular tem-
perature as the starter of turn. This temperature can be controlled on a very
broad beach. The pressure into the tower is carried out by evaporating water
by adjustment with the ventilator downstream of the installation. The flow
of the solution to be dried will be regulated in order to reach the temperature
of the exit air.
For a pressure in the tower corresponding to an air flow, and for an inlet
temperature fixed at a temperature of exit of the air given, will correspond a
moisture of the powder related to the following parameters:
• Concentration of the solution

• Speed of the turbine
• Diameter of the tube
Granulometry is dependent on
• The viscosity of the solution

• Concentration of the starting solution
• Circumferential speed of the turbine
• The temperature of ingoing air
The inlet temperature of air and especially neutral gas injection such as
the nitrogen and carbon dioxide in the solution of entry make it possible to
lighten the powder. This technique is applicable only for atomization by tube.
15.4 Application of Büchi Mini Spray-Dryer B-290 and Büchi

Nano Spray-Dryer B-90 on Nano-Microencapsulation
of Nano-Emulsion of Vitamin E Acetate
As described above, spray-drying is a common technology for nano- and
microencapsulation in the food industry. The spray-drying process to trans-
form liquid substances into a dried powder is widely used for the encapsula-
tion of flavors, vitamins, lipids, probiotic bacteria, and proteins.
Recently, the spray-drying is used as a novel technique for drying lipid
nano-emulsion suspensions to create nano or microparticles in solid form
by using different types of spray-dryer. Encapsulation of nano-emulsion

by using spray-drying technology can conserve the nanometric scale of the

encapsulated droplets entrapped homogeneously in nano- or microcap-
sules. The solid forms ease the conservation and extend the storage time
of nano-emulsions, especially for nano-emulsion containing sensitive com-
pounds. Encapsulation of nano-emulsion by spray-drying is considered as
a new solution for the delivery of nano-emulsion. The spray-dried particles
ensure the delivery of the encapsulated nano-emulsion in their core, with
redispersion of the dried particles in water. Here the nano-emulsion of vita-
min E acetate, which is formulated by the low-energy emulsification method
(described below), is selected as the model and we will discuss the encapsu-
lation of these nano-emulsions by the spray-drying technology.
In this part, we focus on the applications of two spray-dryers developed by
Büchi Company as representative apparatus on nano- or microencapsulation
of nano-emulsion and the different characteristics of encapsulated particles
formulated by these two spray-dryers:
1. The classical spray-dryer of Büchi mini B-290 allows the rapid and
efficient transformation of liquid sample (a solution, a suspension or
an emulsion) in microparticles.
2. The spray-dryer, Büchi Nano Spray-Dryer B-90, that is, producing
submicron particles (nanoparticles) with narrow distributions and
high formulation yield.
15.4.1 Büchi Mini Spray-Dryer B-290

The principle of the Mini Spray-Dryer B-290 is based on the heat and mass
transfers, illustrated in Figure 15.3. The liquid to be atomized is composed of
the core material and the wall material. The core material is homogeneously
solubilized or dispersed in the solution of the wall material. The liquid
sample is fed to the spray head by a pump and dispersed into thin droplets
through the fluid nozzle in the drying chamber. The goal of this stage is to
create a large air and water interface area, thus optimizing the heat and mass
transfers. Thin droplets are atomized in contact with a hot gas flux. The dry-
ing stage of the droplets can be devised into three successive steps: (1) the
temperature of the sprayed droplets increases until a constant value as soon
as the liquid is in contact with the hot air; (2) then, the evaporation of droplets
water is carried out at a constant temperature. The rate of water diffusion
from the core to the droplet surface is considered constant, which is equal
to the surface evaporation speed; (3) then, a dry crust (or shell) is formed on
the surface of droplets, and the drying rate is rapidly decreased and depends
on the water diffusion rate through the crust. The drying process is finished
when the temperature of the particle is equal to that of the air. The spray and
drying processes rapidly finish within a few seconds allowing to work with
sensitive or thermosensitive compounds without resulting in their degra-
dation. Finally, the dried particles are separated with the wet gas through

Compressed
air (N2)
Nano-emulsions
+ wall material
Spray nozzle
Tin
Spray
Hot air
flux Tout
Cyclone Micro-
particles
FIGURE 15.3
Schematic of the Mini Spray-Dryer B-290 (Büchi).
a cyclone and recovered in the collector. The resulting powders are matric
system of microparticles (or microspheres). Depending on the nature of the
wall materials or the operational conditions, such as the inlet temperature,
concentration, or flow rate, the formed microparticles can exhibit a spherical
or hollowed morphology. The formulation yields of the Mini Spray-Dryer
B-290 are around 70%.
15.4.2 Büchi Nano Spray-Dryer B-90

The development of the Nano Spray-Dryer B-90 was an important innova-
tion within the spray-drying technology. This nano spray-dryer based on a
mesh vibration technology allows to produce submicron-sized particles with
narrow distributions and high formulation yields (Li et al. 2010). A complete
diagram of the apparatus is illustrated in Figure 15.4. The liquid sample is
fed into the mesh-based droplet generation system as illustrated in the fig-
ure. The generation of droplets was based on a piezoelectric-driven actuator,
vibrating a thin, perforated, and stainless steel membrane in a small spray
cap. The membrane (spray mesh) features an array of precise, micron-sized
holes (4.0, 5.5, or 7.0 µm). The actuator is driven at around ultrasonic fre-
quency, causing the membrane to vibrate, ejecting millions of precisely sized
droplets per second with a very narrow distribution. These extremely fine
droplets are dried into solid particles by the drying gas, which enters from
the top of the apparatus and heating up to the setting inlet temperature.

Drying gas
Mesh-based droplet
generation system
Heater
Tin
Control
Droplets
Drying
chamber
Product
Top
Collecting
electrode Bott. Tout
Filter
~
Grounded
electrode
FIGURE 15.4
Schematic of the Nano Spray-Dryer B-90 (Büchi).
The drying gas goes through the whole drying chamber to be sucked in at

the bottom and is filtered before leaving the instrument. The spray-dried
particles are finally recovered by electrostatic charging and are deflected to
the collecting electrode. Higher yields up to 90% can be achieved with the
Nano Spray-Dryer B-90.
15.5 Nano-Emulsion
Nano-emulsions are nano-sized emulsions (up to 500 nm) (El-Aasser and
Sudol, 2004; Solans et al. 2005; Anton et al. 2008), generally defined as oil-in-
water emulsion droplets. They are transparent or translucent systems due to
their submicron sizes, shown in Figure 15.5. Nano-emulsions are kinetically
stable systems (not thermodynamically stable) and can remain stable for sev-
eral months due to their small droplet sizes and narrow size distribution.
The reduced gravity forces prevent sedimentation and creaming (Tadros
et al. 2004; Solè et al. 2010). Nano-emulsion systems present many advan-
tages to be as new vehicles in pharmaceutical and food industries:
• Simple in composition (oil-in-water nanodroplets stabilized by sur-

factant) and simple in formulation
• Powerful, great intrinsic suspension stability, adaptability of the for-
mulation processes, incorporation of active ingredients

FIGURE 15.5
Nano-emulsion.
• Size and polydispersity controllable at leisure

• Solvent-free method, using excipients dedicated to the specific
administration routes (parenteral, oral, etc.)
• Facile dispersion of lipophilic compounds in homogeneous and sta-
ble aqueous suspensions
• Nanosize can enhance the penetration in biological media
• Template for nanoparticles and nanocapsules formulations
• Possibilities to encapsulate hydrophilic guest molecules
• Prevention of the inopportune drug leakage
As regards the formulation processes, nano-emulsion preparation meth-

ods can be divided into two groups: high-energy and low-energy emulsifica-
tion methods.
15.5.1 High-Energy Emulsification Methods

These methods use specific equipment to form nano-emulsions, such as
ultrasound generators, high pressure homogenizers, and microfluidiz-
ers (Jafari et al. 2008). High-energy emulsification methods present sev-
eral advantages in industrial applications, such as the flexible control of
droplet size distributions and the ability to produce submicron emulsions
from a large variety of materials. The amount of surfactant is lower than
nano-emulsion produced by low-energy methods. However, high-energy
emulsification methods are more expensive than low-energy methods on
nano-emulsion production and the main limitation of high-energy method
is the “over-processing,” which refers to an increase in the emulsion size by
supplying more energy due to a high rate of re-coalescence of new droplets.

15.5.2 Low-Energy Emulsification Methods

Low-energy method takes intrinsic physicochemical properties of compo-
nents into the formulation, and nano-emulsions can be produced almost
spontaneously (Anton et al. 2008; Anton and Vandamme, 2009). The
emulsification process is characterized by studying the influence of the
surfactant-to-oil ratio (SOR) on the size and polydispersity of the nano-
emulsion. The size of nano-emulsion decreases when the amount of the
surfactant increases and the aqueous phase (water) breaks up the droplets
and dilutes the sample without influencing the size and polydispersity
index (PDI) of nano-emulsion.
The formulation procedure of nano-emulsion of vitamin E acetate is as
follows:
1. The oily phase (vitamin E acetate) and the hydrophilic surfactant

are totally miscible and the mixture is gently homogeneous at room
temperature.
2. The aqueous phase (water or buffer solution) is then added into the
previous mixture.
3. Nano-emulsion is formed spontaneously once these two phases
mixed (the hydrophilic species are immediately solubilized by the
aqueous phase inducing the spinodal demixtion of the oil in the
form of nanometric emulsion droplets). The surfactant stabilizes
immediately the formed nano-droplets.
In order to ensure the efficient nano-emulsion encapsulation of the spray-

dried particles, optimized formulation of nano-emulsion is of the size below
100 nm with a good PDI.
15.5.3 Nano-Microencapsulation of Nano-Emulsion
of Vitamin E Acetate
For the encapsulation of the nano-emulsion, the sample is composed of
nano-emulsion dispersed in an aqueous solution to dissolve the wall mate-
rial. The important parameters to ensure the encapsulation efficiency are the
compatibility of nano-emulsion with the wall material and the respective
proportions of these compounds.
15.5.3.1 Choice of Wall Materials

The choice of a wall material is very important on the encapsulation efficiency
and the stability of the protected molecule, described in the above section of
the wall material for spray-drying. The selection of wall materials is based
on their physicochemical properties. The main criteria to be considered are
the good solubility in water, the emulsifying properties, stability, without

interaction with the nano-emulsion or other ingredients, the tendency to

form a hard, thin crust during the atomization, and the good retention of
nano-emulsion. Based on these criteria, several wall materials are selected:
gum arabic, whey protein, maltodextrin cleargum® (modified starch), klep-
tose® (hydroxypropyl beta cyclodextrin), nutriose® (soluble dietary fiber),
and lycoat® (modified starch).
15.5.3.2 Sample Preparation

The encapsulation process by spray-drying consists of three major steps,
illustrated in Figure 15.6:
1. Nano-emulsion of vitamin E acetate is firstly specifically formulated,

with a size below 100 nm.
2. The formulated nano-emulsion is then mixed with the wall
material solution and the mixture should be homogenous before
spray-drying.
3. Atomization of the mixture of the wall material and nano-emulsion.
In order to obtain suitable powdery samples after spray-drying and

ensure the encapsulation efficiency, optimal spray-drying conditions should
be used. The main parameters are the inlet temperature, the outlet tem-
perature, and the weight ratio between the nano-emulsion (oil + surfactant)
and the wall material. Optimal inlet temperature must be ensured for the
Wall material
solution (polymer)
Water
Vitamin E acetate
+ surfactant
Nano-emulsions Nano-emulsions
+ polymer
Spray-
drying
Powder of
microparticles
FIGURE 15.6
Process of encapsulation of nano-emulsion of vitamin E acetate by spray-drying.

performance and efficacy of the spray-drying process without damaging

the product and the outlet temperature for food ingredients is ideal around
50–80°C. As the microencapsulation efficiency is increased by increasing
the concentration of the wall material, the optimal formulation is a compro-
mise between the nano-emulsion quantity in the final spray-dried particles
and the quantity of the wall material to ensure the encapsulation; thus the
weight ratio of oil + surfactant: wall material is fixed at 1:4 in the study of Li
et al. (2010, 2011). And the concentrations of different wall material solutions
are 15% for Büchi Mini Spray-Dryer B-290 and 1% for Büchi Nano Spray-
Dryer B-90.
15.5.3.3 Nano-Microencapsulation of Nano-Emulsion
of Vitamin E Acetate by Spray-Drying
Comparing the SEM (Scanning electron microscope) pictures of encapsu-
lated nano-emulsion of vitamin E acetate by the Nano Spray-Dryer B-90
and Mini Spray-Dryer B-290 with the identical wall materials, mentioned
in Figure 15.7, we noted that the powders obtained by the Nano Spray-
Dryer B-90 exhibited size distributions with maxima below the 1 µm scale.
And the powders spray-dried by the Mini Spray-Dryer B-290 have size
larger than 1 µm with a relatively narrow size distribution. Comparing
the SEM pictures of encapsulated emulsion to those obtained with pure
wall materials under identical experimental conditions, we can see that
the morphology and size distribution are different. For the Mini Spray-
Dryer B-290, the microparticles of the pure wall material show smooth
surfaces, spherical geometry, and intense invaginations, with a shape
resembling a “deflated ball,” shown in Figure 15.7a,b(A1,B1). However, the
invaginations are generally inhibited by the presence of nano-emulsion in
the spray-dried particles (Figure 15.7a,b(A2,B2)) and the particle sizes are
significantly reduced. This suggests that some of the nano-emulsion drop-
lets place at the water/air interface, decreasing the surface tension and
giving rise to smaller particles. Nevertheless, for the Nano Spray-Dryer
B-90, the SEM pictures demonstrate spherical-shaped particles for both
pure wall material system and wall material + nano-emulsion system, in
Figure 15.7c,d. Comparing the size distribution of encapsulated emulsion
to those obtained with pure wall materials under identical experimental
conditions, we note that nano-emulsion encapsulating particles show a
slight increase in peak size along with a slight decrease in peak width
(Figure 15.7c,d), which demonstrates a nonnegligible impact of the pres-
ence of the oil droplets on the generation of the spray-dried particles.
Thus, we can conclude that the spray-drying technology is obviously able
to encapsulate emulsions smaller than 100 nm into separated solid par-
ticles and the new spray-drying concept of the Nano Spray-Dryer B-90 can
atomize submicron size particles compared with the classical spray-dryer
for producing microparticles.

FIGURE 15.7
Encapsulation of nano-emulsion of vitamin E acetate. (a) Gum arabic particles produced by the
Mini Spray-Dryer B-290: (A1) pure wall material particles, (A2) nano-emulsion encapsulated par-
ticles; (b) whey protein particles produced by the Mini Spray-Dryer B-290: (B1) pure wall material
particles, (B2) nano-emulsion encapsulated particles; (c) gum arabic particles produced by the
Nano Spray-Dryer B-90: (C1) pure wall material particles, (C2) nano-emulsion encapsulated par-
ticles; (d) whey protein particles produced by the Nano Spray-Dryer B-90: (D1) pure wall material
particles, (D2) nano-emulsion encapsulated particles; (e) a schematic illustration of spray-dried
particles encapsulating nano-emulsion of vitamin E acetate.
15.5.4 Redispersion of Nano-Emulsion from the Spray-Dried

Nano- or Microparticles
Nano- or microencapsulation of nano-emulsion requires that the nano-
droplets conserve their size and individual nature after entrapped in the
dried particles. This can be checked by the redispersion of the dried powder

TABLE 15.2
Nano-Emulsion (NE) Size Distribution Mixed with Wall Material before Spray-
Drying and after Spray Drying
Mini Spray-Dryer B-290 Nano Spray-Dryer B-90
NE + Wall NE + Wall NE + Wall NE + Wall
Material Material Material Material
Before After Before After
Spray-Drying Spray-Drying Spray-Drying Spray-Drying
Wall Materials d h (nm) PDI d h (nm) PDI d h (nm) PDI d h (nm) PDI
Gum arabic 86.9 0.130 155.4 0.176 95.49 0.155 105.9 0.511
Modified starch 82.6 0.150 168.3 0.268 91.49 0.229 161.5 0.243
Maltodextrin 81.0 0.110 182.3 0.276 83.49 0.088 140.6 0.167
Whey protein 83.7 0.082 100.6 0.220 88.54 0.261 110.3 0.268
in water. The resolubilization of the wall material in water allows the redis-
persion of the nano-emulsion droplets. Wall materials are rapidly solubilized
due to the huge specific surface of the powder and, if kept intact during the
process, the nano-emulsion droplets can be redispersed in water.
All the nano- or microparticles mentioned above are translucent after redis-
persion, like typical nanometric dispersions. The sizes of nano-emulsion are
measured before and after spray-drying process, and redispersion in the
same volume of water (Table 15.2).
To summarize, for the Nano Spray-Dryer B-90, redissolving the spray-
dried nanoparticles in water obtains nano-emulsion without any significant
degradation and size increase. This result demonstrates the capacity of this
spray-drying process to preserve the integrity of nano-structured systems.
And for the Mini Spray-Dryer B-290, the samples before and after spray-
drying demonstrate a significant increase in size. The results can be simply
due to turbulence and heat during the spray-drying process. The variety
of size after redispersion of different wall materials indicates that the oil/
polymer interactions and affinities could play a role in droplet merging.
However, the average size of the redispersed nano-emulsion droplets is
about 150 nm and, with a PDI around 0.2, such an emulsion can still rightly
be considered as nano-emulsion. The microencapsulation of nano-emulsions
thus appears to have been successful.
15.6 Conclusion
Encapsulation technology for the production of nano- or microcapsules
is widely used in the food industry. It provides the protection against the
degradative reactions of the core materials, enhances the stability of active

ingredients in the core, becomes easier to handle, and gives the controlled
release of the encapsulated active molecules. Spray-drying is an important
technology to produce nano- or microcapsules in the food and nutraceuticals
industries. Depending on the different spray-dryers, both nano- and mic-
roparticles can be produced, and by playing with different parameters, like
the feed rates, the emulsion size, or by using different wall materials, the
encapsulation efficiency can be optimized. Various food ingredients, such as
vitamins, flavors, and lipids, as discussed above can be encapsulated by the
spray-drying process. Since it is very quick drying process and by using the
optimal experimental conditions, such as lower inlet temperature, spray-dry-
ing can even work with thermosensitive materials. The choice of the various
wall materials and the study of the interaction between the wall material and
the core material are necessary to ensure the obtention of the desired prop-
erties of nano- or microcapsules. Recently, the technology of spray-drying
is used for the encapsulation of nano-emulsion, described in this chapter.
Since the nano-emulsion kept their size under 200 nm after redispersion test,
we can conclude that the process of spray-drying preserved the integrity of
nano-emulsion. And the encapsulation of nano-emulsion by spray-drying
appeared to have been successful. Further researches of the spray-drying in
the food industry should be based on the enhancement of the encapsulation
efficiency, the development of new wall materials with a good encapsula-
tion efficiency, low cost, and the encapsulation of some materials that are not
available for the moment.
References
Anton, N., Benoit, J.-P., Saulnier, P. 2008. Design and production of nanoparticles for-
mulated from nano-emulsion templates—A review. Journal of Controlled Release,
128:185–199.
Anton, N., Vandamme, T.F. 2009. The universality of low-energy nano-emulsification.
International Journal of Pharmaceutics, 377:142–147.
Bae, E.K., Lee, S.J. 2008. Microencapsulation of avocado oil by spray drying using
whey protein and maltodextrin. Journal of Microencapsulation, 25:549–560.
Bylaitë, E., Venskutonis, P.R., Mapdpierienë, R. 2001. Properties of caraway (Carum
carvi L.) essential oil encapsulated into milk protein-based matrices. European
Food Research and Technology, 212:661–670.
Chranioti, C., Tzia, C. 2014. Arabic gum mixtures as encapsulating agents of freeze-
dried fennel oleoresin products. Food and Bioprocess Technology, 7:1057–1065.
Cortés-Rojas, D.F., Souza, C.R.F., Oliveira, W.P. 2014. Encapsulation of eugenol rich
clove extract in solid lipid carriers. Journal of Food Engineering, 127:34–42.
De Oliveira, M.A., Maia, G.A., De Figueiredo, R.W. et al. 2009. Addition of cashew
tree gum to maltodextrin-based carriers for spray drying of cashew apple juice.
International Journal of Food Science and Technology, 44:641–645.

Desobry, S.A., Netto, F.M., Labuza, T.P. 1997. Comparison of spray-drying, drum-
drying and freeze-drying for β-carotene encapsulation and preservation.
Journal of Food Science, 62:1158–1162.
Dong, Z., Ma, Y., Hayat, K. et al. 2011. Morphology and release profile of microcap-
sules encapsulating peppermint oil by complex coacervation. Journal of Food
Engineering, 104:455–460.
Drusch, S., Berg, S. 2008. Extractable oil in microcapsules prepared by spray-drying:
Localisation, determination and impact on oxidative stability. Food Chemistry,
109:17–24.
El-Aasser, M.S., Sudol, E.D. 2004. Miniemulsions: Overview of research and applica-
tions. Journal of Coatings Technology Research, 1:20–31.
Fernandes, R.V.D.B., Borges, S.V., Botrel, D.A. 2014. Gum arabic/starch/maltodextrin/
inulin as wall materials on the microencapsulation of rosemary essential oil.
Carbohydrate Polymers, 101:524–532.
Research International, 40:1107–1121.
Jafari, S.M., Assadpoor, E., He, Y., Bhandari, B. 2008. Re-coalescence of emulsion
droplets during high-energy emulsification. Food Hydrocolloids, 22:1191–1202.
Jeong, C.M., Lee, M.K., Lee, H.A., Park, J. 2009. Low-temperature microencapsulation
of sesame oil using fluidized bed granulation. Korean Journal of Food Science and
Kaasgaard, T, Keller, D. 2010. Chitosan coating improves retention and redispers-
ibility of freeze-dried flavor oil emulsions. Journal of Agricultural and Food
Chemistry, 58:2446–2454.
Kagami, Y., Sugimura, S., Fujishima, N. et al. 2003. Oxidative stability, structure, and
physical characteristics of microcapsules formed by spray drying of fish oil
with protein and dextrin wall materials. Journal of Food Science, 68:2248–2255.
Leclercq, S., Harlander, K.R., Reineccius, G.A. 2009. Formation and characterization
of microcapsules by complex coacervation with liquid or solid aroma cores.
Flavour and Fragrance Journal, 24:17–24.
Li, X., Anton, N., Arpagaus, C., Belleteix, F., Vandamme, T.F. 2010. Nanoparticles by
spray drying using innovative new technology: The Büchi Nano Spray Dryer
B-90. Journal of Controlled Release, 147:304–310.
Li, X., Anton, N., Ta, T.M. et al. 2011. Microencapsulation of nanoemulsions: Novel
Trojan particles for bioactive lipid molecule delivery. International Journal of
Nanomedicine, 6:1313–1325.
Minemoto, Y., Hakamata, K., Adachi, S., Matsuno, R. 2002. Oxidation of linoleic
acid encapsulated with gum arabic or maltodextrin by spray-drying. Journal of
Microencapsulation, 19:181–189.
Murugesan, R., Orsat, V. 2012. Spray drying for the production of nutraceutical
i ngredients—A review. Food and Bioprocess Technology, 5:3–14.
Pérez-Masiá, R., López-Nicolás, R., Periago, M.J. et al. 2014. Encapsulation of folic
acid in food hydrocolloids through nanospray drying and electrospraying for
nutraceutical applications. Food Chemistry, 168:124–133.
Rodríguez-Hernández, G.R., González-García, R., Grajales-Lagunes, A., Ruiz-
Cabrera, M.A., Abud-Archila, M. 2005. Spray-drying of cactus pear juice
(Opuntia streptacantha): Effect on the physicochemical properties of powder and
reconstituted product. Drying Technology, 23:955–973.

Solans, C., Izquierdo, P., Nolla, J., Azemar, N., Garcia-Celma, M.J. 2005. Nano-
emulsions. Current Opinion in Colloid and Interface Science, 10:102–110.
Solè, I., Pey, C.M., Maestro, A. et al. 2010. Nano-emulsions prepared by the phase
inversion composition method: Preparation variables and scale up. Journal of
Colloid and Interface Science, 344:417–423.
Soottitantawat, A., Bigeard, F., Yoshii, H. et al. 2005a. Influence of emulsion and pow-
der size on the stability of encapsulated d-limonene by spray drying. Innovative
Food Science and Emerging Technologies, 6:107–114.
Soottitantawat, A., Takayama, K., Okamura, K. et al. 2005b. Microencapsulation of
l-menthol by spray drying and its release characteristics. Innovative Food Science
and Emerging Technologies, 6:163–170.
emulsions. Advances in Colloid and Interface Science, 108–109:303–318.
Tan, L.H., Chan, L.W., Heng, P.W.S. 2009. Alginate/starch composites as wall
material to achieve microencapsulation with high oil loading. Journal of
Microencapsulation, 26:263–271.
Wang, B., Adhikari, B., Barrow, C.J. 2014. Optimisation of the microencapsulation
of tuna oil in gelatin-sodium hexametaphosphate using complex coacervation.
Food Chemistry, 158:358–365.
Wang, R., Tian, Z., Chen, L. 2011. A novel process for microencapsulation of fish oil
with barley protein. Food Research International, 44:2735–2741.
Yuliani, S., Torley, P.J., D’Arcy, B., Nicholson, T., Bhandari, B. 2006. Extrusion of mix-
tures of starch and D-limonene encapsulated with β-cyclodextrin: Flavour
retention and physical properties. Food Research International, 39:318–331.

Section III
Bioprocessing Technology

16
Bioprocessing Technology for Production
of Nutraceutical Compounds
Terry H. Walker, Caye M. Drapcho, and Feng Chen
CONTENTS
16.1 Introduction................................................................................................. 481
16.2 Commodity Biochemicals and Nutraceutical Bioproduction..............484
16.3 Lipid-Based Nutraceuticals....................................................................... 485
16.3.1 Polar Lipids...................................................................................... 487
16.4 Long-Chain Polyunsaturated Fatty Acids.............................................. 490
16.5 Large Molecule Nutraceuticals................................................................. 493
16.5.1 Polysaccharide Compounds.......................................................... 493
16.5.2 Proteins and Nucleotides............................................................... 495
16.5.3 Small Molecular Nutraceuticals................................................... 496
16.6 Bioprocess Design....................................................................................... 498
16.7 Process Analysis......................................................................................... 498
16.8 Process Economics...................................................................................... 499
16.9 Conclusions.................................................................................................. 501
Acknowledgment................................................................................................. 502
References.............................................................................................................. 502
16.1 Introduction
Nutraceuticals are natural compounds used to supplement the diet as a
means to increase uptake of important nutrients in the diet of humans and
other animals. Nutraceutical compounds are becoming an important com-
modity in the United States and globally as market demand increases for
both purified and supplemented forms. In the 1990s, nutraceuticals from
natural sources were most widely explored in “plants, marine organisms
and micro-organisms, in particular actinomycetes and fungi” (Cocks et al.,
1995, p. 115). Development of biopharmaceuticals primarily from animal
tissue culture in serum media has recently enabled obtaining these prod-
ucts in other eukaryotic systems such as plants, algae, and fungi. Many
nutraceuticals are extracted and purified from plants (e.g., antioxidants)
481
and animals (e.g., fish oil rich in essential fatty acids), which are the most
important components of the total market. However, there are vast sources
of microorganisms capable of producing nutraceutical components, and
the database is rapidly increasing in recent years. Nutraceuticals in micro-
organisms are usually produced in highly controlled closed systems capa-
ble of meeting Food and Drug Administration (FDA) approval criteria.
Obvious expansion of this market requires innovation for new products,
and the use of bioprocessing techniques commonly applied to the food
and pharmaceutical industry may provide a means to enhance products
through novel high-density cell culture techniques as well as new fraction-
ation and bioseparation techniques. Researchers in biological and chemical
engineering are finding ways to effectively utilize by-products from food,
seafood, and agricultural industries for first fractionating valuable nutra-
ceutical components and then further processing of co-product to serve as
feedstock for bioenergy compounds such as ethanol, biodiesel, methane,
and hydrogen.
The combined global market of nutraceuticals and functional foods sur-
passed $90 billion in 2004, up approximately 10% per year since 1993 where
nearly half of the market comprised dietary supplements. The combined value
of nutraceuticals and functional foods is expected to reach $240 billion by the
year 2015 (Visiongain, 2015). One example is the Martek Biosciences process
for production of nutraceutical oil from algal and fungal sources containing a
high degree of omega-3 and omega-6 essential fatty acids primarily used as a
supplement in baby formulas.
Development of advanced biological conversion processes (enzymatic,
microbial, and physical/chemical processes) is an important part of the bio-
processing research agenda. Biological processes are preferred paths for con-
verting agricultural-based resources into industrial products. Bioprocesses
tend to have higher reaction specificity and milder reaction conditions and
produce fewer toxic by-products. These characteristics are consistent with the
goal of developing industrial processes and systems that are environment-
friendly. Innovative research is currently underway that links physical/
chemical processes, such as gasification, with microbial conversion processes.
These novel reaction systems are evolving in response to the heterogeneity
of most biomass resources and the realization that there are important auto-
tropic microorganisms that are effective in converting common bio-organic
compounds into more useful industrial or biomedical compounds.
Land grant institutions across the United States represent a great reposi-
tory of scientific and engineering knowledge to catalyze the transition of
society from a fossil fuel to a biobased economy. With the biotechnology
revolution currently in place, applications of new technologies are sought to
address the challenging arenas of specialty biochemical production (e.g., bio-
pharmaceuticals and nutraceuticals), bioenergy compounds (e.g., ethanol),
and biomaterials (e.g., polylactic acid [PLA]). Figure 16.1 illustrates the basic
flow of raw materials to end products proposed for a biorefinery concept

Bioprocessing Technology for Production of Nutraceutical Compounds 483
Raw materials, by-products,

agricultural “wastes”
Bioprocessing,
bioconversion,
bioreactor design and
control, “biorefineries”
Bulk
Specialty
chemicals, Biomaterials
biochemicals
biofuels
FIGURE 16.1
Biorefinery concept for conversion of by-products to bulk chemicals, biomaterials, and spe-
cialty biochemicals with emphasis of integrating the three Es (environment, education, and
economics).
(Kromus et al., 2004) in an initiative established by USDA (Walker, 2012).

Integrated approaches to bioprocessing show the greatest promise to com-
plete the cycle driven by the three Es, including economics, environment, and
educational aspects of the bioindustry. Bottom-line industry economics bal-
anced with environmentally friendly technologies that contribute to carbon
sequestration within the biological system requires educating the increasing
masses of people on the planet of this balance and creating a work-force to
tackle the highly innovative challenges that lie ahead in a widely evolving
biotechnological world. Environmental concerns encourage “green” tech-
nologies, economics requires feasibility of the proposed technologies, and
education addresses the importance of all levels of learning including grade
school, undergraduate, and graduate research opportunities as a mainstay to
achieving the goals to establish a biobased economy.
Current breakthroughs are not limited to only molecular biology and
genetics, but over the past 15 years, biological and chemical engineers have
made notable advances in the fractionation and bioprocessing of biobased
resources of raw materials required to obtain the principal building blocks
for the synthesis of new bioproducts. For example, the critical first step in
bioconversion of lignocellulosic biomass is the fractionation and modifica-
tion of constituent biopolymers to facilitate either enzymatic or microbial
conversion, resulting in the efficient production of fermentable sugars.
Several promising pretreatment technologies, such as ammonia fiber explo-
sion (AFEX), have resulted in improved yields and reactivity (Dale et al.,
2004). In addition, engineering advances in bioseparation technologies are
improving capabilities to identify and separate secondary metabolites and
other bioactive compounds from plant, animal, and microbial materials,
such as simulated moving bed (SMB) chromatography and supercritical

fluid fractionation. Advances in nanobiotechnology such as fabrication of

microfluidic channels in silicon wafers take advantage of differences in
physical and chemical properties such as diffusion, electrophoretic mobil-
ity, or chemical affinity to produce rapid, efficient separations of proteins,
secondary metabolites, and other organic compounds primarily for rapid
identification and synthesis. In analytical biotechnology, chemical and bio-
logical techniques are combined and integrated into engineered devices or
sensors for the detection and quantification of secondary metabolites and
other organic compounds in a variety of bioprocesses.
16.2 Commodity Biochemicals and Nutraceutical Bioproduction

The biorefinery concept utilizes the bioconversion of natural raw materials
or industry by-products to purified specialty biochemicals and bulk fuels
(Figure 16.1). Research to develop bioconversion processes to produce spe-
cialty chemicals and bioenergy consists of several phases: (1) screening for
wild-type strains and/or metabolic engineering of cells to produce desired
products; (2) characterization and pretreatment (hydrolysis) of feedstock
prior to bioconversion; (3) determination of microbial growth, decay, and
product formation kinetics of the target microorganism (filamentous fungi,
bacteria, yeast, algae, etc.); (4) design/optimization of reactor systems to cul-
ture cells and produce desired products based on simulations using mass-
balanced-based models and microbial kinetic parameters determined in
step 3; and (5) downstream processing to separate, concentrate, and purify
products.
The bioprocess involves chemical treatment of feedstocks to hydrolyze
the cellulose and hemicellulose compounds to fermentable sugars, which
consist of mostly glucose and xylose. The fermentable sugars are then fer-
mented to industrial products (e.g., ethanol, lactic acid, or succinic acid) and
nutraceuticals (e.g., amino acids, omega-3 fatty acids, or high-value biophar-
maceuticals). Biopharma compounds range from smaller molecules such as
traditional antibiotic products such as penicillin or insulin from mamma-
lian cell culture or modified Escherichia coli (Eli Lilly process) to larger mol-
ecules that encompass bioactive proteins, lipoproteins, and glycoproteins
ranging from 1000 to 500,000 Da with complex three-dimensional structures
often dictated by their specific levels of glycosylation and chiral chemistry.
Generally, the purity of the substrate is required for increasing product
value. For example, the expense of serum media may exceed the value of
most of the products shown in the table. Therefore, effort to utilize substrates
from conventional agricultural by-products for production of biopharma-
ceuticals with mammalian culture is of great interest to the biotechnology
industry. Carotenoids including β-carotene, astaxanthins, and more recently

increasing markets for lutein and lycopene make up a world market nearing
$1 billion with a growth rate of about 3%. Most carotenoids are produced
synthetically by companies such as BASF and DSM, but recent biotechno-
logical advances for carotenoid metabolism have shown promise in fungi
such as Blakeslea trispora (Vandamme, 2004) and have been extracted for the
nutraceutical market (Nature Source®) from the algae Dunaliella salina (Enes
and Saraiva, 1996; Fuller et al., 2001; Hajazi et al., 2002; Denery et al., 2004;
Leon et al., 2003).
There is a great interest in biological chemicals, functional food ingredi-
ents, and/or nutraceuticals regarding the rapid development in biochemis-
try, genetic engineering, genomics, biological engineering, food chemistry,
and medical research fields. However, bioseparation and downstream pro-
cessing equipment account for nearly half of the total cost of production of
biological products and/or nutraceuticals (Singh and Singh, 1996; Lightfoot
and Moscariello, 2004). Therefore, the competitive nature of biotechnology,
pharmaceutical, and food industries for cost-effective manufacturing has
provided much impetus for the development and use of new biosepara-
tion techniques in large scale, but at lower cost. Except for the conventional
bioseparation techniques such as crystallization, precipitation, distillation,
liquid–liquid extraction, etc., that have been well established and commer-
cialized (Keller et al., 2001), a number of newer bioseparation techniques
have been implemented at the commercial scale including chromatography,
supercritical fluid extraction (SFE), and membrane-based separation. These
techniques have been implemented for purification of proteins and nucleo-
tides, characterization of aromas, whey protein removal from dairy prod-
ucts, extraction of health-benefiting fish oil, and clarification of beverages
including beer, fruit juices, and wine. The following section provides some
examples on analytical- and/or process-scale bioseparation of biological
products/nutraceuticals based on their polarity (lipid-based nutraceuticals)
and molecular size (large and small molecular nutraceuticals). Table 16.1 lists
several potential products from pretreated feedstocks to form high-value
products (Blanch and Clark, 1997).
16.3 Lipid-Based Nutraceuticals

Lipids comprise many broad classes of neutral and partially polar com-
pounds with differing physiological functions. Some contain important
regulatory functions such as sterols (e.g., cholesterol, phytosterol, and ergos-
terol) produced by plants as well as by fungi such as Saccharomyces cerevisiae
and Gibberella fujikuroi (Giordano et al., 1999). Other antioxidants found in
plant materials such as rice bran are the class of phytosterols (Godber et al.,
1994), but also found in microorganisms such as algae (Abalde et al., 1991).

TABLE 16.1
Bioconversions in the Order of Increasing Value and Subsequent Substrate Purity
By-Product Primary Primary Enzyme
Source Substrate (Microbial Strain) Product (Production)
Animal waste Complex (Methylomonas flagellate) Methane
Corn syrup Glucose (S. cerevisiae) Ethanol (26 M ton/year)
Potato/sweet Glucose Alcohol dehydrogenase Ethanol (thermophilic
potato (Kluyveromyces marxianus) pathway)
Wood fibers Xylan (Clostridium sp. SAIV1) Ethanol
Dairy waste Lactose β-Galactosidase Glucose and galactose
Oily waste Lipids Lipase (esterases) Fatty acids
Rice brokens Glucose (Clostridium acetobutylicum) Acetone/butanol (30/60)
Rice straw Xylose Citrate synthase Citric acid (1 M ton/year)
Switchgrass Xylose Lactose dehydrogenase Lactic acid (30 million kg/
(Lactobacillus delbrueckii) year)
Sugarcane Xylose Pyruvate decarboxylase Acetic acid
bagasse pyruvate (Acetobacter sp.)
Acrylonitrile Nitrile hydratase Acrylamide (15,000 ton/year)
Corn stover d-xylulose Xylose reductase (Candida Xylitol
tropicalis)
Corn starch Glucose Glucose isomerase High fructose corn syrup
(8 million tons/year)
Molasses Sucrose Fumarase (Brevibacterium l-malic acid
(sugarcane) ammoniagenes)
Corn syrup Glucose (Schizochytrium sp.) Oil rich in docosahexaenoic
acid ($220 M/year)
Ethanol (P. rhodozyma mut.) Astaxanthin ($2000/kg)
stillage
Glucose Acetyl-CoA carboxylase l-glutamic acid (340,000 ton/
(Corynebacterium year) MSG
glutamicum)-biotin
Glucose Aspartic amino transferase l-phenylalanine (3000 ton/
(E. coli) year) (aspartame synthesis)
Glucose Aspartase (E. coli) l-aspartic acid (4000 ton/year)
Glucose l-aminocaprolactam l-lysine (350,000 ton/year)
hydrolase (Cryptococcus ($2/lb)
laurentii)
Glucose (Pseudomonas fluorescens) l-histidine
Glucose Penicillin amidase 6-Aminopenicilloic acid
(Penicillium chrysogenum) (7500 ton/year)
Glucose (Bacillus lichenformis) Proteases ($236 M/year)
Glucose (Bacillus amyloliquefaciens) Amylases ($70 M/year)
Glucose (Rhizopus and Aspergillus) Other enzymes ($92 M/year)
Glucose (Arthrobacter simplex) Prednisone
Specific Serum Hybridomas Monoclonal antibodies
media Serum Human fibroblasts Interferon
Serum Monkey kidney cells Polio vaccine

5
HO
H3C H H3C H
2
7
8 O
Tocopherol
5
HO
CH3 CH3
2
7
8 O
Tocotrienol
FIGURE 16.2
Antioxidant sterol compounds: tocopherol and tocotrienol.
These antioxidants include tocopherols and tocotrienols (Figure 16.2) benefi-

cial in lowering cholesterol as well as in preventing cardiovascular diseases
(Llyod et al., 2000). Tocopherols are also believed to have anticancer effects
(Tarber and Packer, 1995; Dunford, 2001). Many neutral lipids in mono-, di-,
and triacylglycerol forms act as functional precursors to other metabolites or
act as an energy storage source. Most lipids consumed by humans and other
animals are derived from plants and animals due to their relative abundance
in these sources. Bioproduction of lipids from microbial sources is also well
known, but has only recently been established at the commercial level for
production primarily for lipids with considerable bioactivity such as very
long-chain unsaturated compounds with high degrees of omega-3 fatty
acids or phospholipids and sulfolipids geared toward cancer research from
algal or fungal sources.
16.3.1 Polar Lipids

Polar lipids are often associated with vital membrane structure and func-
tion, depending on the lipid class (e.g., sulfolipid, SL; phosphatidyl choline,
PC; phosphatidyl ethanolamine, PE), length and position of constituent fatty
acids, and degree of saturation (Csabai and Joó, 2003). Sphingolipids (e.g.,
sphingomyelin, a phospholipid, and ceramide) are another special class
of bioactive compounds associated with cell membranes that act as lipid
mediators important to intra- and extracellular signaling, regulation of cell
growth, differentiation, and apoptosis. Breakdown of the abundant mem-
brane phospholipid, sphingomyelin, by sphingomyelinases produces sphin-
gosine and ceramide (Liu et al., 1997). Sphingosine serves as a precursor to
sphingosine-1-phosphate, which has cancer-promoting properties such as
cell proliferation and angiogenesis, whereas ceramide primarily functions

physiologically as a cancer preventative agent with properties of apoptosis

and growth inhibition, thus together placing the cell into a life and death
balance (Ogretment and Hannun, 2004).
Cyanobacteria, similar to higher plants and green algae, have a glycerolipid
composition that is made up of monogalactosyl diacylgylcerol, sulfoquinovo-
syl diacylglycerol (SQDG) shown in Figure 16.3, digalactosyl diacylglycerol
(DGDG), and phosphatidyl glycerol (PG).
SQDGs are structural components of chloroplast membranes in eukary-
otic algae and lamellas in prokaryotic cyanobacteria and appear to be the
only natural lipid with sulfonic acid linkage (Christie, 2005). SQDG is char-
acterized by its unique sulfonic acid head group, a 6-deoxy-6-sulfo-glucose
(Benning, 1998). SQDGs extracted from cyanobacteria have been shown
to inhibit HIV-1 in cultured human lymphoblastoid T-cell lines and thus
were placed as high priority for further research by the National Cancer
Institute (Gustafson et al., 1989). The cyanobacteria evaluated included spe-
cies of Lyngbya, Phormidium, Oscillatoria, Scytonema, Calothrix, and Anabaeana.
Sulfolipids comprised 10% by weight of organic extractables from the cya-
nobacteria tested and all had similar activity, suggesting that the acyl chain
length and degree of unsaturation did not affect potency (Gustafson et al.,
1989). Structurally related acyl glycerols, lipids, and sulfonic acid deriva-
tives did not inhibit HIV-1. SQDG was also found to reduce proliferation and
viability of human gastric carcinoma cells SNU-1 by induction of apoptosis
to direct necrosis at micromolar concentrations (Quasney et al., 2001). These
results indicate the potential of whole algae or algal extracts containing sul-
folipids as chemotherapeutic or preventive dietary supplements.
For the cyanobacterium Anabaena 7120, SQDG content in algal basis at day
20 of batch incubation was unaffected (approximately 11 mg SQDG/g algal
dry weight) by surface light intensities (Archer et al., 1997). SQDG content
in the Anabaena biomass increased throughout exponential and decreasing
–O SH C
3 2
HO O
HO
OH
O
O O
C
O
SQDG O
R1
R2
FIGURE 16.3
Structure of SQDG in which R1 and R2 represent acyl chains of different lengths and degrees
of unsaturation. (Adapted from Benning, C. 1998. Annual Review of Plant Biology, 49: 53–75.)

exponential growth phases, indicating that SQDG may be characterized as a

mixed primary and secondary product. For the green alga Scenedesmus, the
combined SQDG and PG contents of algal biomass did not differ as a func-
tion of light intensity (40–290 μE/m2/s) at day 5 of incubation, but decreased
with increased light intensity by day 25 of incubation when cells had entered
early stationary growth (Conwell, 2005). Full characterization of the interac-
tion among light intensity, growth rate, and SQDG production is needed for
optimization of algal reactors.
Unlike neutral galactolipids, which are easily separated from phospholip-
ids by adsorption chromatography, SQDG is difficult to separate due to its
polar nature (Christie, 2005). Quantification of SQDG in Anabaena extracts
was accomplished by Archer et al. (1997) using total lipid extraction as per
Folch et al. (1957), followed by thin layer chromatography using silica gel
plates and photodensitometric scanning after charring with copper sulfate
and heat. Norman et al. (1996) developed a method for extraction, separa-
tion, and analysis of SQDG from plant (spinach) biomass using the Bligh
and Dyer (1959) extraction method for total lipids using sodium chloride in
two-phase partitioning, separation of lipid classes using silica cartridges
with SQDG and phospholipids (PL) eluted using methanol, and resolution of
SQDG from the PL fraction using normal-phase high-pressure liquid chro-
matography (HPLC) with silica column, heptane–isopropanol–0.001 M KCl
40:52:8 (v/v/v) as the mobile phase, and detector operated at 208 nm. The
sulfolipid yield was found to be 0.18 mg SQDG/g fresh spinach.
Cell lysis of algae and cyanobacteria for extraction of lipids is more diffi-
cult than for plant tissue, and techniques to shear cells include solvent addi-
tion with incubation, agitation, homogenization, and sonication (Wiltshire
et al., 2000). A cell-lysis method developed for the green alga Scenedesmus
that included quartz sand and organic solvent addition, freeze drying, and
extraction in ultrasound bath was reported to provide excellent extraction
efficiency of pigments and fatty acids (Wiltshire et al., 2000).
SFE (CO2) provides an alternative for the extraction and recovery of algal
nutraceuticals and avoids use of toxic organic solvents. SFE of carotenoids
and lipids from the green alga Chlorella has been found to compare favor-
ably with organic solvent extractions (Mendes et al., 1995). Shearing of algal
cells using alternating mechanical and thermal shocking was required for
efficient extraction. Yields of carotenoids and lipids were 5% of dry weight at
35 MPa/55°C for whole algal cells, whereas yields were increased to 13.3% for
disrupted cells. Similar results were achieved using SFE of carotenoids and
lipids from Botryococcus braunii, D. salina, and Arthrospira maxima (Mendes
et al., 2003). SFE of sulfolipids may also prove comparable to organic solvent
extraction. Recently, continuous countercurrent supercritical carbon dioxide
fractionation has been successfully applied to fractionating low molecular
fatty acids (Dunford et al., 2003), and antioxidants in plant oils such as corn
and rice bran oil (Taylor and King, 2000), and could be applied to fungal and
algal systems.

16.4 Long-Chain Polyunsaturated Fatty Acids

Polyunsaturated fatty acids (PUFAs) are a class of compounds that have a
variety of important functions in biological systems. Studies have shown
that longer chain fatty acids, particularly EPA (eicosapentaenoic acid,
C20:5n3; Figure 16.4), ARA (arachidonic acid, C20:4n6), and DHA (docosa-
hexaenoic acid, C22:6n3), have important roles as biosynthetic precursors,
as cellular membrane components, and as protection against oxidative
stress (Iqbal and Valivety, 1997; Holman, 1998). The relative levels of these
compounds have been found to have profound effects on human health.
Growing interest in health benefits of PUFAs has focussed attention on
providing suitable sources of these compounds. Isolation of highly effi-
cient oleaginous microorganisms and development of related fermentation
technologies may lead to fermentation as an alternative to agricultural and
animal processes (Certik and Shimizu, 1999). Some microbial species pro-
duce high yields of certain PUFAs, which include Mortierella alpina (Shimizu
et al., 1988; Higashiyama et al., 1998; Koike et al., 2001), Mortierella elongata
(Bajpai et al., 1991a,b; Bajpai and Bajpai, 1993), Pythium irregulare (Stinson
et al., 1991; O’Brien et al., 1993; Cheng et al., 1999; Hong et al., 2002), Pythium
ultimum (Stredansky et al., 2000), and Entomophthora exitalis (Kendrick and
Ratledge, 1992).
P. irregulare is regarded as one of the most promising microbial species for
possible commercial production of EPA due to its high EPA yield. This micro-
organism can also produce a significant amount of another important PUFA,
ARA (Stinson et al., 1991; O’Brien et al., 1993; Hong et al., 2002). Research
into fungal fermentation kinetics and modeling has been conducted with
Pythium irregulare (Zhu, 2002). SFE and modeling extraction behavior were
implemented to develop a process for fractionation of important components
(essential fatty acids and antioxidants) from both rice bran and fungi after
fermentation (Walker et al., 1999).
Beneficial health effects from consumption of certain fish oils have been
attributed to the presence of the essential PUFA, EPA, and docosapentaenoic
acid (DHA). These omega-3 fatty acids have been linked to a reduced risk
of coronary heart disease, arthritis, inflammation, hypertension, psoriasis,
other autoimmune disorders, and cancer (Simopoulos, 1989). PUFAs are cur-
rently marketed as dietary supplements at health food stores in the form of
19 17 15 13 11 9 7 5 3 1
H3C COOH
cis-5, 8, 11, 14, 17-eicosapentaenoic acid, 20:5 (n-3)
FIGURE 16.4
Chemical structure of EPA.

concentrated fish oils. Supplementation into baby foods has lately received
greater interest. PUFAs are also prescribed medications for humans and pets.
Declining marine resources and an increasing demand for PUFAs have
prompted the search for alternative sources of EPA and DHA. Filamentous
fungi have the potential to produce large amounts of EPA within the myce-
lial walls when grown under optimal conditions (Ghandi and Weete, 1991;
Radwan, 1991). The filamentous fungus P. irregulare converts sweet whey per-
meate into significant amounts of EPA (24.9 mg EPA/g dry biomass), which
could potentially add value to this food-processing byproduct (O’Brien et al.,
1993).
Humans have a limited capacity to synthesize EPA, ARA, and DHA from
shorter chain fatty acids. Additionally, typical American diets are extremely
low in these compounds. The importance of EPA and ARA lies in that they
are biosynthetic precursors of the eicosanoid system that controls inflam-
matory and anti-inflammatory responses. There is increasing evidence that
a variety of disorders such as heart disease and hypertension are related to
malfunctions of the eicosanoid system caused by dietary imbalance of long
chain PUFAs. DHA accumulates preferentially in the brain where it has been
found to have rules both in neuronal impulse transmission and in protect-
ing the brain from oxidative stress. Dietary deficiencies of DHA have been
linked to bipolar disorder and schizophrenia. Connor (2000) noted that diets
rich in n-3 long chain fatty acids tended to offset age-related degenerative
diseases as a result of increased n-3/n-6 ratios in the blood and fatty acid
profile in the brain. As a result, it has been found that dietary supplementa-
tion of PUFAs has considerable efficacy in the treatment of these conditions,
and a considerable dietary supplement market has developed around these
PUFAs (Mace and Halliwell, 2004).
The current commercial supplies of long chain PUFAs primarily come
from various oil seed plants, such as flax or borage, and from marine fish
oil. Plant oils contain only shorter chain essential fatty acids, alpha-linolenic
and linoleic acid, and do not contain the nutritionally important long chain
PUFA. Fish oil is a good source of EPA and DHA, which has major limi-
tations including objectionable “fishy” smell and taste encapsulation and
growing concern of the presence of mercury and heavy metal accumulation
in the extracted oils. Although plants lack the enzymes necessary to create
long chain PUFAs greater than 18 carbon units, microbes particularly in the
algal and fungal classes possess oleaginous properties and an array of essen-
tial fatty acid desaturases required to synthesize short chain PUFAs, which
are elongated to long chain PUFAs. Currently, microalgae are being used
commercially to produce EPA and DHA. Microalgal production, however,
has some drawbacks. Microalgal DHA production requires the use of either
expensive photobioreactors or, in the case of tropically shifted algae, complex
media. Common DHA-producing organisms include the algae Gonyaulax,
Gyrodinium, and Cryptoconidium spp. and the bacteria Rhodopseudomonas and
Shewanella spp.

M. alpina has been commercialized for the production of long-chain PUFAs

primarily rich in ARA for use in infant formulas. Other ARA-producing
fungi include M. elongata, E. exitalis, P. ultimum, and P. irregulare for use as an
ingredient in infant formula to supply essential metabolic precursors for pre-
natal nervous system development. P. irregulare is an attractive candidate for
PUFA production as it produces nearly equal amounts of both ARA and EPA
(Walker et al., 1999). It is relatively easy to grow, can be adapted to both solid-
state and submerged culture techniques, and is capable of utilizing a wide
variety of substrates. Specifically, P. irregulare has a comprehensive comple-
ment of metabolic enzymes and is able to be grown on substrates high in cel-
lulose and hemicellulose. This allows the use of inexpensive waste materials
as substrates for mass production EPA and ARA from P. irregulare.
Research has indicated that culture conditions such as medium compo-
sition, oxygen availability, pH, and temperature could influence microbial
lipid accumulation as well as PUFA composition (Stinson et al., 1991; Koike
et al., 2001; Zhu, 2002). Due to the nature of the individual microorgan-
ism, the optimal conditions may differ from strain to strain. Some of the
culture conditions for P. irregulare have been optimized (Stinson et al., 1991;
O’Brien et al., 1993; Zhu, 2002). Glucose and yeast extract are considered as
the preferred carbon and nitrogen sources for this microorganism (Stinson
et al., 1991). Nitrogen limitation is commonly believed to have a significant
effect on lipid accumulation in microbial fermentation (Ykema et al., 1989).
However, the optimal C/N ratio of P. irregulare for the target PUFA synthe-
sis is not known, and few published studies have addressed this issue with
other species. Therefore, optimizing medium composition and C/N ratio
will be important for target PUFA production and process control. Zhu et al.
(2001) and Cheng et al. (1999) achieved the highest yields of the EPA and
FIGURE 16.5
Photomicrograph of the mycelium of P. irregulare (×1000). The mycelium contains about 20% oil
rich in PUFAs particularly high in EPA and ARA, which occur in about equal amounts.

