Calorimetry in Food Processing by Gonul Kaletunc

Calorimetry in
Food Processing:
Analysis and
Design of Food Systems
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A John Wiley & Sons, Inc., Publication
Calorimetry in
Food Processing:
Analysis and
EDITOR
Gnl Kaletun
A John Wiley & Sons, Inc., Publication
Edition first published 2009

2009 Wiley-Blackwell and the Institute of Food Technologists
Chapter 7 remains with the U.S. Government.
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Library of Congress Cataloging-in-Publication Data
Calorimetry in food processing : analysis and design of food systems/editor Gnl Kaletun.
p. cm.
Includes bibliographical references and index.
ISBN-13: 978-0-8138-1483-4 (alk. paper)
ISBN-10: 0-8138-1483-9 (alk. paper)
1. FoodAnalysis. 2. Thermal analysis. 3. CalorimetryIndustrial
applications. 4. Food industry and trade. I. Kaletun, Gnl
TX544.C35 2009
338.4'7664dc22
2009008348
A catalog record for this book is available from the U.S. Library of Congress.
Set in 11.5 on 13.5 pt Times by SNP Best-set Typesetter Ltd., Hong Kong
Printed in Singapore
1 2009
Titles in the IFT Press series

Accelerating New Food Product Design and Development (Jacqueline H. Beckley, Elizabeth
J. Topp, M. Michele Foley, J.C. Huang, and Witoon Prinyawiwatkul)
Advances in Dairy Ingredients (Geoffrey W. Smithers and Mary Ann Augustin)
Biofilms in the Food Environment (Hans P. Blaschek, Hua H. Wang, and Meredith E. Agle)
Calorimetry and Food Process Design (Gnl Kaletun)
Nondigestible Carbohydrates and Digestive Health (Teresa M. Paeschke and William R.
Aimutis)
Food Ingredients for the Global Market (Yao-Wen Huang and Claire L. Kruger)
Food Irradiation Research and Technology (Christopher H. Sommers and Xuetong Fan)
Food Laws, Regulations and Labeling (Joseph D. Eifert)
Food Risk and Crisis Communication (Anthony O. Flood and Christine M. Bruhn)
Foodborne Pathogens in the Food Processing Environment: Sources, Detection and Control
(Sadhana Ravishankar and Vijay K. Juneja)
Functional Proteins and Peptides (Yoshinori Mine, Richard K. Owusu-Apenten, and Bo
Jiang)
High Pressure Processing of Foods (Christopher J. Doona and Florence E. Feeherry)
Hydrocolloids in Food Processing (Thomas R. Laaman)
Microbial Safety of Fresh Produce (Xuetong Fan, Brendan A. Niemira, Christopher J.
Doona, Florence E. Feeherry, and Robert B. Gravani)
Microbiology and Technology of Fermented Foods (Robert W. Hutkins)
Multiphysics Simulation of Emerging Food Processing Technologies (Kai Knoerzer, Pablo
Juliano, Peter Roupas, and Cornelis Versteeg)
Multivariate and Probabilistic Analyses of Sensory Science Problems (Jean-Franois
Meullenet, Rui Xiong, and Christopher J. Findlay)
Nondestructive Testing of Food Quality (Joseph Irudayaraj and Christoph Reh)
Nanoscience and Nanotechnology in Food Systems (Hongda Chen)
Nonthermal Processing Technologies for Food (Howard Q. Zhang, Gustavo V. BarbosaCnovas, and V.M. Balasubramaniam, Editors; C. Patrick Dunne, Daniel F. Farkas, and
James T.C. Yuan, Associate Editors)
Nutraceuticals, Glycemic Health and Type 2 Diabetes (Vijai K. Pasupuleti and James W.
Anderson)
Packaging for Nonthermal Processing of Food (J. H. Han)
Preharvest and Postharvest Food Safety: Contemporary Issues and Future Directions (Ross
C. Beier, Suresh D. Pillai, and Timothy D. Phillips, Editors; Richard L. Ziprin, Associate
Editor)
Processing and Nutrition of Fats and Oils (Ernesto M. Hernandez and Afaf Kamal-Eldin)
Processing Organic Foods for the Global Market (Gwendolyn V. Wyard, Anne Plotto,
Jessica Walden, and Kathryn Schuett)
Regulation of Functional Foods and Nutraceuticals: A Global Perspective (Clare M. Hasler)
Sensory and Consumer Research in Food Product Design and Development (Howard R.
Moskowitz, Jacqueline H. Beckley, and Anna V.A. Resurreccion)
Sustainability in the Food Industry (Cheryl J. Baldwin)
Water Activity in Foods: Fundamentals and Applications (Gustavo V. Barbosa-Cnovas,
Anthony J. Fontana Jr., Shelly J. Schmidt and Theodore P. Labuza)
Whey Processing, Functionality and Health Benefits (Charles I. Onwulata and Peter J. Huth)
Dedication
For my parents, my son, and my husband for their patience and

encouragement.
Hayatta en hakiki mrsit ilimdir. The truest guide in life is science.
Mustafa Kemal Atatrk, September 22, 1924
vii
Dedication
This book is also dedicated to the memory of the late Professor

Michel Ollivon, a great scientist and an exceptional human being, who
passed away on June 16th, 2007, during the preparation of the book.
viii
Table of Contents
Preface
Contributor List
xiii
xvii
Part 1 Analysis of Food and Biological Materials by

Calorimetry
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Calorimetric Methods as Applied to Food: An

Overview
Gnl Kaletun
Methods and Applications of Microcalorimetry

in Food
Pierre Le Parlour and Luc Benoist
15
High-Pressure Differential Scanning

Calorimetry
Gnther W.H. Hhne and Gnl Kaletun
51
Calorimetry of Proteins in Dilute Solution

G. Eric Plum
Chapter 5 Thermal Analysis of Denaturation and

Aggregation of Proteins and Protein
Interactions in a Real Food System
Valerij Y. Grinberg, Tatiana V. Burova, and
Vladimir B. Tolstoguzov
ix
67
87
Table of Contents
Chapter 6
Chapter 7
Chapter 8
Part 2
Heat-Induced Phase Transformations of Protein

Solutions and Fat Droplets in Oil-in-Water
Emulsions: A Thermodynamic and Kinetic
Study
Perla Relkin
Analysis of Foodborne Bacteria by Differential
Scanning Calorimetry
Michael H. Tunick, John S. Novak, Darrell O.
Bayles, Jaesung Lee, and Gnl Kaletun
Coupling of Differential Scanning Calorimetry
and X-Ray Diffraction to Study the
Crystallization Properties and Polymorphism
of Triacyglycerols
Christelle Lopez, Daniel J.E. Kalnin,
and Michel R. Ollivon
Calorimetry as a Tool for Process Design
Chapter 9
Chapter 10
Chapter 11
Chapter 12
Overview of Calorimetry as a Tool for

Efficient and Safe Food-Processing Design
Alois Raemy, Corinne Appolonia Nouzille,
Pierre Lambelet, and Alejandro Marabi
Shelf Life Prediction of Complex Food
Systems by Quantitative Interpretation of
Isothermal Calorimetric Data
Simon Gaisford, Michael A.A. ONeill, and
Anthony E. Beezer
Use of Thermal Analysis to Design and
Monitor Cereal Processing
Alberto Schiraldi, Dimitrios Fessas, and
Marco Signorelli
Importance of Calorimetry in Understanding
Food Dehydration and Stability
Yrj H. Roos
119
147
169
199
201
237
265
289
Table of Contents
xi
Chapter 13
High-Pressure Calorimetry and Transitiometry

Stanislaw L. Randzio and Alain Le Bail
Chapter 14
Calorimetric Analysis of Starch Gelatinization by

High-Pressure Processing
341
Kelley Lowe and Gnl Kaletun
Chapter 15
Use of Calorimetry to Evaluate Safety of

Processing
Hans Fierz
Index
311
351
369
Preface
The global food industry is very large, producing sales worldwide on

the order of approximately U.S. $1 trillion. To remain competitive in
this complex industry, it is vital that manufacturers optimize foodprocessing conditions, most importantly not only to ensure the safety
of food products but also to produce affordable, healthy, and convenient products, with desired sensory attributes. The global scale of
the food industry brings the new challenges of increasing transport
and export and in turn new requirements for increased shelf life.
Optimization of food-processing conditions as well as development of
new products requires knowledge of the physical properties of the food
products and their components as the variables that are relevant to
processing and storage conditions. Detailed knowledge of physical
properties enables manufacturers to prevent waste of time and resources
caused by trial and error during product formulation and process
design.
Many food-processing protocols involve application of heating or
cooling over a broad range of temperature. Knowledge of a foods
thermal properties as a function of temperature and composition is
necessary for heat transfer and energy balance calculations used to
rationally design these thermal-processing protocols. During processing, the food components go through conformational and phase changes
that affect the state and texture of the final food product.
Temperature-scanning calorimetry provides a useful tool for
detecting, monitoring, and characterizing thermal processes in food
materials. Moreover, calorimetry can be used to evaluate the effects of
various physical and chemical stresses, including nonthermal treatments, on specific components by comparing the thermal profiles of
pre- and post-treated food and biological materials to develop an
xiii
xiv
Preface
understanding of the mechanism of processing-induced changes. The

data generated from thermal analysis techniques also can be used to
develop equations that predict the physical properties of pre- and postprocessed foods as a function of processing and storage conditions.
Although the use of calorimetry to measure the physical properties
of food materials has increased both in academia and in industry over
the past 20 years, the analysis of data frequently is complicated by
multiple overlapping transitions and kinetically controlled events that
occur in food materials.
This book is designed to introduce the basic principles of calorimetry, applications of calorimetry to characterize food products, interpretation of the resultant data, and the use of these data for process
optimization and product development. The book is organized in two
sections. The first section, consisting of eight chapters, focuses on the
basic principles of calorimetry and its use for a wide range of materials
from dilute solutions to solids. The second section, consisting of seven
chapters, emphasizes the use of calorimetric data as a tool for process
design and product development.
Chapter 1 provides an overview of calorimetry and the organization
of the book. Chapters 2 and 3 focus on experimental design principles,
calibration, data collection and analysis for microcalorimetry and highpressure calorimetry. Chapter 4 addresses applications of ultrasensitive
calorimetry to proteins and their interactions in dilute solution to characterize the thermal and thermodynamic stability and the thermodynamic origins of that stability. Chapters 5, 6, and 7 undertake the
characterization of concentrated, multicomponent systems that are
commonly observed in foods and complex biological systems such as
bacteria. The final chapter in this section, Chapter 8, focuses on the
use of an instrument that combines X-ray diffraction and highsensitivity differential scanning calorimetry (DSC) in the same apparatus to simultaneously obtain complementary thermal and structural
information for a sample.
Section Two of the book comprises Chapters 9 through 15. Chapter
9 provides an overview of the use of phase transition information in
development of phase diagrams that can be used for efficient process
design. Chapter 10 covers application of isothermal calorimetry for
analysis of food stability, shelf life, and isothermal cooking processes.
Chapter 11 describes application of thermal analysis to cereal-based
products and mathematical treatment of the complex thermograms to
Preface
xv
deconvolute the contributions from different components of the system.

Chapter 12 reviews the use of calorimetric data for selection of dehydration parameters to produce products with improved storage stability. Chapter 13 describes the relatively new technique of scanning
transitiometry and its specific application to gelatinization of wheat
starch dispersions and for investigation of pressure shift freezing.
Chapter 14 covers the application of calorimetry to characterize the
impact of nonthermal treatment and to determine kinetic parameters
during storage. Chapter 15 reviews the use of calorimetry to quantify
the probability and potential severity of exothermic events such as
formation of hot spots in dryers and to establish safe conditions for
handling materials to prevent accidents in the food industry.
This book is designed to explain the capabilities of calorimetry for
characterization of food and biological systems, which can range from
single component, single-phase systems to multicomponent, multiphase systems. Therefore, information described in the book will
provide comprehensive insight for scientists who have experience with
calorimetry as well as a basic understanding for beginners. This text
may also be used as a textbook for a graduate-level course. The book
is also intended to serve as a resource for food scientists, food technologists, and food engineers working in the area of process design,
optimization, and product development. The descriptions of the basic
principles and potential uses of calorimetry to provide critical information for their respective areas and will serve as a bridge between these
workers and specialists in calorimetry.
Contributors
Bayles, Darrell O. (Chapter 7)

Dairy Processing & Products Research Unit, USDA-ARS-Eastern
Regional Research Center, Wyndmoor, PA, USA
Beezer, Anthony E. (Chapter 10)
The School of Pharmacy, University of London, London, UK
Benoist, Luc (Chapter 2)
SETARAM, Lyon, France
Burova, Tatiana V. (Chapter 5)
Nesmeyanov Institute of Organo-Element Compounds, Russian
Academy of Sciences, Moscow, Russian Federation
Fessas, Dimitrios (Chapter 11)
DISTAM, University of Milan, Milano, Italy
Fierz, Hans (Chapter 15)
Swiss Safety Institute, Basel, Switzerland
Gaisford, Simon (Chapter 10)
The School of Pharmacy, University of London, London, UK
Grinberg, Valerij Y. (Chapter 5)
A.N. Nesmeyanov Institute of Organo-Element Compounds, Russian
Academy of Sciences, Moscow, Russian Federation
xvii
xviii
Contributors
Hhne, Gnther W.H. (Chapter 3)

University of Ulm, Ulm, Germany (Retired)
Kaletun, Gnl (Chapters 1, 3, 7, 14)
The Ohio State University, Department of Food Agricultural and
Biological Engineering, Columbus, OH, USA
Kalnin, Daniel J.E. (Chapter 8)
YKI, Ytkemiska Institutet AB, The Institute for Surface Chemistry,
Stockholm, Sweden
Lambelet, Pierre (Chapter 9)
Nestl Research Center, Nestec LTD, Lausanne, Switzerland
Le Bail, Alain (Chapter 13)
ENITIAA, UMR CNRS GEPEA (6144), Nantes, France
Lee, Jaesung (Chapter 7)
Department of Food Science and Technology, The Ohio State
University, Columbus, OH, USA
Le Parlour, Pierre (Chapter 2)
Thermal Consulting, Caluire, France
Lopez, Christelle (Chapter 8)
UMR Science et Technologie du Lait et de lOeuf, INRA-Agrocampus
Ouest, Rennes Cedex, France
Lowe, Kelley (Chapter 14)
Abbott Nutrition Products Division, Columbus, OH, USA
Marabi, Alejandro (Chapter 9)
Nestl Research Center, Nestec Ltd., Lausanne, Switzerland
Nouzille, Corinne Appolonia (Chapter 9)
Novak, John S. (Chapter 7)
Contributors
xix
Michel Ollivon (Chapter 8, published posthumously)

Universit Paris-Sud, Chatenay-Malabry, France
ONeill, Michael A.A. (Chapter 10)
Department of Pharmacy and Pharmacology, University of Bath, Bath,
UK
Plum, G. Eric (Chapter 4)
IBET Inc., Columbus, OH, USA, and Rutgers, The State University of
New Jersey, Department of Chemistry and Chemical Biology,
Piscataway, NJ, USA
Raemy, Alois (Chapter 9)
Randzio, Stanislaw L. (Chapter 13)
Polish Academy of Sciences, Institute of Physical Chemistry,
Warszawa, Poland
Relkin, Perla (Chapter 6)
UMR 1145 (AgroParisTech, CEMAGREF, INRA), AgroParisTech,
Department of Science and Engineering for Food and Bioproducts,
Massy, France
Roos, Yrj H. (Chapter 12)
Department of Food and Nutritional Sciences, University College
Cork, Ireland
Schiraldi, Alberto (Chapter 11)
Signorelli, Marco (Chapter 11)
Tolstoguzov, V.B. (Chapter 5)
Tolstoguzov consulting.com, Pully, Switzerland.
Tunick, Michael H. (Chapter 7)
Calorimetry in
Food Processing:
Analysis and
Part 1
Analysis of Food and Biological
Materials by Calorimetry
Chapter 1
Calorimetric Methods as Applied to Food:
An Overview
Gnl Kaletun
Introduction
Calorimetry
An Overview of the Book
References
5
6
8
13
Introduction
Several thermal and nonthermal methods are applied to process and
preserve food materials and to manufacture value-added products. The
goals of food processing are to inactivate spoilage and pathogenic
microorganisms and to maintain this status in storage during the
intended shelf life of the product. During processing, changes take
place in food components, including vitamins, lipids, carbohydrates,
and proteins. Such changes lead to structural and functional changes
in foods at the micro- and macromolecular levels that affect the physical, organoleptic, and nutritional properties of the food.
Food materials are complex biological systems. Food products may
have a broad range of structures spanning the three states of matter,
including dilute to concentrated liquids, solids, and mixtures of multiliquid, liquid-solid, liquid-gas, and solid-gas structures. The combination of complex structures making up complex biological compounds
makes the characterization of food systems challenging. To address
the wide variety of compositions and structures, many biophysical
techniques are uesd to characterize the structure and properties of
food materials before and after processing to develop a fundamental
5
Calorimetry in Food Processing
understanding of the impact of processing and storage conditions. The

data resulting from such studies can be used to predict the physical properties of foods so that food processing and storage conditions are
optimized.
Calorimetry
Among biophysical techniques, calorimetry presents itself as particularly well suited for analysis of food materials. Among many reasons,
the first is the relevance of the experimental protocols of calorimetry
to the majority of processes employed in food preservation. Specifically, because many food-processing methods involve thermal treatment (heating, cooling, freezing) of the materials, thermal
characterization of food systems and their components leads to data
that can be related directly to the processing protocols. Determination
of thermal properties of food materials, such as specific heat as a function of temperature, is essential for heat transfer and energy balance
calculations (Kaletun 2007). Generation of a reliable database to
develop equations predicting thermal properties of food materials for
optimization of food processes can be accomplished by using calorimetry. Moreover, food materials and their components go through conformational and phase transitions during processing. Calorimetry data
can be analyzed to evaluate the thermal and thermodynamic stability
of various phases for a rational design of food product formulations
and process conditions.
Differential scanning calorimetry, which measures heat capacity
as a function of temperature, is a well-established thermal analysis
technique that detects and monitors thermally induced conformational
transitions and phase transitions as a function of temperature. During
temperature scanning, depending on the complexity of the material,
many peaks or inflection points (one to several) reflecting the thermally
induced transitions can be observed. The direction of the peak
corresponds to the nature of the transition, being heat absorbing (endotherms) or heat releasing (exotherms). While melting of solids and
denaturation of proteins display endotherms, crystallization of carbohydrates and aggregation of proteins manifest themselves as exotherms.
The temperatures for the endothermic and exothermic transitions and
Calorimetric Methods as Applied to Food: An Overview
the heat involved in such transitions are measured using a calorimeter. Inflection points are indicative of glass transitions; that is,
transitions from a glassy to rubbery state. The transition temperatures
(Tpeak or Tg) reflect the thermal stability of the phase or state going
through the transition. One can extract from calorimetry data values
for the thermal and thermodynamic changes in free energy (G),
enthalpy (H), entropy (S), and heat capacity (Cp) of the various
transitions in addition to determination of the bulk heat capacity of the
material.
The basis for thermodynamic study of food materials is that the
relevant initial and final states (preprocessing and postprocessing
states) can be defined and the energetic and structural differences
between these states can be measured using calorimetric instrumentation. To this end, calorimetry can be used to evaluate the effect of other
physical and chemical variables by comparing the thermograms of the
materials before and after exposure to the variable outside the
calorimetry.
The basics of application of calorimetry to food materials are discussed in detail in this book. However, it is important to start the
discussion with a summary of the advantages of using calorimetry for
study of biological materials. These advantages can be outlined as
follows:
Direct measurement of the energetics of the transition is obtained
(H and Cp). The experimental results are not model dependent.
Calorimetry can be applied to a range of materials, pure or complex.
Materials do not have to be optically transparent or have chromophores as required by spectroscopic methods.
Materials do not have to be uniform or have to be a homogeneous
mixture. In fact, in addition to pure materials, the technique can be
used to evaluate the interactions among the components in a complex
system and how the interactions are altered by the processing.
Calorimetry does not require elaborate or destructive sample
preparation.
Calorimetry is an established technique which has been around since
the 16th century (Haines 1995). Today, the instruments are highly
developed for accurate measurement of thermal events. The theory
behind the technique is well developed, which facilitates interpretation of the data (Hhne et al. 2003).
While the technique is powerful, the validity and utility of the data
depend strongly on the careful use of the equipment and correct interpretation of data. Some analytical methods provide results specific to
materials; however, calorimetry data depends on the conditions used
during the experiment (Haines 1995). One must be careful in choosing
the calorimetry parameters:
1. Time scale: Especially in dynamic measurement systems, for events
to be detected the experimental time scale should match the time
scale of the observed event.
2. Magnitude of the heat flow: If the energy associated with the transition is small, it can lead to ambiguities in its detection. Increasing
the scanning rate enhances the signal; however, it may cause deviation from equilibrium conditions, which requires models beyond
the standard equilibrium thermodynamics treatment of calorimetry
data.
3. Moisture loss during experiment: Biological samples in general are
high-moisture content materials. If the sample cell is not sealed
well, the moisture content of the sample will change due to evaporation during the course of experiment. This may lead to overestimation of the transition temperature as well as the transition enthalpy
change.
4. Interpretation of overlapping peaks: Biological samples may contain
multiple components that undergo thermally induced transitions at
similar temperatures. As a result, overlapping peaks may be observed
on a differential scanning calorimetry (DSC) thermogram. Even if
the origin of the event is known, because the peak temperatures may
shift due to overlap, individual events may appear to happen at different temperatures. The individual peaks can be resolved experimentally (Barrett et al. 2002, 2005), or the complex thermograms
can be deconvoluted by using special software (Fessas and Schiraldi
2000).
An Overview of the Book

This book focuses on the basics of calorimetry and specific applications for characterization of food systems. The material in this book is
designed to provide food scientists, food technologists, and food engi-
neers with knowledge about the potential uses of calorimetry as a tool

in process design and optimization as well as product development and
improvement. The book consists of two sections. The first section
includes eight chapters describing the principles of calorimetry alone
and coupled with other techniques as well as the use of calorimetry to
characterize biological systems ranging from pure single phase to
multicomponent and multiphase systems of solids, dilute and concentrated solutions of macromolecules, emulsions, foams, and bacteria.
The second section of the book is designed to illustrate the use of
calorimetric data to guide engineers and processors in design and optimization of processes.
The multicomponent nature of the food materials presents a challenge in that the specific component undergoing a conformational or
phase transition may be in small quantity relative to the whole, thus
generating an insufficient heat signal to detect. As an alternative to
increasing the heating rate, the heat signal can be enhanced by increasing the sample size. In Chapter 2, the challenges of increasing sample
size and strategies to overcome these challenges by using microcalorimetry are discussed.
The increased interest of consumers in minimally processed foods
pushed the food research community to explore novel technologies that
present alternatives to thermal processing. High hydrostatic pressure
(HHP) processing has become the most promising alternative technology. Currently, HHP processing is implemented for several foods and
has a market value of more than $500 million. The optimization of
HHP processing requires knowledge of physical properties under conditions relevant to the pressures attained during the process. The design
of calorimeters operating under the pressures used in industry is very
challenging. Chapter 3 focuses on the design of a high-pressure calorimeter and the protocols to be followed for calibration, data collection,
and analysis.
Applications of ultrasensitive calorimetry to proteins and their interactions in dilute solution are examined in Chapter 4. Emphasis is
placed on the practical aspects of collecting and analyzing differential
scanning calorimetry (DSC) data to characterize the thermal and thermodynamic stability and the thermodynamic origins of that stability in
a protein in solution. The thermodynamics of association between a
protein and a small molecule or another macromolecule are quantifiable by application of isothermal titration calorimtery (ITC). The
10
design and execution of ITC experiments are described with emphasis

on the information content of titration curves. Together or separately,
DSC and ITC provide valuable tools for developing a predictive understanding of protein stability and interactions as a function of temperature and solution conditions.
Investigation of dilute systems is essential to elucidate the behavior
of macromolecules thermodynamically. However, in biological systems
and foods, dilute systems are rarely encountered. Commonly, macromolecules exist in foods at high concentration and in complexes with
other macromolecules and low-molecular-weight compounds. Heat
denaturation and aggregation of proteins are common during food
processing and affect the quality attributes of food. Therefore, Chapter
5 uses calorimetry to study the effects of pH, salts, alcohols, and polysaccharides on thermal denaturation and aggregation of food proteins
in order to elucidate the mechanisms of structure formation, structuretexture and structure-physical property relationships in foods.
Proteins also play an important role in development of emulsions
and foams that are examples of multicomponent and multiphase food
systems. Both the formation and the stability of such complex systems
depends on the adsorption properties of proteins at oil-in-water or gasin-water interfaces. Chapter 6 reviews the use of DSC in scanning and
isothermal mode for monitoring effects of food composition and physicochemical environment on the conformation and structural modifications of proteins in emulsions under the time-temperature combinations
relevant to processing. The results presented in this chapter illustrates
that a combination of thermodynamic and kinetic data obtained by
using DSC in scanning and isothermal modes provide a better understanding of emulsions and the ability to control structure-forming
mechanisms in food systems.
The main goal of food processing is to manufacture foods that are
stable and safe to consume, which requires the inactivation of bacteria
to prevent spoilage and foodborne diseases. Thermal inactivation of
microorganisms is associated with irreversible denaturation of membranes, ribosomes, proteins, and nucleic acids. DSC can be used to
monitor the reversible and irreversible changes in the cellular components of bacteria. Chapter 7 describes using DSC to provide an insight
into the mechanism of bacterial cell inactivation. Also illustrated is the
utility of DSC data to quantitatively evaluate bacterial inactivation
kinetics. Calorimetry can be used to evaluate the effect of food-
11
processing variables other than heat on bacteria. Chapter 7 describes

the analysis by calorimetry of damage to bacterial cells due to chemical, nonthermal, or antibiotic treatments and the relationship between
the calorimetric data and loss of cell viability.
The data collected by calorimetry are complementary to data collected by other biophysical methods. Thermal analysis is a valuable
tool to observe phase transition, but especially for complex systems,
such as lipids, the thermal observables can be due to a variety of structures forming during the heating or cooling process. Generally, another
technique such as Fourier transform infrared spectroscopy or x-ray
diffraction (XRD) is used in parallel to acquire structural information.
Obtaining complementary data can be further improved by performing
simultaneous DSC-FTIR (Yoshida 1999) or DSC-XRD (Yoshida et al.
1996; Ollivon et al. 2006) measurements on the same sample. Chapter
8 describes in detail the development of a new instrument, called
MICROCALIX, combining XRD at both wide and small angles as a
function of temperature (XRDT) or time (XRDt), and high-sensitivity
DSC, in the same apparatus with scanning or isothermal modes over
the temperature range 30 to +230 C. This approach enables one to
obtain complementary thermal and structural properties information on
the same sample in one experiment.
Foods exhibit thermally induced transitions over a temperature
range between 50 C and 300 C. The thermal behavior of a food is
mainly a reflection of its major component, however, with some change
due to interactions with other components. Chapter 9 focuses on the
use of phase transition information in development of phase diagrams
that can be used for efficient process design. Heat of a solution as a
parameter of great importance for food powder dissolution is also
emphasized. The relevance of calorimetric data to the food industry is
illustrated by specific examples.
Biological samples undergo changes even when they are kept at
constant temperature. Changes, physical or chemical in origin, may
produce heat that can be studied with isothermal calorimetry. However,
detection and monitoring of small quantities of heat, especially at the
initial stage of the physical or chemical event, requires using a highsensitivity calorimeter. Chapter 10 focuses on application of isothermal calorimetry, a relatively less-exploited application of calorimetry
in comparison with DSC, for qualitative and quantitative analysis of
food stability, shelf life, and isothermal cooking processes. Specific
12
examples are discussed, from simple ingredients to complex biological

processes.
Cereal-based products are staple foods all around the world.
Although the main component in such foods is starch, thermally
induced transitions are highly affected by the presence of other compounds in cereals, including proteins, nonstarch carbohydrates, and
lipids, either due to competition for available water or direct interactions. Chapter 11 provides a review of thermal analysis applications to
cereal-based products and cereal processing. This chapter discusses in
detail mathematical treatment of the complex thermograms to deconvolute the contributions from different components in the system.
Drying has been used as a method of food preservation since ancient
times. In modern practice, water is removed by evaporation upon
application of heat or by sublimation from a frozen product under
vacuum. During the drying process, amorphous or partially crystalline
states are formed. The thermal stability of the amorphous state is
defined by the glass transition temperature, which depends strongly on
the amount of water present in the food system. Chapter 12 reviews
the use of calorimetric data for selection of dehydration parameters to
produce products with improved storage stability. This chapter also
discusses the relationship between the glass transition and collapse of
structure in freeze-dried materials, flavor retention by encapsulation of
volatiles in amorphous systems, solids crystallization, lipid oxidation,
nonenzymatic browning, and enzymatic changes.
Chapter 13 describes the relatively new technique of scanning transitiometry developed by Randzio (1996) based on scanning of one of
the three variablespressure, volume, or temperatureand measurement of the other two, as well as the heat signal. This chapter also
discusses the specific application of scanning transitiometry for gelatinization of wheat starch dispersions and for investigation of pressure
shift freezing. In addition, the technique is applied to the study of
water, water in pork muscle, solutions of gelatin in water, and lipids.
Chapter 14 focuses on the application of calorimetry to determine
the effects of high hydrostatic pressure on starch gelatinization as well
as to characterize the recrystallization of the gelatinized starch during
subsequent storage for calculation of starch recrystallization kinetic
parameters. These results are used in selection and optimization of
HHP processing parameters and storage conditions for foods containing starch.
13
Foods show chemical reactivity leading to self-heating and selfignition of hot spots. Especially handling of dry powders in bulk, such
as in milling, drying, and packaging, can be dangerous due to potential
dust explosions. Chapter 15 reviews the evaluation by calorimetry of
the thermal consequences of exothermic decompositions in foods,
describes the methodology for quantifying the risk in terms of its severity and its probability, and discusses methods for collecting the stability data correctly. Specific cases of formation of hot spots in dryers,
storage and hot discharge, and transport safety are discussed. The
importance of establishing safe conditions for handling of materials in
prevention of accidents in the food industry is emphasized.
References
Barrett A., Cardello A., Maguire P., Richardson M., Kaletun G., and Lesher L.
2002. Effects of Sucrose Ester, Dough Conditioner, and Storage Temperature on
Long-Term Textural Stability of Shelf-Stable Bread. Cereal Chem, 79(6):
806811.
Barrett A.H., Marando G., Leung H., and Kaletun G. 2005. Effect of Different
Enzymes on the Textural Stability of Shelf-stable Bread. Cereal Chem, 82(2):
152157.
Fessas D., and Schiraldi A. 2000. Starch Gelatinization Kinetics in Bread Dough,
DSC Investigations on Simulated Baking Processes. J Therm Anal Calorim,
61:411423.
Haines P.J. 1995. Thermal Methods of Analysis, Principles, Applications and
Problems. Glasgow: Blackie.
Hhne G.W.H, Hemminger, W., and Flammersheim, H.J. Differential Scanning
Calorimetry: an Introduction for Practitioners. 2nd Ed. Berlin; New York:
Springer-Verlag, 2003.
Kaletun G. 2007. Prediction of Heat Capacity of Cereal Flours: A Quantitative
Empirical Correlation. J Food Eng, 82(2):589594.
Ollivon M., Keller G., Bourgaux C., Kalnin D., Villeneuve P., and Lesieur P. 2006.
DSC and High Resolution X-Ray Diffraction Coupling. J Therm Anal Calorim,
85:219224.
Randzio S.L. 1996. Scanning Transitiometry. Chemical Society Reviews, 25:383.
Yoshida H., Ichimura Y., Kinoshita R., and Teramoto Y. 1996. Kinetic Analysis of
the Isothermal Crystallization of an N-Alkane and Polyethylene Observed
by Simultaneous DSC/FT-IR/WAXD Measurement. Thermochim Acta, 282/283:
443452.
Yoshida H. 1999. Structure Relaxation of N-Alkanes Observed by the Simultaneous
DSC/FTIR Method. J Therm Anal Calorim, 57(3):679685.
Chapter 2
Methods and Applications of
Microcalorimetry in Food
Pierre Le Parlour and Luc Benoist
Introduction
The Heat Flux Calorimetric Principle
DSC versus Heat Flux Microcalorimetry
Comparison between DSC and Heat Flux Microcalorimetry
The Calvet Principle
Calibration
Description of Different Heat Flux Calorimeters Used for Food
Characterization
High Sensitivity Heat Flux Calorimeter
The Mixing and Reaction Heat Flux Microcalorimeter
Methods of Microcalorimetry in Food
Heat Capacity Determination
Heating Mode
Mixing and Reaction Calorimetry
Pressure Calorimetry
Calorimetry under Controlled Relative Humidity
Conclusion
References
15
17
19
19
22
23
26
26
29
30
30
35
40
43
45
45
46
Introduction
Heat is involved at different steps in the preparation of foods, such as
cooking and processing. During heating, cooling, or freezing, the food
products undergo different types of transformations, including melting,
15
16
crystallization, gelation, gelatinization, denaturation, and oxidation.

All these transformations occur in a certain range of temperature and
are associated with heat variations. The thermal analysis techniques,
and specifically differential scanning calorimetry (DSC), are used as a
main approach for investigating the thermal properties of foods
(Harwalkar and Ma 1990; Farkas and Mohcsi-Farkas Csilla 1996;
Schiraldi et al. 1999; Raemy et al. 2000).
However, in most food processing food ingredients are mixed or
diluted with a liquid (water, milk) or with a powder (sugar, salt, yeast).
For simulation of such transformations and interactions, the limited
volume and the lack of in situ mixing constitute the major drawbacks
of the DSC technique.
For such investigations, microcalorimetry (in the isothermal
and scanning modes) is the ideal solution because it has the capacity
to work on bulk materials and diluted solutions with a very high
sensitivity. Microcalorimeters are found as reaction or solution calorimeters, pressure calorimeters according to the transformation to
be simulated, and provide a wide range of experimental conditions
for applications such as mixing, dilution, wetting, neutralization, and
enzymatic reaction, which have relevance to food industry. For a
food technologist, it is very important to understand various thermal
and functional properties of food components and ingredients for
fundamental research, food quality assurance, and for product
development.
Although many articles have been published in the field, to our
knowledge a book dedicated to the very challenging field of microcalorimetric applications in food science has not been available.
Microcalorimetry, compared with DSC, still remains as a lesserknown technique. For a long time, it has suffered from a reputation as
an old and slow technique (needing days of experimentation), of large
instruments (microcalorimetry meaning microquantity of measured
heat and not microsize instrument), and was used mainly by experts.
Especially with the development of microcalorimetry in the biological
and pharmaceutical fields (Ladbury and Chowdhry 2004; Craig
and Reading 2007), there have been many advances in instrumentation
in the last decade that facilitated the use of calorimeters in laboratories.
Microcalorimetry benefits food research and opens new opportunities
of experiments and applications that are to be described in this
chapter.
Methods and Applications of Microcalorimetry in Food
17
The Heat Flux Calorimetric Principle

Existing calorimeters operate on the following principles:
the heat flux principle
the heat-compensating principle
the heat-accumulating principle
In this chapter, the heat flux calorimetric principle is described as it
is used in most calorimeters for food characterization.
The heat flux calorimeter consists of a measurement chamber surrounded by a detector (thermocouples, resistance wires, thermisters,
thermopiles) to integrate the heat flux exchanged by the sample contained in an adapted vessel. The measurement chamber is insulated
in a surrounding heat sink made of a high thermal conductivity
material.
The heat flux for a given sample at a temperature Ts is equivalent to:
dqs
dh
dT
=
+ Cs s
dt
dt
dt
(2.1)
where dh/dt is heat flux produced by the transformation of the sample

or the reaction and Cs is heat capacity of the sample, including the
container. The heat flux dqs/dt is exchanged with the thermostatic block
at a temperature Tp through a thermal resistance, R, described by the
following relation:
dqs Tp Ts
=
dt
R
(2.2)
Equation 2.1 shows that the thermal contribution due to the heat capacity of the sample and container is very large and will provide a major
disturbance at the introduction of the container in the calorimeter.
From Equation 2.2, it is also evident that any temperature perturbation
of the thermostatic block will affect the calorimetric measurement.
To solve these issues, a symmetrical calorimeter is preferred. Two
identical calorimetric chambers, one housing a container with
the sample and an identical reference container an inert material
18

Ts
Cs
Sample + container
Thermostatic block
R
Tp
Heating elements
Heat sink
Figure 2.1. One-cell calorimetric principle.
(the reference container may also be empty) are placed in the thermostatic block at the same temperature, Tp. The heat flux difference is
measured between the two chambers.
dq dqs dqr
dh
dT
dT
=
=
+ C s s Cr r
dt
dt
dt
dt
dt
dt
(2.3)
Here, Cr is heat capacity of the reference, including the container, and

Tr is temperature of the reference.
Equation 2.2 becomes:
dq Tr Ts
=
dt
R
(2.4)
or by derivation
R
d 2 q dTr dTs
=
dt 2
dt
dt
(2.5)
By combining Equations 2.4 and 2.5, the characteristic equation for

the calorimetric measurement is obtained.
dTp
dh
dq
d 2q
=
+ (Cs Cr )
RCs 2
dt
dt
dt
dt
(2.6)
19
Table 2.1. Some endothermic and exothermic effects for different food types.
Food Type
Endothermic Effect
Exothermic Effect
Fat, oil
Protein
Melting, lipidic transition

Denaturation
Enzyme
Denaturation
Starch
Gelatinization, glass
transition
Melting
Melting
Melting, glass transition
Crystallization, oxidation
Aggregation,
crystallization
Aggregation, enzymatic
reaction
Retrogradation, oxidation
Milk
Hydrocolloid, gelatin
Carbohydrates
Yeast
Bacteria
Crystallization, oxidation
Gelation
Crystallization,
decomposition
Fermentation
Growth, metabolism,
fermentation
If dh/dt corresponds to an endothermic transformation or reaction,

the dh/dt value is positive. If dh/dt corresponds to an exothermic transformation or reaction, the dh/dt value is negative.
If the calorimetry is performed isothermally, the parameter dTp/dt is
null. In a small perturbation of the temperature Tp of the thermostatic
block, the corresponding thermal effect will be minimized if the Cs and
Cr heat capacities are similar. The last term R Cs d2q/dt2 (called as
thermal lag) mostly depends on the thermal resistance or the time of
response of the calorimeter and the heat capacity of the sample and the
container. For a long period (t >> RCs) it will be negligible. Table 2.1
gives an overview of some endothermic or exothermic effects occurring in various types of food.
DSC versus Heat Flux Microcalorimetry
Comparison between DSC and Heat Flux Microcalorimetry
The differences between DSC and heat flux microcalorimetry are
related mainly to the size of the sample and the sensitivity of the measurement but also to interactions between solid and liquid materials.
To clearly understand the difference, it is important to analyze the
technological principles that are behind each technique.
20
International Confederation for Thermal Analysis and Calorimetry

(ICTAC), in its nomenclature, considers two types of DSC: the heat
flux DSC and the power-compensated DSC (www.ictac.org). Even if
the measurement principles are different, the heat transfer from (or to)
the sample is about the same. The detector for each DSC model is a
plate-type design. The sample, contained in a metallic crucible, is
placed and centered on the plate acting as a flat-shaped sensor. A reference crucible (empty or containing an inert material) is placed on the
other plate. In plate DSC (heat flux type and power-compensated type),
the heat exchange between the sample and the detector occurs through
the bottom of the crucible, corresponding to a two-dimensional detection. In fact, only a part of this heat transfer is measured, as a significant
part is dissipated through the walls and the cover of the crucible (Figure
2.2). The ratio of the heat flux measured by the sensor to the total heat
flux produced by the thermal event, calculated by simulation using
Figure 2.2. Schematic of a plate-shaped DSC sensor.
21
50
% Flux
40
30
20
0.01 mm
0.05 mm
0.10 mm
10
0
0
150
300
450
600
Temperature (C)
Figure 2.3. Efficiency ratio of a flat-shaped DSC as a function of the sensor plate
thickness.
thermal modeling software, shows that only around half of the heat
flux is dissipated through the plate (Daudon 1996; Le Parlour and
Mathonat 2005). Figure 2.3 clearly shows that the efficiency rapidly
decreases with the temperature and the thickness of the plate. The
efficiency is also affected by the amount of the sample tested. Therefore,
it is recommended to work with small amount of material (about
510 mg) when using a plate DSC to minimize the heat losses. The
thermal conductivities of the crucible and the gas used in the experimental chambers also are very important parameters to be considered
in the efficiency of the heat exchange. For example, a very heatconductive gas (helium) will favor the heat transfer between the
crucible and the detector, but at the same time increase the heat losses.
Hence, the calibration of a plate-type DSC (heat flux or powercompensated type) is very critical and has to be run with the experimental conditions selected for testing the sample.
The main difference between heat flux calorimetry and DSC (heat
flux or power compensated type) is that in a microcalorimeter, the heat
exchange between the sample and the detector is completely measured.
Such a high efficiency is achieved by applying the technological principle developed by Tian and Calvet.
Calorimeters are also designed on the power-compensating principle using a detector that surrounds the sample in the same way.
MicroCal (www.microcal.com) and CSC (now TA Instruments)
22
(www.tainstruments.com) have developed such ultrasensitive instruments, mostly used for the investigations of dilute liquids. Because
these calorimeters operate with fixed vessels, they are not well-adapted
for the characterization of foods.
The Calvet Principle
The detection is based on a three-dimensional fluxmeter sensor. The
fluxmeter element consists of a ring of several thermocouples in
series (Figure 2.4). The corresponding thermopile of high thermal
conductivity surrounds the experimental space within the calorimetric
block. The radial arrangement of the thermopiles guarantees an almost
complete integration of the heat. This is verified by the calculation of
the efficiency ratio that indicates that an average value of 94% 1%
of heat is transmitted through the sensor on the full range of temperature of the Calvet-type DSC (Figure 2.5). In this setup, the sensitivity
of the DSC is not affected by the type of crucible, the type of purge
gas, or the flow rate. The main advantage of the setup is the increase
Figure 2.4. Schematic of the Calvet type calorimeter.
23
Ratio of energy (%)
Ratio of energy transmitted through the thermopile

95
94.8
94.6
94.4
94.2
94
93.8
93.6
93.4
93.2
93
0
100
200
300
400
500
T (C)
Figure 2.5. Efficiency ratio of a Calvet-type calorimeter versus temperature.
of the experimental vessels size, and consequently the size of the

sample, without affecting the accuracy of the calorimetric measurement.
Calibration
The calibration of the calorimetric detectors is a key parameter and has
to be performed very carefully. In fact, the main purpose of the calibration is to transform the electric signal (emf) provided by the thermocouples of the detector expressed in microvolts (V) in a thermal
power (heat flux) signal expressed in milliwatts (mW). For DSC detectors, this conversion is achieved using metallic reference materials
(Richardson and Charsley 1998). Although this recommended procedure is widely used, it has some limitations:
The calibration can only be performed at the temperature at which
the reference material melts.
At low temperature, it is difficult to find good reference materials.
The calibration is mostly performed in a heating mode, but very
rarely in the cooling mode.
The accuracy of the calibration depends on the purity and quality of
the reference materials.
For Calvet-type calorimeters, a specific calibration, so-called Joule
effect or electrical calibration, has been developed to overcome the
drawbacks described above (Calvet and Prat 1964). A dedicated vessel
24
K=S/P
S
Figure 2.6. Joule effect calibration.
with a built-in electrical heater (platinum resistance) simulating the

experimental vessel that contains the sample is introduced into the
calorimeter at a given temperature. A well-defined electrical power
(between 20 and 200 mW) is applied to the resistance. The calorimeter
gives a corresponding deviation (Figure 2.6). The stabilized signal,
expressed in microvolts, is directly correlated to the applied power,
expressed in milliwatts. The main advantages of this type of calibration
are as follows:
It is an absolute calibration.
The use of standard materials for calibration is not necessary. The
calibration can be performed at a constant temperature, in the heating
mode and in the cooling mode.
It can be applied to any experimental vessel volume.
It is a very accurate calibration.
To understand the direct correlation between the electrical signal and
the heat flux, consider that a power, W, is fully dissipated in a calibration vessel (Figure 2.7) surrounded by a fluxmeter composed of crowns
of thermocouples (Figure 2.3). An elementary power, wi, is dissipated
through each thermocouple giving an elementary variation of temperature Ti between the internal and external weldings:
wi = i Ti
where i is the conductance of the thermocouple.
(2.7)
25
wi
ei g i
q i
Figure 2.7. Joule effect calibration principle.
The corresponding variation of temperature generates an elementary

electromotive force (emf) according to the Oersted law:
ei = i Ti
(2.8)
where i is the thermoelectric constant of the thermocouple.