ARA (20 and 14.7 mg/g, respectively) with medium composed of 2% glucose

and 0.25% yeast extract and C/N ratio of approximately 32. Figure 16.5 shows
the fungal culture of P. irregulare and oil production from P. irregulare grown
on rice bran compared with commercial rice bran oil. Supercritical extraction
of this oil compares favorably with the highly refined oil, compared with
conventional hexane-extracted crude oil (Badal, 2002; Patel, 2005).
Table 16.2 lists the primary fungal and algal sources considered for the
production of oils containing EPA and ARA, which serve as important pre-
cursors to an important class of metabolites including prostaglandins and
leukotrienes. Drapcho et al. (2008) shows detailed list of fungal and algal
lipids with complete fatty acid profiles.
16.5 Large Molecule Nutraceuticals

Large molecular nutraceuticals are generally made up of larger polymers
typically in the range of 103–106 Da for proteins and as high as 1010 Da for
polynucleotides.
16.5.1 Polysaccharide Compounds

Many polysaccharides have been identified for therapeutic activity. Poly-
branched β-d-glucans isolated from the cell walls of S. cerevisiae (Miller
et al., 1997) and Agrobacterium sp. (Kim et al., 2003) have shown potent non-
specific immune activation of macrophage-immune cells, T-cells, NK-cells
and B-cells and have shown high anti-AIDS activity with low side effects
(Kim et al., 2003). A novel sulfated polysaccharide, calcium spirulan (Ca-
SP), isolated from the cyanobacterium Spirulina platensis, has been found to
have both antiviral and anticancer properties (Hiyashi and Hayashi, 1996;
Mishima and Murata, 1998). Chitosan is the deacetylated form of chitin
found in abundance in arthropod exoskeletal layers, but extractable chitosan
is also seen in various fungi such as the industrial microorganism, Rhizopus
oryzae (Tan et al., 1996; Nwe and Stevens, 2002; Hu et al., 2004). Chitosan is
extensively marketed as a weight-loss nutritional supplement that binds to
lipids, lowers serum cholesterol, and improves low-density lipoprotein to
high-density lipoprotein ratios. Chitosan has also shown anticarcinogenic
properties (Kiode, 1998) as well as a tissue scaffold for implants and pro-
motes healing of wounds (Madihally and Matthew, 1999).
Purification of polysaccharides by chromatographic processes are dif-
ferentiated from the laboratory scale in milligrams or even nanograms for
chemical analysis up to the industrial scale in kilograms or tons produc-
tion for drugs and dietary supplements. Conventional gas chromatographic
(GC) technique is commonly used for trace amount of volatile chemical

TABLE 16.2
494
Fungal and Algal Sources of EPA and ARA Grown on Various Carbon Sources Ranging from Pure Glucose to Complex
Carbohydrates and Lipid Components
Lipid EPA ARA
In Dry In In Dry In In Dry
Biomass Lipids Biomass Lipids Biomass
Fungal Cultures Carbon Source T (°C) (% w/w) (% w/w) (mg/g) (% w/w) (mg/g) References
P. irregulare 4% soy oila 12 67 6 42 2 10 Cheng et al. (1999)
P. irregulare 2% glucose 25 29 7 20 5 15
P. irregulare 5% rice bran 25 28 2 6.4 1 2
P. irregulare 1% glucoseb 12 38 13 49 5 14 Stinson et al. (1991)

P. irregulare SWP 14 10 25 25 10 O’Brien et al. (1993)
M. alpina 1S-4 1.8% glucosec 28 47 39 19 Higashiyama et al. (1998)
M. alpina Rice brand 20 8 40
M. alpina 6.6% glucose 25 35 60 21
M. alpina M18 10% glucosee 28 15 50
M. alpina 1S-4 3% linseed oil 12 56 12 67
M. alpina 1S-4 2% linseed oil 20 33 20 66 Jareonkitmongkol et al. (1993)
M. alpina 20-17 1% linseed 28 60 7 42 12 72
M. alpina 5% glucose 25 51
M. elongata 2% linseed oil 15 39 9 35
Saprolegnia sp. 1% olive oil 6 — — 23
Chlorella minutissima 20 45 225
Porphyridium cruentum 8000 lux 32 17 36
a Supplemented with 1% soy meal.
b Spiked with 0.5% glucose 2 days before harvest.
c Supplemented with 0.1% soy oil; SWP, soy whey permeate.
d Solid-state culture and all others are submerged culture.
e Supplemented with 0.8 g/L glutamate.
analysis, and preparative HPLC is suitable to collect biological chemicals

up to milligrams for further chemical structural determination. However,
both aforementioned methods are restricted by their loading capacities that
are not suitable for industrial scale processing of some functional carbohy-
drates. Recently, SMB technique has drawn much attention because of its
high-efficient productivity and suitability for sugar separation. The method
has been found to be suitable for large-scale fractionation of xylitol, man-
nitol, and other specialty sugars (Saska et al., 1999; Saska and Chen, 2002).
In addition, SMB technology has been used in pharmaceuticals, cosmetics,
and fine chemicals. However, highly purified polysaccharides are seldom
used except for their applications in biochemical research and in the biopro-
cessing industry. For instance, cellulose, agarose, and dextran are commonly
used as media of bioseparation such as electrophoresis and chromatography.
16.5.2 Proteins and Nucleotides

In the past decades, the advance in biotechnology has enabled production
of many desirable compounds from animals and plants. Future produc-
tion of novel proteins such as scytovirin isolated from the cyanobacterium
Scytonema varium, which displays anticytopathic activity against labora-
tory strains of HIV-1, is expected to become increasingly important as new
research developments are reported (Boekesch et al., 2003). Such controlled
genetic activities involve the participation of many proteins and nucleotides.
Therefore, recovery and purification of those biological macromolecules are
essential for understanding and/or revealing the genetic activities. Harve
and Bajpai (2000) have reviewed the steps of isolation and purification of
those biological chemicals. In detail, common isolation steps often employ
precipitation and crude adsorption, followed by purification using ion
exchange, affinity, and gel chromatography. Recently, capillary electropho-
resis (CE), which is considered as one of the most powerful separation tech-
niques for bioanalysts, has been commonly used regarding its rapid, high
resolution of water-soluble components. CE has been accepted as a useful
complementary tool for HPLC. However, CE and HPLC are limited by pre-
requisite of several pre-purification steps that often involve losses of use-
ful targets. Therefore, expanded-bed adsorption has appeared to replace the
conventional packed-bed operation to simplify purification of proteins and
other biological chemicals.
Purification of antibodies, in a large degree, is similar to the purification of
other proteins. But its property in antigen-specific recognition makes it also
suitable for bioaffinity chromatography. To date, monoclonal antibodies and
immunoglobulins represent by far the largest class of produced and puri-
fied proteins in numbers and mass. Recently, aqueous two-phase extraction
(ATPE) has been successfully utilized to partition proteins into two partially
immiscible phases in an aqueous environment. Hydrophobic interactions
with proteins are factors in protein folding, transport across membranes,

recognition, and enzyme catalysis. However, these hydrophobic entities are

often buried within the folded lipoprotein structure, and the introduction of
hydrophobic tags (often aromatic rings) to the outer surface has been applied
to increase solvent interactions as a technique to improve bioseparation
(Bulow, 2004). Another recent novel protein purification method particularly
suitable to the class of large glycoproteins utilizes an affinity carbohydrate-
based matrix called bioskin made of a co-culture of Acetobacter xylinum,
S. cerevisiae, and Saccharomyces pombe (Vicente et al., 2001) with gradient elu-
tion with potassium phosphate (Kishino et al., 1997).
Table 16.3 shows the relationship between bioseparation cost relative to
the general class of compounds and global demand value (Ghosh, 2003).
Estimated global market demands for 2002 are also represented for these
classes (Iisakka, 2003; Menrad, 2003).
16.5.3 Small Molecular Nutraceuticals

Many natural nutraceuticals are small molecules with molecular weight
less than 600 Da and typical radius of 0.5 nm. These often include mono-
mer compounds such as sugars, fatty acids, amino acids, and organic acids.
However, they are diverse in chemical characterization in terms of their
chemical polarity, natural existence in plants/animals, and biological activi-
ties. Many important small molecular nutraceuticals often found in plants,
but also present in high concentrations within some microbial communi-
ties, include various classes of antioxidants and alkaloids that stimulate
the immune system by activation of T-cells, scavenge for free radicals, and
inhibit tremors and analgesia (Nalik et al., 2001). Compounds of commer-
cial interest include carotenoids that function as pro-vitamin A, astaxan-
thin from red yeast Phaffia rhodozyma (Lim et al., 2002), canthaxanthin, and
lycopene (Sabio et al., 2003).
TABLE 16.3
Global Demand Value and Bioseparation Cost of Various Compound Classes
Global Demand Market
Value (2014) Expansion Relative % of Total
Product (US$ Billions) Rate (%) Cost Production Cost
Functional foods, e.g., 33 10 1 10–30
casein
Nutraceuticals, e.g., 47 10 2–10 30–50
scytovirin
Industrial enzymes, 4.2 6 5–10 30–50
e.g., cellulases
Speciality enzymes, 2.1 9 50–100 50–70
e.g., glucose oxidase
Biopharmaceuticals 162 10 50–500 60–80

A compound known to have anticonvulsant properties is the extracel-

lular alkaloid compound pimprinine produced by Streptomyces sp. (Nalik
et al., 2001) and processed by filtration, chromatography, and vacuum dry-
ing. Hydroxytyrosol is a powerful antioxidant naturally present in olive oil
and is synthesized in strains of Pseudomonas aeruginosa (Allouche et al., 2004).
Cis-β-carotene is produced in D. salina algal culture and in fungi B. trispora,
Neurospora crassa (Rabbani et al., 1998; Gessler et al., 2002), and P. rhodozyma
when stimulated by light (Ruddat and Garber, 1983).
Due to physiochemical and biological characteristics specific to these bio-
molecules, a variety of extraction and separation methods have been devel-
oped. Solvent extraction is a traditional method to extract phytochemicals or
nutraceuticals, that is, extraction of anticancer polyphenolics from blueberry
(Burns et al., 2005). Recently, SFE has been strongly recommended to extract
nutraceuticals because of the extraction efficacy of SFE and concerns of the
toxicity of solvents. But both extraction methods always bring a mixture of
different classes of chemicals. Therefore, additional steps are often required
for the removal of unwanted interfering substances. For example, solid phase
extraction and open column chromatography are now commonly used for
further chemical fractionation. Successful methods of extraction of antioxi-
dants from algae and fungi with use of supercritical CO2 (Lim et al., 2002;
Sabio et al., 2003) and absolute ethanol (Roukas and Mantzouridou, 2001)
have been identified.
Naczk and Shahidi (2004) reviewed the extraction and analytical meth-
ods for many nutraceutical phytochemicals including phenolics, flavonoids,
anthocyanins, tannins, etc. It was concluded that there are no uniform or
completely satisfactory procedures for extraction of all phytochemicals or a
specific class of phytochemicals in plant material. Considering the volatility of
nutraceuticals, GC and HPLC have been frequently used for volatile and non-
volatile chemicals, respectively. Furthermore, different monitoring detectors
such as ultraviolet and mass spectrometer are often combined with gas and
liquid chromatography for quantitative and qualitative analyses of nutraceu-
ticals (Kim et al., 2004, 2005). Enzyme-linked immunosorbent assay has also
been frequently used in biological and clinical research (Wang et al., 2004).
As a novel emerging technique, magnetic field combined with nanotechnol-
ogy is tried for drug delivery and analysis (Son et al., 2005). Nevertheless, all
above-mentioned methods are limited in laboratory-scale research, and only
SMB has been successfully and commercially used in large industrial scale.
In general, bioseparations are essential in the good manufacturing practice
(GMP) of production of pharmaceuticals and nutraceuticals, which generally
requires the products in an acceptable level of purity.
Aside from basic separation issues of antioxidants from cell biomass in
production systems, extraction procedures within clinical tissues such as
the liver to measure the metabolic effect of antioxidants have hampered the
analysis procedure due to poor sample preparation prior to the HPLC analy-
sis. Burri et al. (1997) demonstrated that use of supercritical carbon dioxide

without modifier proved to generate vitamin A and carotenoid extracts from

calf liver tissue that did not require further pretreatment as typically needed
for organic solvent techniques.
16.6 Bioprocess Design

Bioprocess design for nutraceutical production primarily involves the con-
ceptual work to implement the technology for production and biosepara-
tions, alterations, or scale up to the process plant design. A detailed overview
of the procedures is covered in several texts (Blanch and Clark, 1997). The
process design of food, pharmaceutical, and nutraceutical production is a
marriage of process analysis and cost estimation that includes the life cycle
of the product, use of engineering parameters, and estimates to complete a
series of mass and energy balances in a process flow simulation and envi-
ronmental impact assessment. The economic assessment begins with an
order of magnitude estimation based on previous cost data for similar bio-
process, resulting in large errors (up to 50%) even with experienced teams
of engineers, industrial biotechnologists, and economists (Harrison et al.,
2003). Further estimates include project planning based on major equipment
needed, preliminary and detailed engineering design from process data, and
contractor estimate with estimation error usually less than 10% (Harrison
et al., 2003). The life-cycle analysis for products considered begins with pre-
liminary research findings. Products first undergo a feasibility analysis for
screening, then a project budget development is created, and finally after
product entry into the market, scale-up of commercial production is assessed
on the basis of market demand (Harrison et al., 2003).
Bioreactor design should consider microbial growth and product forma-
tion kinetics, impact of growth-limiting substrate(s) or products, shear sen-
sitivity of cells, temperature effects, immobilized versus suspended culture,
and characterization of products as primary, secondary, or mixed metabolite.
Basic bioreactor design configurations include batch, fed-batch, and continu-
ous flow stirred tank reactors, which are covered in detail in the literature
(Bailey and Ollis, 1986; Shuler and Kargi, 1992; van Dam-Mieras et al., 1992;
Doran, 1995; Blanch and Clark, 1997; Grady et al., 1999).
16.7 Process Analysis

The first step of process analysis is to construct a generalized flow diagram
or flow sheet in which during initial phases of process development, the

unit operations may not be well defined. As research data containing vital
biological information such as substrate (carbon, nitrogen, oxygen, etc.), bio-
mass and product kinetics, and pH range for the desired system (continuous,
fed-batch, or batch mode) are made available, the appropriate material and
energy balances may then be applied to determine reactor volume, product
mass, temperature requirements, air flow rates, etc. Furthermore, molecu-
lar size, polarity, molecular charge, and orientation (degree of glycosylation,
protein folding, etc.) of the bioproduct of interest generally determine the
type of integrated bioprocess regime needed for production and separation
of the product. Whether the biological function of the product is intracellu-
lar, extracellular, or membrane-bound is another important factor to whether
separation may occur during fermentation as done in continuous or fed-
batch situations or additional bioseparation steps are required as in the case
of intracellular and membrane-bound products.
Once reactor volume, mass of products, and product properties are
known, subsequent bioseparation unit operations are placed in the flow
diagram with corresponding material and energy balances conducted for
each unit operation in a similar way conducted for the bioreactor step to
determine equipment sizing and material requirements (Ladisch, 2001). A
cost analysis is then integrated in the design process to narrow down the
type of reactor and separation equipment to further optimize the process
and to enable some important initial order-of-magnitude cost estimates
including capital and operating costs discussed in further detail in the next
section.
Figure 16.6 is an example of a process flow chart constructed in SuperPro
Designer® of a specific biorefinery concept proposed for the co-production
of fungal oil, fiber composites, ethanol, and nanocrystalline cellulose from
conventional lignocellulosic biomass. Ethanol and carbon dioxide from the
simultaneous saccharification and fermentation step may be recycled back to
the oil supercritical extraction unit as well as ethanol used to supply energy
to the process.
16.8 Process Economics

Economics in preliminary bioprocess design typically includes research and
development cost, estimation of capital and operating costs, and profitability
analysis. Research and development cost may range in the millions of dol-
lars, depending on the ability to bring products to market, which is often less
than 10% of many products tested. These efforts typically include screening
of potential microorganisms, genetic modification for increased productiv-
ity, testing for contaminants and toxicity, process production and biosepara-
tion technology, process scale-up studies, clinical trials (e.g., FDA-approved

500
S-104
+
S-101 S-110
S-102 S-113 S-112

P-7 P-8 P-0/GBX-101
Oil S-106 Fiber composite ethanol
S-107
P-3/V-102
S-108 Distillation S-109
S-105 S-114
S-103 P-10
P-6/V-101
Solid-state reactor P-2/DX-101 P-11/CY-1S-111 P-5/MSX-101 Nanocrystalline cellulose
P-4/V-103
SFE Fiber separation Hydrolysis (hot watermild acid) SSF or SHF
FIGURE 16.6
Biorefinery process flow schematic for the production of nutraceutical and bioenergy products from lignocellulosic by-products.
biopharma products), marketability and ethical sensitivity studies, and other

research specific to handling and packaging of the product.
Capital costs are often estimated using multipliers or unit costs that
include total plant direct cost (TPDC) (i.e., installation, process piping,
instrumentation and control mechanisms, electrical, building, and facility
cost), equipment cost (EC), total plant indirect cost (TPIC) (i.e., engineering
and construction), and direct fixed capital (DFC) (TPDC + TPIC + contrac-
tors’ fees and contingency). Building and facility cost range from $2000/m2
for Class 100,000 process areas to as high as $8000/m2 for Class 100 areas
(typical for biopharmaceutical processing subject to strict cGMP and GLP
standards). Individual equipment cost will increase with the key physical
design scale-up parameters such as unit operation volume, membrane sur-
face area, or flow throughput, but generally by a factor of about 52% referred
to as the “economy of scale” (Harrison et al., 2003).
Operating costs include direct costs such as raw materials (10%–80% total
operating cost), consumables (membranes, process seals, etc.), labor (20%–
50% total operating cost), waste disposal (up to $0.5/m3), utilities (approxi-
mately $0.1/kWh, $8/1000 kg steam, up to $50/1000 kg purified water, and
$0.1/1000 kcal heat removed by refrigeration), and laboratory quality control
and quality assurance costs (10%–20% of the labor costs). Indirect operating
costs include depreciation of fixed capital investment, maintenance (up to
10% of equipment cost per year), insurance (up to 1% of DFC), local property
taxes (up to 5% of DFC), and other overhead expenses (accounting, payroll,
security, fire protection, etc., which account for up to 10% of DFC) (Harrison
et al., 2003).
Finally, profitability analysis includes various measures to assess profit-
ability and marketability of the product. These factors include return on
investment or net profit per year per total investment value, net present value,
internal rate of return and payback time that consider cash flows (annual
revenues, operating costs, etc.), and time value of money as a function of time
of the projected life of the processing plant (Peters and Timmerhaus, 1991).
Simulation software programs such as Aspen Plus® and SuperPro Designer
have extensive capabilities to integrate both process analysis and economics
for predicting cost necessary for key decision-making toward conceptual-
izing a processing facility, determining scalability based on key processing
properties and engineering parameters, and estimated profitability of a pro-
duction plant.
16.9 Conclusions
This chapter outlined the relevance of nutraceuticals in a newly developing
biobased economy that is rapidly becoming an economic player in the food

and agricultural industries. Bioprocessing techniques to produce high-value

specialty biochemicals from food and agricultural by-products are empha-
sized, and greater need for bioenergy production to compensate for deficien-
cies in the present oil-based economy is described through the biorefinery
concept. Specific examples of existing and proposed industrial bioprocesses
that show substantial economic potential were outlined in terms of lipid-
based, large and small molecular nutraceutical products. As the world pop-
ulation continues to increase posing an even greater health threat to third
world countries, there is a continuing need for the integration of biology and
engineering to address the issue for optimizing functional food, nutraceuti-
cal and pharmaceutical production that incorporates important principles of
environmentally friendly technologies, and continuing education to support
new advances for a biobased economy.
Acknowledgment
The authors would like to acknowledge undergraduate students (particu-
larly students from the 2005 Biochemical Engineering course) and graduate
assistants (Jack Heaton, Hui Zhu, Kristy Conwel, Jared Powell, Keri Cantrell,
Cheng-Yi Kuan, Meidui Dong, and Xiaohui Yu) in the Clemson University
Biosystems Engineering Program who participated in bioprocessing projects
for contributing to the literature making this chapter possible.
References
Abalde, J., Fabregas, J., Herrero, C. 1991. β-carotene, vitamin C and vitamin E content
of the marine microalga Dunaliella tertiolecta cultured with different nitrogen
sources. Bioresource Technology, 38: 121–125.
Allouche, N., Damak, M., Ellouz, R., Sayadi, S. 2004. Use of whole cells of Pseudomonas
aeruginosa for synthesis of the antioxidant hydroxytyrosol via conversion of
tyrosol. Applied and Environmental Microbiology, 70: 2105–2109.
Archer, S.D., McDonald, K.A., Jackman, A.P. 1997. Effect of light irradiance on the
production of sulfolipids from Anabaena 7120 in a fed-batch photobioreactor.
Applied Biochemistry and Biotechnology, 67: 139–152.
Badal, R. 2002. Supercritical carbon dioxide extraction of lipids from raw and biocon-
verted rice bran. MS thesis, LSU.
Bailey, J., Ollis, D. 1986. Biochemical Engineering Fundamentals, 2nd edition.
McGraw-Hill Book Co., New York, NY.
Bajpai, P., Bajpai, P.K. 1993. Eicosapentaenoic acid (EPA) production from microor-
ganisms: A review. Journal of Biotechnology, 30: 161–183.

Bajpai, P., Bajpai, P.K., Ward, O.P. 1991a. Effects of aging Mortierella mycelium on pro-
duction of arachidonic and eicosapentaenoic acids. Journal of the American Oil
Chemists’ Society, 68: 775–780.
Bajpai, P., Bajpai, P.K., Ward, O.P. 1991b. Eicosapentaenoic acid (EPA) formation:
Comparative studies with Mortierella strains and production by Mortierella
elongata. Mycological Research, 95(11): 1294–1298.
Benning, C. 1998. Biosynthesis and function of the sulfolipid sulfoquinovosyl diacyl-
glycerol. Annual Review of Plant Biology, 49: 53–75.
Blanch, H.W., Clark, D.S. 1997. Biochemical Engineering. Marcel Dekker, Inc., New York.
Bligh, E.G., Dyer, N.J. 1959. A rapid method of total lipid extraction and purification.
Canadian Journal of Biochemistry and Physiology, 37: 911–917.
Boekesch, H.R., O’Keefe, B.R., McKee, T.C., Pannell, L.K., Patterson, G.M.L., Gardella,
R.S., Sowder, R.C. 2003. A potent novel anti-HIV protein from the cultured cya-
nobacterium Scytonema varium. Biochemistry, 42(9): 2578–2584.
Bulow, L. 2004. Hydrophobic peptide tags as tools in bioseparation. Trends in
Biotechnology, 22(10): 511–516.
Burns, K.T.F., Schmidt, B.M., Yousef, G.G., Knight, C.T.G., Cuendet, M., Kang, Y.,
Pezzuto, J.M., Seigler, D.S., Lila, M.A. 2005. Chemopreventive potential of wild
lowbush blueberry fruits in multiple stages in carcinogenesis. Journal of Food
Science, 7093: S159–S166.
Burri, B.J., Neidlinger, T.R., Lo, A.O., Kwan, C., Wong, M.R. 1997. Supercritical fluid
extraction and reversed-phase liquid chromatography methods for vitamin A
and β-carotene heterogeneous distribution of vitamin A in the liver. Journal of
Chromatography A, 762: 201–206.
Certik, M., Shimizu, S. 1999. Biosynthesis and regulation of microbial polyunsatu-
rated fatty acid production. Journal of Bioscience and Bioengineering, 87(1): 1–14.
Cheng, M.H., Walker, T.H., Hulbert, G.J., Raman, D.R. 1999. Fungal production of
eicosapentaenoic and arachidonic acids from industrial waste streams and
crude soybean lipid. Bioresource Technology, 67(2): 101–110.
Christie, W.W. 2005. Mono- and digalactosyldiacylglycerols and related lipids from
plants, www.lipid.co.k.
Cocks, S., Wrigley, S.K., Chicarelli-Robinson, M.I., Smith, R.M. 1995. High-performance
liquid chromatography comparison of supercritical-fluid extraction and solvent
extraction of microbial fermentation products. Journal of Chromatography A, 697:
115–122.
Connor, W.E. 2000. Importance of n-3 fatty acids in health and disease. The American
Journal of Clinical Nutrition, 71: 171S–175S.
Conwell, K. 2005. Green algal production of sulfoquinovosyl diacylglycerol under
various light intensities. MS thesis, Clemson University, Clemson, SC.
Csabai, P., Joó, F. 2003. Reactivity of the individual lipid classes in homogeneous
catalytic hydrogenation of model and biomembranes detected by MALDI-TOF
mass spectrometry. Catalysis Communications, 4(6): 275–280.
Dale, B.E., Leong, C.K., Pham, T.K., Esquivel, V.M., Rios, Lightfoot, E.N., Moscariello,
J.S. 2004. Hydrolysis of lignocellulosics at low enzyme levels: Application of the
AFEX process. Bioseparations, Biotechnology and Bioengineering, 87(3): 259–262.
Denery, J.R., Dragull, K., Tang, C.S., Li, Q.X. 2004. Pressurized fluid extraction of
carotenoids from Haematococcus pluvialis and Dunaliella salina and kavalactones
from Piper methysticum. Analytica Chimica Acta, 501: 175–181.
Doran, P.M. 1995. Bioprocess Engineering Principles. Academic Press, Inc., San Diego, CA.

Drapcho, C.M., Nghiem, P.N., Walker, T.H. 2008. Biofuels Engineering Process
Technology, McGraw Hill, New York, pp. 224–226.
Dunford, N.T. 2001. Health benefits and processing of lipid based nutritional. Food
Technology, 55(11): 38–43.
Dunford, N.T., Teel, J.A., King, J.W. 2003. A continuous countercurrent supercritical
fluid deacidification process for phytosterol ester fortification in rice bran oil.
Food Research International, 36(2): 175–181.
Enes, I., Saraiva, P. 1996. Optimization of operating strategies in β-carotene microal-
gae bioreactors. Computers and Chemical Engineering, 20: S50–S54.
Folch, J., Lees, M., Stanley, G.H.S. 1957. A simple method for the isolation and purifi-
cation of total lipids from animal tissues. The Journal of Biological Chemistry, 226:
497–509.
Fuller, C.J., Butterfoss, M.S., Failla, M.L. 2001. Relative bioavailability of β-carotene
from supplement sources. Nutrition Research, 21: 1209–1215.
Gessler, N.N., Sokolov, A.V., Bykhovshy, V.Y., Belozerskaya, T.A. 2002. Superoxide
dismutase and catalase activities in carotenoid-synthesizing fungi Blakeslea
trispora and Neurospora crassa fungi in oxidative stress. Applied Biochemistry and
Microbiology, 38(3): 205–209.
Ghandi, S.R., Weete, J.D. 1991. Production of the polyunsaturated fatty acids arachi-
donic acid and eicosapentaenoic acid by the fungus Pythium ultimum. Journal of
General Microbiology, 137: 1825–1830.
Ghosh, R. 2003. Protein Bioseparation Using Ultrafiltration Theory, Applications and New
Development, ed. R. Ghosh. Imperial College Press/World Scientific Publishing
Pte Ltd. ISBN 1-86094-317-9. Chapter 1. http://www.worldscientific.com/
worldscibooks/10.1142/p257
Giordano, W., Avalos, J., Fernandez-Martin, R., Cerda-Olmedo, E., Domenech, C.E.
1999. Lovastatin inhibits the production of gibberellins but not sterols or carot-
enoid biosynthesis in Gibberella fujikuroi. Microbiology, 145: 2997–3002.
Godber, J.S., Shin, T.S., Saska, M., Wells, J.H. 1994. Rice bran: As a viable source of
high value chemicals. Louisiana Agriculture, 37(2): 13.
Grady, C.P.L., Daigger, G.T., Lim, H.C. 1999. Biological Wastewater Treatment, 2nd edi-
tion. Marcel Dekker, Inc., New York, NY.
Gustafson, K.R., Cardelina, J.H., II, Fuller, R.W., Weislow, O.S., Kiser, R.F., Snader,
K.M., Patterson, G.M.L., Boyd, M.R. 1989. AIDS-antiviral sulfolipids from
cyanobacterial (blue-green algae). Journal of the National Cancer Institute, 81:
1254–1258.
Hajazi, M.A., de Lamarliere, C., Rocha, J.M.S., Vermue, M., Tramper, J., Wijffels, R.H.
2002. Selective extraction of carotenoids from the microalga Dunaliella salina
with retention of viability. Biotechnology and Bioengineering, 79(1): 29–36.
Harrison, R.G., Todd, P., Rudge, S.R., Petrides, D.P. 2003. Bioseparations Science and
Engineering. Oxford University Press, New York, NY.
Harve, R., Bajpai, R. 2000. Separation of nucleic acids and proteins. In Handbook of Bio
separations, ed. S. Ahuja. Academic Press, San Diego, CA, pp. 365–378. Chapter 8.
Higashiyama, K., Yaguchi, T., Akimoto, K., Fujikawaa, S., Shimizu, S. 1998.
Enhancement of arachidonic acid production by Mortierella alpina 1S-4. Journal
of the American Oil Chemists’ Society, 75(11): 1501–1505.
Hiyashi, T., Hayashi, K. 1996. Calcium spirulan, an inhibitor of enveloped virus replica-
tion, from blue-green alga Spirulina platensis. Journal of Natural Products, 59: 83–87.

Holman, R.T. 1998. The slow discovery of the importance of omega-3 essential fatty
acids in human health. Journal of Nutrition, 128(2): 427S–433S.
Hong, H., Datla, N., Reed, D.W., Covello, P.S., MacKenzie, S.L., Qiu, X. 2002. High-
level production of gamma-linolenic acid in Brassica juncea using a Delta 6
desaturase from Pythium irregulare. Plant Physiology, 129(1): 354–362.
Hu, K.J., Hu, J.L., Ho, K.P., Yeung, K.W. 2004. Screening of fungi for chitosan pro-
ducers and copper adsorption capacity of fungal chitosan and chitosanaceous
materials. Carbohydrate Polymers, 58: 45–52.
Iisakka, K. 2003. Nutraceuticals and functional foods demand for ingredients.
Nutracos, 11: 3–6.
Iqbal, G., Valivety, R. 1997. Polyunsaturated fatty acids, part 1: Occurrence, biological
activities and applications. Trends in Biotechnology, 15(10): 401–409.
Keller, K., Friedmann, T., Boxman, A. 2001. The bioseparation needs for tomorrow.
Trends in Biotechnology, 19(11): 438–441.
Kendrick, A., Ratledge, C. 1992. Lipid formation in the oleaginous mold Entomophthora
exitalis grown in continuous culture—Effects of growth-rate, temperature and
dissolved-oxygen tension on polyunsaturated fatty-acids. Applied Microbiology
and Biotechnology, 37(1): 18–22.
Kim, H.J., Chen, F., Wang, X., Rajapaks, N.C. 2005. Effect of chitosan on biological
properties of sweet basil (Ocimum basilicum L.). Journal of Agricultural and Food
Chemistry, 53: 3696–3701.
Kim, M., Ryu, K., Choi, W., Rhee, Y., Lee, I. 2003. Enhanced production of 1 → 3-β-D-
glucan by a mutant strain of Agrobacterium species. Biochemical Engineering
Journal, 16: 163–168.
Kim, H., Wu, C., Chen, F., Wang, H., Jin, Z. 2004. Evaluation of antioxidant activity of
tea tree (Melaleuca alternifolia) oil and its components. Journal of Agricultural and
Kiode, S.S. 1998. Chitin-chitosan: Properties, benefits, and risks. Nutrition Research,
18: 1091–2002.
Kishino, S., Nomura, A., Saitoh, M., Sugawara, M., Iseki, K., Kitabatake, A., Miyazaki,
K. 1997. Single-stop isolation method of six glycoforms of human α1-acid
glycoprotein by hydroxylapatite chromatography and study of their binding
capacities for disopyramide. Journal of Chromatography B, 703: 1–6.
Koike, Y., Cai, H.J., Higashiyama, K., Fujikawa, S., Park, E.Y. 2001. Effect of consumed
carbon to nitrogen ratio on mycelial morphology and arachidonic acid produc-
tion in cultures of Mortierella alpine. Journal of Bioscience and Bioengineering, 91(4):
382–389.
Kromus, S., Wachter, B., Koschuh, W., Mandl, M., Krotscheck, C., Narodoslawsky, M.
2004. The green biorefinery Austria—Development of an integrated system for
green biomass utilization. Chemical and Biochemical Engineering Quarterly, 18(1):
7–12.
Ladisch, M.R. 2001. Bioseparations Engineering: Principles, Practice, and Economics. John
Wiley and Sons, New York.
Leon, R., Martin, M., Vigara, J., Carlos, V., Vega, J.M. 2003. Microalgae mediated pho-
toproduction of β-carotene in aqueous-organic two phase systems. Biomolecular
Lightfoot, E.N., Moscariello, J.S. 2004. Bioseparations. Biotechnology and Bioengineering,
87(3): 259–273.

Lim, G., Lee, S., Lee, E., Haam, S., Kim, W. 2002. Separation of astaxanthin from red
yeast Phaffia rhodozyma by supercritical carbon dioxide extraction. Biochemical
Liu, B., Obeid, L.M., Hannun, Y.A. 1997. Sphingomyelinases in cell regulation.
Seminars in Cell and Developmental Biology, 8: 311–322.
Llyod, B.J., Sibernmorgen, T.J., Beers, K.W. 2000. Effect of commercial processing on
antioxidants in rice bran. Cereal Chemistry, 77(5), 551–555.
Mace, K., Halliwell, B. 2004. Bioprocesses and Biotechnology for Functional Foods and
Nutraceuticals, eds. J.R. Neeserk and J.B. German. Marcel Dekker, Inc., New York.
Chapter 16.
Madihally, S.V., Matthew, H.W.T. 1999. Porous chitosan scaffolds for tissue engineer-
ing. Biomaterials, 20: 1133–1142.
Mendes, R.L., Fernandes, H.L., Coelho, J.P., Reis, E.C., Cabral, J.M.S., Novais, J.M.,
Palavra, A.F. 1995. Supercritical CO2 extraction of carotenoids and other lipids
from Chlorella vulgaris. Food Chemistry, 53: 99–103.
Mendes, R.L., Nobre, B.P., Cardoso, M.T., Pereira, A.P., Palavra, A.F. 2003. Supercritical
carbon dioxide extraction of compounds with pharmaceutical importance from
microalgae. Inorganica Chimica Acta, 356: 328–334.
Menrad, K. 2003. Market and marketing of functional food in Europe. Journal of Food
Miller, A., Ensley, H., Pretus, H., McNamee, R., Jones, E., McLaughlin, E., Chandley,
W., Browder, W., Lowman, D., Williams, D. 1997. The application of various
protic acids in the extraction of 1 → 3-β-D-glucan from Saccharomyces cerevisiae.
Carbohydrate Research, 299: 203–208.
Mishima, T., Murata, J. 1998. Inhibition of tumor invasion and metastasis by calcium
spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green
algae, Spirulina platensis. Clinical and Experimental Metastasis, 16(6): 541–550.
Naczk, M., Shahidi, F. 2004. Extraction and analysis of phenolics in food. Journal of
Nalik, S.R., Harindran, J., Varde, A.B. 2001. Pimprinine, an extracellular alkaloid pro-
duced by Streptomyces CDRIL-312: Fermentation, isolation and pharmacologi-
cal activity. Journal of Biotechnology, 88: 1–10.
Norman, H.A., Mischke, C.F., Allen, B., Vincent, J.S. 1996. Semi-preparative isolation
of plant sulfoquinovosyldiacylglycerols by solid phase extraction and HPLC
procedures. The Journal of Lipid Research, 37: 1372–1376.
Nwe, N., Stevens, W.F. 2002. Production of fungal chitosan by solid substrate fer-
mentation followed by enzymatic extractions. Biotechnology Letters, 24: 131–134.
O’Brien, D.J., Kurantz, M.J., Kwoczak, R. 1993. Production of eicosapentaenoic
acid by the filamentous fungus Pythium irregulare. Applied Microbiology and
Biotechnology, 40: 211–214.
Ogretment, B., Hannun, Y.A. 2004. Bioactive sphingolipids in cancer pathogenesis
and treatment. Nature Reviews Cancer, 4: 604–616.
Patel, P. 2005. Supercritical fluid extraction of rice bran with adsorption on rice hull
ash. PhD thesis, LSU.
Peters, M.S., Timmerhaus, K.D. 1991. Plant Design and Economics for Chemical Engineers,
4th edition. McGraw-Hill, New York.
Quasney, M.E., Carter, L.C., Oxford, C., Watkins, S.M., Gershwin, M.E., German, J. B.
2001. Inhibition of proliferation and induction of apoptosis in SNU-1 human

gastric cells by the plant sulfolipid sulfoquinovosyl diacylglycerol. The Journal

of Nutritional Biochemistry, 12(5): 310–315.
Rabbani, S., Beyer, P., Johannes, V.L., Hugueney, P., Kleinig, H. 1998. Induced
β-carotene synthesis driven by triacylglycerol deposition in the unicellular alga
Dunaliella bardawil. Plant Physiology, 116: 1239–1248.
Radwan, S.S. 1991. Sources of c-20-polyunsaturated fatty acids for biotechnological
use. Applied Microbiology and Biotechnology, 35: 421–430.
Roukas, T., Mantzouridou, F. 2001. An improved method for extraction of b-carotene
from Blakeslea trispora. Applied Biochemistry and Biotechnology, 90: 37.
Ruddat, M., Garber, E.D. 1983. Secondary Metabolism and Differentiation in Fungi,
eds. J.W. Bennett and A. Ciegler. Marcel Dekker, New York, pp. 95–152.
Sabio, E., Lozano, M., Montero de Espinosa, V., Mendes, R.L., Pereira, A.P., Palavra,
A.F., Coelho, J.A. 2003. Lycopene and β-carotene extraction from tomato pro-
cessing waste using supercritical CO2. Industrial and Engineering Chemistry
Research, 42(25): 6641–6646.
Saska, M., Chen, F. 2002. Process for the separation of sugars. US patent number
6,451,123.
Saska, M., Chen, F., Vatelot, A. 1999. Sugarcane bagasse-to-xylitol process: Preliminary
testing. Journal of International Sugarcane, 101(1207): 353–358.
Shimizu, S., Kawashima, H., Shinmen, Y., Akimoto, K., Yamada, H. 1988. Production
of eicosapentaenoic acid by Mortierella fungi. Journal of the American Oil Chemists’
Society, 65(9): 1455–1459.
Shuler, M.L., Kargi, F. 1992. Bioprocess Engineering: Basic Concepts. Prentice-Hall,
Englewood Cliffs, NJ.
Simopoulos, A.P. 1989. Summary of the NATO advanced research workshop on
dietary Ω-3 and Ω-6 fatty acids: Biological effects and nutritional essentiality.
Journal of Nutrition, 119: 521–528.
Singh, P.C., Singh, R.K. 1996. Review: Choosing an appropriate bioseparation tech-
nique. Trends in Food Science and Technology, 7: 49–58.
Son, S.J., Reichel, J., He, B., Schuchman, M., Lee, S.B. 2005. Magnetic nanotubes for
magnetic-field-assisted bioseparation, biointeraction, and drug delivery. Journal
of the American Chemical Society, 127: 7316–7317.
Stinson, E.E., Kwoczak, R., Kurantz, M. 1991. Effect of cultural conditions on pro-
duction of eicosapentaenoic acid by Pythium irregulare. Journal of Industrial
Microbiology and Biotechnology, 8(3): 171–178.
Stredansky, M., Conti, E., Salaris, A. 2000. Production of polyunsaturated fatty acids by
Pythium ultimum in solid-state cultivation. Enzyme and Microbial Technology, 26:
304–307.
Tan, S.C., Tan, T.K., Wong, S.M., Khor, E. 1996. The chitosan yield of zygomycetes at
their optimum harvesting time. Carbohydrate Polymers, 30: 239–242.
Tarber, M.G., Packer, L. 1995. Vitamin E beyond antioxidant function. The American
Journal of Clinical Nutrition, 62: 1501S–1509S.
Taylor, S., King, J. 2000. Optimization of the extraction and fractionation of corn bran
oil using analytical supercritical fluid instrumentation. Journal of Chromatographic
Science, 38(3): 91–94.
van Dam-Mieras, M.C.E., de Jeu, W.H., de Vries, J., Currell, B.R., James, J.W., Leach,
C.K., Patmore, R.A. 1992. Bioreactor Design and Product Yield. Butterworth-
Heinemann, Boston, MA.

Vandamme, E. 2004. Industrial Biotechnology and Sustainable Chemistry, ed. E.

Vandamme. BACAS Royal Belgian Academy Council of Applied Sciences,
Brussels, Belgium, p. 26.
Vicente, C., Sebastian, B., Fontaniella, B., Marquesz, A., Filho, L.X., Legaz, M.E. 2001.
Bioskin as an affinity matrix for the separation of glycoproteins. Journal of
Walker, T.H. 2012. Multistate Project Annual Report. The Science and Engineering for a
Biobased Industry, eds. K. Rausch, V. Singh, M. Tumbleson. USDA, Washington,
D.C.
Walker, T.H., Cochran, H.D., Hulbert, G.J. 1999. Supercritical carbon dioxide extrac-
tion of lipids from Pythium irregulare. Journal of the American Oil Chemists’ Society,
76(5): 595–602.
Wang, X., Chen, F., Wan, P.J., Huang, G. 2004. Development of monoclonal antibody-
based enzyme linked immunosorbent assay for Gossypol analysis in cottonseed
meals. Journal of Agricultural and Food Chemistry, 52: 7793–7797.
Wiltshire, K.H., Boersma, M., Moller, A., Buhtz, H. 2000. Extraction of pigments and
fatty acids from the green alga Scenedesmus obliquus (Chlorophyceae). Aquatic
Ecology, 34: 119–126.
Ykema, A., Bakels, R.H.A., Verwoert, I.I.G.S., Smit, H., van Verseveld, H.W. 1989.
Growth yield, maintenance requirements and lipid formation in the oleaginous
yeast Apiotrichum curvatum. Biotechnology and Bioengineering, 34(10): 1268–1276.
Zhu, H. 2002. Utilization of rice bran by Pythium irregulare for lipid production. Master
thesis, LSU, Baton Rouge. etd.lsu.edu/docs/available/etd-1111102–205855/…/
Zhu_thesis
Zhu, H., Drapcho, C.M., Walker, T.H. 2001. Bioconversion of rice bran to omega-3
fatty acids of Pythium irregulare submerged culture. ASAE Paper 01-7020.

17
Microbial Modeling as Basis for Bioreactor
Design for Nutraceutical Production
Caye M. Drapcho and Darryl B. Jones
CONTENTS
17.1 Introduction.............................................................................................. 509
17.1.1 Xylitol.............................................................................................. 511
17.2 Summary of Unstructured Microbial Growth Models...................... 513
17.2.1 Unstructured, Single Limiting Nutrient Models..................... 514
17.3 Inhibition Models..................................................................................... 515
17.4 Models for Multiple Limiting Substrates or Nutrients....................... 517
17.4.1 Yield Parameters........................................................................... 520
17.4.2 Temperature Effects..................................................................... 521
17.5 Kinetic Rate Expressions......................................................................... 522
17.6 Bioreactor Design..................................................................................... 524
17.7 Batch Reactors........................................................................................... 525
17.7.1 Continuous Stirred Tank Reactors (CSTRs).............................. 525
17.8 CSTR with Cell Recycle........................................................................... 527
17.8.1 Fed-Batch Systems........................................................................ 529
17.9 Bioreactor Design Strategies................................................................... 530
17.9.1 Modeling of Glucose/Xylose Utilization and Product
Formation by Candida.................................................................. 533
17.10 Summary................................................................................................... 536
References.............................................................................................................. 536
17.1 Introduction
A wide variety of nutraceutical compounds, food additives and foodstuffs
may be produced through the bioconversion of substrates contained in
agricultural crops and residues. Bioconverted products include traditional
foods and beverages such as beer, wine, vinegar, yogurt, tofu, and Baker’s
yeast. Bioconverted nutraceutical and food products used in the food indus-
try range from flavor compounds (roasted, nutty flavor of pyrazine pro-
duced through aerobic solid-state fermentation of soybeans by bacterium
Bacillus subtilis, fruity flavor of citronellol, linalool and geraniol produced
509
by yeast Kluveryomyces lactis, vanillin flavoring produced by Pseudomonas

and Corynebacterium and bioflavors for beer produced by Saccharomyces
cerevisiae [Besson et al., 1997; Drawert and Barton, 1978; Priefert et al., 2001;
Vanderhaegen et al., 2003]); essential fatty acids such as omega-3 and omega-6
fatty acids eicosapentaenoic (EPA) and arachidonic (ARA) acids from aero-
bic submerged fungal cultures of Pythium irregulare, Mortierella elongata, and
Mortierella alpina using soybean and rice bran substrates (Cheng et al., 1999;
Walker et al., 1999; Zhu et al., 2001, 2002; Yu et al., 2003) and omega-3 fatty acid
docosahexaenoic acid (DHA) and EPA from algal sources such as Gonyaulax,
Gyrodinium, and Cryptoconidium (Wen and Chen, 2003; Sijtsma and de Swaaf,
2004); organic acids citric acid produced by aerobic fermentation of sugar-
cane or beet molasses by Aspergillus niger or Candida lipolytica (Zehentgruber
et al., 1985; Grewal and Kalra, 1995; Mayilvahanan et al., 1996; Pazouki et al.,
2000), lactic acid produced by Lactobacillus helveticus from concentrated
cheese whey (Ghaly et al., 2004), and pyruvic acid production by variety of
yeasts and bacteria (Chen and Lun, 2001); pigments the carotenoid pigment
and antioxidant astaxanthin produced by Xanthophyllomyces dendrorhous (Hu
et al., 2005), red pigments produced by fungi orange and red dyes produced
by submerged culture of Monascus purpureus from hard wheat Paecilomyces
sinclairii during aerobic fermentation of sucrose and starch (Cho et al., 2002),
and substrate (Dominquez-Espinosa and Webb, 2003); emulsifiers (biosur-
factants produced by filamentous fungi Curvularia lunata (Paraszkiewics
et al., 2002), and high molecular weight emulsifiers produced by yeast Candida
utilis [Shepherd et al., 1995]); vitamins [vitamin B12 production by prokaryotic
bacteria Propionibacterium shermanii and Psuedomonas denitrificans (Haddadin
et al., 2001; Martens et al., 2002), or mixed cultures of methanogens (Yang
et al., 2004; Zhang et al., 2004), and vitamin K2-7 production by bacterium
Bacillus subtilis using fermented soybeans (Sato et al., 2001)]; and sugar substi-
tutes (xylitol produced from microaerobic fermentation of d-xylose contained
in hydrolysates of sugarcane bagasse, rice straw and other lignocellulosic
material and defined media by yeasts such as Candida and Pichia [Du Preez
et al., 1987; Schneider, 1989; Grootjen et al., 1991; Kastner et al., 1992, 1999,
2001, 2003; Roberto et al., 1995; Pessoa et al., 1996; Pfeifer et al., 1996; Silva
et al., 1996, 1999; Vandeska et al., 1996; Yashashi et al., 1996; Felipe et al., 1997;
Oh and Kim, 1998; Parajo et al., 1998; Rodrigues et al., 1998; Roberto et al.,
1999; Silva, 1999; Sene et al., 2001; Canilha et al., 2003; Martinez et al. 2003;
Mussatto and Roberto, 2003; Virginio et al., 2005]).
Bioprocess design involves consideration of all critical unit operations,
such as feedstock pretreatment and hydrolysis, bioreactor modeling and
design and extraction and separations technologies. Bioreactor modeling
and design based on microbial growth and product formation kinetics may
be used to optimize the production of high-value compounds by a range of
natural microorganisms, such as fungi, bacteria, yeasts and algae and recom-
binant organisms. Steps to the design based on microbial kinetics include
expression of growth, decay and product formation rates; development of

Microbial Modeling as Basis for Bioreactor Design 511
mass-balance models of the bioreactor; simulation of substrate, biomass

and product concentrations and rates of biomass and product formation for
various reactor configurations; selection of reactor configuration; and design
optimization to maximize product formation rate or product concentration
based on simulation. The focus of this chapter is on microbial modeling and
bioreactor design for suspended growth, shear-tolerant and natural cells.
Many bioprocessing examples could serve to illustrate the kinetic approach
to modeling and design; we will highlight the bioconversion of xylose to the
sugar substitute xylitol.
17.1.1 Xylitol
Xylitol, a 5-carbon sugar alcohol (C5H10OH), has a relative sweetness equal to
sucrose and a negative heat of solution and is non-fermentable by cariogenic
bacteria (Makinen, 1978). Once ingested by humans, 80%–85% of xylitol con-
sumed is metabolized by the liver, and 20%–80% of this portion is converted
into glucose depending on metabolic need for glucose (Makinen, 1978). The
slow conversion of xylose into glucose reduces insulin stimulation (Emodi,
1978). Due to these properties, it has found many uses in the food indus-
try, especially in confectionaries, gums, oral hygiene products and diabetic
foods (Van Eys et al., 1974; Emodi, 1978). Xylitol is also a low-cost reactant for
other rare sugar production. These sugars include l-lyxose, l-xylose, l-xylu-
lose, l-ribulose, l-arabinose, and aldopentoses, ketopentoses, and pentitols
(Granstrom et al., 2004).
Xylitol is found naturally at low levels in various natural materials, such
as fruits and vegetables; for example, plums contain 1% xylitol by weight,
while strawberries, raspberries, eggplant, spinach and pumpkin contain
0.3%–0.1% xylitol, respectively (Makinen, 1978). Extraction of xylitol from
woody materials such as jute sticks and beech wood using oxalic acid has
been investigated (Pollar and Muller, 1986; Day et al., 1989). However, the
low concentrations present in these materials make extraction uneconomi-
cal when compared with other methods. Xylitol can be synthesized chemi-
cally through the catalytic dehydrogenation of xylose, a 5-C sugar with a
Raney nickel catalyst (Melaja, 1981). However, chemical production is costly
because it requires several separation and purification steps since only pure
xylose can be used (Harkonen and Nuojua, 1979; Nigam and Singh, 1995).
Therefore, there is a great interest in exploring bioconversion processes for
xylitol production as an alternative.
Bioconversion of xylose into xylitol can be accomplished by yeasts, primar-
ily of the genera Candida and Pichia, and Pachysolen (Du Preez et al., 1987;
Schneider, 1989; Perego et al., 1990; Grootjen et al., 1991; Kastner et al., 1992,
1999, 2001, 2003; Roberto et al., 1995, 1999; Pessoa et al., 1996; Pfeifer et al., 1996;
Silva et al., 1996, 1999; Vandeska et al., 1996; Yashashi et al., 1996; Felipe et al.,
1997; Oh and Kim, 1998; Rodrigues et al., 1998; Parajo et al., 1998; Silva, 1999;
Sene et al., 2001; Martinez et al., 2003; Mussatto and Roberto, 2003; Virginio

et al., 2005). S. cerevisiae has also been utilized for the biotechnological pro-
duction of xylitol but Candida sp. is preferred because Candida are natural
d-xylose consumers and maintain the reduction/oxidation balance during
xylitol accumulation (Granstrom et al., 2007). These yeasts can utilize pentose
and hexose sugars, with hexose sugars as the preferred substrate that inhibit
the use of xylose (Hsiao et al., 1982; Vandeska et al., 1996; Kastner et al., 1999).
The sequential use of glucose than xylose under batch conditions has been
documented extensively (Pfeifer et al., 1996; Oh and Kim, 1998; Rodrigues
et al., 1998; Kastner et al., 1999, 2001; Sene et al., 2001; Walther et al., 2001;
Mussatto and Roberto, 2003). Under aerobic conditions, both glucose and
xylose are metabolized with cell mass as the main product. Under micro-
aerobic conditions, both glucose and xylose can be bioconverted into xyli-
tol, but glucose conversion involves three steps and results in the formation
of an undesirable side product. Even though the yield and productivity are
low compared with the bioconversion of d-xylose into xylitol, investigation
of the glucose pathway is still conducted because of the low price and avail-
ability of d-glucose, the preferred feed source for the biotechnology industry
(Granstrom et al., 2007).
To enhance xylitol production rate and yield, a number of growth con-
ditions can be manipulated or controlled. The aeration rate should be
high at first to maximize biomass formation, and then reduced to create
oxygen-limited (microaerobic) or even anoxic conditions. Under these con-
ditions, species of Candida can form xylitol from d-xylose (Du Preez et al.,
1987; Schneider, 1989; Perego et al., 1990; Grootjen et al., 1991; Kastner
et al., 1992, 1999, 2001, 2003; Roberto et al., 1995; Pessoa et al., 1996; Pfeifer
et al., 1996; Silva et al., 1996, 1999; Vandeska et al., 1996; Yashashi et al., 1996;
Felipe et al., 1997; Oh and Kim, 1998; Parajo et al., 1998; Rodrigues et al.,
1998; Roberto et al., 1999; Silva, 1999; Sene et al., 2001; Martinez et al., 2003;
Mussatto and Roberto, 2003; Virginio et al., 2005). At low dissolved con-
centrations, the production of d-xylulose is minimized and the relatively
high levels of NADH and NADPH reduce d-xylose (Horitsu et al., 1992).
The nitrogen source and C/N ratio are also important in xylitol produc-
tion. Yeast extract and ammonium acetate are excellent nitrogen sources for
Candida (Dahiya, 1991; Horitsu et al., 1992), while ammonium salts are pre-
ferred for S. cerevisiae to maximize xylitol production (Witt and Heilmeyer,
1966; Nigam and Singh, 1995). For Pichia, a low nitrogen content in medium
(high C/N ratio) resulted in greater sugar alcohol formation than an
N-enriched media (Onishi et al., 1980). The concentration of d-xylose is also
important in production rate and yield of xylitol. In general, with increased
concentration of d-xylose, the yield increases because at low concentrations,
xylose is used mainly for cell growth (Meyrial et al., 1991). However, at very
high d-xylose concentrations, there are osmotic effects on cells or substrate
repression of d-xylose-metabolizing enzymes, which results in slower bio-
mass growth rate (Nigam and Singh, 1995). It was shown that Candida sp.
B22 growth rate was sensitive to a high initial xylose concentration (Chen

and Gong, 1985). Because a purely xylose media is rare and the utilization
of agricultural residues containing a mixture of sugars is more economical
as it is important to consider the effects of other sugars on xylose conver-
sion into xylitol. Glucose and other sugars, such as mannose, galactose and
arabinose, found in hemicellulose hydrolysates are rapidly fermented but
are used only for growth and ethanol production (Meyrial et al., 1991). High
concentrations of sugars can also cause osmotic stress and inhibit induction
of xylose reductase enzymes (Walther et al., 2001). Alternative sugars do not
always necessarily inhibit xylitol production. In one study, maltose, arabi-
nose, and d-galactose in a sugarcane bagasse fermentation medium did not
affect xylitol production (Singh et al., 1990).
Hemicellulosic hydrolysates from agricultural residues, such as sugarcane
bagasse, corn-stalks, cotton seed, peanut hulls, coconut husks, flax straw,
rice straw, wheat straw, and wood pulp, are plentiful and contain signifi-
cant quantities of xylose and glucose in their hemicellulose and cellulose
fractions (Counsell, 1977). For example, one ton of milled sugar yields 180–
280 kg of sugarcane bagasse, with the typical bagasse containing 19%–24%
hemicellulose, 32%–48% cellulose, and 23%–32% lignin (Scurlock, nd). This
composition represents typically 71% total reducing sugars, 25% xylose and
41% glucose on a dry weight basis (Pessoa et al., 1996). However, mild acid
pre-treatments with sulfuric or hydrochloric acids result in greater hemi-
cellulose hydrolysis than cellulose hydrolysis (Parajo et al., 1998). Varying
conditions of temperature, time, and acid concentration result in greatly
varied concentrations of sugars in sugarcane bagasse hydrolysate. A typical
sugarcane bagasse hydrolysate may contain 17–105 g/L xylose and 7–30 g/L
glucose, while a typical rice straw hydrolysate may contain 16–79 g/L xylose
and 4–23 g/L glucose (Parajo et al., 1998).
Bioconversion of xylose can achieve xylitol yields as high as 0.7–0.8 g xylitol/g
xylose in hemicellulosic hydrolysates (Roberto et al., 1995; Felipe et al., 1997;
Mussatto and Roberto, 2003) and 0.8–0.9 in defined xylose media (Oh and Kim,
1998; Kastner et al., 2003). The theoretical yield is 0.917 g/g. For example, batch
fermentations of sugarcane bagasse hydrolysate using Candida guilliermondii
FTI20037 achieved maximum xylitol production rates of 0.87 g/L-h with prod-
uct yield of 0.67 g xylitol/g xylose at 0.45 v.v.m. aeration rate (kLa 27 h−1) (Silva
et al., 1997). Reported values of maximum specific growth rate for Candida
range widely from 0.04 to 0.52 h−1 for xylose and glucose/xylose mixtures
(Pessoa et al., 1996; Vandeska et al., 1996; Oh and Kim, 1998).
17.2 Summary of Unstructured Microbial Growth Models

Basic microbial growth models for suspended growth, shear-resistant cells
should consider potential impact of multiple growth-limiting substrate(s) and

inhibitory substrates, products, and/or xenobiotic compounds. Structured

models consider individual reactions occurring within the cell involving spe-
cific components, such as DNA or proteins. Unstructured models view the cell
as an entity and model growth and death of the microorganism. Growth rates
for unstructured models are expressed as v (cell number-specific growth rate)
or μ (biomass-specific growth rate), both in units of time−1. Balanced growth
occurs when v = μ and cell composition is constant with time. Unbalanced
growth occurs as cell composition changes with time, resulting in a change
in cell mass per cell; therefore v ≠ μ (Blanch and Clark, 1997).
17.2.1 Unstructured, Single Limiting Nutrient Models

The Monod model (Monod, 1949) is a widely applied model used to describe
microbial growth. The model was developed for the growth of a single
microorganism (E. coli) growing on a media with a single limiting organic
substrate (glucose) as shown below:
µˆ SS
µ= (17.1)
K S + SS
where μ is the specific growth rate coefficient, h−1; µ̂ the maximum specific
growth rate, h−1; SS the soluble substrate concentration, mg/L; and KS the
half-saturation constant, mg/L. The KS value is the substrate concentration at
which the growth rate μ is equal to 1/2 of µ̂ .
The Monod model has been used successfully to model the growth of
many pure cultures of heterotrophic and autotrophic organisms growing on
single substrates and mixed microbial cultures using mixed substrates, such
as wastewater treatment applications (Henze et al., 1987; Grady et al., 1999).
Estimates of µ̂ and KS parameter values may be obtained by collecting data
of specific growth rate values as a function of soluble substrate concentra-
tion during exponential phase of batch growth, and applying a linearization
technique, such as Lineweaver-Burk or Hanes (Hofstee) equations (Doran,
1995; Blanch and Clark, 1997; Grady et al., 1999).
Zero- and first-order approximations of the Monod model (Equations 17.2
and 17.3) may be applied when the substrate concentration is high and low
relative to the KS value, respectively.
µ ≈ µˆ Zero-order appropriation when SS K S (17.2)

µˆ SS
µ≈ First-order appropriation when SS K S (17.3)
Ks
Due to high substrate concentrations typically found in food/biopro-

cessing applications, many researchers report µ̂ values and not K S (Oh and

Kim, 1998). For Candida tropicalis utilizing xylose under batch microaero-
bic conditions, µ̂ was determined to be 0.52 h−1 at initial xylose concentra-
tions of 50 g/L (Oh and Kim, 1998). However, much lower µ̂ values of 0.04
and 0.11 h−1 were reported for C. guilliermondii (Pfeifer et al., 1996) cultures
using xylose and glucose, respectively, and µ̂ values of 0.057–0.137 h−1 were
reported for C. tropicalis cultures as a function of aeration rate (Pessoa
et al., 1996). In these reports, observed specific growth rate was appar-
ently assumed to be the maximum specific growth rate value. For exam-
ple, Pessoa et al. (1996) reported that µ̂ values were determined from the
growth curve in the exponential phase. These values may represent the
observed specific growth rate for culture conditions present rather than the
maximum specific growth rate.
17.3 Inhibition Models

Expansions of the Monod model to include inhibition by substrate,
product, or xenobiotic compounds have been developed. Many inhibi-
tion models for microbial growth are empirical in nature, while others
apply enzyme kinetics concepts (Blanch and Clark, 1997). In general,
competitive inhibitors compete with substrate for binding site of the
enzyme, and may be xenobiotic compounds or products that accumu-
late in reactor. Uncompetitive inhibition occurs when a compound binds
to the enzyme−substrate (ES) complex. A special form of uncompetitive
i nhibition is substrate i nhibition, where substrate binds to an alternative
site on the enzyme leading to a nonreactive ES complex. Noncompetitive
inhibitors can bind to either the free enzyme or the ES complex. Product
inhibition may be modeled as noncompetitive inhibition (Blanch and
Clark, 1997).
1. Substrate inhibition: High substrate concentrations relative to the KS
value may limit growth. Modifications of the Monod model, such as the
Andrews’ equation (Andrews, 1968), may be used to describe substrate inhi-
bition (Grady et al., 1999):
µˆ SS
µ= (17.4)
K S + SS + (SS2 /K I )
where K I = inhibition coefficient, mg/L.

As the value of K I increases, the above equation simplifies to the Monod
model. In many cases of inhibitory substrate, the value of µ̂ may never
be observed and therefore K S cannot be calculated. In these cases, μ*, the
maximum observed growth rate, and SS* , the substrate concentration at

which μ* occurs, are determined and used to calculate µ̂ and K S (Grady

et al., 1999):
µ
µ* = (17.5)
2(K S /K I )0.5 + 1
and
0.5
K 
SS* =  S  (17.6)
 KI 

Bioprocessing studies have investigated the inhibition of cell growth rates

due to elevated substrate levels. For example, Oh and Kim (1998) found that
increasing the initial xylose concentration above 50 g/L for Candida batch
fermentations on defined xylose media under microaerobic conditions
resulted in reduced exponential-phase-specific growth rates, from 0.52 h−1
at 50 g/L initial xylose concentration, to 0.49, 0.47, 0.35, and 0.23 h−1 at xylose
concentrations of 100, 150, 200, and 250 g/L, respectively (Oh and Kim,
1998). However, Equation 17.5 does not model the response well, which may
indicate that other factor, such as oxygen limitation with increasing sub-
strate, lack of pH control, or product inhibition, may also have influenced
the specific growth rate.
For phototrophic microorganisms, Steele’s model (Steele, 1962) is often
used to describe algal growth considering potentially inhibitory light inten-
sity (Steele, 1962; Lehman et al., 1975; Welch, 1980):
I (1−( I /I ))
µ = µˆ L e L opt (17.7)
I opt

where IL and Iopt = light and optimal light intensity, μmol/m2-s.

2. Product inhibition: High product concentrations may inhibit microbial
growth. Product inhibition may be modeled as
n n
µˆ  P µˆ S  P
µ=  1−  or µ = 1 − (17.8)
1 + ( K S / S)  Pm  K S + S  Pm 

where P = product concentration, mg/L; Pm = product concentration where

growth is completely inhibited; n = coefficient determined by data.
For example, yeast growth under anaerobic conditions may be inhib-
ited by ethanol concentrations above 5% (Shuler and Kargi, 1992; Blanch
and Clark, 1997). The growth of Candida shehatae was completely inhibited
by ethanol concentrations of 25 g/L under microaerobic fermentations
(Kastner et al., 1992) and 37.5 g/L under aerobic conditions (Du Preez et al.,
1987).

Another equation to describe noncompetitive inhibition by product is rep-

resented in several different forms as shown below (Shuler and Kargi, 1992;
Doyle et al., 1995; Blanch and Clark, 1997):
µˆ µˆ SS  K p 
µ= or µ = or
(1 + (K S /SS ))1 + (P/K p ) K S + SS  K p + P 
 S  1
µ = µˆ   (17.9)
 K S + S  1 + (P/K p )

where Kp = product inhibition constant, mg/L.

3. Inhibition by xenobiotic compounds: A general form of uncompetitive
inhibition of enzymes can be used to model inhibition by other compounds
(rearranged from Blanch and Clark (1997)):
µˆ SS
µ= (17.10)
SS (1 + (I/K I ) + K S
where I = inhibitor concentration, mg/L and K I = inhibition constant, mg/L.

Another equation used to model general inhibition is given as
 SS   K I 
µ = µˆ  (17.11)
 K S + SS   I + K I 

Sugarcane bagasse hydrolysate contains compounds such as furfural and

5-hydroxymethylfurfural (HMF) that may inhibit yeast growth at high con-
centrations. Inhibition of cell growth occurred at furfural and HMF concen-
trations of 1.0 and 1.5 g/L, respectively, for Candida guilliermondi (Felipe et al.,
1997). Use of K I value of 0.01 g/L in Equation 17.12 would result in a predicted
99% rate reduction of the specific growth rate at furfural concentration of
1.0 g/L.
17.4 Models for Multiple Limiting Substrates or Nutrients

Substrate or nutrients can be thought of in a broad sense as complementary
or substitutable. Complementary nutrients meet different needs for the micro-
organism; for example, oxygen may serve as an electron acceptor for yeast
growth while glucose serves as an electron donor. Substitutable substrates
meet the same need for the cell. Glucose and xylose used by yeasts such as
Candida may be viewed as substitutable substrates.

1. Complementary substrates: To model multiple complementary nutri-

ents, an interactive form of the Monod model may be used (Henze et al.,
1987; Doyle et al., 1995; Grady et al., 1999):
 S1   S2 
µ = µˆ     (17.12)
 K S1 + S1   K S2 + S2 
The interactive model is based on the assumption that both substrates

influence the growth rate. This model predicts a lower specific growth
rate than the non-interactive model particularly when substrate concentra-
tions are small compared with their K S values. This model is a continuous
function and has been used in many applications, including describing
the growth of hybridoma cells based on substrates glucose and glutamine
with inhibition by products such as lactate and ammonia as follows (Doyle
et al., 1995):
 SGlu  Sglut  K lact  K NH 

µ = µˆ  (17.13)
 K Glu + SGlu   K glut + Sglut   K lact + Plact   K NH + PNH 

with SGlu, Sglut, Plact, and PNH = soluble concentrations of glucose, glutamine,

lactate, and ammonia, respectively mg/L; and Kgl, Kglut, Klact, and K NH = K
values for glucose, glutamine, lactate, and ammonia, respectively.
An alternative approach to model complementary nutrients, called the
non-interactive model, assumes one nutrient limits growth (Grady et al.,
1999):
  S1   S2  
µ = µˆ ∗ minimum of    ,  (17.14)
  K S1 + S1   K S2 + S2  

The predicted growth rate will be the lowest value based on each substrate
concentration. This model is a discontinuous function at the transition from
one nutrient limitation to another. Both models have advantages and have
been used successfully to describe microbial growth.
Neither model was found in the bioprocessing literature investigated
here to describe Candida or Pichia growth utilizing xylose and other sub-
strates. However, these models could be applied for these fermentations
where carbohydrate and oxygen or nutrients may be potentially growth-
rate limiting. The K S value for oxygen, K S, O2, for most aerobic, heterotro-
phic bacterial cultures is quite low, and often is less than 1 mg/L (Grady
et al., 1999).
2. Substitutable substrates: When multiple substitutable substrates are
present in the media, simultaneous use of the substrates may occur. In
this case, one compound may be preferred by the microorganism over

the other. If S1 is preferred, then its presence in the media will inhibit the
use of S2. The growth rate of the microorganism using S2 is modeled as a
function of S2 with inhibition by S1 as follows (Henze et al., 1987; Grady
et al., 1999):
 S1 
µ1 = µˆ  (17.15)
 K S1 + S1 

 S2   KS1 
µ 2 = µˆ  (17.16)
 KS2 + S2   KS1 + S1 

For example, both glucose and xylose may be used by yeasts such as
Candida. Glucose is the preferred substrate, so its presence in media sup-
presses the use of xylose by reversibly inactivating the enzyme xylose
reductase (Pfeifer et al., 1996), while the presence of xylose in the media
does not influence the use of glucose. The sequential use of glucose then
xylose has been documented by many researchers (Pessoa et al., 1996;
Pfeifer et al., 1996; Oh and Kim, 1998; Rodrigues et al., 1998; Kastner et al.,
1999). Thus, the growth rate of Candida based on glucose use may be mod-
eled as follows:
 SGlu 
µ Glu = µˆ  (17.17)
 K Glu + SGlu 

where µ Glu represents the specific growth rate using glucose as organic sub-
strate, h−1.
The growth rate of Candida based on use of xylose may be modeled as
 SXyl  K Glu 
µ Xyl = f * µˆ     (17.18)
 K Xyl + SXyl   K Glu + SGlu 

where µ Xyl represents the specific growth rate using xylose as organic sub-
strate, h−1, and f represents factor for decreased maximum specific growth
rate for non-preferred substrate.
These equations represent the growth of Candida as exclusively using
glucose when the glucose concentration is high. As the glucose concentra-
tion declines, the growth rate based on glucose declines, with concomi-
tant increase in the growth rate based on the use of xylose. Simulations of
Candida-specific growth rate values based on xylose use as a function of glu-
cose and xylose concentrations (Equation 17.18) using representative kinetic
values are shown in Figure 17.1.
If multiple complementary and substitutable substrates are present, the
terms may be combined in multiplicative manner. The following expressions

0.5
Simulated specific growth
rate using xylose, h–1
0 g/L
0.4 glucose
2 g/L
0.3 glucose
5 g/L
0.2 glucose
0.1 50 g/L
glucose
0
0 10 20 30 40 50
Xylose, g/L
FIGURE 17.1
Simulated specific growth rate of Candida as function of glucose and xylose concentrations
using representative kinetic parameter values.
could be used to describe growth of Candida under aerobic conditions if oxy-

gen, glucose, and xylose are growth-rate limiting substrates:
 SGlu  SO2 
µ Glu = µˆ  (17.19)
 K Glu + SGlu   K O2 + SO2 

 SXyl  K Glu  SO2 

µ Xyl = f µˆ     (17.20)
 K Xyl + SXyl   K Glu + SGlu   K O2 + SO2 


Combining Equations 17.8 (product inhibition—ethanol), 17.11 (xenobiotic

inhibition—furfural) 17.12 (complementary nutrients—O2 and glucose), and
17.15/17.16 (substitutable substrates—glucose and xylose) results in the fol-
lowing growth models for Candida:
 SGlu   SO2   Pm,ETOH − PETOH   K furfural 

µ Glu = µˆ  (17.21)
 K Glu + SGlu   K O2 + SO2   PETOH   K
furfural + S 
furfural 

 Sxyl   K Glu   SO2 

µ xyl = f µˆ    K + S   K + S 
K
 xyl + Sxyl  Glu Glu O2 O2
 Pm,ETOH − PETOH   K furfural  (17.22)

   K 
PETOH + S furfural 
furfural

17.4.1 Yield Parameters

Biomass yield (YX/S) is defined as the mass of cells produced per mass of
substrate utilized. Product yield (YP/S) is defined as the mass of product

formed per mass of substrate utilized. Biomass and product yields may be
determined from the stoichiometry of a balanced growth equation, or by
measurement. For the balanced equation describing anaerobic fermentation
of glucose by yeast (Shuler and Kargi, 1992) given below, YX/S and YETOH/S
values are 0.078 mg biomass/mg glucose and 0.33 mg ethanol/mg glucose,
respectively.
1000 C6H12O 6 + 118 NH 3 → 5.9 C100H174 O 45N 20 + 1300 C 2H 5OH

+ 1540 CO 2 + 430 C 3H8 O 3 + 36 H 2O (17.23)
Biomass and product yields are generally assumed to not vary with
respect to substrate concentration, but will vary for different elec-
tron acceptor environments. For example, xylitol yields from xylose
for C. tropicalis were reported by Kastner et al. (2003) as a function of
redox potential of the culture environment as follows: YXylitol/xylose = 0.63,
0.87, 0.15, and 0.10 g/g for redox potentials of –150, −100, −50, and 0 mV,
respectively.
17.4.2 Temperature Effects

Microbial growth models and design should consider impact of tempera-
ture on specific growth rate and decay constant values. Biomass yield, prod-
uct yield, half-saturation constant, and growth-associated product constant
values are often considered independent of temperature. Maximum specific
growth rate and decay constant values should be corrected for temperature
differences using the Arrhenius or modified Arrhenius equation shown
below (Grady et al., 1999):
k = A e− EA /RT Arrhenius equation (17.24)

where k = temperature-dependent reaction rate constant, A = Arrhenius con-

stant, EA = activation energy, kJ/mol, R = gas constant, kJ/mol K, T = absolute
temperature, K.
kT1 = kT2 θ(T1 − T2 ) Modified Arrhenius equation (17.25)

where kT1 and kT2 are the reaction rate constants at temperatures 1 and 2;
θT = temperature correction factor, dimensionless and T = temperature, °C
or K.
The Arrhenius expression is applicable over a wide range of temperatures.
The modified Arrhenius expression should be applied only over a narrow
range of temperatures, typically those encountered for mesophilic organ-
isms (Grady et al., 1999).

17.5 Kinetic Rate Expressions

Once the appropriate relationship to describe the specific growth rate is
determined, the rates of utilization or formation can be developed for impor-
tant reactor compounds. The rate of biomass formation is given as
rXB = µX B where rXB = rate of biomass formation , mg/L-h. (17.26)

For growth rate expressions developed for cells using substitutable sub-
strates, as in the case of xylose and glucose use by Candida, the rates of biomass
formation using both substrates could be described as follows, following mod-
els proposed for wastewater treatment (Henze et al., 1987; Grady et al., 1999):
rXB = µ Glu X B + µ Xyl X B (17.27)

Products formed from bioconversion processes were classified by Gaden

(1959) as one of three types. Type I: growth-associated products arising
directly from metabolism of carbohydrate; Type II: products arising indi-
rectly from carbohydrate metabolism that accumulate under abnormal
metabolism. Type III: nongrowth-associated products formed as a result of
processes other than growth/energy metabolism.
Using this classification, ethanol produced by the anaerobic fermentation
of glucose by yeasts is a Type I product and antibiotics such as streptomy-
cin are Type III products (Gaden, 1959). Citric acid produced as a result of
energy metabolism by aerobic fermentation of molasses and accumulated
under conditions of nutrient-limitation of trace metals such as manganese,
iron, and zinc by A. niger (Zehentgruber et al., 1985) would be characterized
as a Type II product (Gaden, 1959).
This classification may be complex for some bioproducts. For example,
polydroxyalkanoates (PHAs), which are internal energy/carbon storage
products, are of interest for biodegradable polymers in food packaging and
films (Wang and Lee, 1997). PHAs can be formed by two routes: microbes
such as Alcaligenes eutrophus produce and accumulate PHAs under nutri-
ent-limited conditions with excess carbon source, while microbes such as
Alcaligenes latus do not require nutrient limitation for PHA production and
accumulation but will accumulate greater quantities under nutrient-limited
conditions (Wang and Lee, 1997). Therefore, PHA could be classified as a
Type II or Type I product, depending on the microbial culture employed.
Many have adopted a classification that combines the Type I and Type II
products described by Gaden into one category, growth-associated (GA)
products. The Type III products are non-growth-associated (NGA) products.
A middle category, called mixed products, are those that are formed from
both growth and non-growth metabolic processes. Using this convention,
PHA would be considered a GA product (Wang and Lee, 1997).

The rate of growth-associated product formation can be expressed as a

function of the rate of biomass formation (van Dam-Mieras et al., 1992):
rpg = k pg rXB = k pg mX B (17.28)

where rpg = rate of growth associated product formation, mg/L-h; and

kpg = growth associated product formation ratio, mg product/mg biomass.
For example, the kpg value for ethanol based on the anaerobic fermentation
of glucose by yeast (Equation 17.23) (Shuler and Kargi, 1992) can be deter-
mined by the stoichiometry of the balanced equation as 4.26 g ethanol/g bio-
mass. kpg may also be calculated as the ratio of YP/S/YX/S or measured directly.
The rate of nongrowth-associated (NGA) product formation is modeled
solely as a function of the concentration of biomass in the reactor, and not the
rate of biomass formation (van Dam-Mieras et al., 1992).
rpn = k pn X B (17.29)

where
rpn = rate of nongrowth-associated product formation, mg/L-h and
kpn = nongrowth-associated product formation constant, mg product/mg
biomass-h
Products formed from a combination of growth and nongrowth-associ-

ated means may be modeled as (van Dam-Mieras et al., 1992)
rP = k pg µX B + k pn X B (17.30)

Examples of NGA products in food/agricultural bioprocessing include

vitamins such as K2-7 (Sato et al., 2001), pigments produced from Monascus
fermentations (Dominquez-Espinosa and Webb, 2003), and emulsifier pro-
duction by filamentous fungi C. lunata (Paraszkiewics et al., 2002). From
graphs of xylose use and xylitol formation presented by several research-
ers, xylitol appears to be a mixed or an NGA product (Oh and Kim, 1998;
Rodrigues et al., 1998; Kastner et al., 2003).
Other reactions that occur as a result of growth, such as substrate and oxy-
gen utilization, are represented also as functions of the rate of biomass for-
mation. The rate of organic substrate utilization is therefore:
rXB µX B
rS = = (17.31)
YX/S YX/S
The rate of substrate utilization that occurs for products formed during
non-growth processes can be modeled as
rpn
rS = (17.32)
YP/S

The rate of O2 utilization during growth can be expressed as a function of

a stoichiometric factor based on the balanced growth equation or measured
value. For example, in a representative equation describing organotrophic
bacteria (C5H7O2N) grown on glucose, 0.578 mg O2 are required per mg XB
formed (Grady et al., 1999); therefore:
ro = ratio * rXB = 0.578 * µX B (17.33)

where ro = rate of oxygen utilization, mg/L-h O2.

If the stoichiometry is not known, then the rate of oxygen utilization can
be expressed as follows if biomass and organic substrate are measured on
a chemical oxygen demand (COD) basis (Henze et al., 1987; Grady et al.,
1999):
1 − YX/S
ro = (1 − YX/S ) * rS = µX B (17.34)
YX/S
A final expression important to bioreactor modeling is the rate of microbial

decay. This rate of natural death or decay may be modeled as first order with
respect to biomass.
rXB ,d = −bX B (17.35)

where rX B ,d = rate of biomass decay, mg/L-h XB; b = decay constant, h−1.
17.6 Bioreactor Design

The kinetic rate expressions developed above may be used in mass balance
equations with respect to substrate, biomass, and products for different bio-
reactor configurations. To develop the mass balance equations with respect
to products in the reactor, characterization of the product as a GA, NGA, or
mixed metabolite is needed. Basic bioreactor designs for suspended growth
cultures are batch, continuous (flow) stirred tank reactor (CSTR), and CSTR
with biomass recycle. Further characterization of the product as intracellu-
lar versus extracellular, and “soluble” versus “particulate” when compared
with cell separation technique is needed for CSTR with recycle designs. If
the means of cell separation (filtration, centrifugation, and settling) removes
the compound with the biomass, then the product is considered particulate.
Ethanol and xylitol are examples of soluble, extracellular products, while oils
produced by filamentous fungi Pythium and PHAs are intracellular prod-
ucts. Products such as monoclonal antibodies are extracellular, “particulate”
compounds, since they can be separated by ultrafiltration.

17.7 Batch Reactors

The mass balance with respect to biomass for a batch reactor is given as fol-
lows, assuming growth and decay are the only reactions:
dX B
µX B − bX B = (17.36)
dt
Although there is a temporary balancing of the growth and decay terms

during stationary phase, batch reactors never reach true steady-state condi-
tions, where concentrations in the reactor do not change with time. During
exponential growth in a batch reactor, decay is often assumed to be negli-
gible. Integration of Equation 17.36 and substitution of the definition of bio-
mass yield results in the logistic growth equation (Shuler and Kargi, 1992):
 K SYX/S  X  K SYX/S S
(t − t0 )µ =  + 1 ln  Bt  + ln 0 (17.37)
 X B 0 + S0YX/S   X B0  X B0 + S0YX/S St

where t and t0 = time and initial time, h; XBt and XB0 = biomass concentration at
time t and t0, and SSt and SS0 = soluble substrate concentration at time t and t0.
This equation describes how biomass increases while substrate decreases with
time during exponential growth. The length of incubation in a batch reactor
may vary depending on growth kinetics, accumulation of inhibitory products
and product type. Equation 17.37 can also be used to determine the Monod
kinetic parameters for batch growth data, a technique that is primarily utilized
in the wastewater treatment literature (Simkins and Alexander, 1985; Zhang
et al., 1999), but is being applied to bioprocessing applications (Reeves, 2004).
17.7.1 Continuous Stirred Tank Reactors (CSTRs)

For a simple CSTR with a single influent and effluent flow, the mass balance
with respect to biomass is
Q Q dX B
X Bi − XB + µX B − bX B = (17.38)
V V dt
where XBi = biomass concentration in influent flow, mg/L.

For cases with no biomass in the influent flow, growth based on a single
limiting nutrient, and no substrate or product inhibition, the following equa-
tion results at steady state:
1
µ= +b (17.39)
τ
where τ = hydraulic retention time in reactor, h.

Thus, the hydraulic retention time is the main design parameter for a
CSTR, since it controls the microbial growth rate in the reactor. A sim-
ple CSTR can be operated at a short retention time to maintain a high-
specific growth rate to mimic a batch reactor during exponential growth,
with r esulting s ustained formation of growth-associated products, or may
be operated at a long retention time to maintain a low-specific growth
rate to mimic a batch reactor during declining exponential or station-
ary phases, resulting in continuous formation of nongrowth-associated
products.
Substitution of the single Monod model for μ results in
K S (1/τ + b)
SS = (17.40)
µ − (1/τ + b)
The effluent substrate concentration is a function of the hydraulic retention

time and the kinetic parameters values, but not a function of the influent
substrate concentration.
The mass balance with respect to substrate for a simple CSTR is
Q Q µX B dSS
SSi − SS − = (17.41)
V V YX/S dt
After substitution of Equation 17.38, the resulting steady-state concentra-

tion of biomass is given as
YX/S (SSi − SS )
XB = (17.42)
1 + τb
The mass balance with respect to a general product is
Q Q dP
Pi −P + k pg µX B + k pn X B = (17.43)
V V dt
The steady-state solutions for GA and NGA products are
PGA = k pg µX B τ (17.44)

where PGA is growth associated product concentration, mg/L; and
PNGA = k pn X B τ (17.45)

where PNGA is the nongrowth-associated product concentration, mg/L.

At steady state, concentrations of substrate, biomass, and product will
vary as a function of retention time. In general, the specific growth rate and

effluent substrate concentration decline and biomass concentration increases

with increase in hydraulic retention time. Thus, reactor design may be based
on achieving the desired effluent concentrations. Martinez et al. (2003) inves-
tigated a CSTR for xylitol formation using Candida guilliermondi cultures and
sugarcane bagasse hydrolysate. In most comparisons, they found higher
xylitol and biomass concentrations and lower substrate concentrations at a
longer hydraulic retention time (100 vs. 20 h).
To maximize rates of biomass or GA product formation or of substrate uti-
lization, both a high-specific growth rate and a high biomass value must be
achieved. The hydraulic retention time that maximizes these rates can be
estimated from the following (van Dam-Mieras et al., 1992):
1 ˆ  KS  
0.5
= µ 1 −   (17.46)
τ  K S + SSi  
 
For an NGA product, there is no single hydraulic retention time that

maximizes the rate of production. The above equations may be used for
preliminary analysis and design of a CSTR. For more expanded analysis,
and for systems where multiple substrates or inhibition occur, dynamic
modeling may be used to determine the retention time that maximizes
biomass or GA formation rates or the product concentration as a function
of retention time.
17.8 CSTR with Cell Recycle

A CSTR with biomass recycle includes some means to remove biomass from
the reactor effluent flow, through external or internal filter, settling device or
centrifugation, and return biomass flow to the reactor. By separating biomass
from the effluent flow, the length of time that cells remain in the reactor, the
cell retention time, θ, can be controlled independently of the hydraulic reten-
tion time. To control θ, a portion of biomass is harvested or “wasted” from
the reactor. When the harvest flow is pumped directly from the reactor, the
biomass concentration in the reactor is equal to the concentration in the har-
vest flow and θ is calculated as
VX B V
θ= = (17.47)
QH X BH QH
where θ = cell retention time, h; V = reactor volume, L; XB = biomass con-

centration in reactor, mg/L; XBH = biomass concentration in the harvest flow,
mg/L; and QH = harvest flow rate, L/h.

The mass balance equation with respect to biomass for a CSTR with
recycle is
Q Q (Q − Qw ) dX B
X Bi − X B w − X Be + µX B − bX B = (17.48)
V V V dt
where XBe = biomass concentration in final effluent flow, mg/L. XBe ~ 0 if cell

separation step is assumed 100% efficient.
At steady state, the mass balance with respect to biomass simplifies to
1
µ= +b (17.49)
θ
Therefore, θ controls μ for a CSTR with recycle in the same manner as τ
does for a simple CSTR. Substitution of the single Monod model for μ results
in
K S (1/θ + b)
SS = (17.50)
µ − (1/θ + b)
The mass balance with respect to soluble substrate for a CSTR with recycle is
Q Q (Q − Qw ) µX B dSS
SSi − SS w − SS − = (17.51)
V V V YX/S dt
Because soluble organic substrate will not be removed by the cell separa-
tion technique, the same concentration will be present in the wastage and
main effluent flow. The mass balance at steady state simplifies to Equation
17.53, which describes the biomass concentration as a function of both the
hydraulic and cell retention times. Influent organic substrate concentration
and hydraulic retention time can be adjusted to achieve the desired cell con-
centration in the reactor.
θ YX/S (SSi − SS )
XB = * (17.52)
τ 1 + bθ
The mass balance with respect to a general product is given below.
Q Q (Q − Qw ) dP
Pi −P w −P + k pg µX B + k pn X B = (17.53)
V V V dt
Product concentrations at steady state will vary according to whether the

product is GA or NGA, and particulate or soluble, as shown below:
P = k pg (1/θ + b) X B τ If product is GA , soluble (17.54)

P = k pg (1/θ + b) X Bθ If product is GA , particulate (17.55)

P = k pn X B τ If product is NGA , soluble (17.56)

P = k pn X Bθ If product is NGA , particulate (17.57)

In general, the specific growth rate and effluent soluble substrate con-
centration will decline as cell retention time increases, and biomass and
nongrowth-associated product concentrations, especially for particulate
products, will increase with increase in cell retention time. In a CSTR with
recycle, a single optimum cell retention time that maximizes the rate of prod-
uct formation for all types of products does not exist. A model of the system
based on mass balance equations may be used to run simulations to deter-
mine the product concentration and rate of product formation at different
hydraulic and cell retention times. System optimization for a biological CSTR
with recycle is a matter of determining the best combination of cell density
and production rates for the system.
A CSTR with biomass recycle inoculated with C. guilliermondii FTI 20037
was shown to achieve xylitol volumetric productivity rates of 0.91 g/L-h at
a hydraulic retention time of 16.7 h (dilution rate of 0.060/h) and essentially
infinite cell retention time (i.e., all cell mass was filtered through 0.01 μm
ultrafilter and continuously recirculated to reactor so only loss of cell mass
was through decay) (Silva, 1999). This productivity was achieved with defined
media containing 30 g/L xylose and no glucose. A 100% recycle of cell mass
is not typically employed in CSTR with recycle designs, since control of the
specific growth rate through wasting of cell mass is usually desired.
17.8.1 Fed-Batch Systems

The important difference of fed-batch compared with batch and CSTR sys-
tems is that the specific growth rate decreases with time due to increasing
volume at constant feed rates (Blanch and Clark, 1997) as shown by
dµ d  Q  −Q 2
=   = (17.58)
dt dt  V0 + Qt  (V0 + Qt )2

where V0 = initial volume.
However, the steady-state operation may be achieved by exponentially
adjusting the feed rate, Q as shown by
dV
= Q = λV (17.59)
dt
which is integrated to obtain
Q = λV0 eλt (17.60)

where Q is the dilution rate or 1/t.

At steady-state operation, the expression for substrate concentration mim-

ics that developed for the CSTR system (Equation 17.40) as shown below
excluding the decay term
1 ˆ SS K S /τ
µ= =µ and SS ≅ (17.61)
τ K S + SS ˆµ − (1/τ)

Fed-batch operation may be represented at steady-state in terms of bio-

mass and product formation excluding decay (Shuler and Kargi, 1992),
respectively, as
X t = X0t + QYBSSit (17.62)

where Xt is the total biomass produced (mg) and
V0 V t 
P = P0 + kpX B  0 +  t (17.63)
V V 2τ 

where P is the product concentration (mg/L).

A fed-batch bioreactor system has inherent advantages of both batch and
continuous systems since the operation is semi-continuous with only sub-
strate feed and no effluent stream. For this reason, these systems are often
employed in many commercial applications within the food and pharmaceu-
tical industry. The system works well for NGA production typically accom-
plished by introduction of secondary metabolite inducers, specific nutrient
deprivation or substrate inhibition at specific times during the process. The
fed-batch system is also applied to form GA and mixed-growth-associated
(MGA) products, such as heterotrophic algal oils by adjusting substrate feed
rate to obtain maximum conversion to oil stored in high cell density culture.
Other common examples where fed-batch systems are employed in com-
mercial settings include baker’s yeast (GA) and penicillin (NGA) produc-
tion. Fed-batch systems have been used to produce xylitol and ethanol from
mixed sugars, glucose, and xylose, in Candida shehatae (Kastner et al., 1992),
Pichia stipitits (Schneider, 1989), and Candida boidinii (Vandeska et al., 1996)
cultures.
17.9 Bioreactor Design Strategies

Bioreactor design may be based on maximizing the rate of product forma-
tion, biomass production or substrate utilization, or on achieving target
concentrations of product, biomass, or substrate. As revealed by the above
rate expressions, rates of substrate, oxygen and nutrient utilization, and GA

product formation will be high when the rate of biomass formation is high,
while the rates of NGA product formation and decay will be high when
the concentration of biomass is high. In general, rates of primary product
formation in a batch reactor will be highest during mid-to-late exponential
phase, while product concentration will be highest at the end of exponen-
tial phase. Secondary product concentration in a batch reactor will peak at
the end of stationary phase. These responses are illustrated in Figures 17.2
and 17.3 for representative GA and NGA products. For a GA, extracellu-
lar, soluble product such as ethanol, a batch reactor or simple CSTR would
achieve high-product formation rate. But for nongrowth-associated prod-
ucts or reactions that form inhibitory products, batch reactors may not be
advantageous due to the low productivity rate or elevated product con-
centrations that may form. Selection of a simple CSTR allows for control
of the specific growth rate through control of the hydraulic retention time
to maximize the rate of a GA product. In a simple CSTR or CSTR with
biomass recycle, the steady-state-specific growth rate is inversely propor-
tional to the hydraulic or cell retention time, respectively, while biomass
concentration is, in general, directly proportional to the retention times.
Therefore, these designs allow for efficient production of either primary
or secondary products by achieving target-specific growth rate and bio-
mass or product concentrations through control of the retention time(s).
Dynamic modeling and simulation of microbial growth and product for-
mation are needed to optimize the design based on predicted production
rates and concentrations.
1: XB 2: GA product 3: NGA product

60
2 2 2
3
3
30
3
1
1
0 1 2 3
0.00 12.50 25.00 37.50 50.00
Time
FIGURE 17.2
Simulations of representative biomass (XB), growth-associated (GA), and non-growth associ-
ated (NGA) product concentrations (g/L) versus time (h) in a batch reactor. (Simulation con-
ducted with STELLA® 8 software.)

1: XB form 2: GA product form 3: NGA product form

20
10 1
3
3
2 3 3
0 1 2 1 2 1 2
0.00 12.50 25.00 37.50 50.00
Time
FIGURE 17.3
Simulations of representative biomass (XB), GA, and NGA product formation rates (g/L-h)
versus time (h) in a batch reactor. (Simulation conducted with STELLA® 8 software.)
For NGA, intracellular products, such as certain lipids produced by fungi

or NGA extracellular, particulate products such as monoclonal antibodies
produced by hybridoma, rates of formation and product concentrations, will
be maximized by a CSTR with biomass recycle design with fairly long cell
retention time, which easily allows for high biomass concentration to be
achieved without exposing cells to inhibitory high substrate concentrations.
Alternating environments with respect to substrate level, electron acceptor,
or nutrients can be used to maximize product formation or selecting desired
biomass cultures (Blanch and Clark, 1997; Grady et al., 1999). For example, high
yields of the vitamin B12 by the bacterium Propionibacterium require micro-
aerobic conditions; however, biosynthesis of a critical enzyme (5,6-dimethyl-
benzimidazole (DMBI)) requires oxygen. Therefore, biosynthesis of vitamin
B12 requires two reactor environments. Martens et al. (2002) proposed the use
of batch reactors with anaerobic conditions for the first 3 days of incubation
for production of the vitamin B12 precursor cobamide, with subsequent aera-
tion for 3 days for synthesis of DMBI and ultimately, vitamin B12. This biopro-
cesses would be a likely candidate for the use of two-stage CSTR system with
anaerobic/aerobic environment for optimizing vitamin production.
Xylitol fermentations are another example of a bioprocess where alternat-
ing environments can be beneficial. Microorganisms, such as Candida, will
exhibit greater specific growth rate and greater biomass yield under aero-
bic conditions, while the desired products, ethanol or xylitol, are produced
only under anaerobic or microaerobic conditions. Bioreactor designs with
alternating environments allow for full utilization of glucose under aero-
bic conditions for biomass production, followed by anaerobic/microaerobic

conditions to allow for ethanol or xylitol production. For example, the use of
a fed-batch, two phase reactor with aerobic growth phase followed by anaer-
obic conditions to produce ethanol with Candida shehatae cultures produced
yield of 0.4 g ethanol/g xylose and 75%–100% conversion of xylose (Kastner
et al., 1999). Using this design scheme, the specific growth rate for Candida she-
hatae under aerobic utilization of d-xylose was μ = 0.32 h−1 producing 2 × 109
cells/mL, while in the second phase, ethanol yield was 0.23 g/g (Kastner
et al., 1999). Final ethanol concentrations (up to 50 g/L) showed greater suc-
cess than the genetically modified strain of Zymomonas mobilis (Chen and
Gongwhich, 1985) result in 30 g/L ethanol. With a semi-continuous pro-
cess, Rodrigues et al. (1998) used Candida guilliermondi to ferment sugarcane
bagasse hydrolysate. A maximum product yield of 0.79 g xylitol/g xylose
was achieved, and a productivity of 0.66 g/L-h. The authors note that the
critical first step is the rapid production of cell mass in the culture medium,
which can be achieved by aerating throughout the first phase, then allowing
oxygen levels to drop to stimulate xylitol production (Rodrigues et al., 1998).
17.9.1 Modeling of Glucose/Xylose Utilization and Product

Formation by Candida
Fermentation of xylose and glucose by Candida in sugarcane bagasse hydro-
lysate is an excellent example of a bioprocess with complex process kinet-
ics illustrates many of the concepts discussed here. First, the sequential use
of glucose then xylose may be modeled as multiple substitutable substrates;
Second, the impact of xenobiotics, such as furfurals, and products, such as
ethanol, that have been identified as inhibitory may be modeled; Thirdly,
the ability of species such as Candida to metabolize multiple sugars under
aerobic, anaerobic, and microaerobic environments, with resulting products
from each, demonstrates the advantage of using multiple phases of batch
or multiple reactors of continuous flow reactors. To illustrate the xylitol
formation by yeasts, such as Candida, simulations of microaerobic batch,
microaerobic CSTR, and two CSTRs in series design, with first CSTR under
aerobic conditions and second CSTR under microaerobic conditions, were
conducted. For simplicity, the modeling used here considered sequential use
of glucose/xylose but assumed no inhibition by substrates, xenobiotics or
products. A typical concentrated sugarcane bagasse hydrolysate composi-
tion of 62 g/L xylose and 8 g/L glucose (Rodrigues et al., 1998) was used for
these simulations. Representative kinetic and yield parameter values used
were: µ̂ = 0.5 h−1 using glucose for full aerobic conditions; f = 0.4 (assumed);
YXylitol/xylose = 0.87 g/g for microaerobic conditions (Kastner et al., 2001), Xylitol
formation was modeled as a nongrowth-associated product, with kpn value
of 0.3 g/g-h estimated from reported data (Kastner et al., 2001). For microaer-
obic conditions, YX/Glu = 0.18 g/g and YX/Xyl = 0.05 g/g were calculated from
data (Kastner et al., 2001), while biomass yield values for aerobic conditions
(YX/Glu = 0.4 g/g and YX/Xyl = 0.2 g/g) were assumed. Mass balance equations

1: Glucose 2: Xylose 3: Xylitol

62 2
31
3
3
1
3
0 3 1 1 2 1 2
0.00 12.50 25.00 37.50 50.00
Time
FIGURE 17.4
Simulations of glucose, xylose, and xylitol concentrations (g/L) versus time (h) in batch fer-
mentation of sugarcane bagasse hydrolysate. (Simulation conducted with STELLA® 8 software.)
were solved simultaneously using a program written with STELLA® soft-

ware (High Performance Systems, version 8.0).
As shown in Figure 17.4, the sequential use of glucose/xylose in a batch
reactor can be modeled, with xylose consumption beginning at approxi-
mately 12 h, after glucose concentration has dropped considerably. Xylitol for-
mation begins with the use of xylose as substrate at approximately 12 h. The
overall form of the concentration profiles matches those presented for batch
fermentations in mixed substrates (Pfeifer et al., 1996; Rodrigues et al., 1998).
Results obtained by Pfeifer et al. (1996) show that complete glucose utilization
occurred by ~10 h of batch fermentation, and complete utilization of xylose
by 30–35 h. Simulations of rates of substrate utilization and product forma-
tion (Figure 17.5) represent the sequential use of glucose/xylose and the lag in
xylitol formation due to nongrowth-associated nature of product formation.
Simulation of substrate and products concentrations (Figure 17.6) for a sin-
gle microaerobic CSTR with hydraulic retention time of 20 h also represents
the sequential use of glucose followed by xylose. Simulated steady-state
conditions are achieved by approximately 50 h. Simulation of substrate and
product concentrations in a two-stage, aerobic/microaerobic CSTR system
with a total system hydraulic retention time of 20 h is shown in Figure 17.7.
Under aerobic conditions in the first reactor, no xylitol would be formed but
high yields of yeast biomass would be achieved. The simulation suggests
that the two-stage CSTR system would achieve greater xylitol concentration
in the final effluent than the single-stage CSTR, through the use of alter-
nating environments to achieve greater product formation. Optimization of
this system would be accomplished by varying hydraulic retention times to
achieve maximum concentration of xylitol.

1: Rate of glucose util 2: Rate of xylose util 3: Rate of xylitol form

10
1
3
3
0 1 2 3 1 2 1 2 3
0.00 12.50 25.00 37.50 50.00
Time
FIGURE 17.5
Simulation of rates of glucose utilization, xylose utilization, and xylitol formation (g/L-h) ver-
sus time (h) in batch reactor using sugarcane bagasse hydrolysate. (Simulation conducted with
STELLA® 8 software.)
1: Glucose 2: Xylose 3: Xylitol

62 2
31
1 3 3
3
0 3 1 1 2 1 2
0.00 12.50 25.00 37.50 50.00
Time
FIGURE 17.6
Simulation of glucose, xylose, and xylitol concentrations (g/L) versus time (h) in CSTR at
hydraulic retention time of 20 h using sugarcane bagasse hydrolysate. (Simulation conducted
with STELLA® 8 software.)

1: Glucose 1 2: Xylose 1 3: Glucose 2 4: Xylose 2 5: Xylitol 2

62 2
4
2
31
5
5
5
1
2
3 5 2
0 1 3 4 1 3 4 1 3 4
0.00 12.50 25.00 37.50 50.00
Time
FIGURE 17.7
Simulations of glucose, xylose, and xylitol concentrations (g/L) versus time (h) in reactors 1
and 2 of a two-stage CSTR system with hydraulic retention time of 10 h per reactor (20 h system
retention time). (Simulation conducted with STELLA® 8 software.)
17.10 Summary
The kinetics of microbial growth, substrate utilization, and product for-
mation can greatly influence the effectiveness of a bioconversion process.
Modeling involves characterization of growth and product formation kinet-
ics and development of mass balance models of system. Dynamic modeling
and simulation of the bioprocess can be used to select, design, and optimize
a bioreactor for production of nutraceutical products.
References
Andrews, J.F. 1968. A mathematical model for the continuous culture of microorgan-
isms utilizing inhibitory substrates. Biotechnology and Bioengineering, 10: 707.
Besson, I. et al. 1997. Pyrazine production by Bacillus subtilis in solid-state fermenta-
tion on soybeans. Applied Microbiology and Biotechnology, 47: 489.
Blanch, H.W., Clark, D.S. 1997. Biochemical Engineering. Marcel Dekker, New York.
Canilha, L. et al. 2003. Use of response surface methodology to evaluate the optimum
environmental conditions for the bioconversion of xylose into xylitol in eucalyptus
hemicellulosic hydrolysate. Journal of Chemical Technology and Biotechnology, 78: 945.
Chen, L.F., Gong, C.S. 1985. Fermentation of sugarcane bagasse hemicellulose hydro-
zylate to xylitol by hydrozylate-acclimated yeast. Journal of Food Science, 50: 226.

Chen, Y.L., Lun, S.-Y. 2001. Biotechnological production of pyruvic acid. Applied
Microbiology and Biotechnology, 57: 451.
Cheng, M.H. et al. 1999. Fungal production of eicosapentaenoic and arachidonic
acids from industrial waste streams and crude soybean oil. Bioresource
Technology, 67: 101.
Cho, Y.J. et al. 2002. Effect of carbon source and aeration rate on broth rheology and
fungal morphology during red pigment production by Paecilomyces sinclairii in
a batch reactor. Journal of Biotechnology, 95: 13.
Counsell, J.M. (ed.) Xylitol. Papers of Int. Symp., by Roche Products Ltd. & Xyrofin
Ltd. London. 5 May 1977.
Dahiya, J.S. 1991. Xylitol production by Petromyces albertensis grown on medium con-
taining D-xylose. Canadian Journal of Microbiology, 37: 14.
Day, A., Sen, K.K., Sardar, D. 1989. Xylitol from jute stick. Research Industrial, 34: 202.
Dominquez-Espinosa, R.M., Webb, C. 2003. Submerged fermentation in wheat sub-
strates for production of Monascus pigments. World Journal of Microbiology and
Biotechnology, 19: 329.
Doran, P.M. 1995. Bioprocess Engineering Principles. Academic Press, Inc. San Diego, CA.
Doyle, A., Griffiths, J.B., Newell, D.G., eds. 1995. Cell & Tissue Culture: Laboratory
Procedures. John Wiley & Sons, New York.
Drawert, F., Barton, H. 1978. Biosynthesis of flavor compounds by microorganisms. 3.
Production of monterpenes by yeast Kluyveromyces lactis. Journal of Agriculture
and Food Chemistry, 26: 765.
Du Preez, J.C. et al. 1987. Temperature profiles of growth and ethanol tolerance of the
xylose-fermenting yeast Candida shehatae and Pichia stipitis. Applied Microbiology
and Biotechnology, 25: 521.
Emodi, A. 1978. Xylitol: Its properties and food applications. Food Technology, 32: 20.
Felipe, M.G.A. et al. 1997. Environmental parameters affecting xylitol production
form sugar cane bagasse hemicellulosic hydrolysate by Candida guilliermondii.
Journal of Industrial Microbiology and Biotechnology, 18: 251.
Gaden, E.L., 1959. Fermentation process kinetics. Journal of Biochemical Microbial
Technology Engineering, 1: 413.
Ghaly, A.E. et al. 2004. Batch propagation of Lactobacillus helviticus for the production
of lactic acid from lactose concentrated cheese whey with microaeration and
nutrient supplementation. World Journal of Microbiology and Biotechnology, 20: 65.
Grady, C.P.L., Daigger, G.T., Lim, H.C. 1999. Biological Wastewater Treatment, 2nd edi-
tion. Marcel Dekker, Inc. New York, NY.
Granstrom, T.B., Izumori, K., Leisola, M. 2007. A rare sugar xylitol. Part II: biotechno-
logical production and future applications of xylitol. Applied Microbiology and
Granstrom, T.B., Takata, G., Tokuda, M., Izumori, K. 2004. A novel and com-
plete strategy for bioproduction of rare sugars. Journal of Bioscience and
Bioengineering, 97: 89.
Grewal, H.S., Kalra, K.L. 1995. Fungal production of citric acid. Biotechnology Advances,
13: 209.
Grootjen, D.R.J. et al. 1991. Reactors in series for the complete conversion of glu-
cose/xylose mixtures by Pichia stipitis and Saccharomyces cerevisiae. Enzyme and
Microbial Technology, 13: 828.
Haddadin, M.S.Y. et al. 2001. Utilisation of tomato pomace as a substrate for the pro-
duction of vitamin B12—A preliminary appraisal. Bioresource Technology, 78: 225.

Harkonen, M., Nuojua, P. 1979. Eri tekijoiden vaikutus ksyloosin katalyyyttiseen

hydraukseen ksylitoliksi. Kemia-Kemi, 6: 445.
Henze, M., Grady, C.P.L., Jr., Gujer, W., Marais, G.V.R., Matsuo, T. 1987. A general
model for single-sludge wastewater treatment systems. Water Research, 21:
505.
Horitsu, H., Yahashi, Y., Takamizawa, K., Kawai, K., Suzuki, T., Watanabe, N. 1992.
Production of xylitol from D-xylose by Candida tropicalis: Optimization of pro-
duction rate. Biotechnology and Bioengineering, 40: 1085.
Hsiao, H.Y., Chiang, C.L., Ueng, P.P., Tsao, G.T. 1982. Sequential utilization of mixed
monosaccharides by yeasts. Applied Environmental Microbiology, 43: 840.
Hu, Z-C. et al. 2005. Effect of sugar-feeding strategies on astaxanthin production by
Xanthophyllomyces dendrorhous. World Journal of Microbiology and Biotechnology,
21: 771.
Kastner, J.R. et al. 1992. Viability of Candida shehatae in D-xylose fermentations with
added ethanol. Biotechnology and Bioengineering, 40: 1282.
Kastner, J.R., Eiteman, M.A., Lee, S.A. 2001. Glucose repression of xylitol production
in Candida tropicalis mixed-sugar fermentations. Biotechnology Letters, 23: 1663.
Kastner, J.R., Eiteman, M.A., Lee, S.A. 2003. Effect of redox potential on stationary-
phase xylitol fermentations using Candida tropicalis. Applied Microbiology and
Kastner, J.R., Jones, W.J., Roberts, R.S. 1999. Ethanol fermentation of mixed-sugars
using two-phase, fed-batch process: Method to minimize D-glucose repression
of Candida shehatae D-xylose fermentations. Journal of Industrial Microbiology and
Lehman, J.T., Botkin, D.B., Likens, G.E. 1975. The assumptions and rationale of
a computer model of phytoplankton population dynamics. Limnology and
Oceanography, 20: 343.
Makinen, K.K. 1978. Biochemical Principles of the Use of Xylitol in Medicines and Nutrition
with Special Consideration of Dental Aspects. Birkhauser Verlag, Basel.
Martens, J.H. et al. 2002. Microbial production of vitamin B12. Applied Microbiology and
Martinez, E.A., Silva, S.S., Silva, J.B., Solenzal, A.I., Felipe, M.G. 2003. The influence
of pH and dilution rate on continuous production of xylitol from sugarcane
bagasse hemicellulose hydrolysate by C. guilliermondii. Process Biochemistry,
38: 1677.
Mayilvahanan, D. et al. 1996. Citric acid productions: Part 1: Strategies for reduction
in cycle time for targeted yields. Bioprocess Engineering, 15: 323.
Melaja, A., Hamalainen L., Heikkila H.O. Menetelma Ksylitolin suhteen rikastuneen
polyolin vesiliuoksen valmistamiseksi, FI 589388, 1981 (Finish patent).
Meyrial, V., Delgenes, J.P., Moletta, R., Navarro, J.M. 1991. Xylitol production from
D-xylose by Candida guillermondii. Biotechnology Letters, 13: 281.
Monod, J. 1949. The growth of bacterial cultures. Annual Review of Microbiology, 3: 371.
Mussatto, S.I., Roberto, I.C. 2003. Xylitol production from high xylose concentration:
Evaluation of the fermentation in bioreactor under different stirring rates.
Journal of Applied Microbiology, 95: 331.
Nigam, P., Singh, D. 1995. Processes for fermentative production of xylitol—A sugar
substitute process. Biochemistry, 30: 117.
Oh, D.-K., Kim, S.-K. 1998. Increase of xylitol yield by feeding xylose and glucose in
Candida tropicalis. Applied Microbiology and Biotechnology, 50: 419.

Onishi, H., Suzuki, T., Onchi, T. 1980. Mechanism of fermentation conversion from
polyalcohol fermentation to ethanol by Pichia miso. Agricultural and Biological
Chemistry, 44: 1829
Parajo, J.C., Dominguez, H., Dominguez, J.M. 1998. Biotechnological production of
xylitol. Part-3: Operation in culture media made from lignocellulose hydroly-
sates. Bioresource Technology, 66: 25.
Paraszkiewics, K., Kanwal, A., Dlugonski J. 2002. Emulsifier production by steroid
transforming filamentous fungus Curvularia lunata. Growth and product char-
acterization. Journal of Biotechnology, 92: 287.
Pazouki, M. et al. 2000. Comparative studies on citric acid production by Aspergillus
niger and Candida lipolytica using molasses and glucose. Bioprocess Engineering,
22: 353.
Perego, P., Converti, A., Palazzi, E., Del Borghi, M., Ferraiolo, G. 1990. Fermentation of
hardwood hemicellulose hydrolyzate by Pachysolen tannophilus, Candida sheha-
tae, and Pichia stipitis. Journal of Industrial Microbiology, 6: 157.
Pessoa, A., Mancilha, I.M., Sato, S. 1996. Cultivation of Candida tropicalis in sugar
cane hemicellulosic hydrolyzate for microbial protein production. Journal of
Pfeifer, M.J. et al. 1996. Effect of culture conditions on xylitol production by Candida
guilliermondii FTI 20037. Applied Biochemistry and Biotechnology, 57: 423.
Pollar, S., Muller, E. 1986. Experiments for the extraction of xylitol from beech wood
residues under simple conditions. Chemische Technik, 37: 517.
Priefert, H., Rabenhorst, J., Steinbuchel, A. 2001. Biotechnological production of van-
illin. Applied Microbiology and Biotechnology, 56: 296.
Reeves, E.G.M. 2004. Kinetic analysis of Kluyveromyces marxianus yeast strain, M.S.
Thesis, Louisiana State University, Baton Rouge, LA.
Roberto, I.C., Mancilha, I.M., Sato, S. 1999. Kinetics of xylitol fermentation by Candida
guilliermondii grown on rice straw hemicellulosic hydrolysate. Journal of Applied
Biochemistry and Biotechnology, 77: 205.
Roberto, I.C., de Mancilha, I.M., Taqueda, M.E.S. 1995. Influence of media compo-
sition of xylitol fermentation by Candida guiliiermondii using response surface
methodology. Biotechnology Letters, 17: 1223.
Rodrigues D.C.G.A., Silva, S.S., Prata, A.M.R., Felipe, M.G.A. 1998. Biotechnological
production of xylitol from agroindustrial residues. Applied Biochemistry and
Sato, T. et al. 2001. Production of menaquinone (Vitamin K2)-7 by Bacillus subtilis.
Journal of Bioscience and Bioengineering, 91: 16.
Schneider, H. 1989. Conversion of pentoses to ethanol by yeasts and fungi. Critical
Reviews in Biotechnology, 9: 2.
Scurlock, J., Bioenergy feedstock characteristics. Oak Ridge National Laboratory
Publication. http://bioenergy.ornl.gov/papers/misc/biochar_factsheet.html
Sene, L. et al. 2001. Preliminary kinetic characterization of xylose reductase and x
ylitol
dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in
sugarcane bagasse hydrolysate for xylitol production. Applied Biochemistry and
Shepherd, R. et al. 1995. Novel bioemulsifiers from microorganisms for use in foods.
Journal of Biotechnology, 40: 207.
Shuler, M.L., Kargi, F. 1992. Bioprocess Engineering. Prentice Hall PTR, Englewood
Cliffs, NJ.

Sijtsma, L., de Swaaf, M.E. 2004. Biotechnological production and applications of the
omega-3 polyunsaturated fatty acid docosahexaenoic acid. Applied Microbiology
and Biotechnology, 64: 146.
Silva, A.A. et al. 1997. Maximizing the xylitiol production from sugar cane bagasse
hydrolysate by controlling the aeration rate. Applied Biochemistry and Biotechnology,
63: 557.
Silva, S. et al. 1996. Batch fermentation of xylose for xylitol production in stirred tank
bioreactor. Process Biochemistry, 31: 549.
Silva, S.S. 1999. The preliminary information about continuous fermentation
using cell recycling for improving microbial xylitol production rates. Applied
Biochemistry and Biotechnology, 77: 571.
Simkins, S., Alexander, M. 1985. Nonlinear estimation of the parameters of Monod
kinetics that best describe mineralization of several substrate concentrations
by dissimilar bacterial densities. Applied and Environmental Microbiology, 50: 816.
Singh, D., Kumari, P., Dahiya, J.S. 1990. Xylitol production from sugar cane bagasse
by fermentation. In Modernisation of the Indian Sugar Industry, ed. J. K. Gehlawat.
Arnold Publishers, New Delhi, 292.
Steele, J.H. 1962. Environmental control of photosynthesis in the sea. Limnology and
Oceanography, 7: 137.
van Dam-Mieras, M.C.E., de Jeu, W.H., de Vries, J., Currell, B.R., James, J.W., Leach,
C.K., Patmore, R.A. 1992. Bioreactor Design and Product Yield. Butterworth-
Heinemann, Boston, MA.
Van Eys, J., Wang, Y.M., Chan, S., Tanphaichitr, V.S., King, S.M. 1974. In Sugars in
Nutrition, ed. H.L. Sipple & K.W. McNutt. Academic Press, New York, 613.
Vanderhaegen, B. et al. 2003. Bioflavoring and beer refermentation. Applied
Microbiology and Biotechnology, 62: 140.
Vandeska, E. et al. 1996. Fed-batch culture for xylitol production by Candida boidinii.
Process Biochemistry, 31: 265.
Virginio Da Silva, D.D. et al. 2005. Evaluation of inoculum of Candida guilleriermon-
dii grown in presences of glucose on xylose reductase and xylitol dehydroge-
nase activities and xylitol production during batch fermentation of sugarcane
bagasse hydrolysate. Applied Biochemistry and Biotechnology, 121: 427.
Walker, T.H., Cochran, H.D., Hulbert, G.J. 1999. Supercritical carbon dioxide extrac-
tion of lipids from Pythium irregulare. Journal of American Oil Chemists’ Society,
76: 595.
Walther, T., Hensirisak, P., Agblevor, F.A. 2001. The influence of aeration and
hemicellulosic sugars on xylitol production by Candida tropicalis. Bioresource
Technology, 76: 213.
Wang, F., Lee, S.Y. 1997. Poly(3-hydroxybutyrate) productions with high productiv-
ity and high polymer content by a fed-batch culture of Alcaligenes latus under
nitrogen limitation. Applied and Environmental Microbiology, 63: 3703.
Welch, E.B. 1980. Ecological Effects of Waste Water. Cambridge University Press,
Cambridge, Great Britain.
Wen, Z.Y., Chen, F. 2003. Heterotrophic production of eicosapentaenoic acid by
microalgae. Biotechnology Advances, 21: 273.
Witt, I., Heilmeyer, L. 1966. Regulation of pyruvate decarboxylase synthesis by coen-
zyme induction in Saccharomyces cerevisiae. Biochemistry Biophysics Research
Communication, 25: 340.

Yang, Y. et al. 2004. Continuous methane fermentation and the production of vitamin
B12 in a fixed-bed reactor packed with loofah. Bioresource Technology, 92: 285.
Yashashi, Y. et al. 1996. Production of xylitol from D-xylose by Candida tropicalis: The
effect of D-glucose feeding. Journal of Fermentation and Bioengineering, 81: 148.
Yu, L.J., Qin, W.M., Lan, W.Z., Zhou, P.P., Zhu, M. 2003. Improved arachidonic acids
production from the fungus Mortierella alpina by glutamate supplementation.
Bioresource Technology, 88: 265–268.
Zehentgruber, O., Kominek, J., Salzbrun, W. 1985. Citric acid for beverages: A new
economic submerged fermentation process. World Biotechnology Report, 1: 687.
Zhang, C. et al. 1999. Aerobic biodegradation kinetics of four anionic and nonionic
surfactants at sub- and supra-critical micelle concentrations (CMCs). Water
Resources, 33: 115.
Zhang, Z. et al. 2004. Study on the methane fermentation and production of Vitamin
B12 from alcohol waste slurry. Applied Biochemistry and Biotechnology, 113: 1033.
Zhu, H., Drapcho, C.M., Walker, T.H. 2001. Bioconversion of rice bran to omega-3
fatty acids of Pythium irregulare submerged culture. ASAE Paper 01–7020.
Zhu, M., Zhou, P.P., Yu, L.J. 2002. Extraction of lipids from Mortierella alpina and enrich-
ment of arachidonic acid from fungal lipids. Bioresource Technology, 84: 93.