By combining Equations 2.7 and 2.8, for the thermocouples in
series, we obtain:
E = ei =
i
wi
i
(2.9)
Because all the thermocouples are identical, Equation 2.9 can be

expressed as follows:
E=
wi
or
E=
(2.10)
Equation 2.10 shows that the power dissipated in the vessel is directly
correlated with the heat flux. The term / corresponds to the calibration factor of the calorimeter.
26
Description of Different Heat Flux Calorimeters Used for

Food Characterization
According to the Calvet principle, many different calorimeters have
been designed with various temperature ranges, small and large
volumes, with a broad range of sensitivity. In this chapter, we describe
two different Calvet calorimeters (www.setaram.com) that are used
worldwide in many food research laboratories.
High Sensitivity Heat Flux Calorimeter
The development of the very high sensitivity heat flux calorimeter
(www.setaram.com) was mainly motivated by the limitations of
the standard DSCs: small amount of sample, limited sensitivity, no
possibility of interaction or mixing. It was designed to be used as
a multipurpose calorimeter working in isothermal and scanning modes
with batch and flow capacities on a significant volume of sample
(1 cm3).
The calorimetric chamber is made of a highly thermal conductive
block with two cylindrical cavities for the experimental vessels (sample
and reference). The detectors are built with semiconducting Peltier
elements, characterized for their high sensitivity compared with a
standard thermocouple-based detector. For the temperature control of
the calorimeter, two principles are used:
A thermostatic loop of liquid flows around the calorimetric block for
a temperature range from 20 C to 120 C
Different shields with Peltier elements are located around the calorimetric block to extend the use at a lower temperature for a temperature range from 45 C to 120 C.
In both cases, the vessels are easily removed from the calorimetry
block. This is a key point for the cleaning of the vessels when different
types of foods, such as fatty compounds, gels, and proteins, are used.
The tops of the calorimeters are opened to allow the introduction of
fluids (gas, liquid) by means of adapted and dedicated vessels. The
thermostatic loop of liquid provides a prestabilizing ring at the upper
part of the calorimeter that allows the liquid to preheat before entering
the calorimetric chamber. According to the type of experiments to be
27
Figure 2.8. Standard and mixing vessels (batch), fluid circulation vessel (flow).
performed on food components, there are different experimental vessels

for the batch or the flow applications (Figure 2.8).
The standard batch vessel is mainly used to investigate food components in a liquid or solid form in a closed system.
The batch-mixing vessel is composed of two chambers that
allow isolation of each material before mixing in the calorimeter. The
mixing operation is achieved by pushing the rod from outside. The
batch high-pressure vessel is mainly dedicated to investigation of
food components under pressure, especially for modification of structure (glass transition, polymorphism) when high pressure is applied.
For such experiments, the calorimetric vessel is fitted with a highpressure gas panel (maximum pressure: 1000 bar) (Le Parlour et al.
2004).
The fluid-mixing vessel is designed to introduce a gas or a liquid
into the vessel to interact with the sample inside. Before introducing
a liquid, the liquid temperature is stabilized at the temperature of the
calorimeter. The fluid-mixing vessel makes possible the mixing of two
liquids in situ in the calorimetric vessel using an adapted mixer. The
entering liquids are prestabilized at the temperature of the calorimeter
and are introduced through micropumps at variable flow rates. Table
2.2 gives an overview of the variety of applications that can be performed with the different vessels.
Table 2.2. Applications of the MicroDSC technique versus the vessel and the
heating mode.
Vessel
Heating Mode
Component
Application
Batch
Scanning
Isothermal
Scanning
Scanning
Protein (animal,
cereal)
Protein
Enzyme
Starch
Isothermal
Scanning
Starch
Milk
Scanning
Fat
Isothermal
Scanning
Scanning
Fat
Hydrocolloids
Sugar
Isothermal
Scanning
Aroma
Fat, chocolate
Denaturation, aggregation,
lyophilization
Crystallization
denaturation, stability
Gelatinization,
retrogradation, glass
transition
Crystallization (stalling)
Melting, crystallization,
denaturation, aggregation
lipidic transition,
polymorphism
Crystallization
Melting, gelation
glass transition
(amorphism)
Stability
Polymorphism versus
pressure
Glass transition versus
pressure
Enzymatic reaction
Wetting
Yogurt processing
Dough and bread processing
Bacteria growth, food safety
Oxidative stability
Enzymatic reaction
Batch high
pressure
Starch
Batch
mixing
Isothermal
One fluid
vessel
Two-fluid
mixing
vessel
Isothermal
Enzyme
Starch
Dairy bacteria
Yeast
Bacteria
Oil
Isothermal
Enzyme
28
29
The Mixing and Reaction Heat Flux Microcalorimeter

The mixing and reaction microcalorimeter (www.setaram.com) is used
for larger amounts of materials to better fit with the experimental needs
of the food industry. The microcalorimeter can be used as a DSC for
temperature scanning, but with large-volume samples. It is, however,
more suitable for the applications in the isothermal mode. The microcalorimeter has a large experimental volume (15 cm3). It is built around
a metallic conductive block with two cavities that contain the thermopiles, which are made of crowns of thermocouples. The block itself is
surrounded by the heating element and arranged in an insulated
chamber. The calorimeter can be fitted on a rotating mechanism to use
with a special mixing vessel.
The microcalorimeter offers a large choice of experimental vessels
for use with various applications. The most commonly used vessels in
food research are as follows:
The batch standard vessel is designed for investigating transformation during heating or cooling a large volume of samples in the solid
or liquid form. It also can be used to determine heat capacity.
The batch high-pressure vessel is designed for simulation of reaction
and decomposition under pressure in a closed vessel or under controlled pressure (max: 100 bar). It is used to define safety conditions
of some food-processing operations and also for simulation of supercritical gas extraction.
The gas-flow vessel is fitted with two coaxial tubes and is used to
produce a circulation of gas (inert or active) around the sample. It is
used for investigation of oxidative stability of foods.
The mixing vessel using the rotating mechanism is divided into two
chambers and separated by a metallic lid. One of the materials is placed
in the lower chamber (i.e., powder) and the other material is placed in
the upper chamber (i.e., liquid). The mixing of the two components is
provided by rotating the calorimeter, the metallic lid acting as a stirrer.
This mixing vessel is designed for investigation of liquid-liquid mixing
(dilution, neutralization) or solid-liquid mixing (dissolution, hydration,
wetting).
The membrane mixing vessel is used for mixing of viscous samples,
often seen with food components and for applications in which
the rotation of the calorimeter cannot be used. In such a vessel, the
separation between both chambers is achieved with a thin membrane
30
(metal or PTFE). The vessel is fitted with a metallic rod that is operated
from outside the calorimeter. The mixing of components is obtained
by pushing the rod to break the membrane. The rod is also used as a
stirrer during the test.
The ampoule mixing vessel is designed for a slow dissolution
process and for a wetting operation. The sample is sealed under vacuum
in a breakable ampoule. The vacuum operation allows desorbing the
surface of the solid sample for easier dissolution. The sealed ampoule
and the solution are introduced into the vessel. By breaking the ampoule,
the solid and liquid samples are brought into contact.
The Table 2.3 gives an overview of the major calorimetric applications, either in scanning or isothermal modes.
Methods of Microcalorimetry in Food
Microcalorimetry offers a variety of methods that are applied to the
characterization of foods and their components.
Heat Capacity Determination
Heat capacity plays an important role in thermal process and in refrigeration applications. Heat loads, processing times, and industrial equipment sizes are influenced by the heat capacity of the material. Combined
with thermal conductivity and thermal diffusivity, heat capacity data
are needed for modeling of the thermal processes. Heat capacity varies
with temperature and composition, as well as water content (Kaletun,
2007). Because food material can be in solid or liquid form, different
ways of measuring heat capacity using the calorimetric techniques
have been developed.
Heat capacity is thermodynamically defined as the ratio of a small
amount of heat Q added to the substance to the corresponding small
increase in its temperature dT:
C=
Q
dT
(2.11)
For processes at constant pressure, the heat capacity is expressed as:
H
Cp =
T p
(2.12)
31
Table 2.3. Applications of the C80 calorimetric technique versus the vessel
and the heating mode.
Vessel
Mode
Component
Application
Batch standard
Scanning
Starch
Salt
Carbohydrate
Batch high
pressure
Scanning
Coffee
Oil
Gelatinization, retrogradation
Solubility
amorphism, decomposition
Safety (roasting),
supercritical CO2
extraction
Self-ignition, explosion
(powder)
Gelatinization under
pressure, glass transition
versus pressure
Polymorphism versus
pressure
Oxidative stability
Oil
Sugar
Salt
Enzyme
Hydrocolloid
Starch
Yeast
Food powder
Neutralization
Dissolution
Dissolution
Enzymatic reaction
Binding
Wetting, gelatinization
Fermentation
Wetting, dissolution
Cereal
Starch
Fat, chocolate
Gas flow
Mixing
(reversing)
Mixing
(membrane)
Mixing
(ampoule)
Scanning,
isothermal
Isothermal
Isothermal
Isothermal
Although DSC is a technique well suited to measure heat capacity

(Richardson and Charsley 1998), essentially only one procedure has
been developed using a continuous heating mode for solid samples. In
this chapter, another procedure is described using a step-heating mode.
Heat capacity determination in temperature-scanning mode
If there is no conformational or phase transformation for the temperature range considered, the calorimetric signal for a given mass of
sample heated at a constant heating rate dT/dt is relative to the following relation for the sample side:
32

dq = ( m c + m c ) dT
s p( s)
cs p( cs)
dt s
dt
(2.13)
where ms and mcs are, respectively, sample mass and vessel mass
(including the cover) and cp(s) and cp(cs) are, respectively, specific heat
capacity of the sample and its vessel.
For the reference side, an empty vessel is used giving the corresponding signal:
dq = ( m c ) dT
cr p( cr )
dt r
dt
(2.14)
where mcr is reference vessel mass and cp(cr) is specific heat capacity of
reference vessel (equal to cp(cs)).
The resulting differential calorimetric signal dq/dt is given by the
following equation:
dq = ( m c + m c m c ) dT
s p( s)
cs p( cs)
cr p( cr )
dt
dt
(2.15)
To get rid of the thermal effect generated by both vessels, the same
test (called blank test) is run with identical empty containers. The following equation describes the blank test heat flow.
dq = ( m c m c ) dT
cs p( cs)
cr p(cr )
dt b
dt
(2.16)
By subtracting the two calorimetric traces, the specific heat capacity

of the sample is extracted (Figure 2.9).
c p( s) =
1 dq dq dT
ms dt dt b dt
(2.17)
As described in the calibration section, the Joule effect technique

allows conversion of calorimetric signal in milliwatts without the need
of standard reference materials. Therefore, in Equation 2.17, all of the
parameters (sample mass, calorimetric signals, heating rate) are accu-
33
Heat flow (mW)
Ab
As
T
time
Figure 2.9. cp determination in the temperature-scanning mode.
rately known to determine the specific heat capacity of the sample cp(s)
(expressed in J.g1.C1) at a given temperature. For DSC technique, a
third test is needed using a standard reference material (sapphire) that
has a known specific heat capacity.
cp determination in the temperature step mode
The technique described in previous section is easy to use, but has a
drawback regarding the accuracy of the cp determination. Using the
temperature scanning mode, the sample is continuously heated and is
never at the thermal equilibrium. However, cp is a thermodynamical
parameter, defined at the thermal equilibrium. The temperature step
mode has been developed to address this limitation. A temperature step
is applied to the sample, and the thermal equilibrium is established
(characterized by return of the baseline) after each step. If Equation
2.15 is integrated from time t0 (beginning of the step) to time tn (return
to the baseline), the corresponding equation is obtained:
[Q ]tt0n = (ms c p(s) + mcs c p(cs) mcr c p(cr ) ) T
(2.18)
where cp corresponds to the mean cp value between the two temperatures defining the step of temperature. Q is obtained by integrating the
corresponding surface defined by the calorimetric signal between t0
and tn. The signal corresponding to the blank test is subtracted when
34
an identical step of temperature is applied to obtain the final equation

for the mean cp of the sample.
c p( s) =
1
(Q Qb ) T
ms
(2.19)
In Equation 2.19, the result is independent of fluctuations of the

baseline between the tests contrary to that of specific heat determination in temperature scanning mode.
cp determination for liquids
Both methods described above apply mainly for the cp determination
of solid and powder foods. They also can be used for liquids, but
the cp contribution of the vapor above the liquid sample must be
accounted for to have an accurate measurement. The correction can be
obtained by using a vessel designed for the cp determination of liquids
(Cerdeirina et al. 2000). The vessel is a cylindrical container with a
tube welded on the top (Figure 2.10). The liquid is introduced in the
calorimetric vessel via the tube using a syringe with a long needle,
which allows a complete filling of the vessel without a vapor phase.
As the tube is opened, the liquid will freely expand when heating. The
Figure 2.10. Liquid cp vessel and principle.
35
cp determination is run for a given volume V of liquid, located in the

calorimetric detection zone. If Q0 is the differential calorimetric area
corresponding to an increase T of the temperature of the calorimeter
when the two vessels (sample and reference) are empty, Q1 when the
measure vessel is filled with a standard liquid of known heat capacity,
and Q2 with the liquid to be investigated, the following equations are
obtained:
Q1 Q0 = V T S 1 c p1
(2.20)
Q2 Q0 = V T S 2 c p 2
(2.21)
where S is calibration coefficient of the calorimeter, V is volume of the

vessel, 1 and 2 are masses of standard and sample, and cp1 and cp2
are heat capacities of standard and sample. The heat capacity of the
liquid sample, at a given temperature, is obtained without needing to
know and measure the corresponding volume V:
(Q Q0 ) 1
cp2 = 1
c p1
(Q2 Q0 ) 2
(2.22)
The determination of the specific heat capacity requires the measurement of the density of the liquid sample. This cp measurement does
not need vapor phase correction.
Heat capacity of foods
The specific heat of foods depends on their composition, specifically
the water content (Kaletun 2007). Table 2.4 gives an overview of the
specific heat of selected foods above and below freezing (www.
engineeringtoolbox.com).
Heating Mode
Microcalorimetry is used under the various heating modes are described
next.
Scanning calorimetry
The scanning mode (heating or cooling) is the usual method that
applies to the standard DSC technique. A microcalorimeter also can
36
Table 2.4. Heat capacity data for some foodstuffs.

Food Category
Type
Fruit
Apple
Grapefruit
Orange juice
Cabbage
Potato
Pork (bacon)
Pork (ham)
Salmon
Carp
Butter
Cream
Milk (cow)
Milk (coconut)
Ice cream
Vegetable
Meat
Fish
Dairy
Cp before Freezing
(J g1C11)
Cp above Freezing
(J g11C11)
1.76
1.84
1.8
1.88
1.72
1.05
1.42
1.55
1.72
1
1.88
1.97
1.76
1.67
3.64
3.81
3.73
3.94
3.43
1.51
2.6
2.97
3.43
1.26
3.77
3.77
3.98
3.1
be used as a DSC, but with low or very low scanning rates (less than
2 C.min1). Longer time of experimentation may be considered to be
a disadvantage, but it provides a better resolution of different thermal
processes.
Melting and crystallization Physical state transformations (crystallization, melting, polymorphism) in fat samples are associated with
thermal effects that are easily measured by DSC. Microcalorimetry
used in the scanning mode allows improvement of the resolution of
different effects because of low scanning rate, especially for characterization of emulsions (Relkin and Sourdet 2005).
Denaturation and aggregation Proteins are the food components
most studied by the microcalorimetric technique and include studies
of conformation changes of food proteins (animal, vegetable, plant),
food enzymes and enzyme preparations for the food industry, as well
as effects of various additives on their thermal properties.
The denaturation and aggregation processes in thermal gelation of
whey proteins were resolved with the microcalorimetric technique
(Fitzsimons et al. 2007). Numerous previous studies of the thermal
gelation of whey proteins, carried out on conventional (fast-scanning)
37
3.2
3.1
90
100
3.0
Heat flow (mW)
85
2.9
2.8
2.7
2.6
2.5
2.4
40
50
60
70
80
Temperature (C)
90
100
Figure 2.11. DSC heating scans (1.0C/min) of 3.0 wt.% WPI pH 7.0) in the
presence of NaCl at the different concentrations (80, 85, 90, and 100 mM NaCl). From
Fitzsimons et al. (2007).
DSC calorimeters (typical sample mass 1550 mg), have shown only
endothermic transitions. Slow transfer of heat into a large vessel
(850 mg of sample) allows the exothermic heat flow from the slow
aggregation process to keep pace with the endothermic heat flow from
the more rapid denaturation process and give a detectable exotherm
(Figure 2.11). The same resolution effect with a slow scanning rate has
also been noticed on pea storage protein, vicilin (Bacon et al. 1989),
on bovine serum albumin (Barone et al. 1992, 1995), and on ovalbumin
(Hagolle et al. 1997; Relkin 2004).
Gelation Microcalorimetry is applied to investigation of gels formed
by biopolymers, such as carrageenan (Williams et al. 1991a, 1992),
xanthan (Williams et al. 1991a, b), gellan (Miyoshi et al. 1995; Robinson
et al. 1991), agar (Cooke et al. 1996), pectin, and gelatin. Polysaccharides
are widely used for their gelling and thickening properties in the food
industry. In presence of a cation (for example, potassium K+), a solution of kappa-carrageenan gives an aggregate structure during heating.
The temperature of transformation and the reversibility of the reaction
38
(melting/gelation) can be obtained from the calorimetry data.

Furthermore, detection of the transition depends not only on the polysaccharide concentration but also on the product type. For xanthan and
gellan, the energy associated with the transition is very weak, and the
high sensitivity of the microcalorimeter is needed.
Gelatinization and retrogradation Microcalorimetry is used to characterize the gelatinization behavior of starches and interaction of starch
with other food components, as well as phase transitions during baking
processes (Eliasson 2003). Calorimetry in the scanning mode is used
not only to study the order-disorder behavior of starch during gelatinization but also to study the recrystallization (retrogradation) during storage
(Berland et al. 2003). Crystallization can also be investigated in the
isothermal mode. A special calorimetric vessel has been designed to
investigate the starch gelatinization during cooking of pasta (Riva et al.
1991).
Isothermal calorimetry
Isothermal calorimetry is commonly used to simulate a process that
occurs at a constant temperature or to check the storage stability of a
food component (Schffer and Lorinczy 2005). When reactions and
transitions take place within a food system, the kinetic parameters of
reactions and transitions are obtained from analysis of isothermal calorimetric curves (Riva and Schiraldi 1993).
Shelf life Shelf life of foods (Franzetti et al. 1995; Riva et al. 1997,
1998, 2001) is investigated using isothermal calorimetry by continuously monitoring the kinetics of microbial growth or enzymatic activity in fresh foods, such as whole eggs, fresh milk, and fresh carrots
(Figure 2.12), or growth of bacteria in milk (Berridge et al. 1974).
There are studies in the literature reporting the evaluation of bacteriological quality of seafood (Gram 1992), characterization of the thermal
consequences of irradiation of bacteria (Mohcsi-Farkas et al. 1994),
and microbial degradation (Teeling and Cypionka 1997; Andlid et al.
1999) by using isothermal calorimetry.
Oxidative stability Thermal oxidative decomposition of edible oils
examined by calorimetry can be used for predicting oil stability under
normal or high pressure of oxygen.
39
0.14
0.1
A-PASTEURIZED
WHOLE EGG
T=21.7C
exo
HF / mW g1
0.12
0.08
T=14.7C
0.06
0.04
T=9.6C
0.02
0
0
4
6
Time / days
10
0.12
0.08
T=24.6C
B-PASTEURIZED
WHOLE MILK
exo
HF / mW g1
0.1
0.06
T=14.7C
T=19.6C
0.04
0.02
0
0
Time / days
0.14
0.1
0.08
C-FRESH CARROTS
T=24.6C
exo
HF / mW g1
0.12
T=19.6C
0.06
T=14.6C
0.04
0.02
0
0
0.5
1.5
2.5
Time / days
Figure 2.12. Isothermal traces at different temperatures for pasteurized whole egg,
pasteurized whole milk, and fresh carrots. From Franzetti et al. (1995).
Isothermal crystallization The investigation of crystallization in the

isothermal mode requires a high stability of the baseline of the microcalorimeter combined with a high sensitivity because such a test may
last many hours. This type of experimental protocol is applied to isothermal crystallization of proteins, isothermal crystallization of fats, or
isothermal retrogradation of starch.
40

heat flow / mW g1
0
0.10
10
15
20
25
30
35
40
0.08
0.06
0.04
0.02
0.00
40
30
T / C
20
10
0
10
15
20
25
time / hours
30
35
0
40
Figure 2.13. Stepwise heating thermogram (from 0 to 40 C) of 4.5% gelatine (LH1e)

in aqueous 0.1 M NaCl. From Cuppo et al. (2001).
Step heating in calorimetry

Step heating (or cooling) calorimetry is a technique that is between the
two previously described modes. A small variation of temperature is
applied to the sample by step. After each step, the sample is maintained
at a constant temperature for a certain period of time. The relevance
of the stepwise methods resides primarily in the ability to follow step
by step the differential structural changes as a function of the temperature. The technique was applied to follow the kinetics of the gelation
of gelatine (Cuppo et al. 2001) (Figure 2.13).
Mixing and Reaction Calorimetry
For investigation of mixing and reaction processes in foods, the microcalorimetry has major advantages over the DSC. As described previously, the larger capacity of the calorimetric chamber allows the design
of specific mixing vessels. Liquid-liquid or solid-liquid interactions are
evaluated by mixing obtained by stirring or ampoule breaking.
Mixing can be performed by two different modes:
1. Batch mixing: The two components A and B are brought into
contact in the mixing vessel. The heat of mixing corresponds to a
given concentration of A or B.
41
2. Flow mixing: The two components A and B at a given flow rate are
pumped and mixed in the vessel. The concentration of the mixture
can be adjusted by modifying the flow rate of A or B.
Dissolution, solubility
In food industry, solid-liquid and liquid-liquid interactions are often
encountered, such as dissolution of powder (sugar, salt) and solubility
of proteins, lipids, and fibers. For such studies, the batch-mixing vessel
is ideal because it provides information relevant to the start of the
reaction and the corresponding kinetics.
Neutralization
The batch-mixing vessel is also convenient for the investigation of any
reaction occurring during a food process, such as neutralization of
edible oils by soda. Raw edible oils contain free fatty acids that have
to be neutralized before being used. The amount of soda necessary for
neutralization has to be adjusted based on the acidity of the oil. The
simulation of the operation was performed on a microcalorimeter using
edible oil with variable acidities (Figure 2.14).
Binding
A mixing calorimeter is useful to investigate the impact of the weak
nonspecific physical interactions of the food biopolymers (proteins,
heat flow
4 mW
exo
1
1 rape seed 8,8 %
2 peanut
3%
3 peanut
0,85 %
time (mn)
0
10
15
20
25
30
Figure 2.14. Neutralization of free acidity in edible oil by soda (C80).
42

HEAT FLOW
50W
0.5
1.0
1.5
2.0
2.5 TIME (h)
Figure 2.15. Enzymatic reaction (maltose + glucoamylase) at 33 C in batch mode

(MicroDSCIII).
polysaccharides) with each other and with the major low-molecularweight ingredients of the multicomponent food colloids (sugars,
mineral salts, small-molecule surfactants). The structure formation in
the bulk aqueous phase and at the interfaces of colloidal systems, as
well as the functional properties, depend on these weak interactions
(Semenova 2007).
Enzymatic reactions
The example for the enzymatic reaction of transformation of maltose
using glucoamylase illustrates the two modes of mixing. In batch
mode, 30 mg of maltose powder is mixed with 20 l of glucoamylase.
The corresponding exothermic effect indicates the transformation of
maltose for a given concentration of enzyme (Figure 2.15). In flow
mode, maltose (1% in H2O) is circulated in both tubes of the mixing
vessel to establish the baseline of the test. Then, glucoamylase (1% in
H2O) is introduced. A corresponding exothermic deviation measures
the efficiency of the enzyme for maltose transformation at a given
temperature and concentration. When the enzyme flow is replaced by
maltose, the calorimetric signal is returned to the baseline (Figure
2.16). This mixing mode is flexible because various enzyme concentrations can be tested consecutively.
43
HEAT FLOW
20W
maltose
+
maltose
enzyme
+
maltose
0
maltose
+
maltose
10
20
30
Time (min)
Figure 2.16. Enzymatic reaction (maltose + glucoamylase) at 25 C in flow mode

(0.3 ml.min1, MicroDSCIII).
Fermentation, bacterial growth

Isothermal microcalorimetry provides informative data regarding
microbial growth and microbial metabolism involving yeast and bacteria. Following are some examples of applications of isothermal
microcalorimetry:
Dosage of antibiotics or detection of antibiotics in milk

Control of alcoholic fermentation
Control of panification
Control of production of dairy products (lactic acid bacteria)
Control of biomass reaction
Control of bacterial growth
Microcalorimetry was used to investigate the growth of probiotic

cultures (Schffer, Szakaly, and Lorinczy 2004; Schffer and Lorinczy
2005). The process of yogurt production also was simulated by mixing
yogurt containing lactic acid bacteria with milk at 37 C. The exothermic effect is associated with the bacterial growth and the characteristics of the final product, according to the temperature, the pH, and the
quality of the milk.
Pressure Calorimetry
High-pressure processing is an important application for food research.
It is important to understand the pressure-dependent phase change phe-
44
nomena in food to improve and develop new technologies. High hydrostatic pressure can cause denaturation of proteins, solidification of lipids,
inactivation of microorganisms, and destabilization of biomembranes.
The calorimetric technique is used to investigate the transformations
induced by pressure. Because a commercial high-pressure calorimeter is
not available (see Chapter 3) for high hydrostatic pressure applications,
the sample is processed in a specific pressure device, outside the calorimeter. This application was used to investigate the protein denaturation
in egg white after high-pressure processing (Andrassy et al. 2006) to
detect the structural changes in milk protein beta-lactoglobulin induced
by combined effects of pressure and temperature (Tedford and Schaschke
2000; Kolakowski et al. 2001). High-pressure induced modifications of
soy protein in soy milk, studied using microcalorimetry, showed that
denaturation of b-conglycinin and glycinin occurred at 300 MPa and
400 MPa, respectively, as judged by the absence of endothermic peaks in
thermograms of pressure-treated samples (Zhanga et al. 2005).
Microcalorimetry in the scanning mode also was used to evaluate
the relative high hydrostatic pressure resistance of bacterial strains
from Staphylococcus aureus (Figure 2.17) and Escherichia coli in
A
Heat Flow
0.5 mW
20
40
60
80
Temperature (C)
100
120
Figure 2.17. Calorimetric traces of untreated control (A) and pressure-treated

(345 MPa, 35 C, 10 min) (B) Staphylococcus aureus. From Alpas et al. (2003).
45
vivo. The total apparent enthalpy change and thermal stability were
two calorimetric parameters used to compare bacterial strains of
untreated control and pressure-treated bacteria (Alpas et al. 2003).
Pressure can be applied on the sample inside the calorimetric vessel.
A maximum gas pressure of 100 MPa was reported by Le Parlour et
al. (2004) to be used for investigation of polymorphic changes in fatty
compounds, the modification of the glass transition temperature for
food with amorphous phase (sugar, starch, dough, frozen foods), or
oxidative stability of oils. Supercritical extraction process using CO2
as a fluid was simulated in a high-pressure calorimeter with an adapted
vessel (Stassi and Schiraldi 1994). Such a setup allows investigation
either at constant pressure and at variable supercritical CO2 flow rate,
or as batch system with a variable pressure. The latter allows the monitoring of the solubility-pressure relationship.
Calorimetry under Controlled Relative Humidity
Many food processes (extrusion, baking, drying, milling) may generate
amorphous compounds. Their stability upon storage, especially in a
humid atmosphere, has to be controlled. Water induces plasticization and
leads to depression of the glass transition temperature, causing significant changes in the physicochemical and crystallization properties of the
food components containing an amorphous phase. Scanning calorimetry
is recognized as an efficient technique for measurement of the glass transition temperature of hydrated products (Bizot et al. 1997; Borde et al.
2002). Scanning calorimetry was also used to monitor crystallization of
lactose in humidified powders, because lactose crystallisation and
Maillard reaction are two major modifications occurring in milk and
whey powders during processing and storage (Morgan et al. 2005).
Typically, samples are equilibrated outside the calorimeter, and the influence of water content or water activity are investigated using calorimetry.
However, the sample can be prepared with a defined water content, and
thermal properties can be measured inside a special vessel by combining
DSC and humidity generator (Le Parlour and Mathonat 2003).
Conclusion
As in other industrial domains, the food industry is more and more
interested in applying new techniques of investigations to improve the
46
products, to enhance quality and safety, to find new ingredients, and

to better understand the different processes involved in the food production. Although microcalorimetry is not a new technique, the broad
range of applications for calorimetry is not well known in the area of
food technology. As described in this chapter, the possibilities of using
different types of calorimeters are unlimited and can be applied to a
large variety of foods. Because heat is involved in most food transformations during processing and storage, calorimetry will provide
answers to food technologists in their daily research.
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of different yeast strains in doughs. J Thermal Anal Calorim, 52:753.
Riva M., Franzetti L., Galli A., and Schiraldi A. 1997. Growth and fermentation
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bulgaricus in milk: A calorimetric investigation. Ann Microbiol Enzymol,
47:199.
Robinson G., Manning C.E., and Morris E.R. 1991. Conformation and physical properties of the bacterial polysaccharides: Gellan, welan and rhamsan. In: Food
Polymers Gels & Colloids, Dickinson E., editor. Woodhead Publishing, Cambridge,
UK.
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Schffer B. and Lorinczy D. 2005. Isoperibol calorimetry as a tool to evaluate the

impact of the ratio of exopolysaccharide producing microbes on the properties of
sour cream. J Thermal Anal Calorim, 82:537541.
Schffer B., Szakaly S., and Lorinczy D. 2004. Examination of the growth of probiotic
culture combinations by the isoperibolic batch calorimetry. Thermochim Acta,
415:123126.
Schiraldi A., Piazza L., Fessas D., and Riva M. 1999. Thermal analysis in foods and
foods processes. In: Handbook of Thermal Analysis and Calorimetry, Vol. 4,
Kemp R.B., editor. Elsevier Science BV: The Netherlands.
Semenova M.G. 2007. Thermodynamic analysis of the impact of molecular interactions on the functionality of food biopolymers in solution and in colloidal systems.
Food Hydrocolloids, 21(1):2345.
Setaram Instrumentation website: www.setaram.com
Stassi A. and Schiraldi A. 1994. Solubility of vegetable cuticular waxes in supercritical CO2 isothermal calorimetry investigation. Thermochim Acta, 246(2):
417425.
TA Instruments website: www.tainstruments.com
Tedford L.A. and Schaschke C.J. 2000. Induced structural change to -lactoglobulin
by combined pressure and temperature. Biochem Eng J, 5(1):7376.
Teeling H. and Cypionka H. 1997. Microbial degradation of tetraethyl lead in soil
monitored by microcalorimetry. Appl Microbiol Biotechnol, 48:275.
Williams P.A., Clegg S.M., Day D.H., and Phillips G.O. 1991a. In: Food Polymers,
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Chapter 3
High-Pressure Differential
Scanning Calorimetry
Gnther W.H. Hhne and Gnl Kaletun
Introduction
Construction of the High-Pressure DSC
Calibration of the High-Pressure DSC
Temperature Calibration Procedure
Heat Calibration Procedure
Applications of the High-Pressure DSC
Conclusion
References
51
53
57
58
61
63
63
64
Introduction
Pressure is an essential variable in physical chemistry. Measurements
at different pressures are therefore of great importance from the thermodynamic perspective. The change of pressure provides increased
insight into the thermodynamic behavior of materials. The wider the
pressure region, the better description of material response to pressure
is obtained, which enables one to develop predictive capability. Higher
pressure is often used during production and processing of materials,
and the change of properties that occurs with pressure is therefore of
great interest for optimization of processing conditions. In particular,
the latent heat of reactions, phase and conformational transitions, and
their pressure dependence are valuable information for quantitative
51
52
analysis of systems under study both in basic research and in industrial

processing. There is an increasing interest in application of high pressure for food processing and preservation. Although the main goal of
this application is to produce microbiologically safe food, high pressure affects the high-molecular-weight components of a food product,
causing conformational and phase changes. Proteins and starches constitute a large percentage of many food products. The phase or conformational changes in such compounds may cause appearance or textural
changes that might affect the quality of the final food products; therefore, the effects of high-pressure processing on food products and their
individual components need to be determined.
Calorimetry is well-suited for study of food materials because food
processing involves either heating or cooling of materials, which can
be simulated in a calorimeter so that data can be directly related to the
process protocols (Miles, Mackey, and Parsons 1986; Mackey et al.
1991; Lee and Kaletun 2002a, 2000b). If the food products are processed by other means, such as using high pressure or chemicals,
temperature-scanning calorimetry may used to compare thermograms
of food product before and after exposure to treatment to evaluate its
effect (Niven, Miles, and Mackey 1999; Alpas et al. 2003; Kaletun
et al. 2004; Lee and Kaletun 2005). However, this approach provides
information about only irreversible changes that occur in the food
product. Therefore, there is great interest in high-pressure calorimetry
to characterize the changes in a sample and to determine the thermal
properties, such as heat capacity, under the conditions relevant to highpressure processing.
Some commercially available calorimeters that operate at rather
moderate pressures of up to 100 MPa (1000 bar) exist. Unfortunately,
there is no truly high-pressure calorimeter on the market (i.e., working
at pressures well above 100 MPa). One major limitation is the need for
a fluid medium (gas or liquid) to transfer the pressure to the sample,
and any liquid or highly compressed gas has a rather large thermal
conductivity (compared with a gas at ambient pressure). The heat flows
through the pressure medium rather than through the thermal pathway
constructed inside the calorimeter to measure the heat flow.
Consequently, a large thermal leakage occurs, leading to much reduced
calorimeter sensitivity.
These problems and the fact that high pressures up to 500 MPa
(5 kbar) or even more are not easy to handle due to the need for special
High-Pressure Differential Scanning Calorimetry
53
equipment and their potential danger have impeded the development

of a commercially available high-pressure calorimeter. Currently,
researchers in the field construct their own equipment (see Chapter 13).
Although high-pressure differential thermal analysis (DTA), a nonquantitative caloric method, exists in several laboratories worldwide
at pressures up to 1 GPa (e.g., see Szab et al. 1969; Shulgin and
Godovsky 1992; Schmidt et al. 1994; Nakafuku and Sugiuchi 1996),
the number of high-pressure calorimeters remains small. The most
widely used calorimeter type is the differential scanning calorimeter
(DSC; see Chapter 1), and some high-pressure differential scanning
calorimeters (HP-DSC) have been constructed during the last three
decades. Different research groups have approached the several problems of HP-DSC in different ways (Schmidt et al. 1994; Arntz 1980;
Kamphausen 1975; Sandrock 1982a; Eichler and Gey 1979; Mellander,
Baranowski, and Lundn 1981; Randzio 1983; Schneider 1985; Zhu
et al. 2004a). To our knowledge, only one power-compensated
DSC (based on the PerkinElmer DSC-7, PerkinElmer, Waltham,
Massachusetts) exists, and it works up to a pressure of 500 MPa (5 kbar)
(Blankenhorn and Hhne 1991). Hhne and co-workers (Blankenhorn
and Hhne 1991; Ledru et al. 2006) modified a commercial powercompensated DSC (PerkinElmer DSC 7) by building a new highpressure measuring head, rather than building a completely new
high-pressure DSC. In this chapter, we focus on the construction of
this calorimeter because it was demonstrated that it can generate calorimetric data at high pressures successfully (Hhne and Blankenhorn
1994; Hhne, Schawe, and Shulgin 1997; Hhne 1998; Rastogi, Hhne,
and Keller 1999; Hhne, Rastogi, and Wunderlich 2000; Ingram et al.
2008), and it is possible to build similar ones without significant difficulty (Ledru et al. 2006).
Construction of the High-Pressure DSC

The HP-DSC presented here operates on the power compensation
principle, making use of a commercial DSC (PerkinElmer). The original measuring head was replaced with a head built by Hhne and
co-workers (1991) with the same electrical and sensor properties but
positioned inside an autoclave that can be pressurized by means of a
hand-operated spindle pump (Figure 3.1).
54
Figure 3.1. High-pressure DSC setup. (a) DSC; (b) spindle pump; (c) autoclave
(Ledru et al. 2006 in accordance with Blankenhorn and Hhne 1991).
High-pressure handling is a dangerous task. To illustrate, note that

a pressure of 500 MPa (5 kbar) is nearly two times the pressure inside
a gun being shot. If a part of the autoclave fails during an experiment
under high pressure, the effect may potentially be worse than that from
a bullet. Consequently, there are high safety demands on high-pressure
experiments. Usually, an autoclave driven with gas as the pressure
medium must be operated in a separate high-pressure shelter room, and
the operator must be outside in a safe place. To avoid such trouble, we
gave preference to silicone oil as the pressurizing medium. Using a
liquid pressure medium (usually oil) for high-pressure experiments is
still rather dangerous, and proper steps to avoid accidents have to be
taken, but the measures are by far less expensive than that used with
highly compressed gas.
Because of these safety reasons, the complete high-pressure system,
including the autoclave (Figure 3.2), the spindle pump, high-pressure
lines, valves, and transducers, were provided by a high-pressure specialist (SITEC-Sieber Engineering AG, Switzerland). The HP-DSC
head consists of two silver furnaces constructed by the research group
and is located within ceramic housings (Figure 3.3). To avoid greater
heat loss, the size was chosen in order to fit closely within the autoclave
but without direct solid-to-solid contact. The furnaces (Figure 3.4)
Figure 3.2. High-pressure DSC: Autoclave unit (Ledru 2006 in accordance with
Blankenhorn and Hhne 1991).
Figure 3.3. High-pressure DSC: Ceramic housing with the silver furnace inside
(Ledru et al. 2006 in accordance with Blankenhorn and Hhne 1991).
55
56
Figure 3.4. High-pressure DSC: Silver furnace with sample pan inside (Ledru et al.
2006 in accordance with Blankenhorn and Hhne 1991).
were electrically isolated with special ceramic glue. They were provided with platinum wire windings for both the heaters and sensors to
match the resistance of the original DSC cell and with leads through
the autoclave closing caps to the calorimeter control system.
The purpose of locating the two furnaces within the ceramic housings is to isolate them, preventing cross talk between the sample and
reference and minimizing the disturbance of the heat flow signal caused
by convection currents in the oil. These housings contain holes for oil
entry and escape during the filling/emptying and pressure change
process, and they have screw caps (Figure 3.3) to allow their volume
to be adjusted in order to balance the two furnaces to obtain a flat
baseline.
Using a branched silicone oil of approximately 100 mPa/s (Wacker
AS 100, Wacker-Chemie GmbH, Germany), the HP-DSC may be
operated in a temperature range from 20 C to 300 C at pressures from
ambient to 500 MPa and with various heating and cooling rates (from
0.5 K.min1 to 20 K.min1). The actual pressure value within the autoclave is measured using a pressure transducer close to the reference
cell, which can be connected to a data logger for pressure recording.
The sample must, of course, be encapsulated to avoid any contact
with the pressurizing medium. This is not an easy matter, as the encap-
57
sulation must be oil-tight on the one hand and free from air bubbles
and empty space on the other. The latter would lead to large deformation of the sample container when the pressure rises; thus, the container
possibly would not remain oil-tight and the measurement would be
faulty. There are several possible ways to solve the problem. One is to
prepare the sample to fit exactly between two aluminum crucibles that
then are welded together with a proper press. Another possibility is to
put the samples into crucibles of a plastic metal such as indium or lead
and close the crucible hermetically.
In operation, the furnaces are placed within the autoclave with their
axes horizontal. The autoclave has a lid (Figure 3.4) that screws down
onto the crucible, ensuring that it is firmly located within the silver
furnace and that good thermal contact is made.
The HP-DSC constructed this way had the following properties:
pressure and temperature ranges from 0.1 to 500 MPa and from ambient
to 600 K (330 C), respectively, and thermal noise 50100 W peak to
peak (much larger than in normal DSCs because of the oil convection).
The detection limit for transitions (peak area) is about 5 mJ (i.e., 1 J g1).
Compared to common DSCs, the baseline repeatability of the HP-DSC
is poor (23 mW) because of unavoidable small differences in oil
volume between the sample and reference cell when a new sample is
remounted. This makes it impossible to determine heat capacities of a
sample with the usual method, which is to subtract an empty pan run
from the sample run. The change in baseline after remounting a new
sample is often larger than the expected difference of the heat flow rate
between the two runs. This unavoidable effect is a serious disadvantage
of the high-pressure DSC.
Calibration of the High-Pressure DSC