Section IV
Stability and Bioactivity of

Antioxidative Components
during Food Processing

18
Dehydration Technologies for
Functional Foods and Nutraceuticals
Robert V. Parsons and Stefan Cenkowski
CONTENTS
18.1 Introduction..............................................................................................546
18.2 Functional Foods, Nutraceuticals, and Bioactivity..............................546
18.3 Dehydration Technology Principles and Options............................... 547
18.4 Dehydration Kinetics............................................................................... 550
18.5 Thermal Degradation Reactions............................................................ 553
18.5.1 Enzyme/Protein Denaturation................................................... 554
18.5.2 Microbial Inactivation.................................................................. 554
18.5.3 Enzymatic Degradation............................................................... 555
18.5.4 Maillard Reaction......................................................................... 555
18.5.5 Caramelization.............................................................................. 556
18.5.6 Oxidation....................................................................................... 556
18.6 Engineering and Physical Properties.................................................... 557
18.7 Predrying and Water Removal............................................................... 560
18.8 Atmospheric Drying................................................................................ 562
18.8.1 Spray Drying................................................................................. 562
18.8.2 Tray Drying...................................................................................564
18.8.3 Fluid Bed and Jet Spouted Bed Drying..................................... 565
18.9 Freeze Drying/Lyophilization............................................................... 567
18.10 Superheated Steam Drying..................................................................... 568
18.11 Microwave Drying................................................................................... 569
18.12 Vacuum Drying........................................................................................ 570
18.13 Heat-Pump Drying................................................................................... 571
18.14 Osmotic Dehydration............................................................................... 571
18.15 Solar Drying.............................................................................................. 572
18.16 Hybrid Drying.......................................................................................... 572
18.17 Optimizing Technology Selection......................................................... 574
18.18 Future Directions..................................................................................... 574
Acknowledgments............................................................................................... 575
References.............................................................................................................. 575
545
18.1 Introduction
Through the course of producing functional foods or nutraceuticals, as
described throughout this book, it is often desirable or necessary to employ
dehydration. Although the terms “dehydration” and “drying” are often used
interchangeably, there are subtle differences that can be meaningful in cer-
tain cases. Dehydration is a broader term for the removal of water from any
given material via any medium, whereas drying more specifically refers to
the removal of liquid from a solid material by evaporation. In either case, the
end point is generally a dry final product material with low moisture content
and low water activity (aw).
The intent of this chapter is not to comprehensively review the current
status of dehydration and drying. Rather, it is to provide more up-to-date
information on dehydration technologies that are specifically relevant for
functional foods and nutraceuticals. Essential details are provided, includ-
ing a number of important older references, but in general, the focus is on
recent applications. A key observation by Betoret et al. (2011) has been that so
far the overall emphasis in terms of technologies is a reliance of existing pro-
cesses, or, secondarily, finding protection against detrimental effects, rather
than trying to create new technologies. As is obvious from this chapter, their
observation is borne out with respect to dehydration.
18.2 Functional Foods, Nutraceuticals, and Bioactivity

Functional foods and nutraceuticals have become increasingly prominent
over the last two decades, but in terms of nomenclature, they have never
been defined in an entirely precise or consistent manner. As outlined by
Doyon and Lebreque (2008) for functional foods and by Palthur et al. (2010)
for nutraceuticals, at least 25 different definitions can be put forward for each
term. The intricacies of functional foods and nutraceuticals are discussed
elsewhere in this book. For the purposes of this chapter, the relatively simple
but broad definitions put forth early on by Health Canada (1998) are sufficient.
Both functional foods and nutraceuticals can be described as sharing the
characteristic of having demonstrable health-related benefits beyond basic
nutritional functions, either in terms of physiology or in terms of reduction
of risks from or protection against chronic diseases. Whereas functional
foods are similar in appearance to, or indeed may be, conventional foods con-
sumed as part of a usual diet, nutraceuticals involve isolation or purification
of some kind. They are more generally sold in medicinal forms, not neces-
sarily directly associated with foods. For the sake of brevity, functional foods
and nutraceuticals are often described as just “products” in this chapter.

Dehydration Technologies for Functional Foods and Nutraceuticals 547
Functional foods and nutraceuticals are generally associated with plant-

based phytochemicals, but not exclusively. Nonplant examples include
fish-oil-derived omega-3 fatty acids and cultures of functional microorgan-
isms known as probiotics. The plant-based origins of phytochemicals are
important to note, as well as the large number of phytochemicals known to
exist. An earlier compendium by Harborne and Baxter (1993) listed roughly
3000 different identified constituents, grouped into five main classes of
compounds: carbohydrates; nitrogen-containing compounds, excluding
alkaloids; alkaloids; phenolics; and terpenoids. Liu (2003) indicated a rough
increase to more than 5000 known phytochemicals by roughly a decade
later. More recently, DeLuca et al. (2012) suggest that both the number and
importance of phytochemicals for health-related applications have contin-
ued to expand.
One group of constituents that has received significant recent attention is
antioxidants, that is, compounds that can delay, inhibit, or prevent oxida-
tion by scavenging free radicals and diminishing oxidative stress (Dai and
Mumper, 2010). These tend to be primarily, but not exclusively, polypheno-
lics. The latter include compounds within five chemical categories: cinnamic
acids, chalcones, flavonoids, procyanidins, and anthocyanins (Stevenson
and Hurst, 2007), again a broad group of constituents.
The possession of some sort of beneficial bioactive effect is common to
all functional foods and nutraceuticals, but beyond that they are highly
diverse in nature, in particular regarding their structural form and chemi-
cal makeup. They can involve protein, carbohydrate, phenolic or lipid con-
stituents, or some combination. They may be more solid in nature or may be
present in solution. They may be strongly bound to other constituents or not.
They may be soluble in water or not. They may be temperature sensitive and
readily subject to degradation or not. As described throughout this chapter,
there are no general rules of thumb regarding their characteristics. This has
important implications in that there is no single best way to undertake dehy-
dration. Selection of a suitable process is specific to each case.
18.3 Dehydration Technology Principles and Options

Unlike functional foods and nutraceuticals, which are modern product con-
cepts, various forms of dehydration and drying processes have been already
well developed. At the same time, dehydration technologies are extremely
diverse. There are literally more than 100 different variants of industrial dry-
ing technologies alone, as well as many new technology developments.
For the vast majority of cases, dehydration involves the removal of water
to produce a sufficiently dry product, although in some special cases, it
can involve the removal of nonaqueous solvents. Moisture content is thus

important, and for dehydration and drying, it is typically expressed on a

mass basis rather than on a volumetric basis. Mass-based moisture content
(MC) can further be expressed in two different ways. The first is on a wet
basis (MCwb), which is calculated according to Equation 18.1, and is frequently
used for agricultural and food products. The second is on a dry basis (MCdb),
which is calculated according to Equation 18.2. The MCdb value is typically
used in drying operations, specifically for the reason that it varies linearly
with the mass of water present in the material. On the other hand, MCwb var-
ies nonlinearly. These two bases can be interrelated, but their slight differ-
ences need to be acknowledged.
masswater masswater
MCwb = = (18.1)
masswater + massdry matter masstotal

masswater
MCdb = (18.2)
massdry matter

The final moisture content is typically specified for each particular prod-
uct, based on necessary quality requirements, and this determines the extent
of dehydration that will be required. The change in moisture content from
the feed material to the product, combined with mass flow rate of material,
determines the total water removal requirements, which essentially dictates
the dehydration capacity required. There is no single correct method for
determining moisture content, with a variety of moisture content measure-
ment standards available. Selecting the appropriate standard depends on
both the specific product and the specific market location.
A number of different driving forces can be employed to remove water
(or other solvent) away from the feed material. All are based primarily
on partial pressure differences for water. Most drying systems employ
t hermal-based evaporation. For conventional atmospheric drying, the driv-
ing force is related to psychrometry, that is, the ability of a heated air-flow
to carry away significantly increased quantities of water vapor (Devres,
2004). In the case of superheated steam drying (SSD), which is still ther-
mally driven but with no air-flow, the driving force is de-superheating of
steam. For vacuum-based drying systems, it is still the partial pressure
of water, but based on reducing the partial pressure externally to create
a driving force, typically by mechanical means rather than by thermal
means. For freeze drying, or lyophilization, the vacuum of systems again
creates the driving force, but water is removed via sublimation rather than
by evaporation. In the case of radio-frequency or microwave-based drying,
it is the selected excitation of water molecules, leading to elevated partial
pressure. In the case of osmotic dehydration, it is osmotic pressure, again
related to the partial pressure of water.
Irrespective of the mechanism employed, the movement of water out of
a feed material being dehydrated or dried under batch conditions tends to

Moisture content (db) of sample

(generic)
Time of batch operation (generic)
FIGURE 18.1
Generic dehydration curve for sample processed under batch conditions.
follow a reasonably consistent pattern, as illustrated in Figure 18.1. The key

common characteristic involves an initial period of more rapid d ehydration
followed by a subsequent period of slowed dehydration. The curve in
Figure 18.1 is discussed further in terms of drying kinetics in the next section.
Additional insights can be derived from the same generic dehydration
curve by recalculating the same data and considering the rate of moisture
removal, first with respect to time, as illustrated in Figure 18.2a, and second
as a function of MCdb, as illustrated in Figure 18.2b. These more clearly illus-
trate an essentially constant-drying rate period followed by a falling-rate
drying period, with the transition occurring at the critical moisture content
(MCcrit), which is a function of the particular material involved. Conventional
atmospheric drying absolutely follows this precise pattern. The dramatic
change in the evaporation rate is understood to be caused by a transition
from surface evaporation to diffusional drying, where water molecules first
have to diffuse to the surface before being evaporated. For other drying pro-
cesses, the curves may not precisely follow the same characteristic, but are
still very similar, as discussed later.
Dehydration, as alluded to in the previous discussion, is by its nature
extremely complex. It typically involves simultaneous heat transfer and
mass transfer, physical and chemical transformations, and transient opera-
tions. For a more complete overview of dehydration technologies and their
associated complexities, a useful starting point is provided by Kudra and
Mujumdar (2009), Mujumdar and Law (2010), and Jangam and Mujumdar
(2013).
Dehydration can be an expensive process, in terms of both equipment
costs and ongoing energy costs, but one for which there are no simple alter-
natives. Three major categories of benefits can be associated with dehydra-
tion in relation to foods in general, which are also relevant to functional
foods and nutraceuticals. These are (i) enhanced preservation, in terms of
product safety from detrimental microorganism, and longevity; (ii) reduced

(a)
Rate of moisture removal (generic)

Constant rate
period
Falling rate period
Time of batch operation (generic)
(b)
Rate of moisture removal (generic)
Falling rate Constant rate period

period
Critical moisture content

(MCcrit)
Moisture content (db) of sample (generic)
FIGURE 18.2
Generic dehydration curve in terms of rate of moisture removal. (a) Rate of moisture removal
as function of time. (b) Ratio of moisture removal as function of moisture.
product mass/volume, in terms of simplified storage requirements and

reduced transportation costs; and (iii) enhanced product consistency, in
terms of simplified material handling and uniformity in any subsequent
reformulations.
18.4 Dehydration Kinetics

Dehydration kinetics relate to the rate of moisture removal from a feed mate-
rial, that is, how quickly a material will dry. The nature of drying kinetics deter-
mines the time duration required for a dehydration process to reach the desired

level of dryness for the final product. As discussed in the previous section,
dehydration involves two distinct characteristic time frames, an initial period
of more rapid (and typically constant) rate of moisture removal, followed by a
remaining period involving reduced (and typically declining) rate of moisture
removal. This was outlined in the generic example curve in Figure 18.1.
The declining moisture content can be expressed mathematically. For the
declining rate period, it is typically in the form of a first-order differential
equation, as outlined in Equation 18.3, with the rate of moisture removal at
any time (t) proportionate to the instantaneous mass of moisture present,
with an associated rate constant (k):
d( MCdb )
= − k( MCdb ) (18.3)
dt
Note that in some papers the term MCdb is represented as just M or W as a

practical means to reduce the number of subscripts required. The standard
first-order kinetic relationship in Equation 18.3 can be readily solved to show
an overall negative exponential decline in sample moisture content as out-
lined in Equation 18.4:
MCdb
= exp(− kt) (18.4)
( MCdb )initial
A negative exponential model form of some kind is frequently used to

describe experimental drying or dehydration data. At the same time, more
than a dozen slightly different specific mathematical models have been cited
for use with data. These models include Lewis/Newton (simple exponential);
Page; modified Page; Henderson and Pabis (biparametric exponential); mod-
ified Henderson and Pabis; logarithmic; logistic; two term; two-term expo-
nential; sigmoidal; Wang and Singh; Midilli-Kucuk; Verma; and Hii (Midilli
and Kucuk, 2003; Simal et al., 2005; Hii et al., 2009; Bal et al., 2010, CalÍn-
Sanchez et al., 2014). In practice, no single model is obviously preferable to
any other in terms of data fitting for all cases. That said, in order to avoid
problems associated with overfitting, the model of Page (1948, unpublished,
as cited in CalÍn-Sanchez et al., 2014) represents a useful starting point for
data analysis. This model is outlined in Equation 18.5, expressed in terms of
the dimensionless moisture ratio:
MCdb − ( MCdb )equil (18.5)

= exp(− kt n )
( MCdb )initial − ( MCdb )equil

Common with all kinetic drying models, the equilibrium moisture con-
tent ((MCdb)equil) is introduced into the dimensionless ratio. This is given that
residual moisture content will never reach zero, but instead will approach an
equilibrium level for the given conditions. A power-law exponent (n) is also

added to the drying time (t). The latter is strictly an empirical modification to
achieve better fit of data, and has no fundamental basis.
Individual drying tests are undertaken based on constant conditions, par-
ticularly temperature. The relationship between temperature (T) and the
drying rate constant (k), based on multiple test runs at different conditions,
is expressed through the Arrhenius equation, presented in Equation 18.6,
involving the universal gas constant (R), a general constant (a), and the acti-
vation energy (Ea):
 E 
k = a exp −  a  (18.6)
 RT 

Reviewing data of six recent studies on drying kinetics of relevant prod-

ucts using a variety of models (Xiao et al., 2010; Ioannou et al., 2011; Ahmad-
Qasem et al., 2013, Vega-Galvez et al., 2014, Wu et al., 2014; Zhu and Shen,
2014) shows activation energies ranging from 15 to 67 kJ per mol, with a mean
value of 40 ± 18 kJ per mol. The latent heat of evaporation for water is well
known, and tends to decline from about 45 kJ per mol at 0°C down to about
41 kJ per mol at 100°C. Not surprisingly, activation energy values cluster rela-
tively consistently around the value of the latent heat, albeit with some vari-
ability, in part due to different products and different models. These values
are also relatively consistent with broader results from convective air-drying
tests on 18 different food materials summarized by Kholmanskiy et al. (2013).
Their data shows activation energy to range from 22 to 83 kJ per mol, with a
mean value of 48 ± 14 kJ per mol.
Kinetic models provide useful information but also tend to make drying
or dehydration operations appear simple, masking underlying complex-
ity. As outlined by Simal et al. (2005), there are four prevailing transport
phenomena in drying operations: internal heat transfer; external heat trans-
fer; internal mass transfer; and external mass transfer. And, further, these
transport phenomena are occurring simultaneously. Given that diffusive
internal mass transfer tends to become the limiting factor during the fall-
ing-rate drying period, a natural extension of initial kinetic modeling is to
attempt to evaluate effective moisture diffusivity. This is described later in
Section 18.6 regarding engineering and physical properties.
A last important implication of drying kinetics for conventional atmo-
spheric drying is that during the falling-rate period, the external temper-
ature of the air-flow must keep increasing in order to maintain adequate
driving force to continue moisture removal. It is thus this last component of
conventional drying that is most dangerous to consider for thermal-sensitive
products, as discussed in the next section. This situation also leads to the
notion for functional foods and nutraceuticals that one single drying tech-
nology may be not necessarily preferred. Combining different drying tech-
nologies is a useful potential solution to avoid excessive thermal stress on
sensitive products.

18.5 Thermal Degradation Reactions

The maintenance of bioactivity is critical in the dehydration of any product
intended as a functional food or nutraceutical. Bioactivity can be adversely
impacted in a variety of different ways (Lee and Ho, 2002), but a particular
concern during dehydration is degradation due to elevated t emperatures.
The starting point for selection of a suitable dehydration process is to under-
stand how and to what extent bioactive constituents may be impacted.
Nutrient loss and formation of adverse contaminants have been both
well recognized as general problems in dehydration and drying of foods
and, in the latter case, certain regulatory restrictions on
processing
conditions depending on jurisdiction (Benali, 2004).
Considerations for individual functional foods or nutraceuticals are
more specific. Importantly, bioactive constituents outlined earlier in
Section 18.2 are not uniform in their sensitivity. Six different categories of
reactions can be identified that could lead to reductions in desired bioac-
tivity at elevated temperatures. These are (i) enzyme/protein denaturation,
involving the irreversible denaturing of desirable proteins, particularly
enzymes; (ii) microbial inactivation, involving the inactivation of desirable
microbes; (iii) enzymatic degradation, involving accelerated undesirable
degradation of constituents by enzymes that may be present, this includ-
ing enzymatic browning reactions; (iv) Maillard reaction, involving non-
enzymatic browning reactions of reducing sugar(s) with amino acid(s); (v)
caramelization, involving nonenzymatic browning reactions of reducing
sugar(s); and (vi) oxidation, involving a variety of product-specific oxida-
tive breakdown reactions. Each of these is described in more detail in sub-
sequent sections.
At the same time, there are a number of generally common features of
these degradation reactions. First, although the reactions outlined can lead
to negative consequences, sometimes their results can be beneficial, such as
for the creation of antioxidant compounds through the Mallaird reaction,
discussed later. This apparent contradiction is present in all cases.
Second, degradations are usually reasonably characterized, mathemati-
cally, as first-order reactions in the form of a differential equation. In this
case, the rate of disappearance of the bioactive as a function of time (t) is
proportional to the instantaneous bioactive concentration (C), represented in
Equation 18.7 with reaction rate constant (k):
dC
= −k C (18.7)
dt
This is the same general relationship as shown earlier in Equation 18.3 for
drying rate. Upon integration, it also leads to a classical exponential decline

equation over time, based on maintained consistent conditions with initial

bioactive concentration (C0), as represented by Equation 18.8:
C
= exp(− k t) (18.8)
C0
In the absence of any other data, a first-order reaction assumption is a

reasonable starting point; however, not all reactions will necessarily follow
first-order kinetics. As such, experimental confirmation is necessary. Third,
temperature dependency of degradation reactions is also generally well
described through the application of the Arrhenius equation, presented ear-
lier in Equation 18.6, in terms of the degradation reaction rate constant, just
as in the case of the drying rate constant. Putting together the Arrhenius
relationship with the degradation rate equation indicates that the extent of
degradation for a given case will depend not only on the nature of the prod-
uct, but also significantly on how high is the temperature and how long is the
exposure time. Lastly, it is generally understood that success in mitigation
tactics to enhance bioactive stability during the course of a given dehydra-
tion process depends on the specific product and process involved.
18.5.1 Enzyme/Protein Denaturation

Bioactive proteins, in particular enzymes, are subject to thermal degrada-
tion. This is characterized as initially involving a reversible mechanism that
subsequently progresses to irreversible (Cornish-Bowden, 2012). Irreversible
degradation is termed denaturation, and is often considered to result physi-
cally from inappropriate folding of proteins that in turn disrupt highly ste-
reospecific active sites. The mathematics of enzyme degradation is more
complex than the simple models outlined above (Cornish-Bowden, 2012). In
practical terms, increasing temperature causes an increase in enzyme activ-
ity, but as the temperature continues to increase, enzyme activity peaks and
then declines, that is, overtaken by reversible (and irreversible) degradation.
Limiting temperatures for enzyme denaturation tend to be in the range of
60–100°C, depending on the specific enzyme, and when water activity is
reduced to around 0.2 (Van Den Burg, 1986).
18.5.2 Microbial Inactivation

The deliberate inactivation of detrimental microbes is important in nor-
mal food processing to ensure safety and preserve quality (VanGarde and
Woodburn, 1994). However, if it is desired to have active organisms within
a final product, for example, as in the case of emergent probiotic products
(Granato et al., 2012), dehydration requirements become trickier, given the
need to selectively maintain viability. The nature of microbial death due to
various inactivation methods, including thermal processes, is outlined in

detail by Casolari (1988). A series of more advanced inactivation methods

are covered by Manas and Pagan (2005), that is, irradiation, ultrasonics, high
pressure, and pulsed electric fields. Thermal inactivation, which is most
relevant in this case, generally follows classic first-order characteristics as
outlined earlier (i.e., exponential decline). However, in practice, microbial
survivor curves can vary significantly depending on specific conditions and
organisms, necessitating confirmation. In terms of potential stabilization, a
variety of options considered specifically for probiotic products are outlined
by Goderska (2012).
18.5.3 Enzymatic Degradation

As noted earlier, increased temperature will lead to increased enzyme activ-
ity. A variety of enzymes can be present incidentally within a processed food
product stream. At elevated temperatures, these can lead to accelerated con-
versions that may be unintended or even detrimental, including effects on
bioactive constituents. This is one aspect of the generic effect termed enzy-
matic browning. The kinetics of an enzyme-mediated conversion is gener-
ally described by the Michaelis–Menten equation (Cornish-Bowden, 2012).
When a susceptible substrate is present at small concentrations, degradation
can be described as first-order with respect to the substrate, corresponding
to the simple relationships noted earlier. If the susceptible substrate is pres-
ent at large concentrations, the relationship approximates zero order. This is
indicative of enzyme saturation, with a constant maximum rate of conver-
sion. A cautionary note is that it has been known for some time that not all
enzyme systems necessarily follow Michaelis–Menten kinetics (Bailey and
Ollis, 1977).
18.5.4 Maillard Reaction

The Maillard reaction is not a single reaction as implied by its name, but
rather a complex network of interrelated reactions (O’Brien et al., 1998). It
involves constituents having a carbonyl group, typically reducing sugars
whether in aldose or ketose form, reacting with constituents having an
amino group, typically amino acids from proteins, although other types
of constituents can also be involved. The Maillard reaction sequence is pri-
marily understood in terms of brown pigment development and associated
product color change at elevated temperatures, being a well-identified form
of nonenzymatic browning. It can also positively lead to antioxidant forma-
tion. At the same time, this reaction sequence can lead to negative product
degradations or the formation of negatively impacting chemical species. For
example, the formation of the toxicant acrylamide in food products at ele-
vated temperatures is related to the Maillard reaction (Mottram et al., 2002).
The Maillard reaction is notoriously difficult to control, being both strongly
temperature dependent, beginning to initiate rapidly at temperatures in the

range of about 140–160°C, and pH dependent. Given its complexity, analyses

of kinetics for the Maillard reaction sequence show significant variability. It
cannot be neatly quantified as being of any particular simple reaction order
(Martins et al., 2001).
18.5.5 Caramelization
Caramelization, like the Maillard reaction, represents a well-identified form
of nonenzymatic browning in food products, and also involves not a single
reaction but a series of related reactions (Eskin et al., 2013). While caramel-
ization and Maillard reaction can occur simultaneously, their mechanisms
are distinct. Caramelization involves reactions of reducing sugars when
heated above the melting point, typically at higher temperatures than the
Maillard reaction, and involves no amino group reactions. Fructose, with
a lower melting point than other sugar monomers, is generally more sus-
ceptible. Caramelization can proceed under acid or alkaline conditions or
without catalysis. Acid conditions result in degradation to sugar dehydration
products, including hydroxymethylfurfural, levulinic acid, and humin. Not
surprisingly, these happen to be similar to the by-products of conventional
acid hydrolysis processes for cellulosics. Alkaline conditions are noted to
produce fragmentation to smaller molecules with possible recombination
leading to a plethora of potential end products. Degradation by carameliza-
tion also essentially follows first-order reaction kinetics (Quintas et al., 2007).
As in the case of the Maillard reaction, caramelization can produce positive
attributes, but can, depending on circumstances, lead to negative impacts.
For example, caramelization reactions have been directly related to forma-
tion of toxicant furans (Blank, 2008).
18.5.6 Oxidation
Oxidation reactions represent the final and most diverse form of degrada-
tion. The sensitivity of individual constituents to oxygen and temperature,
however, is highly specific. As such, oxidation is also the most difficult form
of degradation to describe in generic terms. A good example illustrating
inherent complexity is vitamin C. It involves a complex of l-ascorbic acid
with its reversible oxidation product, dehydro-l-ascorbic acid. Vitamin C has
been long established to have beneficial bioactive properties, and thus is con-
sistent with the term “nutraceutical” even though significantly predating it.
It has been known for a long time that vitamin C is destroyed by oxidation
(Hodges, 1980). Exposure to heat, air (oxygen), alkaline medium, and cer-
tain metals ions (copper and iron) first hastens reversible oxidation, and then
irreversible oxidation to end products, including oxalic acid and l-threonic
acid. The oxidation of l-ascorbic acid not only represents a third major form
of nonenzymatic browning but can also be linked to the initiation of the
Maillard reaction, noted earlier (Erkin et al., 2012). A review of vitamin C

retention in fruit and vegetable products was recently undertaken by Santos

and Silva (2008). Various other recent studies suggest that l-ascorbic acid
degradation is first order in nature (Marfil et al., 2008; Paul and Ghosh, 2012;
Rahman et al., 2013; Summen and Erge, 2014), but these reflect specific prod-
ucts and temperature conditions. For atmospheric drying, Kaya et al. (2010)
showed that vitamin C retention is affected not just by the temperature but
also by the relative humidity of the inlet drying air.
A key comment by Hodges (1980) continues to be borne out over time that,
despite the prominence of vitamin C in terms of both the level of interest
and prolonged study, much less is known about it than would be antici-
pated. This caution applies to other less studied products regarding oxida-
tion. Other product-specific examples of investigation of oxidation in the
literature include anthocyanin (Tonon et al., 2010; Summen and Erge, 2014);
lycopene (Shi and LeMaguer, 2000; Xianquan et al., 2005); oleyl and linoleoyl
residues (Le Grandois et al., 2010); phenolics (Miranda et al., 2010); and sesa-
mol (Yeo et al., 2011).
18.6 Engineering and Physical Properties

In the selection of a suitable dehydration technology, understanding the
engineering and physical properties of the materials to be handled are just
as important as the drying characteristics. It is useful to note that physi-
cal properties and material handling issues for drying operations have been
well identified for some time. As such, as a starting point, Keey (1991) pro-
vides an overview of relevant properties.
From a practical perspective, four different forms of feed materials can be
considered that cover the breadth of rheological characteristics, from liquid
to intact solid. These are (i) soluble product contained within a liquid stream,
usually Newtonian in nature, and with a lower viscosity; (ii) product in a
paste, representing a non-Newtonian liquid, typically with a higher effective
viscosity; (iii) product as part of or in the form of semisolid, for example, flex-
ible gel; or (iv) product as part of or in the form of solid. In dealing with func-
tional foods and nutraceuticals, any of these forms could be encountered.
An obvious complication involved with dehydration is that the physical
nature of the material being processed is deliberately changed, in some cases
quite significantly. The feed material may start as a liquid, paste, semisolid,
or moist solid, whereas the final product is usually a dry solid, typically in
particulate form. As such, it is not just the physical characteristics of the feed
material that need to be understood, but also of the final product, and often,
critically, the transition state(s) between the two (Roos, 2004).
The volumetric throughput requirements and scale of the dehydration
system are determined by the feeding rate, combined with the density (or

specific gravity) in the case of liquid or paste feed, or the bulk density in the
case of semisolid or solid feed. Liquid or paste feed typically can be pumped,
such that material handling is relatively straightforward. The feed-pump-
type and pumping energy requirements are determined by the viscosity (or
effective viscosity) as well as possible non-Newtonian characteristics of the
feed stream.
The handling of semisolid or solid feed is more complicated, and depends
on understanding a broader range of properties, given that such feedstock
is usually present as particulate or loose bulk-solid material. This can be
defined as composed of discrete units that are too small to be handled indi-
vidually. The topic of bulk-solids materials is not new and is well established,
but is still relevant to new products such as functional foods and nutraceuti-
cals, which may exhibit such properties.
Bulk solids may agglomerate into larger lumps or tangles, but can be nor-
mally broken back down into the smaller discrete units again (Keey, 1991).
Relevant characteristics to describe particulate material include (i) particle
shape; (ii) particle size or size distribution; (iii) internal friction coefficient
(or angle) and associated cohesion (particle-to-particle); and (iv) wall fric-
tion coefficient (or angle) and associated adhesion (particle-to-wall surface).
Bulk solid characteristics are particularly important for drying systems,
such as spray drying, that are intended to produce product in the form of
particulates.
Particle shape can be characterized in several ways. A useful approach is
outlined by Feda (1982). This employs three characteristic orthogonal par-
ticle dimensions, length (L), thickness (Th), and breadth (B), to define two
self-descriptive dimensional ratios: elongation (Equation 18.9) and flatness
(Equation 18.10):
L
Elongation = (18.9)
B
B
Flatness = (18.10)
Th
Plotting the inverse of one ratio against the inverse of the other yields a
two-by-two matrix, defining four quadrants with distinctive types of particle
shapes: isometric (or bulky); disk-like; flake-like; or needle-like (Figure 18.3).
Particle size characterization is most typically undertaken using sieving
(Keey, 1991). However, sieving is also well recognized to be problematic
when nonisometric particles are involved. Sieves are only two-dimensional
in nature while particles are three-dimensional. If one dimension is much
longer, as with needle-like or fibrous material, sieves cannot distinguish
(Bartl et al., 2004). Particle friction characterization, including both internal
and wall friction, is determined using a series of direct shearing tests with
samples subjected to varying normal force (Mohsenin, 1986; Shamlou, 1988).

Disk-like Isometric
1/Elongation ratio
2/3
Flake-like Needle-like
0
0 2/3 1
1/Flatness ratio
FIGURE 18.3
Particle shape characterization matrix.
This information in turn is used to assess the extent to which a particulate

material will free-flow as a bulk solid, and thus to select appropriate convey-
ance systems (Williams et al., 2008). The angle of repose is noted as important
for solids conveyance within drying systems (Keey, 1991), but this is really a
special case of the angle of internal friction (Reimbert and Reimbert, 1987).
The impacts of liquid content also need to be considered in terms of particle
friction, and can be evaluated at different liquid levels to confirm effects. As
outlined by Parsons et al. (2008), internal friction is increased when liquid
is present, irrespective of the liquid type. Wall friction is also increased by
liquid addition, but is affected differently by different types of liquids, for
example, water versus alcohols.
The heat capacity of the whole material being processed can be adequately
estimated by combining, according to mass proportion, the heat capacity of
the dry matter and that of water (Dupont et al., 2014). The heat capacity of dry
biomass has been shown to increase somewhat at highly elevated tempera-
tures, but at more modest temperatures of 40–80°C is in the range of only
30%–40% that of water. In most situations, thus, it is the component mass of
the water present that dominates this property for the overall material.
Solid materials in dehydration processes are observed in different forms,
exhibiting crystalline, glassy, or rubbery characteristics (Roos, 2004). The
form depends on both the liquid content and temperature. Of particular
importance in spray drying (Roos, 2004) and freeze drying (Santivarangkna
et al., 2011) is glass transition, which is observed for polymeric materials, and
which is characterized by the glass transition temperature (Tg). Shrinkage
is a common phenomenon observed in thermal drying (Mayor and Sereno,
2004). This directly impacts size and shape, but more subtly heat transfer and
mass transfer.
Dehydration and drying are by nature mass transfer processes for mois-
ture removal. And as noted earlier, moisture diffusion typically becomes the
limiting parameter. As such, understanding mass transfer characteristics of

the solids involved is important. Mass diffusivity characterization is often

associated with studies on drying kinetics. However, estimating diffusivity
typically involves assumptions that may not be realistic, including uniform
shape and isometric properties. For the same six drying kinetics studies
noted earlier in Section 18.4, moisture diffusivity values were also estimated.
These ranged widely from 0.16 × 10−9 m2/s to 292 × 10−9 m2/s, with a mean
value of 40.3 × 10−9 ± 102 × 10−9 m2/s. The variability observed for diffusiv-
ity was substantially higher than that for activation energy. This partially
reflects differences not only in materials but also in the idealized model.
Moisture diffusivity in foods and biological materials, and associated
issues are discussed by Chen (2007). A known concern is that mass transfer
is impacted by both shrinkage and variation in diffusivity (Ruiz-Lopez and
Garcia-Alvarado, 2007). An additional concern is that diffusion can sometimes
be non-Fickian in nature, paralleling the nonlinearity seen in flow for non-
Newtonian fluids. Normally, the rate of mass transfer is linearly proportion to
the concentration gradient. However, in some cases, it is not. This is a strongly
associated diffusion through polymeric solids exhibiting viscoelastic behavior
(Edwards, 1996), which is relatively common in food products. Non-Fickian
diffusion has been observed of drying of food products (Jemai, 2013).
A last subtlety to note is stickiness. This is a frequently observed character-
istic of materials in dehydration processes. Although practically important,
stickiness itself is not per se a property on its own. Mechanisms of sticki-
ness in food products, and associated underlying properties are discussed
by Adhikari et al. (2001).
18.7 Predrying and Water Removal

Although dehydration is essential for the production of a dry final prod-
uct, it is well understood that such processes are extremely energy intensive
(Kemp, 2012). The latent heat of evaporation for water at 100°C is about 41 kJ
per mol or 2.3 MJ per kg. At the same time, energy inputs to industrial dry-
ing systems to evaporate moisture range from about 3.0 to 16.0 MJ per kg, or
effective efficiencies ranging from only about 75% down to 15%. Hence, there
is a strong incentive to remove as much water as may be practical prior to
final dehydration. Four major generic methods are practically available for
this purpose: (i) evaporation; (ii) membrane concentration; (iii) mechanical
dewatering; and (iv) selective solvent extraction.
Evaporation is a well-established industrial technology, although one that
continues to undergo extensive evolution (Minton, 1986). Given the abil-
ity to employ multiple effects with cascading energy use, an evaporator
system can be four to eight times more energy efficient for per-unit water
removal than a typical final dehydration system. Evaporation can be used

to concentrate soluble or fine insoluble constituents in a liquid feed solution.

The major constraints are that the product cannot be volatile and must be
heat stable at temperatures used for evaporation, which in turn, thermody-
namically, are set by the pressures involved. Evaporation temperatures can
be reduced by employing vacuum evaporation, but typically at higher cost.
Other constraints that could limit the use of evaporation can include exces-
sive foaming, salting, scaling, fouling, corrosion, or other fluid properties.
As noted later, water evaporation is essential for continuous operation of
osmotic dehydration processes.
Membrane technologies involve semipermeable membranes, with general
characteristics described by Cheryan (1986) or Sutherland (2008). Membrane
systems are categorized by the increasingly smaller particle or molecular
size involved. In sequence from largest to smallest size, these are microfil-
tration, ultrafiltration, nanofiltration, and ultimately reverse osmosis, which
only permits water to pass. They exclude solutes based on a combination
of molecular size or, at extremely small size, molecular adsorption charac-
teristics. Although membrane systems can technically be used for selective
separations, they are more practically used to concentrate a desired product
in a liquid feed stream. They can be readily operated at ambient temperature
conditions. As such, they are desirable for preconcentration of heat-sensitive
materials. As such, they are quite commonly cited in the recent literature for
a range of different bioactive products, for example, Xu et al. (2006), Diaz-
Reinoso et al. (2011), Gurak et al. (2013), and Murakami et al. (2013).
Mechanical dewatering is typically employed when the product is part of
a wet solid, including fine, but not colloidal, particles. Such techniques are
based on the use of size or density differences or some combination. The
technologies include centrifuges of various types, hydrocyclones, and vari-
ous types of filtration (Tarleton and Wakeman, 2007). In the latter case, sim-
ple filtration is distinguished from membrane technology when the particles
involved are no smaller than about 5–10 µm in size. Mechanical dewatering
requires much less energy than drying, but the extent of water removal is
limited. Typically, a solid concentration of no more than about 30%–50% can
be achieved depending on the technique involved.
A last preconcentration technology to note is solvent extraction. The use
of selective solvents for the recovery of bioactive constituents is an entire
area on its own that is beyond the scope of this chapter, but importantly,
such techniques can be employed to achieve preconcentration. Solvents are
by nature product specific, and are usually characterized based on both their
capacity to hold the product and their solvent selectivity to remove and con-
centrate the product. They can be used to extract a selected product from a
solid or from another immiscible liquid phase. Although actual testing is
ultimately required, a variety of predictive techniques exist to help identify
suitable candidates (Sheikholeslamzadeh and Rohani, 2012). A major con-
straint in the case of nutraceutical and functional food products that lim-
its the ability to employ selective solvents is toxicity. A variety of organic

liquids are commonly used for solvent extraction but most are not accept-
able in terms of toxicity. As noted by Turner (2006), for this reason, advanced
techniques focus on three main solvents suitable for food products: water or
ethanol, under a variety of specialized conditions, and supercritical carbon
dioxide. Supercritical carbon dioxide in particular continues to be investi-
gated extensively in the literature for the recovery of bioactive constituents
(e.g., Martinez, 2008; Sovova and Stateva, 2011). It can also achieve preconcen-
tration of desired bioactives. At the same time, the high cost of this approach
means that energy savings alone by this technique may not necessarily be
justifiable.
18.8 Atmospheric Drying

Atmospheric drying represents the most common approach employed for
industrial drying overall. This is for the obvious reasons of comparative low
cost and familiarity. There are many diverse atmospheric drying technolo-
gies available. All of them share the key characteristic that heated air is used
under atmospheric conditions to drive off moisture from the feed material.
The critical property thus controlling suitability of such technologies is the
thermal stability of the bioactive ingredient(s) involved, or the ability to miti-
gate thermal degradation.
The intent in this chapter is not to comprehensively review all available
atmospheric dryer types, which are extensive, but instead to highlight those
that have been recently cited for products incorporating bioactive ingredi-
ents. Despite diversity, only four major types of atmospheric dryers have
been significantly applied in recent times to functional foods and nutraceu-
ticals. These are in approximate order of priority: (i) spray drying; (ii) tray
drying; (iii) fluid bed drying; and (iv) spouted bed drying. Each of these is
described in more detail in subsequent sections.
In all cases for atmospheric drying, the final product moisture content
is typically around 10% (db) or so, but can vary depending on the specific
product. In order to be systematic, three major factors are noted to catego-
rize these various types of atmospheric drying systems. First is whether the
dryer is batch or continuous. Second is the nature of materials handling and
suitability for different types of feed materials. Third is the primary nature
of heat transfer involved, whether mostly convective, mostly conductive, or a
combination of the two.
18.8.1 Spray Drying

Spray drying is the most common type of atmospheric drying technique
cited in the current literature regarding functional foods and nutraceuticals.

Such popularity is not surprising. Although it can be used at a reduced scale

for pilot testing, spray drying is commonly used in the industry at a large
scale, including both for food products and for conventional pharmaceu-
ticals (Ameri and Maa, 2006). A general overview of this technology, as it
relates to bioactives, is provided by Murugesan and Orsat (2012). A pho-
tograph of a small-size commercial spray drying system is illustrated in
Figure 18.4.
In spray drying, which involves continuous processing, a liquid feedstock
is used to produce ultimately a powdered solid product, primarily based on
convective heat transfer. Although typically the feed involves water-soluble
constituents, it can incorporate lipid materials if adequately homogenized
before drying. An additional obvious reason for significant attention on this
technology is the intrinsic linkage of spray drying with encapsulation as a
bioactivity preservation technique. Such use of spray drying for encapsula-
tion has been common in the conventional pharmaceutical industry (Tewa-
Tagne et al., 2007).
FIGURE 18.4
Small-scale commercial spray drying system. (Photograph by J. Viafara. With permission.
Courtesy of the Food Development Centre, Portage la Prairie, MB.)

Encapsulation of food-related bioactive constituents is reviewed in a num-

ber of recent papers (Gharsallaoui et al., 2007; Fang and Bhandari, 2010;
Kwak, 2014; Mahdavi et al., 2014). By incorporating suitable matrix materials
into the feed, a protective encapsulation layer is formed during the drying
of globules that limits accessibility to oxygen. This protects the materials
within the formed solid particle, reducing oxidation-related degradation.
This layer is not only protective of the bioactive materials relative to the tem-
perature conditions encountered during drying itself, but also, later, making
the products stable over longer period.
A variety of materials incorporating bioactives have been recently inves-
tigated in terms of spray drying. These include the following products, in
alphabetical order: acai extract (Tonon et al., 2008, 2010); amla extract (Mishra
et al., 2014); blueberry (Lim et al., 2011); carrot (black) (Ersus and Yurdagel,
2007); citrus fiber extract (Chiou and Langrish, 2007); fish oil (Jafari et al.,
2008); flaxseed oil (Quispe-Condori et al., 2011; Carneiro et al., 2013); gac
(Tran et al., 2008; Kha et al., 2010); honey (Nurhadi et al., 2012); lactic culture
(Peighambardoust et al., 2011; Paez et al., 2012); linseed oil (Gallardo et al.,
2013); mango (Cano-Chauca et al., 2005); medicinal plant extract (Sansone
et al., 2011); rosemary extract (Couto et al., 2012); soy protein (Tang and Li,
2013); soybean extract (Georgetti et al., 2008); sumac extract (Caliskan and
Dirim, 2013); sweet potato (Ahmed et al., 2010); whey isolate (Zhu and
Damodaran, 2011); wine lees extract (Perez-Serradilla and Luque de Castro,
2011); yacon (Scher et al., 2009); and yeast broth (Teixeira et al., 2011).
18.8.2 Tray Drying

The second most commonly cited drying methods is simple tray drying. This
is typically a batch process used for drying of solid or semisolid feedstock,
and based primarily on convective heat transfer. Most of such cited examples
for bioactive products involve testing of impacts on degradation, particularly
in comparison to alternative drying techniques, or as part of evaluations of
comparative drying kinetics. As such, tray drying is used more as a research
tool than for industrial applications. Nevertheless, tray drying is used for
selected products even at an industrial scale. A photograph of a commercial
tray drying system is presented in Figure 18.5.
A variety of materials incorporating bioactives have been recently investi-
gated in terms of tray drying. These include the following products, in alpha-
betical order: aloe vera (Miranda et al., 2009); apple (Vega-Galvez et al., 2012);
berry fruit (Asami et al., 2003); blueberry (Lohachoompol et al., 2004; Lopez
et al., 2010); broccoli (Mahn et al., 2011); cape gooseberry (Vega-Galvez et al.,
2014); grape pomace (Vashisth et al., 2011); mint (Therdthai and Zhou, 2009);
mushroom (Giri and Prasad, 2007); orange (Garau et al., 2007); peach (Zhu
and Shen, 2014); pear (Mrad et al., 2012); pineapple (Ramallo and Macheroni,
2012); quinoa (Miranda et al., 2010; Vega-Galvez et al., 2010); and yacon (Scher
et al., 2009).

FIGURE 18.5
Commercial tray drying system. (Photograph by J. Viafara. With permission. Courtesy of the
Food Development Centre, Portage la Prairie, MB.)
18.8.3 Fluid Bed and Jet Spouted Bed Drying

The last two drying systems cited in the recent literature are fluid bed and
spouted bed drying.
Just as in the case of spray drying, both such technologies have been
already used extensively for industrial-scale drying of food products and
within the conventional pharmaceutical industry (Srivastava and Mishra,
2010; Souza and Oliveira, 2012). Both are continuous processes applied to a
pumpable feed stream, that is, liquid or paste. Unlike spray drying or tray
drying, however, heat transfer involves a combination of convective and con-
ductive mechanisms, the latter achieved through the presence of solid or
inert particles. Photographs of small-scale fluid bed drying and jet spouted
bed drying systems are presented in Figures 18.6 and 18.7, respectively.
Recent investigations of fluid bed–related drying applications include the
following products, in alphabetical order: blueberry (Chen and Martynenko,
2013); broccoli (Reyes et al., 2012); and soybean (Niamnuy et al., 2011). Recent

FIGURE 18.6
Small-scale fluid bed drying system.
FIGURE 18.7
Small-scale jet spouted bed drying system. (Copyright Her Majesty the Queen in Right of
Canada as represented by the Minister of Natural Resources, 2014.)

investigations of jet spouted-bed-related drying applications include the fol-

lowing products, in alphabetical order: blueberry (Pallas et al., 2012); grape
(Xiao et al., 2010); and prepared wheat (Kahyaoglu et al., 2012).
18.9 Freeze Drying/Lyophilization

Freeze drying or lyophilization is a relatively complex dehydration process,
but one that also has been well established in the conventional pharmaceuti-
cal industry for the dehydration of heat-sensitive products (Nireesha et al.,
2013). This also includes the drying of viable microorganisms. As such, it is
not surprising that the technology has also recently been investigated exten-
sively for functional foods and nutraceuticals. A photograph of a commercial
freeze drying system is presented in Figure 18.8.
Detailed background on freeze drying is provided by Rey and May (2004).
Procedures and protocols for pharmaceuticals are described in detail by
Tsinontides et al. (2004), with applications to food-related products reviewed
by Ratti (2001). Dehydration occurs under vacuum conditions to create the
driving force for water removal. Water removal in this case, however, is not
by evaporation, but instead via sublimation. The feed material with all asso-
ciated water is first rapidly frozen to form a solid state. Rapid freezing itself
can cause damage to products; hence, there has been extensive work in the
development of cryoprotectant agents (Day and Stacey, 2007).
Freeze drying, even at the industrial scale, is still typically undertaken in
a batch manner, using two stages of drying. The first stage, called primary
FIGURE 18.8
Commercial freeze drying system. (Photograph by J. Viafara. With permission. Courtesy of the
Food Development Centre, Portage la Prairie, MB.)

drying, involves sublimation under moderate vacuum conditions to remove

free-water. The rate of water removal is more rapid and relatively constant
during this stage (Nireesha et al., 2013). The second stage, called secondary
drying, involves higher vacuum in order to more slowly remove absorbed
water remaining within the material. Even though the mechanisms are a bit
different from thermal drying technologies, the familiar two-stage nature of
drying kinetics is still observed. The residual moisture content after freeze
drying is very low, typically only in the range of 1%–3% (db). Freeze-dried
products are also typically readily reconstituted with water.
Freeze drying can be described as essentially a gentle treatment of heat-
sensitive products. It is, for example, used for preservation of biologi-
cal standards and bacterial culture standards. It is not, however, without
issues. A particular concern is the glass transition temperature (Tg) of the
material through the course of the dehydration process (Kumar et al., 2011;
Santivarangkna et al., 2011). A glassy to vitreous state transition is often
observed, which can be important when it is desired that the product be crys-
talline in nature. This depends on the specific product involved. Potential
damage to proteins may also occur, which is understood to involve misfold-
ing (Pikal, 2004). Lastly, freeze drying by its nature tends to be an expensive
process, which requires sufficient product value to justify its application.
A variety of materials incorporating bioactives have recently been inves-
tigated in terms of freeze drying. These include the following products,
in alphabetical order: apple juice (Raharitsifa and Ratti, 2010); chili pepper
(Toontom et al., 2012); date fiber (Borchani et al., 2011); fruits (Asami et al.,
2003); grape juice (Gurak et al., 2013); grape pomace (Vashisth et al., 2011);
hara leaf (Lin et al., 2012); lemon balm leaf (Antal et al., 2014); milk (Zhu and
Damodaran, 2011); mushroom (Zhang et al., 2009; Lau et al., 2013); pepper-
mint (Rohloff et al., 2005); polysaccharides (Chin et al., 2011); and star fruit
(Shui and Leong, 2006).
18.10 Superheated Steam Drying

SSD is a counterintuitive approach that is best known as one of the energy-
efficient forms of dehydration available. At the same time, the technology
can be no longer considered entirely novel, and has been implemented for a
variety of applications at an industrial scale, most prominently in the beet-
sugar industry. The basic principles of the technology are outlined by Pronyk
et al. (2004) for food applications in general.
It is possible for steam to be superheated above its normal, saturated-
equilibrium conditions. But if such steam comes into contact with liquid
water, de-superheating occurs. The energy of the steam is reduced back to
saturated conditions, in the process evaporating some of the water to create

additional saturated steam. Such a technique is routinely applied in conven-

tional steam boiler systems, but can also be deliberately harnessed for drying,
by exposing a moist solid material to superheated steam. Rather than just
evaporating water into air that is lost to the atmosphere, as in conventional
atmospheric thermal drying, SSD creates a dried product and excess satu-
rated steam. The latter can be usefully employed for heating elsewhere in an
industrial process. Hence, there is a dramatic advantage in terms of efficiency.
Thermal stability of any bioactive is an important constraint for SSD, but
with a subtle difference. Temperatures are typically above 100°C, although
they can be lowered using vacuum-SSD conditions, but in all cases, there is
no exposure to air or oxygen, only steam. Thus, no oxidative degradation
reactions occur, as described earlier. As such, improved color and the lack of
browning are well-recognized advantages of SSD (Ioannou and Ghoul, 2013).
There are, however, a number of more subtle disadvantages. The first relates
to the glass transition temperature (Tg) of the material in question, given the
relatively high and uniform temperature environment. The second relates to
associated steam-stripping of the material being processed, whereby poten-
tially desirable constituents, such as phenolics, may be inadvertently removed.
A variety of materials incorporating bioactives have been recently inves-
tigated in terms of SSD. These include the following products, in alphabeti-
cal order: bamboo shoots (Choudhary et al., 2012); carrot-based carotenoids
(Devahastin and Niamnuy, 2010); edible chitosan (Mayachiew and
Devahastin, 2010; Mayachiew et al., 2010); distillers dried grain (Cenkowski
et al., 2012); Indian gooseberry (Kongsoontornkijkul et al., 2006); longam
(Somjai et al., 2009); oats (Head et al., 2011); paneer (Shrivastav and Kumbhar,
2011); coriander and pepper seed (Kozanoglu et al., 2006, 2012); soybean
(Niamnuy et al., 2011); and yam (Xiao et al., 2012). SSD represents a technol-
ogy that is worth considering for specific functional foods or nutraceuticals.
18.11 Microwave Drying

Microwave drying is more accurately described as radio-frequency-based
drying; however, the common name has generally stuck, given its enormous
popularity for consumer convenience heating and cooking. Importantly, given
that microwaves are more frequently used for an assistive role, the technology
is also considered later in Section 18.16 regarding hybrid drying techniques.
A number of recent reviews have been undertaken regarding both micro-
wave and microwave-assisted drying (Zhang et al., 2006; Orsat et al., 2007;
Chandrasekaran et al., 2013). Water molecules possess dipole moment, which
can be selectively excited, thus causing heating, at specific radio frequencies.
This leads to elevated temperature and increased partial pressure of water,
and hence to drying.

Microwave-based drying does not rely on an external temperature-

gradient, which has advantages. At the same time, the creation of micro-
waves is relatively expensive, such that the process is not applied extensively
alone at an industrial scale. The convenience of microwave heating also
makes it a useful research tool for laboratory investigations. Recent investi-
gations of microwave-alone-based drying applications include the following
products, in alphabetical order: bamboo shoots (Bal et al., 2010, 2011); olive
fruit (Mahdhaoui et al., 2014); Shiitake mushroom (Zhang et al., 2009); and
spinach (Dadali et al., 2007).
18.12 Vacuum Drying

Vacuum drying, which is briefly described by Ratti (2001), involves the appli-
cation of vacuum in order to reduce the exposure temperature for materials
being dried, while still evaporating moisture. A photograph of a small-scale
vacuum drying system is presented in Figure 18.9.
The reduction of pressure also reduces the presence of oxygen associ-
ated with air. The technology can be comparatively expensive, and is often
employed in conjunction with other drying technologies, as discussed later
in Section 18.16. Recent investigations of vacuum-based drying applications
include the following products, in alphabetical order: apple (Joshi et al.,
2011); blueberry (Kim and Kerr, 2013); carrots (Panyawong and Devahastin,
2007); edible chitosan (Mayachiew and Davahastin, 2010); grape pomace
(Vashisth et al., 2011); honey (Nurhadi et al., 2012); Indian gooseberry tea
(Kongsoontornkijkul et al., 2006); mangosteen peel (Suvarnakuta et al., 2011);
soybean (Niamnuy et al., 2011); and sweet potato (Xu et al., 2013).
FIGURE 18.9
Small-scale vacuum drying system.

18.13 Heat-Pump Drying

Heat-pump drying involves moisture evaporation to an air stream, just as in
the case of atmospheric thermal-based drying, but relies on a slightly differ-
ent and subtle manipulation of the air-flow. Instead of significantly heating
the air stream, the heat-pump system is used to cool and condense moisture
from the air prior to exposure to the material being dried. As such, the driv-
ing force is still based on psychrometry, but achieved via a dehumidified air
stream at moderate temperature. Details of heat-pump drying systems, as
well as the application to food products, are provided by Chua et al. (2002).
With all heat-pump applications more broadly, a key limitation of the tech-
nology for drying is high cost. Given the cost, but also the adaptability of
the technology, heat pumps are often employed in conjunction with other
drying technologies, as discussed later in Section 18.16. Recent investigations
of heat-pump-based drying applications include the following products, in
alphabetical order: ginger (Hawlader et al., 2006); herbs (Shaw et al., 2005);
macadamia nut (Borompichaichartkul et al., 2009, 2013); mushroom (Juan
et al., 2013); and protein (Alves-Filho et al., 2008).
18.14 Osmotic Dehydration

Osmotic dehydration is an industrial process used for the dehydration of
fruits, vegetables, meats, and seafood (Khan, 2011; Akbarian et al., 2014).
Unlike other technologies discussed earlier, it directly involves only mass
transfer, and is undertaken at moderate temperatures. Solid feed materials
are immersed in a hypertonic solution, typically involving either sucrose or
salt (NaCl). Moisture moves from the feed material into the hypertonic solu-
tion, resulting in dehydration.
This movement of water causes obvious dilution of the hypertonic solu-
tion. In order to restore concentration to permit continuous operation, an
evaporator system is typically employed on a side flow stream. This removes
water and regenerates the hypertonic solution. As such, a thermal process is
involved, but only indirectly. Also, given the high efficiency of evaporation
systems, net overall energy requirements for dehydration are low.
Osmotic dehydration is a well-established technology (Torreggiani,
1993; Raoult-Wack, 1994), and per se is not novel. What has changed is the
recognition of fruits and vegetables in particular as key sources of ben-
eficial bioactive constituents. As such, the focus has moved to addressing
and managing impacts on bioactive constituents. Recent relevant investi-
gations of osmotic dehydration applications include the following prod-
ucts, in alphabetical order: aloe vera (Pisalkar et al., 2014); apple (Fazli and

Fazli, 2011; Moreno et al., 2013); beet root (Nazni and Thara, 2011); blue-
berry (Lohachoompol et al., 2004; Stajanovic and Silva, 2007); cherry tomato
(Heredia et al., 2009); green pepper (Ozen et al., 2002); kiwi fruit (Peinado
et al., 2011); mango (Khan et al., 2011); oyster mushroom (Ramya et al., 2014);
pumpkin (Abraao et al., 2013); radish (Herman-Lara et al., 2013); squid (sea-
food) (Uribe et al., 2011); sweet potato (Antonio et al., 2008); and tropical
fruit (Pereira et al., 2006).
There is little problem regarding thermal damage in this case. Rather, the
concern is associated with leaching and diffusional losses of nutrients and
bioactives, including antioxidants (Stojanovic and Silva, 2007; Heredia et al.,
2009). In an example comparative study by Lohachoompol et al. (2004) on
blueberry, losses of anthocyanin from osmotic dehydration were roughly
49% compared to 41% for convective drying, that is, just as significant even
without elevated temperature exposure. At the same time, the back diffu-
sion of product solutes from the hypertonic solution into the feed material
has been known. This has begun to be considered for deliberate applica-
tion, termed osmotic infusion, in order to add desired constituents (Jacob
and Paliyath, 2012).
18.15 Solar Drying

Solar drying, as inherent in its name, involves the use of incident solar radia-
tion for drying. The technology has been extensively reviewed (Belessiotis
and Delyannis, 2011; Bala and Janjai, 2012), and is a well-established and
often traditional approach for drying. What has changed, similar to the situ-
ation with osmotic dehydration, is the recognition of the materials involved,
particularly fruits, vegetables, and herbs, as sources of beneficial bioac-
tive constituents. Again, the focus is on addressing and managing impacts
on bioactives. Recent relevant investigations of solar drying applications
include the following products, in alphabetical order: banana (Janjai et al.,
2009); fruits (Delgado et al., 2011); grapes (Fadhel et al., 2005); longan (Janjai
et al., 2009); mint leaf (Akpinar, 2010); okra pod (Ismail and Ibn Idriss, 2013);
pistachio (Midilli and Kucuk, 2003); prickly pear peel (Lahsasni et al., 2004);
seafood (Wang et al., 2011); and tomato (Ringeisen et al., 2014).
18.16 Hybrid Drying

Hybrid drying involves a combination of dehydration technologies to achieve
a better overall result. As noted by Mujumdar and Law (2010), the nature of

drying kinetics lends itself to potential combinations of technologies, in par-

ticular to address the falling-rate drying period. In conventional atmospheric
drying it is this period that requires ever-increasing temperatures to remove
residual moisture, with obvious detrimental effects. The concept of hybrid
drying, however, is no longer novel (Chou and Chua, 2001). Development
obviously continues on a case-by-case basis.
A variety of relevant recent work has been undertaken on hybrid drying,
involving 11 different combinations of technologies. Most hybrid approaches
involve two distinct technologies, but several involve three. An important
observation is how prominently microwave is involved in hybrid approaches.
The overwhelming majority of identified citations involve microwave com-
bined with another technology. The reasons for considering microwave were
noted earlier in Section 18.12.
A second important observation is the diversity of technology combi-
nations that are possible and have been considered. One of the technolo-
gies included in one of the combinations noted is infrared. Infrared has
been cited in the literature for application to foods, but is typically more
suitable for thermal processing rather than drying of sensitive materials,
given elevated temperatures involved. As such, it has not been otherwise
noted.
As noted, microwave has been employed in most of the cited applications,
investigating a variety of different relevant products. Atmospheric hot-air
drying combined with microwave has been considered for moringa (Dev
et al., 2011); longan fruit (Chaikham et al., 2013); and macadamia fruit (Silva
et al., 2006). Spray drying combined with microwave has been considered for
wine lees extract (Perez-Serradilla and Luque de Castro, 2011). Spouted bed
drying combined with microwave has been considered for prepared wheat
(Kahyaoglu et al., 2012). Vacuum drying combined with microwave has been
considered for apple (Erle and Schubert, 2001); carrot (Cui et al., 2004); clove
(Figiel, 2009); garlic (Figiel, 2009); mint (Therdthai and Zhou, 2009); mush-
room (Giri and Prasad, 2007; Zecchi et al., 2011); potato (Bondaruk et al.,
2007); parsley (Zecchi et al., 2011); and strawberry (Erle and Schubert, 2001).
Freeze drying combined with microwave has been considered for collagen
(Duan et al., 2013). Osmotic dehydration combined with microwave has been
considered for pineapple (Botha et al., 2012). Lastly, heat-pump drying com-
bined with microwave has been considered for green pea (Zielinska et al.,
2013) and yacon (Shi et al., 2014).
A number of other hybrid combinations have also been investigated for a
variety of products. Osmotic dehydration combined with atmospheric hot-
air drying has been considered for chayote (Ruiz-Lopez et al., 2010). Solar
drying combined with heat pump has been considered by Fadhel et al. (2011).
A combination of atmospheric hot air drying, heat pump, and infrared has
been considered for longan (Nathakaranakule et al., 2010). Lastly, a combina-
tion of atmospheric hot air, freeze drying, microwave, and vacuum has been
considered for blueberry (Mejia-Meza et al., 2008).

18.17 Optimizing Technology Selection

Given the complexity of dehydration processes, and the diverse range of
technologies or technology combinations available, all with different attri-
butes, there are typically no obvious best choices. At the same time, the
characteristics of and the processing requirements for functional foods and
nutraceuticals are diverse and variable. There are many options and much
uncertainty. Researchers can often make selections based on familiarity,
rather than what may be indeed optimal. Thus, the use of computer software
has a logical application in the analysis and selection of dehydration options
for functional foods and nutraceuticals.
Gong and Mujumdar (2008) provided a relatively recent review of avail-
able software packages.
Unfortunately, commercial packages tend to be detailed and expensive,
more oriented to high-end users, that is, it simply does not pay to write
simple software oriented to early-stage researchers. Fortunately, a number
of somewhat earlier papers are available that outline the logical basis used
behind analysis and selection processes. These can provide guidelines for
researchers or early-stage developers. Key relevant papers include Sadykov
(2004), Lababidi and Baker (2003), and Baker and Lababidi (2000).
18.18 Future Directions

An important trend noted early in this chapter is that dehydration activities
for functional foods and nutraceuticals have tended recently still to focus
primarily on already-existing technologies or associated adaptations. That
said, dehydration remains a complicated, energy-intensive, and expensive
operation, with significant scope for improvement and new opportunities.
A number of recent reviews have identified consistent overall themes and
directions into the future.
Galanakis (2013) succinctly summarized seven ongoing requirements that
continue to motivate improvement and the development of new technologies
for dehydration. These include needs to (i) shorten process and residence
times; (ii) accelerate heat and mass transfer; (iii) control Maillard reactions;
(iv) improve product quality; (v) enhance functionality; (vi) protect from
environmental stresses; and (vii) extend preservation.
Into the near-term future, Jangam (2011), Dev and Raghavan (2012), and
Moses et al. (2014) commonly identified four significant areas of develop-
ment. These are (i) heat-pump-assisted drying, including adaptation of heat
pumps to already-existing dryer systems; (ii) microwave-assisted drying;
(iii) intermittent- or pulsation-based drying adaptations; and (iv) variants

of SSD. A number of new technologies are also beginning to emerge, for

example, refractance-window drying (Nindo and Tang, 2007). Moses et al.
(2014), in particular, identified a variety of emergent dehydration concepts
that merit investigation over the longer-term period.
Acknowledgments
Photographs of selected drying systems were taken by Jairo Viafara, with
arrangements for photographs at the Food Development Centre in Portage
La Prairie, Manitoba provided by Alphonsus Utioh. The authors thank
Idaresit Ekaette for her preliminary gathering of literature.
References
Abraao, A.S., Lemos, A.M., Vilela, A., Sousa, J.M., Nunes, F.M. 2013. Influence of
osmotic dehydration process parameters on the quality of candied pumpkins.
Food and Bioproducts Processing, 91: 481–494.
Adhikari, B., Howes, T., Bhandari, B.R., Truong, V. 2001. Stickiness in foods: A
review of mechanisms and test methods. International Journal of Food Properties,
4: 1–33.
Ahmed, M., Akter, M.S., Lee, J.-C., Eun, J.-B. 2010. Encapsulation by spray drying
of bioactive components, physicochemical and morphological properties from
purple sweet potato. LWT—Food Science and Technology, 43: 1307–1312.
Ahmad-Qasem, M.H, Barrajon-Catalan, E., Micol, V., Carcel, J.A., Garcia-Perez, J.V.
2013. Influence of air temperature on drying kinetics and antioxidant potential
of olive pomace. Journal of Food Engineering, 119: 516–524.
Akbarian, M., Ghasemkhani, N., Moayedi, F. 2014. Osmotic dehydration of fruits in
food industrial: A review. International Journal of Biosciences, 4: 42–57.
Akpinar, E.K. 2010. Drying of mint leaves in a solar dryer and under open sun:
Modelling, performance analyses. Energy Conversion and Management, 51:
2407–2418.
Alves-Filho, O., Eikevik, T.M., Goncharova-Alves, S.V. 2008. Single and multistage
heat pump drying of protein. Drying Technology, 26: 470–475.
Ameri, M., Maa, Y.F. 2006. Spray drying of biopharmaceuticals: Stability and process
considerations. Drying Technology, 24: 763–768.
Antal, T., Chong, C.H., Law, C.L., Sikolya, L. 2014. Effects of freeze drying on reten-
tion of essential oils, changes in glandular trichomes of Lemon Balm leaves.
International Food Research Journal, 21: 387–394.
Antonio, G.C., Azoubel, P.M., Murr, F.E.X., Park, K.J. 2008. Osmotic dehydration of
sweet potato (Ipomoea batatas) in ternary solutions. Food Science and Technology
(Campinas), 28: 696–701.

Asami, D.K, Hong, Y., Barrett, D.M., Mitchell, A.E. 2003. Comparison of the total
phenolic and ascorbic acid content of freeze-dried and air-dried marionberry,
strawberry, and corn grown using conventional, organic, and sustainable agri-
cultural practices. Journal of Agricultural and Food Chemistry, 51: 1237–1241.
Bailey, J.E., Ollis, D.F. 1977. Biochemical Engineering Fundamentals. McGraw-Hill,
New York.
Baker, C.G.J., Lababidi, H.M.S. 2000. Development of a fuzzy expert system for the
selection of batch dryers for foodstuffs. Drying Technology, 18: 117–141.
Bal, L.M., Kar, A., Satya, S., Naik, S.N. 2010. Drying kinetics and effective moisture
diffusivity of bamboo shoot slices undergoing microwave drying. International
Journal of Food Science and Technology, 45: 2321–2328.
Bal, L.M., Kar, A., Satya, S., Naik, S.N. 2011. Kinetics of colour change of bamboo
shoot slices during microwave drying. International Journal of Food Science and
Bala, B.K., Janjai, S. 2012. Solar drying technology: Potentials and developments. In
Energy, Environment and Sustainable Development, ed. M.A. Uqaili and K. Jarijan,
Springer, New York, pp. 69–98.
Bartl, A., Mihalyi, B., Marini, I. 2004. Applications of renewable fibrous materials.
Chemical and Biochemical Engineering Quarterly, 18: 21–28.
Belessiotis, V., Delyannis, E. 2011. Solar drying. Solar Energy, 85: 1665–1691.
Benali, M. 2004. Thermal drying of foods: Loss of nutrient content and spoilage
issues. In Dehydration of Products of Biological Origin, ed. A.S. Mujumdar. Science
Publishers, Enfield, pp. 137–152.
Betoret, E., Betoret, N., Vidal, D., Fito, P. 2011. Functional foods development: Trends
and technologies. Trends in Food Science and Technology, 22: 498–508.
Blank, I. 2008. Furan in processed foods. In Bioactive Compounds in Foods, ed. J. Gilbert
and H.Z. Senyuva. Blackwell Publishing, Singapore, pp. 291–322.
Bondaruk, J., Markowski, M., Błaszczak, W. 2007. Effect of drying conditions on the
quality of vacuum-microwave dried potato cubes. Journal of Food Engineering,
81: 306–312.
Borchani, C., Besbes, S., Masmoudi, M., Blecker, C., Paquot, M., Attia, H. 2011. Effect
of drying methods on physico-chemical and antioxidant properties of date
fibre concentrates. Food Chemistry, 125: 1194−1201.
Borompichaichartkul, C., Chinprahast, N., Devahastin, S., Wiset, L., Poomsa-ad, N.,
Ratchapo, T. 2013. Multistage heat pump drying of macadamia nut under mod-
ified atmosphere. International Food Research Journal, 20: 2199–2203.
Borompichaichartkul, C., Luengsode, K., Chinprahast, N., Devahastin, S. 2009.
Improving quality of macadamia nut (Macadamia integrifolia) through the use
of hybrid drying process. Journal of Food Engineering, 93: 348–353.
Botha, G.E., Oliveira, J.C., Ahrne, L. 2012. Quality optimization of combined osmotic
dehydration and microwave assisted air drying of pineapple using constant
power emission. Food and Bioproducts Processing, 90: 171–179.
Calin-Sanchez, A., Figiel, A., Szaryez, M., Lech, K., Nuncio-Jauregui, N., Carbonell-
Barrachina, A.A. 2014. Drying kinetics and energy consumption in the dehy-
dration of pomegranate (Punica granatum L.) arils and rind. Food and Bioprocess
Caliskan, G., Dirim, S.N. 2013. The effects of the different drying conditions and the
amounts of maltodextrin addition during spray drying of sumac extract. Food
and Bioproducts Processing, 91: 539–548.

Cano-Chauca, M., Stringheta, P.C., Ramos, A.M., Cal-Vidal, J. 2005. Effect of the
carriers on the microstructure of mango powder obtained by spray dry-
ing and its functional characterization. Innovative Food Science and Emerging
Carneiro, H.C.F., Tonon, R.V., Grosso, C.R.F., Hubinger, M.D. 2013. Encapsulation effi-
ciency and oxidative stability of flaxseed oil microencapsulation by spray dry-
ing using different combinations of wall materials. Journal of Food Engineering,
115: 443–451.
Casolari, A. 1988. Microbial death. In Physiological Models in Microbiology: Volume II,
ed. M.J. Bazin and J.I. Prosser. CRC Press, Boca Raton, pp. 1–44.
Cenkowski, S., Sosa-Morales, M.E., Flores-Alvarez, M. 2012. Protein content and anti-
oxidant activity of distillers’ spent grain dried at 150°C with superheated steam
and hot air. Drying Technology, 30: 1292–1296.
Chaikham, P., Kreungngern, D., Apichartsrangkoon, A. 2013. Combined microwave
and hot air convective dehydration on physical and biochemical qualities of
dried longan flesh. International Food Research Journal, 20: 2145–2151.
Chandrasekaran S., Ramanathan, S., Basak, T. 2013. Microwave food processing—A
review. Food Research International, 52: 243–261.
Chen, X.D. 2007. Moisture diffusivity in food and biological materials. Drying
Technology, 25: 1203–1213.
Chen, Y., Martynenko, A. 2013. Computer vision for real-time measurements of
shrinkage and color changes in blueberry convective drying. Drying Technology,
31: 1114–1123.
Cheryan, M. 1986. Ultrafiltration Handbook. Technomic, Lancaster.
Chin, S.K., Law, C.L., Cheng, P.G. 2011. Effect of drying on crude ganoderic acids
and water-soluble polysaccharides content in Ganoderma lucidum. International
Journal of Pharmacy and Pharmaceutical Sciences, 3: 38–43.
Chiou, D., Langrish, T.A.G. 2007. Development and characterization of novel nutra-
ceuticals with spray drying technology. Journal of Food Engineering, 82: 84–91.
Chou, S.K., Chua, K.J. 2001. New hybrid drying technologies for heat sensitive food-
stuffs. Trends in Food Science and Technology, 12: 359–369.
Choudhary, D., Sahu, J.K., Sharma, G.D. 2012. Bamboo shoot: Microbiology, biochem-
istry and technology of fermentation—A review. Indian Journal of Traditional
Knowledge, 11: 242–249.
Chua, K.J., Chou, S.K., Ho, J.C., Hawlader, M.N.A. 2002. Heat pump drying: Recent
developments and future trends. Drying Technology, 20: 1579–1610.
Cornish-Brown, A. 2012. Fundamentals of Enzyme Kinetics, 4th edition. Wiley-Blackwell,
Singapore.
Couto, R.O., Conceicao, E.C., Chaul, L.T., Oliveira, E.M.S., Martins, F.S., Bara, M.T.F.,
Rezende, K.R., Alves, S.F., Paula, J.R. 2012. Spray-dried rosemary extracts:
Physicochemical and antioxidant properties. Food Chemistry, 131: 99–105.
Cui, Z.-W., Xu, S.-Y., Sun, D.-W. 2004. Microwave-vacuum drying kinetics of carrot
slices. Journal of Food Engineering, 65: 157–164.
Dadali, G., Demirhan, E., Ozbek, B. 2007. Color change kinetics of spinach undergo-
ing microwave drying. Drying Technology, 25: 1713–1723.
Dai, J., Mumper, R.J. 2010. Plant phenolics: Extraction, analysis and their antioxidant
and anticancer properties. Molecules, 15: 7313–7352.
Day, J.G., Stacey, G.N. 2007. Cryopreservation and Freeze-Drying Protocols, 2nd edition.
Humana Press, Totowa.

Delgado, E.S., Martinez-Flores, H.E., Garnica-Romo, M.G., Aranda-Sanchez, J.I., Sosa-

Aguirre, C.R., Cortes-Penagos, C.J., Fernandez-Munoz, J.L. 2011. Optimization
of solar dryer for the dehydration of fruits and vegetables. Journal of Food
Processing and Preservation, 37: 489–495.
DeLuca, V., Salim, V., Atsumi, S.M., Yu, F. 2012. Mining the biodiversity of plants: A
revolution in the making. Science, 336: 1658–1661.
Dev, S.R.S., Geetha, P., Orsat, V., Gariepy, Y., Raghavan, G.S.V. 2011. Effects of
microwave-assisted hot air drying and conventional hot air drying on the
drying kinetics, color, rehydration, and volatiles of Moringa oleifera. Drying
Technology, 29: 1452–1458.
Dev, S.R.S., Raghavan, V.G.S. 2012. Advancements in drying techniques for food,
fiber and fuel. Drying Technology, 30: 1147–1159.
Devres, O.Y. 2004. Psychrometry. In Dehydration of Products of Biological Origin, ed.
A.S. Mujumdar. Science Publishers, Enfield, pp. 43–92.
Devahastin, S., Niamnuy, C. 2010. Modelling quality changes of fruits and vegetables
during drying: A review. International Journal of Food Science and Technology, 45:
1755–1767.
Diaz-Reinoso, B., Moure, A., Dominguez, H., Parajo, J.C. 2011. Membrane concen-
tration of antioxidants from Castanea sativa leaves aqueous extracts. Chemical
Doyon, M., Labrecque, J. 2008. Functional foods: A conceptual definition. British Food
Journal, 110: 1133–1149.
Duan, X., Liu, L.L., Ren, G.Y., Zhu, W.X. 2013. Microwave freeze drying of Type I col-
lagen from bovine bone. Drying Technology, 31: 13–14.
Dupont, C., Chiriac, R., Gauthier, G., Toche, F. 2014. Heat capacity measurements of
various biomass types and pyrolysis residues. Fuel, 115: 644–651.
Edwards, D.A. 1966. Non-Fickian diffusion in thin polymer films. Journal of Polymer
Science, Part B: Polymer Physics, 34: 981–997.
Erle, U., Schubert, H. 2001. Combined osmotic and microwave-vacuum dehydration
of apples and strawberries. Journal of Food Engineering, 49: 193–199.
Ersus, S., Yurdagel, U. 2007. Microencapsulation of anthocyanin pigments of black
carrot (Daucus carota L.) by spray drier. Journal of Food Engineering, 80: 805–812.
Eskin, N.A.M., Ho, C.-T., Shahidi, F. 2013. Browning reactions in foods. In Biochemistry of
Foods, 3rd edition, ed. N.A.M. Eskin and F. Shahidi. Elsevier, San Diego, pp. 245–289.
Fadhel, A., Kooli, S., Farhat, A., Bellghith, A. 2005. Study of the solar drying of grapes
by three different processes. Desalination, 185: 535–541.
Fadhel, M.I., Sopian, K., Daud, W.R.W., Alghoul, M.A. 2011. Review on advanced of
solar assisted chemical heat pump dryer for agriculture produce. Renewable and
Sustainable Energy Reviews, 15: 1152–1168.
Fang, Z., Bhandari, B. 2010. Encapsulation of polyphenols—A review. Trends in Food
Fazli, F.A., Fazli, N.A. 2011. Mass transfer of fruit slices in hypertonic solution. In
Proceedings of the 11th International Congress on Engineering and Food (ICEF11):
Food Process Engineering in a Changing World, Athens, Greece, Volume III,
eds. P.S. Taoukis, N.G. Stoforos, V.T. Karathanos, and G.D. Saravacos. May
22–26, 2011, pp. 1609–1610.
Feda, J. 1982. Mechanics of Particulate Materials. Elsevier, Prague.
Figiel, A. 2009. Drying kinetics and quality of vacuum-microwave dehydrated garlic
cloves and slices. Journal of Food Engineering, 94: 98–104.

Galanakis, C.M. 2013. Emerging technologies for the production of nutraceuticals

from agricultural by-products: A viewpoint of opportunities and challenges.
Gallardo, G., Guida, L., Martinez, V., Lopez, M.C., Bernhardt, D., Blasco, R., Pedroza-
Islas, R., Hermida, L.G. 2013. Microencapulation of linseed oil by spray drying
for functional food application. Food Research International, 52: 473–482.
Garau, M.C., Simal, S., Rossello, C., Femenia, A. 2007. Effect of air-drying tempera-
ture on physio-chemical properties of dietary fibre and antioxidant capac-
ity of orange (Citrus aurantium v. Canoneta) by-products. Food Chemistry, 104:
1014–1024.
Georgetti, S.R., Casagrande, R., Fernandes Souza, C.R., Pereira Oliveira, W., Vieira
Fonseca, M.J. 2008. Spray drying of the soybean extract: Effects on chemi-
cal properties and antioxidant activity. LWT—Food Science and Technology, 41:
1521–1527.
Giri, S.K., Prasad, S. 2007. Drying kinetics and rehydration characteristics of
microwave-vacuum and convective hot-air dried mushrooms. Journal of Food
Goderska, K. 2012. Different methods of probiotic stabilization. In Probiotics, ed. E.C.
Rigobelo. InTech, Rijeka, pp. 541–550.
Gong, Z.-X., Mujumdar, A.S. 2008. Software for design and analysis of drying sys-
tems. Drying Technology, 26: 884–894.
Granato, D., Branco, G.F., Nazzaro, F., Cruz, A.G., Faria, J.A.F. 2010. Functional foods
and nondairy probiotic food development: Trends, concepts, and products.
Comprehensive Reviews in Food Science and Food Safety, 9: 292–392.
Gurak, P.D., Cabral, L.M.C., Rocha-Leao, M.H. 2013. Production of grape juice pow-
der obtained by freeze-drying after concentration by reverse osmosis. Brazilian
Archives of Biology and Technology, 56: 1011–1017.
Harborne, J.B., Baxter, H. 1993. Phytochemical Dictionary: A Handbook of Bioactive
Compounds from Plants. Taylor & Francis, London.
Hawlader, M.N.A., Pererab, C.O., Tian, M. 2006. Comparison of the retention of
6-gengerol in drying of ginger under modified atmosphere heat pump drying
and other drying methods. Drying Technology, 24: 51–56.
Head, D., Cenkowski, S., Arntfield, S., Henderson, K. 2011. Storage stability of oats
processed commercially with superheated steam. LWT—Food Science and
Health Canada. 1998. Therapeutic Products Programme and the Food Directorate
from the Health Protection Branch. Policy Paper: Nutraceuticals/Functional Foods
and Health Claims on Foods. November 2.
Heredia, A., Peinado, I., Barrera, C., Andres Grau, A. 2009. Influence of process vari-
ables on colour change, carotenoids retention and cellular tissue alteration
of cherry tomato during osmotic dehydration. Journal of Food Composition and
Analysis, 22: 285–294.
Herman-Lara, E., MartÍnez-Sanchez, C.E., Pacheco-Angulo, H., Carmona-GarcÍa, R.,
Ruiz-Espinosa, H., Ruiz-Lopez, I.I. 2013. Mass transfer modeling of equilibrium
and dynamic periods during osmotic dehydration of radish in NaCl solutions.

Hii, C.L., Law, C.L., Cloke, M. 2009. Modeling using a new thing layer drying model
and product quality of cocoa. Journal of Food Engineering, 90: 191–198.
Hodges, R.E. 1980. Ascorbic acid. In Modern Nutrition in Health and Disease, 6th edition,
ed. R.S. Goodhart and M.E. Shils. Lea and Febiger, Philadelphia, pp. 259–273.
Ioannou, I., Ghoul, M. 2013. Prevention of enzymatic browning in fruit and vegeta-
bles. European Scientific Journal, 9: 310–341.
Ioannou, I., Guiga, W., Charbonnel, C., Ghoul, M. 2011. Frozen Mirabelle plum
drying: Kinetics, modelling and impact on biochemical properties. Food and
Bioproducts Processing, 89: 438–448.
Ismail, M.A., Ibn Idriss, E.M. 2013. Mathematical modelling of thin layer solar dry-
ing of whole okra (Abelmoschus esculentus (L.) Moench) pods. International Food
Research Journal, 20: 1983–1989.
Jacob, J.K., Paliyath, G. 2012. Infusion of fruits with nutraceuticals and health regulatory
components for enhanced functionality. Food Research International, 45: 93–102.
Jafari, S.M., Assadpoor, E., Bhandari, B., He, Y. 2008. Nano-particle encapsulation of
fish oil by spray drying. Food Research International, 41: 172–183.
Jangam, S.V. 2011. An overview of recent developments and some R&D challenges
related to drying of foods. Drying Technology, 29: 1343–1357.
Jangam, S.V., Mujumdar, A.S. 2013. Recent developments in drying technologies
for foods. In Advances in Food Process Engineering Research and Applications, ed.
S. Yanniotis, P. Taoukis, N.G. Stoforos and V.T. Karathanos. Springer, New York,
pp. 153–172.
Janjai, S., Lamlert, N., Intawee, P., Mahayothee, B., Bala, B.K., Nagle, M., Muller, J. 2009.
Experimental and simulated performance of a PV-ventilated solar greenhouse
dryer for drying of peeled longan and banana. Solar Energy, 83: 1550–1565.
Jemai, A.B. 2013. Characterizing the drying kinetics of high water content agro-
food particles exhibiting non-Fickian mass transport. Chemical Engineering
Transactions, 32: 1759–1764.
Joshi, A.P.K., Rupasinghe, H.P.V., Khanizadeh, S. 2011. Impact of drying processes on
bioactive phenolics, vitamin C and antioxidant capacity of red-fleshed apple
slices. Journal of Food Processing and Preservation, 35: 453–500.
Juan, W., Chong, Z., Zhentao, Z., Luwei, Y. 2013. Performance analysis of heat-pump
dryer to dry mushroom. Advance Journal of Food Science and Technology, 5: 164–168.
Kaya, A., Aydin, O., Kolayli, S. 2010. Effect of different drying conditions on the
vitamin C (ascorbic acid) content of Hayward kiwifruits (Actinidia deliciosa
Planch). Food and Bioproducts Processing, 88: 165–173.
Kahyaoglu, L.N., Sahin, S., Sumnu, G. 2012. Spouted bed and microwave-assisted
spouted bed drying of parboiled wheat. Food and Bioproducts Processing, 90:
310–308.
Keey, R.B. 1991. Drying of Loose and Particulate Materials. Taylor & Francis, New York.
Kha, T.C., Nguyen, M.H., Roach, P.D. 2010. Effects of spray drying conditions on the
physicochemical and antioxidant properties of the Gac (Momordica cochinchi-
nensis) fruit aril powder. Journal of Food Engineering, 98: 385–392.
Khan, M., Ahrne, L., Oliveira, J. 2011. Analysis of the quality attributes of osmotically
dehydrated mango. In Proceedings of the 11th International Congress on Engineering
and Food (ICEF11): Food Process Engineering in a Changing World, Athens, Greece,
Volume III, ed. P.S. Taoukis, N.G. Stoforos, V.T. Karathanos, G.D. Saravacos,
May 22–26, 2011, pp. 1975–1976.

Khan, M.R. 2012. Osmotic dehydration technique for fruits preservation—A review.
Pakistan Journal of Food Science, 22: 71–85.
Kholmanskiy, A.S., Tilov, A.Z., Sorokina, E.Y. 2013. Drying kinetics of plant prod-
ucts: Dependence on chemical composition. Journal of Food Engineering, 117:
378–382.
Kim, M., Kerr, W.L. 2013. Vacuum-belt drying of Rabbiteye blueberry (Vaccinium
ashei) slurries: Influence of drying conditions on physical and quality proper-
ties of blueberry powder. Food and Bioprocess Technology, 6: 3227–3237.
Kongsoontornkijkul, P., Ekwongsupasarn, P., Chiewchan, N., Devahastin, S. 2006.
Effects of drying methods and tea preparation temperature on the amount of
vitamin C in Indian gooseberry tea. Drying Technology, 24: 1509–1513.
Kozanoglu, B., Flores, A., Guerrero-Beltran, J.A., Welti-Chanes, J. 2012. Drying of pep-
per seed particles in a superheated steam fluidized bed operating at reduced
pressure. Drying Technology, 30: 884–890.
Kozanoglu, B., Vazquez, A.C., Welti-Chanes, J., Patino, J.L. 2006. Drying of seeds
in a superheated steam vacuum fluidized bed. Journal of Food Engineering, 75:
383–387.
Kudra, T., Mujumdar, A.S. 2009. Advanced Drying Technologies. CRC Press, Boca Raton.
Kumar, G.P., Prashanth, N., Kumari, B.C. 2011. Fundamentals and applications of
lyophilization. Journal of Advanced Pharmaceutical Research, 2: 157–169.
Kwak, H.-S. 2014. Nano- and Microencapsulation for Foods. Wiley Blackwell, Hoboken.
Lababidi, H.M.S., Baker, C.G.J. 2003. Web-based expert system for food dryer selec-
tion. Computers and Chemical Engineering, 27: 997–1009.
Lahsasni, S., Kouhila, M., Mahrouz, M., Idlimam, A., Jamali, A. 2004. Thin layer con-
vective solar drying and mathematical modeling of prickly pear peel (Opuntia
ficus indica). Energy, 29: 211–224.
Lau, C.-C., Abdullah, N., Aminudin, N., Shuib, A.S. 2013. Effect of freeze-drying pro-
cess on the property of angiotensin I-converting enzyme inhibitory peptides in
grey oyster mushrooms. Drying Technology, 31: 1693–1700.
Lee, T.-C., Ho, C.T. 2002. Bioactive Compounds in Foods: Effects of Processing and Storage.
ACS Symposium Series 816. American Chemical Society, Washington.
Le Grandois, J., Marchoni, E., Zhao, M., Giuffrida, F., Ennahar, S., Bindler, F. 2010.
Oxidative stability at high temperatures of oleyol and linoleoyl residues in the
forms of phosphatidylcholines and triacylglycerols. Journal of Agricultural and
Lim, K., Ma, M., Dolan, K.D. 2011. Effects of spray drying on antioxidant capacity
and anthocyanidin content of blueberry by-products. Journal of Food Science, 76:
H156–H164.
Lin, L., Lei, F., Sun, D.-W., Dong, Y., Yang, B., Zhao, M. 2012. Thermal inactivation
kinetics of Rabdosia serra (Maxim.) Hara leaf peroxidase and polyphenol oxi-
dase and comparative evaluation of drying methods on leaf phenolic profile
and bioactivities. Food Chemistry, 134: 2021–2029.
Liu, R.H. 2003. Health benefits of fruit and vegetables are from additive and syner-
gistic combinations of phytochemicals. American Journal of Clinical Nutrition, 78:
5175–5205.
Lohachoompol, V., Srzednicki, G., Craske, J. 2004. The change of total anthocyanins
in blueberries and their antioxidant effect after drying and freezing. Journal of
Biomedicine and Biotechnology, 5: 248–252.

Lopez, J., Uribe, E., Vega-Galvez, A., Miranda, M., Vergara, J., Gonzalez, E., Di Scala, K.
2010. Effect of air temperature on drying kinetics, vitamin C, antioxidant activ-
ity, total phenolic content, non-enzymatic browning and firmness of blueber-
ries variety O’Neil. Food and Bioprocess Technology, 3: 772–777.
Mahdavi, S.A., Jafari, S.M., Ghorbani, M., Assadpoor, E. 2014. Spray-drying micro-
encapsulation of anthocyanins by natural biopolymers: A review. Drying
Mahdhaoui, B., Mechlouch, R.F., Mahjoubi, A., Brahim, A.B. 2014. Microwave drying
kinetics of olive fruit (Olea europeae L.). International Food Research Journal, 21:
67–72.
Mahn, A.V., Antoine, P., Reyes, A. 2011. Optimization of drying kinetics and quality
parameters of broccoli florets. International Journal of Food Engineering, 7: Article 14.
Manas, P., Pagan, R. 2005. Microbial inactivation by new technologies of food preser-
vation. Journal of Applied Microbiology, 98: 1387–1399.
Marfil, P.H.M., Santos, E.M., Telis, V.R.N. 2008. Ascorbic acid degradation kinetics in
tomatoes at different drying conditions. LWT—Food Science and Technology, 41:
1642–1647.
Martinez, J.L. 2008. Supercritical Fluid Extraction of Nutraceuticals and Bioactive
Compounds. CRC Press, Boca Raton.
Martins, S.I.F.S., Jongen, W.M.F., van Boekel, M.A.J.S. 2001. A review of Maillard reac-
tion in food and implications to kinetic modeling. Trends in Food Science and
Mayachiew, P., Devahastin, S. 2010. Effects of drying methods and conditions on
release characteristics of edible chitosan films enriched with Indian goose-
berry extract. Food Chemistry, 118: 594–601.
Mayachiew, P., Devahastin, S., Mackey, B.M., Niranjan, K. 2010. Effects of drying
methods and conditions on antimicrobial activity of edible chitosan films
enriched with galangal extract. Food Research International, 43: 125–132.
Mayor, L., Sereno, A.M. 2004. Modelling shrinkage during convective drying of food
materials: A review. Journal of Food Engineering, 61: 373–386.
Mejia-Meza, E.I., Yanez, J.A., Davies, N.M., Rasco, B., Younce, F., Remsberg, C.M.,
Clary, C. 2008. Improving nutritional value of dried blueberries (Vaccinium
corymbosum L.) combining microwave-vacuum, hot-air drying and freeze dry-
ing technologies. International Journal of Food Engineering, 4: Article 5.
Midilli, A., Kucuk, H. 2003. Mathematical modeling of thin layer drying of pistachio
by using solar energy. Energy Conversion and Management, 44: 1111–1122.
Minton, P.E. 1986. Handbook of Evaporation Technology. Noyes Publications, Park Ridge.
Mishra, P., Mishra, S., Mahanta, C.L. 2014. Effect of maltodextrin concentration
and inlet temperature during spray drying on physicochemical and antioxi-
dant properties of amla (Emblica officinalis) juice powder. Food and Bioproducts
Processing, 92: 252–258.
Miranda, M., Maureira, H., RodrÍguez, K., Vega-Galvez, A. 2009. Influence of tempera-
ture on the drying kinetics, physicochemical properties, and antioxidant capac-
ity of aloe vera (Aloe barbadensis Miller) gel. Journal of Food Engineering, 91: 297–304.
Miranda, M., Vega-Galvez, A., Lopez, J., Parada, G., Sanders, M., Aranda, M., Uribe,
E., Di Scala, K. 2010. Impact of air-drying temperature on nutritional properties,
total phenolic content and antioxidant capacity of quinoa seeds (Chenopodium
quinoa Willd.). Industrial Crops and Products, 32: 258–263.

Mohsenin, N. 1986. Physical Properties of Plant and Animal Materials: Structure, Physical
Characteristics, and Mechanical Properties, 2nd edition. Gordon and Breach, New
York.
Moreno, J., Simpson, R., Pizarro, N., Pavez, C., Dorvil, F., Petzold, G., Bugueno, G.
2013. Influence of ohmic heating/osmotic dehydration treatments on poly-
phenoloxidase inactivation, physical properties and microbial stability of
apples (cv. Granny Smith). Innovative Food Science and Emerging Technologies,
20: 196–207.
Moses, J.A., Norton, T., Alagusundaram, K., Tiwari, B.K. 2014. Novel drying tech-
niques for the food industry. Food Engineering Reviews, 6: 43–55.
Mottram, D.S, Wedzicha, B.L., Dodson, A.T. 2002. Acrylamide is formed in the
Maillard reaction. Nature, 419: 448–449.
Mrad, N.D., Boudhrioua, N., Kechaou, N., Courtois, F., Bonazzi, C. 2012. Influence
of air drying temperature on kinetics, physicochemical properties, total phe-
nolic content and ascorbic acid of pears. Food and Bioproducts Processing, 90:
433–441.
Mujumdar, A.S., Law, C.L. 2010. Drying technology: Trends and applications in post-
harvest processing. Food and Bioprocess Technology, 3: 843–852.
Murakami, A.N.N., Dias de Mello Castanho Amboni, R., Schwinden Prudencio,
E., Amante, E.R., Beddin Fritzen-Freire, C., Bremer Boaventura, B.C., de
Bona Munoz, I., dos Santos Branco, C., Salvador, M., Maraschin, M. 2013.
Concentration of biologically active compounds extracted from Ilex paraguar-
iensis St. Hil. by nanofiltration. Food Chemistry, 141: 60–65.
Murugesan, R., Orsat, V. 2012. Spray drying for the production of nutraceutical
ingredients—A review. Food and Bioprocess Technology, 5: 3–14.
Nathakaranakule, A., Jaiboon, P., Soponronnarit, S. 2010. Far-infrared radiation
assisted drying of longan fruit. Journal of Food Engineering, 100: 662–668.
Nazni, P., Thara, D.K. 2011. Optimization of beetroot peel osmotic dehydration
process using response surface methodology. International Journal of Current
Research, 3: 27–32.
Niamnuy, C., Nachaisin, M., Laohavanich, J., Devahastin, S. 2011. Evaluation of bioac-
tive compounds and bioactivities of soybean dried by different methods and
conditions. Food Chemistry, 129: 899–906.
Nindo, C.I., Tang, J. 2007. Refractance window dehydration technology: A novel con-
tact drying method. Drying Technology, 25: 37–48.
Nireesha, G.R., Divya, L., Sowmya, C., Venkateshan, N., Niranjan Babu, M.,
Lavakumar, V. 2013. Lyphilization/freeze drying—An review. International
Journal of Novel Trends in Pharmaceutical Sciences, 3: 87–98.
Nurhadi, B., Andoyo, R., Indiarto, M.R. 2012. Study the properties of honey powder
produced from spray drying and vacuum drying method. International Food
Research Journal, 19: 907–912.
O’Brien, J., Nursten, H.E., Crabbe, M.J.C., Ames, J.M. 1998. The Maillard Reaction in
Foods and Medicines. Royal Society of Chemistry, Cambridge.
Orsat, V., Yang, W., Changrue, V.,Raghavan, G.S.V. 2007. Microwave-assisted drying
of biomaterials. Food and Bioproduct Processing, 85: 256–263.
Ozen, B.F., Dock, L.L., Ozdemir, M., Floros, J.D. 2002. Processing factors affecting the
osmotic dehydration of diced green peppers. International Journal of Food Science
and Technology, 37: 497–502.

Paez, R., Lavari, L., Vinderola, G., Audero, G., Cuatrin, A., Zaritzky, N., Reinheimer,
J. 2012. Effect of heat treatment and spray drying on lactobacilli viability and
resistance to simulated gastrointestinal digestion. Food Research International,
48: 748–754.
Page, G.-E. 1949. Factors influencing the maximum rates of air drying shelled corn in
thin layer. Master’s thesis, Purdue University (unpublished).
Pallas, L.A., Pegg, R.B., Shewfelt, R.L., Kerr, W.L. 2012. The role of processing con-
ditions on the color and antioxidant retention of jet tube fluidized bed-dried
blueberries. Drying Technology, 30: 1600–1609.
Palthur, M.P., Palthur, S.S.S., Chitta, S.K. 2010. Nutraceuticals: A conceptual defini-
tion. International Journal of Pharmacy and Pharmaceutical Sciences, 2: 19–27.
Panyawong, S., Devahastin, S. 2007. Determination of deformation of a food product
undergoing different drying methods and conditions via evolution of a shape
factor. Journal of Food Engineering, 78: 151–161.
Parsons, R.V., Cenkowski, S., Zhang, Q. 2008. Effects of alcohols versus water addi-
tion on the bulk solids frictional behaviour of flax shive. Canadian Biosystems
Engineering, 50: 3.67–3.75.
Paul, R., Ghosh, U. 2012. Effect of thermal treatment on ascorbic acid content of
pomegranate juice. Indian Journal of Biotechnology, 11: 309–313.
Peighambardoust, S.H., Tafti, A.G., Hesari, J. 2011. Application of spray drying for
preservation of lactic acid starter cultures: A review. Trends in Food Science and
Peinado, I., Rosa, E., Heredia, A., Andres, A. 2011. Influence of dry and wet osmotic
dehydration on colour and texture of a spread kiwi. In Proceedings of the 11th
International Congress on Engineering and Food (ICEF11): Food Process Engineering
in a Changing World, Athens, Greece, Volume III, eds. P.S. Taoukis, N.G. Stoforos,
V.T. Karathanos, G.D. Saravacos. May 22–26, 2011, pp. 1995–1996.
Pereira, L.M., Ferrari, C.C., Mastrantonio, S.D.S., Rodrigues, A.C.C., Hubinger, M.D.
2006. Kinetic aspects, texture, and color evaluation of some tropical fruits dur-
ing osmotic dehydraton. Drying Technology, 24: 475–484.
Perez-Serradilla, J.A., Luque de Castro, M.D. 2011. Microwave-assisted extraction
of phenolic compounds from wine lees and spray-drying of the extract. Food
Chemistry, 124: 1652–1659.
Pikal, M.J. 2004. Mechanisms of protein stabilization under freeze-drying and
storage: The relative importance of thermodynamic stability and glassy

state relaxation dynamics. In Freeze-Drying/Lyophilization of Pharmaceutical
and Biological Products, 2nd edition, ed. L. Rey and J.C. May. Marcel Dekker,
New York, pp. 63–108.
Pisalkar, P.S., Jain, N.K., Pathare, P.B., Murumkar, R.P., Revaskar, V.A. 2014. Osmotic
dehydration of aloe vera cubes and selection of suitable drying model.
International Food Research Journal, 21: 373–378.
Pronyk, C., Cenkowski, S., Muir, W.E. 2004. Drying foodstuffs with superheated
steam. Drying Technology, 22: 899–916.
Quintas, M., Guimaraes, C., Baylina, J., Brandao, T.R.S., Silva, C.L.M. 2007.
Multiresponse modelling of the caramelisation reaction. Innovative Food Science
and Emerging Technologies, 8: 306–315.
Quispe-Condori, S., Saldana, M.D.A., Temelli, F. 2011. Microencapsulation of flax oil
with zein using spray and freez drying. LWT—Food Science and Technology, 44:
1880–1887.

Raharitsifa, N., Ratti, C. 2010. Foam-mat freeze drying of apple juice. Part 1:
Experimental data and ANN simulations. Journal of Food Process Engineering,
33(2010): 268–283.
Rahman, M.S., Al-Rizeiqi, M.H., Guizani, N., Al-Ruzaiqi, M.S., Al-Aamri, A.H.,
Zainab, S. 2013. Stability of vitamin C in fresh and freeze-dried capsicum stored
at different temperatures. Journal of Food Science and Technology, 52: 1691–697.
Ramallo, L.A., Mascheroni, R.H. 2012. Quality evaluation of pineapple fruit during
drying process. Food and Bioproducts Processing, 90: 275–283.
Raoult-Wack, A.L. 1994. Recent advances in the osmotic dehydration of foods. Trends
in Food Science and Technology, 5: 255–260.
Ramya, H.G., Kumar, S., Kapoor, S. 2014. Optimization of osmotic dehydration pro-
cess for oyster mushrooms (Pleurotus sajor-caju) in sodium chloride solution
using RSM. Journal of Applied and Natural Science, 6: 152–158.
Ratti, C. 2001. Hot air and freeze-drying of high-value foods: A review. Journal of Food
Rey, L., May, J.C. 2004. Freeze-Drying/Lyophilization of Pharmaceutical and Biological
Products, 2nd edition. Marcel Dekker, New York.
Reyes, A., Mahn, A., Guzman, C., Antoniz, D. 2012. Analysis of drying of broccoli
florets in a fluidized pulsed bed. Drying Technology, 30: 1368–1376.
Ringeisen, B., Barrett, D.M., Stroeve, P. 2014. Concentrated solar drying drying of
tomatoes. Energy for Sustainable Development, 19: 47–55.
Rohloff, J., Dragland, S., Mordal, R., Iversen, T.-H. 2005. Effect of harvest time and
drying method on biomass production, essential oil yield, and quality of
peppermint (Mentha × piperita L.). Journal of Agricultural and Food Science, 53:
4143–4148.
Roos, Y.H. 2004. Phase and state transitions in dehydration of biomaterials and
foods. In Dehydration of Products of Biological Origin, ed. A.S. Mujumdar. Science
Publishers, Enfield, pp. 3–24.
Ruiz-Lopez, I.I., Garcia-Alvarado, M.A. 2007. Analytical solution for food-drying
kinetics considering shrinkage and variable diffusivity. Journal of Food
Ruiz-Lopez, I.I., Huerta-Mora, I.R., Vivar-Vera, M.A., Martínez-Sánchez, C.E.,
Herman-Lara, E. 2010. Effect of osmotic dehydration on air-drying characteris-
tics of chayote. Drying Technology, 28: 1201–1212.
Sadykov, R.A. 2004. Optimal technology for drying labile bioproducts. In Dehydration
of Products of Biological Origin, ed. A.S. Mujumdar. Science Publishers, Enfield,
pp. 437–466.
Sansone, F., Mencherini, T., Picerno, P., d’Amore, M., Aquino, R.P., Lauro, M.R. 2011.
Maltodextrin/pectin microparticles by spray drying as carrier for nutraceutical
extracts. Journal of Food Engineering, 105: 468–476.
Santivarangkna, C., Aschenbrenner, M., Kulozik, U., Foerst, P. 2011. Role of glassy
state on stabilities of freeze-dried probiotics. Journal of Food Science, 76:
R152–R156.
Santos, P.H.S., Silva, M.A. 2008. Retention of vitamin C in drying processes of fruits
and vegetables—A review. Drying Technology, 26: 1421–1437.
Scher, C.F., De Oliveira Rios, A., Norena, C.P.Z. 2009. Hot air drying of yacon
(Smallanthus sonchifolius) and its effect on sugar concentrations. International
Journal of Food Science and Technology, 44: 2169–2175.
Shamlou, P.A. 1988. Handling of Bulk Solids: Theory and Practice. Butterworths, London.

Shaw, M., Meda, V., Tabil, L., Leduc, P. 2005. Development and trends in drying
of herbs and specialty crops in Western Canada. CSAE Paper No. 05–030.
Canadian Society for Bioengineering.
Sheikholeslamzadeh, E., Rohani, S. 2012. Solubility prediction of pharmaceutical and
chemical compounds in pure and mixed solutions using predictive models.
Industrial and Engineering Chemistry, 51: 464–475.
Shi, J., LeMaguer, A. 2000. Lycopene in tomatoes: Chemical and physical proper-
ties affected by food processing. Critical Reviews in Food Science and Nutrition,
40: 1–42.
Shi, Q., Zheng, Y., Zhao, Y. 2014. Optimization of combined heat pump and micro-
wave drying of yacon (Smallanthus sonchifolius) using response surface method-
ology. Journal of Food Processing and Preservation, 38: 2090–2098.
Shrivastav, S., Kumbhar, B.K. 2011. Drying kinetics and ANN modeling of paneer
at low pressure superheated steam. Journal of Food Science and Technology, 48:
577–583.
Shui, G., Leong, L.P. 2006. Residue from star fruit as valuable source for functional
food ingredients and antioxidants. Food Chemistry, 97: 277–284.
Silva, F.A., Marsaioli, A., Maximo, G.J., Silva, M.A.A.P., Goncalves, L.A.G. 2006.
Microwave assisted drying of macadamia nuts. Journal of Food Engineering, 77:
550–558.
Simal, S., Femenia, A., Garau, M.C., Rossello, C. 2005. Use of exponential, Page’s and
diffusional models to simulate the drying kinetics of kiwi fruit. Journal of Food
Somjai, T., Achariyaviriya, S., Achariyaviriya, A., Namsanguan. K. 2009. Strategy
for longan drying in two-stage superheated steam and hot air. Journal of Food
Souza, C.F., Oliveira, W.P. 2012. Drying of phytochemical preparations in a spouted
bed: Perspectives and challenges. Drying Technology, 30: 1209–1226.
Sovova, H., Stateva, R.P. 2011. Supercritical fluid extraction of vegetable materials.
Reviews in Chemical Engineering, 27: 79–156.
Srivastava, S., Mishra, G. 2010. Fluid bed technology: Overview and parameters
for process selection. International Journal of Pharmaceutical Sciences and Drug
Research, 2: 236–246.
Stevenson, D.E., Hurst, R.D. 2007. Polyphenolic phytochemicals—Just antioxidants or
much more? Cellular and Molecular Life Sciences, 64: 2900–2916.
Stojanovic, J., Silva, J.L. 2007. Influence of osmotic concentration, continuous high
frequency ultrasound and dehydration on antioxidants, colour and chemical
properties of Rabbiteye blueberries. Food Chemistry, 101: 898–906.
Summen, M.A., Erge, H.S. 2014. Thermal degradation kinetics of bioactive com-
pounds and visual color in raspberry pulp. Journal of Food Processing and
Preservation, 38: 551–557.
Sutherland, K.S. 2008. Filters and Filtration Handbook, 5th edition. Elsevier Science,
Burlington.
Suvarnakuta, P., Chaweerungrat, C., Devahastin, S. 2011. Effects of drying meth-
ods on assay and antioxidant activity of xanthones in mangosteen rind. Food
Chemistry, 125: 240–247.
Tang, C.-H., Li, X.-R. 2013. Microencapsulation properties of soy protein isolate and
storage stability of the correspondingly spray dried emulsions. Food Research

Tarleton, E.S., Wakeman, R.J. 2007. Solid/Liquid Separation Equipment Selection and
Process Design. Elsevier Science, Burlington.
Tewa-Tagne, P., Briancon, S., Fessi, H. 2007. Preparation of redispersible dry nanocap-
sules by means of spray-drying: Development and characterization. European
Journal of Pharmaceutical Sciences, 30: 124–135.
Teixeira, C.C.C., Taixeira, G.A., Freitas, L.A.P. 2011. Spray drying of extracts from red
yeast fermentation broth. Drying Technology, 29: 342–350.
Therdthai, N., Zhou, W. 2009. Characteristics of microwave vacuum drying and
hot air drying of mint leaves (Mentha cordifolia Opiz ex Fresen). Journal of Food
Tonon, R.V., Brabet, C., Hubinger, M.D. 2008. Influence of process conditions on the
physicochemical properties of acai (Euterpe oleraceae Mart.) powder produced
by spray drying. Journal of Food Engineering, 88: 411–418.
Tonon, R.V., Brabet, C., Hubinger, M.D. 2010. Anthocyanin stability and antioxidant
activity of spray-dried acai (Euterpe oleracea Mart.) juice produced with differ-
ent carrier agents. Food Research International, 43: 907–914.
Toontom, N., Meenune, M., Posri, W., Lertsiri, S. 2012. Effect of drying method on
physical and chemical quality, hotness and volatile flavor characteristics of
dried chili. International Food Research Journal, 19: 1023–1031.
Torreggiani, D. 1993. Osmotic dehydration in fruit and vegetable processing. Food
Tran, T.H., Nguyen, M.H., Zabaras, D., Vu, L.T.T. 2008. Process development of Gac
powder by using different enzymes and drying techniques. Journal of Food
Tsinontides, S.C., Rajniak, P., Pham, D., Hunke, W.A., Placek, J., Reynolds, S.D. 2004.
Freeze drying—Principles and practice for successful scale-up to manufactur-
ing. International Journal of Pharmaceutics, 280: 1−16.
Turner, C. 2006. Modern Extraction Techniques: Food and Agricultural Samples. ACS
Symposium Series 926. American Chemical Society, Washington.
Uribe, E., Miranda, M., Vega-Galvez, A., Quispe, I., Claveria, R., Di Scala, K. 2011.
Mass transfer modeling during osmotic drying of jumbo squid (Dosidicus gigas)
influence of temperature on diffusion coefficients. Food Bioprocess Technology, 4:
320–326.
Van Den Burg, C. 1986. Water activity. In Concentration and Drying of Foods, ed.
D. MacCarthy. Elsevier Applied Science, New York, pp. 11–51.
VanGarde, S.J., Woodburn, M. 1994. Food Preservation and Safety: Principles and Practice.
Iowa State University Press, Ames.
Vashisth, T., Singh, R.K., Pegg, R.B. 2011. Effects of drying on the phenolics con-
tent and antioxidant activity of muscadine pomace. LWT—Food Science and
Technology, 44: 1649–1657.
Vega-Galvez, A., Ah-Hen, K., Chacana, M., Vergara, J., MartÍnez-Monzo, J., GarcÍa-
Segovia, P., Lemus-Mondaca, R., Di Scala, K., 2012. Effect of temperature and air
velocity on drying kinetics, antioxidant capacity, total phenolic content, colour,
texture and microstructure of apple (var. Granny Smith) slices. Food Chemistry,
132: 51–59.
Vega-Galvez, A., Puente-Diaz, L., Lemus-Mondaca, R., Miranda, M., Torres,
M.J. 2014. Mathematical modelling of thin-layer drying kinetics of Cape
Gooseberry (Physalis peruviana L.). Journal of Food Processing and Preservation,
38: 728–736.

Vega-Galvez, A., San Martin, R., Sanders, M., Miranda, M., Lara, E. 2010. Characteristics
and mathematical modeling of convective drying of Quinoa (Chemopodium qui-
noa willd.): Influence of temperature on the kinetic parameters. Journal of Food
Processing and Preservation, 34: 945–963.
Wang, Y., Zhang, M., Mujumdar, A.S. 2011. Trends in processing technologies for
dried aquatic products. Drying Technology, 29: 382–394.
Williams, G.D., Jofriet, J.C., Rosentrater, K.A. 2008. Biomass storage and handling:
Status and industry needs. ASABE Paper No. 08–2081. St. Joseph: American
Society of Agricultural and Biological Engineers.
Wu, B., Pan, Z., Wang, W.Q.B., Wang, J., Ma, H. 2014. Effect of simultaneous infrared
dry-blanching and dehydration on quality characteristics of carrot slices. Food
Xianquan, S., Shi, J., Kakuda, Y., Yueming, J. 2005. Stability of lycopene during food
processing and storage. Journal of Food Medicine, 8: 413–422.
Xiao, H., Pang, C., Wang, L., Bai, J., Yang, W., Gao, Z. 2010. Drying kinetics and quality
of Monukka seedless grapes dried in an air-impingement jet dryer. Biosystems
Xiao, H.-W., Yao, X.-D., Lin, H., Yang, W.-X., Meng, J.-S., Gao, Z.-J. 2012. Effect of
SSB (superheated steam blanching) time and drying temperature on hot air
impingement drying kinetics and quality attributes of yam slices. Journal of
Food Process Engineering, 35: 370–390.
Xu, L., Kumar, A., Lamb, K., Layton, L. 2006. Recovery of isoflavones from red clo-
ver flowers by a membrane-based process. Innovative Food Science and Emerging
Xu, S., Pegg, R.B., Kerr, W.L. 2013. Sensory and physicochemical properties of sweet
potato chips made by vacuum-belt drying. Journal of Food Process Engineering,
36: 353–363.
Yeo, J.-D., Park, J.-W., Lee, J.-H. 2011. Evaluation of antioxidant capacity of sesamol
and free radical scavengers at different heating temperatures. European Journal
of Lipid Science and Technology, 113: 910–915.
Zecchi, B., Garreiro, Martínez, J., Gerla, P. 2011. Modeling and minimizing process
time of combined convective and vacuum Drying of mushrooms and parsley.
Journal of Food Engineering, 104: 49–55.
Zhang, M., Tang, J., Mujumdar, A.S., Wang, S. 2006. Trends in microwave related dry-
ing of fruits and vegetables. Trends in Food Science and Technology, 17: 524–534.
Zhang, Z.F., Lv, G.Y., Pan, H.J., Wu, Y.Z., Fan, L.F. 2009. Effects of different dry-
ing methods and extraction condition on antioxidant properties of shiitake
(Lentinus edodes). Food Science and Technology Research, 15: 547–552.
Zhu, A., Shen, X. 2014. The model and mass transfer characteristics of convection
drying of peach slices. International Journal of Heat and Mass Transfer, 72: 345–351.
Zhu, D., Damodaran, S. 2011. Composition, thermotropic properties, and oxidative
stability of freeze dried and spray dried milk fat globule membrane isolated
from cheese whey. Journal of Agricultural and Food Chemistry, 59: 8931–8938.
Zielinska, M., Zapotoczny, P., Alves-Filho, O., Eikevik, T.M., Blaszczak, W. 2013. A
multi-stage combined heat pump and microwave vacuum drying of green
peas. Journal of Food Engineering, 115: 347–356.

19
Biological Antioxidation Mechanisms:
Quenching of Peroxynitrite
Takashi Maoka and Hideo Etoh
CONTENTS
19.1 Antioxidative Activity of Carotenoids..................................................... 589
19.2 Reactive Nitrogen Species and Production of Peroxynitrite................ 591
19.3 Scavenging Peroxynitrite........................................................................... 592
19.4 Carotenoids and RNS................................................................................. 594
19.5 Reaction of Carotenoids with Peroxynitrite............................................ 595
19.5.1 Astaxanthin..................................................................................... 595
19.5.2 β-Carotene....................................................................................... 597
19.5.3 Lutein................................................................................................ 597
19.5.4 Capsanthin and Fucoxanthin........................................................ 598
19.5.5 Lycopene.......................................................................................... 599
19.6 Inhibition of Nitration of Tyrosine with Peroxynitrite by
Carotenoids..................................................................................................600
19.7 Biological Activity of Nitrocarotenoids................................................... 601
19.7.1 Quenching Effects of Singlet Oxygen (1O2)
by Nitrocarotenoids........................................................................ 601
19.7.2 Antitumor-Promoting and Anticarcinogenesis Activities
of Nitrocarotenoids......................................................................... 602
19.8 Summary......................................................................................................604
References.............................................................................................................. 605
19.1 Antioxidative Activity of Carotenoids

Carotenoids exhibit red, orange, and yellow colors, and are distributed in
microorganisms, plants, and animals. They are tetraterpene pigments con-
sisting of eight isoprenoid units having 40 carbon skeletons. Their structures
commonly consist of polyene chains with nine conjugated double bonds near
the center of the molecule and end groups at both sides of the polyene chain.
Carotenoids are divided into two groups, that is, carotenes and xanthophylls
(Britton et al., 2004). Carotenes are hydrocarbons consisting of carbon and
hydrogen atoms. Therefore, they are nonpolar compounds. Typical carotenes
589
are β-carotene, widely distributed in plants and animals, and lycopene a

characteristic carotenoid in tomato. On the contrary, xanthophylls contain
oxygen atoms, which can be represented by hydroxyl, carbonyl, carboxylic,
epoxide, and/or lactone groups in the carotene skeleton. Typical xantho-
phylls are lutein, which is widely distributed in plants, astaxanthin, which is
a characteristic red pigment in salmon and crustacean, capsanthin, a charac-
teristic red pigment in paprika, and fucoxanthin a characteristic pigment in
sea weeds. These carotenoids occur in several vegetables and sea food and
are important functional nutrients for humans (Maoka and Etoh, 2011).
Carotenes are nonpolar compounds, therefore they orient the hydrophobic
core of the cell membrane. On the contrary, xanthophylls such as astaxanthin
have polar 3-hydroxy-4-keto-β-end groups on both sides of the nonpolar
polyene chain. This polar–nonpolar–polar layout also allows the astaxan-
thin molecule to take a trans-membrane orientation, making a precise fit into
the polar–nonpolar–polar span of the cell membrane (Shibata et al., 2001;
Gruszecki, 2009). Figure 19.1 shows the structure of a typical carotene and
xanthophylls.
Carotenoids have excellent quenching activity for singlet oxygene and
lipid peroxidation. Foote and Denny (1968) were first to demonstrate that
β-carotene has a strong quenching activity for singlet oxygen. Since then,
it has been shown that several natural carotenoids with more than 11 con-
jugated double bonds show excellent quenching activity for singlet oxygen.
Among them, astaxanthin, lycopene, and capsorubin exhibited the strongest
quenching activities for singlet oxygen (Hirayama et al., 1991; Miki, 1991;
Shimizu et al., 1996). The mechanism for quenching of singlet oxygen is a
physical reaction. Carotenoids take up thermal energy from singlet oxygen
and release this energy by polyene vibration. Singlet oxygen is always gen-
erated during a photosynthetic reaction. Therefore, carotenoids are essen-
tial compounds in plants and photosynthetic organisms. Singlet oxygen
is also generated in humans during phagocytolysis of macrophages and
β-Carotene Lycopene
O
OH OH
HO Astaxanthin HO Lutein
O OH OH
O .
O
HO OH
Capsanthin O
HO
Fucoxanthin
FIGURE 19.1
Structure of typical carotene and xanthophylls.