To get precise and reliable thermodynamic data, a careful calibration
of every calorimeter is necessary. For normal DSCs, this includes both
temperature and heat calibration or heat flow rate calibration using
standard procedures (Hhne, Hemminger, and Flammersheim 2003)
and certified reference substances with well-known temperature and
heat of transition values. Based on calibration procedure, a function or
table of corrections is generated to obtain the true temperature and the
true heat of transition from the measured quantities. As a rule, the
58
correction depends on temperature but may also be influenced by other

parameters, such as sample mass, pan type, and heating rate (for details,
see the textbook by Hhne, Hemminger, and Flammersheim [2003]).
Various reference substances with different transition temperatures are
needed for a complete calibration. For the HP-DSC, however, the
change of the calibration with the change of pressure must be taken
into consideration, too. Sensitivity of every thermometer and every
heat flow rate sensor is affected by pressure. This is particularly important for very high pressure levels used in the HP-DSC. Consequently,
the resulting correction function for the HP-DSC always depends on
at least two parameters, namely, temperature and pressure.
Unfortunately, there is no existing certified data for the influence of
pressure on the temperature and heat of transition for the recommended
calibration reference substances. Among common reference substances
such as indium, tin, lead, and zinc, only for indium is there reliable
information in literature (Hhne et al. 1996) regarding the pressure
dependence of temperature and enthalpy of fusion. The respective
literature for the high-pressure dependence of the melting point of
tin (McDaniel, Babb, and Scott 1962; Sandrock 1982b) and lead
(McDaniel, Babb, and Scott 1962) differs a little. The pressure dependence of the melting enthalpy for tin and lead has, to our knowledge,
not been reported; therefore, we assume that the melting enthalpy of
tin and lead is almost pressure independent, similar to that of indium
for the calibration of HP-DSC.
As a result, the calibration of the HP-DSC cannot be as precise as
the calibration of normal DSCs. Consequently, the uncertainty of
HP-DSC enthalpy measurements must be considered much higher than
the uncertainty of common DSC measurements at ambient pressure.
Temperature Calibration Procedure
The measured temperature Tmeas has to be corrected (Tcorr) by adding a
correction term based on temperature and pressure dependence:
Tcorr ( p, T ) = Tmeas ( p, T ) + Tcorr ( p, T )
(3.1)
Normally, the correction Tcorr depends on the heating rate, too. This
influence is, however, of minor importance if the same heating rate is
always used for all measurements.
59
Calibration means the determination of the Tcorr(p,T) function (for

a certain heating rate) either as a best fit function or, more often, in
form of a table or array. The respective calibration procedure compares
the measured temperature of transition at different temperatures and
pressures with the true value of certified reference substances. To
check possible nonlinear dependences, at least three different transitions must be measured at more than three different pressures. Indium
is the only reference substance for which the pressure dependence of
the melting point is well known (Hhne et al. 1996):
In
In
Tfus
K ] + [( 0.0507 0.003) K MPa 1 ][ p MPa ]
( p ) K = [Tfus,0
(3.2)
In
= 429.7485 K is the fixed melting point of the ITS-90 for
where Tfus,0
indium at normal pressure (Preston-Thomas 1990) and p is the
pressure. The given standard deviation defines the limits of this best
value estimation and leads to a maximum uncertainty of 1.5 K at
500 MPa.
For tin, to our knowledge two reliable publications exist for the
pressure dependence of the melting point (McDaniel, Babb, and Scott
1962; Sandrock 1982b). The best value approximation for these data
reads:
Sn
Sn
Tfus
K ] + [(0.0324 0.0025) K MPa 1 ][ p MPa ]
( p ) K = [Tfus,0
(3.3)
[(1.45 4.86)106 K MPa 2 ][ p MPa ]2
Sn
where Tfus,0
= 505.078 K , the fixed melting point of the ITS-90 for tin
at normal pressure (Preston-Thomas, 1990) and p the pressure. The
standard deviations were defined from this best value estimation and,
according to the literature data, lead to a maximum uncertainty of 2.5 K
at 500 MPa.
For the pressure dependence of the melting point of lead, to our
knowledge only one publication exists (McDaniel, Babb, and Scott
1962). Any best value estimation, therefore, cannot be performed for
the pressure dependence of its melting point, and the uncertainty is not
known. Consequently, lead cannot be used for calibration of the HPDSC. Because we have two reference substances only, we have to
restrict ourselves to a linear approximation of the temperature dependence of the correction function.
60
To calibrate the temperature of the HP-DSC the first time at least

three different In as well as Sn samples with a mass of 110 mg have
to be weighted precisely (0.01 mg) and encapsulated as described
above in hermetically sealed pans. With these samples, heating and
cooling runs must be performed at different pressures and typical
heating rates. To detect possible differences between the reference and
the sample side of the HP-DSC, a small sample of the same calibrant
should also be positioned on the reference side.
Tcorr(p,T) is defined as the reference temperature minus the
measured temperature at each pressure for both indium and tin, where
the reference values are defined by Equations 3.2 and 3.3 for indium and
tin, respectively. This then permits the construction of a calibration
diagram in which the Tcorr values are plotted against the measured
temperature for both indium and tin for pressures from 0.1 MPa to
500 MPa, as shown in Figure 3.5. In this figure, a linear relationship
between the temperature correction and the measured temperature
has been assumed, as is usual for two-point calibration, to enable an
extrapolation to be made over a wider temperature range. For the
temperature correction, a maximum uncertainty of 1.6 K at 500 MPa
has been calculated from Equation 3.2 from the indium values. For
15
Tcoor/K
10
0.1 MPa
50 MPa
100 MPa
150 MPa
200 MPa
250 MPa
300 MPa
350 MPa
400 MPa
450 MPa
500 MPa
5
330
380
430
480
530
Measured temperature/K
Figure 3.5. Example of temperature correction function for high-pressure DSC

(according to Ledru et al. 2006).
61
tin values, the maximum uncertainty of the correction is larger, namely,

3.1 K at 500 MPa.
This rather time-consuming procedure is absolutely necessary for a
correct temperature calibration of the HP-DSC. The calibration should
be verified in regular periods. This can be done in a shortened procedure with only one sample and one heating rate at two or three pressures. If the result of the respective correction remains unchanged, no
further action is needed. If not, the whole calibration procedure must
be repeated. As noted, a small indium sample may be placed and left
in the reference cell to verify the temperature calibration online, and
then equation 3.1, valid for the sample cell, may be rewritten for the
reference cell as:
Ttrue ( p ) = Tmeas ( p ) + Tcorr ( p, T ) + Ts-r
(3.4)
where Tcorr(p,T) is the correction obtained from the calibration procedure and Tsr is an additional correction that corresponds to the
difference between the reference and sample temperature sensor. This
difference is often temperature and pressure independent and can be
taken as a constant value compared with the overall uncertainty of the
corrected temperatures, which are within the range of 14 K (depending on temperature and pressure) for our calorimeter.
Heat Calibration Procedure

The measured heat or heat flow rate in a DSC is related to the true
value as follows:
fus H true ( p ) = fus H meas ( p ) Rcorr ( p )
(3.5)
where Rcorr(p) is the calibration factor. For power-compensated DSC,

the calibration factor does not depend highly on temperature, but rather
it is a function of pressure. The heat calibration is performed similarly
to the usual procedure for common power-compensated DSCs. An
indium sample is positioned in the HP-DSC, and the melting peak is
measured at different pressures. The calibration factor k(p) is then
determined by comparing the measured value with the reference value
at the respective pressure:
62

Rcorr ( p ) =
In
fus H ref
( p)
In
fus H meas ( p )
(3.6)
For the pressure dependence of the latter, the best value estimation
from Preston-Thomas (1990) is used:
In
fus H ref
J g 1 = fus H 0In, ref J g 1 + [(3.3 2 )10 3 g 1 MPa 1 ][ p MPa ]
[(2.6 2)107 J g1 MPa 2 ][ p MPa ]2
(3.7)
where fus H 0Inref = 28.62 0.11J g 1 is taken as the best value for the
heat of fusion of indium at normal pressure and p the pressure. This
best value estimation includes an uncertainty of 0.1 J g1 at ambient
pressure increasing to 1.1 J g1 at 500 MPa for the best value of the heat
of fusion of indium. A best value estimation of the pressure dependence of the melting enthalpy for tin has not been reported; therefore,
we did not use the melting enthalpy of tin for calibration purposes. The
total uncertainty of a heat measurement with the HP-DSC is the sum
of the uncertainties of the reference material and the respective measurement, which is about 0.92 J g1. In Figure 3.6 Rcorr(p) resulting
from such a heat calibration is given as an example.
Figure 3.6. Calibration factor in dependence on pressure for a high-pressure DSC

(according to Ledru et al. 2006).
63
Following the calibration of the HP-DSC, the measured values can

be reliably corrected. The calibration must be repeated if any essential
part of the high-pressure calorimeter is changed or replaced. This is
especially necessary if the oil is changed, because the heat transfer conditions become different. It may be sufficient to verify the calibration
from time to time, at least when a different type of sample pan and scanning rate are used. To be on the safe side, it is recommended to always
have a small indium sample in the otherwise empty reference pan to
verify the calibration online during every measurement.
Applications of the High-Pressure DSC

To our knowledge, HP-DSC has rarely been applied to studying food
samples to date (Zhu et al. 2004b) because there are no HP-DSCs
commercially available. The few working groups that have constructed
such a device (Sandrock 1982a; Blankenhorn and Hhne 1991; Ledru
et al. 2006) were mainly interested in polymer science (Hhne 1999;
Ledru et al. 2006) or organic chemistry (Sandrock 1982b). Another
problem arises from the demand to seal the samples hermetically in
such a way that any cavity is avoided. For solid materials, this is rather
easy, as described above. But it seems to be impossible to seal liquids
or solutions, which often are matter of interest in food science, in this
way. For liquid samples, special hermetically sealed pans must be
developed and filled in such a way that no cavity or bubble remains
inside after closure to avoid a huge deformation of the pan when the
pressure rises.
Conclusion
High-pressure differential scanning calorimeters are not available
commercially. They are, however, very valuable instruments for obtaining essential thermodynamic data, including food applications. If highpressure measurements are needed, one must build such an instrument
oneself. This is not an easy task and needs experience. As described
above, the high-pressure power compensated DSC has been built
and works well. The advantages of this type of high-pressure calorimeter are as follows:
64
the well-tested construction,

well-established calibration procedures, and
compatibility with the widely used PerkinElmer DSCs and the wellknown power compensation method.
The disadvantages are the following:
Sample size (and mass) is limited to 30 l.
The samples must be hermetically sealed.
The air-free sealing of liquid samples is very difficult.
Nevertheless, we believe that the high-pressure power compensated
DSC can be very useful in food research applications. It should be
possible to overcome its disadvantages, which will be easier than constructing a new high-pressure calorimeter better suited for demands of
food science or to modify other types of high-pressure calorimeters
that currently exist.
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Sandrock R. 1982a. High-pressure high-temperature differential scanning calorimeter. Rev Sci Instrum, 53(7):10791081.
Sandrock R. 1982b. Differentialkalorimetrie (DSC) bei hohen Drcken; Phasenverhalten
und Umwandlungsenthalpien sowie daraus abgeleitete thermodynamische Gren
von Polyethylen und Diamantan bis 6000 bar und 600 K. Dissertation, RuhrUniversitt: Bochum.
Schmidt C., Rittmeier-Kettner M., Becker H., Ellert J., Krombach R., and Schneider
G.M. 1994. Differential thermal analysis (DTA) and differential scanning calorimetry (DSC) at high pressures. Experimental techniques and selected results.
Schneider G.M. 1985. Recent developments of microcalorimetry at high pressures.
Shulgin A.I. and Godovsky Y. 1992. DTA measurements on polymers under high
pressurepolyethylene and poly(diethylsiloxane). J Thermal Anal, 38:
12431250.
Szab J., Luft G., and Steiner R. 1969. Anwendung der Differentialthermoanalyse
zu reaktioiiskinetischen Untersuchungen von Hochdruckreaktionen. Chemie Ing
Technik, 41:1007101.
Zhu S., Bulut S., Le Bail A., and Ramaswamy H.S. 2004a. High pressure differential
scanning calorimetry (DSC): Equipment and technique validation using water-ice
phase-transition data. J Food Process Eng, 27:359376.
Zhu S., Ramaswamy H.S., and Le Bail A. 2004b. High-pressure differential scanning
calorimetry: Evaluation of phase transitions in pork muscle at high pressures.
J Food Proc Eng, 27: 377.
Chapter 4
G. Eric Plum
Introduction
Differential Scanning Calorimetry
Isothermal Titration Calorimetry (ITC)
Conclusion
References
67
68
77
83
84
Introduction
As the food industry begins to take advantage of recent developments
in protein chemistry by introducing enzymes and structural proteins
into modern food materials and their processing, detailed understanding of protein chemical and physical properties becomes increasingly
important. Development of a predictive understanding of the energetics-structure-function relationships will be required to fully exploit the
possibilities presented to engineer proteins with novel substrate specificity or enhanced physical properties, including thermal stability, pH,
and ionic strength optima.
Calorimetry provides several valuable tools for the characterization
of the thermal properties of proteins and their interactions with
other macromolecules and small-molecule affectors. The objective
of this chapter is to introduce and summarize the methods of modern
67
68
ultrasensitive calorimetry, their application to purified protein samples,

and interpretation of the resultant data.
In its broadest terms, calorimetry involves measurement of heat
effects in a system in response to some perturbation. Modern ultrasensitive calorimetry comprises two distinct techniques, each of which
requires specialized instrumentation to affect the perturbation to the
system. In differential scanning calorimetry (DSC), that perturbation
is a change in temperature of the sample. In isothermal titration calorimetry (ITC), the perturbation is the introduction of new material into
the sample.

Information content
To fully exploit the structural and catalytic properties of proteins it
is critical to develop a predictive understanding of their functions
and stability as a function of temperature and solution conditions.
Monitoring the unfolding of a macromolecule induced by exposure
to elevated temperature is a classical method for evaluating stability.
DSC is particularly well suited to characterization of protein stability
because no chromophores are required, nor are optically clear solutions
required. Most importantly, interpretation of the resultant data is
not dependent on any model of the unfolding process. The thermodynamic characterization of the protein unfolding process derived from
DSC data can be used to predict the stability of the protein at any
temperature.
With the techniques of modern molecular biology and biochemistry
one can manipulate the structure of biological macromolecules almost
at will. Site-directed mutagenesis permits substitution or deletion of
amino acids at the polypeptide sequence level. Techniques have been
developed to include, in addition to the naturally occurring amino
acids, a variety of nonnatural amino acid variants into proteins. Because
they are state functions, thermodynamic quantities (heat capacity,
enthalpy, entropy, free energy) represent sums of contributions from
many sources. Systematic comparison of protein variants allows, in
principle, for the quantification of contributions of particular amino
acid side chain interactions to the measured thermodynamic quantities.
The increasingly wide availability of detailed structural models for
69
proteins from x-ray crystallography and nuclear magnetic resonance

permit one to examine mutation-induced changes in atomic detail.
Unfortunately, a simple substitution of one amino acid for another may
have effects that propagate well beyond the particular interactions that
appear to change in the three-dimensional structure. Because many of
the forces that determine the thermodynamic parameters operate on
scales of distance smaller than can be reliably determined by the structural methods, these assignments may not be reliable.
Instrumentation
During scanning calorimetric examination of a biological macromolecule in dilute aqueous solution, most of the energy introduced into
the system goes toward heating the solvent. A concentration of about
0.1 mg/ml is typically used for protein DSC. Differential scanning
calorimeters of sufficient sensitivity to study biological macromolecules in dilute solution typically are based on the power compensation
method. Modern ultrasensitive DSC instruments are capable of
detecting signals deviating from the baseline of well under 100 nW.
While commercial instruments vary in the means by which they make
the measurement (Privalov et al. 1995; Plotnikov et al. 1997), the
general concept is described here. The power compensation DSC
instrument comprises two matched cells, both fixed in position and
in thermal contact with a thermopile, housed in an adiabatic chamber.
In one cell is placed the sample solution. In the second cell is
placed a reference solution. A small (23 atm) pressure is maintained
on the cells to suppress bubble formation and evaporation of the
samples.
As the instrument is heated, the thermopile responds to differences
in temperature of the two cells. Heat is applied to the lagging cell to
zero the temperature difference. The amount of energy required to
compensate for the thermal event that causes the cell to lag in temperature is directly related to the applied heat, which is quantified in terms
of power (energy per time). Thus, the primary data collected by the
power compensated DSC instrument is a curve of power versus temperature. Division by the heating rate converts the curve to the heat
capacity difference between the sample and reference cells, frequently
referred to as excess heat capacity, as a function of temperature.
Normalization by the amount of analyte yields a curve in terms of
specific or molar heat capacity.
70
The sample must be prepared so that the only the difference between
the sample and reference solutions is the presence of the macromolecule of interest. This is generally best accomplished by exhaustive
equilibrium dialysis of the sample solution (dialysand) against the
buffer solution (dialysate) and subsequent use of the dialysate as the
reference solution. Even with the greatest care, the solutions in the
sample and reference cells cannot be matched exactly. The macromolecule under study displaces solution that contains solvent, buffers,
salts, etc., which have temperature-dependent heat capacities. These
effects, coupled with imperfect matching of the calorimeter cells, mean
that a small but nontrivial baseline correction must be made to separate
the contribution of the macromolecule order-disorder transition from
the solution displacement and instrumental effects. Small errors in
sample preparation and baseline correction can lead to large errors in
the calculated thermodynamic parameters. Some attempts have been
made to standardize DSC experimental and analysis procedures to
reduce interlaboratory variability (Hinz and Schwarz 2001), but
care should be exercised when comparing data from different
laboratories.
Basic equations
With modern instrumentation, complex multidomain DSC thermograms can be resolved into individual transitions (Vlker et al. 1999).
However, for this discussion it is assumed that the thermogram is of a
single transition, which is typically observed for single domain globular proteins in solution. Thus, only monomolecular unfolding processes, those involving only a single polypeptide chain, are considered.
Methods to address multisubunit proteins exist but are beyond the
scope of this discussion.
Figure 4.1 shows a simulated DSC thermogram of the temperatureinduced unfolding of a small globular protein. Differential scanning
calorimetry data are analyzed using a set of standard thermodynamic
relations (Privalov and Potekhin 1986). All thermodynamic relations
herein are at constant pressure. The DSC curve is analyzed in terms of
the relationships between the measured heat capacity and the thermodynamic parameters of interest.
The excess heat capacity, C pex, is the difference in heat capacity
between the sample solution containing the macromolecule of interest
relative to the reference solution. Tm is the temperature at the mid-
71
12,000
Tm
Cp cal mol1 K1
10,000
8,000
H(Tm)
6,000
4,000
2,000
Cp
0
300
325
350
375
400
Temperature (K)
Figure 4.1. Simulated DSC thermogram of a small globular protein. The excess heat
capacity versus temperature curve is calculated using Tm = 350, H(Tm) = 100 kcal/
mol, and Cp = 1.5 kcal/mol K.
point of the transition; that is, the temperature at which the concentrations of the folded and unfolded forms of the protein are equal. The
maximum of the C pex versus temperature curve will correspond to Tm
only when the unfolding unit is monomolecular and the difference in
heat capacity between the folded and unfolded forms of the protein is
negligible. The enthalpy change at Tm is determined from integration
of the DSC curve
H (Tm ) = C pex dT
(4.1)
where the integration covers the entire temperature range of the denaturation transition. The free energy change at temperature T, which is
a measure of the proteins stability at that temperature, depends on the
enthalpy and entropy changes.
G (T ) = H (T ) T S (T )
(4.2)
To describe the thermodynamics of the system at any temperature, it

is necessary to adjust the DSC determined enthalpy and entropy
changes determined at Tm. The heat capacity change associated with
the order-disorder transition, Cp, which comes directly from the DSC
curve, is used for the temperature extrapolation.
72

T
H (T ) = H (Tm ) + T C p dT
(4.3)
C p
dT
T
(4.4)
S (T ) = S (Tm ) + T
For a monomolecular process, G(Tm) = 0 and therefore from Equation

4.2
S (Tm ) =
H (Tm )
Tm
(4.5)
For higher order complexes, additional statistical effects must be

included (Marky and Breslauer 1987). Assuming that the heat capacity
change is independent of temperature, by integrating and combining
the expressions above, the enthalpy, entropy, and free energy changes
are approximated by
H (T ) H (Tm ) (Tm T )C p
(4.6)
T
S (T ) S (Tm ) C p ln m
T
(4.7)
Combining Equations 4.2, 4.6, and 4.7 permits calculation of the free
energy change at any temperature from the three parameters, H(Tm),
Tm, and Cp, obtainable from the DSC curve.
G (T )
Tm T
T
H (Tm ) (Tm T ) C p + T C p ln m
T
Tm
(4.8)
Using these expressions one can predict the stability of the protein and
its thermodynamic origins at any temperature.
The vant Hoff enthalpy change
While the enthalpy change measured by DSC does not depend on a
model, comparison with models can provide insight into the nature of
the order-disorder transition. Consider an equilibrium constant, Keq.
K eq =
[ unfolded ]
[folded ]
(4.9)
73
Because the shape of the DSC curve reflects the change in the equilibrium constant as a function of temperature, the vant Hoff model
can be applied to DSC data as an alternate means of enthalpy change
determination. The vant Hoff model is based on the temperature
dependence of the dimensionless equilibrium constant.
ln K eq
ln K eq
H vH = RT 2
= R
T
(1 / T )
(4.10)
Note that the units of the vant Hoff enthalpy, HvH, are defined by the
units of the constant R.
Comparison of the model independent calorimetrically determined
H(Tm) value with the HvH value assesses the validity of the assumptions employed in the derivation of the vant Hoff relation. Specifically,
it is assumed that the transition from the ordered, low temperature form
to the disordered, high temperature form passes through no thermodynamically significant intermediate states (two-state assumption); that
is, there is no partial unfolding of the protein in the denaturation
pathway. The HvH reports the enthalpy change associated with disruption of a single cooperative unit, the fraction of the protein that acts as
a single thermodynamic unit.
There are several methods used to extract HvH from the shape of
equilibrium denaturation curves, which are frequently based on indirect observations such as temperature-dependent spectroscopic measurements (Marky and Breslauer 1987). The equation below comes
most directly from the DSC curve. Because the equation, as written
here, does not account for changes in heat capacity, a DSC curve from
which the contribution of Cp has been subtracted should be used.
2
H vH = 4 RTmax
C pmax
H (Tmax )
(4.11)
For small globular proteins, it is expected that HvH = H(Tm). When

HvH H(Tm), an error in the determination of the folded protein
concentration may be indicated, the baseline may be assigned incorrectly, or the origins of the deviation may depend on a more fundamental property of the protein system. While in practice they must be
examined and eliminated, for this discussion errors in concentration
74
determination and assignment of the baseline will be discounted. When

HvH < H(Tm), a deviation from the two-state assumption is indicated;
the cooperative unit is smaller than the entire protein. The transition
involves either unfolding intermediates or independent domains. When
HvH > H(Tm), aggregation of the unfolded polypeptide has sharpened
the DSC observed transition.
Origins of the heat capacity change
The molecular origins of the heat capacity of protein and its change
with denaturation are still a matter of active study and debate (Prabhu
and Sharp 2005). About 30 years ago, Sturtevant (1977) enumerated
the underlying contributions to the heat capacity of proteins; these
include the hydrophobic effect, electrostatic effects, hydrogen bonds,
intramolecular vibrations, and changes in equilibria. The heat capacity
and its change with denaturation can be roughly divided into contributions from hydration (protein-solvent and solvent-solvent interactions) and from intraprotein interactions, C p = C pHydration + C pProtein.
Relatively little progress has been made in the ensuing years in understanding the magnitudes of the various contributions to the heat capacity. Because it is widely believed that the contribution of hydration
dominates, most theoretical and experimental work has been directed
at the C pHydration term.
Record and coworkers (Spolar, Ha, and Record 1989) described an
empirical correlation between the change in solvent-accessible nonpolar surface area of a protein Anp and the heat capacity change associated with thermal denaturation. Numerous workers have extended the
empirical model by inclusion of terms to account for differences in the
contributions from hydration of polar and nonpolar surface elements
(Prabhu and Sharp 2005).
C pHydration = c p Ap + cnp Anp
(4.12)
where cp and cnp are empirical coefficients, Ap and Anp are the differences between the folded and unfolded forms of the protein in polar and
nonpolar surface area in contact with solvent. Although the structure of
the folded form of most proteins is stable and the relevant surface areas
readily calculated, the fluctuating structure of the unfolded form is
poorly defined, and thus different methods of calculating the surface
areas of the unfolded form lead to different empirical equations.
75
Cold denaturation
Due to the sign and magnitude of the heat capacity change relative to
the changes in enthalpy and entropy observed for globular proteins, at
some temperature a maximum in the free energy change is observed
(Privalov 1990). This further implies that, in addition to the high temperature (Tm) at which G = 0, there is a low temperature that satisfies
the same condition. In most cases, this implied cold denaturation temperature is below the freezing point of water; however, there are
examples of cold denaturation observable within the aqueous liquid
temperature range accessible experimentally with fixed cell instrumentation, wherein the cells must be filled completely (Privalov 1990). The
cold denaturation phenomenon may be particularly important in freezing and lyophilization processes.
DSC data can quantify high-affinity binding
Binding equilibria between a protein and a small molecule effector,
such as a cofactor or drug, or a second protein subunit, can alter
the proteins denaturation temperature. If the second molecule binds
more tightly to the folded form of the protein than to the unfolded
form, the denaturation will shift to higher temperature. Conversely,
preferential binding to the unfolded form shifts the denaturation to
lower temperature. DSC thermograms are particularly well suited
to measure very tight binding based on the observed bindinginduced changes in the heat capacity versus temperature profiles.
Brandts and Lin (1990) present methods and models for analysis of
the shapes of DSC thermograms to quantify binding affinities for
protein-small molecule and protein-protein interactions up to 1040 M1,
whereas most methods cannot quantify binding constants greater than
1010 M1.
Assumptions
The above described analysis of DSC thermograms in terms of equilibrium thermodynamic parameters depends on a number of assumptions about the system and the design of the experiment. It is
important to evaluate the validity of the assumptions to assure the
quality of the derived thermodynamic data and because deviations
from the assumed behavior can provide insight into the protein unfolding process.
76
Equilibrium, two-state, reversible transitions

The above analysis of the DSC curve assumes that the system is in
equilibrium. The DSC measurement is that of power (energy/time).
The observed signal increases with scanning rate; therefore, it is advantageous to scan the temperature at a high rate. This advantage is tempered by the necessity to maintain equilibrium conditions in the sample.
If the scanning rate is too high, the H value will still be correct, but
the shape and position of the curve will be compromised. Thus, G,
S, and Tm will be incorrect. To ensure that the equilibrium assumption
is valid, it is advisable to conduct the DSC experiment as a function
of scanning rate. If the resulting thermograms are independent of scanning rate, the equilibrium assumption is satisfied.
As part of the equilibrium assumption, it is further assumed that the
process is fully reversible. Generally, protein denaturation is described
by a reaction involving a reversible unfolding of the native state (N)
to form a soluble unfolded form (U), which may subsequently irreversibly form an aggregated state (D) (Privalov and Potekhin 1986).
N
U
mD
(4.13)
Even if the U D transition had no effect on Cp, it would manifest

itself in the shape of the transition, reflected in an increase in HvH. If
the U D transition is not monomolecular, that is m 1, HvH will
increase with increasing concentration.
It is commonly assumed that subsequent to the temperature induced
unfolding process the protein exhibits a random coil confirmation.
This, however, is an oversimplification in most cases. Due to their
implication in some diseases, unfolded proteins are receiving intensive
structural study (Mittag and Forman-Kay 2007). Some proteins refold
into an alternate confirmation, whereas others form aggregates or precipitates. Upon unfolding, most soluble proteins exhibit reduced solubility in aqueous solutions and tend to aggregate. The extent of this
aggregation varies widely, depending on the specific amino acid composition and sequence as well as the solution conditions. Minor aggregate formation may not be readily visualized in the DSC trace but will
affect the shape of the transition resulting in a difference between the
observed calorimetric and vant Hoff enthalpy changes. Extensive
77
aggregation resulting in precipitation is often apparent by wild fluctuation in the high temperature baseline.
Concentration and purity
The enthalpy and heat capacity measurements conducted using DSC
are based on the amount of material thought to be in the sample.
Whether the objective is molar or specific values, the measured thermal
effects are divided by the quantity of the analyte protein. Therefore,
accurate DSC results depend on accurate determination of the amount
of analyte protein present, as well as its purity. The best means of
determining concentration of the analyte will vary with the properties
of the particular protein under study.
It is generally assumed that the concentrations of all components in
the solution are sufficiently low that all activity coefficients may be
satisfactorily approximated by unity. While this assumption may not
be correct, in most cases there is little alternative.
Selection of hydrogen ion buffer
The selection of hydrogen ion buffer is critically important in DSC
experiments. Buffers with high heats of ionization lead to temperature
dependent changes in the pH of the solution. Thus, any pH dependent
changes in the protein will be superimposed onto the temperature
dependent changes, which the DSC experiment is designed to measure.
Unfortunately, some of the most widely used buffers for general biological macromolecule studies exhibit high ionization heats. A particularly egregious example is tris buffer, although most buffers carrying
amine groups are problematic.
A second important issue in buffer selection for DSC studies is the
buffers propensity for metal ion chelation. Because proteins frequently
carry anionic functionalities on their surfaces or require metal ion
cofactors, interactions with metal ions are often important in stabilizing
their structures. Competition for metal ion binding between the protein
and the buffer can compromise the DSC experiment.
Isothermal Titration Calorimetry (ITC)
Information content
Most biological processes involve one or more binding events. The
types of binding reactions are varied and include, but are not limited
78
to, assembly of protein subunits into functional enzyme complexes,

formation of enzyme-inhibitor complexes, formation of protein-nucleic
acid complexes, enzyme-substrate binding, and enzyme-cofactor
binding. These binding processes can be described in terms of the
standard thermodynamic parameters. A predictive understanding of the
binding process can be achieved by measurement of the binding free
energy change G, enthalpy change H, entropy change S, and their
temperature dependence Cp.
All of the binding processes enumerated above are amenable to
analysis by some variation of the ITC experiment. Because the binding
sites for small molecules to proteins tend to be well defined and small
in number, and because most of the binding reactions involve a nonzero
enthalpy change, protein-small molecule interactions frequently are
particularly well suited to examination by isothermal titration calorimetry. Here, we consider a simple association (without reaction) defined
by the equilibrium nL + M MLn of a small molecule, L, with a
protein or other macromolecule, M, with n identical, noninteracting
binding sites. The ITC method can be applied to more complex equilibria; however, that is beyond the scope of this discussion.
Instrumentation
The design of the power compensation isothermal titration calorimeter
is conceptually similar to the power compensation DSC but is adapted
for isothermal operation and for introduction of liquid into the sample
cell (Wiseman et al. 1989). The instrument comprises a thermostated
chamber housing inverted lollipop-shaped sample and reference cells.
The cell volumes are on the order of 1 ml. A small constant amount of
heat is applied to the reference cell. A sensor detects differences in the
temperature between the cells, and heat is applied to the lagging cell.
The energy applied per unit of time is recorded.
Rather than by heating as in the DSC, the system is perturbed by
addition of material into the sample cell by means of a syringe. Usually,
the titrant solution contains the small molecule L and the sample cell
contains the macromolecule M. Typically, upon introduction of an
aliquot of titrant (a few microliters) into the sample cell, an identical
volume of the previous solution is expelled from the measuring volume
of the cell. Stirring of the sample cell provides efficient mixing of
the titrant solution into the titrate solution. In a typical experiment,
about 20 injections of titrant are made. The concentration of L in
79
the syringe is selected to provide a final ratio of concentrations

[L]/[M] 2n.
The primary data from an ITC experiment is a plot of applied power
as a function of time. Upon addition of an aliquot of titrant, any thermal
response in the sample cell is compensated by heating the sample or
reference cell, as appropriate. Sufficient time must elapse between
injections to return the instrument response to the baseline. Thus, for
each addition of titrant to the sample cell, a peak is observed in the
power-versus-time profile. Each of these peaks is integrated to yield a
value for the thermal response (heat) due to the titrant injection. The
resultant plot of the measured heat due to injection of L, dQ/d[L],
versus the concentration of added binding species, or more typically
the molar ratio of the binding species in the cell, that is [L]/[M], is
analyzed to determine the thermodynamic parameters that characterize
the binding process. See Figure 4.2 for examples of the raw and integrated data plots. For the model described here, the resultant curve is
sinusoidal beginning at approximately H for tight binding or less for
lower-affinity binding and declining to a small value that includes
dilution effects (see below), with an inflection point at [L]/[M] = n.
Thermal effects observed in the ITC experiment include those
associated with macromolecule-macromolecule, macromolecule-small
molecule interactions, small molecule-small molecule interactions, and
heats of dilution, as well as temperature differences between the solution in the syringe and the solution in the sample cell. Therefore, it is
critical that additional experiments identical to the first, except for
selective absence of the binding species in the sample cell or syringe,
be performed to correct for dilution heats and artifacts due to temperature differences between the syringe and sample cell. The corrected
heat is determined by subtraction of dilution heats for L and for M as
well as a buffer dilution that corrects for injection artifacts and mismatched buffers.
Qcorrected = Qmeasured QL_dilution QM_dilution Qbuffer
(4.14)
ITC data analysis

While in some advanced applications DSC data are model dependent,
determination of the basic thermodynamic functions from DSC
does not depend on a model of the process under study. In contrast,
Figure 4.2. Isothermal titration calorimetry of binding of two inhibitors to glucosidase (Zechel et al. 2003). (a) The raw data (upper panel) and the integrated
data with fitted curve (lower panel) for an inhibitor with K = 1.25 105 M1. (b) An
inhibitor with K = 1.5 107 M1. Reprinted with permission from J. Am. Chem. Soc.
2003, 125, 1431323. Copyright 2003 American Chemical Society.
80
81
isothermal titration calorimetry is model dependent. To interpret the

ITC curve, expressions must be derived to relate the change in heat,
dQ, as a function of change in added small molecule, d[L], to the
amount of complex formed, d [LM], in the cell volume, V.
dQ
d [ LM ]
= HV
d [L]
d [L]
(4.15)
For the reaction described herein where n = 1, a closed form expression

d [ LM ]
for
can be written in terms of K, [M], [L] (Wiseman et al.
d[L]
1989).
1 [L]
1 1 +
2
[ M ] K [ M ]
d [ LM ] 1
(4.16)
= +
1
2
2
2 [L] 2
d [L]
1
1
[L]
[ M ] 2 [ M ] 1 [ M ] K + 1 + [ M ] K
The ITC curve can then be fitted by standard nonlinear least
squares techniques for H and K. The free energy and entropy changes
are determined by the standard relations G(T) = RTlnK and
H (T ) G (T )
. A series of titrations as a function of
T
temperature provide the heat capacity change associated with the
binding reaction via the temperature dependence of the enthalpy
dH
.
change, C p =
dT
Only for the most simple models can a closed form expression be
written relating the change in heat accompanying introduction of an
aliquot of titrant to the parameters of the binding reaction. More elaborate models can be evaluated numerically. Software provided with the
instruments can analyze ITC data in terms of several different models,
including the single binding site case considered here, multiple identical binding sites that may or may not interact, and multiple classes of
distinct binding sites.
Many, if not most, binding events involving macromolecules and
small molecules or other macromolecules are coupled to changes in
ionization state of one or more charged groups. Thus, the measured
S (T ) =
82
heat associated with the binding event includes the net ionization heats
of the groups for which protonation or deprotonation occurs as well as
compensating effects from the included hydrogen ion buffer. It is
therefore advisable to measure the association heat in two or more
hydrogen ion buffers, which differ in ionization heat, at identical pH
values (Baker and Murphy 1996). The plot of the measured association
heat as a function of the buffer ionization heat permits determination
of the intrinsic heat of binding and the net number of protons taken up
or released upon binding. The intercept at zero buffer ionization heat
corresponds to the intrinsic enthalpy of binding; the slope corresponds
to the net change in protonation.
Due to the strong model dependence of the ITC method, challenges
arise that are not part of the analysis of DSC data. Most ITC-binding
isotherms comprise a small number of features: specifically, the intercept on the enthalpy axis and one or two inflection points. Thus, the
number of fitting parameters that the data can support is limited. Many
processes of interest involve competing equilibria with multiple binding
species and binding sites that require elaborate multiparameter models
to describe. Caution, and appropriate statistical tests, should be applied
to ensure that the data can support the applied model. It is relatively
easy to devise a model that cannot be adequately addressed by ITC
isotherms alone; however, if complementary data are available to fix
some of the fitting parameters, such models may become tenable.
Range of applicability
The classical ITC experiment described here is limited to binding
affinities and concentrations that produce a titration curve with a shape
that can be fit accurately by nonlinear least squares methods. A rough
estimate of this range defined by the macromolecule concentration and
the equilibrium association constant is 1 < [M]K < 1000 (Wiseman et
al. 1989), with 10 < [M]K < 100 being preferred. Note that while the
macromolecule concentration could always be adjusted to place [M]K
in range, the heat produced by the binding reaction must be detectable.
This places an effective limit on the range of binding affinities accessible to direct ITC measurement.
Figure 4.3 shows how the shape of the ITC titration curve varies
with [M]K. As [M]K , the ITC curve becomes a step function with
essentially all the L injected binding to M, until all of the available
binding sites are exhausted. So dQ/d[L] = H for [L]/[M] < n and
83
Figure 4.3. Dependence of the shape of the ITC titration curve on [M]K for the reaction nL + M MLn.
dQ/d[L] = 0 for [L]/[M] > n. There is no information about K except

that it is large. As [M]K 0, the ITC curve approximates a flat line
and provides no information on H, n, or K.
Linkage to other equilibria can be used to measure indirectly binding
affinities that are too tight or too weak to measure by standard ITC
experiments (Doyle et al. 1995; Sigurskjold 2000). If n is known,
reasonable estimates of K may be obtained by extension of the titration
range so the final [L]/[M] >> 2n (Turnbull and Daranas 2003). Note
that this method provides only an estimate of K and will not provide
accurate H values.
Conclusion
Modern ultrasensitive calorimetry provides powerful tools for understanding the stability of proteins in solution, the forces that maintain
their folded structures, and their interactions with other macromolecules and small molecules.
Differential scanning calorimetry quantifies the thermal (Tm) and
thermodynamic (G) stabilities of the protein. The thermodynamic
origins of the stability (H, S, and Cp) can be interpreted to dissect
the forces maintaining the folded structure and how they depend on
84
the structure of the protein and the solution conditions. The modelindependent values derived directly from the DSC thermogram can
be compared to model-dependent values derived from the shape of
the DSC curves to gain insight into the unfolding mechanism and
aggregation.
Isothermal titration calorimetry provides a means to directly measure
the heat of interaction (H) of a protein with another macromolecule
or with small molecules. Application of models for the association
reaction provides detailed thermodynamic characterization (K, G, S,
and Cp) of the binding process.
Taken together, the techniques of modern solution calorimetry
provide a predictive understanding of the stability of a protein and its
interactions with other molecules as a function of temperature and
solution conditions.
References
Baker B.M. and Murphy K.P. 1996. Evaluation of linked protonation effects in
protein binding reactions using isothermal titration calorimtery. Biophys J,
71:204955.
Brandts J.F. and Lin L. 1990. Study of strong to ultra tight protein interactions using
differential scanning calorimetry. Biochemistry, 29:692740.
Doyle M.L., Louie G., Dal Monte P.R., and Sokoloski T.D. 1995. Tight binding
affinities determined from thermodynamic linkage to protons by titration calorimetry. Methods Enzymol, 259:18394.
Hinz H.J. and Schwarz F.P. 2001. Measurement and analysis of results obtained on
biological substances with differential scanning calorimetry. Pure Appl Chem,
73(4):74559.
Marky L.A. and Breslauer K.J. 1987. Calculating thermodynamic data for transitions
of any molecularity from equilibrium melting curves. Biopolymers, 26:160120.
Mittag T. and Forman-Kay J.D. 2007. Atomic-level characterization of disordered
protein ensembles. Curr Opin Struct Biol, 17:314.
Plotnikov V.V., Brandts J.M., Lin L., and Brandts J.F. 1997. A new ultrasensitive
scanning calorimeter. Anal Biochem, 250:237244.
Prabhu N.V. and Sharp K.A. 2005. Heat capacity in proteins. Annu Rev Phys Chem,
56:52148.
Privalov G., Kavina V., Freire E., and Privalov P.L. 1995. Precise scanning calorimeter for studying thermal properties of biological macromolecules in dilute solution. Anal Biochem, 232:7985.
Privalov P.L. 1990. Cold denaturation of proteins. Crit Rev Biochem Mol Biol,
25(4):281305.
Privalov P.L. and Potekhin S.A. 1986. Scanning microcalorimetry in studying tem-
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perature-induced changes in proteins. Methods Enzymol, 131:451.