Biological Antioxidation Mechanisms 591
neutrophiles and causes some inflammation and arteriosclerosis. Oshima

et al. (1999) reported on a carotenoid-rich, low-density lipoprotein (LDL)
that was prepared by humans ingesting long-term supplementation with
tomato juice, which inhibited the formation of singlet oxygen-mediated lipid
peroxidation better than the LDL devoid of carotenoid. Furthermore, it was
reported that supplementation with astaxanthin could increase the antiox-
idative activity of human LDL (Iwamoto et al., 2000). Therefore, it can be
strongly suggested that carotenoids suppress oxidative damage induced by
singlet oxygen, in humans.
Active oxygen and free radicals induce a chain reaction of lipid peroxi-
dation. Burton and Ingold (1984) found that carotenoids showed excellent
antioxidative activities against lipid peroxidation at low oxygen pressure.
Oxygen pressure in tissues and cells is 40 and 1 mmHg, respectively.
Therefore, it was assumed that carotenoids might act as excellent antioxi-
dants in human tissues and cells. The antioxidative activity of carotenoids
is influenced by the number of conjugated double bonds. Carotenoids hav-
ing long conjugated double bond systems showed excellent inhibitory effects
on lipid peroxidation. It was reported that astaxanthin, canthaxanthin, cap-
santhin, and capsorubin having conjugated carbonyl groups showed the
strongest quenching activities for lipid peroxidation, among the naturally
occurring carotenoids (Terao, 1989; Miki, 1991; Lim et al., 1992).
The molecular mechanisms for the reaction of active oxygen and free rad-
icals with carotenoids have been discovered. Carotenoids take up oxygen
from active oxygen into their conjugated double bond system to form apocar-
otenals, apocarotenones, and epoxy carotenoids (Kim et al., 2001).
Until now, it has been assumed that carotenoids do not directly scav-
enge superoxide and hydroxyl radicals (Trevithick-Sutton et al., 2006).
However, recent studies have shown that β-carotene, lycopene, zeaxan-
thin, lutein (Trevithik-Sutton et al., 2006), fucoxanthin, fucoxanthinol,
and halocynthiaxanthi (Sachindra et al., 2008) could scavenge superoxide
and hydroxyl radicals. Furthermore, some carotenoids could inhibit the
generation of superoxide or nitric oxide (NO) radicals in cells (Murakami
et al., 2000).
19.2 Reactive Nitrogen Species and Production

of Peroxynitrite
As well as reactive oxygen species (ROS), reactive nitrogen species (RNS)
such as nitric oxide (.NO), nitrogen dioxide (.NO2), peroxynitrite (ONOO−),
and peroxynitrous acid (ONOOH) also cause several oxidative damages to
bio-molecules such as lipid, amino acid, protein, nucleic acid and induced
inflammatory and progression to cancer, etc. Among them, peroxynitrite

(ONOO−), which is formed from superoxide ( O −2 ) and nitric oxide (NO•)

in vivo, is a highly reactive oxidant as shown below.
•
NO (nitric oxide) + • O −2 (superoxide ) → ONOO − (peroxynitrite)

Peroxynitrite can react directly with proteins that contain transition

metal centers. Therefore, it can modify proteins such as hemoglobin,
myoglobin, and cytochrome c by oxidizing ferrous heme into its corre-
sponding ferric forms. Peroxynitrite may also be able to change protein
structure through the reaction with various amino acids in the peptide
chain. The most common reaction with amino acids is cysteine oxida-
tion. Another reaction is tyrosine nitration; however peroxynitrite does
not react directly with tyrosine. Tyrosine reacts with other RNS that are
produced by peroxynitrite. All of these reactions affect protein structure
and function and thus have the potential to cause changes in the catalytic
activity of enzymes, altered cytoskeletal organization, and impaired cell
signal transduction. Peroxynitrite also react with phenolic bio-molecules
such as tyrosine, guanine, tryptophan, etc., to form corresponding nitro-
compounds. These compounds caused several biological damages such
as inflammation and progression of cancer (Halliwell, 1994; Wiseman
and Halliwell, 1996; Scheidegger et al., 1998; Pannala et al., 1999; Valko
et al., 2007).
19.3 Scavenging Peroxynitrite
Peroxynitrite was formed by superoxide and nitric oxide in vivo. Therefore,
superoxide and/or nitric oxide scavengers can inhibit the formation of
peroxynitrite. It was reported that Apocynin (4-hydroxy-3-methoxy-
acetophenone) (Muijsers et al., 2000) and hydrazine (Daiber et al., 2005)
effectively inhibited peroxynitrite formation. Uric acid, a purine metabo-
lite, is a well-known peroxynitrite scavenger (Hopper et al., 1998, 2000).
Furthermore, phenolic compounds such as zingerone (Shin et al., 2005),
flavonids (Pannala et al., 1999), cinapic acid (Niwa et al., 1999), antocyanin
(Tsuda et al., 2000), and polyphenol (Ferroni et al., 2004) can directly scav-
enge peroxynitrite. For example, phenolic compounds such as p-coumaric
acid and pelargonidin could scavenge peroxynitrite by the formation of
nitro-p-coumaric acid and nitro-pelargonidin, respectively, and protect
against the nitration of tyrosine (Kato et al., 1997; Niwa et al., 2001; Tsuda
et al., 2000). This mechanism was shown in Figure 19.2. Similar results were
reported for γ-tocopherol (Goss et al., 1999; Mortion et al., 2002; Williamson
et al., 2002).

Biological Antioxidation Mechanisms

OH OH
CH2 CH2 HO
NO2 Nitro-γ-tocopherol
R N CH C R R N CH C R
H H
O O
Nitration
Tyrosine Tyrosine
ONOO– O
HO
γ-Tocopherol
OH
Oxidation Nitration
HO O+ Oxidation
OH
OH OH
OH O
HO HO
NO2
H2O –H2O O
O O
OH O Tocored
OH p-Hydroxybenzoic acid 4-Hydroxy-3-nitrobenzoic acid
HO O
OH
OH Pelargonidin
FIGURE 19.2
Inhibitory of nitration of tyrosine by pelargonidin and γ-tocopherol. (Adapted from Tsuda, T., Kato, K., Osawa, T. 2000. FEBS Letters, 484: 207; Goss,
593
S.P.A. Hogg, N., Kalyanaraman, B. 1999. Archives of Biochemistry and Biophysics, 363: 333.)
19.4 Carotenoids and RNS

It has been reported that β-carotene is an effective scavenger of nitrogen
dioxide (NO2), peroxynitrous acid (ONOOH), and peroxynitrite, and that
the nitrogen atoms derived from nitrogen dioxide were tightly bound to
the β-carotene molecule (Kikugawa et al., 1997). Scheidegger et al. (1998)
studied the reaction of peroxynitrite with zeaxanthin in liposomes and
reported that zeaxanthin was reacted with peroxynitrite. Furthermore, they
hypothesized that zeaxanthin plays a major role in protection of macular
tissue from oxidative damage. Interaction of peroxynitrite with carotenoids
and tocopherols within LDL were studied by Pannala et al. (1999). They
found that carotenes, lycopene, and β-carotene in LDL were consumed
when exposed to peroxynitrite. Among them, lycopene showed more reac-
tivity than β-carotene. Panasenko et al. (2000) studied the interaction of
peroxynitrite with carotenes (lycopene, α-carotene, and β-carotene) and
xanthophylls (β-cryptoxanthin, zeaxanthin, and lutein) in a homogeneous
solution and in human LDLs. They found that all carotenoids prevented the
formation of rhodamine 123 from dihydrorhodamine 123 caused by per-
oxynitrite. Xanthophylls were as effective as biothiols, known as scavengers
of peroxynitrite, whereas lycopene, α-carotene, and β-carotene exhibited a
considerably more pronounced effect. These findings suggest that carot-
enoids can efficiently react with peroxynitrite and perform the role of scav-
engers of peroxynitrite in vivo. Muzandu et al. (2006) studied the effect of
the carotenoids, lycopene, and β-carotene, on peroxynitrite-mediated modi-
fications in DNA and proteins. These carotenoids strongly inhibited DNA
strand breaks caused by peroxynitrite protein tyrosine nitration at the phys-
iological concentrations. The results suggest that carotenoids may allevi-
ate some of the deleterious effects of peroxynitrite and possibly other RNS
as well in vivo. Lutein, zeaxanthin, and astaxanthin were found to protect
against DNA damage in SK-N-SH human neuroblastoma cells induced by
RNS (Santocono et al., 2007). Rodrigues et al. (2012) studied that scavenging
capacity of marine carotenoids against ROS and RNS in a membrane-mim-
icking system. They found that the carotenoid-bearing hydroxyl groups
were generally more potent ROS scavengers than the carotenes, whilst
β-carotene was the most efficient ONOO− scavenger. The role of astaxanthin
as an antioxidant should be highlighted, since it was a more potent scaven-
ger of ROO•, HOCl, and ONOO− than α-tocopherol.
Furthermore, Maoka et al. (2006) studied prevention activity of paprika
carotenoids and capsanthin on nitric oxide and peroxynitrite-induced car-
cinogenesis of mouse. They found that paprika carotenoids and capsanthin
inhibited the initiation stage of two-stage carcinogenesis, initiated by nitric
oxide and peroxynitrite on the skin of mice. These reports suggested that
carotenoid could inhibit oxidative damage caused by active nitrogen species
in vivo.

19.5 Reaction of Carotenoids with Peroxynitrite

19.5.1 Astaxanthin
Yoshioka et al. (2006) and Hayakawa et al. (2008) investigated the reaction of
carotenoids with peroxynitrite in vitro. The HPLC of reaction products with
astaxanthin is shown in Figure 19.3.
The major reaction products of astaxanthin with peroxynitrites are
aposataxanthins, nitroastaxanthins, and cis-astaxanthins as shown in
Figure 19.3. Several apoastaxanthinals such as 8′-, 10′’-, and 12′-apoastaxan-
thin were formed by reaction. Furthermore, nitrated astaxanthins, named
nitroastaxanthins, were obtained from this reaction as major products.
They were determined to be 14′-s-cis-15′-nitroastaxanthin (1), 10′-s-11′-cis-11′-
nitroastaxanthin (2), 9-cis-14′-s-cis-15′-nitroastaxanthin (3), 13-cis-14′s-cis-15′-
nitroastaxanthin (4), and 13-,15-,13′-tri-cis-15′-nitroastaxanthin (5) as shown
in Figure 19.4. Structures of these compounds were determined based on
FAB MS and NMR spectral data. All nitroastaxanthins obtained by this
reaction showed cis configurations in the polyene chain. These stereo struc-
tures were elucidated based on NOE data (Yoshioka et al., 2006; Hayakawa
et al., 2008). Among the nitroastaxanthins, 14′-s-cis-15′-nitroastaxanthin was
found to be the main product. Due to the bulky size of the nitro-group,
nitration mainly occurred at C-11 (11′) or C-15 (15′) positions in astaxan-
thin because these positions are unhindered spaces in the polyene chain.
Furthermore, nitration occurred through isomerization of the polyene chain
to form a sterically stable cis conformation (Yoshioka et al., 2006; Hayakawa
et al., 2008).
Apoastaxanthins Nitroastaxanthins
Astaxanthin
cis-astaxnthins
0 10 20 30 40 50 min
FIGURE 19.3
HPLC of peroxynitrite reaction products of astaxanthin. (From Hayakawa, T. et al. 2008.
Bioscience, Biotechnology, and Biochemistry, 72: 2716.)

OH
NO2 O
15′
HO
O
HO NO2
O
10′-s-11′-cis-11′-nitroastxanthin (2)
OH
O
OH O
14′-s-cis-15′-nitroastxanthin (1)
HO
OH O NO2
O 13-cis-14′-s-cis-15′-nitroastxanthin (4)
HO O
O NO2 OH
HO
O
NO2
9-cis-14′-s-cis-15′-nitroastxanthin (3) 13,15,13′-tri-cis-15′-nitroastxanthin (5)
FIGURE 19.4
Structure of nitroastaxanthins.
Furthermore, aponitroastaxanthins were also obtained as minor com-

ponents. They were 12′-apo-15′-nitroastaxanthinal (6) and 13-apo-8-
nitroastaxanthinone (7) (Figure 19.5). They were assumed to be further
oxidative cleavage product of nitroastaxanthins or nitration products of
apoastaxanthins (Hayakawa et al., 2008).
From the results, the following three pathways can be considered for
the scavenge mechanism of peroxynitrite by astaxanthin. In the first one,
astaxanthin accepts energy from peroxynitrite and goes to an excited
state (biradical), and while returning to the ground state produces cis-
isomers. In the second one, astaxanthin directly reacts with peroxynitrite
NO2 O
15′
HO NO2
O HO
H O O
12′-Apo-15′-nitroastxanthinal (6) 13-Apo-8-nitroastaxanthinone (7)
FIGURE 19.5
Structure of apo-nitroastaxanthins.

to produce a dioxetane that cleaves to apo-astaxanthinals. The third path-

way, astaxanthin uptakes nitro group from peroxynitrite by the formation
of nitroastaxanthins.
The formation mechanism of nitrocarotenoid can be considered as follows
(Yoshioka et al., 2006; Hayakawa et al., 2008).
The first •NO2 was formed from peroxynitrite as shown in the following
reaction:
ONOO − + ONOOH → ONOO − + • NO 2 + OH −
Then, •NO2 removed hydrogen from astaxanthin (AST) to form astaxan-

thin radical.
AST + • NO 2 → • AST + • NO 2 + • H
Next, •NO2 reacted with astaxanthin radical to form nitroastaxanthin.
• AST + •NO2 → AST -NO2
β -Carotene
19.5.2
Similar to astaxanthin, β-carotene also produced nitro-β-carotenes, 14′-s-cis-
15’-nitro-β-carotene (8) and 10’-s-11’-cis-11’-nitro-β-carotene (9) (Figure 19.6),
as a major reaction product with peroxynitrite along with apo-β-carotenes
and cis-β-carotenes (Yoshioka et al., 2006).
19.5.3 Lutein
Lutein has an asymmetric structure. As well as astaxanthin and
β-carotene, lutein also formed nitroluteins, 14-s-cis-15-nitrolutein (10)
NO2
15′
11′
NO2
10′-s-11′-cis-11′-nitro-β-carotene (9)
14′-s-cis-15′-nitro-β-carotene (8)
FIGURE 19.6
Structure of nitro-β-carotene.

HO
NO2
HO
NO2
HO OH
14-s-cis-15-nitro-lutein (10) 14′-s-cis-15′-nitro-lutein (11)
OH
N
HO O
Lutein-6H-1,2-oxzazine (12)
FIGURE 19.7
Structures of nitroluteins and lutein-6H-1,2-oxazine.
and 15’-s-cis-15’-nitrolutein (11), by reaction with peroxynitrite (Tsuboi

et al., 2010). In addition to these nitroluteins, lutein-6H-1,2-oxazine (12)
(Figure 19.7) was also formed by this reaction. This compound was a first
report of the oxazine derivatives of carotenoids. The formation mecha-
nism of lutein-6H-1,2-oxazine was uncertain. However, it was assumed
that this compound was formed through 8-nitrolutein, which was pro-
duced by the nitration of lutein. 8-Nitrolutein was assumed to be unsta-
ble. Therefore, it was quickly converted into lutein-6H-1,2-oxazine
(Tsuboi et al., 2010). These results indicate that lutein is able to capture
peroxynitrite and n itrogen dioxide radicals from their molecules to form
nitrocarotenoids.
19.5.4 Capsanthin and Fucoxanthin

Capsanthin is a characteristic carotenoid in red pepper. Fucoxanthin is
a major carotenoid in sea weed. These carotenoids have conjugated car-
bonyl group at the eng position of the conjugated double bonds. They also
formed nitrocompounds by reaction with peroxynitrite (Tsuboi et al., 2011).
These are 14’-cis-15’-nitrocapsanthin (13), 12-nitrocapsanthin (14), 14-cis-15-
nitrofucoxanthin (15), 11-cis-11-nitrofucoxanthin (16), and 14,9’-di-cis-15-
nitrofucoxanthin (17) as shown in Figure 19.8.

15
NO2
15′
HO 14′ O
12
HO NO2 OH
12-Nitrocapsanthin (14)
O
HO
(14′Z)-15′-nitrocapsanthin (13)
NO2
O O O
11
HO
O
HO .
NO2
15
NO2
. 15
O
14 .
HO
O
OH O
O
O O
O O
HO
(11Z)-11- (14Z,9′Z)-15-
HO nitrofucoxanthin (16) nitrofucoxanthin (17)
(14Z)-15-
nitrofucoxanthin (15)
FIGURE 19.8
Structures of nitrocapsanthins (13), (14) and nitrofucoxanthins (15)–(17).
19.5.5 Lycopene
Lycopene has 11 conjugated double bonds in the polyene chain with
an acyclic end group. Contrary to astaxanthin, β-carotene, lutein,
capsamthin, and fucoxanthin, having a cyclic end group, lycopene does
not produce a nitrated compound by this reaction, the reason is uncer-
tain. Detailed thermodynamic kinetic studies will be needed to reveal
this problem. The major reaction products were apolycopenals (18)–(21),
methoxy compounds, such as 5,5’-dimethoxylycopene (23) and 2,6-cyclo-
1,5-dimethoxylycopene (24) (Figure 19.9) and cis lycopenes (Yokota et al.,
2004). Apolycopenes were formed by oxidative cleavage through the diox-
etane structure. Methoxy compounds were assumed to be artifacts formed
by the nucleophilic addition of methanol, which was used as a solvent of
the reaction.

O O
6′-Apolycopenal (18) 8′-Apolycopenal (19)
O O
10′-Apolycopenal (20) 12′-Apolycopenal (21)
O
12′-Apolycopenal (22)
OCH3
OCH3
5,5′-Dimethoxylycopene (23)
H3CO
2,6-Cyclo-1,5-dimethoxylycopene (24)
OCH3
FIGURE 19.9
Structures of reaction products lycopene (18)–(24) with peroxynitrite.
19.6 Inhibition of Nitration of Tyrosine with

Peroxynitrite by Carotenoids
As described previously, peroxynitrite induces nitration of the aromatic ring
of free tyrosine and protein tyrosine residues and causes various diseases
(Halliwell, 1994; Scheidegger et al., 1998; Wiseman and Halliwell, 1996; Valko
et al., 2007; Pannala et al., 2008). It was reported that phenolic compounds
such as p-coumaric acid and pelargonidin could scavenge peroxynitrite by
the formation of nitro-p-coumaric acid and nitro-pelargonidin, respectively,
and protect against nitration of tyrosine (Kato et al., 1997; Tsuda et al., 2000;
Niwa et al., 2001). Similar results were reported for tocopherols (Goss et al.,
1999; Moriton et al., 2002; Williamson et al., 2002). Recently, Maoka et al.
(2012) reported that astaxanthin suppressed the formation of nitrotyrosine
to about 10.0% of that of the control group. This effect was almost the same
as that of γ-tocopherol, which is a well-known nitration inhibitor of tyro-
sin. Similar results were also obtained in the case of lutein, capsanthin,
and fucoxanthin (Tsuboi et al., 2011). These results indicate that astaxan-
thin, lutein, capsanthin, and fucoxanthin are able to capture peroxynitrite
to form nitroastaxanthin and nitrolutein and inhibit the tyrosine nitration.
•NO was formed from peroxynitrite as shown in the following reaction:
2
ONOO− + ONOOH → ONOO− + •NO2 + OH−

OH OH
NO2
CH2 Inhibit CH2

H2N C COOH H2N C COOH
H H
Tyrosine 3-Nitrotyrosine
ONOO–
Easliy react with carotenoid
Carotenoids Nitrocarotenoids
FIGURE 19.10
Inhibition of nitration of tyrosine with carotenoid.
As described previously, carotenoid uptakes •NO2 to form nitrocarot-

enoid. Carotenoids might have higher reactivity with proxynitrite and/or
•NO than phenol compounds such as tyrosine. Therefore, carotenoid could
2
inhibit nitration of tyrosine (Figure 19.10).
19.7 Biological Activity of Nitrocarotenoids

19.7.1 Quenching Effects of Singlet Oxygen (1O2) by Nitrocarotenoids
It is well known that carotenoids such as astaxanthin have an excellent
singlet oxygen activity. Nitrocarotenoids, which are reaction products of
carotenoids with peroxynitrite, also showed similar singlet oxygen activ-
ity. Maoka et al. (2012) examined the 1O2 quenching activity of nitroca-
rotenoids using the methylene blue-sensitized photooxidation method.
The IC50 values of 1O2 quenching activity were as follows: astaxanthin
7.0 μM, 15-nitroastaxanthin 20.0 μM, and β-carotene 100 μM. As well as
astaxanthin, 15-nitroastaxanthin also showed excellent 1O2 quenching
activity. This activity was slightly weaker than that of astaxanthin and
higher than that of β-carotene. It is reported that carotenoids with more
than 11 conjugated double bonds show excellent quenching activity for 1O2
and that the 1O2 quenching activity of carotenoids depends on the num-
ber of conjugated double bonds, polyene chain structures, and functional
groups. 15-Nitroastaxanthin has the same conjugated double bond system
as that of astaxanthin. Therefore, 15-nitroastaxanthin exhibited strong 1O2

quenching activity. Similar results were obtained in the case of 15-nitrolu-

tein (Maoka et al., 2012).
19.7.2 Antitumor-Promoting and Anticarcinogenesis

Activities of Nitrocarotenoids
The in vitro antitumor-promoting activity of some nitrocarotenoids was
examined using an Epstein–Barr virus early antigen (EBV-EA) activa-
tion assay in Raji cells (Tsuboi et al., 2011; Maoka et al., 2012). This assay
was used to estimate the antitumor-promoting effects of several natural
carotenoids (Tsushima et al., 1995). The results are shown in Table 19.1.
Both 15-nitroastaxanthin and 15-nitrolutein showed inhibitory effects on
the EBV-EA induction of Raji cells without significant cytotoxicity (more
than 60% viability of Raji cells) in this assay. 15-Nitroastaxanthin showed
slightly higher activity than astaxanthin. Furthermore, 15-nitrolutein also
showed slightly higher activity than lutein. Similar results were obtained
by nitrofucoxanthin and nitricapsanthins. Furthermore, nitrocapsanthins
and nitrofucoxanthins inhibited the proliferation of human pancreatic can-
cer cells (Tsuboi et al., 2011).
Furthermore, Maoka et al. (2012) examined the inhibitory effects of astaxan-
thin and 15-nitroastaxanthin on two-stage mouse skin carcinogenesis (Figure
19.11). There was a marked delay in the formation of papillomas and a reduction
in the average numbers of papillomas per mouse compared with the control
TABLE 19.1
Relative Rate of EBV-EA Activationa with Respect to the Positive Control
(100%) in the Presence of Carotenoids and Nitrocarotenoids
Concentration (mol ratio/TPA)b 1000 500 100 10 IC50
Compounds Values
Astaxanthin 5.0 (>70)c 29.0 78.0 100 307
15-Nitroastaxanthin 4.1 (>60) 28.5 76.9 100 300
Lutein 2.9 (>70) 24.5 75.4 97.6 283
15-Nitrolutein 1.6 (>60) 23.3 74.1 95.3 277
Capsanthin 5.5 (>60)c 29.4 78.1 100 310
12-Nitrocapsanthin 7.4 (>60) 30.9 79.7 100 346
(14′Z)-15′-Nitrocapsanthin 4.6 (>60) 26.9 76.8 100 305
Fucoxanthin 3.1 (>60) 26.8 76.6 100 296
(11Z)-11-Nitrofucoxanthin 1.3 (>60) 23.6 72.0 95.0 275
(14Z)-15-Nitrofucoxanthin 2.6 (>60) 25.0 74.2 100 282
Note: There are significant differences (P < 0.01) in inhibitory capacity in treat-

ment groups of compounds 1–5 compared with the control group.
a Values represent the percentage relative to the positive control values (100%).
b TPA concentration was 20 ng (32 pmol)/mol.
c Values within parentheses are percentage viability of Raji cells.

(a) 8
6
Papillomas/mouse
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Weeks after promotion
(b) 100
90
80
70
Papillomas (%)
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Weeks after promotion
FIGURE 19.11
Inhibitory effects of astaxanthin and 15-nitroastaxanthin on DMBA-induced mouse skin car-
cinogenesis. (a) Average number of papillomas per mouse; (b) percentage of mice-bearing
papillomas. ◆-◆ DMBA (390 nmol) + TPA (1.7 nmol), ▪-▪ Astaxanthin (85 nmol) + DMBA
(390 nmol) + TPA (1.7 nmol), ▴-▴15-Nitroastaxanthin (85 nmol) + DMBA (390 nmol) + TPA
(1.7 nmol).

group. Among them, 15-nitroastaxanthin showed slightly higher inhibitory

activity than astaxanthin. These results indicated that nitrocarotenoids them-
selves showed anticarcinogenesis effect. Therefore, it was assumed that carot-
enoids could inhibit peroxynitrite-induced damages such as oxidation of lipids
and protein, DNA strand scission, and induction of carcinogenesis.
19.8 Summary
Carotenoids having cyclic end groups such as β-carotene, astaxanthin,
lutein, capsanthin, and fucoxanthin scavenge peroxinitrite by the following
mechanism:
1. Carotenoids accept energy from peroxynitrite and go to an excited

state (biradical), and while returning to the ground state produce
cis-isomers.
2. Carotenoids directly react with peroxynitrite to produce a dioxetane
that cleaves to apo-carotenals.
3. Carotenoids uptake nitro group from peroxynitrite by the formation
of nitrocarotenoids.
Lycopene having a cyclic end group does not form nitrogenated com-
pounds. Lycopene scavenges peroxynitrite through (1) and (3) mechanisms.
These mechanisms are compiled in Figure 19.12.
Carotenoids uptake peroxynitrite by the formation of nitrocarotenoids
and inhibit nitration of tyrosine. Furthermore, nitrocarotenoids themselves
show singlet oxygen-quenching activity and anticancer-promoting activity.
Therefore, carotenoids might scavenge RNS in vivo and have the potential to
reduce the risk of disease induced by RNS.
ONOO– ONOO–
nitration oxidative cleavage
Nitrocarotenoids
Aponitrocarotenoids
Carotenoid with
Oxidative cleavage (Oxazine carotenoid)
cyclic end group Apocarotenals Nitration
(β-carotene, astaxanthin, Apocarotenones Aponitrocarotenoids
lutein, capsanthin) Isomerization
Cis-carotenoids
Oxidation nucleophilic
addition of MeOH
Cyclo-methoxylycopenes
Carotenoid with Oxidative cleavage Apolycopenals
acyclic end group
Apolycopenones
lycopene Isomerization
Cis-lycopenes
FIGURE 19.12
Reaction pathways of carotenoids with peroxynitrite.

References
Britton, G., Liaaen-Jensen, S., Pfander, H. 2004. Carotenoids Hand Book. Birkhauser,
Basel, Switzerland.
Burton, G. W., Ingold, K. U. 1984. Beta-carotene an unusual type of lipid antioxidant.
Science, 224: 569.
Daiber, A., Oetze, M., Coldewey, M., Kaiser, K., Huth, C., Schildknecht, S., Bachschmid,
M. et al. 2005. Hydrazine is a powerful inhibitor of peroxynitrite formation as
a possible explanation for its beneficial effects on prognosis in patients with
congestive heart failure. Biochemical and Biophysical Research Communications,
338: 1865.
Ferroni, F., Maccaglia, A., Pietraforte, D., Turco, L., Minetti, M. 2004. Phenolic antioxi-
dants and the protection of low density lipoprotein from peroxynitrite-mediated
oxidations at physiologic CO2. Journal of Agricultural and Food Chemistry, 52: 2866.
Foote, C.S., Denny, R.W. 1968. Chemistry of singlet oxygen. VII, Quenching by
β-carotene. Journal of the American Chemical Society, 90: 6233.
Goss, S. P. A., Hogg, N., Kalyanaraman, B. 1999. The effect of α-tocopherol on the
nitration of γ-tocopherol by peroxynitrite. Archives of Biochemistry and Biophysics,
363: 333.
Gruszecki, W.I. 2009. Carotenoids in lipid membranes. In Carotenoids: Physical,
Chemical, and Biological Functions and Properties, ed. J.T. Landrum. CRC Press,
New York, NY, p. 19.
Halliwell, B. 1994. Free radicals, antioxidants, and human disease: Curiosity, cause or
consequence? Lancet, 344: 721.
Hayakawa, T., Kulkarni, A., Terada, Y., Maoka, T., Etoh, H. 2008. Reaction of Astaxan
thin with peroxynitrite. Bioscience, Biotechnology and Biochemistry, 72: 2716.
Hirayama, O., Nakamura, K., Hamada, S., Kobayashi, Y. 1991. Singlet oxygen quench-
ing ability of naturally occurring carotenoids. Lipids, 29: 149.
Hopper, D.C., Scott, G.S., Zborek, A., Mikheeva, T., Kean, R. B., Koprowski, H.,
Spitisin, S. 2000. Uric acid, a peroxynitrite scavenger, inhibits CNS inflamma-
tion, blood–CNS barrier permeability changes, and tissue damage in a mouse
model of multiple sclerosis. FEBS Letters, 14: 691.
Hopper, D.C., Spitsin, S., Kean, R. B., Champion, J. M., Diskson, G. M., Chaudhry, L.,
Koprowski, H. 1998. Uric acid, a natural scavenger of peroxynitrite, in experi-
mental allergic encephalomyelitis and multiple scleroris. Proceedings of the
National Academy of Sciences, USA, 95: 675.
Iwamoto, T., Hosoda, K., Hirano, R., Kurata, H., Matsumoto, A., Miki, W., Kamiyama,
M., Itakura, H., Yamamoto, S., Kondo, K. 2000. Inhibition of low-density lipo-
protein oxidation by astaxanthin. Journal of Atherosclerosis and Thrombosis, 7: 216.
Kato, Y., Ogino, Y., Aoki, T., Uchida, T. K., Kawakishi, S., Osawa, T. 1997. Phenolic anti-
oxidants prevent peroxynitrite-derived collagen modification in vitro. Journal of
Agricultural Food Chemistry, 45: 3004.
Kikugawa, K., Hiramoto, K., Tomiyama, S., Asano, Y. 1997. β-Carotene effectively
scavenges toxic nitrogen oxides: Nitrogen dioxide and peroxynitrous acid.
FEBS Letters, 404: 175.
Kim, S. J., Nara, E., Kobayashi, H., Terao, J., Nagao, A. 2001. Formation of cleavage
products by autoxidation of lycopene. Lipids, 36: 191.

Lim, B. P., Nagao, A., Terao, J., Tanaka, K., Suzuki, T., Tanaka, T. 1992. Antioxidant
activity of xanthophylls on peroxy radical-mediated phospholipid peroxida-
tion. Biochemica et Biophysica Acta, 1126: 178.
Maoka, T., Etoh, H. 2011. Some biological functions of carotenoids in Japanese food.
In Functional Foods of the East, ed. John Shi, Chi-Tang Ho, Fereidoon Shahidi.
CRC Press, Boca Raton, FL, p. 85.
Maoka, T., Mochida, K., Kozuka, M., Enjo, F., Kuchide, M., Nobukuni, Y., Tokuda, H.,
Nishino, H. 2006. Chemopreventive activity of paprika extract and capsanthin
on nitric oxide or peroxynitrite induced carcinogenesis (in Japanese). Food and
Clinical Nutrition, 1: 7.
Maoka, T., Tokuda, H., Suzuki, N., Kato, H., Etoh, H. 2012. Anti-oxidative, anti-tumor-
promoting, and anti-carcinogenesis activities of nitroastaxanthin and nitrolu-
tein, the reaction products of astaxanthin and lutein with peroxynitrite. Marine
Drugs, 10: 1391.
Miki, W. 1991. Biological functions and activities of animal carotenoids. Pure and
Applied Chemistry, 63: 141.
Mortion, L. W., Ward, N. C., Croft, K. D., Puddey, I. B. 2002. Evidence for the nitration
of γ-tocopherol in vivo: 5-Nitro-γ-tocopherol is elevated in the plasma of sub-
jects with coronary heart disease. Biochemistry Journal, 364: 625.
Muijsers, R. B. R., van den Worm, E., Folkerts, G., Beukelman, C. J., Koster, A. S.,
Postma, U. S., Nijkamp, F. P. 2000. Apocynin inhibits peroxynitrite formation
by murine macrophage. British Journal of Pharmcology, 130: 932.
Murakami, A., Nakashima, A., Koshiba, T., Maoka, T., Nishino, H., Yano, M.,
Sumida, T., Kim, K., Koshimizu, K., Ohigashi, H. 2000. Modifying effects of
carotenoids on superoxide generation from stimulated leukocytes. Cancer
Letters, 149: 115.
Muzandu, K., Ishizuka, M., Sakamoto, K. Q., Shaban, Z., Ei Bohi, K., Kazusako, A.,
Fujita. S. 2006. Effect of lycopene and β-carotene on peroxynitrite-mediated cel-
lular modification. Toxicology and Applied Pharmacology, 215: 330.
Niwa, T., Doi, U., Kato, Y., Osawa, T. 1999. Inhibitory mechanism of sinapinic acid
against peroxynitrite-mediated tyrosine nitration of protein in vitro. FEBS
Letters, 459: 43.
Niwa, T., Doi, U., Kato, Y., Osawa, T. 2001. Antioxidative properties of phenolic antiox-
idants isolated from corn steep liquor. Journal of Agriculture and Food Chemistry,
49: 177.
Oshima, S., Ojima, F., Sakamoto, H., Ishiguro, Y., Terao, J. 1999. Supplementation with
carotenoids inhibits singlet oxygen-mediated oxidation of human plasma low-
density lipoprotein. Journal of Agricultural Food Chemistry, 44: 2306.
Panasenko, O. M., Sharov, V. S., Briviba, K., Sies, H. 2000. Interaction of peroxynitrite
with carotenoids in human low density lipoproteins. Archives of Biochemistry
and Biophysics, 373: 302.
Pannala, A. S., Rice-Evansb, C., Sampsona, J., Singha, S. 2008. Interaction of peroxyni-
trite with carotenoids and tocopherols within low density lipoprotein. FEBS
Letters, 423: 297.
Pannala, A. S., Singh, S., Rice-Evans, C. 1999. Flavonoids as peroxynitrite scavengers
in vitro. Methods in Enzymology, 299: 207.
Rodrigues, E., Mariutti, L. R. B., Mercadante, A. Z. 2012. Scavenging capacity of
marine carotenoids against reactive oxygen and nitrogen species in a mem-
brane-mimicking system. Marine Drugs, 10: 1784.

Sachindra, N. M., Sato, E., Maeda, H., Hosokawa, M., Niwano, Y., Kohno, M.,
Miyashita, K. 2008. Radical scavenging and singlet oxygen quenching activity
of marine carotenoid fucoxanthin and its metabolites. Journal of Agricultural and
Food Chemistry, 55(21): 8516–22.
Santocono, M., Zumia, M., Berrettini, M., Fedeli, D., Falcioni, G. 2007. Lutein, zeaxan-
thin and astaxanthin protect against DNA damage in SK-N-SH human neuro-
blastoma cells induced by reactive nitrogen species. Journal of Photochemistry
and Photobiology B, 88: 1.
Scheidegger, R., Pande, A. K., Bounds, P. L., Koppenol, W. H. 1998. The reaction of
peroxynitrite with zeaxanthin. Nitric Oxide: Biology and Chemistry, 2: 8.
Shibata, A., Kiba, Y., Akali, N., Futakami, K., Terada, H. 2001. Molecular characteris-
tic of astaxanthin and beta-carotene in the phospholipid monolayer and their
distribution in the phospholipid bilayer. Chemstry and Physics of Lipids, 113: 11.
Shimizu, N., Goto, M., Miki, W. 1996. Carotenoids as singlet oxygen quenchers in
marine organisms. Fisheries Science, 62: 134.
Shin, S-G., Kim, J. Y., Chung, H. Y., Jeong, J. C. 2005. Zingerone as an antioxidant
against peroxynitrite. Journal of Agricultural Food Chemistry, 53: 7617.
Terao, J. 1989. Antioxidative activity of beta-carotene-related carotenoids in solution.
Lipids, 24: 659.
Trevithick-Sutton, C. C., Foote, C. S., Collinus, M., Trevithick, J. R. 2006. The retinal
carotenoids zeaxanthin and lutein scavenge superoxide and hydroxyl radicals:
A chemiluminescence and ESR study. Molecular Vision, 12: 1127.
Tsuboi, M., Etoh, H., Kato, K., Nakatugawa, H., Kato, H., Maejima, Y., Matumoto,
G. et al. 2011. Nitrocapsanthin and nitrofucoxanthin, respective products of
capsanthin and fucoxanthin reaction with peroxynitrite. Journal of Agricultural
Food Chemistry, 59: 10872.
Tsuboi, M., Etoh, H., Yomoda, Y., Kato, K., Kato, H., Kulkarni, A., Terada, Y., Maoka
T, Mori, H., Inakuma, T. 2010. Nitration reaction of lutein with peroxynitrite.
Tetrahedron Letters, 51: 676.
Tsuda, T., Kato, K., Osawa, T. 2000. Mechanism for the peroxynitrite scavenging
activity by anthocyanins. FEBS Letters, 484: 207.
Tsushima, M., Maoka, T., Katsuyama, M., Kozuka, M., Matsuno, T., Tokuda, H., Nishino,
H., Iwashima, A. 1995. Inhibitory effect of natural carotenoids on Epstein–Barr
virus activation activity of a tumor promoter in Raji cells. A screening study for
antitumor promoters. Biological and Pharmaceutical Bulletin, 18: 227.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T. D., Mazur, M., Telser, J. 2007. Free
radicals and antioxidants in normal physiological functions and human dis-
ease. International Journal of Biochemistry and Cell Biology, 39: 44.
Williamson, K. S., Gabbita, S. P., Mou, S., West, M., Pye, Q. N., Markesbery, W. R.,
Cooney, R. V. et al. 2002. The nitration product 5-nitro-tocopherol is increased
in the Alzheimer brain. Nitric Oxide: Biology and Chemistry, 6: 221.
Wiseman, H., Halliwell, B. 1996. Damage to DNA by reactive oxygen and nitrogen
species: Role in inflammatory disease and progression to cancer. Biochemistry
Journal, 313: 17.
Yokota, T., Ohtake, T., Ishikawa, H., Inakuma, T., Ishiguro, Y., Terao, J., Nagao, A.,
Etoh, H. 2004. Chemistry Letters, 33: 80.
Yoshioka, R., Hayakawa, T., Ishizuka, K., Kulkarni, A., Terada, Y., Moaka, T., Etoh,
H. 2006. Nitration reaction of astaxanthin and β-carotene by peroxynitrite.
Tetrahedron Letters, 47: 3637.

20
Bioactive Stability and Antioxidative
Property of Lycopene from
Tomatoes during Processing
John Shi, Sophia Jun Xue, Lishui Chen, Wenliang

Wang, Hetong Lin, Ying Ma, and Gauri S. Mittal
CONTENTS
20.1 Introduction................................................................................................. 609
20.2 Physical and Chemical Properties of Lycopene..................................... 611
20.3 Lycopene Degradation during Processing.............................................. 613
20.3.1 Effect of Temperature on Lycopene Degradation...................... 614
20.3.2 Effect of Light-Irradiation on Lycopene Degradation............... 619
20.3.3 Effect of Oxygen on Lycopene Degradation............................... 620
20.4 Lycopene Isomerization in Food Processing.......................................... 623
20.5 Synergistic Effects....................................................................................... 627
20.6 Stabilization Technologies of Lycopene...................................................630
20.7 Summary...................................................................................................... 631
References..............................................................................................................633
20.1 Introduction
Lycopene is a natural pigment that contributes red color to fruits and vegeta-
bles. There are numerous epidemiological studies which have showed lyco-
penes possess strong antioxidant to provide protection against a broad range
of chronic diseases and cancers, such as cardiovascular disease, hyperten-
sion, atherosclerosis, prostate cancer, lung cancer, and diabetes among others
(Micozzi et al., 1990; Kohlmeier et al., 1997; Campbell et al., 2004; Etminan
et al., 2004; Kun et al., 2006; Kong et al., 2010; Ried and Fakler, 2011; Ankita
et al., 2012). Therefore, the content and stability of lycopene in food has taken
on added importance. Lycopene is a lipophilic compound, and belongs to the
family of carotenoids. Moreover, among carotenoids, the quenching constant
of lycopene was found to be more than double that of β-carotene and 10 times
more than that of α-tocopherol, which makes its presence in the diet of con-
siderable interest (Böhm et al., 2001; Cao-Hoang et al., 2011). Levy et al. (1995)
609
studied the inhibitory effect of lycopene, comparing it with that of α- and

β-carotenes on the growth of several human cancer cells, and found lycopene
to be the most potent inhibitor than others. Lycopene is the primary carot-
enoid present in tomatoes, thus tomatoes (especially deep-red fresh tomato
fruits) and their products are considered to be the most important sources of
lycopene in the human diet (Rao et al., 1998; Rao and Agarwal, 1999).
Lycopene is known to exist in a variety of isomeric forms, including the
all-trans, mono-cis, and poly-cis forms. Lycopene is present naturally in the
trans-form in fresh tomato, the formation of cis-forms of lycopene by isom-
erization that is probably due to processing (Nguyen and Schwartz, 1999).
All-trans isomer lycopene (C40H56) is an acyclic, open-chain polyene hydro-
carbon with 13 double bonds, of which 11 are conjugated in a linear array
(Figure 20.1). But seven bonds can isomerize from the trans-form to the
mono- or poly-cis forms. Lycopene undergoes degradation via isomeriza-
tion and oxidation. It is generally accepted that the all-trans-isomers have
the highest stability among all isomeric forms, but the cis-isomers are likely
to have higher bioactivity and bioavailability because of their shorter length,
the greater solubility of cis-isomers in mixed micelles, and/or as a result of
the low tendency of cis-isomers to aggregate (Clinton et al., 1996). Bioactivity
potency is dependent on the extent of isomerization and oxidation. A true
assessment of the nutritional quality of tomato products depends not only on
the total lycopene content but also on the distribution of lycopene isomeriza-
tion (Yi et al., 2009).
The cis-isomers are easily degraded compared with trans-form. The isom-
erization of trans-isomers to cis-isomers can be eversible under certain con-
ditions. Food processing and environmental conditions, such as heat, light,
oxygen, pH, ions, and different food matrices, are factors that have a marked
effect on its isomerization and autoxidation (Shi et al., 2003, 2008, 2009). The
oxidation of lycopene splits the molecule, which causes a loss in color and the
production of off-flavors. Thus, lycopene degradation in commercial tomato-
based products could affect the attractive color, nutritional and bioactive or
functional value of the final product (Re et al., 2002; Chen et al., 2009). It is
important to address the lycopene level characterization in current tomato
H3C CH3
CH3 CH3 CH3
CH3 CH3 CH3
H3C CH3
FIGURE 20.1
Molecular structure of lycopene. (Adapted from Ankita, J., Vinod, J., Samir, M. 2012. International
Journal of Nutrition, Pharmacology, Neurological Disease, 2: 167–170.)

Bioactive Stability and Antioxidative Property of Lycopene 611
commercial products as well as the control of its potential degradation dur-

ing its shelf-life storage, at room temperature, and avoiding direct light or
heat, according to the labeling instructions (Sharma and Le Maguer, 1996;
Colle et al., 2010; Kessy et al., 2011; Lavelli and Torresani, 2011). With increas-
ing interest and awareness of the health benefits of lycopene, lycopene con-
centrates and lycopene-rich food products have been used in functional
foods and its stability during food processing and storage has drawn more
and more attention. In order to ensure lycopene stability, there is a need to
understand the influence of processing parameters and conditions on the
oxidation and isomerization of lycopene. The characterization and quantifi-
cation of isomers would be desirable to more accurately assess the bioactiv-
ity than relying on only the total lycopene content with no knowledge of its
isomeric composition.
20.2 Physical and Chemical Properties of Lycopene

Lycopene is the major carotenoid pigment found in tomatoes. It is a polyene
hydrocarbon characterized by a symmetrical and acyclic structure contain-
ing 13 double bonds of which 11 are conjugated double bonds arranged in a
linear array and having a molecular formula of C40H56. Color and antioxidant
activities of lycopene are a consequence of its unique structure, an extended
system of conjugated double bonds. In nature, lycopene exists in the all-trans
form, the most thermodynamically stable form. However under certain con-
ditions, seven of these bonds can isomerize from the trans-form to the less
stable mono or poly-cis form. Lycopene is soluble in oils and apolar organic
solvents. In aqueous systems, they tend to aggregate and precipitate as crys-
tals; such reactions are thought to hinder the bioavailability of lycopene in
humans. In ripe tomato fruits, lycopene takes the form of elongated, needle-
like crystals, which are responsible for the typical bright red color of ripe
tomato fruits. Lycopene is more soluble in chloroform, benzene, and other
organic solvents than in water. Lycopene is also very sensitive to light, heat,
oxygen, and acids, and some metallic ions such as Cu2+, Fe3+ which catalyzes
its oxidation.
Lycopene exhibits many unique and distinct biological properties. One of
its main activities is to function as an antioxidant. Through its conjugated
double-bound system, lycopene is able to quench efficiently the energy of
deleterious forms of oxygen (singlet oxygen) and to scavenge a large spec-
trum of free radicals. Lycopene is among the most efficient singlet oxy-
gen quenchers of the natural carotenoids (Di et al., 1989; Conn et al., 1991).
The quenching activity of the different carotenoids depends essentially on
the number of conjugated double bonds. It is modulated by the end groups
or the nature of the substituents in the carotenoids that contain cyclic end

groups (Stahl and Sies, 1992). There are considerable differences in the
quenching rate constants (kq) for various carotenoid species (Table 20.1). The
antioxidant activities of lycopene and other carotenoids are highlighted by
their singlet oxygen quenching properties and their ability to trap peroxyl
radicals (Burton and Ingold, 1984).
Lycopene is known to exist in a variety of geometric forms, including all-
trans, mono-cis, and poly-cis forms. Lycopene can undergo trans-to-cis isom-
erization during food processing and storage. In various tomato-based foods,
the all-trans isomer is composed of 35%–96% of the total lycopene (Schierle
et al., 1996). In general, cis-isomers are more polar than their all-trans coun-
terparts, and are less prone to crystallization due to their kinked forms. The
cis-isomers are also more soluble in oil and hydrocarbon solvents than the
all-trans isomers (Table 20.2). The bioactive potency of the cis-isomers is not
the same as the all trans-isomers, because of the differences in their struc-
tural shapes (Table 20.2). Most stability studies of lycopene in food systems
focused on its degradation. Lycopene is a conjugated polyene and is suscep-
tible to at least two reactions during tomato processing, that is, isomeriza-
tion and oxidation. Lycopene may be partially destroyed in food products
by heating, light, oxygen, or in the presence of metallic ions (Cu2+, Fe3+, etc.).
The isomerization of lycopene occurs when pure lycopene is treated, and
when the lycopene in food products is subjected to processing conditions.
On the other hand, the conversion of the cis-isomer to the trans-isomer, which
is the more favorable reaction, occurs during the storage of the product. The
cis-isomers are the less stable form, while the trans-isomers are in the more
stable ground state.
TABLE 20.1
Comparison of Antioxidant Activities of Carotenoids: Singlet Oxygen
Quenching
Lycopene
a. Singlet oxygen quenching, kq × 109 (m−1 s−1) 31
b. Radical scavenging (Trolox equivalents) 2.9
c. Reaction of carotenoid radical anions with O2; k × 108 (m−1 s−1) 2
Other Carotenoids’ Singlet Oxygen Quenching 109 × k q (m −1 s −1)

γ-Carotene 25
α-Carotene 19
β-Carotene 14
Lutein 8
Astaxanthin 24
Bixin 14
Canthaxanthin 21
Zeaxanthin 10
Source: Data from Di, M.P., Kaiser, S., Sies, H. 1989. Archives of Biochemistry and Biophysics,
274: 532–538; Conn, P.F., Schalch, W., Truscott, T.G. 1991. Journal of Photochemistry
and Photobiology B, 11: 41–47; Miller, N.J. et al. 1996. FEBS Letter, 384: 240–242.

TABLE 20.2
Effect of Heating Treatment on Lycopene Trans- to Cis-Isomerization in Aqueous
and Oily Dispersions of Tomato Paste (70°C)
Heating Time All-trans 5-cis 9-cis 15-cis Other cis
(min) (%) (%) (%) (%) (%)
In Water
0 92.6 4.5 0.9 1.6 0.5
15 92.3 4.4 0.9 1.6 0.5
30 88.1 5.1 2.1 2.3 2.5
60 87.1 5.2 2.2 2.7 3.0
120 86.2 5.5 2.7 2.6 3.1
180 83.4 6.1 3.6 3.2 3.8
In Olive Oil
0 87.4 4.8 4.3 3.0 0.5
30 85.2 5.8 5.5 2.9 0.5
90 83.5 6.2 5.9 3.3 1.2
120 80.3 7.0 6.9 3.2 2.6
180 76.7 8.1 8.8 3.1 3.3
Source: Data from Schierle, J. et al. 1996. Food Chemistry, 59(3): 459–465.
20.3 Lycopene Degradation during Processing

More than 80% of the tomatoes produced are consumed in the form of
processed products such as tomato juice, paste, puree, catsup, sauce, and
salsa. The concentration of lycopene in tomato-based products depends on
the original raw material and technology that are used. Thus, the lycopene
content in concentrated tomato products is generally lower than expected
because of losses during tomato processing (Tavares and Rodriguez-
Amaya, 1994; Chen et al., 2009; Maiani et al., 2009; Cámara et al., 2012).
The main causes of lycopene degradation in food processing are isomeri-
zation and oxidation. It is widely assumed that lycopene in general under-
goes isomerization during thermal processing. These changes in lycopene
content and in the distribution of trans–cis isomers result in a change in
biological activity. Extensive studies indicate that lycopene bioavailability
of tomato-based products is higher than the lycopene of raw tomatoes,
probably because the release of lycopene from cellular matrix and isom-
erization to its cis-isometric form caused by the heating process (Agarwal
et al., 2001; Chen et al., 2009; Yi et al., 2009). Physical and chemical factors
known to degrade lycopene include elevated temperature, exposure to
light, oxygen, extremes in pH, and active surfaces (Nguyen and Schwartz,
1999; Shi et al., 2008).

20.3.1 Effect of Temperature on Lycopene Degradation

Tomato products including tomato juice, soup, sauce, puree, paste, powder,
and catsup are usually prepared by thermal processing or dehydration pro-
cessing. Thermal processing with increased heating temperature and cook-
ing period has been shown to decrease total and all-trans lycopene content
with an increase in lycopene cis-isomers and oxidation products in puree
samples. The native lycopene is located within the tomato cell matrix which
gives it some protection from degradation during heat processing. The results
of lycopene retention in tomato juice are presented in Table 20.3. The length
of cooking time has less effect on the degradation of lycopene if the heating
temperature is less than 100°C. However, temperatures greater than 120°C
will result in more lycopene loss. It has also been reported that serious losses
of lycopene can occur when the holding times at high temperature are long.
Figure 20.2 showed the degradation trends on total lycopene and cis-
isomer content in tomato puree with elevating temperature. Increasing the
temperature from 100°C to 150°C increased the degradation of both the trans-
and cis-isomers of lycopene. It was observed that the cis-isomers increased
with thermal treatment at 100°C, but dropped significantly with treatment
at 150°C. An increase in temperature from 100°C to 150°C caused a 76%
decrease in the total lycopene content. A 90 min treatment at 150°C resulted
in lycopene degradation and a greater loss in total lycopene, compared to
cis-isomer formation. Total lycopene concentration decreased at all treatment
times, but the cis-isomers appeared only during the first hour of heating.
After 1 h of heating, the rate of cis-isomer accumulation decreased. It was
observed that increasing the temperature from 100°C to 150°C or increasing
thermal treatment time would increase the degradation of trans-isomer and
TABLE 20.3
Lycopene Loss Rate in Tomato Juice during Heating
Lycopene Loss (%)
Heating
Temperature Heating Time Heating Time Heating Time
(°C) 1 min 3 min 7 min
90 0.6 0.9 1.1
100 0.9 1.4 1.7
110 2.2 3.2 4.4
115 2.7 4.5 7.0
118 3.7 6.0 9.1
121 4.6 7.3 10.6
124 5.5 8.5 12.5
127 6.5 9.9 14.6
130 7.4 11.5 17.1
Source: Data from Miki, N., Akatsu, K. 1970. Journal of Japanese Food
Industry, 17: 175–181.