Sigurskjold B.W. 2000. Exact analysis of competition ligand binding by displacement
isothermal titration calorimetry. Anal Biochem, 277:26066.
Spolar R.S., Ha J., and Record, Jr. M.T. 1989. Hydrophobic effect in protein folding
and other noncovalent processes involving proteins. Proc Natl Acad Sci USA,
86:838285.
Sturtevant J.M. 1977. Heat capacity changes in processes involving proteins. Proc
Natl Acad Sci USA, 74:223640.
Turnbull W.B. and Daranas A.H. 2003. On the value of c: Can low affinity systems
be studied by isothermal titration calorimetry? J Am Chem Soc, 125:1485966.
Vlker J., Blake R.D., Delcourt S.G., and Breslauer K.J. 1999. High-resolution calorimetric and optical melting profiles of DNA plasmids: Resolving contributions
from intrinsic melting domains and specifically designed inserts. Biopolymers,
50:30318.
Wiseman T., Williston S., Brandts J.F., and Lin L. 1989. Rapid measurement of
binding constants and heats of binding using a new titration calorimeter. Anal
Biochem, 179:1317.
Zechel D.L., Boraston A.B., Gloster T., Boraston C.M., Macdonald J.M., Tilbrook
D., Matthew G., Stick R.V., and Davies G.J. 2003. Iminosugar glycosidase inhibitors: Structural and thermodynamic dissection of the binding of isofagomine and
1-deoxynojirimycin to -glucosidases. J Am Chem Soc, 125:1431323.
Chapter 5
Thermal Analysis of Denaturation and
Aggregation of Proteins and Protein
Interactions in a Real Food System
Valerji Y. Grinberg, Tatiana V. Burova, and Vladimir B. Tolstoguzov
Introduction
Effects of pH on Thermal Denaturation of Food Proteins
Effects of Salts on Thermal Denaturation of Food Proteins
Effects of Alcohols on Thermal Denaturation of Food Proteins
Effects of Odorants on Thermal Denaturation of Food Proteins
Effects of Polysaccharides on Thermal Denaturation of Food
Proteins
Postdenaturation Aggregation of Food Proteins
Conclusion
References
87
89
95
99
102
105
110
112
113
Introduction
Normally, food contains a heterogeneous, heterophase mixture of
high- and low-molecular-weight components and their aggregates and
complexes. Among food macromolecules, proteins and polysaccharides are largely responsible for the structural changes accompanying
food processing and for mechanical and other physical properties of
foods.
Proteins are both most-multifunctional biopolymers and mostversatile food macromolecules. Normally, a functional protein has a
unique ordered (crystal-like) molecular structure, which appears to be
87
88
responsible for a high specificity and efficiency of its functioning. Food

proteins greatly influence the structure-property relationship in foods.
Heating is one of the most important treatments of food processing.
Heat denaturation and aggregation of proteins are therefore the most
typical events in food processing. For instance, the difference between
raw and boiled eggs is caused by the denaturation and aggregation of
the denatured egg proteins. The heat denaturation involves a cooperative or noncooperative transition of a protein from its folded to its
unfolded state. It is related to some structural disorganization of the
three-dimensional structure of native molecules. The unfolding changes
the interaction of the protein with aqueous medium and induces aggregation of the unfolded protein molecules. Consequently, the denaturation governs the structure, flavor, texture, and other qualities of food.
It also contributes to the nutritional qualities and physical stability of
the foods during storage.
The heat effects of denaturation and aggregation of proteins are
usually small and have the opposite sign, namely, heat absorption
(endothermic) and release (exothermic), respectively. The key role of
heating in food processing determines a high efficiency of thermal
analysis techniques, in particular, differential scanning calorimetry
(DSC), for food system investigations. Among various DSC methods,
the high-sensitivity differential scanning calorimetry is of most significance. It was developed and substantially evaluated in the USSR
Academy of Sciences and later extensively applied and improved in
many laboratories. Its high sensitivity provides the heat capacity measurements in dilute solutions of proteins and other biopolymers. In the
field of food science, the high-sensitivity DSC was first systematically
applied at the A.N. Nesmeyanov Institute of Organo-Element division
of the USSR Academy of Sciences in the early 1980s (Tolstoguzov
et al. 1985; Tolstoguzov 1988, 1991; Grinberg et al. 1989). A systematic investigation in the field of food protein denaturation was begun
with thermal denaturation of individual proteins under physicochemical conditions typical of food processing (Grinberg et al. 1988, 1989,
2000; Burova et al. 1989a,b; Burova et al. 1991). These conditions are
pH, salt composition (Bikbov et al. 1983; Danilenko et al.
1986a, 1987), the presence of lipids and flavor compounds (Mikheeva
et al. 1998; Burova et al. 1999; Grinberg et al. 2002; Burova et al.
2003), other low-molecular weight compounds (Danilenko et al.
Thermal Analysis of Denaturation and Aggregation
89
1986b), and polysaccharides (Tolstoguzov 1988, 1991; Burova et al.

1992a, 2002b). The applied objective of calorimetry was to elucidate
the mechanisms of structure formation and structure-texture and structure-physical property relationships in foods.
These investigations resulted in (1) general methodological
approaches to study denaturation of proteins; (2) basic information on
the protein behavior upon heating and processing into different kinds
of food; (3) food flavorings; (4) thermodynamic compatibility of
denatured proteins with native proteins and polysaccharides and
phase behavior of macromolecular components in biological and food
systems; and (5) thermodynamic aspects of composition-property relationship in formulated food (Tolstoguzov 1988, 1998, 2000, 2002).
The results have been reported in many reviews and research publications (Tolstoguzov et al. 1985; Tolstoguzov 1988, 1991, 2000, 2002;
Grinberg et al. 1989, 2000; Burova et al. 2003).
This chapter is concentrated on the effects of pH, neutral salts,
alcohols, and polysaccharides on thermal denaturation of food proteins. Another objective of this chapter is to consider the potential of
high-sensitivity DSC for investigation of protein aggregation.
Effects of pH on Thermal Denaturation of Food Proteins

Among food proteins, the seed storage oligomeric proteins and, primarily, 11S globulins that represent the main proteins of most oil and
legume seeds, are of particular importance. The molecule of 11S globulins has a molecular weight of about 300 kDa and consists of six
subunits located in the vertices of a trigonal antiprism. Each subunit
contains two polypeptide chains linked by a disulfide bond. Upon
thermal denaturation, the molecule of 11S globulin behaves as an
ensemble of 12 independent cooperative units (domains). The unfolding mechanism of domains is consistent with the two-state model
(Grinberg et al. 1988). This two-state model can therefore be used to
analyze the denaturation of 11S globulins.
The quaternary structure of 11S globulins depends on pH. The pHinduced changes in the quaternary structure of 11S soybean globulin
(taken as an example) were compared with changes in conformational
stability of the protein (Danilenko et al. 1987).
90
The sedimentation velocity data show that at room temperature

11S globulin undergoes the following structural changes upon decreasing pH from 7.6 to 2.0: 11S (dodecamer) 7S (hexamer) 3S
(dimer).
The thermograms of 11S globulin at different pH values are given
in Figure 5.1a. With deceasing pH, the denaturation heat capacity peak
splits into two peaks that are shifted to lower temperatures. The hightemperature peak decreases, and the low-temperature peak increases;
only the low-temperature peak is observed at pH 3.0. The 11S globulin
converts into the 3S dimer at pH 2.75, and the thermograms do
not display any peaks. Hence, the low-temperature and hightemperature peaks in the bimodal thermograms of 11S globulin can be
assigned to the denaturation of the hexamer and the dodecamer,
respectively.
Figure 5.1b shows that the denaturation temperatures of both forms
of 11S globulin decrease with decreasing pH.
A deconvolution of the thermogram of 11S globulin can be performed in terms of the two-state model if one considers the dodecamer
and the hexamer as ensembles of 12 and 6 identical cooperative units,
respectively. At pH 3.5, it provides values of the denaturation enthalpy
and heat capacity increment for both forms of the protein:
dH7S = 12.2 0.5 J g1, dCp,7S = 0.50 0.08 J g1 K1 and
dH11S = 20.7 1.5 J g1, dCp,7S = 0.46 0.08 J g1 K1.
A difference in the denaturation heat capacity increments does not
seem to be significant. However the denaturation enthalpy of the
dodecamer is significantly larger than that of the hexamer. This difference reflects the dependence of the denaturation enthalpy on temperature. According to Kirchhoff s law, it is a linear function with a slope
Figure 5.1. Thermal denaturation of 11S soybean globulin at different pH values.
(a) Thermograms at different pH values (shifted arbitrary along the heat capacity
axis). Points represent approximation of the thermogram at pH 7.6 by a two-state
model considering the protein molecule as an ensemble of 12 thermodynamically
equivalent domains. (b) Denaturation temperatures of dodecamer and hexamer forms
of the protein versus pH. The insert shows the pH dependences of weight fractions
of the dodecamer and hexamer forms, 11S and 7S, respectively, from velocity sedimentation data. (c) Correlation between values of the denaturation enthalpy and the
denaturation temperature for different forms of the protein according to Kirchhoff s
law (see explanations in the text). (d) Excess denaturation free energy per constituent
chain of the protein versus pH at temperature T0 = 352 K.
91
92
equal to the denaturation heat capacity increment, dCp (Privalov

1979). Figure 5.1c demonstrates a correlation between the denaturation
enthalpy and the denaturation temperature of the different forms of
11S globulin at pH 3.5 and pH 7.6. This dependence is approximated
by a straight line with a slope of 0.46 0.10 J g1 K1. It coincides with
the average value of the denaturation heat capacity increment of 11S
globulin, cCp = 0.46 0.04 J g1 K1. This result reveals a thermodynamic consistency of the denaturation parameters of 11S globulin.
The denaturation parameters of the 11S globulin forms can be used
to calculate their denaturation free energies at a standard temperature
as a function of pH (Prigogine and Defay 1954):
T0
d G (T0 , pH ) = d H ( pH ) 1
+ d C p [T0 Td ( pH )0 ]
Td ( pH )
T0
T0 d C p ln
Td ( pH )
(5.1)
Here, the standard temperature T0 = 352 K is the denaturation temperature at a reference value of pH (pH0 3.5).
The excess denaturation free energy, that is, a general criterion of
pH effects on protein denaturation, can then be determined:
d G E ( pH ) = d G (T0 , pH ) d G (T0 , pH 0 )
(5.2)
Dependences cGE(pH) of the dodecamer and hexamer forms of 11S

globulin seem to be in close agreement and can be approximated by a
single straight line (Figure 5.1d). According to the two-state model
(Ptitsyn and Birstein 1967), a slope of this line is directly linked to a
change in the number of the protein-bound protons upon denaturation,
dH+:
1 d G E
H+
= 2.303 d
RT pH T
(5.3)
In the case of 11S globulin, dH+ 3 per each constituent polypeptide chain. This value is close to the similar estimates for small globular
proteins (Tanford 1968, 1970; Nicoli and Benedek 1976).
The origin of the denaturation proton adsorption by proteins at acid
pH values is well-known (Tanford 1968, 1970). In the native protein
93
molecule, there are often abnormal carboxyl groups with pKa 1.5
(Nicoli and Benedek 1976) localized in the nonpolar interior of the
molecule. Their ionized state is stabilized by hydrogen bonds with
neighboring residues of tyrosine, lysine, or histidine. Several of these
bonds decrease with decreasing pH, which leads to a decrease in the
conformational stability of the protein globule. In the course of the
denaturation, the hydrogen bonds tyrosyl (histidyl)-carboxylate are
broken, and the abnormal carboxyl groups become normal groups with
pKa 3 to 4. They turn to the nonionized state by the adsorption of
protons. Typically, several of the abnormal carboxyl groups in the
protein molecule are rather small; for example, they do not exceed 2
to 3 for some small globular proteins (Nicoli and Benedek 1976).
The considered approach was used to analyze the pH effects on the
stability of a number of other oligomeric and small food proteins, such
as ribulose 1,5 biphosphate carboxylase (RBPC) of alfalfa (Burova et
al. 1989B), tobacco (Burova et al. 1991), and other green leaves; 7S
globulin of French beans (Burova et al. 1989b, 1992); soybean trypsin
Kunitz inhibitor (Varfolomeeva et al. 1989; Burova et al. 1990;
Grinberg et al. 2000); and porcine -lactoglobulin (Burova et al.
2002a).
The 7S globulin of French beans, phaseolin, is an oligomeric storage
protein with the molecular weight of about 150 kDa. It contains three
subunits (Paaren et al. 1987). Each subunit involves two domains
(Lawrence et al. 1990). The thermal denaturation of 7S globulin was
studied in the range of pH 2.0 to 10.9 (Burova et al. 1992). The quaternary structure of the protein is stable within this pH range; however
the denaturation thermogram of phaseolin has a complex profile. In
addition to the main heat capacity peak, there is a lower temperature
shoulder. The thermogram recalculated per polypeptide chain can be
deconvoluted into two independent two-state transitions that represent
unfolding of the domains of phaseolin. Dependences of the denaturation temperature and enthalpy of both domains on pH pass through a
maximum at pH 5.4. The latter corresponds to the isoelectric point of
phaseolin. For both domains, the temperature dependences of the denaturation enthalpies strictly follow the Kirchhoff s law. The excess
denaturation free energies of the domains are maximal in the vicinity
of the isoelectric point of phaseolin. The analysis of the excess denaturation free energies of the domains as a function of pH showed
that there are at least two types of the side-chain hydrogen bonds:
94
tyrosyl-carboxylate and histidyl-carboxylate. There are six bonds in

the high-temperature domain and four in the low-temperature
domain. All hydrogen bonds are presumably localized in the hydrophobic interiors of the domains.
The Kunitz inhibitor (KI) is one of several trypsin inhibitors of
soybean (Kunitz 1947; Koshiyama et al. 1981). It is a small globular
protein with the molecular weight of 21.5 kDa (Wu and Scheraga 1962;
Koide and Ikenaka 1973) belonging to globulins. Its polypeptide chain
consists of 181 amino acid residues and contains two disulfide bonds.
A remarkable feature of this protein is a very low rate of the thermal
denaturation (Kunitz 1948). For example at 45 C, a time of the half
conversion of the denaturation process is about 6 h, that is, of 1 to 2
orders of magnitude longer than that of other globular proteins (Joly
1965). The thermal denaturation of KI was studied in the range of pH
212 (Varfolomeeva et al. 1989; Burova et al. 1990; Grinberg et al.
2000). Due to the slow denaturation rate of KI, the heating rate affects
calorimetric curves within a wide region of pH values. An increase in
the heating rate from 0.01 up to 2.0 K min1 increases the apparent
denaturation temperature by about 20 C without any significant
changes in the denaturation enthalpy and heat capacity increment. It is
important that an increment of the apparent denaturation temperature
induced by pH alteration does not depend on the heating rate. This
allows one to calculate the denaturation free energy of KI as a function
of pH. The dependence of the true thermodynamic denaturation temperature on pH has a rather wide plateau at pH 4.48.0 and rapidly
decreases at both lower and higher pH values. It is important that a
point of the maximal conformational stability of KI (about pH 7) does
not coincide with its isoelectric point (pH 4.5). It means that a contribution of electrostatic effects to the conformational stability of KI is
rather small. The pH dependence of the excess denaturation free energy
of KI shows that the native protein molecule contains two pH-sensitive
side-chain hydrogen bonds of the tyrosyl-carboxylate and lysylcarboxylate types.
Bovine -lactoglobulin is one of the most important milk proteins
(Hambling et al. 1992). Its thermal behavior, including the thermal
denaturation and postdenaturation aggregation, is of significance for
many food technologies (Relkin 1996; Holt 2000). The presence of the
free thiol (Cys121) is a structural feature of bovine -lactoglobulin.
This functional group of high reactivity to thiol-disulfide exchange
95
becomes accessible in the course of the thermal denaturation and

induces the postdenaturation aggregation (Burova et al. 1998). Porcine
-lactoglobulin is a close homolog of bovine -lactoglobulin (66%
identity of their primary structures), but it does not contain a free thiol
group (Hoedemaeker et al. 2002). It was therefore of interest to compare
the thermal denaturation behavior of porcine and bovine -lactoglobulins
(Burova et al. 2002a).
The thermal denaturation of porcine -lactoglobulin is reversible
at pH 210, while that of bovine -lactoglobulin is reversible only
below pH 3.5. This difference supports the assumed postdenaturation
aggregation of -lactoglobulin initiated by the free accessible thiol in
the unfolded protein. The aggregation is responsible for irreversibility
of the thermal denaturation of bovine -lactoglobulin. The denaturation temperature and enthalpy of porcine -lactoglobulin are maximal
at a pH of about 6.5. With increasing or decreasing pH relative to
pH 6.5, both denaturation parameters decrease, and more rapidly in
the acid region. The maximal stability of bovine -lactoglobulin coincides with its isoelectric point (pH 4.5), diminishes upon increase
and decrease in pH values, and does so more rapidly in the alkaline
region. Thus the denaturation parameters of bovine -lactoglobulin
exceed the denaturation parameters of porcine -lactoglobulin in
the acid region and are significantly lower within the alkaline region.
The analysis of pH dependence of the excess denaturation free energy
of both proteins shows their considerable difference in the conformational stability. The latter seems to reflect the different role of
carboxyl groups in the formation of pH-sensitive hydrogen bonds
stabilizing the native proteins. These carboxyl groups are proton
donors in bovine -lactoglobulin and proton acceptors in porcine
-lactoglobulin.
Effects of Salts on Thermal Denaturation of Food Proteins

Normally, the effects of salts on thermal denaturation of food protein
corresponds to the Hoffmeister lyotropic series (Danilenko et al. 1986a;
Yamasaki et al. 1991; Komsa-Penkova et al. 1996; Pico 1996; Kim et
al. 2004). The kosmotropic salts (salting-out salts) increase the denaturation temperature of protein, whereas chaotropic salts (salting-in
salts) decrease it.
96
Effects of neutral salts on the protein thermal denaturation were

studied using 11S globulin of broad beans as an example (Danilenko
et al. 1986A).
Figure 5.2a shows thermograms of the 11S globulin at different
NaCl concentrations. When the salt concentration increases, the denaturation heat capacity peak shifts to higher temperatures, increases in
height, and becomes narrower. Both the denaturation temperature and
enthalpy increase with increase in the salt concentration (Figure 5.2b).
There is a strict linear correlation between values of the denaturation
enthalpy and the denaturation temperature obtained at different salt
concentrations (Figure 5.2c). Its slope, 0.42 0.02 J g1 K1, agrees well
with the denaturation heat capacity increment, 0.40 0.02 J g1 K1,
determined directly in accordance with the Kirchhoff s law.
A general criterion of salt effects on protein denaturation is the
excess denaturation free energy:
s G E (Cs ) = d G (T0 , Cs ) d G (T0 , 0 )
(5.4)
where Cs is the salt concentration and T0 is the reference temperature.

It can be calculated as a function of the salt concentration at the reference temperature by Equation 5.1 using experimental values of the
denaturation temperature, enthalpy, and heat capacity increment. The
excess denaturation free energy of 11S globulin is plotted against the
NaCl concentration in Figure 5.2d. It is positive and increases with
increasing salt concentration. Thus the salt enhances the conformational stability of 11S globulin.
The salt-induced changes in the conformational stability of 11S
globulin are determined by the effect of screening of electrostatic
interactions of surface charges of the native (N) form of the protein
Figure 5.2. Thermal denaturation of 11S broad bean globulin in the presence of
sodium chloride at pH 7.6. (a) Thermograms at the different salt concentrations, Cs.
(b) Denaturation temperature and enthalpy versus the salt concentration. (c) Correlation
between values of the denaturation enthalpy and the denaturation temperature obtained
at the different salt concentrations, according to Kirchhoff s law. The slope of the
correlation line coincides with the denaturation heat capacity increment, dCp, determined directly. (d) Excess denaturation free energy per constituent chain of the
protein versus NaCl concentration at temperature T0 = 352 K. The solid line curve is
calculated by a two-state model, taking into account the electrostatic screening and
lyotropic effects.
97
98
and by the lyotropic effect of the salt on the structure of water. The
excess denaturation free energy can be determined as a sum of contributions of these two effects:
d G E (Cs ) = d GeE (Cs ) + d GsE (Cs )
(5.5)
where d GeE (Cs ) and d GsE (Cs ) are the contributions of the electrostatic and lyotropic effects, respectively. The contribution of the electrostatic effect can be expressed in the Debye-Hckel approximation
(Tanford 1965):
d GeE (Cs ) = RT Be [ F ( ) F ( 0 )]
(5.6)
where
F ( ) =
2 1 + RN
(5.7)
and Be is the electrostatic interaction parameter; n = 12 is the number

of constituent chains of 11S globulin; = (Cs) and 0 = (Cs = 0) are
the values of the Debye-Hckel parameter; RN 5 nm is the molecular
radius of 11S globulin. The contribution of the lyotropic effect can be
considered in the form (Schellman 1978) equivalent to the Setschenow
equation (Setschenow 1889):
d GsE = RT d BsCs
(5.8)
where dBs is the difference in the second virial coefficients of the

unfolded (D) and native (N) forms for salt-protein interactions.
Equations 5.5 to 5.8 describe well the dependences of the excess
denaturation free energy of 11S globulin on salt concentration for
NaCl, KCl and (NH4)2SO4 (for example, see Figure 5.2d). The determined parameters Be and dBs are presented in Table 5.1.
The values of the parameter Be correspond to an apparent 11S globulin charge of 46 proton units per constituent chain. Note that many
globular proteins carry about the same charge in the vicinity of the isoelectric point (Tanford 1965). The parameter Be for (NH4)2SO4 is larger
than that for NaCl. It reflects an increase in the protein charge due to
more strong binding of sulfate anions to the protein (Record et al. 1978).
99
Table 5.1. Salt-protein interaction parameters Be and dBs for 11S globulin
from broad beans.
dBs, L mol1
Salt
NaCl
KCl
(NH4)2SO4
106Be, cm
Experimental
Group contribution
method
MelanderHorvath theory
1.2
1.2
2.3
8.2
8.2
10.6
8.4
8.8
7.8
11.0
According to the obtained dBs values, the salts can form a series
(NH4)2SO4 >> NaCl = KCl in agreement with general features of the
lyotropic effect (von Hippel and Schleich 1969).
It is possible to calculate the parameter dBs by the group contribution method (Nandi and Robinson 1972) and the Melander-Horvath
theory (Melander and Horvath 1977). In both cases, the theoretical
estimates of the parameter dBs of 11S globulin are very close to its
experimental values (Table 5.1).
Effects of Alcohols on Thermal Denaturation of Food Proteins

Effects of alcohols on thermal denaturation of food proteins can be of
importance for food technology, specifically, for control of functional
properties of food proteins (Grozav et al. 1985). Generally, alcohol can
decrease the denaturation temperature and, consequently, the protein
conformational stability (Grozav et al. 1985; Danilenko et al. 1986b;
Stepuro et al. 1991; Cinelli et al. 1997; Grinberg et al. 1998; van
Koningsveld et al. 2002; Michnik 2007). It is, however, noteworthy that
an extrapolation to low temperatures of calorimetric data on thermal
protein denaturation reveals that the presence of low alcohol concentrations can stabilize the native conformation of protein (Danilenko et al.
1986B). This effect can be undoubtedly of interest for food storage.
The most detailed information about alcohol effects on thermal
denaturation of food proteins was obtained for the 11S globulin from
broad beans (Danilenko et al. 1986b).
In aqueous ethanol solutions, the denaturation heat capacity peak of
11S globulin decreases, broadens, and moves to lower temperatures
100
(Figure 5.3a). A new, small high-temperature peak is resolved at the

ethanol concentrations higher than 0.5 M. It can be assumed that this
peak is associated with a molten globule-coil transition, since it has
shown a significant increase in lifetime of the molten globule structure
of proteins in the presence of alcohols (Biringer and Fink 1982; Burova
et al. 2000). The denaturation temperature and enthalpy decrease linearly with the ethanol concentration (Figure 5.3b). The denaturation
heat capacity increment does not depend practically on the ethanol
concentration. Its average value amounts to 0.31 0.02 J g1 K1. The
dependence of the denaturation enthalpy on the denaturation temperature is generally in accordance with the Kirchhoff s law (Figure 5.3c).
Figure 5.3d shows the dependence of the excess denaturation free
energy of 11S globulin on the ethanol concentration. It can be approximated by a linear function that is convenient to represent in a form
equivalent to the Setschenow equation:
d G E = RT d BAC A
(5.9)
where dBA is the difference in the second virial coefficients of the D

and N forms of the protein for alcohol-protein interactions and CA is
the molar ethanol concentration. Here, dBA = 3.46 0.06 L mol1.
Hence, ethanol considerably decreases the conformational stability of
11S globulin in the temperature range, where the thermal denaturation
can be observed.
Figure 5.3. Thermal denaturation of 11S broad bean globulin in the presence
of ethanol at pH 7.6. (a) Thermograms at the different alcohol concentrations, CA.
(b) Denaturation temperature and enthalpy versus the alcohol concentration. (c)
Correlation between values of the denaturation enthalpy and the denaturation temperature obtained at the different alcohol concentrations according to Kirchhoff s law.
The solid line corresponds to the average value of the experimental denaturation heat
capacity increment, dCp = 0.31 0.02 J g1 K1. The dashed lines represent a prediction range of the correlation. (d) Excess denaturation free energy per constituent chain
of the protein versus ethanol concentration at temperature T0 = 352 K. The line is
calculated by a two-state model using the linear approximation of alcohol-protein
interactions in terms of the denaturation increment of the second virial coefficient,
dBA. The insert gives a correlation between values of dBA determined experimentally and those calculated by an additive scheme based on independent group contributions of amino acids for lysozyme, ribonuclease, and 11S broad bean globulin. The
correlation coefficient is more than 0.999.
101
102
Effects of Odorants on Thermal Denaturation of Food Proteins

Binding of odorants to proteins is one of the key factors of food flavoring. Normally, binding of odorant is due to its hydrophobic interactions
with the accessible apolar groups of the protein. Consequently, a close
link should exist between the odorant affinity to the protein and
the conformational state of the protein. Odorant-protein interactions
could therefore be studied using the effect of odorant binding on
thermal denaturation of proteins (Burova et al. 1999, 2003; Grinberg
et al. 2002).
Figure 5.4a shows that vanillin decreases, broadens, and moves the
denaturation heat capacity peak of ovalbumin to lower temperatures
(Grinberg et al. 2002). The denaturation temperature and enthalpy of
the protein decrease linearly with the vanillin concentration (Figure
5.4b). Since the thermal denaturation of ovalbumin is a nonequilibrium
process, its denaturation parameters determined by DSC depend on the
heating rate. The kinetic factor complicates interpretation of the calorimetric data on the ovalbumin thermal denaturation. Nevertheless,
Figure 5.4c shows that there is a good linear correlation between values
of the denaturation enthalpy and the denaturation temperature determined at different heating rates, pH values, and vanillin concentrations.
Its slope is close to the denaturation heat capacity increment of ovalbumin (Sochava and Smirnova 1993). This is a rare example of validity
of the thermodynamic Kirchhoff s law concerning the nonequilibrium
data.
Figure 5.4. Thermal denaturation of ovalbumin in the presence of vanillin. (a)

Thermograms at the different vanillin concentrations, L, at pH 6.7. (b) Denaturation
temperature and enthalpy versus vanillin concentration at pH 6.7. (c) Generalized
correlation between values of the denaturation enthalpy and the denaturation temperature obtained at different vanillin concentrations (075 mM), heating rates (0.125
2.0 K min1), and pH values (pH 3.0 and pH 6.7). The slope of the correlation line,
0.43 0.1 J g1 K1, is close to the denaturation heat capacity increment of ovalbumin
(Sochava and Smirnova 1993). The dashed lines represent a prediction range of the
correlation. (d) Excess denaturation free energy of ovalbumin versus the vanillin
concentration at pH 3.0 (T0 = 333.4 K) and pH 6.7 (T0 = 351.2 K). The solid line
curves are calculated by a model of ligand binding to polymer matrix with independent identical sites at the following binding parameter values: the denaturation increment of the number of binding sites, d = 3.0 0.1 (pH 3.0) and 20.0 0.1 (pH
6.7); the binding constant, Kb = 32.9 1.3 M1 (pH 3.0) and 5.3 0.1 M1 (pH 6.7).
103
104
The Lumry-Eyring model (Lumry and Eyring 1954) can be applied

for estimation of equilibrium values of the denaturation temperature
and enthalpy of ovalbumin at different vanillin concentrations. Using
Equation 5.1, these data can be converted into the dependence of
excess denaturation free energy on the vanillin concentrations, dGE(L)
(Figure 5.4d). According to the model of ligand binding to identical
independent sites, this dependence has a form (Schellman 1975):
d G E = RT d n ln (1 + K b L )
(5.10)
where dn is the denaturation increment of the binding site number;

Kb is the binding constant; L is the free ligand concentration that is
approximately equal to the total ligand concentration at an excess of
the ligand. This equation fits well to the experimental dependence of
the excess denaturation free energy of ovalbumin on the vanillin concentration at the following values of the binding parameters:
dn = 20.0 0.1; Kb = 5.3 0.1 M1 (pH 6.7), and dn = 3.0 0.1;
Kb = 32.9 1.3 M1 (pH 3.0) (Figure 5.4d). A positive value of the
parameter dn means an increase in the number of binding sites caused
by the denaturation and also indicates that vanillin binds preferentially
to the unfolded D form of the protein. The relative low value of this
parameter at pH 3.0 could, however, indicate a noticeable binding of
vanillin to the N form of the protein. This may possibly be due to the
conformational transition of ovalbumin into a molten globule-like state
at the acid pH values (Tatsumi and Hirose 1997). The vanillin binding
to ovalbumin is nonspecific because of the extremely low values of the
binding constants. Apparently, it can be liken to solubilization of
apolar compounds by surfactant micelles.
In contrast to ovalbumin, bovine serum albumin (BSA) binds the
odorants, vanillin, and octanone at pH 6.4 preferentially in the native
state (Burova et al. 2003). Therefore, the denaturation temperature and
enthalpy, and hence the free energy of the protein, increase upon
binding of these ligands. Analysis of the experimental dependence of
the excess denaturation free energy of BSA on the vanillin or octanone
concentration by Equation 5.10 shows that the native form of the
protein carries two to three sites of strong binding of these odorants
with the binding constants of about 103 M1. These estimates agree well
with the results of direct determination of the binding parameters using
the data of equilibrium dialysis.
105
Effects of Polysaccharides on Thermal Denaturation of

Food Proteins
Biopolymers, polysaccharides in particular, are the most widespread
food ingredients that can crucially modify functional properties of
proteins and foods. Accordingly, the effects of polysaccharides on
functional properties of food proteins were intensively studied (Burova
et al. 1992; Delben and Stefancich 1998; Baeza and Pilosof 2002;
Burova et al. 2002b; Zhang et al. 2004; Ibanoglu 2005; Ibanoglu and
Ercelebi 2007). A large variety of partially contradictory data were
described, however. These contradictory data are due to the fact that
the interaction of polysaccharides with food proteins greatly depends
on the polysaccharide nature (charged or neutral) and on the solution
conditions, such as pH, salt, and polymer concentration. For example,
polysaccharides can be either thermodynamically incompatible or able
to form soluble and insoluble complexes with proteins (Grinberg and
Tolstoguzov 1997). We consider effects of polysaccharides on
the conformational stability of proteins under conditions of both
thermodynamic incompatibility and complexation of proteins with
polysaccharides.
The thermal denaturation of 11S globulin from broad beans was
investigated in the presence of various anionic (carboxyl- and sulfatecontaining) and neutral polysaccharides (Burova et al. 1992). It has
been shown that 11S globulin forms polyelectrolyte complexes with
anionic polysaccharides (both carboxyl- and sulfate-containing) at pH
below the protein isoelectric point (pI 4.8). In neutral and weakly basic
medium (pH 7.6) at low salt concentration (0.01 M NaCl) 11S globulin
is incompatible with the neutral and carboxyl-containing polysaccharides. It is however able to form noncooperative complexes with
the sulfate-containing polysaccharides. At higher salt concentration
(0.4 M NaCl), the complexation is suppressed, and 11S globulin
becomes incompatible with the sulfate-containing polysaccharide.
Table 5.2 represents the denaturation temperature of 11S globulin
in the presence of polysaccharides at pH 7.6 and pH 4.2. It is noteworthy that under these conditions the thermograms of 11S globulin alone
have a single denaturation peak. In the presence of polysaccharides,
two different situations are observed. First, the denaturation peak (peak
1) is close to that of free 11S globulin. Second, there is an additional
peak (peak 2) that is shifted to lower temperatures relative to peak 1.
106
Table 5.2. Denaturation temperature of 11S globulin from broad beans (C) in
the presence of polysaccharides.*
pH 7.6; q = 20
Polysaccharide
Dextran
Sodium alginate
Pectin
Carboxymethylcellulose
Methyl cellulose
Arabic gum
Dextran sulfate
-Carrageenan
-Carrageenan
Free protein
0.01 M NaCl
0.4 M NaCl
Peak 1
Peak 2
Peak 1 Peak 2 Peak 1 Peak 2
74.9
75.1
74.3
75.4
74
74.3
76.3
77.9
76.4
76.0
63.0
61.0
64.0
90.3
90.8
93.1
93.0
pH 4.2; q = 1
77.0
59.0
62.0
50.5
* q is the protein/polysaccharide weight ratio.
Table 5.2 shows that the polysaccharides incompatible with 11S globulin (pH 7.6; 0.01 M NaCl), such as dextran, alginate, pectin, methyland carboxymethylcellulose, and Arabic gum, do not affect the thermal
denaturation of the protein. Under the same conditions, the sulfatecontaining polysaccharides can form complexes with 11S globulin.
The result is a destabilized form of 11S globulin coexisting with the
free protein. When the complexation is inhibited by 0.4 M NaCl, the
peak 2 disappears, and only the peak 1, corresponding to the denaturation of the free 11S globulin, remains in the thermogram. These data
imply that the 11S globulin-polysaccharide incompatibility does not
significantly affect the protein unfolding. On the contrary, the 11S
globulin-polysaccharide complexation results in the destabilization of
the protein.
At pH 4.2 11S globulin is able to form cooperative electrostatic
complexes with anionic polysaccharides, both sulfate- and carboxylcontaining. Under these conditions in the presence of alginate, pectin,
and dextran sulfate, the thermograms of 11S globulin have a single
denaturation peak (peak 2) at temperatures well below the denaturation
temperature of the free 11S globulin (Table 5.2). No free protein (peak
107
1) was observed in studied systems. Consequently, 11S globulin bound

to the polysaccharide matrix is substantially destabilized.
The effects of incompatibility and complexation of various polysaccharides on the thermal denaturation and renaturation of a small globular protein from soybean seeds (the Kunitz trypsin inhibitor, KI) were
investigated (Burova et al. 2002b). It was shown that such polysaccharides as dextran, pectin, Arabic gum, dextran sulfate, and - and
-carrageenans do not affect the denaturation parameters of KI under
conditions of the protein-polysaccharide incompatibility (pH 8,
0.1 M NaCl). Variation of the protein/polysaccharide weight ratio from
0.01 to 20 did not change this tendency.
The effects of interpolyelectrolyte complex formation of KI with
anionic polysaccharides (dextran sulfate, pectin) upon the denaturation
of the protein were studied at pH 3.0. Figure 5.5a shows the denaturation thermograms of KI in the presence of dextran sulfate at different
values of the protein/polysaccharide weight ratio, q. With increasing
content of the protein, the denaturation peak is shifted to the lower
temperatures at small q and moves back at higher q.
According to the velocity sedimentation data, the composition of
the system was characterized by two components (Figure 5.5b). The
sedimentation coefficient of component 1 exceeded that of the free KI
(2S). It increased abruptly as the parameter q increased. Obviously,
component 1 corresponded to the KI-dextran sulfate complex. The
sedimentation coefficient of component 2 did not depend significantly
on the parameter q and was approximately equal to 3S. It may correspond either to light complexes of approximately constant composition (i.e., having a relatively low content of the bound protein) or to
the free dextran sulfate that was characterized by the sedimentation
coefficient of 2S. It is important that at high q values free KI could not
be detected in the system. The result implied that the denaturation
transition observed calorimetrically (Figure 5.5a) could be attributed
to the protein bound to the polysaccharide.
The denaturation temperature and enthalpy of KI in complexes
depended on the parameter q (Figure 5.5c). At low protein content, the
denaturation temperature of KI was about 15 C lower than that of the
free protein. When the protein concentration increased, the denaturation temperature of KI raised monotonically and at high protein concentrations reached the value close to the denaturation temperature of
free KI. The denaturation enthalpy of KI in complexes with dextran
108
109
sulfate was lower than that of the free protein in the whole range of q
values and decreased slightly with increasing q values (Figure 5.5c).
Let us analyze possible mechanisms responsible for changes in
conformational stability of a protein bound to a polymer matrix. The
protein stability to denaturation is determined by the free energy of
denaturation, dG, which is the difference between free energies of the
protein in unfolded, GD, and native, GN, form: dG = GD GN. When
N or D forms of the protein are bound to a matrix, the free energy of
each form decreases by a value of the free energy of binding, bGN or
bGD, respectively. Reasonably, the free energy of binding depends on
the number of contacts formed by the protein upon its fixation on the
matrix. As a rule, the D form of a protein possesses a higher number
of accessible binding sites than the N form, consequently bGD << bGN
(the case of a preferential binding of the D form). This situation is the
most probable in complexes with a low protein occupancy on the
matrix (Figure 5.5d). A gradual saturation of the complex with protein
results in a decrease in the number of free binding sites on the polysaccharide matrix (Figure 5.5e). This leads to a decreasing probability of
preferential binding of the D form. In such a densely occupied complex,
the stability of bound protein approaches that of the free protein, but
does not exceed it.
One could expect that thermodynamic incompatibility of proteins
and polysaccharides may result in an increase in the denaturation temperature of protein due to the excluded volume effect (Grinberg and
Tolstoguzov 1997). The minor manifestation of this effect in the case
Figure 5.5. Thermal denaturation of soybean trypsin (Kunitz) inhibitor in the presence of dextran sulfate under the protein-polysaccharide complexation conditions (pH
3.0, ionic strength 0.005). (a) Thermograms at the different protein-polysaccharide
weight ratios, q. (b) Sedimentation coefficients of the components of the proteinpolysaccharide mixtures at the different protein-polysaccharide weight ratios; 1:
heavy protein-polysaccharide complex; 2: light protein-polysaccharide complex.
The dashed line corresponds to the free protein (sw,200 2S). (c) Denaturation
temperature and enthalpy of the protein in the complexes versus the proteinpolysaccharide weight ratio. The dashed lines represent the corresponding denaturation parameters of the free protein. (d and e) Schematic presentation of the
denaturation of a protein (P) bound to a polymer matrix (M) at the loose (d) and dense
(e) protein occupancy. bGN and bGD are free energies of binding of the protein in
the native and denatured states to the matrix. Td is the change in the denaturation
temperature of the protein due to binding.
110
of 11S globulin and KI could probably signify small changes in the

volume of protein molecule upon denaturation. Possibly this is because
the thermally denatured proteins adopt a compact conformation of the
molten-globule type or globule clusters. Their molecular volume is
apparently not substantially larger than that of the native globule.
Effects of the protein-polysaccharide incompatibility on the protein
denaturation could be better pronounced when concentration of the
macromolecules is markedly increased (up to 10% and more). The
convenient DSC method was applied to a concentrated mixed solution
of -lactoglobulin with - and -carrageenans, guar gum, xanthan,
propylene glycol, and alginate at neutral pH (Baeza and Pilosof 2002).
In the presence of the polysaccharides, a slight increase in the denaturation temperature of the protein (of about 23 C) was detected.
Similar results were reported for concentrated mixtures of lactoglobulin with dextran sulfate and -carrageenan (Zhang et al.
2004). For these polysaccharides, an increase in the denaturation temperature was about 4.6 C and 1.2 C, respectively. The results imply
certainly that under conditions of thermodynamic incompatibility of
proteins with polysaccharides, the conformational stability of proteins
does not change significantly.
Postdenaturation Aggregation of Food Proteins
Some qualitative features of the postdenaturation protein aggregation
are especially pronounced in comparative DSC studies of reversible
and irreversible thermal denaturation in concentrated protein solutions
(Tsereteli 1982; Sochava et al. 1985).
When the postdenaturation aggregation is minimal, the protein denaturation is reversible. In this case, the denaturation temperature and
enthalpy as well as the thermogram profile do not practically depend
on the heating rate. On the contrary, the irreversible denaturation is
accompanied by significant aggregation of the protein. In this case, the
denaturation temperature and enthalpy decrease, and the thermograms
are considerably narrowed, with a decrease in the heating rate or an
increase in the protein concentration. The aggregation of protein is
accompanied by heat evolution and is slower than the protein unfolding. A significant difference in the rate of protein unfolding and aggregation permits reducing to zero the heat contribution of protein
aggregation at sufficiently high heating rates.
111
When the rate of protein denaturation is much less than that of the
aggregation of the unfolded protein molecules, the aggregation rate is
determined by the denaturation rate. In this case, the aggregation
process could be considered as a first-order reaction. Under these conditions, information about the aggregation kinetics can be directly
derived from the DSC denaturation thermograms obtained at different
heating rates.
Such an approach was used to study the postdenaturation aggregation of ovalbumin (Weijers et al. 2003). From the denaturation temperature-heating rate dependence (Sanchez-Ruiz et al. 1988) the
activation energy and the aggregation frequency factor were determined. As a result, the aggregation rates were calculated for the temperature range (6787 C), which covers the denaturation heat capacity
peak of the protein in the DSC thermogram. The aggregation constant
of ovalbumin increases by more than 4 orders of magnitude over the
temperature range under investigation.
An attempt was made to extract kinetic parameters of the postdenaturation aggregation of a protein directly from its denaturation thermograms (Remmele et al. 2005). It was suggested that the unfolded form
of protein, U, participates in the aggregation. Its concentration is generally determined by the conformational equilibrium. During the beginning stage of the aggregation, dimers of unfolded protein molecules
(the form D) are mainly formed by two paths of dimerization according
to the mono- and bimolecular mechanisms. An essence of the model
is illustrated by the scheme:
k
k1
D1
k2
D2
U
N
]= D
(5.11)
where D1, D2 are the fraction of protein molecules aggregating by the

mono- and bimolecular mechanisms (D1 + D2 = D); and k1, k2, k3, k4
are the rate constants of denaturation, renaturation, mono- and bimolecular aggregations, respectively. Temperature dependences of the
rate constants are expressed in the spirit of the transition state theory,
but taking into account the activation heat capacity increments. An
expression for excess heat capacity of protein as a function of temperature and heating rate is derived. This expression in combination with
high-performance liquid chromatography (HPLC) data on the aggregation kinetics was used for description of the denaturation thermograms
112
of a small pharmaceutical protein, interleukin-1 receptor (type II), at

different heating rates. The calculated denaturation temperature and
enthalpy coincided with the experimental equilibrium values of these
parameters determined in 2 M urea when the aggregation is completely
suppressed. The activation parameters of the bimolecular aggregation
exceed significantly those of the monomolecular aggregation.
The simplest approach for the application of DSC to study kinetics
of the postdenaturation aggregation of proteins is to estimate the apparent denaturation enthalpy after heating the protein solution at a given
temperature for some time. The protein solution may be considered a
mixture of the native and denatured forms. Because in the DSC experiment the native protein only gives a heat feedback, a relative content
of the native protein can be found from the value of the apparent denaturation enthalpy, as
d Happ ( wN ) = d H wN
(5.12)
where wN is the apparent weight fraction of the native form, and dH

is the specific denaturation enthalpy of the protein. Hence, it is possible
to determine a degree of protein aggregation, wa, for the given preheating time, t (Grinberg et al. 1993):
wa (t ) 1 w (t ) = 1
d Happ (t )
d H
(5.13)
This approach was used by Wang et al.(2006) to study kinetics of

the aggregation of -lactalbumin at 90 C. It was found previously that
the amount of the native protein determined by DSC correlates well
with that obtained by direct HPLC determination. A kinetic curve of
the aggregation wa(t) obtained at t = 225 min could be described by
the first-order reaction equation with the rate constant of about 104 s1.
This result seems to signify that unfolding of the protein is a limiting
stage of the aggregation.
Conclusion
Thermodynamic analysis of the DSC data on thermal denaturation of
food proteins highlighted some key relationships between structure,
113
interactions, and functional properties of the protein systems. Common

tendencies were found in the denaturation mechanism of large
oligomeric multisubunit and small globular proteins. Most of them
unfold in accordance with the two-state model on the level of a structural domain. Conformational stability of food proteins is first of all
affected by pH. Sensitivity of a protein conformation to decrease or
increase in pH is mainly defined by the number of specific side-chain
H bonds between ionogenic groups in protein structure. Neutral lyotropic salts stabilize native protein conformation in result of two main
effectsscreening of electrostatic repulsions and lyotropic action of
salts on the structure of water. Alcohols decrease the protein conformational stability at high temperatures but are able to stabilize proteins
at low temperatures. Interpolyelectrolyte complexation of food proteins with polysaccharides results in reduced stability of protein native
conformation because of the preferential binding of the unfolded
protein form with the polysaccharide matrix. Alternatively, under conditions of thermodynamic incompatibility of these biopolymers the
polysaccharides do not affect significantly the stability of food
proteins.
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Chapter 6
Heat-Induced Phase Transformations of
Protein Solutions and Fat Droplets in Oilin-Water Emulsions: A Thermodynamic
and Kinetic Study
Perla Relkin
Introduction
Heat-Induced Transformations in Protein Solutions
Protein Structures
Thermodynamics of Protein Heat-Induced Transformations
Denaturation-Aggregation of Globular Proteins in Bulk Phase
System
Thermodynamics and Kinetics of Heat-Induced Transformations
Heat-Induced Transformations in Oil-in-Water Emulsions
Crystallization and Melting of Fat Droplets
Kinetics of Fat Droplet Crystallization in Oil-in-Water
Emulsions
Conclusion
References
119
121
121
123
124
129
132
132
136
141
141
Introduction
The thermomechanical treatments applied for food manufacturing
involve batch or continuous heating and cooling steps for mixing, aging,
pasteurization, cooking, or storage. Monitoring the effects of heatinduced transformations in raw ingredients and additives can help to
optimize such food processing or storage conditions in terms of macroscopic properties, quality attributes, and shelf life of the final products.
119
120
Proteins are built from 20 amino acids constituting polypeptide

chains in various spatial arrangements and reactivities that impart
structure functionality in food systems. Besides their nutritional role,
proteins are used for their ability to form macroscopic structures such
as gels or aggregates (Clark and Lee-Tuffnell 1986; Donovan and
Mulvihill 1987; Relkin and Launay 1990; Morr and Ha 1993; Relkin
et al. 1998; Mulvihill and Ennis 2003; Singh and Hevea 2003) or to
stabilize emulsions and foams (Walstra 1988; Dickinson 1992, 1997;
Dalgleish 1996; Sourdet et al. 2002). Particular attention has been paid
to heat sensitivity of proteins and consequences for molecular interactions in bulk phases in relation to denaturation-aggregation mechanisms (De Wit and Klarenbeek 1984; Hagolle, Launay, and Relkin
1998; Galani and Apenten 1999) and on adsorption properties at oilin-water or gas-in-water interfaces in relation to stability of emulsions
and foams (Lefbvre and Relkin 1996; Relkin et al. 1999; Dalgleish,
Van Mourik, and Corredig 1997; Sourdet, Relkin, and Cesar 2003).
The degree to which the initial conformation state of a protein may be
changed is highly dependent on several intrinsic and extrinsic factors.
Structural changes that occur during heating vary with time and temperature attributes of the process and also with protein characteristics,
such as initial conformation state (globular, fibrillar, micellar), concentration, and environmental conditions (ionic strength, pH).
Emulsions used for the preparation of whipped cream or ice cream
are multicomponent and multiphase systems (Pelan et al. 1997;
Bolliger, Goff, and Tharp 2000). Numerous studies showed that formation of fat crystals from liquid emulsions plays a major role in the
stabilization of desired structure-texture and mouth-feel properties of
such complex food emulsions (Barfod et al. 1991; Walstra and van
Beresteyn 1997; Boode, Walstra, and de Groot-Mostert 1993; Abd El
Rahman et al. 1997; Bolliger, Goff, and Tharp 2000; Relkin and
Sourdet 2005; Bazmi, Duquenoy, and Relkin 2007). In complex
systems, fat droplets are stabilized against coalescence-aggregation by
using proteins in combination with small-molecular-weight surfactants
and with gelatin or polysaccharides. Proteins and surfactants are used
for their competitive adsorption properties at the oil-water interface,
whereas gelatin or polysaccharides are used for their structuring or
thickening properties of the continuous aqueous phase.
Differential scanning calorimetry (DSC), in scanning or isothermal
mode, is one of the frequently used techniques to study heat-induced
Heat-Induced Phase Transformations
121
structural changes in food materials (Wright 1984; Ruegg, Morr, and