Lycopene content, mg/100 g

7
6
5
4
3
2
1 90°C 100°C 120°C 150°C
0
0 1 2 3 4 5 6
Time (h)
FIGURE 20.2
Effect of heat treatment on total lycopene degradation. (Adapted from Shi, J. et al. 2003. Journal
of Food Process Engineering, 25: 485–498.)
cis-isomer of lycopene. It was suggested that the degradation of lycopene was

the main mechanism of lycopene loss when heated above 100°C, and that
lycopene in general underwent isomerization with thermal processing. An
increase in temperature from 90°C to 150°C caused a 35% decrease in the
total lycopene content. The high temperature and large amount of air incor-
porated in the tomato juice during the breaking and straining operations can
quickly destroy substantial amounts of lycopene. The results suggest that the
length of heating is a critical factor controlling the degradation of lycopene.
It appears that “high temperature–short duration” heat treatment of tomato
juice can have beneficial effects on the tomato juice quality.
Research by Agarwal et al. (2001) showed that subjecting tomato juice to
cooking temperatures in the presence of corn oil resulted in the formation of
cis-isomeric forms. According to the study by Ax et al. (2003), the total lyco-
pene content in oil-in-water emulsions decreased during thermal treatment
with and without exposure to oxygen. Higher temperatures were directly
correlated with increasing lycopene losses, and thermal treatment leads to
a significant decrease in the concentrations of all-trans and 13-cis isomers,
while the concentration of the 9-cis isomer increased.
Chen et al. (2009) studied the lycopene stability in water- and oil-based
food model systems under thermal- and light-irradiation treatments. The
results showed that 80°C and 100°C heating favored the stability of lycopene
in oil-based tomato products. The high heating temperatures (120°C and
140°C) increased the isomerization of lycopene and resulted in the degrada-
tion of total lycopene and cis-isomers in both water- and oil-based tomato
products. More cis-isomers were generated and also degraded faster in the
water-based samples than in the oil-based samples (Figures 20.3 and 20.4).
As compared with in oil media, these cis-isomer formations in water-based
tomato samples could be attributed to the effects of combining the high heat
transfer rates and the high polarity of the water medium that promoted the

0.8
Cis-lycopenes content (mg/100 g)
0.6
0.4
0.2
0.0
0 1 2 3 4
Time (h)
FIGURE 20.3
Effects of thermal treatments on the cis-isomer contents of water-based samples (◊, 80°C;
◽, 100°C, ▵, 120°C; and ⚪, 140°C). (Adapted from Shi, J. et al. 2003. Journal of Food Process
Engineering, 25: 485–498.)
conversion of more all-trans lycopene into the cis-isomers, and some poly-cis
isomers were subsequently converted into di-cis or mono-cis isomers that
more easily degraded. Consequently, the higher oxygen content present in
the water systems further induced the cis-isomers undergoing oxidative deg-
radation. Moreover, the levels of degradation of total lycopene contents and
0.8
Cis-lycopenes content (mg/100 g)
0.6
0.4
0.2
0.0
0 1 2 3 4
Time (h)
FIGURE 20.4
Effects of thermal treatments on the cis-isomer contents of oil-based samples (◊, 80°C; ◽ 100°C;
▵, 120°C; and ⚪, 140°C). (Adapted from Shi, J. et al. 2003. Journal of Food Process Engineering, 25:
485–498.)

cis-isomers were greater in water-based samples than in oil-based model sys-

tems under different treatments.
Zanoni et al. (1999) performed a study on the loss of lycopene during
air-drying. Tomato halves were dried in a cabinet air dryer and the dry-
ing was carried out at 80°C and 110°C, with an airflow rate of 1.5 m/s.
Lycopene losses of 12% occurred at 110°C. According to Sharma and Le
Maguer (1996), heating tomato pulp at 100°C for 120 min decreased lyco-
pene content from 185.5 to 141.5 mg/100 g total solid, and the freeze-dry-
ing and oven-drying (25–75°C) of tomato pulp solids did not cause any
loss in lycopene content. Anguelova and Warthesen (2000) found that 70%
of all-trans lycopenes were retained in tomato products at 6°C, but storage
at 45°C affected lycopene stability to a greater extent as only about 40% of
all-trans lycopenes were retained under the same conditions of 6 weeks of
light irradiation.
Lycopene in the pure form without plant matrix protection will undergo
degradation more quickly than in tomato tissue. Lee and Chen (2002) studied
the stability of lycopene by heating lycopene (lycopene dissolved in hexane)
under 50°C, 100°C, and 150°C. For the 50°C treatment, there was no signifi-
cant change in the all-trans-lycopene content within the first 12 h; however,
the content began to decline thereafter. The levels of the mono-cis forms of
lycopene were found to decrease with increasing heating time, which indi-
cates that the degradation rate of mono-cis lycopene may be greater than
the formation rate. Unlike the mono-cis forms of lycopene, the percentage
change of two di-cis isomers showed increasing trends, which were probably
due to the conversion of mono-cis-lycopene. There was a significant decrease
in total lycopene after heating at 150°C for 9 h. The result revealed that isom-
erization was the main reaction during heating in the first 9 h, after which
the degradation reaction dominated. Similar results were observed for the
concentration change of all-trans-lycopene and its cis-isomers during heating
at 100°C, the level of all-trans-lycopene decreased by 78% after 120 min heat-
ing. The mono-cis forms of lycopene showed a decreasing trend. The levels
of two di-cis isomers were found to rise in the first 60 min and then decrease,
implying that di-cis-lycopene might be converted into the mono-cis-lycopene
or undergo degradation after prolonged heating. The result indicated that
the isomerization reaction was favored at the beginning and then the deg-
radation dominated after prolonged heating at 150°C. When comparing the
results, it appears that with increasing temperature and heating time, degra-
dation dominated over isomerization.
Shi et al. (2002) dissolved extracted lycopene into canola oil, and heated the
sample at 25°C, 100°C, and 180°C. It was observed that increasing the tem-
perature from 100°C to 180°C or increasing thermal treatment time increased
the degradation of trans-isomer and cis-isomer of lycopene (Figure 20.5a
through c). Compared with the treatment at 25°C, the cis-isomers increased
with thermal treatment at 100°C, but dropped significantly with the treat-
ment at 180°C.

40
30 Cis-isomers All-trans
mAU
20
10
5-cis
0
0 5 10 15 20
Minutes
40
30
mAU
20 Cis-isomers
10 All-trans
5-cis
0
0 5 10 15 20
Minutes
All-trans
40
30
mAU
20
Cis-isomers
10 5-cis
0
0 5 10 15 20
Minutes
FIGURE 20.5
Lycopene oxidation and isomerization during thermal treatment. (Adapted from Shi, J. et al.
2002. Nutraceutical and Food, 7: 179–183.)
It is also possible to form peroxyl radical capable of acting as a pro-oxidant

and propagate autoxidation. The proposed degradation pathway of lycopene
is shown in Figure 20.3. It is widely assumed that lycopene in general under-
goes isomerization with thermal processing. This isomerization results in
the conversion of the all-trans isomer into cis-isomers. The cis-isomers are
present in processed food samples and their concentration increased with
increasing temperature and time of thermal treatment. A large loss of lyco-
pene during processing would indicate a longer and more drastic thermal

procedure. The changes in lycopene content and the formation of cis-isomers

may result in a reduction in bioactive potency (Wilberg and Rodriguez-
Amaya, 1995; Stahl and Sies, 1996; Lee and Chen, 2002).
20.3.2 Effect of Light-Irradiation on Lycopene Degradation

Increasing illumination causes increases in the loss of lycopene. Lee and Chen
(2002) studied lycopene stability by dissolving standard lycopene in hexane
and illuminating (illumination intensity ranged 2000–3000 lx) the samples
for 6 days at 25°C. The content of all-trans lycopene was found to decrease
by increasing the illumination time. After 144 h of exposure to light, the total
lycopene losses amounted to 94% (Figure 20.6). All the mono-cis isomers of
lycopene showed an inconsistent change. For instance, the level of 5-cis-lyco-
pene was found to increase in the beginning and then decrease after the illu-
mination time reached 2 h. Likewise, a similar trend was observed for 9-cis-,
13-cis-, and 15-cis-lycopenes. These results indicate that isomerization and deg-
radation of lycopene and its cis-isomers, during illumination, were proceeding
simultaneously. The increased level of mono-cis-lycopene was probably due
to the initial conversion of all-trans lycopene. With continued illumination, a
decrease in the mono-cis lycopene could occur due to a further conversion into
another cis-form through intermediate all-trans lycopene or undergo degrada-
tion. The percentage changes of all-trans lycopene and its cis-isomers showed
a different trend when compared to the changes in the total amount of lyco-
pene. The all-trans lycopene may have been isomerized to form mono-cis or
di-cis lycopene. These results may account for the percentage increase in all the
mono-cis and di-cis forms of lycopene during illumination.
0.50
0.00
–0.50
y = –0.0176x + 0.1435
R2 = 0.9609
ln (CA/CAD)
–1.00
–1.50
–2.00
–2.50
–3.00
0 20 40 60 80 100 120 140 160
Time (h)
FIGURE 20.6
First-order plot for the degradation of the total amount of lycopene during illumination at 25°C
for 144 h. (Adapted from Lee, M.T., Chen, B.H. 2002. Food Chemistry, 78: 425–432.)

The effects of light irradiation on the content of total lycopene, trans-iso-

mers, and cis-isomers in pure lycopene samples under different light irradia-
tion intensities of 2010 (outdoor), 900, 650, and 140 (indoor): mol m−2 s−1 for 24 h
are shown in Figure 20.7a through d. The results indicate that isomerization
and degradation of lycopene and its cis-isomers proceed simultaneously dur-
ing illumination. The loss in total lycopene, trans-isomers, and cis-isomers
increased significantly as the intensity of the light irradiation increased. The
small amount of cis-isomer formed during light irradiation indicates that
either less cis-isomers were formed, or that the oxidation reactions were pre-
dominating. It is possible that any cis-isomer formed was quickly degraded
into oxidative by-products, indicating that the rate of cis-isomer oxidation
was much greater than the formation of cis-isomers by light irradiation. This
would suggest that cis-isomer oxidation is the main reaction pathway. The
content of total lycopene, trans-isomers, and cis-isomers in lycopene concen-
trates decreased under all light irradiation conditions, which indicate that
light irradiation induces lycopene oxidation.
Chen et al.’s (2009) study results showed that the total lycopene content
decreased with increasing light intensity and treatment time. However, dif-
ferent phenomena have found in water-based and oil-based samples. The
light-induced reduction in total lycopene in the water-based samples seems
to follow an exponential function as determined by a best-fit line with the
correlation constants greater than 0.956, showing that the total lycopene con-
tent decreased with increasing light intensity and treatment time. The sam-
ple’s final residual lycopene concentrations were 2.77 ± 0.71, 1.66 ± 0.62, and
0.76 ± 0.17 mg/100 g after they underwent irradiation intensity of 300, 500, and
700 mmol m−2 s−1, respectively, in water-based samples. However, this trend
was not as pronounced in the oil-based samples. Instead, the changes in total
lycopene followed the same pattern, where the lycopene contents decreased to
a certain level, but then no significant changes were found thereafter during
the rest of the treatments. The residual values for total lycopene in the treated
oil-based samples were 4.50 ± 0.66, 4.12 ± 0.28, and 3.37 ± 0.44 mg/100 g under
irradiation intensities of 300, 500, and 700 mmol m−2 s−1, respectively. The
increase in cis-isomers appeared at the very beginning of light treatment in all
the samples. However, after one-day treatment for the water-based samples,
there was a great increase in cis-isomer loss associated with increasing irra-
diation treatment time and light intensity. The amount of cis-isomer gained
in the water-based samples during light-irradiation treatments appeared to
rapidly decrease as the light intensity increased, suggesting that cis-isomer
oxidation was occurring. In contrast to the water-based samples, a gradual
decline in cis-isomer content was observed in the oil-based samples.
20.3.3 Effect of Oxygen on Lycopene Degradation

Henry et al. (1998) studied the effect of oxygen on the degradation of lyco-
pene in an aqueous model system. The standard lycopene was exposed to

(a)
17.5
15
12.5
10
mAU
7.5
Cis-isomers
5 All-trans
2.5 5-cis
0
0 5 10 15 20
Minutes
(b)
17.5
15
12.5
10
mAU
7.5
Cis-isomers All-trans
5
2.5 5-cis
0 5 10 15 20 min
Minutes
(c)
17.5
15
12.5
10
mAU
All-trans
7.5 Cis-isomers
5
2.5 5-cis
0
0 5 10 15 20
Minutes
FIGURE 20.7
(a) HPLC chromatogram of lycopene in oil solution after irradiation by light at 2010 μmol m−2 s−1
intensity (outdoor) for 24 h. (b) HPLC chromatogram of lycopene in oil solution after irradiation
by light at 900 μmol m−2 s−1 intensity (outdoor) for 24 h. (c) HPLC chromatogram of lycopene in
oil solution after irradiation by light at 650 μmol m−2 s−1 intensity (indoor) for 24 h. (d) HPLC
chromatogram of lycopene in oil solution after irradiation by light at 140 μmol m−2 s−1 intensity
(indoor) for 24 h. The changes of all trans- and cis-isomers of lycopene of oxidation and isom-
erization during sunlight irradiation. (Adapted from Shi, J. et al. 2003. Journal of Food Process
Engineering, 25: 485–498.) (Continued)

(d)
17.5 All-trans
15
12.5
10
mAU
7.5
Cis-isomers
5
5-cis
2.5
0
0 5 10 15 20
Minutes
FIGURE 20.7 (Continued)

(a) HPLC chromatogram of lycopene in oil solution after irradiation by light at 2010 μmol m−2 s−1
intensity (outdoor) for 24 h. (b) HPLC chromatogram of lycopene in oil solution after irradia-
tion by light at 900 μmol m−2 s−1 intensity (outdoor) for 24 h. (c) HPLC chromatogram of lyco-
pene in oil solution after irradiation by light at 650 μmol m−2 s−1 intensity (indoor) for 24 h. (d)
HPLC chromatogram of lycopene in oil solution after irradiation by light at 140 μmol m−2 s−1
intensity (indoor) for 24 h. The changes of all-trans- and cis-isomers of lycopene of oxidation
and isomerization during sunlight irradiation. (Adapted from Shi, J. et al. 2003. Journal of Food
Process Engineering, 25: 485–498.)
continuous flow of water saturated with oxygen at 30°C, and the result showed
that 90% of the lycopene was lost after 2 h. Ax et al. (2003) dissolved lycopene
in the oil phase of oil-in-water emulsions, and the emulsions were poured
inside a glass flask with a sintered-glass frit allowing continuous flushing
with either synthetic air or nitrogen gas, thus providing oxygen saturation
or oxygen-free conditions. At 25°C, about 25% of the lycopene was degraded
within 30 h in the oxygen-removed emulsions, whereas 80% was loss in the
oxygen-saturated emulsion. In the presence of oxygen, lycopene destabiliza-
tion was about three times higher than in the absence of oxygen. A nitrogen
or argon headspace can be employed to keep the exposure to atmospheric
oxygen to a minimum (Nguyen and Schwartz, 1999). However, according to
Ribeiro et al. (2003), removing oxygen in water by flushing the system with
nitrogen, no improvement of stability was achieved. Instead, lycopene deg-
radation was accelerated. On the other hand, complete exclusion of oxygen
using the enzyme glucose oxidase was shown to produce far better stability.
Anguelova and Warthesen (2000) subjected tomato powder to three
treatments: light exposure at room temperature, 6°C and 45°C in the dark.
Differences among the storage treatments are not obvious from the data,
but the amount of cis-isomers as a percent of the total lycopene increased
to the 14%–18% range, regardless of the storage conditions (Table 20.4). The
treatments at 6ºC significantly increased the amount of 5,5′ di-cis lycopene
after 4 weeks storage time, compared to the other two treatments that were

TABLE 20.4
Contents of Presumptive 5,5′ di-cis Lycopene in Tomato Powders
(% of the Total Lycopene in the Sample after a Given Storage Period
under Fluorescent Light 38,500 lux)
Tomato Powder T1 Tomato Powder T2
Light Light
Weeks Exposure 6°C 45°C Exposure 6°C 45°C
0 4.3 4.3 4.3 6.2 6.2 6.2
1 5.6 4.8 6.5 6.7 6.1 5.9
2 5.8 5.4 5.3 5.2 7.6 6.3
3 7 5.5 5.4 5.5 6.4 7
4 8.6 9.4 9.1 7.7 6.8 7.8
5 8.1 11 12.8 11.8 11.7 12.1
6 14.2 18.4 14.6 14.1 18.2 14.1
Source: Data from Anguelova, T., Warthesen, J. 2000. Journal of Food Science,
65(1): 67–70.
explained as the degradation of cis-isomer due to autoxidation, since cis-iso-

mers were more susceptible to autoxidation than the all-trans isomer.
20.4 Lycopene Isomerization in Food Processing

Bioactive potency depends on the content of total lycopene and the extent of
isomerization (Sies et al., 1992; Emenhiser et al., 1995; Wilberg and Rodriguez-
Amaya, 1995). Thus, isomerization can lead to a change in the bioactivity of
lycopene. The characterization and quantification of lycopene isomers would
provide better insights into the potential nutritional quality and healthy ben-
efit of the processed tomato products, and more accurately predict the lyco-
pene bioactivity than just the total lycopene content with no knowledge of
its isomeric composition. Controlling isomerization and oxidation of lyco-
pene during the preparation of a lycopene-based food would be of benefit in
improving the product quality.
Different isomers of lycopene behave differently, particularly concern-
ing their bioavailability. It was suggested that cis-lycopenes may be better
absorbed than the all-trans parent structure. This may be explained by a
greater solubility of cis-isomers as observed in bile acid micelles, and this
could explain why in serum and tissues, lycopene is more than 50% cis-
lycopene contrasting with the composition of food sources (Clinton et al.,
1996; Boileau et al., 1999). Cis-isomers of lycopene have distinct physical
characteristics and chemical behaviors from their all-trans counterpart.
The trans–cis isomerization in lycopene can be induced by different proce-
dures. Food processing, heating, illumination, and also the interaction with

TABLE 20.5
Lycopene Isomers in Various Thermally Processed Tomato
Products
Total Lycopene Cis-Isomers
Sample (mg/100 g, Dry Basis) (%)
Peeled tomato 149.89 5.37
Tomato juice (hot-break) 161.23 5.98
Tomato juice (retorted) 180.10 3.56
Tomato (whole, retorted) 183.49 3.67
Tomato paste (concentrated) 174.79 5.07
Tomato paste (retorted) 189.26 4.07
Tomato soup (retorted) 136.76 4.34
Tomato sauce (retorted) 73.33 5.13
Source: Data from Nguyen, M.L., Schwartz, S.J. 1998. Experimental
Biological and Medicine, 218: 101–105.
molecular carriers would definitely facilitate the trans-to-cis isomerization of

lycopene, and thus would alter the bioactivity of these compounds (Shi and
Le Maguer, 2000). Some of the differences observed as a result of a trans- to
cis-isomerization reaction include decreased color intensity and lower melt-
ing points (Zechmeister and Polgar, 1944). The decrease in color intensity is
of paramount importance taken into account during quantitative analysis of
lycopene isomers to avoid underestimation.
Nguyen and Schwartz (1999) assessed the effect of several different heat
treatments on lycopene’s isomeric distribution in a variety of tomato prod-
ucts (Table 20.5). Schierle et al. (2003) showed that heating tomato-based
food in oil caused increased lycopene isomerization than heating in water as
shown in Table 20.2. Lycopene isomerization and the amount of cis-isomers
increased as a function of processing time during heating of tomatoes. Heat
treatment clearly increased the percentage of the cis-isomers. It is obvious
from these results that food processing can enhance cis-isomerization in
tomato-based foods. This indicates that not only the duration and tempera-
ture of heat treatment, but also the food matrix components such as oil or
fat influence lycopene isomerization. It was observed that lycopene loss and
the rate of thermal isomerization were less while heating tomato pulp than
when heating pure lycopene in an organic solution. The tomato tissue matrix
may offer protection for lycopene.
It is generally accepted that the all-trans form of lycopene has the highest
stability and the cis-isomers have the lowest stability. Although the typical
processes such as cooking, freezing, or canning do not usually cause signifi-
cant changes in the total lycopene content, it is widely assumed that lyco-
pene undergoes isomerization upon thermal processing. Heat, light, acids,
and other factors have been reported to cause the isomerization of lycopene

(Schierle et al., 1996; Nguyen and Schwartz, 1998; Ax et al., 2003). The changes
in lycopene content and the distribution of trans–cis isomerization will result
in a reduction in biological potency, when lycopene concentrates are sub-
jected to processing (Emenhiser et al., 1995; Wilberg and Rodriguez-Amaya,
1995; Stahl and Sies, 1996).
The key question now is how to maintain high bioactive property dur-
ing food processing and storage. The isomerization and oxidation of lyco-
pene greatly depends on the treatments used since each treatment produces
a different form of energy (such as heat and light). It was found that the
light irradiation caused more losses in total lycopene than the heating treat-
ment. In all treatments, the rate of trans-isomer loss was greater than the
cis-isomer formation, which suggests that degradation through oxidation of
lycopene was the predominant mechanism, rather than the isomerization of
trans-isomer to cis-isomer. The losses could be coupled to the conversion of
trans-isomers to cis-isomers, followed by the direct degradation into smaller
oxidized by-products which are not isomers of lycopene (Boskovic, 1979).
Because lycopene is a highly unsaturated molecule, comprising many conju-
gated double bonds, it is very susceptible to oxidation. The pathway of lyco-
pene degradation is described in Figure 20.8.
Extended trans–cis-isomerization of lycopene correlated with antioxidant
property changes of lycopenes. The generation of cis-lycopene caused an
Lycopene
CHO
o o
Pseudo-ionone
Geranial
2-Methy-2-hepten-6-one o
CHO
6-Methyl-3.5-heptadien-2-one Neural
FIGURE 20.8
Molecular structural changes of lycopene during heating. (Adapted from Kanasawud, P.,
Crouzet, J.C. 1990. Journal of Agriculture and Food Chemistry, 38: 1238–1242.)

increase in the antioxidant capacity of the treated samples. Böhm et al. (2001)
found that at a higher temperature of 80°C, the isomerization was more
important, the antioxidant capacity increase was significantly faster than at
50°C. The antioxidant capacity of the mixture of lycopene isomers depends
on the structure and the amount of each cis-isomer. A significant amount of
13-cis-lycopene after 240 min at lower temperature made the lycopene iso-
mer solution significantly more antioxidant than the one composed of all-
trans-lycopene alone. Particularly, 9-cis-lycopene was shown to be more
antioxidant than the 13-cis-isomer. Indeed, lycopene solutions exhibited a
strong increase in the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic
acid) bleaching activity after 120 min at 80°C when they contained a signifi-
cant percent of the 9-cis isomer (9%).
A true assessment of the relationship between nutritional quality and health
benefits of dietary lycopene depends not only on the total lycopene content, but
also on the distribution of lycopene isomers. All-trans, 5-cis, 9-cis, 13-cis, and
15-cis are the most commonly identified isomeric forms of lycopene, with the
stability sequence being 5-cis > all-trans > 9-cis > 13-cis > 15-cis > 7-cis > 11-cis,
so that the 5-cis form is thermodynamically more stable than the all-trans-
isomer (Chasse et al., 2001). The control of lycopene oxidation and isomer-
ization during production and storage can be of benefit in improving the
retention of biological activity and health-promoting effect. Lycopene iso-
mers in various thermally processed tomato products are listed in Table 20.6.
TABLE 20.6
Lycopene Isomers in Various Commercial Tomato Products
Total Lycopene
(mg/100 g Wet All-trans 5-cis 9-cis 13-cis Other cis
Sample Basis) (%) (%) (%) (%) (%)
Tomato paste (“Tomatenmark,” 52 96 4 <1 <1 <1
Panocchia, Italy)
Tomato paste (“Maracoli,” Kraft, 3.7 91 5 1 2 <1
Germany)
Tomato Ketchup (“Hot Ketchup,” 9.5 88 7 2 3 1
Del Monte, Italy)
Tomato Ketchup (“Hot Ketchup,” 3.0 77 11 5 7 1
Heinz, The United States)
Instant Meal (“Eier-Ravioli,” 0.6 76 8 5 6 5
Hero, Switzerland)
Sauce (“Hamburger Relish,” 3.0 93 5 <1 3 <1
Heinz, The Netherlands)
Sauce (“Sauce Bolognaise,” 9.2 67 14 6 5 8
Barilla, Italy)
Canned tomatoes (“Chris,” Roger 7.1 84 5 3 5 3
Sud, Italy)
Source: Data from Schierle, J. et al. 1996. Food Chemistry, 59(3): 459–465..

20.5 Synergistic Effects

In the functional formulation development, it has been shown that the com-
bination of certain bioactive compounds can produce synergistic effects to
enhance bioactivity and bioavailability based on the studies of synergistic
effects of multiple bioactive compounds. Although considerable attention
has been concentrated on the effect of lycopene, tomato contains a mix-
ture of many other antioxidant components, including vitamins C and E,
b-carotene, lutein, and smaller amounts of flavonoids. Interaction of struc-
turally different antioxidants may contribute to the final beneficial action
of tomato-based products. The protective effect of a high intake of tomato
products against the risk of a range of degenerative/chronic diseases may
not rely on the action of a single antioxidant but rather on a concerted effect
of several antioxidant nutrients. The synergistic interaction of valuable nutri-
ents is becoming an interesting topic of many research investigations.
Information of antioxidants interaction in the molecular level is very lim-
ited. There is an evidence that indicated that α-tocopherol prevents β-carotene
and lycopene degradation during lipid oxidation (Shi et al., 2004). Experiments
of some research groups have observed synergistically improved antioxi-
dant activity during lipid oxidation when vitamin C and vitamin E were
used together (Lambelet et al., 1985; Chan et al., 1991). It has been suggested
that vitamin C (ascorbic acid) is able to interact with vitamin E (a-tocopherol)
radicals and regenerate vitamin E. It has also been reported that although
water-soluble vitamin C is less reactive with lipid radicals than hydrophobic
lycopene, it is able to interact with the oxidized forms of lycopene in a time-
dependent reaction and to regenerate lycopene. Therefore, lycopene would
recycle to exert their protective action against free radicals (Biacs and Daood,
2000). A previous work by the same group found that α-, β-, and γ-tocopherol
reduced all the carotenoid radical cations tested, whereas the δ-tocopheroxyl
radical was reduced by lycopene and beta-carotene (Mortensen et al., 1997).
Lutein or β-carotene in combination with γ-tocopherol was found to be able to
enhance the activity of γ-tocopherol in preventing triglycerides from oxida-
tion (Haila et al., 1996). Palozza and Krinsky (1992) reported that β-carotene
and α-tocopherol interacted cooperatively to provide a higher inhibition of
lipid peroxidation than the addition activity of the individual antioxidant
in a membrane system. Lycopene and α-tocopherol have been reported to
inhibit the proliferation of different prostate carcinoma cell lines. However,
the effective concentration of lycopene, when used alone, is above 50 μM,
much higher than the physiological concentration of lycopene in human
plasma or prostate, which is about 1 μM (Clinton et al., 1996). Pastori et al.
(1998) reported that lycopene of low concentration alone did not efficiently
inhibit prostate carcinoma cell proliferation. However, addition of low con-
centration of lycopoene together with α-tocopherol significantly increased
the inhibition effect of the later. The use of various concentration of lycopene

from 0.1 to 5 μM together with α-tocopherol showed a synergistic phenome-

non, rather than a simple additive effect. It is interesting to note that simulta-
neous addition of lycopene with β-tocopherol and ascorbic acid did not show
the synergistic effect. And ascorbic acid did not exhibit the same stimulation
to α-tocopherol as lycopene. The inhibition of proliferation by lycopene in
association with α-tocopherol was not potent in other cell lines as in prostate
cell, suggesting a specific function of lycopene in prostate tissue.
Amir et al. (1999) have investigated the inhibition effect of lycopene and
1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the proliferation of HL-60 pro-
myelocytic leukemia cell line. Both lycopene and 1,25(OH)2D3 inhibited cell
growth by slowing cell cycle progression at the G0/G1 phase. After two-day
incubation, low concentration of lycopene and 1,25(OH)2D3 alone produced
only a small effect on cell proliferation. However, simultaneous admission of
low concentration of these two compounds showed a significant synergistic
inhibitory effect on cell proliferation.
Limited information is available so far on the interaction and synergistic
antioxidant properties of carotenoids, especially lycopene. There was evidence
in indicating that the mixture of carotenoids exhibited higher protection on
lipid oxidation than one single carotenoid, especially when lycopene and lutein
were present in the mixture (Stahl et al., 1998; Anguelova and Warthesen, 2000).
Combination of lycopene and vitamin E at various concentrations was added
to linoleic acid methyl ester (LAME) solution in order to investigate whether
the synergistic effect of these two antioxidants was affected by the concentra-
tion, and if so to obtain the optimum concentrations that show the strongest
synergistic effect among the tested antioxidant concentrations. With 0.5 mM of
vitamin E and increasing levels of lycopene (2.5–25 mM) and 1.0 mM of vita-
min in the current model system, no significant synergistic effect was observed
(Figure 20.9). However, with 1.0 mM of vitamin E, a synergistic maximum was
obtained with 12.5 mM of lycopene (Figure 20.10). The synergistic effect was
not linearly dependent on lycopene concentration as the highest level (25 mM
lycopene) did not show synergism. There appears to be an optimum concen-
tration for the two antioxidants as well as a specific ratio (vitamin E to lyco-
pene). High or low concentration of either antioxidant removes the synergistic
effect. The combination of b-carotene and vitamin E did not produce a similar
synergistic effect at the levels tested. The values of the measured inhibition
effect (%MIE) were not significantly different from the expected inhibition
effect (%EIE) at time intervals. At 60 min, the %MIE was 48.6% and the %EIE
was 48.1% with an MIE/EIE value of 1.01. The calculated MIE/EIE values at 30,
40, and 50 min were 0.97, 1.02, and 1.02, respectively.
The synergistic effect of an antioxidant was shown to be dependent on
the type of antioxidant and its concentration (Liu et al., 2008). The results
indicated that lycopene interacted synergistically with vitamin E at a spe-
cific concentration and ratio, whereas β-carotene showed no synergistic
effect with vitamin E at the levels used in this study. There are many fac-
tors that may contribute to synergy of antioxidants in a biological system.

10,000 LAME
LAME + 0.5 μM Vit E
LAME + 12.5 lycopene
8000
LAME + 0.5 Vit E + 12.5 lycopene
Peak area at 234 nm
6000
4000
2000
0
0 10 20 30 40 50 60
Time (min)
FIGURE 20.9
Effect of lycopene together with 0.5 μM vitamin E on the oxidation of LAME. (⚪) without anti-
oxidant; (▴) 12.5 mM lycopene; (▪) 0.5 mM vitamin E; and (⚫) 12.5 mM lycopene + 0.5 mM vita-
min E. (Adapted from Shi, J. et al. 2003. Journal of Food Process Engineering, 25: 485–498.)
The appropriate concentration and combination of antioxidants appear to be

important factors for maximum synergistic action of antioxidative property.
In addition, the antioxidant activity of various dietary nutrients may be
affected by the concentration and hydrophobicity of the antioxidants, the
location of free radical species, and the generating rate of free radicals.
Therefore, the specific combination and concentration of various antioxi-
dants are crucial for the total antioxidant activity of dietary nutrients.
10,000
8000 LAME
Peak area at 234 nm
Lycopene
6000
Vit E
4000
2000 Lycopene + Vit E
0
0 10 20 30 40 50 60
Time (min)
FIGURE 20.10
Effect of lycopene together with 1.0 μM vitamin E on the oxidation of LAME. (⚪) without anti-
oxidant; (▪) 12.5 mM lycopene; (▴) 1 mM vitamin E; and (⚫) 12.5 mM lycopene + 1 mM vitamin
E. (Adapted from Shi, J. et al. 2003. Journal of Food Process Engineering, 25: 485–498.)

20.6 Stabilization Technologies of Lycopene

The incorporation of lycopene into food and beverages has been limited due
to its poor solubility in water and low stability when exposed to environmen-
tal stresses such as heat, light, and oxygen (Shi et al., 2008). Numerous stud-
ies have suggested that the bioavailability of lycopene could be enhanced
when mixed with dietary fats. This would imply that the amount and type
of dietary fat in the food product will increase the incorporation of free
lycopene and enhance its bioavailability (Deming et al., 2000; Hoppe et al.,
2003). Encapsulation or emulsion technology can be used to protect com-
pounds from oxidation, evaporation, and off-flavor. The technologies have
also shown to improve the solubility, stability, and bioavailability of lipo-
philic compounds (Pedrolas Islas, 2002; McClements, 2012). The stability
test revealed that microencapsulation offered greater protection to lycopene
compared to its free form and it was observed that the microcapsules were
able to release pigment and color the studied food system in a homogenous
manner (Gracia et al., 2007). The oil-formed lycopene in the emulsion would
enhance their bioavailability after consumption. Faisala et al. (2013) studied
the enhancement of oral bioavailability of lycopene by a novel lipid-based
solid dispersion (LBSD); they reported that a novel LBSD formulation was
developed to enhance the oral bioavailability of the model lipophilic com-
pound, lycopene, by enhancing dissolution in the gastrointestinal tract and
promoting intestinal lymphatic uptake utilizing digestible lipid excipients.
Moreover, the emulsifier layer surrounding the oil droplets that contains
lipophilic lycopene can be designed to protect the lycopene against oxida-
tion and degradation by reducing their exposure to oxygen, interacting with
metal ions such as Fe2+, heat, and light (McClements and Decker, 2000; Mao
et al., 2009).
A number of researchers have prepared emulsions containing carot-
enoids. Ax et al. (2003) used a high-pressure homogenizer to prepare lyco-
pene emulsions, and evaluated the thermal stability and oxidative stability
of the lycopene-emulsion system. They found that the total lycopene content
of the oil-in-water emulsions decreased during thermal treatment with and
without exposure to oxygen. However, the addition of tocopherol contain-
ing sunflower oil to the emulsion system reduced lycopene degradation dur-
ing thermal treatment. Qian et al. (2012) studied the physical and chemical
stability of β-carotene-enriched nano-emulsions under different pHs, ionic
strengths, and temperature and emulsifier types. They reported that encap-
sulated β-carotene had a tendency to chemically degrade which led to color
fading over time during storage. The rate of color fading increased with
increasing storage temperature, had the fastest rate at the lowest pH value
(pH = 3), and was largely independent of salt concentration (0–500 mM NaCl).
Several studies reported that microencapsulation offered greater protec-
tion to lycopene by atomization and spry-drying methods using modified

starch gum arabic and sucrose, gum arabic and maltodextrin, and gelatin
and sucrose, and Capsul® modified starch as encapsulants materials (Matioli
and Rodriguez-Amaya, 2002; Shu et al., 2006; Glaucia et al., 2012). Gracia et al.
(2007) stabilized all-trans-lycopene from tomato by encapsulation using a-, b-,
and c-cyclodextrins (CDs). In their study, two different encapsulation meth-
ods were comparatively studied: a conventional method and a supercritical
fluid extraction (SFE) process. All-trans-lycopene employed was obtained
by SFE with purity around 90%–95%. As a result, a molar ratio CD/lyco-
pene of 1/0.0026 was selected as it provided the best complexation yields
(93.8%), while b-CD seemed to be the most favorable to be used to stabilize
lycopene. Lycopene microcapsules were prepared by a spray-drying method
using a wall system consisting of gelatin and sucrose. Shu et al. (2006) stud-
ied the effects of technological parameters including the ratio of core and
wall materials, ratio of gelatin and sucrose, homogenization pressure, inlet
temperature, feed temperature, and lycopene purity on encapsulation yield
(EY) and encapsulation efficiency (EE). The resulted microcapsules were
also characterized in terms of lycopene isomerization, storage stability, and
outer and inner structures. The results showed that EY and EE were signifi-
cantly affected by the ratio of core and wall materials, ratio of gelatin and
sucrose, homogenization pressure, inlet temperature, feed temperature, and
lycopene purity. The optimal condition was determined as follows: the ratio
of gelatin/sucrose of 3/7 and the ratio of core and wall material of 1/4, feed
temperature of 55°C, inlet temperature of 190°C, homogenization pressure
of 40 MPa, lycopene purity of not less than 52%, at which the microencap-
sulated lycopene showed some isomerization, but a good storage stability.
In study by Nunes and Mercadante (2007), the encapsulated lycopene in a
powder form, using either spray-drying or molecular inclusion with b-cyclo-
dextrin (b-CD) followed by freeze-drying. Lycopene-b-CD complexes were
only formed at a molar ratio of 1:4, and irregular structures of different sizes
that eventually formed aggregates, similar to those of b-CD, were observed
after freeze-drying. Lycopene purity increased from 96.4% to 98.1% after
spray-drying, whereas lycopene purity decreased from 97.7% to 91.3% after
complex formation and freeze-drying. Therefore, the studied microcapsules
presented good functionality and good potential for use in lycopene-rich
functional ingredients since they were able to release the lycopene during
preparation of the studied system and to color it homogenously.
20.7 Summary
Consumers, researchers, and the leaders in the food industry have become
dramatically more interested and aware of the health benefits of lycopene
from tomatoes. Lycopene in the human diet has significant protective

and beneficial physiological effects, because lycopene has many unique

and distinct biological properties. Among these is its strong antioxidant
activity, which plays an important role in improving human health and
providing protection against a broad range of epithelial cancers. The anti-
oxidant activities of lycopene and other carotenoids are highlighted by
their singlet oxygen- quenching properties and their ability to trap per-
oxyl radicals. Lycopene is known to exist in a variety of geometric forms,
such as the all-trans, mono-cis, and poly-cis isomers. The all-trans isomer
of lycopene is the most predominant geometrical isomer in fresh toma-
toes. Lycopene can undergo trans-to-cis isomerization during tomato pro-
cessing and storage. Lycopene can undergo at least two changes during
tomato processing, that is, isomerization and oxidation. Lycopene may be
partially destroyed in processed tomato products by heating, light, oxy-
gen, and by the presence of metallic ions. The opposite reaction, the con-
version of the cis-isomer to the trans-form can occur during the storage of
the product. Cis-isomers are less stable than the all trans-isomer. Lycopene
is a very efficient antioxidant that quenches highly reactive singlet oxygen
(O2−) and traps peroxyl radicals (ROO). In processed tomato products, oxi-
dation is a complex process and that depends upon many factors, such as
processing conditions, moisture, temperature, and the presence of pro- or
antioxidants. Certain steps can be taken to minimize lycopene degrada-
tion during food processing and storage. Lycopene-containing products
need to be protected from excessive heat, extreme pH conditions, and
exposure to oxygen and light. Processing technology should be optimized
to prevent lycopene oxidation. The reduction in lycopene contents and
trans to cis isomerization would result in a reduction in biological activity.
It is generally accepted that the all-trans form of lycopene has the highest
stability and the cis-isomers have the lowest stability. Bioactive potency
depends on the extent of isomerization and oxidation. A true assessment
of the nutritional quality and healthy benefit of processed tomato-based
food depends not only on the total lycopene content, but also on the dis-
tribution of lycopene isomers. Lycopene can undergo degradation via
isomerization and oxidation under different processing conditions, which
impacts on its bioactivity and reduces its health-promoting functional-
ities. How to maintain the high bioactive properties of lycopene during
food processing and storage is a key question. Controlling isomerization
and oxidation of lycopene during tomato processing of lycopene-based
food products can improve product quality. The mechanism of lycopene
destruction depends on many parameters during food processing and stor-
age. The main cause of damage to lycopene during food processing and
storage is oxidation. Low storage temperature, low oxygen content, low
light, low water activity, and low moisture content will help to limit the
oxidation of lycopene. Microemulsions and microencapsulation technolo-
gies can be used as potential lycopene protective systems, and enhance its
bioavailability after consumption, because of their transparency, ease of

in preparation and long-term stability. Microemulsions are self-assembled

mixtures of water, oil, and surfactants and have the advantages of being
optically isotropic and thermodynamically stable.
References
Agarwal, A., Shen, H., Agarwal, S., Rao, A.V. 2001. Lycopene content of tomato prod-
ucts: Its stability, bioavailability and in vivo antioxidant properties. Journal of
Medicinal Food, 4(1): 9–15.
Amir, H., Karas, M., Giat, J., Danilenko, M., Levy, R., Yermiahu, T., Levy, J., Sharoni,
Y. 1999. Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of
cell cycle progression and induction of differentiation in HL-60 leukemic cells.
Nutrition and Cancer, 33: 105–112.
Anguelova, T., Warthesen, J. 2000. Lycopene stability in tomato powders. Journal of
Food Science, 65(1): 67–70.
Ankita, J., Vinod, J., Samir, M. 2012. Role of lycopene in the prevention of cancer.
International Journal of Nutrition, Pharmacology, Neurological Disease, 2: 167–170.
Ax, K., Mayer-Miebach, E., Link, B., Schuchmann, H., Schubert, H. 2003. Stability of
lycopene in oil-in-water emulsions. Engineering in Life Sciences, 4: 199–201.
Biacs, P.A., Daood, H.G. 2000. Lipoxygenase-catalysed degradation of carotenoids
from tomato in the presence of antioxidant vitamins. Biochemical Society
Transactions, 28: 839–845.
Böhm, V., Puspitasari-Nienaber, N.L., Ferruzzi, M.G., Schwartz, S.J. 2001. Trolox
equivalent antioxidant capacity of different geometrical isomers of a-carotene,
b-carotene, lycopene, and zeaxanthin. Journal of Agricultural and Food Chemistry,
50(1): 221–226.
Boileau, A.C., Merchen, N.R., Wasson, K., Atkinson, C.A., Erdman, J.W. 1999. Cis-
lycopene is more bioavailable than trans-lycopene in vitro and in vivo in lymph-
cannulated ferrets. Journal of Nutrition and Metabolism, 129: 1176–1181.
Boskovic, M.A. 1979. Fate of lycopene in dehydrated tomato products: Carotenoid
isomerization in food system. Journal of Food Science, 44: 84–86.
Burton, G.W., Ingold, K.U. 1984. β-carotene: An unusual type of lipid antioxidant.
Science, 224: 569–573.
Cámara, M., Fernández-Ruiz, V., Redondo, D.F., Sánchez-Mata, M.C., Torrecilla, J.S.
2012. Radial basis network analysis to estimate lycopene degradation kinetics
in tomato-based products. Food Research International, 49: 453–458.
Campbell, J.K., Canene-Adams, K., Lindshield, B.L., Boileau, T.W.M., Clinton, S.K.,
Erdman, J.W. Jr. 2004. Tomato phytochemicals and prostate cancer risk. The
Journal of Nutrition, 134: 34,86S–34,92S.
Cao-Hoang, L., Phan-Thi, H., Osorio-Puentes, F.J., Waché, Y. 2011. Stability of
carotenoid extracts of gâc (Momordica cochinchinensis) towards cooxidation—
Protective effect of lycopene on b-carotene. Food Research International, 44:
2252–2257.
Chan, A.C., Tran, K., Raynor, T., Ganz, P.R., Chow, C.K. 1991. Regeneration of v itamin E
in human platelets. The Journal of Biological Chemistry, 266: 17,290–17,295.

Chasse, G.A., Chasse, K.P., Kucsman, A., Torday, L.L., Papp, J.G. 2001. Conformational
potential energy surfaces of a lycopene model. Journal of Molecular Structure
(Theochem), 571(1): 7–26.
Chen, J., Shi, J., Xue, J., Ma, Y. 2009. Comparison of lycopene stability in water- and
oil-based food model systems under thermal and light irradiation treatments.
LWT—Food Science and Technology, 42(3): 740–747.
Clinton, S.K., Emenhiser, C., Schwartz, S.J. 1996. Cis–trans-Lycopene isomers, carot-
enoids, and retinol in human prostate. Cancer Epidemiol Biomarkers Prevention,
5: 823–833.
Conn, P.F., Schalch, W., Truscott, T.G. 1991. The singlet oxygen and carotenoid inter-
action. Journal of Photochemistry and Photobiology B, 11: 41–47.
Colle, I., Van Buggenhout, S., Van Loey, A., Hendrickx, M. 2010. High pressure
homogenisation followed by thermal processing of tomato pulp: Influence on
microstructure and lycopene in vitro bioaccessibility. Food Research International,
43: 2193–2200.
Deming, D.M., Boileau, A.C., Lee, C.M., Erdman, J.W. 2000. Amount of dietary fat
and type of soluble fiber independently modulate postabsorptive conversion
of β-carotene to vitamin A in Mongolian Gerbils. Journal of Nutrition, 130:
2789–2796.
Di, M.P., Kaiser, S., Sies, H. 1989. Lycopene as the most efficient biological carotenoid
singlet oxygen quencher. Archives of Biochemistry and Biophysics, 274: 532–538.
Emenhiser, C., Sander, L.C., Schwartz, S.J. 1995. Capability of a polymeric C30 sta-
tionary phase to resolve cis-trans carotenoids in reversed phase liquid chroma-
tography. Journal of Chromatography A, 707: 205–216.
Etminan, M., Takkouche, B., Caamano-Isorna, F. 2004. The role of tomato products
and lycopene in the prevention of prostate cancer: A meta-analysis of observa-
tional studies. Cancer Epidemiology, Biomarkers & Prevention, 13: 340–345.
Faisala, W., Ruane-O’Horab, T., O’Driscolla, G.M., Griffin, B.T. 2013. A novel lipid-
based solid dispersion for enhancing oral bioavailability of Lycopene—In vivo
evaluation using a pig model. International Journal of Pharmaceutics, 453: 307–314.
Glaucia, A.R., Carmen, S.F., Carlos, R.F.G. 2012. Microencapsulation of lycopene by
spray drying: Characterization, stability and application of microcapsules. Food
and Bioproducts Processing, 90: 37–42.
Gracia, P.B., Maria, L.R.C., Maria, M.C., Mercedes, P.M., Santiago, S.C. 2007.
Stabilization of all-trans-lycopene from tomato by encapsulation using cyclo-
dextrins. Food Chemistry, 105: 1335–1341.
Haila, K., Lievonen, S.M., Heinonen, M. 1996. Effects of lutein lycopene, annatto, and
γ-tocopherol on autoxidation of triglycerides. Journal of Agriculture and Food
Chemistry, 44: 2096–2100.
Henry, L.K., Catignani, G.L., Schwartz, S.J. 1998. Oxidative degradation kinetics of
lycopene, lutein, 9-cis and all-trans beta-carotene. Journal of the American Oil
Chemists’ Society, 75: 823–829.
Hoppe, P.P., Krämer, K., van den Berg, H., Steenge, G., van Vliet, T. 2003. Synthetic
and tomato-based lycopene have identical bioavailability in humans. European
Journal of Nutrition, 42(5): 272–278.
Kanasawud, P., Crouzet, J.C. 1990. Mechanism of formation of volatile compounds by
thermal degradation of carotenoids in aqueous medium. 2. Lycopene degrada-
tion. Journal of Agriculture and Food Chemistry, 38: 1238–1242.

Kessy, H.H., Zhang, H., Zhang, L. 2011. A study on thermal stability of lycopene
in tomato in water and oil food systems using response surface methodology.
International of Food Science and Technology, 46: 209–215.
Kohlmeier, L., Kark, J.D., Gomez-Gracia, E., Martin, B.C., Steck, S.E., Kardinal, A.F.M.
1997. Lycopene and myocardial infarction risk in the EURAMIC study. American
Journal of Epidemiology, 146: 618–626.
Kong, K.W., Khoo, H.E., Prasad, K.N., Ismail, A., Tan, C.P., Rajab, N.F. 2010. Revealing
the power of the natural red pigment lycopene. Molecules, 15: 959–987.
Kun, Y., Lule, U.S., Xiao-Lin, A.D. 2006. Lyocpene: Its properties and relationship to
human health. Food Reviews International, 22: 309–333.
Lambelet, P., Saucy, F., Loliger, J. 1985. Chemical evidence for interactions between
vitamins E and C. Experientia, 41: 1384–1388.
Lavelli, V., Torresani, M.C. 2011. Modeling the stability of lycopene-rich by-products
of tomato processing. Food Chemistry, 125: 529–535.
Lee, M.T., Chen, B.H. 2002. Stability of lycopene during heating and illumination in a
model system. Food Chemistry, 78: 425–432.
Levy, J., Bisin, E., Feldman, B., Giat, Y., Minster, A., Danilenko, M. Sharoni, Y. 1995.
Lycopene is a more potent inhibitor of human cancer cell proliferation then
either α-carotene or β-carotene. Nutrition and Cancer, 24: 257–266.
Liu, D.H., Shi, J., Ibarra, A.C., Kakuda, Y., Xue, S.J. 2008. The scavenging capacity and
synergistic effects of lycopene, vitamin E, vitamin C, and β-carotene mixtures
on the DPPH free radical. Food Science and Technology—LWT, 41(7): 1344–1349.
Maiani, G., Periago Castón, M.J., Catasta, G., Toti, E., Goñi Cambrodón, I., Bysted, A.
2009. Actual knowledge on food sources, intakes, stability and bioavailability
and their protective role in humans. Molecular Nutrition & Food Research, 53:
S194–S218.
Mao, L., Xu, D., Yang, J., Yuan, F., Gao., Y., Zhao, J. 2009. Effects of Small and large
molecule emulsifiers on the characteristics of b-carotene nanoemulsions pre-
pared by high pressure homogenization. Food Technology and Biotechnology, 47:
336–342.
Matioli, G., Rodriguez-Amaya, D.B. 2002. Licopeno encapsulado em goma arábica
e maltodextrina: Estudo da estabilidade. Brazil Journal and Food Technology, 5:
197–203.
McClements, D.J. 2012. Nanoemulsions versus microemulsions: Clarification of dif-
ferences, similarities and terminology. Soft Matter, 8(6): 1719–1729.
McClements, D.J., Decker, E.A. 2000. Lipid oxidation in oil-in-water emulsions:
Impact of molecular environment on chemical reactions in heterogeneous food
system. Journal of Food Science, 65: 1270–1282.
Micozzi, M.S. Beecher, G.R., Taylor, P.R., Khachik, F. 1990. Carotenoid analyses of
selected raw and cooked foods associated with a lower risk for cancer. Journal
of the National Cancer Institute, 82: 282–288.
Miki, N., Akatsu, K. 1970. Effect of heating sterilization on color of tomato juice.
Journal of Japanese Food Industry, 17: 175–181.
Miller, N.J., Sampson, J., Candeias, L.P., Bramley, P.M., Rice-Evans, C.A.: 1996.
Antioxidant activities of carotenes and xanthophylls. FEBS Letter, 384: 240–242.
Mortensen, A., Skibsted, L.H., Sampson, J., Rice-Evan, C., Everett, S.A. 1997.
Comparative mechanisms and rates of free radical scavenging by carotenoid
antioxidants. FEBS Letters, 418: 91–97.

Nguyen, M.L., Schwartz, S.J. 1998. Lycopene stability during food processing.
Experimental Biological and Medicine, 218: 101–105.
Nguyen, M.L., Schwartz, S.J. 1999. Lycopene: Chemical and biological properties.
Food Technology, 53: 38–44.
Nunes, I.L., Mercadante, A.Z. 2007. Encapsulaion of lycopene using spray-drying and
molecular inclusion process. Brazilian Archives of Biology and Technology, 50(5):
893–900.
Palozza, P., Krinsky, N.I. 1992. Antioxidant effects of carotenoids in vivo and in vitro:
An overview. Methods in Enzymology, 213: 403–420.
Pastori, M., Pfander, H., Boscoboinik, D., Azzi, A. l998. Lycopene in association with
alpha-tocopherol inhibits at physiological concentrations proliferation of pros-
tate carcinoma cells. Biochemical and Biophysical Research Communications, 250:
582–585.
Pedroza-Islas, R. 2002. Alimentos microencapsulados: Particularidades de los pro-
cesos para la microencapsulación de alimentos para larvas de especies acuíco-
las. In: Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Gaxiola-Cortés,
M.G., Simoes, N. (eds.). Avances en Nutrición Acuícola VI. Memorias del VI
Simposium Internacional de Nutrición Acuícola. 3 al 6 de Septiembre del 2002.
Cancún, Quintana Roo, México.
Qian, C., Decker, E.A., Xiao, H., McClemments, D.J. 2012. Physical and chemical sta-
bility of β-carotene-enriched nanoemulsions: Influence of pH, ionic strength,
temperature, and emulsifier type. Food Chemistry, 132: 1221–1229.
Rao, A.V., Agarwal, S. 1999. Role of lycopene as antioxidant carotenoid in the preven-
tion of chronic diseases: A review. Nutrition Research, 19(2): 305–323.
Rao, A.V., Waseem, Z., Agarwal, S. 1998. Lycopene content of tomatoes and tomato
products and their contribution to dietary lycopene. Food Research International,
31: 737–741.
Re, R., Bramley, P.M., Rice-Evans, C. 2002. Effects of food processing on flavonoids
and lycopene status in a Mediterranean tomato variety. Free Radical Research,
36: 803–810.
Ribeiro, H.S., Ax, K., Schubert, H. 2003. Stability of lycopene emulsions in food sys-
tems. Journal of Food Science, 68: 2730–2734.
Ried, K., Fakler, P. 2011. Protective effect of lycopene on serum cholesterol and blood
pressure: Meta-analyses of intervention trials. Maturitas, 68: 299–310.
Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner, K., Schuep, W. 1996.
Content and isomeric ratio of lycopene in food and human blood plasma. Food
Chemistry, 59(3): 459–465.
Schierle, J., Klipel, J.-D. Pietch, B. 2003. Determination of added lycopene and B-carotene in
foods-version 1.1. DSM Nutritional Products (registered as Roche Vitamins Ltd),
December 12, 2003.
Sharma, S.K. Le Maguer, M. 1996. Kinetics of lycopene degradation in tomato
pulp solids under different processing and storage conditions. Food Research
Shi, J., Dai, Y., Kakuda, Y., Mittal, G., Xue, J. 2008. Effect of heating and light irradia-
tion on the stability of lycopene in tomato purée. Food Control, 19(5): 514–520.
Shi, J., Kakùda, Y., Yeung, D., Jiang, Y. 2004. Stability and synergistic effect of antioxi-
dative properties of lycopene and other active components. Critical Reviews in
Food Science and Nutrition, 44: 559–573.

Shi, J., Le Maguer, M. 2000. Lycopene in tomatoes: Chemical and physical properties
affected by food processing. Critical Reviews in Food Science and Nutrition, 40(1):
1–42.
Shi, J., Le Maguer, M., Bryan, M., Kakuda, Y. 2003. Kinetics of lycopene degradation
in tomato puree by heat and light irradiation. Journal of Food Process Engineering,
25: 485–498.
Shi, J., Wu, Y., Bryan, M., LeMaguer, M. 2002. Oxidation and isomerization of lycopene
under thermal treatment and light irradiation in food processing. Nutraceutical
and Food, 7: 179–183.
Shi, J., Yi, C., Xue, J., Jiang, Y., Ma, Y., Li, D. 2009. Effects of modifier on lycopene
extract profile from tomato skin using supercritical-CO2 fluid. Journal of Food
Shu, B., Yu, W., Zhao, Y., Liu, X. 2006. Study on microencapsulation of lycopene by
spray-drying. Journal of Food Engineering, 76: 664–669.
Sies, H., Stahl, W. Sundquist, A.R. 1992. Antioxidant function of vitamins. Vitamins E
and C, β-carotene, and other carotenoids. Annals of the New York Academy of
Sciences, 669: 7–20.
Stahl, W., Junghans, A., de Boer, B., Driomina, E.S., Briviba, K., Sies, H. 1998.
Carotenoid mixtures protect multilamellar liposomes against oxidative dam-
age: Synergistic effects of lycopene and lutein. FEBS Letters, 427: 305–308.
Stahl, W., Sies, H. 1992. Uptake of lycopene and its geometrical isomers is greater
from heat-processed than from unprocessed tomato juice in humans. Journal of
Nutrition, 122: 2161–2166.
Stahl, W., Sies, H. 1996. Perspectives in biochemistry and biophysics. Archives of
Biochemistry and Biophysics, 336(1): 1–9.
Tavares, C.A., Rodriguez-Amaya, D.B. 1994. Carotenoid composition of Brazilian
tomatoes and tomato products. LWT—Food Science and Technology, 27: 219–224.
Wilberg, V.C., Rodriguez-Amaya, B.D. 1995. HPLC quantitation of major carotenoids
of fresh and processed guava, mango and papaya. LWT—Food Science and
Yi, C., Shi, J., Xue, J., Jiang, Y., Li, D. 2009. Effects of supercritical fluid extraction
parameters on lycopene yield and antioxidant activity. Food Chemistry, 113(4):
1088–1094.
Zanoni, B., Peri, C., Nani, R., Lavelli, V. 1999. Oxidative heat damage of tomato halves
as affected by drying. Food Research International, 31: 395–401.
Zechmeister, L., Polgar, A. 1944. Cis-trans isomerization and cis-peak effect in the
α-carotene set and in some other stereoisomeric sets. JAOCS, 66(1): 137–144.