Blanc 1987; Harwalkar and Ma 1990; Roos 1995; Lrinczi 2004). It
is particularly used for monitoring effects of food composition (nature,
concentration, and physicochemical environment of ingredients and
additives) on the conformation and structure modifications under the
time-temperature combinations relevant to processing conditions.
Several high-sensitivity microcalorimeters are commercially available
(see Chapter 2). Although they differ in their characteristics (temperature and heat flow detection principles, response time, scanning rate,
cell volume), all of them can efficiently be used to receive signals
related to heat-induced transformations in the system being investigated. This chapter summarizes some of our previous reviews obtained
on heat-induced protein denaturation in model solutions (Relkin and
Launay 1990; Relkin 1996, 2004; Relkin et al. 1998, 1999, 2007) and
presents some new results on fat droplet crystallization in oil-in-water
emulsions.
Heat-Induced Transformations in Protein Solutions
Protein Structures
Proteins are of particular concern in a variety of food applications for
their structure-forming properties. Their polypeptide chains are more
or less tightly packed in different spatial arrangements, depending on
the vegetable or animal species from which they are extracted, on the
physicochemical environmental parameters used for extraction, and on
manufacturing processes, including time-temperature parameters. The
amino acid composition of proteins (primary structure) determines
their nutritional value, whereas the higher structural organizations of
the polypeptide chains (secondary, tertiary, quaternary structures) are
related to protein conformational stability, solubility in aqueous
medium, and structure-forming properties. The conformation stability
of proteins results from a balance of attractive and repulsive forces
within the polypeptide chain itself and also between polypeptide amino
acids and cosolvent/cosolute molecules or gas or oil-solution interfaces. Globular proteins, in their native state, are compact particles
with dimensions in the order of magnitude 110 nm. Their polypeptide
chains form secondary structures (-helices, -strands, -sheets formed
between neighboring antiparallel -strands), high-ordered tertiary
122
structures (characterized by a hydrophobic core from which H2O molecules are squeezed and surface-exposed charged amino acids), and
eventually quaternary structures resulting from non-covalent bonding
between monomers. Under the effect of heat treatment, the initial
structure of globular proteins is altered without hydrolysis of primary
covalent bonds. The thermal transition between an initial low-temperature state to a high-temperature state is called denaturation (Privalov
1979; Brandts and Lin 1990; Privalov and Potekin 1996). Compared
with the protein initial state (native protein), the newly created conformational state called denatured is characterized by a lower proportion
of high-order structures and a higher exposure to the solvent environment of hydrophobic groups initially buried in the protein core (Mills
1976; Cooper 1999). In addition, globular proteins possessing disulfide, thiol groups prone to SH/S-S interchange, and intermolecular
disulfide bonds have S-S reactions at neutral or basic pH values, especially in denaturing conditions (Liu, Relkin, and Launay 1994).
Multimeric proteins may dissociate into monomers, before or during
denaturation. In such conditions, thermodynamic laws are not relevant
to the study of heat-induced transformations, which may occur successively or simultaneously with interaction mechanisms between
unfolded proteins themselves or between other solute or surfaces,
contributing to aggregation or gelation, ligand binding, and interfacial
properties (Lefbvre and Relkin 1996).
Gelatin molecules derive from chemical transformation of collagen.
They display extended overall shapes, but likely as for the other polypeptides they present and secondary structures sharing basically
similar conformational change properties under heating and cooling.
But contrary to globular proteins, and likely for linear polysaccharide
chains, gelatin molecules undergo heat-induced reversible-ordered
helix-to-disordered random coil transitions in most of the conditions
used in a variety of food applications (low-fat yogurt or creams,
mousses). Due to their thickening and gelling properties, they are
added to milk proteins to improve the texture and firmness of dairy
products (Fiszman, Lluch, and Salvador 1999).
Caseins, the major milk protein component, is not susceptible to
heat-induced denaturation. When considered as individual molecules,
they are much less compact and organized than other proteins, such as
gelatin (helical secondary structure), or globular proteins (high-order
tertiary structure). However, the degree to which globular proteins
123
(e.g., whey, blood plasma, egg white, soya proteins) or extended proteins (gelatin) can interact with casein is also related to their structural
properties (Haque, Kristjansson, and Kinsella 1987; Kinsella and
Whitehead 1989).
Thermodynamics of Protein Heat-Induced Transformations
The denaturation mechanism of small-molecular-weight globular proteins may occur after a reversible two-state model (Privalov 1979;
Brandts and Lin 1990; Relkin 1996; Cooper 1999):
K eq
N ( native ) U(denatured unfolded )

K eq (T ) =
[U ]
[N ]
GNU (T ) = H NU (T ) T SNU (T ) = RT ln K (T )
(6.1)
(6.2)
(6.3)
where, GNU (T ), H NU (T ), and SNU (T ) are the variations of Gibbs free

energy, of enthalpy, and of entropy, respectively, upon unfolding.
When globular proteins are exposed to denaturing conditions, the
equilibrium constant Keq (T) is shifted to favor the unfolded state. At
T = Tmax, the temperature at which approximately half of the initial
amount of the proteins have altered structures, the equilibrium constant
Keq 1 (Equation 6.2) and the free energy change under denaturation
G (Tmax) 0 (Equation 6.3). Tmax depends on several parameters,
including protein initial state and concentration, pH, ionic strength, and
presence of cosolvent.
In practice, all protein preparations used in food applications are
mixtures of several protein species, and they may contain other solutes
(salt, sugar, traces of polysaccharides) that have effects on the proteins
thermal behavior. In this case, protein heat-induced denaturation in
food systems does not take place after the reversible two-state model,
and other consecutive or successive reactions may be triggered by
unfolding or compete with protein refolding (Lefbvre and Relkin
1996).
In DSC methodology, the reversibility of the denaturation process
is typically monitored by a second heating scan of the sample. If the
second heating thermogram does not show a peak, then the thermal
reaction may proceed according to the scheme in Equation 6.4:
124

K eq
k1
k2
N x ( native ) xD1 xD2 xDi (denatured unfolded state )

aggregated
A(
)
(6.4)
If ki Keq, most of denatured proteins are converted irreversibly into

A species (aggregates) and the thermal behavior of the system is kinetically controlled by the slowest conversion reaction.
Denaturation-Aggregation of Globular Proteins in Bulk Phase
System
In food-manufacturing conditions, the reversibility of the process is
hindered by high protein concentrations and added salts that may
increase protein-protein interactions. Other chemical reactions, such as
deamination of amino acid residues, hydrolysis of peptide bonds, disruption of disulphide bonds, and isomerization of proline residues, may
also hinder the refolding of the polypeptide chain into the native folded
conformation for stereoisomeric reasons (Kinsella and Whitehead
1989). The lack of protein refolding may be related to the loss of solubility and to modification of protein functionality in food products
(Relkin 1996). Over a temperature range between 60 C and 80 C,
protein denaturation is caused by weakening of hydrophilic interactions (hydrogen bonds, van der Waals interactions, electrostatic interactions between charged groups, specific binding) and by strengthening
of hydrophobic interactions. The hydrophobic interactions are exothermic whereas the breaking of the other bonds is endothermic (Relkin
and Launay 1990).
Identification and evaluation of parameters related to conformation
stability and functionality of food components are of great importance
in monitoring effects of manufacturing parameters and particularly in
optimizing food processing and storage conditions for improving
quality of food products. DSC, a noninvasive technique, is particularly
interesting for monitoring protein conformational changes from the
native initial state to another one through the change of one thermodynamic parameter: the temperature (Brandts and Lin 1990, Privalov
and Potekin 1996; Lefvre and Relkin 1996). Commercially available
calorimeters working on the basis of different measuring principles
(power compensation or heat flux calorimeters) determines the heat
flow difference between a sample and reference containers during the
125
heat-induced reactions occurring in the sample material. Calorimetric

parameters associated with a protein thermal transition from the initial
conformation state to another one are extracted from the DSC signal
obtained from the protein solution after subtraction of the baseline
DSC signal. This signal, corresponding to the equipment baseline, is
obtained by using two pans filled with reference materials (buffer for
study of transitions in protein solutions). The transition temperature
mostly used for evaluation of protein denaturation is that of peak
maximum (Tmax) temperature of maximum deviation of the heat flow
signal. For a solution containing one protein, Tmax corresponds to temperature of the maximum rate of the protein reaction, and it is close to
50% denaturation reaction. For a mixture of proteins in solution, Tmax
corresponds to the reaction of the major component, and it can be
preceded or followed by shoulders due to the presence of less or more
conformationally stable proteins.
A DSC study of protein heat-induced transformations was performed
from solutions of a whey protein isolate that was obtained by ultrafiltration of skimmed milk. We used highly sensitive DSC equipment
(micro-DSC III; SETARAM, Caluire, France), working with 800 L
volume of samples and scanning rates ranging from 0.1 C.min1 to
1.2 C.min1, from 20 C to 120 C. The thermograms in Figure 6.1
were obtained at a low heating rate (0.1 C.min1) using a whey protein
isolate that was dispersed in distilled water at protein concentrations
ranging from 2% and 10% at pH 6.6.
The maximum deviation of heat flow corresponds to the denaturation of -lactoglobulin (major whey protein component) and the
shoulder located at T < Tmax corresponds to the denaturation of lactalbumin (25% protein content). The apparent heat of reaction, Qcal,
required for the thermal transition is determined from the area between
the peak and a sample baseline drawn from temperatures corresponding to pre- and post-transitions (maximum amount of proteins in the
initial and final states, respectively) divided by the amount of reacting
materials in the sample pan. Heat-induced transformations in protein
solutions used in food manufacturing occur without a significant shift
between the pre- and post-transition region, and approximation of a
straight baseline drawn by interpolation between the beginning and the
end points of the transition is usually used.
Peak temperature and total enthalpy change of protein solutions
depend on several factors. Changes in heating rate, protein concentra-
Endothermic heat flow, Wg1
126
2%
4%
6%
10%
20
30
40
50
60
70
80
Figure 6.1. Heating curves obtained from solutions of whey proteins at different
concentrations (pH 6.6, 0.1 C.min1).
tion, or other extrinsic factors (pH, added salts or other cosolutes)

could be applied to resolve superimposed phenomena (Relkin 1996).
For example, an exothermic reaction (aggregation) may be superimposed with an endothermic reaction (dissociation of polymeric proteins
to monomers, unfolding process) during the DSC run, as described
below. DSC curves obtained at heating rates ranging from 1 C to
0.1 C.min1 for a solution containing 4.15% protein, 1.2% ash, and
2.5% lactose at pH 6.6 are shown in Figure 6.2.
The shape of the DSC signals reveals one single endothermic peak
for dT/dt < 0.5 C.min1. For higher scan rates, the DSC signals present
a major endothermic peak and a slight exothermic reaction event at a
temperature lower than Tmax, the temperature of maximum deviation
of the endothermic signal. Due to different heat transfer properties,
depending on the heating rate, heat-induced protein transformations
within the sample volume seem to behave differently. The DSC curves
shown in Figure 6.1 were registered from protein solutions at higher
concentrations and a low heating rate (0.1 C.min1), at which thermal
equilibrium within the sample volume could be expected. In these
experimental conditions, heat-induced transformations were appar-
127
Endothermic heat flow
1
20 mWg1
endo
0.75
0.5
0.25
0.1
40
50
60
70
Temperature, C
80
90
Figure 6.2. Heating curves obtained at different scanning rates from a solution of
whey proteins at 4.15% protein concentration (pH 6.6).
ently reflected by one single endothermic signal, and calorimetric

parameters (Tmax and QD) of apparent heat of reaction obtained from
solutions at protein concentrations ranging from 2% to 25% are reported
in Figure 6.3.
As suggested in previous studies (Lefbvre and Relkin 1996), the
decrease in the apparent heat of reaction and the increase in the peak
temperature values with increasing protein concentration may be
assumed to be due to enhancement of hydrophobic interactions, as a
result of simultaneous unfolding (endothermic) reactions of proteins
and protein-protein interactions (exothermic). Considering the simplified scheme represented by Equation 6.4, if k > Keq, most of denatured
proteins can be converted irreversibly into aggregates and the thermal
behavior of the system is kinetically controlled by the rate-limiting
denaturation step reaction. Following this mechanism, the decrease in
QD (apparent heat of reaction) and increase in Tmax (major peak temperature) observed from solutions containing increased dry matter
compositions (of which 2% to 25% protein) could be explained by
increasing values of the equilibrium constant (Keq) and increased con-
128

24
74
Peak temperature, C
72
20
18
70
16
68
14
12
66
10
15
20
% Protein concentration
25
Apparent heat of denaturation, Jg1
22
10
30
Figure 6.3. Variations of peak temperatures (Tmax in C) and heat of reaction (Qcal in
J.g1) obtained from solutions of whey proteins at the indicated concentrations (pH
6.6, 0.1 C.min1).
centration of denatured states (shift to the right of the equilibrium

reaction). Similar trends were observed for globular protein solutions
at a pH close to the protein isoelectric pH or in the presence of added
salts for which the net protein surface charge is either close to zero or
screened by electrolyte opposite charges, respectively (Relkin and
Launay 1990; Relkin 1996; Fiszman, Lluch, and Salvador 1999). In
both of these cases, peak temperature could increase and QD decreases.
Aggregation and denaturation mechanisms involving proteinprotein interactions (exothermic reaction) and breaking down of internal low-energy forces between amino acids (endothermic reaction) can
be superimposed in the same temperature range. This may explain the
overall trends in the calorimetric energy, Qcal, and temperatures, Tmax,
as due to denaturation-aggregation changes during the DSC runs.
Thus, among the heat-induced reactions occurring in globular protein solutions, aggregation and denaturation mechanisms may be
overlapped, leading to either one single endothermic curve at scan
rate < 0.5 C.min1 for protein concentrations up to 25%, or to successive
endothermic and exothermic reactions, which became distinguishable at
higher heating rates. At neutral pH, whey proteins are negatively
charged, and increasing the protein and salt concentrations may favor
129
interaction properties between proteins as they denature during the DSC

run. Thus, dissociation of noncovalently bound protein aggregates and
their unfolding mechanism followed by irreversible aggregation could
explain the increase in peak temperature in parallel with the decrease in
the apparent heat of reaction with increasing protein concentration.
Thermodynamics and Kinetics of Heat-Induced Transformations
If the heat involved in the aggregation step D A is much lower
compared to that of the first denaturation step N D, then the calorimetric heat of reaction could be very close to that of the enthalpy
change value of denaturation. In an earlier study, the activation energy
of protein heat-induced denaturation was determined by using peak
temperature values obtained from thermograms registered at different
scan rates or by using partial completion of DSC reaction as a function
of temperature (Relkin and Launay 1990). The activation energy (kJ.
mol1) of heat-induced denaturation of whey protein varied with protein
concentration (400 < EA < 550 kJ.mol1 for protein concentrations
ranging between 3.5% and 24%). Considering the effects of protein
concentration on Tmax, Qcal, and EA, it was suggested that the reaction
mechanism of denaturation may involve a fast initial step of partial
dissociation-unfolding, followed by a slow interchain hydrophobic
reaction. In another study (Sanchez-Ruiz 1992), the recording of thermograms at different scan rates () and corresponding Tmax values were
applied to the Lumry-Eyring model (Lumry and Eyring 1954) for
evaluation of the activation energy (EA) and pre-exponential factor (Z)
using the following relation:
ZR
EA

= ln
ln
Tmax
E A RTmax
(6.5)
Activation enthalpy (H#) and entropy (S#) were deduced from the
following relations:
H # = E A RT
(6.6)
S #
= ln( Zh ) ln(ekbT )
R
(6.7)
130
where, kB is the Boltzmann constant (1.38 1023 J.K1), T temperature

in K, and h is the Planck constant (6.62 1034 J.s). Plots of ln
(/Tmax) = f(1000/Tmax) shown in Figure 6.4 were obtained by application of Equation 6.5 to Tmax recorded from a protein solution (5.15%;
pH 6.6) using a small volume sample (45 L) and scanning rates
ranging from 15 C.min1 to 2.5 C.min1 (curve a), or using a large
volume sample (750 L) and scanning rates ranging from 1 C.min1
to 0.1 C.min1 (curve b). By using the same protein solution but two
different calorimeters (classical DSC working at high scan rates and
small volume pans or highly-sensitive DSC working at low scan rates
and large-volume vessels), EA (activation energy) values (Table 6.1)
deduced from the slope of the linear parts of these two representations
were very similar. However, the differences between the calorimetric
parameter, Qcal (determined from the surface area under the transition
peak), and the thermodynamic parameter, H# (deduced from Equations
6.5 and 6.6), were very much lower when evaluated from DSC measurements at scanning rates ranging between 0.1 1 C.min1 than
between 2.5 15 C.min1. Following the Lumry-Eyring theory,
3
ln (/Tmax)
8
2.86
2.88
2.9
1000/Tmax
2.92
2.94
Figure 6.4. Lumry-Eyring representation obtained from protein solutions (4.15%,

pH 6.6) at two different ranges of scanning rates: classical DSC working in a high
scan rate range (empty circles), and highly sensitive DSC working in a low scan rate
range (filled circles). See text.
131
Table 6.1. Examples of calorimetric (Tmax, Qcal) and thermodynamic (EA, H#)
parameters, deduced from DSC heating curves obtained from a protein solution
(4.15% concentration, pH 6.6) using two weight samples and two ranges of
scanning rates. Lumry-Eyring theory was applied to DSC curves obtained at
the indicated scan rates, and activation energy values, EA, were calculated from
the linear part of plots in Fig. 5.4 (see text).
Sample
weight
(mg)
750
45
dt/dT
(C.min1)
Tmax
(C)
Qcal
(kJ.mol11)
EA
(kJ.mol11)
H#
(kJ.mol11)
0.1
5
68.2
76.3
283
232
342
345
340
342
these differences could indicate differences in heat-induced conformation transitions and reaction mechanisms, depending on the heating
rate and constant rate of reactions, as suggested previously (Relkin and
Launay 1990; Sanchez-Ruiz 1992; Relkin 2004).
Milk proteins are composed by approximately 80% caseins in
micelle form and 20% whey proteins, of which half are -lactoglobulin.
The DSC curves (1 C.min1) in Figure 6.5 were obtained from solutions in simulated milk ultrafiltrate (SMUF, pH 6.6) of a whey protein
isolate, alone or in mixture with 20% casein or 40% casein at 5.3%
total protein concentration. These curves showed the presence of an
exothermic reaction occurring at T > 80 C. Partial replacement of
whey proteins by 20% or 40% casein micelles gave DSC curves
composed of a major endothermic peak at Tmax, accompanied by an
exothermic effect at T > Tmax.
The intensity of the exothermic event seems to increase, whereas
Tmax seems to decrease with increasing casein-to-whey protein weight
ratio. The lowering of Tmax with increased proportion of casein to whey
proteins could be explained by an increase in the rate of irreversible
aggregation mechanism between casein and unfolded whey proteins.
Upon heating, some of the hydrophilic interactions (hydrogen bonds,
van der Waals interactions, electrostatic interactions between charged
groups, specific binding) are weakened, whereas some of the hydrophobic amino acids (initially buried in the interior core of whey proteins) become more exposed at the surface. In the example of Figure
6.1 (2% protein in water; > 0.5 C.min1), the exothermic signal is
shown at T < Tmax, whereas in the example of Figure 6.5 (5.3% protein
132

Heat flow (mW)
3.5
3
(c)
2.5
(b)
(a)
1.5
1
40
60
80
Temperature (C)
Figure 6.5. Heating curves obtained from solutions of whey proteins, alone (a), or
in mixtures with either 20% casein (b), or 40% casein (c). 5.3% total protein, pH 6.6
in simulated milk ultrafiltrate, 1 C.min1.
in SMUF, = 1 C.min1) it is seen at T > Tmax. This difference may

be due to the presence of lactose, which increases the protein resistance
to heat-induced denaturation (Park and Lund 1984; Ruegg, Morr, and
Blanc 1987).
Heat-Induced Transformations in Oil-in-Water Emulsions

Crystallization and Melting of Fat Droplets
Oil-in-water emulsions are constituted by a dispersing aqueous medium,
oil-water interface, and dispersed fat droplets (Dickinson 1992;
Dalgleish 1996; Walstra 1998). Globular proteins, due to their amphiphilic (polar/nonpolar) nature and their marginal conformational stability, may adsorb from aqueous solutions to solid surfaces and fluid-fluid
interfaces. They act as surfactants by reducing the interfacial tension
and forming a cohesive film (Walstra 1988; Dickinson 1997; Sourdet
et al. 2002). Denatured proteins, compared with native proteins,
which are characterized by a less-ordered structure related to higher
flexibility and surface hydrophobic index (due to a greater exposure to
the aqueous medium of initially buried hydrophobic), were shown to
accommodate easier to oil-solution interfaces.
Monitoring heat-induced transformations of fat droplets in oil-inwater emulsions as a function of their composition is of great technological interest in relevance to their physical stability against coalescence
(Walstra and van Beresteyn 1975; Boode Walstra and de Groot-Mostert
1993; Relkin and Sourdet 2005). In the formulation of many oil-in-
133
water food emulsions, proteins are used in combination with smallmolecular-weight emulsifiers (surfactants) and polysaccharides
(Dickinson 1998). Proteins compete with surfactant molecules for the
adsorption to the oil-water interface, giving appropriate interfacial
properties, whereas polysaccharides are used as thickeners of the
aqueous continuous phase. In addition to these parameters that have
effects on colliding properties of fat droplets and their resistance to
coalescence, crystallization behavior of fat droplets was shown to play
a major role in stability and instability of food emulsions. Several
techniques may be used to study thermal behavior of fat in bulk and
emulsified phases (Dickinson and McClements 1995; Hindle, Povey,
and Smith 2000; Garti and Sato 2001). Because crystallization of fat
releases a large amount of heat, numerous studies were performed
using DSC in nonisothermal and isothermal modes. It was shown that
crystallization temperature of dispersed fat droplets is lowered, compared to bulk fat (Skoda and van den Temple 1963; Walstra and van
Beresteyn 1975; Walstra, Kloeck, Vliet Ton van 2001). In addition to
droplet curvature, supercooling needed to initiate crystallization of fat
globules depends on several other factors, including the origin and
composition of fat, adsorbed materials, and, particularly, added lipophilic or hydrophilic emulsifiers (Dickinson and McClements 1995;
Garti and Sato 2001; Relkin and Sourdet 2005; Relkin et al. 2008).
Besides oil-in-water food emulsions, such as sauce or mayonnaise
where polyunsaturated lipids are used, there are other types of emulsions prepared from anhydrous milk fat (AMF) that are used for fabrication of dairy whipped cream or ice creams. AMF is constituted by
a wide diversity of saturated and unsaturated triacylglycerols (TG),
each characterized by its own melting temperature (Hartel and
Kaylegian 2001). The physical properties of AMF, resulting mainly
from its extraction processing and TG composition, have different
temperature dependency. AMF has broad melting and crystallization
temperature ranges from approximately 40 C to 45 C, and it may
contain more than 50% crystalline fat when stored at 5 C (refrigerator
temperature). Therefore, the manufacturing process of dairy emulsions
consists of successive steps. In the first step, AMF is heated above its
melting temperature (50 C), and lipophilic emulsifiers are dispersed
in the lipid melt. In the second step, this lipid-melt phase is mixed by
stirring with the aqueous phase, which contains water-soluble ingredients (proteins and polysaccharides). In the third step, the premix is
134
passed through a high-pressure homogenizer, and the resulting emulsion is cooled down to a storage or ageing temperature (Relkin, Sourdet,
and Fosseux 2003). Emulsions used to prepare dairy whipped cream
or ice creams are aged at 4 C for a time ranging from 10 to 18 h.
During the aging step, in addition to complete hydration of polysaccharides and partitioning properties of surfactants and proteins between
the oil-in-water interface and the aqueous continuous phase, the behavior of fat droplets and their heat-induced transformations are considered as key factors for the development of desired structures in the
final product (Abd El Rahman et al. 1997; Goff 2002).
DSC is used to study thermal behavior of ingredients (fat, surfactant,
polysaccharide) as a function of processing parameters, and especially
for evaluation of supercooling and kinetics of fat crystallization from
a liquid emulsified phase. Supercooling, needed to initiate fat crystallization from melted systems can be evaluated from the cooling and
re-heating DSC signals registered in a scanning mode. Examples of
DSC curves obtained from AMF in bulk phase (20 mg) or fat droplets
in protein-stabilized emulsions (80 mg) are shown in Figures 6.6 and
6.7. All the samples were heated to 50 C (crystal melting) before
cooling. The curves in Figures 6.6 and 6.7 were obtained from cooling
and reheating cycles at 0.5 C.min1. In the Figure 6.7, besides the DSC
cooling (a) and reheating (b) curves (0.5 C.min1), we present also the
melting curve (c) obtained after cooling to 4 C and holding the emulsion at this temperature for 10 h.
These curves present distinguishable exothermic and endothermic
events corresponding to crystal formation and melting of crystals (or
polymorphs), respectively. Comparison of the temperatures of the
initial scan and scans after cooling and reheating indicated a higher
supercooling in emulsified samples than in bulk fat samples (Table
6.2). The shape of the melting curves obtained by reheating (0.5 C.
min1) just after cooling at the same scan rate was very similar for all
the fat samples (Figures 6.6 and 6.7). However, the shape of the crystallization curves differed depending on the fat sample (bulk- or emulsified-fat sample) and ingredient composition. Melting curves (0.5 C.
min1) obtained after a holding step at 4 C for 10 h applied to bulk
(AMF-0 and AMF-S) or emulsified (E-0 and E-S) fat samples in the
absence or presence of surfactant, respectively, show a broad endothermic curve (Figure 6.7, curve c), with Tmax (maximum peak temperature) located at around 20 C and Qcal (apparent heat of reaction)
Heat flow, mW
AMF
endo
AMF-S
10
20
Temperature, C
30
40
Figure 6.6. Cooling and heating curves (0.5 C.min1) obtained from anhydrous milk
fat alone (AMF) or with 1.75 wt% added surfactant (AMF-S).
Heat flow, mWg1
endo
(c)
(b)
(a)
10
20
Temperature, C
30
40
Figure 6.7. Cooling (a) and first reheating (b) curves, and second reheating curve (c)
observed at 0.5 C.min1 from protein-stabilized AMF emulsion containing added
surfactant (E-S). The second reheating curve (c) was registered after 10 h holding of
the emulsion at 4 C.
135
136
Table 6.2. Calorimetric parameters observed from cooling and heating

thermograms (0.5 C.min1) obtained from anhydrous milk fat sample in the
absence of added surfactant (AMF-0) or in presence of 1.75% surfactant
(AMF-S), from protein-stabilized emulsion in the absence of surfactant (E-0),
and in protein-stabilized emulsion in the presence of surfactant E-S. Tmax values
correspond to temperature of peak maximum observed in the melting curve of
emulsions, which were held at 4 C for 10 h (see text).
Sample
Tcris
(C)
Tend
(C)
Tmax (10 h)
(C)
AMF
AMF-S
E-0
E-S
22.4
19.7
19.9
19.1
38.0
38.0
38.0
35.5
20.6
20.6
21.1
20.2
close to 65 J.g1. This indicates that for both bulk and emulsified fat
samples there was formation of a similar amount of crystalline fat
during the 10-h aging at 4 C.
Application of DSC in isothermal mode (4 C) to the same bulk fat
and emulsions led to observation of a single exothermic heat flow
signal as seen in Figures 6.8 and 6.9. The maximum heat flow deviation of this exothermic peak occurs after different holding times at
4 C. Compared with the bulk AMF-0 sample, this event seemed to
occur after a similar holding period (27 min) in E-S (emulsion with
surfactant). However, it seems to be anticipated for AMF-S (bulk fat
in presence of surfactant) and more delayed (5 min) in the proteinstabilized emulsion without added surfactant.
Kinetics of Fat Droplet Crystallization in Oil-in-Water Emulsions
Analysis of the heat flow pattern involved during the isothermal step
could be used for evaluation of crystal growth characteristics, such as
the induction time and growth rate values. From the determination of
the partial apparent heat of reaction at time t (calculated from the
partial area under the exothermic heat flow), it is possible to obtain the
fractional fat crystallization, X(t), from the following relation:
X (t ) =
A(t )
Qcal
(6.8)
Endothermic heat flow, mWg1
AMF-0
AMF-S
10
40
60
Holding time at 4C, min
80
Figure 6.8. Isothermal curves registered at 4 C from anhydrous milk fat alone
(AMF-0) or with 1.75 wt% added surfactant (AMF-S).
E-0
2
E-S
20
40
60
Holding time at 4C, min
80
Figure 6.9. Isothermal curves registered at 4 C from protein-stabilized emulsion,

without added surfactant (E-0) or with added surfactant (E-S).
137
138
where Qcal is the total calorimetric heat of reaction calculated from the
area under the exothermic peak registered during the holding time.
The mechanism of fat crystal growth can be described by the Avrami
equation (Avrami 1939):
n
X ( t ) = 1 exp ( t )
(6.9)
or can be linearized:
ln [ ln(1 X ( t ))] = ln( ) + n ln( t )
(6.10)
In this expression, represents the nucleation rate of homogeneous

crystallization and n (Avrami index) represents the rate of crystal
growth, with n 3 for a disklike crystal growth mechanism and n 4
for a spherulic crystal growth mechanism (Tore-Vazquez et al. 2002).
Avrami plots obtained by applying Equation 6.10 to DSC data obtained
for AMF-0, E-0, and E-S fat samples under isothermal condition at
4 C are shown in Figure 6.10. They present a linear variation in a short
time region, with different slope values (2.7 < n 4), suggesting different crystal growth mechanisms. Application of the Avrami model
to fat crystallization is valid for a homogeneous mechanism, whereas
noninteger values might suggest heterogeneous and secondary nucleation. Results in Table 6.3 could suggest a spherulic crystal growth
mechanism (n 4) in fat droplets in the protein-stabilized emulsion,
without added surfactant.
The growth of fat crystals with a sigmoidal time variation may also
be modeled using the modified Gompertz equation, as follows (Walstra,
Kloeck, Vliet Ton van 2001):
e
X( t ) = X max exp exp max ( t ind t ) + 1
X max
(6.11)
In this model, Xmax is the asymptotic value of fractional completion of

crystallization, max is the slope at the time when the growth of crystals
becomes exponential (steepest ascent of the sigmoid curve), and tind is
the induction time (intersecting this line with the t-axis). max and tind
are adjustable parameters determined from partial integration of the
heat flow signal registered by the DSC isothermal method.
139
ln (ln (1-TS))
8
1.5
2.5
3
ln (t), min
3.5
4.5
Figure 6.10. Avrami plots obtained from anhydrous milk fat alone (square symbols),
from protein-stabilized emulsion without added surfactant (diamonds), or with added
surfactant (circles).
Table 6.3. Kinetic parameters of fat crystallization at 4 C in anhydrous milk

fat sample (AMF-0), in protein-stabilized emulsion (E-0), without addition of
surfactant, and in protein-stabilized emulsion with added surfactant (E-S). The
kinetic parameters were deduced by application of Gompertz and Avrami
models (see text).
Gompertz model
AMF-0
E-0
E-S
Avrami index
tmax
min
tinduction
min
max
min1
R2
27.2 2.1
32.7 1.2
27.0 3.0
14.2 0.12
22.4 0.04
13.5 0.02
3.48
4.00
3.28
3.461
4.038
2.654
0.999
0.992
0.999
The experimental curve obtained from partial integration of the heat

flow signal as a function of holding time and the sigmoid curve,
obtained by applying Equation 6.11 to X(t), are compared in Figure
6.11. Values of tmax (maximum deviation of the exothermic DSC
signal), n (Avrami index), and Gompertz parameters (values of
induction time, tind, and maximum growth rate, max) are reported in
140

120
50
80
60
X (t)
100
40
20
20
40
60
Holding time at 4C min1
80
0
100
Figure 6.11 Examples of time evolution of X(t) and partial completion of crystallization reaction at 4 C, determined from anhydrous milk fat sample (experimental
values, circles) and its Gompertz representation (sigmoidal dashed curve), as deduced
from the endothermic heat flow signal (see text).
Table 6.3. According to the Gompertz method, AMF in the bulk phase
and in the emulsion containing milk proteins with surfactant (E473)
have very close tmax (27 min) and tind values (14 min), an n value of 3.5
and 2.7, respectively, and an max value of 3.5 min1 and 3.3 min1,
respectively. On the other hand, the protein-stabilized emulsion (E-0)
without added surfactant exhibits a higher value of n 4 (indication
of a spherulic crystal growth) and higher values of tmax, tind (delay in
nucleation), and max (increase in the crystal growth rate).
Emulsification procedure and ingredient complexity have a dominant role in characteristics of fat droplets, such as particle average
diameters and size distributions, composition and physical properties
of surrounding surface layers, and crystalline fat content and polymorphism (Skoda and van den Tempel 1963; Walstra 1975; McClements
et al. 1993; Dickinson and McClements 1995; Kaneko et al.1999;
Hindle, Povey, and Smith 2000; Relkin, Sourdet, and Fosseux 2003;
Relkin and Sourdet 2005). The supercooling effect (temperature needed
to initiate fat crystallization in globules) has been shown to differ
depending on fat composition and mean droplet size of fat droplets and
on the concentration and lipophilic or hydrophilic nature of emulsifiers
141
(Garti and Jano 2001). D50 values (average median diameters of fat
droplets) determined from laser light scattering measurements (Relkin
and Sourdet 2005) were close to 3.1 m for E-0 and E-S emulsions.
However, whereas a similar supercooling was determined by DSC in
scanning mode (Table 6.2), kinetic parameters (tmax, tind, max) and n
(Avrami index) deduced from the DSC isothermal method were different (Table 6.3). These results indicate, as expected from numerous
studies, that hydrophobic surfactant acts as a catalyzer for crystal
nucleation in fat globules, where crystallization mechanism is considered as homogeneous.
Conclusion
DSC has been used for several years to investigate heat-induced conformational or structural changes of a broad range of food ingredients
(biopolymers, proteins, fats, sugars, emulsifiers) in various physicochemical conditions. Most of the previous DSC studies showed the
ability of food ingredients in heat-induced structural or physical state
changes, which are of great importance for manufacturing of food
products with controlled structures. The examples described in this
chapter indicate that combining DSC in nonisothermal and isothermal
methods can provide thermodynamic and kinetic data to contribute to
better understanding and control of structure-forming mechanisms in
food systems.
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Tore-Vazquez J.F., Dibildox-Alvarado E., Charo-Alonso, M.,Herea-Coronado

V.,Gome-Aldapa C.A. 2002. The Avrami index and the fractal dimension in vegetable oil crystallization. J Am Oil Chem Soc, 79:855866.
Walstra P. 1988. The role of proteins in the stabilization of emulsions. In: Gums and
Stabilisers for the Food Industry 4, G.O. Phillips, D.J. Wedlock, P.A. Williams,
editors, pp. 323336. IRL Press: Oxford, England.
Walstra P. and van Beresteyn E.C.H. 1975. Crystallization of milk fat in the emulsified state. Netherlands Milk Dairy J, 29:35-65.
Walstra P., Kloeck W., Vliet Ton van. 2001. Fat crystal network In: Crystallization
Processes in Fats and Lipid Systems, N. Garti and K. Sato, editors, pp. 289. Marcel
Dekker: New York.
Wright D.J. 1984. Thermo-analytical methods in food research. In: Biophysical
Methods in Food Research, H.W.S. Chan editor, p 136. Blackwell Scientific
Publications: Oxford.
Chapter 7
Analysis of Foodborne Bacteria by
Michael H. Tunick, John S. Novak, Darrell O. Bayles,
Jaesung Lee, and Gnl Kaletun
Introduction
C. perfringens and L. monocytogenes Analysis by DSC
Sample Preparations
C. perfringens Results
L. monocytogenes Results
Effect of Antibiotics on Bacteria
E. coli and Lactobacillus plantarum Analysis by DSC
Sample Preparations
E. coli and L. plantarum Results
Application of DSC for Evaluation of Food-Processing Treatments
Determination of Heat Inactivation Parameters of Bacteria from
Calorimetric Data
Determination of Efficacy of Nonthermal Treatments from
Calorimetric Data
Determination of Impact of Antimicrobials on Bacteria from
Calorimetric Data
Conclusions
References
147
149
149
150
152
153
155
156
156
158
158
161
163
164
164
Introduction
The World Health Organization estimates that 325,000 hospitalizations
and 5000 deaths result from foodborne illness in the United States each
147
148
year (WHO 2007). Tens of thousands of cases of foodborne illness in

the United States each year are the result of contamination by
Clostridium perfringens (Mead et al. 1999), a spore-forming anaerobe
that may initiate spore production in response to acidic conditions in
the gastrointestinal tract (Novak, Tunick, and Juneja 2001). Illness due
to Listeria monocytogenes is much less prevalent but far more serious,
leading to 500 deaths in the United States annually (Mead et al. 1999).
These and other foodborne pathogens can be inactivated by heat or
antibiotics, which alter the efficacy of protein synthesis in ribosomes.
Ribosomes, which are organelles found in the cytoplasm of all cells,
assemble amino acids into proteins by using the directions supplied by
messenger RNA molecules (Borman 2007). In bacteria, ribosomes
consist of a small 30S subunit and a large 50S subunit about twice the
size of the smaller subunit, which fit together to form the 70S ribosome. An Escherichia coli cell contains thousands of ribosomes, each
made up of three RNA components and over 50 proteins weighing
2.5 106 Da (Borman 2007). Stressing microorganisms at relatively
high or low temperatures, known as heat shocking or cold shocking,
decreases their thermal tolerance by impairing the 30S subunit
(Stephens and Jones 1993). This decreased thermal tolerance can be
measured by determining the microorganisms D60 value, which is the
length of time required for the viable population to decrease 10-fold
at 60 C.
About 35%40% of the mass of the ribosome consists of proteins,
which are analyzable by differential scanning calorimeter (DSC) if the
sample is sufficiently concentrated. Ribosomal proteins are similar to
many other proteins in that they are irreversibly denatured when heated,
producing an endothermal effect that disappears upon reheating. In
addition to ribosomes, bacterial cells contain other macromolecular
components, such as the cell envelope, nucleic acids, and proteins.
These components in whole cells go through conformational transitions upon exposure to heating in DSC. The transitions are recorded
as endothermic (heat absorption) or exothermic (heat release) peaks in
the thermogram. The area under the peak (enthalpy of transition, H)
and the thermal stability (transition temperature, Tm), of each cellular
component present on a typical DSC thermogram have been used to
characterize bacterial cells.
The first application of DSC on bacterial thermal analysis was the
study on the physical properties of biomembranes. The physical prop-
Analysis of Foodborne Bacteria
149
erties of lipids in cell membranes of Mycoplasma laidlawii were investigated by Steim et al. (1969) with DSC, by using whole cells, cell
membranes, and extracted lipids. DSC thermograms of both isolated
cell membranes and extracted membrane lipids showed an endothermic transition around 40 C, suggesting extraction of lipids did not
change the stability. However, whole-cell thermogram did not exhibit
any distinguishable peaks (Bach and Chapman 1980).
The first successful DSC on whole cells was the study on heat inactivation and spontaneous germination of bacterial spores. Maeda and
colleagues (1974) observed that germinated Bacillus megaterium
spores had endothermic peaks at about 100 C and 130 C. For vegetative cells, Verrips and Kwast (1977) reported eight endothermic peaks
on the whole-cell thermogram of Citrobacter freundii.
It is necessary to obtain distinguishable and reproducible transitions
to identify the origin of the transitions and to examine their stability.
The resolution of peaks can be enhanced by increasing viable cell
density in the sample, by increasing sample size, and by improving the
sensitivity of DSC instrument. Recent studies showed that larger and
more distinguishable peaks can be obtained by using cell pellets instead
of cell suspensions and by using the cells at a late logarithmic growth
stage (Mackey et al. 1991; Lee and Kaletun 2002a,b).
This chapter focuses on the characterization of bacterial inactivation
by using DSC-relevant conditions on precooking, refrigerating, or
high-pressure processing of food to ensure its safety.
C. perfringens and L. monocytogenes Analysis by DSC