Index
A Atomization, 463–464; see also

Nano- and microparticle
ABTS, see 2,2-Azino-bis-3-
drying process
ethylbenzothiazoline-6-
ATPE, see Aqueous two-phase
sulfonic acid (ABTS)
extraction (ATPE)
Acentric factor, 92; see also Solubility in
2,2-Azino-bis-3-ethylbenzothiazoline-6-
supercritical-CO2 fluid
sulfonic acid (ABTS), 626
Active packaging, 415–416; see also
Azulene, 200
Functional food packaging
AFEX, see Ammonia fiber explosion
(AFEX) B
Agricultural residues, 513 Bactofugation, see Centrifugation
AHA, see American Heart Association Batch reactors, 525; see also Bioreactor
(AHA) design
ALA, see α-Linolenic acid (ALA) Batch-stirred tank reactor, 146, 147; see
Algal oil, 358 also Enzyme reactors
α-Carotene solubility, 68, 75 BCAAs, see Branched-chain amino acids
α-Linolenic acid (ALA), 359, 376 (BCAAs)
American Heart Association (AHA), b-CD, see b-Cyclodextrin (b-CD)
375 β-Carotene, 325
Ammonia fiber explosion (AFEX), 483 extraction yields, 74, 78
Antioxidant sterol compounds, 487; see nitrogen dioxide scavenging, 594
also Nutraceuticals solubility, 69–72
Antisolvent, 361 superoxide scavenging, 591
Aqueous two-phase extraction (ATPE), β-Cyclodextrin, 335
495 Better Than Fresh (BTF), 443
ARA, see Arachidonic acid(ARA) BHT, see Butylated hydroxytoluene (BHT)
Arachidonic acid(ARA), 510; see also BICs, see Broken and intact cells (BICs)
Polyunsaturated fatty acids Binary diffusion coefficient, 115; see also
(PUFAs) Supercritical fluid extraction
algal sources, 494, 510 (SFE)
fungal sources of, 494 Bioactive components, 8; see also
producing fungi, 492 Supercritical-CO2 fluid
Arrhenius equation, 521 extraction technology
Astaxanthin, 590; see also Carotenoids extraction, 29–30
carcinogenesisinhibition, 603 solubility, 60–61
quenching of lipid peroxidation, 591 Bioactive proteins, 236, 554; see also
Atmospheric drying, 562; see also Thermal degradation reactions
Dehydration technologies Biochemical reactions, 128, 149; see also
fluid-bed-related drying, 565 Enzymatic catalysis; Enzyme
jet spouted bed drying system, reactors; Supercritical fluids
566, 567 (SCFs)
spray drying, 562–564 SCFs as solvents, 128–129
tray drying, 564, 565 water in, 128, 144
639
640 Index
Biocompounds, 57 C
Bioconversions, 486; see also
Caffeine, 36–39; see also
Nutraceuticals; Microbial
Supercritical-CO2 fluid
modeling
extraction technology
products, 509
CALB, see Candida antarctica lipase B
Biodiesel, 139
(CALB)
Biopharma compounds, 484; see also
Candida antarctica lipase B (CALB), 135
Nutraceuticals
Canthaxanthin, 591
Biopolymers, 285–289; see also
CAP, see Cellulose acetate propionate
Emulsifiers
(CAP)
of plant, 362
Capillary electrophoresis (CE), 495
Bioprocess, 482, 484; see also
Capsanthin, 590, 591, 598
Nutraceuticals; Microbial
Capsorubin, 591
modeling
Caramelization, 556; see also Thermal
design, 510
degradation reactions
Bioreactor design, 511, 524; see also
Carbamates, 140
Continuous stirred tank
Carbon dioxide, 7, 105
reactors (CSTRs); Microbial
polarizability of, 63
modeling; Nutraceutical
Carcinogenesis inhibition, 603, 604
batch reactors, 525
Carotenes, 589–590
strategies, 530–533
Carotenoids, 226–227, 484–485; see also
xylitol fermentations, 532
Lycopene; Nitrocarotenoids;
xylose and glucose fermentation,
Nutraceuticals; Peroxynitrite;
533–536
Singlet oxygen
Biorefinery, 483; see also Nutraceuticals
antioxidative activities of, 589–591,
process flow, 500
612
Biphasic mixtures, 132
apo-nitroastaxanthins, 596
BNF, see British Nutrition Foundation
with astaxanthin, 595–597
(BNF)
with β-carotene, 597
Bovine; see also Emulsifiers; Milk
with capsanthin and fucoxanthin,
casein, 287
598–599
milk, 237, 238
conjugated double bond, 591
Branched-chain amino acids (BCAAs),
EBV-EA activation, 602
244
free radical mechanisms, 591
British Nutrition Foundation (BNF),
with lutein, 597–598
375
lutein-6H-1,2-oxazine, 598
Broken and intact cells (BICs), 107;
with lycopene, 599–600
see also Supercritical fluid
nitroastaxanthins, 596
extraction (SFE)
nitroluteins, 598
models, 116–117
nitro-β-carotene, 597
BTF, see Better Than Fresh (BTF)
with peroxynitrite, 604
Büchi mini spray-dryer B-290, 465,
quenching of singlet oxygen, 590
466–467; see also Spray-drying
and RNS, 594
of nano- and microcapsules
tyrosine nitration inhibition by,
Büchi nano spray-dryer B-90, 465,
600–601
467–468; see also Spray-drying
Casein, 287–288; see also Emulsifiers;
of nano- and microcapsules
Milk
Buriti fruit extraction data, 58
micelles, 243
Butylated hydroxytoluene (BHT), 299

Index 641
CDs, see c-Cyclodextrins (CDs) Coaxial microcapillary fluidic device,

CE, see Capillary electrophoresis (CE); 341
Co-encapsulation (CE) Co-crystallization, 335; see also
Cell disruption, 42; see also Micro-nanoencapsulation
Supercritical-CO2 fluid technology
extraction technology COD, see Chemical oxygen demand
Cellulose acetate propionate (CAP), 343 (COD)
Centrifugation, 246; see also Milk protein Co-encapsulation (CE), 298; see
extraction also Coating materials;
CFUs, see Colony-forming units (CFUs) Microencapsulation
Charged UF membranes, 225; see also applications, 298
Nutraceutical production efficiency, 300
Chemical effect, 74; see also Solubility in of gallium, 298
supercritical-CO2 fluid techniques, 299
Chemical oxygen demand (COD), 524 Coextrusion, 332; see also Micro-
Chilli (Capsicium species), 34 nanoencapsulation technology
Chitosan, 493; see also Nutraceuticals Colloidosomes, 339; see also
Cholesterol, 32; see also Supercritical-CO2 Micro-nanoencapsulation
fluid extraction technology technology
-free food products, 32–33 Colony-forming units (CFUs), 357
Chrastil equation, 88, 145 Color-masking techniques, 353; see also
CLA, see Conjugated linoleic acid Salt fortification
(CLA) Commodity biochemical
CO2 thermodynamic solubility bioproduction, 484–485; see
correlation, 111; see also also Nutraceuticals
Supercritical fluid extraction Compressed gas model, 84; see also
(SFE) Solubility in supercritical-CO2
Coacervation, 320, 332, 391; see fluid
also Microencapsulated Compressibility equation, 91; see also
omega-3 ingredient; Micro- Solubility in supercritical-CO2
nanoencapsulation technology fluid
advantages, 333 Concentration-driven membrane
complex, 333, 392 processes, 220; see also
droplets, 332 Membrane technology
in food industry, 392 Conjugated linoleic acid (CLA), 141,
limitations, 334 413–414
Coating materials, 342, 343; see also Continuous high-pressure
Co-encapsulation (CE); enzyme membrane
Micro-nanoencapsulation reactor, 148–149; see also
technology Enzyme reactors
classification, 342 Continuous packed-bed reactor, 146, 147;
enteric coatings, 346 see also Enzyme reactors
Eudragit® E, 347–348 Continuous stirred tank reactors
HPMC-based encapsulants, (CSTRs), 525; see also Bioreactor
345, 346 design
hydrophilic coatings, 344–345 with cell recycle, 527–529
hydrophobic coatings, 345–346 fed-batch systems, 529–530
reverse-enteric coatings, 347–349 hydraulic retention time, 526
selection of, 344 mass balance, 525, 526, 528

642 Index
Controlled release technology, 336; see Dehydration technologies, 546, 547–550;

also Micro-nanoencapsulation see also Atmospheric drying;
technology Thermal degradation reactions
biodegradable system, 337, 338 benefits, 549–550
in food industry, 338 bioactivity, 547
matrix-based, 337 dehydration curve, 549, 550
microencapsulation coupling with, dehydration kinetics, 550–552
338–339 dimensionless moisture ratio, 551
swelling-controlled release, 337 drying rate constant, 552
Conventional extraction processes, 4 evaporation, 560–561
Correlation models, 162–163; see also freeze drying, 567–568
Mass transfer coefficient functional food, 546–547
Cosolvent, 19–21; see also Solubility in future directions, 574–575
supercritical-CO2 fluid heat-pump drying, 571
effects, 64–66 hybrid drying, 572–573
modification, 64, 73 mass diffusivity characterization,
Creaming, 283; see also Emulsions 560
Critical point, 10 mathematical models, 551
Crossover point, 56 mechanical dewatering, 561
Crossover pressure operation, 95; see also membrane technologies, 561
Solubility in supercritical-CO2 microwave drying, 569–570
fluid moisture content, 548
CSTRs, see Continuous stirred tank moisture diffusivity, 560
reactors (CSTRs) nutraceuticals, 546
Cyanobacteria, 488 osmotic dehydration, 571–572
Cyclodextrin, 334, 384–385 particle friction characterization, 558
b-Cyclodextrin (b-CD), 631 particle shape and size
c-Cyclodextrins (CDs), 631 characterization, 558, 559
Cylindrical extractor, 106; see also predrying and water removal,
Supercritical fluid extraction 560–562
(SFE) properties, 557–560
Cysteine oxidation, 592 solar drying, 572
solid materials, 559
solvent extraction, 561
D
superheated steam drying, 568–569
Dairy product extraction, 235, 260; technology optimization, 574
see also Milk fat extraction; vacuum drying, 570
Milk fat globule membrane Denaturation, 554; see also Thermal
(MFGM); Milk protein degradation reactions
extraction Dense gases, 130
DCB, see 1,2-Dichlorobenzene (DCB) Dense-phase carbon dioxide (DPCD),
DCMD, see Direct contact membrane 428, 436; see also High-pressure
distillation (DCMD) processing of foods
DE, see Dextrose equivalent (DE) advantages, 436
Decosahexaenoic acid (DHA), 39 applications, 442–445
Degree of separation, 202; see continuous, 437
also Vacuum fractional drawback of, 436
distillation effects on foods quality, 441–442

Index 643
microbial inactivation by, 438–440 freeze drying, 293

Porocrit LLC scCO2 system, 444 methods, 278
potentials of, 445 rate constant, 552
principles and equipment, 437 DSD, see Droplet size distribution (DSD)
state-of-the-art, 438
supercritical-CO2 extrusion process,
E
444
Dextrose equivalent (DE), 290; see also EBV-EA, see Epstein–Barr virus early
Emulsifiers antigen (EBV-EA)
DFC, see Direct fixed capital (DFC) EC, see Equipment cost (EC)
DFS, see Double-fortified salt (DFS) ED, see Electrodialysis (ED)
DGDG, see Digalactosyl diacylglycerol Edible nanocoatings, 418; see also
(DGDG) Nanotechnology
DHA, see Decosahexaenoic acid (DHA) EDTA, see Ethylenediaminetetraacetic
1,2-Dichlorobenzene (DCB), 170 acid (EDTA)
Digalactosyl diacylglycerol (DGDG), 488 EE, see Encapsulation efficiency (EE)
5,6-Dimethylbenzimidazole (DMBI), 532 EEC, see European Economic
Direct contact membrane distillation Commission (EEC)
(DCMD), 221; see also EFAs, see Essential fatty acids (EFAs)
Membrane technology Eicosapentaenoic acid (EPA), 39,
Direct fixed capital (DFC), 501 272, 358, 375, 490; see also
Distillation, 199, 213–214; see also Orange Polyunsaturated fatty acids
peel oil purification; Vacuum (PUFAs)
fractional distillation algal sources, 494, 510
fractional, 201 alternative sources of, 491
simple, 201 disorders associated with, 491
steam, 200–201 fungal sources of, 494
DMBI, see 5,6-Dimethylbenzimidazole Electrodialysis (ED), 219; see also
(DMBI) Membrane technology
Docosapentaenoic acid (DHA), 272, 358, Electrospun nanofibers, 418; see also
375; see also Polyunsaturated Nanotechnology
fatty acids (PUFAs) Emulsification techniques, 290–291; see
algal sources, 510 also Microencapsulation of
alternative sources of, 491 LBCs
disorders associated with, 491 Emulsifiers, 284, 339; see also
producing organisms, 491 Microencapsulation of LBCs
Double-fortified salt (DFS), 351; see also biopolymers, 285–289
Salt fortification complex emulsifier systems, 289–290
Downstream processing, 159 glycerophospholipid, 286
DPCD, see Dense-phase carbon dioxide low-molecular-weight surfactants,
(DPCD) 285
Droplet size distribution (DSD), 281 polysorbate, 286
Drying, 77–78, 278, 291, 546; see also Emulsions, 278, 281; see also
Dehydration technologies; Microencapsulation of LBCs
Microencapsulation of LBCs; coalescence, 283
Solubility in supercritical-CO2 continuous phase, 279
fluid; Spray drying creaming, 283
disadvantages, 294 dispersed phase, 279

644 Index
Emulsions (Continued) in SCFs, 134–137

droplet, 281–282 SC technology, 138
flocculation, 284 solvent systems, 134
interfacial properties, 282–283 technological advances, 133
multiple, 280–281 temperature effect, 143–144
Ostwald ripening, 283–284 water activity effect, 144–146
O/W emulsion, 279 Enzymatic ring-opening polymerization
stability, 283 (e-ROP), 136; see also Enzymatic
Enantioselective hydrolysis, 135; see also catalysis
Enzymatic catalysis Enzyme−substrate (ES), 515
Encapsulants, 382; see also Enzyme, 131
Microencapsulated omega-3 Enzyme reactors, 146; see also
ingredient Biochemical reactions
carbohydrates, 384–385 batch-stirred tank reactor, 146, 147
classes of, 383 continuous high-pressure enzyme
lipid materials, 385 membrane reactor, 148–149
protein–carbohydrate conjugates, 385 continuous packed-bed reactor, 146,
proteins, 383–384 147
selection of, 344 downstream processing, 146
Encapsulated premix-based processing costs, 149
fortification, 354; see also Ultra EOS, see Equation of state (EOS)
Rice® technology EPA, see Eicosapentaenoic acid (EPA)
Encapsulation, 459; see also Epstein–Barr virus early antigen
Nano-microencapsulation (EBV-EA), 602
Encapsulation efficiency (EE), 327–328, 631 Equation of state (EOS), 56, 88–90;
Encapsulation yield (EY), 631 see also Solubility in
Enhancement factor, 76, 77; see also supercritical-CO2 fluid
Solubility in supercritical-CO2 Equipment cost (EC), 501
fluid e-ROP, see Enzymatic ring-opening
Enteric coatings, 346; see also Coating polymerization (e-ROP)
materials Error-in-variable model (EVM),
Entrainer, 73, 74, 130; see also Solubility 164–165; see also Mass transfer
in supercritical-CO2 fluid coefficient
Enzymatic catalysis, 127; see also ES, see Enzyme−substrate (ES)
Biochemical reactions Essence Recovery System, 206; see also
advantages of, 131 Orange peel oil purification
Chrastil equation, 145 Essential fatty acids (EFAs), 39, 510
dense gases, 130 Essential oils, 117; see also
enantioselective hydrolysis, 135 Supercritical-CO2 fluid
enzyme stability, 137–139 extraction technology
esterification of secondary alcohols, separation of, 34–36, 37, 38
136 Ethylenediaminetetraacetic acid
in nonconventional media, 131–134 (EDTA), 328
organic vs. aqueous media, 135 Eudragit® E, 347–348; see also Coating
poly-l-lactide production, 136 materials
pressure effect, 139–141 European Economic Commission (EEC),
pressurization–depressurization 37
steps, 141–143 Evaporation, 560–561; see also
ring-opening polymerization, 136 Dehydration technologies

Index 645
EVM, see Error-in-variable model (EVM) Fractional distillation, 201, 213; see also
External mass-transfer resistance, Distillation
113–114; see also Supercritical Free fatty acid (FFA), 141
fluid extraction (SFE) Freeze drying, 78, 293, 331, 567–568;
Extraction, 4, 8 see also Dehydration
Extrusion, 331–332; see also technologies; Drying; Micro-
Microencapsulated nanoencapsulation technology;
omega-3 ingredient; Micro- Solubility in supercritical-CO2
nanoencapsulation technology fluid
modifications to, 389–390 FTNF (from the named fruit), 208
EY, see Encapsulation yield (EY) Fucoxanthin, 590, 591, 598
Fugacity coefficient, 57; see also
Solubility in supercritical-CO2
F
fluid
FAO, see Food and Agriculture calculation, 89
Organization (FAO) Functional food, 267, 374,
FDA, see Food and Drug Administration 409, 546–547; see also
(FDA) Dehydration technologies;
FFA, see Free fatty acid (FFA) Microencapsulation;
Fish oil, 358, 491; see also Nutraceuticals; Omega-3
Polyunsaturated fatty acids fatty acid; Functional food
(PUFAs); Supercritical-CO2 packaging
fluid extraction technology global demand value and
concentration, 39–40 bioseparation cost, 496
Flavonoids, 178, 359 shelf-life of, 419
Flavor and fragrance fractionation, Functional food packaging, 409, 419
30–32; see also Supercritical-CO2 active packaging, 415–416
fluid extraction technology fruits and vegetables, 410
Flavor Tec process, 208; see also Orange intermediate moisture products, 413
peel oil purification material choice, 414–415
Flocculation, 284; see also Emulsions nanotechnology, 416–419
Fluid-bed-related drying, 565; see also oils and fats, 413–414
Atmospheric drying probiotics, 411–413
small-scale fluid bed drying system, requirements of, 410, 414
566 Functional ingredients, 268; see also
Fluidized-bed coating, 331, 391; see also Microencapsulation
Micro-nanoencapsulation
technology
G
Folded oils, 204; see also Orange peel oil
purification Galactooligosaccharides (GOSs), 138
Food and Agriculture Organization GA products, see Growth-associated
(FAO), 357 products (GA products)
Food and Drug Administration (FDA), Gas chromatography (GC), 493
342, 376, 482 Gastrointestinal tract (GI tract), 325
Food components solubility, 55–58; GC, see Gas chromatography (GC)
see also Solubility in Gelation, 334; see also Micro-
supercritical-CO2 fluid nanoencapsulation technology
Fouling, 221; see also Membrane Generally recognized as safe (GRAS), 5,
technology 342, 436

646 Index
Gibbs–Thomson effect, 283; see also effects on food quality, 432–435

Emulsions generation, 429
Ginger (Zingiber officinalis), 34 inactivation mechanisms, 430–431
GI tract, see Gastrointestinal tract (GI microbial inactivation, 431–432, 433
tract) potentials of, 445
Globular proteins, 340, 460 principles and equipment, 429–430
Glycerophospholipid, 286; see also state of art of, 430
Emulsifiers types of, 430
GMP, see Good manufacturing practice High-methylester-pectin (HMP), 288,
(GMP) 360
Good manufacturing practice (GMP), High-performance liquid
497 chromatographic (HPLC), 191,
GOSs, see Galactooligosaccharides 461, 489
(GOSs) High-power ultrasounds (HPU), 445
Granulometry, 465 High-pressure batch-stirred tank
GRAS, see Generally recognized as safe reactor (HP BSTR), 149; see also
(GRAS) Enzyme reactors
Greek letters, 121 High-pressure continuously operated
Green separation technologies, 4; see reactor (HP COR), 149; see also
also Supercritical-CO2 fluid Enzyme reactors
extraction technology High-pressure processing of foods,
Growth-associated products (GA 427; see also Dense-phase
products), 522 carbon dioxide (DPCD); High
Gum arabic (GA), 288 hydrostatic pressure (HHP)
Gums, 384 High resolution gas chromatography
(HRGC), 209; see also Orange
peel oil purification
H
High vacuum distillation, 202; see
Halocarbons, 63 also Vacuum fractional
Halocynthiaxanthi superoxide distillation
scavenging, 591 High-value functional substances, 40
HDL, see High-density lipoprotein HLB value, see Hydrophilic–lipophilic
(HDL) balance value (HLB value)
Heat-pump drying, 571; see also HMF, see 5-Hydroxymethylfurfural
Dehydration technologies (HMF)
Height Equivalent per Theoretical Plate HMP, see High-methylester-pectin
(HETP), 201 (HMP)
HETP, see Height Equivalent per Horseradish peroxidase (HRP), 142; see
Theoretical Plate (HETP) also Enzymatic catalysis
HHP, see High hydrostatic pressure HP BSTR, see High-pressure batch-
(HHP) stirred-tank reactor (HP
High-density lipoprotein (HDL), 32, 278 BSTR)
High-energy emulsification methods, HP COR, see High-pressure
469; see also Nano-emulsion continuously operated reactor
(NE) (HP COR)
High hydrostatic pressure (HHP), HPLC, see High-performance liquid
428; see also High-pressure chromatographic (HPLC)
processing of foods HPMC, see Hydroxypropyl
applications, 435–436 methylcellulose (HPMC)

Index 647
HPU, see High-power ultrasounds J

(HPU)
Jet spouted bed drying system, 566, 567;
HRGC, see High resolution gas
see also Atmospheric drying
chromatography (HRGC)
HRP, see Horseradish peroxidase (HRP)
Hybrid drying, 572–573; see also K
Dehydration technologies
Hydrophilic coatings, 344–345; see also Kelvin effect, see Gibbs–Thomson
Coating materials effect
Hydrophilic–lipophilic balance value
(HLB value), 389
L
Hydrophobic coatings, 345–346; see also
Coating materials LAME, see Linoleic acid methyl ester
Hydrophobic materials, 220; see also (LAME)
Membrane technology LBCs, see Lipophilic bioactive
Hydrosol, 200 components (LBCs)
5-Hydroxymethylfurfural (HMF), 517 LBSD, see Lipid-based solid dispersion
Hydroxypropyl methylcellulose (LBSD)
(HPMC), 344, 345; see also LDL, see Low-density lipoprotein
Coating materials (LDL)
-based encapsulants, 346 Likens–Nickerson method, 30
Hydroxytyrosol, 497; see also d-Limonene, 205; see also Orange peel oil
Nutraceuticals purification
Linear least-squares method, 164; see
also Mass transfer coefficient
I
Linoleic acid methyl ester (LAME), 628
IMF, see Intermediate moisture foods Lipid, 485; see also Nutraceuticals
(IMF) peroxidation, 591
Inclusion complexation, 393; see also Lipid-based solid dispersion (LBSD), 630
Microencapsulated omega-3 Lipophilic bioactive components
ingredient (LBCs), 268; see also
Inhibition models, 515; see also Microbial Microencapsulation of LBCs;
modeling Microencapsulation
Institute of Medicine (IOM), 375 Lipoproteins, 32
Interactive model, 518; see also Microbial Liposomes, 334, 385; see also
modeling Microencapsulated
Intermediate moisture foods (IMF), 413 omega-3 ingredient; Micro-
Internal mass-transfer resistance, 114; nanoencapsulation technology
see also Supercritical fluid encapsulation, 393
extraction (SFE) entrapment, 334
International Society of the Study Liquid–liquid extraction (LLE), 221; see
of Fatty Acids and Lipids also Membrane technology
(ISSFAL), 375 LLE, see Liquid–liquid extraction (LLE)
IOM, see Institute of Medicine (IOM) LMW, see Low-molecular-weight
Ion-exchange separations, 252–253; see (LMW)
also Milk protein extraction Low-density lipoprotein (LDL), 32, 272
ISSFAL, see International Society of antioxidative activity, 591
the Study of Fatty Acids and Low-energy emulsification methods, 470;
Lipids (ISSFAL) see also Nano-emulsion (NE)

648 Index
Low-molecular-weight (LMW), 285; see correlation equation, 161

also Emulsifiers correlation parameters, 162
compounds, 251 data and predictions, 168, 169
surfactants, 285, 286 error-in-variable model, 164–165
Lutein, 590, 597–598 errors for Sherwood number
superoxide scavenging, 591 prediction, 171
Lycopene, 604, 609, 631–633; estimation, 164, 171
see also Carotenoids; JCR of every two parameters, 172
Microencapsulation kinetic variables, 161
antioxidant activities of, 612 model development, 162–163
bioactive potency, 623 prediction, 161
contents in tomato powders, 623 Sherwood number, 162, 173
degradation by heat, 613, 614–619 3D view of model prediction, 174
degradation by light, 619–620 verification, 166–174
degradation by oxygen, 620–623 weighted linear least-squares, 164
density and solubility, 93 Matrix encapsulation, 331; see also
first-order plot, 619 Micro-nanoencapsulation
HPLC chromatogram of, 621–622 technology
isomeric forms, 610 MC, see Membrane contactors (MC);
isomerization, 618, 623–626 Moisture content (MC)
isomers in tomato products, 624, 626 MD, see Membrane distillation (MD)
on LAME oxidation, 629 Measured inhibition effect (MIE), 628
microemulsion, 298 Mechanical dewatering, 561; see also
properties, 611–612 Dehydration technologies
relative recoveries of, 67, 82 Membrane contactors (MC), 220; see also
solubility and yield, 83 Membrane technology
solubility verification, 90–94 Membrane distillation (MD), 220; see also
stabilization, 630–631 Membrane technology
structural changes during heating, Membrane-specific proteins, 237
625 Membrane technology, 217, 230, 561; see
structure, 610 also Dehydration technologies;
superoxide scavenging, 591 Nutraceutical production
synergistic effects, 627–629 concentration-driven, 220
Lyophilization, 567–568 electrodialysis, 219
family of processes, 220–221
fouling, 221
M
investigation, 223
Maillard reaction, 246, 555–556; see also modeling and simulation, 223
Milk; Thermal degradation optimized process, 222
reactions polarization, 221
Maillard reaction products (MRPs), 280 process analysis, 223
Maltodextrin, 460 separation processes, 218–222
MAP, see Modified atmosphere solution-diffusion model, 219
packaging (MAP) MF, see Microfiltration (MF)
Martek Biosciences process, 482 MFGM, see Milk fat globule membrane
Mass balance equations, 108–109; see also (MFGM)
Supercritical fluid extraction MGA products, see Mixed-growth-
(SFE) associated products (MGA
Mass transfer coefficient, 161, 173 products)

Index 649
MI, see Micronutrient Initiative (MI) growth rate of Candida, 520

Micro-nanoencapsulation technology, inhibition models, 515–517
320, 361–363; see also Coating interactive model, 518
materials; Controlled kinetic rate expressions, 522–524
release technology; microbial growth models, 513
Microencapsulated delivery; Monod model, 514–515
Nano-microencapsulation; for multiple limiting substrates,
Salt fortification; Spray drying; 517–520
Ultra Rice® technology non-interactive model, 518
chemical methods, 332–336 product inhibition, 516
coacervation, 332–334 rate of GA and NGA product
coaxial microcapillary fluidic device, formation, 523
341 rate of oxygen utilization, 524
co-crystallization, 335 rate of substrate utilization, 523
coextrusion, 332 single limiting nutrient models, 514
colloidosomes, 339 Steele’s model, 516
concept and history, 320–321 temperature effects, 521
controlled release, 338–339 xylitol, 511–513
equipment development, 341–342 yield parameters, 520–521
extrusion, 331–332, 354–355 Microcapsules, 272, 325, 326, 330; see
for ferrous fumarate, 350–353 also Microencapsulation;
fluidized-bed coating, 331 Micro-nanoencapsulation
in food applications, 322, 349 technology
for food ingredients, 323–324 morphology and size, 328–329
in fortified and functional food, Microemulsion, 340; see also Nanolevel
322–326 delivery systems
freeze drying, 331 Microencapsulated delivery, 326; see
gelation, 334 also Micro-nanoencapsulation
liposome entrapment, 334 technology
methods, 330–332 bioavailability, 326–327
microcapsules, 330 digestion process, 327
molecular inclusion, 334–335 encapsulation efficiency, 327–328
nano-level delivery systems, 339–341 microcapsule morphology and size,
polymerization, 335 328–329
processing techniques, 329 of nutraceuticals, 356–357
relationship between material and of omega-3 and omega-6 oils,
technique, 321 358–359
self-assembly process, 339 of phytochemicals, 359–361
solvent extraction, 335 of probiotics, 357–358
spray chilling, 331 storage stability, 329
surface defects, 352 technologies, 356–357
for vitamins and minerals, 349–350 Microencapsulated food ingredients,
Microbial modeling, 511, 536; see 323–324; see also Microcapsules
also Bioreactor design; benefits of, 456
Nutraceutical Microencapsulated omega-3 ingredient,
Arrhenius equation, 521 394; see also Encapsulants;
concentration simulation, 535, 536 Microencapsulation; Omega-3
of glucose/xylose utilization, fatty acid; Omega-3 oil
533–536 coacervation, 391–392

650 Index
Microencapsulated omega-3 ingredient bioavailability of, 326–327

(Continued) encapsulation efficiency of, 327–328
delivery format, 381–382 malnutrition, 351
emulsion and stabilization, 386–387 storage stability of, 329
encapsulant selection, 379–380 Microparticles, 457
extrusion, 389–390 morphologies of, 458
gelation and particle formation, Microspheres, 330; see also Micro-
390–391 nanoencapsulation
inclusion complexation, 393 technology
liposome encapsulation, 393 Microwave drying, 569–570; see also
manufacture, 378, 381, 385–386, 391 Dehydration technologies
material selection, 383 MIE, see Measured inhibition effect (MIE)
nanoencapsulation, 393–394 Milk, 235, 260; see also Dairy product
production elements, 379, 380 extraction
spray-drying, 387–389 acidic serum proteins, 245
supercritical fluid spraying, 393 bacteriological quality of, 246
Microencapsulation, 268, 300–301, bovine, 237, 238
320, 374–375, 377, 456; casein micelles, 243
see also Functional food; components, 236
Microencapsulation of fat, 237–240
LBCs; Microencapsulated polar lipids, 240
omega-3 ingredient; Micro- proteins, 245
nanoencapsulation technology skimmed, 245
applications of, 321 whey proteins, 244
of bioactive food ingredients, 273–277 Milk fat extraction, 237; see also Milk;
co-encapsulation, 298–300 Milk protein extraction
coupling with controlled release, bovine milk, 237, 238
338–339 enrichment and extraction, 240
elements of, 380 milk constituent particle size, 239
entrapped material, 320 milk fat characteristics, 237–240
in food industry, 378 polar lipid content, 241
in foods, 269 Milk fat globule membrane (MFGM),
methods, 270 236; see also Milk
of omega-3 oils, 378 composition, 239
types of microcapsules, 272 isolation, 241–243
wall materials, 269–270 Milk protein extraction, 243; see also
Microencapsulation of LBCs, 272; see Milk; Milk fat extraction
also Emulsifiers; Emulsions; casein coagulation, 247–249
Microencapsulation; Spray casein fractionation, 249–250, 259–260
drying casein isolation, 247
challenges, 296–298 ion-exchange separations, 252–253
delivery, 278 milk pretreatment, 245–247
drying, 278, 291–294 milk proteins, 243
emulsification, 290–291 protein fractionation, 245, 246
lycopene microemulsion, 298 protein separation, 248
Microfiltration (MF), 218, 246; see also serum protein concentration, 250
Milk protein extraction serum protein fractionation, 253
Micronutrient Initiative (MI), 351 UF membrane processes, 251–252
Micronutrients, 322 whey composition, 248

Index 651
Mixed-growth-associated products material for spray-drying, 458–460

(MGA products), 530 nano- and microparticle structure,
MMM, see Mohsen–Nia–Moddaress– 457–458
Mansoori (MMM) vitamins, 461
Modern food production practice, 363 Nano-based composite packaging
Modified Arrhenius equation, 521 materials (NCP materials), 417;
Modified atmosphere packaging (MAP), see also Nanotechnology
411 Nanobiotechnology, 484; see also
Mohsen–Nia–Moddaress–Mansoori Nutraceuticals
(MMM), 90 Nano-emulsion (NE), 468, 474; see also
Moisture content (MC), 548; see also Nano-microencapsulation;
Dehydration technologies Nano- and microparticle
Molecular inclusion, 334–335; see also drying process; Spray-drying
Micro-nanoencapsulation of nano- and microcapsules
technology advantages, 468–469
Monod model, 514–515; see also choice of wall materials, 470–471
Microbial modeling high-energy emulsification, 469
M-O/W, see Multilayer oil-in-water low-energy emulsification, 470
(M-O/W) redispersion of, 473–474
MRPs, see Maillard reaction products sample preparation, 471–472
(MRPs) size distribution, 474
Mucor miehei lipase, 138 vitamin E acetate encapsulation, 476,
MULMICAP, see Multi Microcapsule 470, 472
(MULMICAP) Nanoencapsulation, 300–301, 393–394;
Multilayer oil-in-water (M-O/W), 279; see also Microencapsulated
see also Emulsions omega-3 ingredient;
Multi Microcapsule (MULMICAP), 350 Microencapsulation
Nanofiltration (NF), 218
Nanolevel delivery systems, 339–341; see
N
also Micro-nanoencapsulation
Nanomaterials (micro materials), 462 technology
Nano- and microparticle drying Nanoparticulate delivery systems, 340;
process, 462; see also Micro- see also Nanolevel delivery
nanoencapsulation technology; systems
Nano-microencapsulation; Nanotechnology, 416; see also Functional
Nano-emulsion; Spray-drying food packaging
of nano- and microcapsules edible nanocoatings, 418
atomization, 463–464 electrospun nanofibers, 418
effect on properties, 464 innovations, 418–419
granulometry, 465 inorganic, 417
temperature effect on powder, 464–465 NCP materials, 417
Nano-microencapsulation, 457; see also smart packaging, 418
Nano-emulsion; Nano- and National Cash Register Corp. (NCR
microparticle drying process; Corp.), 320
Spray-drying of nano- and NCP materials, see Nano-based
microcapsules composite packaging materials
encapsulation methods, 459 (NCP materials)
flavors, 460–461 NCR Corp., see National Cash Register
lipids, 462 Corp. (NCR Corp.)

652 Index
NE, see Nano-emulsion (NE) carotenoids, 484–485

NF, see Nanofiltration (NF) and food products, 509
NGA products, see Non-growth- global demand value and
associated products (NGA bioseparation cost, 496
products) global market of, 482
Nitric oxide, 591, 592 hydroxytyrosol, 497
15-Nitroastaxanthin, 603, 604 large molecular, 493
Nitrocarotenoids, 601; see also lipid-based, 485
Carotenoids long-chain polyunsaturated fatty
anticarcinogenesis, 602–604 acids, 490–493
quenching effects of singlet oxygen, in microorganisms, 482
601–602 pimprinine, 497
NLLS, see Nonlinear least-squares polar lipids, 487–489
(NLLS) polysaccharide compounds, 493, 495
Non-flavonoid polyphenols, 359 process analysis, 498–499, 501
Non-growth-associated products (NGA profitability analysis, 501
products), 522, 523 proteins and nucleotides, 495–496
Non-interactive model, 518; see also simulation software programs, 501
Microbial modeling small molecular, 496–498
Nonlinear least-squares (NLLS), 170; see
also Mass transfer coefficient
O
Nonpolar solvents, 63; see also Solubility
in supercritical-CO2 fluid OD, see Osmotic membrane distillation
Non-water-soluble phytochemicals, 360 (OD)
Nutraceutical production, 224, 230; see OE, see Osmotic evaporation (OE)
also Membrane technology Oil-in-water (O/W), 279; see also
carotenoids, 226–227 Emulsions
charged UF membranes, 225 Omega-3 fatty acid, 358; see also
lipid-soluble compounds, 226 Microencapsulated omega-3
other compounds, 229–230 ingredient; Microencapsulated
peptides, 224 delivery; Microencapsulation;
phenolics, 227–229 Omega-3 oil
pressure-driven process, 228 drivers for delivery of, 375–376
size range of bioactive compounds, incorporation into foods, 376–377
224 microencapsulated omega-3
terpenoids, 229 ingredient properties, 394
tocopherols, 227 microencapsulation of, 378
Nutraceuticals, 322, 481, 501–502, 546; oxidation of, 374, 376–377
see also Bioreactor design; Omega-3 oil, 395, 397; see also
Dehydration technologies; Microencapsulated
Microbial modeling; omega-3 ingredient;
Polyunsaturated fatty acids Microencapsulation; Omega-3
(PUFAs) fatty acid
antioxidant sterol compounds, 487 bakery and cereal products, 396
bioconversions, 486 confectionery, 397
biopharma compounds, 484 dairy products, 396
bioprocess design, 498 drinks and beverages, 397
bioproduction, 484–485 enriched products, 395
biorefinery process, 483, 500 infant formula, 395

Index 653
interactions with other ingredients, Particles from gas saturated solutions

398–399 (PGSS), 360–361
meat and fish products, 397 PBAC, see Polyol and phenylboronic acid
microencapsulated ingredient, 398 (PBAC)
properties of final product, 399 PC, see Phosphatidyl choline (PC)
protection and release requirements, PDI, see Polydispersity index (PDI)
398 PE, see Phosphatidyl ethanolamine (PE);
regulatory standards, 398 Polyethylene (PE)
Omega-6 fatty acids, 358; see also PEG, see Poly-ethylene-glycol (PEG)
Microencapsulated delivery Peng–Robinson equation (P–R
Optimized membrane process, 222; see equation), 84, 88–89
also Membrane technology Pepper (Piper nigrum L.), 34
Orange peel oil purification, 203–204; see Peroxynitrite, 591
also Distillation carotenoids with, 604
applications, 205–206 inhibitory of nitration of tyrosine,
area % by HRGC, 212, 213 593
chromatographic profile orange oil, quenching of, 604
211, 212, 214 reaction products of astaxanthin, 595
citrus industry, 203, 209 reaction with proteins, 592
deterpenation, 204 scavenging peroxynitrite, 592
essence product, 208 Peroxynitrous acid, 591, 594
essence recovery system, 206 Pervaporation (PV), 220; see also
Flavor Tec process, 208, 210 Membrane technology
folded oils, 204–205 PES, see Polyethersulfone (PES)
FTNF orange aromas, 214 PG, see Phosphatidyl glycerol (PG)
recovery system, 207 PGSS, see Particles from gas saturated
sweet orange oil, 203 solutions (PGSS)
TASTE evaporators, 206 PHAs, see Polydroxyalkanoates (PHAs)
Organic solvents, 4, 178 pH-dependent polymers, 347
Osmotic dehydration, 571–572; see also Phenolics, 29, 177, 410–411
Dehydration technologies bio-molecules, 592
Osmotic evaporation (OE), 221; see also Phosphatidyl choline (PC), 487
Membrane technology Phosphatidyl ethanolamine (PE), 487
Osmotic membrane distillation (OD), Phosphatidyl glycerol (PG), 488
221; see also Membrane Phospholipids (PL), 489
technology Phytochemicals, 177–178, 359, 410, 547;
Ostwald ripening, 283–284; see also see also Microencapsulated
Emulsions delivery; Pressurized low-
Ovalbumin, 434 polarity water extraction
O/W, see Oil-in-water (O/W) (PLPW extraction)
Pimprinine, 497; see also Nutraceuticals
PL, see Phospholipids (PL)
P
PLA, see Poly-lactic acid (PLA)
Partial molar volume, 57 Plant
Particle formation, 390–391; see also -derived biopolymers, 362
Microencapsulated omega-3 particles, 115
ingredient PLPW extraction, see Pressurized
Particle porosity, 115; see also Supercritical low-polarity water extraction
fluid extraction (SFE) (PLPW extraction)

654 Index
Polar cosolvent, see Entrainer P–R equation, see Peng–Robinson

Polarization, 221; see also Membrane equation (P–R equation)
technology Pressure-driven membrane processes,
Polar lipids, 237, 487; see also Milk; 218; see also Nutraceutical
Nutraceuticals production
in cyanobacteria, 488 Pressure effect, 76; see also Solubility in
sphingolipids, 487 supercritical-CO2 fluid
sphingosine, 487 Pressurization–depressurization
SQDGs, 488–489 steps, 141; see also
Polar solvents, 63; see also Solubility in Enzymatic catalysis
supercritical-CO2 fluid Pressurized low-polarity water
Polydispersity index (PDI), 470 extraction (PLPW extraction),
Polydroxyalkanoates (PHAs), 522 177, 178, 180, 195–196
Polyethersulfone (PES), 226 applications of, 184–187
Polyethylene (PE), 220; see also benefits, 196
Membrane technology of bioactives, 193–195
Poly-ethylene-glycol (PEG), 343 chromatograms of frozen black
Poly-lactic acid (PLA), 343, 482 currant extracts, 192
Poly-l-lactide production, 136; see also components, 182
Enzymatic catalysis equipment, 180, 181
Polymerization, 335; see also Micro- flow rate effect on thymol yields, 195
nanoencapsulation fractionation of compounds of
technology different polarity, 190–193
Polymers, 342 kinetic model, 193
Polyol and phenylboronic acid (PBAC), phenolic yield and extract
130 concentration, 191
Polyphenols, 359; see also Phenolics plant material for, 184
Polypropylene (PP), 220; see also pressure–enthalpy chart of water,
Membrane technology 179
Polysaccharide-based polymers, 342 process, 178
Polysorbate, 286; see also Emulsifiers of SDG from flax meal, 183
Polytetrafluoroethylene (PTFE), 220; see temperature and pressure effect,
also Membrane technology 187–190
Polyunsaturated fatty acids (PUFAs), thermodynamic model, 193
39, 272, 358, 375, 490; see also Probiotics, 357, 411–413, 547; see also
Nutraceuticals Microencapsulated delivery
alternative sources of, 491 Product inhibition, 516; see also
disorders associated with, 491 Microbial modeling
EPA, 490 PTFE, see Polytetrafluoroethylene
fish oil, 491 (PTFE)
microbes producing, 490, 491–492 PUFAs, see Polyunsaturated fatty acids
P. irregulare, 492–493 (PUFAs)
Polyvinyl-alcohol (PVA), 343 PV, see Pervaporation (PV)
Polyvinyl alcohol-polyethylene glycol PVA, see Polyvinyl-alcohol (PVA)
(PVA-PEG), 353 PVA-PEG, see Polyvinyl alcohol-
Polyvinylidene fluoride (PVDF), 220; see polyethylene glycol (PVA-PEG)
also Membrane technology PVDF, see Polyvinylidene fluoride
Porocrit LLC scCO2 system, 444 (PVDF)
PP, see Polypropylene (PP) Pythium irregulare, 490, 492–493

Index 655
Q SCFs, see Supercritical fluids (SCFs)

SCLE, see Supercritical fluid–liquid
Quenching of singlet oxygen, 590
extraction (SCLE)
SDG, see Secoisolariciresinol diglucoside
R (SDG)
Secoisolariciresinol diglucoside (SDG),
Rapid expansion of supercritical 183
solution (RESS), 360 Secondary alcohol esterification, 136; see
Ratio of binary solubility, 61 also Enzymatic catalysis
Reactive nitrogen species (RNS), 591 Self-microemulsifying drug delivery
Reactive oxygen species (ROS), 591 system (SMEDDS), 340–341;
Redlich–Kwong equation (R–K see also Nanolevel delivery
equation), 84, 89 systems
Reflux, 201 SEM, see Scanning electron microscopy
Relative humidity (RH), 348 (SEM)
Resident time, 24 Separation technique, 41, 159; see
RESS, see Rapid expansion of also Supercritical-CO2 fluid
supercritical solution (RESS) extraction technology
Reverse-enteric coatings, 347–349; see conventional, 159
also Coating materials newer, 160
Reverse osmosis (RO), 218, 219 Sesquiterpeneless oils, 204; see also
RH, see Relative humidity (RH) Orange peel oil purification
R–K equation, see Redlich–Kwong SFE, see Supercritical fluid extraction
equation (R–K equation) (SFE)
RNS, see Reactive nitrogen species SFEE, see Supercritical fluid extraction
(RNS) of emulsions (SFEE)
RO, see Reverse osmosis (RO) Sherwood number, 162; see also Mass
ROS, see Reactive oxygen species (ROS) transfer coefficient
Shrinking core model, 116; see also
Supercritical fluid extraction
S
(SFE)
SAA, see Supercritical-assisted Simple distillation, 201; see also
atomization (SAA) Distillation
Salt fortification, 350; see also Simulated moving bed (SMB), 483
Micro-nanoencapsulation Singlet oxygen
technology in humans, 590–591
color-masking techniques, 353 quenching of, 590
double-fortified salt, 351, 353 Skimmed milk, 245; see also Milk
encapsulation, 351 SL, see Sulfolipid (SL)
extrusion agglomeration, 352 SLP, see Solid lipid particle (SLP)
ferrous fumarate premix, 353 SMB, see Simulated moving bed (SMB)
fluidized-bed agglomeration, 351–352 SMEDDS, see Self-microemulsifying
malnutrition, 351 drug delivery system
SASP, see Supercritical antisolvent (SMEDDS)
precipitation (SASP) Sodium tripolyphosphate (STPP), 354
Scanning electron microscopy (SEM), Solar drying, 572; see also Dehydration
329, 472 technologies
SC-CO2, see Supercritical carbon dioxide Solid lipid particle (SLP), 279; see also
(SC-CO2) Microencapsulation of LBCs

656 Index
Solubility correlation, 111; see also solvent selectivity, 61–63

Supercritical fluid extraction tea leaf analyte recovery, 62
(SFE) temperature, 81–83
Solubility in supercritical-CO2 fluid, 53, vapor pressures, 77
94–95; see also Supercritical-CO2 virial EOS, 90
fluids effect of water content, 77–80
acentric factor, 92 Solubility prediction, 84–88; see also
α-carotene solubility and yield, 68 Solubility in supercritical-CO2
β-carotene extraction yield, 74, 78 fluid
β-carotene solubility and yield, 69–72 Soluble proteins, see Whey proteins
binary solubility ratio, 61 Solution-diffusion model, 219; see also
bioactive component solubility, 60–61 Membrane technology
carotenoid solubility and yield, 75 Solvent; see also Dehydration
chemical effect, 74 technologies; Micro-
composition in carbon dioxide rich nanoencapsulation technology;
phase, 79 Solubility in supercritical-CO2
compressed gas model, 84 fluid
compressibility equation, 91 evaporation/extraction, 335
cosolvents, 64–67, 73 extraction, 561
crossover point, 56 modifiers, 67
crossover pressure operation, 95 selectivity, 61–63
density near critical point, 76 SOR, see Surfactant-to-oil ratio (SOR)
drying process, 77–78 Soy stearin, 346
enhancement factor, 76, 77 Spherosil process, 253; see also Milk
entrainer effect, 73 protein extraction
equation of state, 88–90 Sphingolipids, 487; see also Polar lipids
expression for chemical potential, 85 Sphingosine, 487; see also Polar lipids
extraction data, 58 Spices separation, 33–36; see also
factors affecting, 59 Supercritical-CO2 fluid
food components solubility, 55–58 extraction technology
freeze-drying, 78 Spinning disk system, 391
fugacity coefficient, 57, 89 Spray chilling, 331
lycopene recovery, 67, 82 Spray drying, 291, 330, 387, 456, 562;
lycopene solubility, 83, 90–94 see also Atmospheric drying;
mole fraction vs. pressure, 94 Drying; Microencapsulated
oil solubility vs. water concentration, omega-3 ingredient;
81 Micro-nanoencapsulation
partial molar volume, 57 technology
Peng–Robinson equation, 88–89 advantages, 388
physical effect, 74 emulsion size, 295–296
polar and nonpolar solvents, 63 emulsion stability, 295
pressure effect, 56, 73–77, 80 factors influencing, 294
Redlich–Kwong equation, 89 flow rate of dry air, 296
solubility data and cosolvent effects, formulations, 386, 388
65–66 inlet and outlet temperatures, 296
solubility prediction, 84–88 materials incorporating bioactives, 564
solute solubility behavior, 55 for omega-3 oils, 389
solvating power of fluids, 73 for powder premixes, 360
solvent modifiers, 67 solid content of infeed emulsion, 294

Index 657
spray-dried microencapsulated carbamate formation, 140

ingredients, 388 drawback of, 130
spray driers, 292, 563 extrusion process, 444
viscosity of infeed emulsion, 295 fluid extraction, 160–161
volatility of core material, 294 Mucor miehei lipase, 138
wall materials, 294–295 water solubility in, 145
Spray-drying of nano- and microcapsules, Supercritical-CO2 fluid extraction
456, 474–475; see also technology, 4, 40–43; see also
Nano-microencapsulation; Supercritical-CO2 fluids
Nano-emulsion; Nano- and advantages, 4
microparticle drying process applications in food industry, 24
Büchi mini spray-dryer B-290, 465, bioactive compound extraction,
466–467 29–30
Büchi nano spray-dryer B-90, 465, cholesterol extraction, 18
467–468 cholesterol-free food products, 32–33
core materials for, 460 cosolvent, 19–21
encapsulation of nano-emulsion of critical point, 10
vitamin E acetate, 471 decaffeination, 36–39
flavors, 460–461 essential oil separation, 33–36, 37, 38
lipids, 462 extraction yield, 15, 16, 17, 23, 24, 30
vitamins, 461 fish oil concentration, 39–40
wall material for, 458–460 flavor compounds, 25, 30–32
SQDG, see Sulfoquinovosyl flow rate, 23–24
diacylglycerol (SQDG) moisture content, 18–19
SSD, see Superheated steam drying (SSD) multistage extraction, 12, 13–14
Starches, 384 particle size, 21–22
Steam distillation, 200–201; see also pressure, 16–17
Distillation pressure–temperature phase
Steele’s model, 516; see also Microbial diagram, 6
modeling principle of, 5, 9
STPP, see Sodium tripolyphosphate process parameters, 26–28
(STPP) process system, 8–12
Structured O/W emulsion systems, 279; single-stage extraction, 11, 13
see also Emulsions temperature, 17–18
Subcritical water extraction, see Supercritical-CO2 fluids, 14; see also
Pressurized low-polarity water Solubility in supercritical-CO2
extraction (PLPW extraction) fluid; Supercritical-CO2 fluid
Sulfolipid (SL), 487 extraction technology
Sulfoquinovosyl diacylglycerol (SQDG), advantages, 7
488; see also Polar lipids characteristic traits of, 7
Supercritical antisolvent precipitation compared to hydrodistillation
(SASP), 361 extraction, 22
Supercritical-assisted atomization comparison with liquid solvents, 5
(SAA), 341 drawback of, 41
Supercritical carbon dioxide (SC-CO2), phase diagram, 14
128, 134; see also Biochemical physical properties, 15
reactions; Mass transfer Supercritical fluid extraction (SFE), 105,
coefficient; Separation 485, 631
technique apparent solubility in CO2, 113

658 Index
Supercritical fluid extraction (SFE) Superheated steam drying (SSD), 548,

(Continued) 568–569; see also Dehydration
BIC models, 116–117 technologies
binary diffusion coefficient, 115 Superoxide, 592
correlation for CO2 thermodynamic Surfactant material, 279
solubility, 111 Surfactant-to-oil ratio (SOR), 470
cylindrical extractor, 106 Sweet orange oil, 203; see also Orange
equilibrium concentration of peel oil purification
extracted solute, 111
external mass transfer, 113–114
T
extraction kinetics, 106
flow patterns, 109–110 TASTE evaporators, 206; see also Orange
Greek letters, 121 peel oil purification
linear driving force, 114 Terpenefree, 204; see also Orange peel oil
mass balance constraint, 110 purification
mass balance equations, 108–109 Terpenoids, 229; see also Nutraceutical
mass transfer, 113, 114 production
of mixtures, 119–120 Tetraterpene pigments, 589
models, 112 TFS, see Triple-fortified salt (TFS)
multicomponent models, 119 Thermal degradation reactions, 553; see
nomenclature, 120–121 also Dehydration technologies
of oilseeds, 107 caramelization, 556
particle porosity, 115 enzymatic degradation, 555
porous particles, 115 enzyme/protein denaturation, 554
relationships used in, 112 Maillard reaction, 555–556
shrinking core model, 116 microbial inactivation, 554–555
S-shaped dependence, 113 oxidation, 556–557
thermodynamic and apparent reaction categories, 553
solubility, 110–113 Time-of-flight secondary ion mass
of vegetable oils from seed, 115–117 spectrometry (TOF-SIMS), 328
of volatile oils, 117–119 Time–temperature indicators (TTI), 418
Supercritical fluid extraction of TMN, see 2,6,8-Trimethyl-4-nonanol
emulsions (SFEE), 361 (TMN)
Supercritical fluid–liquid extraction Tocopherol (Vitamin E), 227, 487; see also
(SCLE), 221; see also Membrane Nutraceutical production
technology Tocotrienol, 487
Supercritical fluids (SCFs), 7, 127, TOF-SIMS, see Time-of-flight secondary
149; see also Biochemical ion mass spectrometry
reactions; Enzymatic catalysis; (TOF-SIMS)
Microencapsulated omega-3 Total plant direct cost (TPDC), 501
ingredient; Solubility in Total plant indirect cost (TPIC), 501
supercritical-CO2 fluid TPDC, see Total plant direct cost (TPDC)
critical properties of commonly TPIC, see Total plant indirect cost (TPIC)
used, 54 TPP, see Tripolyphosphate (TPP)
as enzymatic reaction media, 135, 141 Tray drying, 564, 565; see also
enzyme stability in, 137–139 Atmospheric drying
properties of, 129 2,6,8-Trimethyl-4-nonanol (TMN), 135
spraying, 393 Triple-fortified salt (TFS), 350; see also
Supercritical solvent, 54, 105 Salt fortification

Index 659
Tripolyphosphate (TPP), 349 W

TTI, see Time–temperature indicators
Wall material, 269, 294–295; see also
(TTI)
Microencapsulation
Tyrosine nitration, 592
common, 270
effect on microencapsulation
U efficiency and stability, 300
properties, 271
UF, see Ultrafiltration (UF)
ratio of oil to, 295
Ultrafiltration (UF), 218; see also
selection of, 269
Milk protein extraction;
for spray-drying, 458–460
Nutraceutical production
types of, 271
membrane processes, 251–252
Water-in-oil (W/O), 279; see also
membranes, 225
Emulsions
Ultra Rice® technology, 354; see also
Water-in-oil-in-water (W/O/W), 279;
Micro-nanoencapsulation
see also Emulsions
technology
Whey protein, 244, 250, 288; see also
antioxidant system, 354
Emulsifiers; Milk; Milk protein
process flow, 355
extraction
surface-coating process, 354
Whey protein isolates (WPIs), 251,
UNIFAC model, 85
288; see also Milk protein
Uniform Transmembrane Pressure
extraction
(UTP), 247; see also Milk protein
Whey proteins concentrates (WPCs),
extraction
251, 360; see also Milk protein
United States Patent and Trademark
extraction
Office (USPTO), 356
WIPO, see World Intellectual Property
United States Pharmacopeia (USP), 327
Organization (WIPO)
USP, see United States Pharmacopeia
W/O, see Water-in-oil (W/O)
(USP)
World Intellectual Property
USPTO, see United States Patent and
Organization (WIPO),
Trademark Office (USPTO)
356
UTP, see Uniform Transmembrane
W/O/W, see Water-in-oil-in-water
Pressure (UTP)
(W/O/W)
WPCs, see Whey proteins concentrates
V (WPCs)
WPIs, see Whey protein isolates
Vacuum drying, 570; see also
(WPIs)
Dehydration technologies
Vacuum fractional distillation, 201; see
also Distillation X
enhanced purity of distillate, 202
at Flavor Tec., 210 Xanthophylls, 590
greater fractionation efficiency, 202 Xylitol, 511–513; see also Bioreactor
method of deterpenation, 204 design
thermal hazard reduction, 202 fermentations, 532
Virial EOS, 90; see also Solubility in
supercritical-CO2 fluid
Z
Vistec process, 253; see also Milk protein
extraction Zeaxanthin, 591


Functional Food Ingredients and Nutraceuticals - Processing Technologies Volume in Functional Foods and Nutraceuticals (2nd Ed.) - CRC-Taylor & Francis (PDFDrive)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Functional Food Ingredients and Nutraceuticals - Processing Technologies Volume in Functional Foods and Nutraceuticals (2nd Ed.) - CRC-Taylor & Francis (PDFDrive)

Uploaded by

Copyright:

Available Formats

SECOND EDITION

© 2016 by Taylor & Francis Group, LLC

Functional Food Ingredients and Nutraceuticals: (2015)

Marine Products for Healthcare: Functional and Bioactive (2009)

Methods of Analysis for Functional Foods and Nutraceuticals, (2008)

Handbook of Fermented Functional Foods, Second Edition (2008)

Functional Food Carbohydrates (2007)

Dictionary of Nutraceuticals and Functional Foods (2006)

Handbook of Functional Lipids (2006)

Handbook of Functional Dairy Products (2004)

Herbs, Botanicals, and Teas (2002)

Functional Foods: Biochemical and Processing Aspects, Volume 2 (2002)

Functional Foods: Biochemical and Processing Aspects, Volume 1 (1998)

© 2016 by Taylor & Francis Group, LLC

Boca Raton London New York

CRC Press is an imprint of the

© 2016 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4822-4065-8 (eBook - PDF)

© 2016 by Taylor & Francis Group, LLC

Section I “Green” Separation/Extraction/

2. Solubility of Food Components in the Supercritical-CO2

3. Mathematical Modeling of Supercritical Fluid Extraction................. 105

4. Biochemical Reactions in Supercritical Fluids...................................... 127

5. Mass Transfer Coefficient of Plant Oil in Supercritical-CO2

6. Pressurized Low-Polarity Water Extraction of Biologically

7. Purification of Orange Peel Oil and Oil Phase as Functional

8. Membrane Technology for Production of Nutraceuticals................... 217

9. Extraction of Functional Food Ingredients and Nutraceuticals

Section II Nano-Microencapsulation, Delivery

11. Nano-Microencapsulation Technology and Applications in

12. Microencapsulation and Delivery of Omega-3 Fatty Acids............... 373

13. Packaging Functional Foods: From Basic Requirements to Nano

14. High-Pressure Processing of Foods toward Their

15. Spray-Drying of Nano- and Microcapsules of Nutraceuticals........... 455

Section III Bioprocessing Technology

16. Bioprocessing Technology for Production of Nutraceutical

17. Microbial Modeling as Basis for Bioreactor Design for

© 2016 by Taylor & Francis Group, LLC

Section IV Stability and Bioactivity of Antioxidative

19. Biological Antioxidation Mechanisms: Quenching

20. Bioactive Stability and Antioxidative Property of Lycopene

© 2016 by Taylor & Francis Group, LLC

and food ingredients, as well as college and university students majoring in

Siddika Mithani, PhD

Gilles Saindon, PhD

© 2016 by Taylor & Francis Group, LLC

John Shi, PhD

This book is a product of an extensive multidisciplinary collaborative

Nicolas Anton Lishui Chen

Feng Chen Geneviève Gésan-Guiziou

Darryl B. Jones Ying Ma

Željko Knez Giuseppe (Joe) Mazza

Maja Leitgeb Gauri S. Mittal

© 2016 by Taylor & Francis Group, LLC

John Shi Terry H. Walker

© 2016 by Taylor & Francis Group, LLC

© 2016 by Taylor & Francis Group, LLC

John Shi, Sophia Jun Xue, Lamin S. Kassama, and Xingqian Ye

© 2016 by Taylor & Francis Group, LLC

1.2 Principle of Supercritical-CO2 Fluid Extraction Technology

© 2016 by Taylor & Francis Group, LLC

of temperature, pressure, and density (Mukhopadhyay, 2000). Under ­constant

Section I “Green” Separation/Extraction/

Section II Nano-Microencapsulation, Delivery

Section IV Stability and Bioactivity of Antioxidative

of temperature, pressure, and density (Mukhopadhyay, 2000). Under constant