DSC was used to examine changes in temperatures of endothermal
effects of ribosomal proteins under cold- and heat-shocked conditions
to determine thermal tolerance of ribosomes in C. perfringens and
L. monocytogenes. In addition, L. monocytogenes cells were exposed
to several antibiotics that bind to ribosomes to mimic cold-shock
responses.
Sample Preparations
Enterotoxin-producing strains of C. perfringens were grown in fluid
thyoglycolate bacteriological medium. The ribosomes from C. perfrin-
150
gens were isolated according to procedure of Novak, Tunick, and

Juneja (2001). Harvested vegetative cells were concentrated by centrifugation and resuspended in buffer consisting of 25 mM Tris (pH
7.5), 1 mM EDTA (pH 7.5), 5 mM -mercaptoethanol, 6 mM MgCl2,
and 30 mM NH4Cl. Cells were broken in a French pressure cell at
82.7 MPa, and DNase was added. Pellets of crude ribosomes were
produced by further centrifugation at 32,500 g.
Whole cells of L. monocytogenes were concentrated by centrifugation and resuspended in buffer consisting of 10 mM Tris (pH 7.5),
6 mM MgCl2, and 30 mM NH4Cl using the procedure of Bayles et al.
(2000). Investigations of antibiotic-treated cultures were performed
after exposing cells to antibiotics for 30 min at 37 C and then centrifuging and resuspending in buffer. Antibiotics used included chloramphenicol, erythromycin, kanamycin, puromycin, rifampin, streptomycin,
and tetracycline (Sigma Chemical Co., St. Louis, MO). Cold shocking
was performed by incubating at the specified temperature for 3 h.
C. perfringens cells and ribosomes were analyzed using a PerkinElmer DSC-7 equipped with an intercooler cooling accessory, and
DSC of L. monocytogenes cells was performed in a Perkin-Elmer Pyris
I with a liquid nitrogen cooler (Perkin-Elmer Corp., Norwalk, CT).
Samples weighing approximately 1220 mg were hermetically sealed
in volatile sample pans, and the appropriate Tris buffer was used as a
reference. After placing the pan in the instrument, C. perfringens
samples were cooled to 10 C and L. monocytogenes samples were
cooled to 0 C. After 2 min, samples were scanned to 100 C at 10 C/
min, and the baseline obtained from scanning the sample a second time
was subtracted, producing the final curve. At least three replicate
analyses of each sample were performed. Peak temperatures were
calculated by using the instruments software. Helium was used as the
flow gas in both instruments, which were regularly calibrated with an
indium standard.
Thermal tolerance studies and determination of D60 values were
conducted by dilution, submerged-coil heating, plating, and enumeration as described previously (Bayles et al. 2000).
C. perfringens Results
A DSC scan of ribosomes isolated from C. perfringens vegetative cells
is shown in Figure 7.1, curve A. There was an endothermal effect with
151
Figure 7.1. DSC of C. perfringens vegetative cells. Curve A, isolated ribosomal

proteins; curve B, whole cells treated at 46 C for 60 min; curve C, whole cells treated
at 28 C for 60 min; curve D, technique for B followed by storage at 4 C for several
days; curve E, technique for C followed by storage at 4 C for several days.
a peak around 72 C, which corresponded to the 50S subunit and 70S

particle (Miles, Mackey, and Parsons 1986). A shoulder at 66 67 C
was due to the 30S subunit (Mackey et al. 1991). The peak and shoulder disappeared with a subsequent scan without a change in baseline,
proving that the ribosomal proteins were denatured by heat. The denaturation peaks of whole cells kept at 46 C (heat-shocked) and 28 C
(control) were several degrees higher (Figure 7.1, curves B and C).
The heat-shocked sample exhibited an increased resistance to heat,
indicating that the structure or conformation of the protein was altered
at elevated temperatures (Novak, Tunick, and Juneja 2001). Additional
endothermal effects were observed around 81 85 C, which have
been attributed to bacterial DNA (Miles, Mackey, and Parsons 1986).
Storage at 4 C for several days, mimicking refrigeration in a
152
supermarket or by a consumer, caused the peaks of both the heatshocked and control samples to flatten and shift to lower temperatures
(Figure 7.1, curves D and E). The increased heat resistance of the heatshocked cells was lost, supporting the theory that this resistance is
transient (Heredia, Labb, and Garca-Alvarado 1998). Heat is uniformly distributed in a cell, resulting in damage to the most sensitive
molecules within it. The results suggest that conformational changes
in ribosomal proteins in response to temperature differences alter
protein synthesis in C. perfringens and that refrigeration will destroy
this organism in food. These conformational changes, which may
involve changing the shape and structure of the protein, are readily
discerned by evaluation of DSC scans.
L. monocytogenes Results
The DSC curve of L. monocytogenes cells (Figure 7.2A) exhibited
melting transitions at 67.5 0.4 C, corresponding to thermal denaturation of the 30S subunit, and at 73.4 0.1 C, corresponding to the
combined 50S subunit and 70S particle (Bayles et al. 2000). Cold
shocking the cells at 0 C for 3 h caused a shift in the 50S/70S peak
denaturation temperature to 72.1 0.5 C (Figure 7.2B). The position
Figure 7.2. DSC of L. monocytogenes cells. Curve A, control grown at 37 C; curve

B, grown at 37 C and cold shocked at 0 C for 3 h.
153
of the 30S peak did not shift significantly. Similar results were observed
with a cold shock to 5 C. Peak shoulders observed around 81 C were
due to bacterial DNA (Miles, Mackey, and Parsons 1986), as with the
Clostridium samples. The results indicate that intracellular changes in
the ribosomes, such as an alteration in the association status of the 70S
particles, are correlated with changes in the thermal properties of L.
monocytogenes. The 30S and 50S subunits are more thermally labile
than the associated 70S particle, so any change that causes dissociation
of 70S would make the ribosome more sensitive to heat (Stephens and
Jones 1993).
Effect of Antibiotics on Bacteria
Certain antibiotics inhibit protein synthesis by selectively targeting
bacterial 70S ribosomes while leaving eukaryotic ribosomes unaffected (Weisblum and Davies 1968). The effects of seven antibiotics,
six active against the ribosome and one (rifampin) active against RNA
polymerase, were tested on the cells to determine whether the antibiotic treatment produced alterations in peak denaturation temperatures
corresponding to ribosomes or their subunits. Figure 7.3, curve A, is
Figure 7.3. DSC of L. monocytogenes cells treated with antibiotic. Curve A, control;
curve B, kanamycin-treated cells; curve C, tetracycline-treated cells.
154
the DSC curve of a control similar to that in Figure 7.2, curve A, and
Figure 7.3, curve B, is the curve of cells treated with kanamycin. The
50S/70S peak shifted from 73.3 0.1 C to 72.1 0.7 C, a shift
that was similar to the temperature reduction in cells that had been cold
shocked (Figure 7.2). Treatment with tetracycline removed the 30S
transition that had been observed around 67 C (Figure 7.3, curve C).
Thus, DSC analysis showed evidence of structural changes in the
ribosomal protein. Treatment with chloramphenicol, erythromycin,
puromycin, rifampin, or streptomycin produced results that were
similar to those of the control.
Cells were also cold shocked from 37 to 0 C for 3 h and then
thermally challenged at 60 C to determine thermal tolerance. Previous
research revealed that the D60 value of L. monocytogenes is 75.6 s
(Miller, Bayles, and Eblen 2000). Kanamycin and tetracycline, which
measurably altered the DSC curves of L. monocytogenes cells, were
the antibiotics that caused reductions in thermal tolerance; chloramphenicol, erythromycin, puromycin, rifampin, and streptomycin did
not alter the D60 values. Compared with the controls, kanamycin and
tetracycline each reduced the D60 value by 20 s. These 26% reductions
were approximately the same as those observed following cold shocks
of 37 0 C (Miller, Bayles, and Eblen 2000) and 37 5 C. The antibiotic treatment data indicate that ribosomal changes have a significant
impact on the thermal resistance of L. monocytogenes. Cold shock and
certain antibiotics alter the state and modify the structure of ribosomes,
as reflected by changes in the DSC curves. The results are probably
due to disassociation of the 30S subunits, which are more thermally
labile and more effectively denatured by heat.
Similar results were observed in Dr. Kaletuns laboratory when
erythromycin-treated E. coli cells were analyzed by DSC (Figure 7.4).
E. coli cells suspended in HEPES buffer were treated with erythromycin for 40 min. With increasing concentration of erythromycin, it
appears that major ribosomal transition shifts to a higher temperature
in comparison with the thermogram of untreated cells. Furthermore,
the shape of the peak changes and becomes less broad. Erythromycin
is known to bind the 50S of bacterial ribosome, blocking the exit of
the growing peptide chain, thus inhibiting the translocation of peptide.
It can be speculated that treatment with erythromycin removes the 50S
transition from the 50S/70S peak observed in the control cell thermo-
155
Figure 7.4. Thermograms of whole cells of E. coli treated with erythromycin, control
(thick dashes), 10 /ml erythromycin (thin dashes), 50 g/ml erythromycin (dots).
gram as one broad peak. The thermogram after a treatment at a 50 g/

ml erythromycin level therefore shows the endothermic transition
being shifted to a higher temperature because it belongs to denaturation
of 70S ribosomes, which is expected to have the highest thermal stability among the ribosomal subunits.
E. coli and Lactobacillus plantarum Analysis by DSC

When microorganisms are heated in DSC, thermograms exhibit a
number of overlapping transitions with a net endothermic effect (Miles,
Mackey, and Parsons 1986; Anderson et al. 1991; Mackey et al. 1991;
Mohacsi-Farkas et al. 1999; Lee and Kaletun 2002a). Mackey et al.
(1991) investigated the origins of apparent individual transitions on the
thermogram of E. coli. Individual peaks observed in thermograms of
whole cells of E. coli were assigned to cell components by comparing
the transition temperatures of isolated cell components with corresponding transitions in whole cells.
156
Sample Preparations
E. coli cells were grown in trypticase soy broth, and Lactobacillus
plantarum cells were grown in MRS broth at 37 C to late exponential
growth phase. The final concentration of cells in the medium was
1.3 0.1 109 cfu ml1 for E. coli and 9.0 0.1 108 cfu ml1 for L.
plantarum. The cells were harvested by centrifugation at 10,000 g for
10 min at 4 C. The supernatant was discarded and the pellets were
washed with sterile distilled water and centrifuged for a second time
before transferring into DSC crucibles.
A differential scanning calorimeter (DSC 111, Setaram, Lyon,
France) was used to record thermograms of microorganisms heated at
a 3 C min1. All DSC measurements were conducted using fluid-tight,
stainless steel crucibles. For each DSC run, the reference crucible was
filled with distilled water equivalent to the water content of the sample.
After heating in the DSC, samples were cooled rapidly by liquid nitrogen and rescanned to evaluate the reversibility of transitions. DSC
thermograms were corrected for differences in the empty crucibles by
subtracting an empty crucible baseline.
E. coli and L. plantarum Results

DSC thermograms for E. coli and L. plantarum whole cells are shown
in Figure 7.5 (Lee and Kaletun 2002a). The peaks on the thermograms
correspond to the thermally induced transitions of cellular components.
Several differences exist between the DSC thermograms of E. coli and
L. plantarum. The major peak, peak a2, shows up at a higher temperature in the E. coli thermogram (70 C) in comparison with the L. plantarum thermogram (63 C). Another visible difference between the E.
coli and L. plantarum thermograms is a high-temperature endothermic
transition (peak d) observed only in the DSC thermogram of E. coli
whole cells. Based on the other DSC studies in Dr. Kaletuns laboratory for Gram-negative (Pseudomonas fluorescens) and Gram-positive
(Staphylococcus aureus and Leuconostoc mesenteroides) bacteria, Lee
and Kaletun (2002a) suggested the origin of this peak is a cellular
component of Gram-negative bacteria, more likely to be due to lipopolysaccharide transition.
Lee and Kaletun (2002a) also evaluated the thermal stabilities and
the reversibility of individual transitions by a second temperature scan
157
Figure 7.5. Thermograms of whole cells of E. coli (dashes) and L. plantarum (dots)
obtained by DSC (1 to 150 C with 3 C min1 heating rate). From Lee and Kaletun
(2002a).
after preheating in the DSC to various temperatures between 40 C and

130 C. They correlated with calorimetric data viability of bacteria
subsequent to a heat treatment between 55 C and 70 C in the DSC.
The fractional viability based on calorimetric data defined as the
reduced apparent enthalpy [(H Hf)/(H0 Hf)] and plate count
data defined as (N/N0) show a linear relationship. Viability loss and the
irreversible change in DSC thermograms of pretreated whole cells are
highly correlated between 55 C and 70 C. Comparison of DSC scans
for isolated ribosomes shows that the thermal stability of ribosomes
from E. coli is greater than the thermal stability of L. plantarum ribosomes, consistent with the greater thermal tolerance of E. coli observed
from viability loss and DSC scans of whole cells. The denaturation of
the ribosomal subunits occurred at the 50 80 C range in both thermograms. The result indicated that the ribosomal denaturation by the
DSC was associated with the 30S and 50S ribosomal subunits in
increasing order of thermal stability. This study demonstrated that
calorimetric data can be used to evaluate the viabilities of microorganisms exposed to thermal treatments. Furthermore, the relative thermal
stabilities of different organisms to heat treatment can be compared.
The calorimetric data also show that the heat denaturation of DNA
might not be a major factor of vegetative cells death because the event
158
is only partially irreversible and requires a higher temperature (85

100 C) than bacterial death (Lee and Kaletun 2002a).
Application of DSC for Evaluation of Food-Processing

Treatments
Food preservation treatments are used to inactivate microorganisms
and to enhance the shelf life of food products. The food industry uses
thermal processing as the main technology for food preservation.
However, alternative thermal processes, nonthermal processes, and
processes using mild heating in conjunction with antimicrobial agents
also have been used to preserve nutritional and textural qualities of
food materials. Preservation treatments affect cellular components of
foodborne microorganisms, resulting in physiological changes in cells
and eventually the death of bacteria. DSC thermograms of whole bacterial cells exhibit differences in thermally induced transitions, revealing the response of bacteria to heat. Thus, DSC technique allows one
to monitor and to detect the impact of thermal treatment on cellular
components of bacterial cells, including ribosomal subunits, nucleic
acids, and cell wall components. The differences in ribosomal thermal
stabilities of various bacteria are shown to be related to the thermal
tolerances of bacterial cells to heat (Lee and Kaletun 2002a; Mackey
et al. 1993; Miles, Mackey, and Parsons 1986). In this section, we will
focus on the quantitative evaluation of cell viability from calorimetric
data and the evaluation of impact of nonthermal treatments using calorimetric data.
Determination of Heat Inactivation Parameters of Bacteria from
Calorimetric Data
The efficacy of a given treatment for inactivation of foodborne pathogenic and spoilage microorganisms depends on the inactivation kinetics
of a target microorganism. In general, bacterial inactivation is considered as a first-order kinetics process. Therefore, the bacterial inactivation kinetics can be described by the D value (the time needed to reduce
the population by 1 log) and z value (temperature change required for a
1-log reduction in D value. The D and z values are determined under
isothermal conditions. However, in industrial applications, processing
159
temperature is reached over a period of time during that a significant

reduction of microbial population may occur as the temperature rises
(Peleg 1999). Therefore, it is important to determine the D and z values
under conditions similar to those used in processing.
There are several studies in the literature modeling microorganism
inactivation during increasing temperature protocols (Reichart 1979;
Thompson et al. 1979a,b; Van Impe et al. 1992). DSC is ideally suited
to achieve heat treatment under controlled conditions of linearly
increasing temperature. Some investigators have used DSC to determine the thermally induced transitions and to evaluate the relationship
between the stability of cellular components and cell injury or death
(Miles, Mackey, and Parsons 1986; Mackey et al. 1988, 1991, 1993).
An equation describing the rate of microorganism inactivation as a
function of linearly increasing temperature was used to determine the
temperature at which the maximum death rate occurred for vegetative
cells (Miles, Mackey, and Parsons 1986) and to predict the number of
surviving microorganisms as a function of temperature at a constant
heating rate (Miles and Mackey 1994). The results demonstrated that
the temperatures required to inactivate L. monocytogenes increased
with the heating rate. Miles and Mackey (1994) stated that the derived
equation can also be used to calculate the D and z values under linearly
increasing temperature protocols.
Lee and Kaletun (2002b) used a novel approach to obtain the
kinetic parameters of E. coli K12 inactivation using calorimetric data.
E. coli pellets were preheated in the DSC to preset temperatures, were
cooled immediately by liquid nitrogen, equilibrated at 1 C, and were
rescanned to 140 C. The rescan contained the thermally induced transitions associated with the bacterial cells surviving after the preheat.
Peak areas (apparent enthalpies, H, J g1) corresponding to the contributions of survivors were determined from the apparent heat capacity versus temperature profile by integrating the area under the curve
(Figure 7.6).
Miles and Mackey (1994) derived a mathematical model describing
the number of surviving cells under linear heating conditions (Equation
7.1).
N
2.303
z
2.303
ln ln =
T + ln
Te
N0
z
Der
z
(7.1)
160

0.75
1.00
1.25
1.50
1.75
2.00
2.25
10
20
30
40
50
60
70
80
90 100 110 120 130
Figure 7.6. DSC thermogram for whole cells of E. coli K12 displaying curve baseline
used to determine the apparent enthalpy value. From Alpas et al. (2003).
where N is the number of survivors at time t, N0 is the initial number

of viable cells, r is the heating rate, and De is the D value at an arbitrary
temperature Te. The value N/N0 represents the fraction of survivors as
a result of heat treatment.
Lee and Kaletun (2002b), assuming that H is proportional to the
number of survivors, wrote Equation 7.2 to describe the fraction of
surviving cells in terms of the DSC observable:
H H f
N
N 0 H 0 H f
(7.2)
By substituting Equation 7.2 into Equation 7.1, Lee and Kaletun

(2002b) obtained an equation that enables one to obtain kinetic parameters of bacterial inactivation from calorimetric data.
z
2.303
H H f 2.303
ln ln
=
T + ln
Te
H 0 H f
z
Der
z
(7.3)
This novel approach demonstrated that calorimetric data obtained

with linearly rising temperature in DSC can be used not only for qualitative evaluation of bacterial inactivation kinetics but also quantitative
evaluation. The D and z values for E. coli K12 determined from the
calorimetric data and the corresponding values from plate count data
161
obtained after heat treatment in the DSC and after isothermal treatment
displayed close agreement. This approach provides reproducible and
accurate results in a short time compared with the plate count technique
because the DSC approach eliminates the incubation time normally
used for plating, which might take 2 days or more.
Determination of Efficacy of Nonthermal Treatments from
Calorimetric Data
There is a growing interest in using techniques alternative to thermal
processing for food preservation to enhance safety and shelf life of
perishable foods (Hoover et al. 1989; Knorr 1993). Among nonthermal
treatment processes, high hydrostatic pressure (HHP) appears to be the
most promising technology. HHP processing has the advantage over
conventional heat treatments in that, while this technique is effective
in inactivation of nonspore-forming microorganisms, substantial food
quality retention can be retained by avoiding the destruction of small
molecular compounds such as vitamins.
It is reported that cell death increases as the level of the pressure
applied increases, implying that critical cellular activities or processes
have been irreversibly damaged (Hoover et al. 1989; Cheftel 1995).
However, the pressure tolerance varies among the species of bacteria
and even among the various strains of the same species (Styles et al.
1991; Patterson et al. 1995; Hauben et al. 1997; Alpas et al. 1999;
Benito et al. 1999).
Although DSC is a thermal analysis technique, it has been applied
to evaluate the impact of HHP processing on inactivation of bacteria
by comparing the pre- and postprocess thermograms (Niven, Miles,
and Mackey 1999; Alpas et al. 2003; Kaletun et al. 2004). The comparison of various final states as a function of various physical and
chemical factors, starting from the same initial state, makes it possible
to use DSC to predict the effectiveness of methods to inactivate
microorganisms.
Niven and colleagues (1999) demonstrated by DSC studies that cell
death due to high-pressure treatment may also be related to irreversible
ribosomal damage. Alpas et al. (2003) confirmed quantitatively that
cell viability decreases as the extent of ribosomal denaturation assessed
by calorimetry increases. The ribosomal denaturation was evaluated
by comparing the total apparent enthalpy of the control and pressure-
162
treated cells and was related to the log reduction in viability.

Furthermore, they demonstrated quantitatively that the relative sensitivities to high hydrostatic pressure treatment of bacterial strains from
E. coli O157:H7 and S. aureus can be assessed from calorimetric data
(Table 7.1). The results showed that pressure and thermal tolerances
of bacteria can be different as can be the mechanism of denaturation.
Table 7.1. Apparent enthalpy and viability data for untreated control and
pressure-treated cells.
Bacteria
S. aureus 485
Control
S. aureus 485
345 MPa
S. aureus 765
Control
S. aureus 765
345 MPa
E. coli
O157:H7
933
Control
E. coli
O157:H7
933
275 MPa
E. coli
O157:H7
931
Control
E. coli
O157:H7
931
275 MPa
Apparent
enthalpy
(J/g wet
weight)
Fractional
reduction in
apparent
enthalpy
(H0
H)/H0
4.0
2.7
0.32
3.8
2.4
0.37
3.7
2.8
0.24
3.7
2.7
From: Alpas et al. 2003
0.27
Viable cells
(cfu/ml)
Log reduction
in viability
log10(N/N0)
1.6 109
5.0 106
2.5
2.0 109
1.6 106
3.1
2.0 109
2.0 107
2.0
1.3 109
6.3 106
2.3
163
Whereas S. aureus 765 had a relatively higher resistance to thermal

treatment in comparison with S. aureus 485, S. aureus 485 was determined to be more resistant to pressure than S. aureus 765. This information can be used in the design of processes specific to targeting
certain cellular components by using different physical stresses.
Determination of Impact of Antimicrobials on Bacteria from
Calorimetric Data
Hurdle technology, which involves mild heating in conjunction with
antimicrobial agents, has been used by the food industry to preserve
nutritional and textural qualities of food while maintaining its extended
shelf life (Leistner 2000). Acids, salt, and ethanol are the most commonly employed preservatives used to reduce the intensity of the heat
treatment (Cameron, Leonard, and Barret 1980; Adams et al. 1989;
Casadei et al. 2001). The effectiveness of hurdle technology can be
enhanced if hurdles target different cellular components, thereby
reducing the tolerance of bacteria to heat treatment and preventing
cellular repair mechanisms during the storage of the food product. DSC
can be used to monitor changes in cellular components induced by
chemical agents in vivo by comparing the thermograms of bacteria
before and after treatment.
Lee and Kaletun (2005) investigated the influence of organic (acetic
acid) and inorganic (hydrochloric acid) acids, ethanol, or NaCl treatment on the cellular components of E. coli by using calorimetry and
compared the calorimetric data with viability results obtained by the
plate count method. All chemical treatments resulted in shifting of
ribosomal denaturation transition to a lower temperature, an indication
of the increasing sensitivity of the bacteria prior to heat treatment. The
comparison of the DSC thermograms of control cells with the thermograms of ethanol or acetic acid-treated cells showed, in addition to
thermal stability decrease, a major reduction in size of the ribosomal
subunit transition peak, which can be interpreted as the lower energy
requirement for denaturation of ribosomes. The observed changes in
the DSC profiles were irreversible and were associated with the loss
of viability assessed by a plate count method. The decrease in thermal
tolerance of the bacterial cell to heat treatment was chemical-specific
and a function of the chemical concentration. The heat sensitivity of
bacterial cells following an acid treatment was observed to be greater
164
than in the cells treated with ethanol and salt. Differences also were
observed in the DSC profiles of bacterial cells treated with organic or
inorganic acid, suggesting that the mechanism of reduced thermal
tolerance of bacterial cells by these acids may be different. For design
of hurdle technology application in food processing, DSC studies in
vivo provide valuable information relevant to the effectiveness of
hurdles.
Conclusions
DSC is a valuable tool when investigating the effect of physical or
chemical treatments applied during food preservation on inactivation
of bacteria. Among the cellular components in a bacterial cell, the
damage to ribosomal proteins due to thermal, nonthermal, chemical,
or antibiotic treatments appears to be related to loss of cell viability.
DSC scans show that protein synthesis in C. perfringens and L. monocytogenes ribosomes is more efficiently destroyed during heating when
conformational changes and disassociation of the 30S subunits are
induced by temperature shocks. DSC thermograms display information
about the cellular components affected by various preservation treatments, thereby providing insight into the mechanism of bacterial inactivation. Furthermore, the calorimetric data can be analyzed to obtain
quantitative information about bacterial inactivation, including thermal
stability, thermal energy required for bacterial inactivation, and the
kinetic parameters of inactivation. Calorimetric data can be used to
optimize the processing conditions of food preservation in a rational
manner.
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Chapter 8
Coupling of Differential Scanning
Calorimetry and X-Ray Diffraction to
Study the Crystallization Properties and
Polymorphism of Triacylglycerols
Christelle Lopez, Daniel J.E. Kalnin, and Michel R. Ollivon*
Introduction
Thermal and Crystallographic Properties of Triacylglycerols
X-Ray Diffraction
Coupling of XRD and DSC: MICROCALIX
Applications and Results
Cocoa Butter and Its Components
Milk Fat
Lard
Conclusion
References
169
170
170
173
175
176
179
179
184
190
193
194
Introduction
Lipids from vegetable or animal origins are widely consumed in food
products, for example, in chocolate, shortenings, margarine, and butter.
The composition of triacylglycerols (TG), which are the main constituents of natural fats and oils or hydrogenated and interesterified fats
(i.e., such as in margarine), their suprastructure, and the physical prop*This chapter is dedicated to Michel Ollivon who passed away on June 16th, 2007.
169
170
erties of fat determine the mouth-feel, flavor release, and functional

properties of high-fat content food products. Moreover, the lipid phase
of food stuff is sometimes partially crystallized at the temperature of
storage (in a freezer or fridge at 20 and 4 7 C, respectively) and
consumption, including cocoa butter in chocolate, hydrogenated margarine, and milk fat in dairy products.
Studying the properties of TG is important to better understand and
then control the physical properties of fats. Increasing the knowledge
of both the physical and thermal properties of fats (i.e., solid fat content
and type of crystals as a function of temperature) in anhydrous state
as well as in situ in food products is of tremendous importance with
respect to functional, sensorial, and nutritional properties. Moreover,
the increased knowledge of TG crystallization and polymorphism in
fats is of value for technical applications as well as for the development
of new processes and products.
The polymorphism of TG renders the study of the thermal and
structural properties of lipids very complex. Both types of properties,
largely depending on sample history, are conveniently determined
using differential scanning calorimetry (DSC) and X-ray diffraction
(XRD) techniques. In this chapter, we focus on the thermal and crystallographic properties of TG investigated by these two techniques, which
are coupled using the microcalorimeter MICROCALIX.
Thermal and Crystallographic Properties of Triacylglycerols
Fatty acids have various melting points, which mainly depend on the
number of carbon atoms and their level of unsaturation (Table 8.1).
The melting point of a TG molecule (triester of fatty acids and glycerol) depends on the three fatty acids esterified and on their position
on the glycerol (sn-1, sn-2, sn-3) (Table 8.1).
Thus, the thermal behavior of natural fats, constituted by several
types of TG molecules, is really complex. Moreover, the assignment
of the thermal properties of fats is complicated by the existence of a
polymorphism of monotropic type for each TG (Small 1986; Ollivon
and Perron 1992).
Each TG can exhibit several crystalline forms, the occurrence of
which strongly depends on its thermal history. Each polymorphic form
171
Table 8.1. Melting point of the main fatty acids found in natural fats and oils
and of triacylglycerols.
Fatty Acids
Formula
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1 9c
C18:2 9c, 12c
C18:3 9c,
12c, 15c
Triacylglycerols
Name
(Abbreviation)
Melting
Point (C)
TG
Abbreviation
Melting
Point (C)
Butyric acid
(B)
Caproic acid
Caprylic acid
Capric acid
Lauric acid
(L)
Myristic acid
(M)
Palmitic acid
(P)
Stearic acid
(St)
Oleic acid (O)
Linoleic acid
Linolenic acid
BBB
75
4
16
31
44
OOO
StOO
StStO
StOSt
5
24
38
44
54
PPO
34
63
POP
36
70
LLL
47
16
5
14
MMM
PPP
StStSt
58
66
73
of a given TG molecule is characterized by its own melting point

(Table 8.2). Then, TG mixtures exhibit multiple melting points depending on their composition, which makes the overall melting behavior of
fat even more complex because some TG can cocrystallize.
TG polymorphism relates to the ability of molecules to arrange
themselves within a crystal lattice in several different ways of lateral
packing of the fatty acid chains (Figure 8.1B) and of longitudinal stacking of molecules (Figure 8.1B) in lamellar structures (Hagemann
1988). Thus, pure TG and mixtures of TG can adopt several crystalline
arrangements. TG molecules have a polymorphism of monotropic
type, which means that the transitions between the polymorphic forms
are irreversible and are only possible from the least to the most stable
species as characterized by a higher melting point. Moreover, polymorphic transitions are only possible by way of a liquid phase (Small
1986). The three main polymorphic forms frequently observed for the
lateral packing of fatty acid chains correspond to different subcells that
172
Table 8.2. Some crystallographic and energetic properties of the three main
polymorphic forms of a selection of TGs.
Polymorphic Form
Property of TG*
Main short
spacings ()
Melting point
(C) of StStSt
Enthalpy of
fusion (J g1) of
StStSt
Melting point
(C) of OOO
Melting point
(C) of LLL
Melting point
(C) of POP
Hexagonal
4.15
Orthorhombic
Perpendicular
Triclinic Parallel
3.8 and 4.2
4.6
55
64
72
163
180
230
32
12
15
35
46
21
30
36
*TG, triacylglycerols; St, stearic acid; O, oleic acid; L, lauric acid; P, palmitic acid.
Adapted from Mulder and Walstra 1974.
have been described in detail (Small 1986; Ollivon and Perron 1992):
hexagonal ( form), orthorhombic perpendicular ( form), and triclinic parallel ( form) (Figure 8.1B). The density, enthalpy of fusion,
melting point, and stability increase in the order , , and , according
to the monotropic character of the polymorphism. In the form, the
lateral packing of the fatty acid chains is not very tight and the chains
have considerable rotational freedom, whereas in the form, the chains
are very densely packed. TG crystals are made by the stacking of TG
molecules layers, the thickness of which depends on the length and
unsaturation of the fatty acid chains and their angle of tilt with respect
to the basal planes formed by the methyl end groups of the TG (Figure
8.1C). The longitudinal organization of TG in lamellar structures is
primarily related to the number of chains stacked in the crystalline cell.
For TG in natural fats, the number of fatty acid chains frequently
observed is two or three and corresponds to the stacking of double
(2L)- or triple (3L)-chain length lamellar structures (Small 1986;
Hagemann 1988). Roughly, 3L forms are usually related to lowmelting, long-chain monounsaturated and mixed long- and short-chain
173
Figure 8.1. Main types of triacylglycerol (TG) packings. (A) Lamellar structure
formed by TG molecules in the solid state: for example, form of trilaurin. (B) Left:
The stable conformation of the hydrocarbon chains of saturated fatty acid (FA) is a
planar zigzag shown here as a 3D view along its main axis. Right: Three main types
of lateral chain packings (only carbon atoms are drawn): hexagonal, orthorhombic
perpendicular (O + upside down T), and triclinic parallel (T//) subcell in the order of
their stability, which are named , , and for TG. (C) Two main types of TG
longitudinal chain stacking (fatty acids are drawn as straight lines), 2L and 3L.
TG, whereas 2L forms are generated mostly by similar long-chain,

high-melting, trisaturated TG (Small 1986).
The techniques most frequently used for the study of the thermal
and crystallographic properties of TG are DSC and XRD.
The thermal properties of fats are generally studied using DSC. In
DSC, the difference between the heat flow (J/s or W) of a reference and
a sample is measured as a function of temperature or time while they
are subjected to a controlled temperaturetime program. DSC is thus a
form of differential thermal analysis (DTA). In any DSC instrument,
the sample and reference are placed in small individual pans or cruci-
174
bles, which may be opened or hermetically sealed, of 10100 l capacity. Because sample size is small, accurate weighing is essential for
quantification of the thermal properties of fats. The reference is often
an empty pan, so as to not exhibit thermally induced transitions within
the temperature range of interest. Transitions that occur in the sample
during the applied temperature program appear as peaks or troughs,
depending on whether they are exothermic or endothermic, on the plot
of differential heat flow versus temperature or time (the thermogram)
that is the output of the instrument. Conventionally, the temperature
program used in DSC is a linear change in temperature with time, with
various cooling and heating rates. After heating a lipid sample to 20 C
over its final melting point to erase its thermal history/memory (Ollivon
and Perron 1992), different rates of cooling permit the investigation of
the crystallization properties of TG, polymorph formation, and transformation. Cooling and heating can also be performed from a temperature at which the fat is partially crystallized (e.g., 4 C for milk fat).
Isothermal DSC, in which the temperature is kept constant at a value at
which a transition of interest is known to occur, is especially useful for
studying the polymorphic evolutions as a function of time (Lopez et al.
2002a). The crystallization properties of fats depend on their thermal
history and on the rates of cooling and heating applied. A combination
of cooling, heating, and isothermal DSC scans can be used to study
polymorphism of TG and fats in complex products.
DSC is a useful tool (1) to record the crystallization and melting
profiles recorded on cooling and heating, respectively; (2) to determine
the characteristic temperatures, such as the temperatures of initial
crystallization (Tonset) and final melting (Toffset); (3) to monitor polymorphic evolutions and measure the heat of transitions; and (4) to quantify
the solid fat content that is proportional to the enthalpy of melting (H)
of fat.
The effects of different rates of cooling and heating on polymorph
formation and polymorphic transitions recorded as a function of temperature or time are easily studied by DSC. DSC studies have given
an insight into the thermodynamics of fat phase transition in bulk and
in emulsions. However, because the complex DSC recordings are often
difficult to interpret and it is not possible to identify unequivocally
polymorphs using DSC, the experiments must be coupled with other
techniques, such as XRD or other techniques yielding structural information. Moreover, DSC allows the characterization of the physical
175
state changes if a change of energy is involved. However, this technique does not provide information on the structure that exists before
and after the phase transition.
X-Ray Diffraction
XRD is a powerful technique to use to provide structural information.
As explained above, characteristic lengths of structures formed by TG
range from atomic distances up to hundreds of ngstroms. The whole
size range can be investigated by means of X-ray scattering. Generally,
X-ray scattering reflects periodical differences of the electron density
within a sample. Thus, X-rays are the ideal direct probe for determining the internal structure of crystalline material because they provide
information on the repetitive patterns of the electron density of the
array of atoms. In their solid state, TG molecules are arranged periodically in planes at repetitive distance d, which can be identified using
XRD (Figure 8.1A).
Wide-angle X-ray scattering (WAXS) monitors the structure at
atomic scale (from about 1 to 10 ). It provides information on intraand intermolecular distances called short spacings. For crystallized
TG, the hydrocarbon chains are arranged in regularly spaced planes.
Crystallographic planes give rise to a reflection line at a distinct angle
, satisfying the well-known Bragg relation: 2d sin = n, where is
the X-ray wavelength, d is the repetitive distance between planes, n is
an integer, and is half the angle between the incident and diffracted
beam (Guinier 1964; Small 1986).
The actual data registered during an XRD experiment is the scattered intensity as a function of 2. It is often convenient to use the
scattering vector q instead of the scattering angle 2 because the former
is independent of the wavelength of the incident beam. They are related
as follows: q = (4/) sin . Thus, the distance d between planes can
be deduced from the position q of the diffraction peak by d = 2/q.
The different packing of aliphatic chains in the three crystalline TG
subcells leads to characteristic wide-angle X-ray reflections enabling
their identification. Short spacings are widely used for identifying the
various crystalline subcells characterizing the polymorphic forms
(Figure 8.1B). A single line around 4.15 characterizes the form
(hexagonal subcell). A strong line at 4.6 and two small lines at about
3.85 and 3.7 identifies the form (triclinic parallel subcell), whereas
176
the form (orthorhombic subcell) pattern exhibits two lines at about

4.2 and 3.8 .
Small-angle X-ray scattering (SAXS) monitors the lamellar organization (e.g., 2L or 3L) of a TG sample in a range from 10 to about
1000 . Reflection maxima appear in the scattering profile. The Bragg
relation applies again, where d yields the mean thickness of adjacent
lamellae (called TG long spacing). From the measurement of d () and
with the knowledge of the fatty acid composition (chain length, unsaturation), it is then possible to deduce if the stackings correspond to 2L
or 3L organization (Figure 8.1C).
In conclusion, the two levels of organization of crystallized TG, for
example, the lateral packing of the fatty acid chains and the longitudinal stacking of TG molecules in lamellae, are easily identifiable from
the short and long spacings observed by X-ray scattering at wide and
small angles, respectively.
Recent use of synchrotron radiation, which provides X-ray flux
103106 times more intense than that generated by usual X-ray sources,
permits recordings to be performed in times ranging from a few milliseconds to seconds. Thus, direct continuous recordings can be
achieved as a function of time (XRDt) or temperature (XRDT).
Moreover, synchrotron radiation permits studying the organization of
TG in water-dispersed systems such as emulsions and complex food
products and to quantitatively monitor phase changes within emulsion
droplets. This is especially interesting when relating the textural and
rheological properties of fats and high-fat food products to the thermal
and crystallographic properties of TG. Therefore, the functionality of
fat in many food products cannot be understood without knowing both
its composition and physical properties their dependencies.
Coupling of XRD and DSC: MICROCALIX

A new differential microcalorimeter, called MICROCALIX, has been
developed within Centre National de la Recherche Scientifique (CNRS)
group UMR 8612 (Ollivon et al. 2006) to perform simultaneous thermal
and X-ray measurements (Figure 8.2). Small volumes of samples (from
about 1 to 20 l) are loaded in glass or quartz capillaries (external
diameter 1.5 mm) ensuring minimum attenuation of the X-ray beam
and parasitic scattering. The microcalorimeter allows, in its last version,
177
Figure 8.2. Experimental setup of the microcalorimeter MICROCALIX in the timeresolved synchrotron XRD environment. (A) Schematic representation: The cell is
positioned with the capillary containing the sample perpendicular to the beam in such
a way that the diffraction patterns are recorded in the vertical plane by one or two
one-dimensional proportional detectors (LD) at small and wide angles. Counting
electronic (Counting Elect.), nanovoltmeter (nVmeter), and temperature controller
(T Ctrl) are monitored by a single computer. The temperature-controlled cryostat
(TCC) is kept at constant temperature (e.g., 6 C). (B) Setup on the D22 bench
of synchrotron (LURE, Orsay, France). (C) Setup on the D24 bench of synchrotron
(LURE, Orsay, France).
thermal scans in the temperature range 30 C to +230 C, with scanning rates between 0.01 and 10 C/min and with sensitivity comparable with that of a modern commercial apparatus (>100 V/mW).
Scanning temperature is controlled with a resolution of 0.01 C, and
the microcalorimeter is calibrated with lauric acid.
MICROCALIX was used on synchrotron radiation X-ray benches.
The lastest version of the instrument has been adapted for laboratory
bench and conventional source, but it is preferably used with rotating
anode or multilayered mirrors.
MICROCALIX inserted in a laboratory conventional X-ray source
The microcalorimeter can be installed on a laboratory source designed
for simultaneous SAXS and WAXS measurements, such as that of
CNRS group at UMR 8612 in Chtenay-Malabry (France; Lopez et al.
178
2008). The X-ray source of this setup is a Diffractis 586 generator

(ENRAF-NONIUS) equipped with a long-fine-focus, Cu anode, sealed
tube operated at 40 kV and 20 mA. CuK ( = 1.54 ) radiation is
selected and the line focused by a graded, elliptically bent, multilayer
mirror (OSMIC-Rigaku, Troy, Michigan). SAXS and WAXS patterns
are recorded by two linear, position-sensitive, gas detectors using
ASA2.4 software (HECUS-Braun, Graz, Austria). The detector recording SAXS data is placed at the focus point of the mirror. The scattered
intensity is reported as a function of the scattering vector q = 4 sin /,
where is half the scattering angle and the wavelength. The detectors
are calibrated at wide angles with the crystalline form of high-purity
tristearin (characteristic repeat spacing 4.59, 3.85, 3.70 0.01 )
(Ollivon and Perron 1992) and at small angles with silver behenate
(long spacing of 58.380 0.001 ) (Blanton et al. 2000). At small
angles, this instrument covers the range of scattering vectors
0.05 < q < 0.4 1 and is thus well suited for measurements on lipids.
Line focusing of the beam increases the flux on the sample in such a
way that measuring time can be reduced to a few minutes (about
2 min), allowing study of lipid phase transition kinetics and mechanism
at a rather low scanning rate.
MICROCALIX inserted in synchrotron radiation XRD bench
The availability of a synchrotron radiation source with a brilliant beam
of variable wavelength with very small vertical angular divergence has
opened new opportunities. The high collimation of the beam allows
the investigation of much larger structural features, with better spatial
resolution. Increase in the flux by several orders of magnitude, compared to flux of conventional sources, enables the study of very dilute
or weakly scattering samples such as TG emulsions (Lopez et al. 2007)
and aerated food products (Kalnin et al. 2002). For bulk TG samples,
time-resolved measurements can be performed with a time resolution
down to a few milliseconds; thus, possible intermediate states in the
course of a transition can be assessed (Lopez et al. 2006b).
Generally, the timescale that may be reached during a time-resolved
experiment depends on instrumental factors such as source and detector characteristics and sample properties. For conventional sources, the
time resolution is usually determined by the flux at the sample, whereas
for synchrotron radiation source it is rather limited by the counting rate
of detectors, because statistically significant information must be col-
179
lected. Note that the time resolution is also limited by the heat conductivity of the sample. If the high flux of synchrotron radiation source
is used to follow very fast transitions, very good heat transfer to a small
sample must be ensured.
Experiments using MICROCALIX have been carried out at three
synchrotron radiation sources, Laboratoire pour lUtilisation du
Rayonnement Electromagntique (Orsay, France), European
Synchrotron Radiation Facility (Grenoble, France), and Elettra (Trieste,
Italy); further experiments are planned at SOLEIL (Saclay, France).
Applications and Results

Examples of the study of crystallization properties and polymorphism
of TG in natural fats and complex food products are presented below
as major applications of the coupling of DSC with XRD.
Cocoa Butter and Its Components
Polymorphism of cocoa butter (CB), which is a vegetable fat used
mainly by chocolate manufacturers, has often been discussed in the
literature because it is related to the organoleptic and physical characteristics of the final products (snap, molding contraction, gloss, and
blooming during the storage). In fact, the quality of chocolate bars and
pralines strongly depends on their physicochemical properties and on
the polymorphic form of CB.
The polymorphism of CB can be compared with that of the main
TG, POP, POSt and StOSt (where P is palmitic acid, O is oleic acid,
St is stearic acid), which represent about 17%, 37%, and 27% of CB
composition, respectively, and that of their mixtures (Wille and Lutton
1966; Kunutsor and Ollivon 1983; Sato 1987; Sato et al. 1989; Arishima
et al. 1991; Loisel et al. 1998a). CB polymorphism is commonly
described in the literature in terms of six different polymorphic forms,
noted as forms I to VI in Figure 8.3 (which are in fact sub-, , ,
and forms) in increasing order of melting points, according to the
nomenclature of Wille and Lutton (1966). These six forms have been
confirmed by other authors (Loisel et al. 1998a; Chapman et al. 1971;
Adenier et al. 1975; Huyghebaert and Hendrickx 1971; Lovegren
et al. 1976; Merken and Vaeck 1980; Davis and Dimick 1986). However,
180
Figure 8.3. Summary of the possible arrangements of cocoa butter. Upper left shows
a table resuming the notation, melting point, lateral and longitudinal packing of TGs.
An overlay of two DSC recordings shows the closeness of two thermal events, notably
the occurrence for V and form VI. Underneath is the XRD pattern of what is believed
to be the pure crystalline species of forms V to VI.
the existence of some of them is debated, as well as the fact of the

purity of some forms, especially I, III, and VI. In fact, it is very hard
to obtain monocrystalline samples; rather, one obtains polycrystalline
powders from pure TG as a solid state (Van Malssen 1994). Thus, it
should be considered that complex mixtures of TG will occur either at
a molecular level. It also has to be considered that lipid crystals of
sub-, , and do not necessarily correspond to one homogenous
crystalline state (Marangoni and McGauley 2003). Lipid crystals can
be seeded to achieve the commercially desired form V (Figure 8.3)
(Davis and Dimick 1989a,b; Chaiseri and Dimick 1995a,b). However,
it is apparent from Figure 8.3 that this crystalline form is not the
most stable one, and it is necessary to avoid polymorphic transition
toward the more stable form VI, which is responsible for fat bloom.
This can be achieved by the addition of minor components such as
glycolipids, phospholipids, and saturated TG, which promote the
181
crystallization of CB. However, good practice has to be used during

tempering since seeding can also hinder formation of the desired form
V of CB.
To better understand the phase behavior of CB, a MICROCALIX
calorimeter has been used, together with synchrotron radiation, for the
characterization of coexisting organizations resulting from the phase
separations of TG and to follow the competition between the different
polymorphic species quantitatively, even at fast scanning rates.
Cocoa butter polymorphism
Using temperature-resolved XRD with MICROCALIX, it has
been shown that phase separation systematically occurs during CB
crystallization (Loisel et al. 1998). A trisaturated fraction of TG partially phase-separates from the mono- and polyunsaturated TGs by
crystallizing first on cooling. Segregation of TG molecules in the solid
state has been observed when CB crystallizes under the forms II and
V (the form under which chocolate is usually commercialized). It
also can occur with some other forms, such as VI (Loisel et al. 1998a).
This behavior results from the poor solubility of trisaturated TG within
the monounsaturated ones. The use of MICROCALIX for the monitoring of the formation and then transformation of the different forms,
including form III, during heating of CB confirmed unambiguously
the results of phase transitions previously reported by Wille and Lutton
(1966).
Other approaches for obtaining the desired form V have been undertaken using the knowledge of these phase transitions upon heating and
using the influence of shear and additives (Loisel et al. 1997a, 1998b),
providing a better understanding of the polymorphism of CB.
The resulting knowledge is also relevant for the study of fat bloom
(Loisel et al. 1997b), which must be hindered by means other than
additives.
The very fast cooling (about 100 C/s) of melted CB results in the
formation of a phase that is less organized than the form, since it
transforms irreversibly into the latter on heating (Loisel et al. 1998a).
It is believed that metastable forms can be used for the rapid formation
of desired polymorphic form V. Partly liquid crystalline structure, in
which hydrocarbon chains are organized as in a brush may occur
even in a partially crystalline state (Loisel et al. 1998a). Such an organization also can be compared with that shown by phospholipids, such
182
as phosphatidylcholine, in the liquid crystalline state (L). This degree

of freedom in the molecular moiety left by the liquid part of the organization allows the other moiety to crystallize very rapidly and into a
very compact subcell. On the other hand, the presence of a liquid
crystalline moiety in the structure would also explain its progressive
transition into the form, since it is well-known that the presence of
a liquid favors the transition of unstable species toward more stable
forms. This is well-known for fats (Timms 1984). This preorganization
assumes that (1) the liquid state of CB, as other liquid fats, is liquid
crystalline and already organized in lamellae in their liquid state (van
den Tempel 1979), and (2) its organization corresponds to layers (or
flat aggregates) made from saturated chains while other parts contain
the unsaturated ones.
Polymorphism of 1,2-dipalmitoyl-3-oleoylglycerol
As an example of the rapid liquid-mediated phase transitions, the
heating of a crash-cooled sample of 1,2-dipalmitoyl-3-oleoylglycerol
(PPO) illustrates the importance of using the coupled techniques such
as MICROCALIX (Ollivon et al. 2006). With the cooling of PPO from
a melted state at 65 C down to 0 C, about 20 mg of pure PPO (99%)
at the rate of 5 C/min leads to the crystallization of a metastable crystalline variety (type 3L). On heating, this metastable form transforms
into more stable varieties (Figure 8.4, bottom). The DSC thermograms
and the XRD patterns observed at small and wide angles on heating at
1 C/min are presented in Figure 8.4. Four steps can be identified on
both DSC and XRD traces (Figure 8.4, middle). Three endothermic
and one exothermic event are observed in the domain 20 to 40 C on
the DSC heating scan. The metastable structure (3L) initially formed
with a period of about 75 (only order 2 line at 37.637.7 is shown
on Figure 8.4) transforms into a mixture of two forms (both of type).
One form has intermediate stability and the other form with smaller d
spacing is stable (close to 33 and 40 ). The two forms melt consecutively (vertical dashed lines delimit the domain of existence of phases;
Figure 8.4, middle). DSC thermograms display several overlapping
thermal events. As a consequence, in the intermediate part of this
double transition, between the maximum of both peaks, only the result
of both phenomena is recorded and schematically explained (Figure
8.4, top). Such behavior is frequently observed in monotropic systems
during melting of metastable crystalline forms when more stable nuclei

20C
unstable
form
a (3L) 75
intermediate
form
b2 (2L) 3941 30C
183
liquid
35C
50
3
2
10
40
30
20
10
0
0
1000
0
3000
2000
Temperature (C)
Peak Area (A.U)
21C
15
Long Spacings ()
stable form
b1 (3L ?) 6466
42
3
1
32
0
10
time (sec)
3
2
1
0.2
q (1)
30
40
50
endo
50
45
40
35
30
25
20
15
10
5
0
60
45
40
35
30
25
20
15
10
5
0
0.1
20
temperature (C)
0.3
50
Temperature (C)
37
1.4
1.6
q (1)
1.8
40
3
2
30
20
10
0
100 50
50
100
Heat flow (mV)
Figure 8.4. MICROCALIX recording of SAXS/WAXS and DSC. Heating from 0

to 50 C at 1 C/min of a 20-mg sample of PPO. Three-dimensional representation of
the evolutions of diffraction patterns recorded at small (left) and wide (middle) angles
and shown as intensity as a function of scattering vector q and temperature T during
the heating of sample. The four types of structures that are clearly visible in the
domain 0 to 50 C are delimited by the transitions evidenced. The line corresponding
to order 2 of the structural period of 75 is voluntarily cut to allow a better visualization of other lines at high temperature.
formerly entrapped in this metastable variety are allowed to grow at

the expense of the metastable variety. This type of complicated thermal
recording can be elucidated only if coupling of a structural technique
is provided.
By using MICROCALIX, it is confirmed that the transition toward
the more stable crystalline form goes through an intermediate stability
form (Ollivon et al. 2006). This monotropic transition is liquid mediated. This intermediate form, more stable than the initial form, melts
between 27 and 30 C. A schematic of the proposed mechanism of
184
the first reaction is seen in Figure 8.4 (top); however, mechanism of

the second reaction is more complex. Only the quantification of the
evolution of the line intensities (Figure 8.4, middle), corresponding to
the long spacings and measured as peak surface areas, allows to interpret the second transition. It can then be seen that two forms coexist
in the 20 to 30 C range, while the periods of the crystalline arrangement change (Figure 8.4). In fact, the mechanism of transition is
similar to the preceding one, except no exothermic event is recorded.
The existence of a liquid crystalline phase obtained by very fast cooling
was not found in pure TG such as triolein, tristearin, or even POP,
which is nevertheless one of the major constituents of CB, because
they all readily crystallize in phase.
The usefulness of MICROCALIX is underlined by the explanation
of complex phase changes as they frequently occur in lipids. Only clear
attribution of thermal events as they are given in the examples above
can lead to a thorough understanding of polymorphism of lipids and
other materials showing polymorphism.
Milk Fat
Milk fat is consumed in dairy products, that is, milk, cream, whipped
cream, cheeses, and butter, and also in powders, pastries, and cooked
foods. Milk fat can in be partially crystallized form (e.g., a mixture of
crystals and oil) over a wide range of temperatures, including the temperature of storage (4 7 C) and consumption. This thermal behavior
results from its fatty acid composition and polymorphism of TG. Milk
fat is the most complex fat found in nature, with more than 400 different fatty acids (about 70% of saturated fatty acids and 25% of
monounsaturated fatty acids, mainly oleic acid) and 200 different TG
identified.
DSC and XRD studies have given an insight into the thermodynamics of milk fat phase transition in bulk (Timms 1980; Lavigne 1995;
ten Grotenhuis et al. 1999), in emulsions (Lopez et al. 2002b), and in
complex food products such as cheese (Rowney et al. 1998; Famelart
et al. 2002; Lopez et al. 2006a).
Anhydrous milk fat
Anhydrous milk fat (AMF), which is the fat isolated from butter, has
a broad melting range, from 40 C to +40 C, and no true melting
185
point as do pure compounds. DSC was used to demonstrate the

presence of polymorphism in anhydrous milk fat (Mulder and Walstra
1974). When polymorphism is present, the thermograms for samples
of the same fat preconditioned thermally in different ways will have
different features. DSC studies of anhydrous milk fat show that it
crystallizes and melts in several steps (Lopez et al. 2007). A typical
melting curve of AMF shows three endothermic peaks, corresponding
to low melting point (LMP), medium melting point (MMP), and high
melting point (HMP) fractions (Timms 1980). These peaks correspond
to large groups of TG that melt separately and behave as solid
solutions. The number of thermal transitions in DSC thermograms,
the partial overlapping of the melting peaks, and their respective
enthalpies and transition temperatures, depend strongly on the thermal
treatments (e.g., heating and cooling rates, tempering) and on the entire
thermal history of the sample (Ollivon and Perron 1992; Ali and
Dimick 1994).
Recently, the use of DSC coupled to synchrotron radiation XRD
with MICROCALIX allowed identification of the crystalline structures formed by TG molecules as a function of temperature and
time in anhydrous milk fat (Lopez et al. 2001a,b, 2005) and its fractions (Lavinge 1995; Lopez 2006b). The samples were melted completely (heated to 60 C for 5 min) to ensure that all crystals
and nuclei were melted and to erase the thermal history of fat. The
samples were then cooled with cooling rates in the range of 0.15 C.
min1 Rcooling 1000 C.min1. The most rapid Rcooling was obtained
by rapid introduction of the capillary into the calorimeter MICROCALIX
precooled to the temperature, for example, 4 C. Tempering in isothermal conditions were also performed, for example, at 8 C, 4 C, and
20 C. Then, XRD patterns were recorded as a function of time and on
subsequent cooling or heating (in general at 2 C.min1). Figure 8.5
shows the crystallization curve of anhydrous milk fat recorded on
cooling at 1 C/min, with the XRD patterns recorded as a function of
temperature, which allows the relation between the thermal events and
the crystalline structures.
The crystallization properties of milk TG were studied after quenching (1000 C.min1) to characterize the most unstable crystalline
structures and their reorganization as a function of time (Figure 8.6).
The samples were cooled rapidly from 60 C to 4 C to ensure
crystallization of fat, and after temperature equilibration, the thermal
186
Figure 8.5. Crystallization properties of anhydrous milk fat. Left: Three-dimensional

plots of the XRD patterns recorded at small and wide (insert) angle during cooling
from 60 C to 7 C at 1 C.min1. Right: Evolution of the maximal intensity of the
XRD peaks recorded at small angles, allowing relation of the structural data to the
thermal properties recorded simultaneously by DSC.
properties were investigated as a function of time under isothermal

conditions. Isothermal DSC was performed, with the temperature being
kept constant at 4 C to study the thermal and the structural properties
of TG. The heat of crystallization released as a function of time resulted
in exothermic signals corresponding to the polymorphic evolution in
the fat ( to polymorphic transition as indicated in Figure 8.6). The
nucleation time (the time at which a peak starts forming), time of
maximum crystallization rate (the time of peak maximum), and heat
of crystallization (proportional to peak area) can all be determined
from the thermogram. The absence of exotherm recorded by DSC for
cream at 4 C indicated that no polymorphic reorganizations occurred
in milk fat globules during the 30 min after their quenching from 60 C
(Figure 8.6). These studies indicated differences in the polymorphic
behavior of TG as a function of their organization, in bulk or dispersed
in emulsion.
187
Figure 8.6. Left: Three-dimensional plot of the isothermal evolution of small-angle

and wide-angle (insert) XRD patterns recorded at 4 C after rapid quenching from
60 C of anhydrous milk fat (AMF). Upper left: Time evolution of the intensities,
taken at the peak maximum and normalized to 100%, of the XRD patterns recorded
at small angles; DSC recordings of AMF and cream obtained simultaneously with
XRD experiments.
DSC and XRD investigations showed that the fat phase of dairy
products displays a complex polymorphism. Depending on the cooling
rate, six different types of crystals were identified, several of them in
coexistence, and their time- and temperature-dependent evolutions
were quantitatively monitored. They correspond to lamellar structures
with 2L (40.548 ) and 3L (5472 ) organizations of TG. At least
five crystalline subcell species were observed at wide angles: and
sub-, two , and one . All these crystalline structures coexist with
a liquid phase even at low temperature (T < 4 C). Thermal events
recorded by DSC were related to the structural information on the
organization of TG obtained by XRD. These experiments focusing on
the crystallization properties of whole milk fat and fat fractions characterized by different composition and thermal properties contributed
to the development of spreadable butters with defined solid fat content
as a function of temperature.
188
Polymorphism of TG in milk fat globules

In milk, lipids are naturally dispersed as small droplets (4 m) called
the milk fat globules. Studying the crystallization of TG in milk fat
globules is of prime importance because it affects many properties,
such as (1) rheological properties, (2) resistance of fat globules to
disruption and then to coalescence, (3) susceptibility of globules to
churning for the manufacture of butter, (4) stability of whipped cream,
and (5) consistency and mouth feel of high-fat products. Thus, it is
important to understand better the physical properties of fat globules,
for example, their thermal and crystallographic properties, for industrial applications and to improve the quality of food products.
Moreover, it is interesting to compare crystallization of fat dispersed
in an emulsion such as milk or cream (which is the concentration of
fat globules from milk) in which fat globules are surrounded by a
membrane rich in phospholipids with crystallization of bulk milk fat.
Lopez et al. (2002a,b) showed that the dispersion state of milk fat, for
example in bulk as anhydrous milk fat or dispersed in fat globules,
alters both its thermal and structural properties. The use of DSC
coupled to synchrotron radiation XRD allowed identification of the
crystalline structures formed by TG molecules as a function of temperature and time in dispersed systems such as milk fat globules (Lopez
et al. 2000, 2001c, 2002a, b). Figure 8.7 shows that slow cooling of
cream (0.15 C.min1) leads to the recording of a single exotherm corresponding to crystallization of TG in fat globules. The organization
of TG molecules in the solid state (e.g., in fat crystals) investigated
using XRDT allowed the identification of four crystalline structures
that are successively formed as a function of the decrease in temperature (Lopez et al. 2001c).
Studies on milk fat globules showed that the temperature of the
beginning of crystallization is lowered as a function of the decrease of
their size (Lopez et al. 2002b; Michalski et al. 2004). XRD permitted
the identification of different crystallization behavior in natural milk
fat globules with different sizes, which could be implicated in the
manufacture of dairy products involving tempering periods in the technological process (butter, ice cream, whipped products).
The examination of TG polymorphism in milk fat globules is much
more challenging than for fat in bulk and especially difficult because
(1) both small- and wide-angle XRD should be considered at the same
time and compared to determine the evolution of each of the species

Small-angle XRD
2L1
46.5
3L2
65
0.15C/min
3L2(002)
3L1(003)
10
20
Temperature (C)
30
3L1(005)
16
27
T (C)
22C
Liquid
16
37
26
47
0.1
0.2
0.3
q (1)
0.4
0.5
T (C)
3L1(001) 2L2
71.3 40
Wide-angle XRD
Endo > (u.a.)
3L1(002)
12C 20C
DSC
189
36
1.1
1.3
1.5
1.7
q (1)
Figure 8.7. Structural evolution, expressed in scattering vector q (1), of TGs dispersed in milk fat globules during cooling from 60 C to 8 C at 0.15 C.min1.
Three-dimensional plots of the XRD patterns recorded at small angles (left) and at
wide angles (right). DSC curve recorded simultaneously.
as a function of time; (2) the X-ray intensity diffracted by each of the

crystalline structures is proportional to the fraction of particular crystal
in the structure; (3) the whole XRD signal is largely absorbed by the
surrounding water and its solutes (e.g., casein micelles, minerals,
lactose); and (4) the peak broadening results from the crystallization
constraints in dispersed systems and the smaller size of the crystals.
Crystallization properties of fat in dairy products
The crystallographic and thermal properties of fat in complex food
products have also been investigated. The melting properties of butters
with different fatty acid composition showed different DSC profiles,
which have been related to the textural properties of the butters (Lopez
at al. 2007). Lopez et al. (2008) identified the crystalline structures
formed by TG in Emmental cheese at 4 C and their melting behavior
upon heating. Recently, DSC was used to investigate the thermal properties of fat in cheese. Lopez et al. (2006a) showed that the liquid-tosolid phase transition recorded by DSC upon cooling is sensitive to the
190
destabilization of fat globules and the formation of nonemulsified fat

during the manufacture of Emmental cheese. Moreover, these authors
developed a protocol to determine the solid fat content in cheese at
4 C and the evolution of the ratio of solid to liquid fat as a function
of temperature (Lopez et al. 2006a).
Lard
Pork production is estimated at about 93 106 tons, of which approximately 50% and 22% are produced in China and Europe, respectively.
Pork represents almost 40% of worldwide daily meat protein intake.
Lard is the fat obtained by rendering fatty tissue of the hog, the domestic pig. Natural lard has a characteristic waxy texture and exhibits
unsatisfying bakery qualities that are frequently corrected by fat blending, partial hydrogenation, or interesterification in making commercial
shortenings. The composition of lard varies with the hogs food and is
mainly composed of a few long-chain major fatty acids, including
C16:0 (24%), C18:0 (14%), C18:1 (41%), and C18:2 (10%).
Although composed of only a few TGs, lard, as many other fats,
exhibits complex thermal properties. DSC thermograms of lard exhibit
several peaks upon heating or cooling of samples (Figure 8.8). These
peaks reflect the occurrence of numerous thermal transitions, the temperatures and enthalpies of which vary as a function of sample thermal
history. The underlying polymorphic transitions of DSC peaks have
been identified (Table 8.3) and shall be illustrated for fast cooling and
heating rates at 5 C/min.
For each of the thermal events recorded upon crystallization, a distinct structure of TG molecules is evident (Figure 8.9, top). Even at
fast cooling rates, the c1 thermal event is associated with the formation
of a 2L form, whereas c2 and c3 can be attributed to two 2L structures from the evolution of the peak intensities as a function of temperature (Figure 8.9, bottom). It is important to point out that that c1
crystallization did not occur at cooling rates lower than 2 C/min. In
addition, at lower cooling rates the structure formed during c2 exotherms crystallizes in a 2L structure (Kalnin et al. 2005). Upon
heating, more than seven endotherms have been observed and attributed to the polymorphic forms (Figure 8.9) by using MICROCALIX
(Kalnin et al. 2005). Two main melting endotherms, the temperature
positions of which vary widely, are observed around 0 and 30 C.
e4 e3
5.0
e2
0.2 ( 12)
Normalized Heat Flow (W/g)
4.0
0.5 ( 5)
3.0
1 ( 2.5)
2.0
2 ( 2.5)
endo
cooling
1.0
c2
c3
Tonset (c3)
0.0
5 ( 2)
c1
Tonset (c1)
Tonset (c2)
10
cooling rate (rc) in K/min
1.0
40
20
0
20
Temperature (C)
40
60
Figure 8.8. Characteristic DSC curves of the crystallization of lard. The influence of
the cooling rate (rc) is evidenced in the range of 0.2 up to 10 K/min as indicated.
The normalized heat flow is scaled to the cooling rate of 10 K/min. DSC curves are
shifted relatively to each other and multiplied for clarity as indicated in brackets.
When not associated to Tonset, arrows indicate minor exothermic events.
Table 8.3. Main crystallographic parameters of the fat crystals in lard and
their attribution to major DSC peaks.
Crystalline Form/
Subcell
(hexagonal)
1
(orthorhombic)
2
(pseudoorthorhombic
triclinic)
SAXS
Peaks d ()
WAXS
Peaks d ()
DSC
Exotherms
48.2
35.1
4.18
4.14, 3.83
c1
c2
h3
h4, h5
43.8
3.95, 4.4,
and 4.3
c3
h7, h6
43.8
4.6
c2 (only
upon slow
cooling
rates)
h1, h2
191
DSC
Endotherms
40.0C
500
34.6C
40.0C
34.4C
29.2C
18.9C
29.2C
400
18.9C
8.4C
l (cps)
300
Heating
at rH = 5 K/min
c2
U-shaped
200
43.6
c2
35.1
100
c1
48.2
0
0.05
0.10
0.15
4.14
c3
20.5C
Crystallization
at rC = 5 K/min 15.9C
12.7C
4.8C
8.9C
13.9C
19.2C
0.20
0.25
0.30
c1
4.18
1.10
1.20
1.30
1.40
1.50
8.4C
WAXS
c2
q (1)
c2
= 3.83
1.60
20.5C
12.7C
4.8C
8.9C
13.9C
19.2C
1.70
1.80
q (1)
60
DSC
SAXS
WAXS
h 2 + h1
40
h3
h4
h5
20
Temperature (C)
c3
3.95 = 3.79
c3
rH = 5 K/min
h6
h7
20
0
c3
rc = 5 K/min
c1
c2
20
48.2
43.8
35.1
endo
40
4.15
3.8
3.95.1 + 4.3 + 4.4
60
6
0
Heat Flow (mW)
1.0
0.5
0.0 1.0
0.5
Relative Peak Intensity (%)
0.0
Figure 8.9. A selection of small and wide angle X-ray scattering (SAXS) patterns at
the average temperature indicated is redrawn on top to illustrate crystallographic
properties of lard. Pure tristearin (SSS) WAXS pattern is shown for comparison
(dashed line). All X-ray patterns were recorded for 60 s and shifted relatively to each
other for clarity. The corresponding DSC curves are drawn for comparison next to
SAXS and WAXS relative peak intensity plots vs. T. Evolution of three SAXS and
five WAXS lines were followed and plotted with normalizations on the peak maximum
intensity (figure redrawn from Kalnin et al. 2005). Main crystallographic parameter
of the crystals in lard and their attribution to major DSC peaks (right).
192
193
They could be attributed to the mono- and di-unsaturated fractions

crystallizing in 2L forms according to the variation of the peak
intensities as a function of temperature (Figure 8.9, bottom). The
two overlapping endotherms, h1 and h2, the importance of which
increases with decreasing cooling rate, are only recorded at T > 40 C
and show that transition of 2L 2L form. The observed transition
at high temperatures, however, is dependent on the whole thermal
history. Thus, h1 is only observed at low cooling rates or after a longer
time.
It can be concluded from this study that, in general, saturated, monosaturated, and polyunsaturated TGs do not cocrystallize at all cooling
rates, but intersolubility is temperature and time dependent. The phase
separation between layers of saturated-saturated-saturated TG, saturated-saturated-oleic acid TG, and saturated-oleic acid-saturated TG
was supposed to lead to an alternate structure due to oleic acid esterified in sn-2 and sn-3 positions, which might explain the diffraction
patterns for the observed pseudo-orthorhombic structure (Kalnin et al.
2005). Moreover, structural transitions take place after rapid crystallizations even at very low temperatures due to to transition
based on isothermal recordings using MICROCALIX (Kalnin et al.
2005).
Coupling of DSC and XRDT using MICROCALIX allows the
partial identification of the structures developed during the thermal
treatments, both fast and slow rates, and permits the assignment of the
thermal events recorded by DSC. Once this identification has been
established, conventional not coupled DSC analysis can be undertaken using commercially available DSC. This knowledge is further
used for fractionation of lard.
Conclusion
Elucidating the polymorphism of lipids is important to better understand the properties of fat-rich food products and to improve food
technology.
The presence of one or several long fatty acid chains and their different packings lead to a variety of polymorphic forms that cause the
thermal and structural behaviors of lipids to be rather complex to study.
Both types of properties are currently measured by XRD and DSC
194
alone. The thermal and structural characterizations of fats are generally

obtained on an independent apparatus, which does not facilitate the
correlation between both types of phenomena. The new instrument,
MICROCALIX, allowing simultaneous (time-resolved synchrotron)
XRD at both wide and small angles as a function of temperature
(XRDT) or time (XRDt), coupled with high-sensitivity DSC, permits
the study of lipid properties for food applications. The technique has
demonstrated the power of coupling for the investigation of the properties and structures of lipids. MICROCALIX has also shown its usefulness in domains such as chemistry and biology for cosmetic and
pharmaceutical applications.
References
Adenier H., Ollivon M., Perron R., and Chaveron H. 1975. Le blanchiment gras. I.
Observations et commentaries. Chocolaterie Confiserie France, 315:714.
Ali M.A.R. and Dimick P.S. 1994. Thermal analysis of palm mid-fraction, cocoa
butter, and milk fat blends by differential scanning calorimetry. J Am Oil Chem
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Part 2
Calorimetry as a Tool for Process Design
Chapter 9
Overview of Calorimetry as a Tool for
Efficient and Safe Food-Processing Design
Alois Raemy, Corinne Appolonia Nouzille, Pierre Lambelet,
and Alejandro Marabi
Introduction
Generalities About Thermal Analysis and Calorimetry
Techniques
Methods
Samples
Thermal Behavior of Food Constituents
Carbohydrates (sugars)
Lipids
Proteins
Water
Thermal behavior of Raw and Reconstituted Food
Safety Aspects
Other Thermodynamic Parameters
Heat of Solution
Specific Heat
Heat of Combustion
Related Techniques
Interest of Calorimetry for the Food Industry
Conclusion
References
201
202
203
203
205
206
206
206
208
214
216
217
217
218
218
224
225
225
226
226
227
202
Introduction
In the investigation of foods using thermal analysis and calorimetric
techniques, many physicochemical effects can be observed in the temperature range between 50 C and 300 C. These thermal phenomena
may be either endothermic, such as melting, denaturation, and vaporization, or exothermic, such as crystallization, oxidation, and fermentation. Some exothermic reactions present a hazard in industrial
operations or during storage. They can lead either to self-ignition,
causing fires or dust explosions in open systems such as spray-dryers,
or to pressure increase and bursting in closed vessels such as autoclaves or extraction cells. Glass transitions are observed as a shift in
the baseline; this information, associated with humidity and water
activity determination, is of particular interest in relation to storage of
food powders, but also for gas retention in powders predicted to foam
when dissolved. This is the safe-processing aspect of thermal analysis
and calorimetry.
The thermal behavior of food strongly depends on its composition.
We therefore consider primarily thermal characteristics of the major
food constituents: carbohydrates, lipids, proteins, and water. Specific
thermal phenomena of minor constituents (e.g., caffeine), as well as
those of additives such as emulsifiers, are mentioned. Raw and reconstituted foods and finally interactions between food constituents will
be considered. Some of these aspects will only be mentioned and not
fully discussed. The reader should refer to previous work by the same
authors (Raemy and Lambelet 1991; Raemy et al. 2000, 2004).
Emulsifiers (endogenous or exogenous) are often used in the food
industry to stabilize interfaces in emulsions and foams. When added
to an aqueous phase, emulsifiers often spontaneously form self-assembly structures. Such structured fluids can be used as active ingredients
for encapsulation or as microreactors for flavor formation. In this
context, differential scanning calorimetry (DSC) instruments, especially micro-DSC, can help detect liquid crystal phase transitions and
establish phase diagrams. This is the material science aspect of thermal
analysis and calorimetry.
Other thermodynamic parameters, such as heat of solution, specific
heat, and heat of combustion can be determined; they are also important for efficient food-processing design. Here, we focus on heat of a
solution that is of great interest for food powder dissolution.
Overview of Calorimetry as a Tool
203
Some related or complementary techniques also are mentioned to

situate thermal analysis and calorimetry in the physicochemistry
domain.
Generalities About Thermal Analysis and Calorimetry

Thermal analysis and calorimetry are extensively described in the literature (Miller 1982; Hemminger and Hhne 1984; Sestak 1984;
Widmann and Riesen 1987; Hemminger and Cammenga 1989; Haines
2002; Claudy 2005). See also Chapter 1.
Techniques
The most currently used technique today is DSC, which often replaces
the older differential thermal analysis (DTA). DSC instruments are
classified into power-compensated DSC instruments (Perkin-Elmer
instruments) and heat flow calorimeters. Heat flow calorimeters can in
turn be classified into Calvet-type calorimeters, where the thermopiles
surround the sample and reference cells (Setaram Instruments, Caluire,
France) and those where the thermopiles are below the sample and
reference crucibles (suppliers being Mettler-Toledo AG, Schwerzenbach,
Switzerland; Netzsch-Gertebau GmbH, Selb, Germany; TA Instruments, New Castle, DE). For isothermal dissolution measurements a
Calvet-type calorimeter equipped with a specially designed membrane
cell can be used as shown in Figure 9.1.
The liquid and the powder sample are placed in the measuring cell
separated by a membrane. The same amount of liquid is also placed
in the reference cell. The cells are then introduced in the calorimeter,
and thermal stabilization is achieved after some minutes. The measurement is then initiated, and after checking for a stable baseline, the
membrane is pierced and mixing is started, bringing the solid and the
liquid into close contact. The heat released or absorbed is measured in
relation to the reference cell. A calorimetric curve is obtained, and the
area under the curve is automatically integrated, yielding the heat (J/g)
absorbed or released during dissolution of the solid sample. A negative
value for the enthalpy of dissolution indicates the emission of heat
(exothermic process), and positive values indicate an absorption of
heat (endothermic process).
204
a

b
Mixing rod
Liquid reservoir
Aluminum
membrane
Recipient for solid
sample
Figure 9.1. Instrumentation used for performing heat of solution measurements

(Calorimeter Setaram C80 and cell with membrane): (a) general view, (b) the heating
block showing the geometry for the reference and measurement cells, and (c) the
membrane mixing cell. The solid sample and the liquid are efficiently separated
during the thermal stabilization step, without risk of moisture transfer. From Marabi
et al. (2007b). Courtesy of Setaram.
Other calorimeters are used for specific applications, for example,

adiabatic calorimeters and the accelerating rate calorimeters, or ARC
(Thermal Hazard Technology, Piscataway, NJ). These are particularly
useful for process safety as adiabatic conditions are the most dangerous
thermal conditions for a product.
The more recent technique of modulated DSC (MDSC) or alternating DSC (ADSC) is that in which a modulated temperature signal is
superimposed on the temperature ramp to help separate reversible
phenomena (e.g., glass transition) from nonreversible phenomena (e.g.,
relaxation).
Adiabatic or isoperibolic bomb calorimeters are used to determine
the heat of combustion of foods. Although the values may be important
in the context of process safety, they are mainly used to calculate the
caloric value of food for human nutrition or when foods are used as
energy sources (e.g., bio-ethanol) for engines.
Some thermal analysis instruments or microcalorimeters allow
either working under virtually fixed pressures (a high-pressure autoclave surrounds the cells) or give thermomanometric information (the
cells are linked to a high-pressure system or even directly fitted with
pressure sensors). Even if thermomanometric information is rarely
given in the literature, these techniques are of great interest for process
205
safety applications, as increased pressure is responsible for bursting of

autoclaves.
For high-sensitivity measurements, various microcalorimeters,
working only in isothermal mode (Thermometric AB, Jarfalla,Sweden),
or micro-DSC, working in isothermal and scanning mode (Setaram
Instruments) are available today. Another way to increase sensitivity
is to use high heating rates: 10 C/min with a standard DSC instrument
or up to 500 C/min with new DSC instruments (sometimes called
hyperDSC).
For high-resolution measurements, best results are obtained with
small samples and slow heating (cooling) rates. Resolution of DSC
instruments can be checked with the help of chemical products (Marti
et al. 2004).
A more extensive classification of the available calorimeters is given
elsewhere in the literature (Rouquerol et al. 2007).
The criteria for choosing an instrument include temperature range,
type of application, ability to work under pressure or under gas flow,
sample size, resolution or sensitivity needed, software performances,
and budget.
Methods
DSC measurements can be performed in isothermal or scanning
(heating and cooling) mode depending on the instrument and on the
goals of the study. The temperature range of the scans has to be decided
according to the phenomena of interest. Heating food samples above
100 C can lead to pressure increase due to water vaporization; there
is therefore a risk of cell rupture if sealed cells are used. Cooling food
below 0 C also can provoke a cell rupture due to volume expansion
upon crystallization.
Generally, to obtain clearer interpretation of the thermal transitions,
consecutive scans (generally first and second scans) are performed;
they allow identification of which phenomena are reversible and which
are not. Sequences of heating-cooling-heating scans are often helpful.
Concerning heating rates, the specialist will generally select the
value giving the best possible curves (often 5 C/min with a DSC and
0.5 C/min with a micro-DSC instrument). However, to use (Arrhenius
type) kinetic models to fit the curves, measurements at different heating
rates are sometimes required (Roduit 2002). The ARC uses a special
206
heat-search procedure to find exothermic phenomena (Raemy and

Ottaway 1991).
Calibration is checked with metals (e.g., In, Sn, Pb) or chemicals
(naphthalene, which has been contested due to health issues; benzoic
acid). With Calvet-type calorimeters Joule effect measurements can
also be performed with specially designed cells.
Samples
Generally, food products are available in large quantities and are easy
to handle. The size of the samples to study is very important. The
samples must be representative of the food of interest; if it is a lipid,
a very thin layer (25 mg) is sufficient, so most DSC instruments can
be used. If the samples are beans (e.g., coffee, cocoa, cereals), large
cells are required, thus there is a need for special instruments allowing
study of some hundreds of milligrams or even grams.
When small sample sizes are used, the reference cell can be empty.
When the sample size is larger, the reference cell must be loaded with
a material that is inert in the temperature range of interest (generally
Al2O3 when studying powders or sometimes water when studying
carbohydrate or protein solutions). Reference and sample cells are thus
equilibrated.
Thermal Behavior of Food Constituents

Foods are mainly composed of carbohydrates, lipids, proteins, and
water. In addition, they contain small proportions of minerals and
various organic substances. Minerals are often analyzed globally as
ash. The organic substances can be vitamins, caffeine, emulsifiers,
acids, antioxidants, pigments, polyphenols, or flavors. Before presenting the thermal behavior of raw and reconstituted foods, we first
describe the thermal behavior of the main food constituents.
Carbohydrates (Sugars)
The main phenomena observed during heating of carbohydrates are
release of crystallization water, melting, decomposition, gelatinization
of starch in the presence of water, and retrogradation of the gel. In

Exo
207
55
50
45
dQ/dt [mW]
40
35
30
25
20
15
Aw 0.078
Aw 0.112
Aw 0.176
Aw 0.225
10
5
20
40
60
Temperature [C]
80
100
Figure 9.2. Calorimetric curves of amorphous sucrose at increasing water activities.

Setaram Micro-DSC III, 1 C/min. From Raemy et al. (1993), with permission.
addition, glass transition, relaxation, and crystallization of amorphous

samples occur (Raemy and Schweizer 1983; Blanchard and Lillford
1993; Raemy et al. 1993; Roos 1995; Vuataz 2002). Glass transition
indicates that amorphous carbohydrates change from the glassy state
to the rubbery state during heating; glass transition is often superimposed on the relaxation phenomenon. A glass transition is reversible
and observed as a change in baseline, whereas relaxation is a nonreversible endothermic transition.
As shown in Figure 9.2, the glass transition (superimposed with
relaxation in the first scan) temperature and the crystallization temperature diminish rapidly with increasing water activity. Amorphism, even
at low levels (down to about 0.5%), can be detected and quantitatively
determined on the basis of the crystallization enthalpies (Raemy et al.
1993), sometimes from the height of the glass transition.
In Figure 9.3, the phenomena observed for these crystalline carbohydrate samples are melting followed by decomposition. Tables with
melting and decomposition temperatures of carbohydrates as well as
corresponding enthalpies are given in the literature (Raemy and
Schweizer 1983).
Gelatinization of starch-water systems is an endothermic nonreversible phenomenon easily observed by DSC. Retrogradation, which is a
slow and low-energy recrystallization process, can be followed by
isothermal microcalorimetry (Raemy et al. 1990; Silverio et al. 1996)
208
Heat flow, J/s
1C/min
Exo
Galactose
f
Sucrose
f
d
Cellobiose
f
50
100
150
200
250
Temperature, C
Figure 9.3. Calorimetric curves of crystalline galactose, sucrose, and cellobiose, all
three heated in sealed cells up to 260 C. Calorimeter Setaram C80, 1 C/min. From
Raemy and Schweizer (1983), with permission.
but is more often characterized after a storage period by measuring the

melting transition of the retrograded gel.
Lipids
Oils and fats reveal many thermally induced transitions as a result of
heating or cooling. These transitions are fundamental and can be used
to elucidate chemical and physical properties of oils and fats.
Thermal analysis and calorimetric techniques (mainly DTA, DSC)
have been methods of choice for studying thermal transitions in bulk
lipids for more than 60 years. They have been proven to be the most
efficient techniques for studying thermal effects that occur during
melting, crystallization, and oxidation of lipids. More recently, DSC
measurements have also been applied for investigating properties of
lipids in dispersed systems; thus, numerous recent calorimetric studies
concern membrane lipids (phospholipids and glycolipids). Interactions
209
between lipids and other macronutrients are also a subject of recent

calorimetric investigations.
Bulk systems
Melting profile DSC melting curves give valuable information on the
melting profile of triacylglycerols (TAGs) and fats, for example, how
they melt in the mouth. The complexity of thermal profiles of oils and
fats is essentially due to their great variety of TAGs. Calorimetry has
long been used to determine the melting profiles of TAGs and fats for
controlling technological processes such as blending (Bartsch et al.
1990), chemical or enzymatic interesterification (Dian et al. 2006; Vu
et al. 2007), fractionation (Herrera and Anon 1991; Bhaskar et al.
1998), and hydrogenation (Daniels et al. 2006). Changes in melting
temperature and enthalpy also have been correlated to fat composition
(Tan and Man 2000).
Solid fat content (SFC), which represents the ratio of solid to liquid
in a partially crystallized lipid at a given temperature, can be obtained
from the calorimetric melting curve by sequential peak integration
(Lambelet et al. 1986; Kaisersberger 1989; Bhaskar et al. 1998). SFC
values are currently used in the fat industry for quality control. Accurate
determinations of SFC values require, however, knowing the exact
melting enthalpy of each phase or of the various fractions present in a
sample, which is very difficult to assess for most fats.
Polymorphism Polymorphism of TAGs and fats as well as phase
transitions between the various polymorphic forms have been extensively studied by calorimetry (Wille and Lutton 1966; Huyghebaert
and Hendrickx 1971; Dimick and Manning 1987; Garti and Sato 1988;
Arishima et al. 1991; Loisel et al. 1998; Lovegren et al. 1976; Merken
and Vaeck 1980; Minato et al. 1997; Rousset 1997; Rousset and
Rappaz 1996; Sato 1996; Spigno et al. 2001). These studies have been
conducted by measuring the melting enthalpy and temperature (pure
components) or temperature range (complex mixtures such as fats) of
the phases present in a lipid sample, as shown for cocoa butter in
Figure 9.4.
For binary or ternary mixtures of TAGs or fats, DSC has been used
to determine real or pseudo phase diagrams, or iso-solid diagrams,
by identifying the domains of the various phases formed (Knoester
210

3.5
Melting curves
Exo
VI
dH/dt (W/g)
2.5
2
1.5
II
III
IV
0.5
0
15
20
25
30
T (C)
35
40
45
Figure 9.4. DSC heating curves of five polymorphs of cocoa butter. Mettler FP900,
5 C/min. From Rousset (1997).
1972; Lambelet and Raemy 1983; Ali and Dimick 1994; Culot 1994;
Elisabettini et al. 1998; Koyano et al. 1992; Rousset et al 1998; Timms
1994).
Kinetics of crystallization Crystallization kinetics of bulk lipids and
the formation and stability of their various polymorphs as a function
of time and temperature are another domain in which DSC is very
useful. For these measurements, the lipid sample has first to be heated
to at least 20 C above the melting temperature of its stable polymorph
to erase all memory effects. Kinetic information has been obtained by
measuring either isothermally after quenching at the desired temperature (Rousset and Rappaz 1997; Metin and Hartel 1998; Toro-Vazquez
et al. 2005) or at constant cooling rate under various cooling conditions
(Kawamura 1980; Cebula and Smith 1991). Complex thermal paths
such as tempering stages also were studied by calorimetry to understand precisely the mechanisms that induce the appearance of stable
crystalline forms (Rousset and Rappaz 1997).
Precise kinetic parameters can be determined from isothermal
experiments. The variation of SFC as a function of time can be obtained
by sequential integration of the crystallization peak. This SFC function
is then used to estimate crystallization parameters with the help of the
Avrami or more complex models (Kloek et al. 2000; Foubert et al.
2002; Rousset 2002). Nucleation induction times, which are periods
211
of time before nucleation appears, also can be determined from isothermal crystallization experiments (Rousset and Rappaz 2001). These
kinetic parameters are useful indicators of the nucleation rate and may
serve as a basis for predicting how to crystallize lipids in the desired
form.
Kinetic studies also help to understand the effects of compositional
changes (TAGs or other minor components) on crystallization (Garti
et al. 1988; Wahnelt et al. 1991; Tan et al. 2000; Vanhoutte et al.
2002a,b). However, as samples are not mixed, results from DSC crystallization studies are often difficult to interpret directly in terms of
process operating conditions (Rousset and Rappaz 2001; Hartel 2001).
DSC curves are often complex because various modes of crystallization and solid state transformations can contribute to a single peak. To
define unambiguously the key signal characteristics such as minima
and maxima, as well as end and start points, a method based on the
determination of the first and the second derivative of the DSC raw
signal has been proposed (Bouzidi et al. 2005).
Quality control DSC crystallization and melting profiles of lipids
have been used to assess the quality of oils, in particular of heated oils
(Gloria and Aguilera 1998; Tan and Man 1999, 2000, 2002). Similarly,
contamination (adulteration) of fats and fat-based products can be
detected by calorimetry (Lambelet and Ganguli 1983; Bringer et al.
1991; Marikkar et al. 2002). Adulteration was demonstrated by the
appearance or a modification (shift in the peak position and peak area)
of a thermal transition occuring in the heating or cooling DSC curves
of lipid mixtures.
Oxidative stability of oils and fats Lipid oxidation is an exothermic
phenomenon that can be observed by continuous monitoring of total
thermal effect either under isothermal or nonisothermal conditions of
measurements (Raemy et al. 1987; Kowalski 1989; Tan and Man 2002;
Ulkowski et al. 2005). Measurements can be performed under a static
air atmosphere or, preferably, under oxygen flow or oxygen pressure
(Litwinienko and Kasprzycka-Guttman 1998).
In isothermal experiments, oxidation induction times correspond to
the time at which a rapid exothermic reaction between the lipid and
oxygen occurred. Tables of oxidation induction times measured by
isothermal heat flux calorimetry around 100 C are reported in the
212
literature (Raemy et al. 1987). For edible oils, induction times obtained
by calorimetry were shown to correlate well with corresponding values
determined by traditional methods (Tan et al. 2002). DSC can therefore
be used to assess the oxidative stability of lipids (Raemy et al. 1987;
Kowalski 1989; Tan and Man 2002).
In the nonisothermal mode, Arrhenius kinetic parameters can be
deduced from the shape of the oxidation curves, and these parameters
can in turn be applied for calculation of the overall first-order rate
constant of oxidation at various temperatures (Litwinienko and
Kasprzycka-Guttman 1998; Ulkowski et al. 2005).
Antioxidant efficacy Food antioxidant activity can be measured by
calorimetry. The efficiency of an antioxidant to protect an oil is measured by the increase of induction time after incorporation of the test
antioxidant into the oil. (Raemy et al. 1987; Irwandi et al. 2000; Tan
et al. 2002; Giuffrida et al. 2006). A good correlation between DSC
oxidative induction time and oxidative stability index determined by
other analytical techniques was found (Tan et al. 2002; Gouveia et al.
2006; Giuffrida et al. 2006).
Also, the radical scavenging activities of antioxidants can be investigated by DSC monitoring of the polymerization of substrates initiated
by radical reactions (Fujisawa and Kadoma 2006).
Dispersed systems
Emulsifier-water systems Lipid-water systems that can be regarded
as models of the lipid matrix of cell membranes include emulsifiers
that may exhibit highly ordered self-assembly structures, which are
liquid crystalline phases. DSC has been applied to the study of endothermic phase transitions occurring in lipid-water systems (Blume 1991;
Figure 9.5. (a) Presentation of the calorimetric curve of a saturated MAG with 20%
water showing melting of different crystalline forms up to 70 C and weak liquid
crystal transitions at 85 C and 110 C. (b) Presentation of the following cooling curve,
which shows that there is practically no hysteresis between the temperatures of the
phenomena; however, the crystalline form melting at 45 C has disappeared.
(c) Presentation of the second heating curve, which confirms the reversible character
of most transitions. (ac) Setaram Micro-DSC III, 0.2 C/min. From Raemy et al.
(2005), with permission.
Heat flow/mW
Exo
(a)
0.5
0.5
1.5
2.5
3.5
4.5
5.5
6.5
7.5
liquid crystal
phase transitions
melting
10 20 30 40 50 60 70 80 90100
Temperature (C)
Heat flow/mW Exo
(b)
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
crystallization
liquid crystal
phase transitions
10 20 30 40 50 60 70 80 90 100
Temperature (C)
Exo
(c)
0.5
melting
liquid crystal
phase transitions
Heat flow/mW
1.5
2.5
3.5
4.5
5.5
6.5
7.5
10 20 30 40 50 60 70 80 90 100
Temperature (C)
213
214
Brandenburg et al. 2006) when they transform from the gel to the liquid
crystal phase (Chapman et al. 1974; Tlgyesi et al. 1985) and to determine thermotropic and lyotropic behavior of these systems (Briggs et
al. 1996; Qiu and Caffrey 1999). Both gel to liquid crystalline
acyl chain melting transitions (Mellier 1988) and structural transitions
between different three-dimensional structures (Willumeit et al. 2005)
were associated with transitions observed in DSC curves. The most
pronounced enthalpy changes were observed for acyl chain melting,
whereas structural transitions, which were not accompanied by acyl
chain melting, exhibited much lower enthalpy changes.
Micro-DSC can be used at low heating and cooling rates to detect
liquid crystalline transitions of exogenous emulsifier-water systems
(Figure 9.5a, b, and c).
Similarly, DSC has been applied to investigate the thermal behavior
of several emulsifier-water systems modified by changing the pH
value, the ionic composition of the environment, or by chemical agents
(Tlgyesi et al. 1985; Forte et al. 1998; Fournier et al. 1998).
Emulsions DSC is useful as it is sufficiently sensitive to measure
transformations in dispersed phases, in particular when used simultaneously with synchrotron X-ray diffraction (XRD; Kalnin et al. 2002).
Thermal behavior of lipids in a dispersion or emulsified form has been
shown to be quite different from that of the same fat in bulk, for
example, crystallization of milk fat (Lopez et al. 2002). Lipid polymorphism in dispersed systems can also be investigated by calorimetry. As shown in Figure 9.6, polymorphism of colloidal suspensions
of TAG has been determined based on DSC melting transitions (Bunjes
et al. 2007).
Proteins
The main phenomena observed by DSC, and especially micro-DSC,
during heating of protein solutions are endothermic phenomena associated with protein denaturation. These phenomena were first described
by Privalov (Privalov and Khechinashvili 1974). These phenomena
were then also observed for products containing large amounts of
proteins and enough water to allow protein mobility; for example, for
proteins of dairy products where small exotherms associated to protein
aggregation also can be detected (Unterhaslberger 2006), fish and meat
Heat flow
2 mW
Heating
Cooling
S100 / SGC
10
20
30
40
50
60
70
80
Heat flow
Temperature (C)
2 mW
Heating
Cooling
S100-3 / SGC
10
20
30
40
50
60
70
80
Temperature (C)
Figure 9.6. DSC heating (10 C/min) and cooling (5 C/min) curves of tristearin
nanoparticles stabilized with phospholipid/bile salt blends containing unmodified
(top) or hydrogenated soybean lecithin (bottom) shortly after preparation. The dashed
arrow in the bottom panel indicates the thermal range of the exothermic event prior
to crystallization. Pyris 1 from Perkin Elmer. Endothermic is upward. From Bunjes
et al. (2007), with permission.
215
216

1
Heat Flux [mW/g]
91.9C
54.9C
0.5
Endo
0.5
Denaturation Enthalpy: 1.82 J/g
1
1.5
2
2.5
3
10
Instrument: Micro DSC III Setaram

Scan rate: 1C/min
Sample weight: 0.4152 g
20
30
40
50
60
Temperature [C]
70
80
90
100
Figure 9.7. DSC curve of the ovalbumin fraction of egg (first minus second run).
Setaram Micro-DSC III, 1 C/min. From Ferreira et al. (1997), with permission.
(Harwalkar and Ma 1990), egg (as shown in Figure 9.7; Ferreira et al.
1997; Grinberg et al. 2002), and cereals (Ellepola and Ma 2006).
Denaturation can be quantified by the determination of the denaturation enthalpy. As the native protein sample is considered to be not
denatured at all, its enthalpy in a first scan corresponds to 100% of
denaturation. Thermally processed products can then be considered as
partially or totally denatured, and a percentage of denaturation can thus
be calculated from the determined enthalpies.
The pH is important because it influences denaturation temperature
and thus allows finding protective conditions for industrially treated
proteins. Protein denaturation can also be partially or even totally
reversible according to the results of second runs.
For dry proteins, glass transition and oxidation also can be observed
with thermal analysis techniques.
Water
Crystallization (undercooling) of water, melting of ice, and vaporization can be observed with thermal analysis and calorimetry. Since the
enthalpies corresponding to these phenomena are quite high (333 J/g
for ice melting and 2255 J/g for water vaporization), they can be
217
observed easily with standard DSC instruments, even in samples with

low water content.
Thermal Behavior of Raw and Reconstituted Food

Most physicochemical effects observed with the main food constituents are also found in the calorimetric curves of raw and reconstituted
foods; examples are coffee beans, chicory roots, cereals, milk powders,
and infant formulas (Raemy 1981; Raemy and Lambelet 1982; Raemy
and Lliger 1982; Raemy et al. 1983; Raemy and Schweizer 1983),
where carbohydrate decomposition is observed systematically. For
milk powders, lactose crystallization and lipid oxidation also are
detected. The thermal phenomena observed with pure minor constituents will not be observed, however, once these constituents (e.g., caffeine) are dispersed in a food matrix. Many raw and reconstituted foods
contain water. Therefore, measurements of such products in sealed
cells above 100 C must only be performed with great precautions
because of pressure increase due to water vapor and gas release during
decomposition.
In addition to these phenomena, some reactions between food constituents, such as the Maillard reactions, which occur between proteins
and reducing sugars, may be observed, for example, as an exothermic
phenomenon in calorimetric curves of milk powders or infant formulas
(Morgan et al. 2005) obtained with sealed cells.
Safety Aspects
Carbohydrate decomposition, which sometimes immediately follows
melting, lipid oxidation (especially if oil is present as a layer or at the
surface of the product) as well as protein oxidation, and even Maillard
reactions may present a hazard in industrial operations (e.g., roasting,
high-temperature drying).
The role of thermal analysis and calorimetry for determining safe
conditions of industrial processes has already been explained elsewhere (Raemy and Lliger 1985; Raemy et al. 1985; Raemy and
Gardiol 1987; Raemy 1988, 2001). The application of adiabatic
calorimetry to the study of cellulose decomposition, cellulose being
218
considered as a model for other foods, has been described in detail

(Raemy and Ottaway 1991). Pressure increase due to water vapor pressure, gases evolved during roasting or decomposition, and air compression (the pressure increase due to dilatation of the pressure sensor has
to be deduced) can be monitored by thermomanometry, for example,
with the C80 calorimeter or with the ARC.
In the case of safety studies, thermal analysis and calorimetric techniques must sometimes be applied unconventionally as measurements
have to be carried out under conditions close to those of the process
to be studied (Raemy 1992; Raemy et al. 2000).
Other Thermodynamic Parameters

In addition, to observe the thermal behavior of food as a function of
temperature, calorimetric techniques can also be used to determine
thermal parameters such as heats of solutions, specific heats, and heats
of combustion.
Heat of Solution
Solution calorimetry is a suitable technique for the study of liquidliquid and liquid-solid interactions (Hogan and Buckton 2000). It
allows quantifying the thermodynamic effects that occur during the
dissolution process and can potentially give information on the kinetics
and the mechanism of dissolution. In a calorimetric experiment addressing the dissolution process, the output is a composite of wetting, liquid
penetration, dissolution phenomena (disruption of the solid, removal
of surface molecules, and incorporation of the solute molecules in
cavities in the solvent), and any other interactions that might occur
(Buckton 1995; Gao and Rytting 2006).
This is a widely used technique, mostly in the pharmaceutical field.
More specifically, solution calorimetry is used for quantifying the heat
as a solid dissolves in a liquid. The theory behind the technique has
been explained in detail previously (Gao and Rytting 2006).
Some examples include the determination of amorphous content of
lactose (Hogan and Buckton 2000; Harjunen et al. 2004; Dilworth et
al. 2004; Katainen et al. 2005), sucrose and drugs (Gao and Rytting
2006), the enthalpy of solution of -cyclodextrin (Bastos et al. 2004),
219
various saccharides (Miller and de Pablo 2000), and caffeine (Pinto

and Diogo 2006), to name a few. The swelling of seaweed and green
tea has been studied by means of calorimetry (Miyagawa et al. 1995a,b),
and the swelling and dissolution behavior of different polymers and
polymer blends used as pharmaceutical excipients have also been
reported (Conti et al. 2006).
The significantly different dissolution behavior of crystalline and
amorphous saccharides has also been studied (Miller et al. 1997; Miller
and de Pablo 2000; Salvetti et al. 2007). All crystalline samples examined presented endothermic enthalpies of dissolution, ranging from
about 17 to 90 J/g. Conversely, the same samples in the amorphous
state showed an exothermic response, with enthalpies between 30 and
80 J/g. This difference is often attributed to the higher entropy and
internal free energy of the metastable amorphous material, leading to
enhanced dissolution rate and chemical reactivity relative to the thermodynamically more favorable and stable crystalline state (Hancock
and Zografi 1997; Hancock and Parks 2000; Hancock 2002; Wong et
al. 2006). In the case of lactose, it was proposed that the interactions
within the crystalline material were stronger than the hydration process;
thus, its dissolution resulted in an endothermic response (Harjunen et
al. 2004). In the case of an amorphous material, the solid interactions
might be weaker compared with the crystalline counterpart, resulting
in a more spontaneous dissolution characterized by a release of energy
during the process. In all cases, crystallization leads to a change of the
physical structure, possibly leading to impaired rehydration properties
(Roos and Karel 1991).
The heats of solution (or dissolution) of many pharmaceutical substances also have been measured directly by solution calorimetry,
generally with a different experimental setup. Quantitative analysis of
polymorphs, solvates, and amorphous forms in pharmaceutical substances has been performed using solution calorimetry (Giron et al.
2004). Studies of polymorphism by solution calorimetry also have
been reviewed (Giron 1995).
The effect of moisture content on the thermodynamic response of
dissolving powders was studied, and a significant change in the enthalpy
of dissolution (less exothermic) for amorphous lactose samples stored
at increasing humidities was reported (Hogan and Buckton 2000). For
-cyclodextrin, the enthalpy of solution reverted from exothermic for a
dry sample to endothermic for a hydrated sample (Bastos et al. 2004).
220
This effect was explained in terms of the exothermic nature of the

wetting process, resulting in a less exothermic response for a sample
that has already adsorbed moisture from the environment, compared
with a completely dry sample. It is often argued that the sorption of
water by a dry powder is the first stage of wetting and that the first few
molecules adsorbed on the surface are responsible for the greatest part
of the overall exothermic wetting response (Buckton 1995; Hancock
and Shamblin 1998; Hancock and Dalton 1999; Hogan and Buckton
2000). This effect might be observed and can be quantified when a solid
sample is exposed to water vapor. However, if the solid is undergoing
dissolution, the distribution of moisture in the bulk of the material will
govern the calorimetric response as consecutive layers of the solid are
exposed to the liquid medium (Marabi et al. 2007b). Assuming that the
moisture is uniformly distributed in the solid matrix, a less exothermic
response is expected as the moisture content increases, and a transition
from an exothermic to an endothermic process might be observed at a
limiting moisture content (Bastos et al. 2004). In a dry glassy material,
the measured enthalpy might result from the bonds created between the
water molecules and the hydrogen-bonding sites (Miller and de Pablo
2000; Lechuga-Ballesteros et al. 2002). Haque and Roos (2006) recently
speculated that freeze-dried materials might have a higher amount of
hydrogen bonding sites available for sorption of water molecules than
spray-dried materials. Indeed, even a small increase in moisture content
of a drug above a critical value of 3% was shown to significantly
decrease its dissolution rate (Li et al. 2004). Consequently, a less exothermic response due to either higher moisture content of the powder or
a reduced number of available hydrogen bonding sites could have major
implications in the wetting mechanism of food powders, which in turn
might slow down their dissolution process (Marabi et al. 2007b). Faster
dissolution kinetics were also reported to be correlated with more exothermic processes under different conditions (Hancock and Parks 2000;
Terada et al. 2000; Marabi et al. 2007a).
Isothermal solution calorimetry was also used to derive the wettability of finely divided solids (Lazghab et al. 2005) and the surface energy
of solids such as silica, quartz, kaolinite, and illites (Zoungrana et al.
1994; Medout-Marere et al. 1998), fluorinated carbons (Spagnolo
et al. 1996), talc and quartz (Malandrini et al. 1997a,b), and fumed
silica (Yan et al. 2000). By using nondissolving liquids, it is possible
to measure the enthalpy of immersion that can then be used to derive
221
0.16
Fat content
0.7%
14.3%
29.3%
35.7%
45.0%
Normalized Heat Flow [W/g]
0.14
0.12
0.10
0.08
0.06
0.04
EXO
0.02
0.00
0.02
500
1000
1500
2000
2500
Time [s]
Figure 9.8. Typical dissolution calorimetry curves of a model food powder with
increasing amounts of fat. From Marabi et al. (2008), with permission.
the contact angle between the particulate solid and the liquid (Spagnolo
et al. 1996; Adamson and Gast 1997). The application of immersion
calorimetry could therefore circumvent the difficulties associated with
assessing contact angles of food powders. Several reviews on the use
of isothermal calorimetry in different (e.g., pharmaceutical, microbiological) applications are available (Buckton 1995; Wads 1997), indicating the wide applicability and useful information that can be obtained
from this technique, which surprisingly, has not been exploited in the
field of food science.
The effects of fat content on the dissolution enthalpy and kinetics
of a model food powder were reported previously (Marabi et al. 2008).
Typical dissolution calorimetry curves for five freeze-dried samples
with increasing fat contents are shown in Figure 9.8.
Dissolution of all the powders resulted in an exothermic response.
Increasing the amount of fat in the samples is clearly related to a
decrease in the amount of heat released during the dissolution process.
The decrease in the enthalpy of dissolution ranged from 61.9 to 32.4 J/g
for samples with 0.7% and 45.0% of fat, respectively (Figure 9.9).
This, in turn, is related to the dissolution kinetics of the powders, which
also was shown to be significantly affected by increasing amounts of
fat in the samples (Marabi et al. 2008). However, when the enthalpy
of dissolution is normalized by the amount of fat material, it can be
222

0.7
14.3
FAT (%)
29.3
35.7
45.0
0
10
20
Hdiss (J/g)
30
40
50
60
70
Enthalpy of dissolution normalized by fat content
80
Total Enthalpy of dissolution
90
Figure 9.9. Enthalpy of dissolution of a model food powder with increasing amounts
of fat expressed as J/g of sample and as J/g of nonfat material.
seen that very similar values are obtained for all the samples tested
(Figure 9.9). This fact indicates that the fat has a minimal contribution
to the overall enthalpy measured, which is in agreement with the slight
endothermic response measured when pure fat is mixed with water
(Marabi et al. 2008).
The effects of different moisture contents and the physical state of
maltodextrin (MD) and skim milk powder (SMP) also were studied
(Marabi et al. 2007b). Namely, three different conditions were studied:
after samples were freeze-dried (FD), after equilibration at 54.4% relative humidity, and after FD (water activity again less than 0.01) of the
equilibrated sample. The calorimetric curves are shown in Figure 9.10a
and b. The dissolution was found to be exothermic for all the tested
samples, and the curves showed similar shapes. For both samples, a
clear decrease in the response was observed when the FD and the
equilibrated samples are compared. When the latter were FD again,
the MD resulted in a curve almost identical to that obtained with the
original FD samples. In contrast, the SMP sample showed only an
intermediate response between those of the FD and the equilibrated
samples. The enthalpy of dissolution decreased (became less exothermic) about 12- and 20-fold from the FD state compared with the
equilibrated state for the MD and SMP samples, respectively.
0.16
223
a
MD DE21 - FD
MD DE21 - aW 0.54
MD DE21 - aW 0.54 & FD
0.14
0.12
0.10
0.08
0.06
0.04
Exo
0.02
0.00
500
1000
1500
2000
2500
3000
Time [s]
0.14
b
SMP - FD
SMP - aW 0.54
SMP - aW 0.54 & FD
0.12
0.10
0.08
0.06
0.04
0.02
Exo
0.00
0
500
1000
1500
2000
2500
3000
Time [s]
Figure 9.10. Typical dissolution calorimetry curves of the maltodextrin DE21 (a) and
skim milk powder (b) at different conditions. All the samples showed an exothermic
response; however, increased moisture content or recrystallized lactose led to a less
exothermic dissolution process. From Marabi et al. (2007b), used with permission.
After freeze-drying the equilibrated MD sample, more than 98% of

the original enthalpy was recovered. These data indicate that the
enthalpy of dissolution is a strong function of the water content and
that it is reversible to some extent upon drying of the powder, provided
a phase change does not occur in the solid. The part of enthalpy not
recovered could be explained by the residual amount of water in the
224
sample. In contrast to the MD powder, the equilibrated SMP sample

that was subsequently FD recovered only approximately 50% of the
value observed for the originally FD sample. This effect is related to
the nonreversible process of lactose crystallization that occurred in the
SMP sample. The enthalpy of dissolution was significantly reduced
due to the presence of the crystalline material for which an endothermic effect is expected upon dissolution, with reported values between
approximately 52 and 60 J/g (Miller and de Pablo 2000; Harjunen et
al. 2004). An approximate reduction of about 8 J/g in the calorimetric
response for each 1% of adsorbed water was calculated when both the
MD and SMP FD samples were compared with the equilibrated
ones. These values are comparable with those observed for amorphous
lactose (Hogan and Buckton 2000), sucrose and trehalose (Miller and
de Pablo 2000) having different moisture contents, for which an
approximate reduction of 35 J/g in the net enthalpy for each 1% of
water that is adsorbed was reported.
Clearly, the physical state of the samples affected the enthalpy of
dissolution, with amorphous samples showing a more exothermic
response than partially crystalline samples. It was observed optically
that the dissolution kinetics were hastened by more exothermic
responses, which contributed to an overall spontaneous process,
whereas less exothermic responses clearly resulted in a much slower
dissolution rate.
In conclusion, the current approach demonstrated that the thermodynamic aspect has a crucial importance in the dissolution of food
powders and that isothermal calorimetry should be implemented if
optimization of the dissolution process is required.
Specific Heat
Calorimeters are often used to determine specific heats of foods because
process engineers request such information when installing new equipment. The methods and a synthesis of results have been presented in
the literature (Mohsenin 1980). The values obtained vary between
1.25 J g1 K1 for very dry food products (without fat) and 4.18 J g1 K1
for water. The moisture content of a food has thus a strong influence
on its specific heat value. The specific heat values of a solid increase
with temperature; for water, the value is approximately constant
between 0 C and 100 C.
225
For high-precision measurements, synthetic sapphire is used as the

standard (Raemy and Lambelet 1982).
Sometimes specific heat measurements are combined with glass
temperature measurements for food-water systems (Pyda 2002) as well
as for polymers (Marti et al. 2006).
Heat of Combustion
During burning of a food product, a large amount of energy is liberated. Values determined with calorimetric bombs are about 39 kJ g1
for fat, 23 kJ g1 for protein, and 17 kJ g1 for carbohydrate.
Related Techniques
Some related thermal analysis techniques, such as dynamical mechanical analysis (DMA) or dynamical mechanical thermal analysis (DMTA)
give rheological rather than thermal information. Also, thermogravimetry, sometimes coupled with gas analysis instruments, can provide a
better interpretation of calorimetric curves (e.g., when studying
hydrated carbohydrates) by indicating weight losses associated with
the observed thermal phenomena.
Microscopy techniques, sometimes performed at different temperatures with a hot stage microscope, are often also very helpful in obtaining a clear interpretation of calorimetric curves.
Concerning lipids, as assignments of DSC signals may be ambiguous due to the high number of thermal events, calorimetry often needs
to be combined with XRD (Chung and Caffrey 1992; Keller et al.
1996) or even synchrotron XRD when transformations are rapid.
Recent experiments combining DSC and synchrotron XRD have revolutionized the study of lipid crystallization (Ollivon et al. 2001; Kalnin
et al. 2002; Lopez et al. 2002). DSC can also be used simultaneously
with microscopy to identify morphologies associated with polymorphs
(Rousset et al. 1998).
Fat crystallization in oil-in-water emulsions has been followed by
DSC in combination with either nuclear magnetic resonance spectroscopy (zilgen et al. 1993) or XRD (Awad et al. 2001; Ollivon et al.
2001).
226
Interest of Calorimetry for the Food Industry

The main reason for studying exothermic phenomena (carbohydrate
decomposition, fermentation, oxidation) of food with calorimetric
techniques is to improve process safety. A precise knowledge of the
temperature ranges of these phenomena and the corresponding enthalpies allows adequate safety measures to be taken and helps to diminish
the number of fires, explosions, and bursting of autoclaves in foodprocessing plants (see Chapter 15). It leads thus to a favorable context
for avoiding personnel injuries and for loss prevention. Exothermic
reactions are desired in some processes, for example, roasting, and
have to be avoided in others, such as high-temperature drying.
Endothermic phenomena, such as melting, must be avoided or monitored to obtain the correct product. For example chocolate should melt
in the mouth but not on the hand; thus, the cocoa butter in chocolate
should mainly be in the crystalline form V (melting temperature range
between 27 C and 37 C).
Glass transition temperatures are, in combination with water activity
and moisture content, of great interest for studying adequate storage
conditions of milk powders, for example by avoiding browning and
caking. But this concept also has allowed the development of coffee
and milk products with desired amount of foam at the top when reconstituted in water or milk. These products, which are typical examples
of the so-called glass transition technology, are presently very popular
with consumers.
Specific heats of foods are requested by process engineers for the
design and installation of new thermal equipment (Kaletun 2007).
Heat of solution of food powders in water or milk is of major importance for the food industry because dissolution speed, which is directly
related, is one of the main criteria of the consumer when selecting a
food powder for preparing an instant drink.
Conclusion
In addition to this information, which can be obtained with the help of
calorimetric techniques, some databases such as the European database
EVITHERM can be used to find thermal parameters about food and
nonfood products (www.evitherm.org). The Internet sites of the commercial instrument suppliers also give much valuable information.
227
Even so, new products, new conditions, and new processes push
performance of more calorimetric measurements and testing of new
instrumentations, such as isothermal titration calorimetry (ITC), which
is of interest for studying such interactions as that between proteins
and active substances, or dielectric spectroscopy, which is of interest
in the study of proteins or the amorphous state of carbohydrates.
A new trend is to use coupled systems for performing evolved gas
analysis in relation to the thermal signal; for example, thermogravimetry coupled with mass spectrometry is commonly used today.
Thermal analysis and the study of foods have been mutually beneficial: thermal analysis and calorimetry in general have brought much
information to food science and processing technology, whereas the
study of foods, as the samples are easily available, has allowed the
development and the promotion of many calorimetric techniques.
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Chapter 10
Shelf Life Prediction of Complex
Food Systems by Quantitative Interpretation
of Isothermal Calorimetric Data
Simon Gaisford, Michael A.A. ONeill, and Anthony E. Beezer
Introduction
Qualitative Studies
Quantitative Studies
Empirical Model Fitting
Modeling Based on Reaction Kinetics
Reactions That Proceed to Completion
Calculation of the Initial Calorimetric Signal 0
Calculation for the Reaction Order
Calculation for the Total Heat Released for Complete Reaction
Calculation for Reaction Half-Life
Calculation for Rate Constant
Calculation for Reaction Enthalpy
Reactions That Proceed to a Point of Equilibrium
Test for Complete Reaction
Determination of K
Calculation of QT
Summary
References
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Introduction
The analysis of foods and food components presents a considerable
challenge, not least because they may contain many ingredients, be
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difficult to handle, and may derive from one or more natural sources
(which inherently introduces batch-to-batch variability in composition). Indeed, in many respects foods may be considered as complex
biomaterials. While qualitative analyses may suffice for some quality
assurance protocols (e.g., texture, feel, density), it is often desirable to
obtain quantitative data; this is particularly true where food authenticity (i.e., whether a food substance conforms to its label claim) is
required. In this context, it is often difficult or complex to use classical
assay techniques. It is not necessarily straightforward, for example, to
quantify banned additives in foodstuffs using high-performance liquid
chromatography or spectroscopy because of the need to isolate the
analyte prior to analysis. A discussion of the merits of various analytical tools for determination of food authenticity can be found in Reid
et al. (2006).
We have long argued that calorimetry, in particular isothermal calorimetry, is ideally suited to the study of complex samples because it
offers many unique advantages. First, the measured parameter is heat.
This is advantageous because heat can be considered as a universal
indicator of change (and note here that change in this context can mean
both physical and chemical processes). Thus, it will unquestionably be
the case that a sample can in principle be studied with calorimetry.
Whether a meaningful interpretation can be made depends only upon
the magnitude of the heat change and the number of events occurring
(discussed further below). Second, the instrument requires no sample
treatment or preparation. The entire sample is housed within an ampoule
and monitored in situ; or, if the sample is too large for the ampoule, a
fraction is enclosed that is representative of the whole. Thus, the need
to isolate a particular analyte, as in a chromatographic assay, is obviated. Finally, the technique does not require optical clarity of a sample
and is invariant to physical form, which means that any complex material can be studied in its entirety.
There are two principal calorimetric techniques; isothermal calorimetry (IC) and differential scanning calorimetry (DSC). With the former,
the sample is monitored at a constant temperature; and with the latter,
the sample is subjected to a controlled temperature ramp (usually
increasing). Unsurprisingly many of the calorimetric studies of foodstuffs have used DSC (see Raemy et al. 2004; Schiraldi 2004) or
temperature-modulated DSC (De Meuter et al. 1999). This is, perhaps,
not unexpected since such instruments are uniquely well suited to
Shelf Life Prediction of Complex Food Systems
239
evaluating the consequences of cooking on foodstuffs. There have been

much fewer such detailed studies devoted to IC investigationsthe
subject of this chapterand yet the type of information obtainable
from isothermal investigation can be vastly more enlightening because
it is, of course, just such studies of storage over time that may define
food stability and shelf life. It may, too, allow investigation of those
reactions that occur isothermally at high temperature, such as the
cooking temperature. In addition to these patent benefits, recent work
has resulted in a new range of analysis methodologies with which to
recover quantitative reaction parameters from IC data; these data can
be used to inform sample design and improvement. A review of the
application of isothermal calorimetry to food stability is thus the topic
of this chapter.
Qualitative Studies
Quantitative data interpretation usually requires some prior knowledge
of the properties of the system under investigation (e.g., number of
reacting systems, reaction pathway) as well as a model with some
factual basis. While for simple (one- or two-component) systems quantitative interpretation may be possible, inevitably with more complex
systems there will be instances in which qualitative outcomes can be
indicative of change despite the absence of detailed interpretation of
the experimental data. Much of the literature in this field thus reports
qualitative data, and it is here that this discussion starts.
Qualitative analyses are usually applied to complex systems because
in such cases it is not technically possible to interpret multifaceted
power-time data. Consequently, this discussion starts with applications
of calorimetry to bioprocesses and bioprocessing in which microorganisms are used in the production of, or as an ingredient in, foodstuffs.
It is not a simple matter to measure efficacy of bioprocesses in situ
because of the heterogeneity of the system (which may change viscosity, density, and optical transparency). Conventional microbiological
assay techniques, such as plating and counting, are time consuming,
do not provide information on real-time growth in the actual process
environment, do not compensate for the fraction of the microbial load
that is dead or not viable, and exclude any contribution to efficacy
caused by physical effects (such as thickening of the medium).
240
With calorimetry, the power signal is quantitatively proportional to

the number of viable organisms in the sample, which means the technique is immediately appropriate for counting cell numbers. Indeed,
this is one of its primary advantages over plate-counting methods,
because the calorimeter only reports heat changes from living organisms and is not subject to error from inclusion of nonviable cells. The
technique addresses all of the concerns raised earlier because it is not
dependent on the physical form of the sample and does not impose a
requirement for optical clarity.
An important consideration when using calorimetry to monitor
growth of organisms is the repeatability of the growth curves. There
can be enormous variation between bacteria cultured on different days;
if this variability results in greater heat changes than the process under
investigation, no conclusions can be drawn. In attempting to overcome
this limitation, Beezer et al. (1976) and Cosgrove (1979) developed
procedures to allow storage of frozen inocula of various organisms,
including Saccharomyces cerevisiae and Pseudomonas aeruginosa. In
this method, a batch of organisms is grown overnight in a bacterial
culture medium. Late exponential growth phase cells are harvested,
washed in phosphate-buffered saline (PBS), resuspended in 15% v/v
glycerol to an organism density of 108 cfu/ml, and frozen in aliquots
(1 ml) over liquid nitrogen. Organisms can be stored for more than 6
years in this frozen state and remain viable after thawing with less than
1% decrease in viability.
The benefit of using frozen inocula is very tight reproducibility of
the growth curves. An example (in this case for P. aeruginosa) is
shown in Figure 10.1. Taking the total area under the growth curve
(total heat output) as an indicator of organism numbers shows reproducibility to 6.3% (3.51 0.22 J). For S. cerevisiae, the reproducibility
is normally no greater than 1.5% from the mean and never greater
than 2.5% from the mean (Perry et al. 1979). The growth curve represented in Figure 10.1 is complex and characteristic of organism
growth in an undefined medium with restricted oxygen (the ampoule
is sealed and the oxygen level is limited to that dissolved in the medium
and present in the headspace). In brief, the initial exponential phase
represents aerobic metabolism, which is then followed by a switch to
anaerobic metabolism. Subsequent peaks and troughs represent sequential use of the major carbohydrate sources typically found in a complex
growth medium.
241
Figure 10.1. Power-time data showing the growth curves for six repeats of P.
aeruginosa.
A good example of the very valuable data that can be produced from
qualitative analysis of isothermal calorimetric data is provided by
studies of the growth of yeasts on a variety of substrates (Perry et al.
1981, 1983). Yeasts play an important role in many food industries,
including baking and brewing, and are used in other industries as well,
for example, as a means of producing ethanol from renewable sources
or for use in single-cell protein (SCP) production. The authors took
three commercial strains of S. cerevisiae (D1, distilling; D2, pressed
baking; and D3, active dried baking) and two NCYC strains (87, distilling and 239, brewing) and analyzed their growth curves with flow
microcalorimetry (an isothermal calorimeter equipped with a cell that
allows medium to be flowed through from an external reservoir). In a
glucose medium the growth curves of the three baking strains showed
little differentiation, although the growth curve of the brewing strain
was notably different. In a maltose medium, good differentiation was
observed between all strains. One immediate outcome from these data
is the ability to use a simple medium (maltose) to characterize the
properties of a new strain of S. cerevisiae and select it as appropriate
for either baking or brewing.
When complex media are considered the utility of calorimetric study
increases further. Perry et al. (1983) showed that growth of yeast in
242
glucose-maltose media did not show sequential use of the carbohydrates; rather, both sugars were exhausted after the initial growth
phase. Discrimination of strains was achieved by adding maltotriose
to the medium.
Molasses, a waste product of the sugar industry, is a raw material
used in both baking and brewing because it is highly suitable for yeast
growth. The suitability of a particular batch of molasses is dependent
upon the technical processes by which the sugar was manufactured and
also by agronomic factors of the cane and beet production. As a result,
the growth of yeast in a particular molasses batch can be highly variable, and there will be a direct impact on the production costs and
quality of the baked or brewed product. A method that allows rapid
assessment of the quality and suitability of a batch of molasses is thus
highly desirable and difficult to achieve by classical physical and
chemical means. It is known that only part of the carbohydrate reservoir in molasses is of nutritive value to the yeast. As a consequence,
analytical data are not sufficient to characterize molasses batches from
a bioavailability point of view. Perry et al. (1981) show how interpretation of calorimetric growth curves of S. cerevisiae in molasses samples
could be used rapidly to identify optimal growth media, growth curves
in molasses identified as poor being distinct from growth in molasses
typed as adequate-to-good.
A similar approach was used more recently by Alklint et al. (2005)
to predict the shelf life of carrot juice. Here, growth of the mesophilic
and psychrotrophic flora in the carrot juice was monitored after manufacture at 17 C (the highest permitted temperature for elevated stability tests of chilled foodstuffs in Sweden) with an isothermal calorimeter.
It was found that the heat outputs recorded correlated with plate counts,
indicating that the calorimetric approach was valid. The initial cause
of spoilage was found to be the same at 17 C as at 8 C (the maximum
permitted storage temperature of chilled foodstuffs in Sweden), and
the data were found to be suitable for predicting shelf lives.
An area of growing interest is foods that offer some health benefits,
so-called functional foods. Prime among these are products that aim
to modulate the microflora of the gastrointestinal (GI) tract. Bacterial
numbers vary along the human GI tract, increasing from about 103 g1
of gastric content to about 106107 g1 of content at the terminal ileum
(Gorbach et al. 1967). The colon, in particular, is a complex and
diverse microbial ecosystem, bacterial numbers reaching 10111012 g1
243
of gut content (Cummings and Macfarlane 1991). This makes the colon
the most metabolically active organ in the body. In addition, the number
of bacterial species is large (several hundred), creating a diverse
microflora. The numerically predominant bacteria (30%) are the
bacteroides, although other genera, such as bifidobacteria, eubacteria,
lactobacilli, streptococci, and clostridia, are also present (Fooks et al.
1999).
The microflora has many roles and is important to general health
and well-being. A primary function is in fermenting undigested foodstuffs that have not been absorbed in the upper GI tract. Typically,
substrates are carbohydrates (including starches, dietary fiber, and oligosaccharides). The principal fermentation products are short-chain
fatty acids (SCFA); these are subsequently absorbed by the body and
metabolized, which contributes to the energy gain of the host
(Cummings 1995) and means the relationship between host and microflora is symbiotic. Another important function is to inhibit the growth
of pathogenic organisms. However, in the absence of sufficient carbohydrate, certain bacteria, such as clostridia, switch to protein fermentation, which produces harmful nitrogenous metabolites (including
biogenic amines, indoles, and ammonia).
To minimize this effect, the body excretes a number of mucins,
which are high in carbohydrates and encourage the growth of certain
microbial species. The UK food market is currently replete with products designed to modulate the gut microflora; usually, such products
are supplemented with either prebiotics or probiotics. Probiotics are
live bacteria of species deemed to be beneficial to health when ingested;
usually, lactic acid bacteria (LAB) are indicated and they are commonly found in yogurts, where they convert lactose to lactic acid,
giving the product its distinctive sour taste and acting as a preservative.
Prebiotics are nondigestible food ingredients that stimulate the growth
of one or more beneficial bacteria in the colon and were first defined
by Gibson and Roberfroid (1995). Several potential nondigestible
foodstuffs have been investigated for prebiotic efficacy, but to date the
only substances for which credible data showing a favorable effect are
available are the oligosaccharides (Delzenne and Roberfroid 1994).
However, it is not a simple matter to show a beneficial prebiotic
effect in vivo, primarily because of the sheer complexity of the gut
microflora and its effect on physiological response. Typically,
an increase in the number of bifidobacteria excreted in feces has
244
historically been accepted as proof of efficacy, but it remains to be

proven either that an increased level of bifidobacteria in the gut correlates with an increase in health or well-being or that the number of
Bifidobacteria is a good biomarker for gut health (Ouwehand et al.
2005).
In addition to the complexity of determining an in vivo effect, many
probiotic foodstuffs contain multiple cultures, and it is difficult simply
to demonstrate that these do not compete with each other. While classical microbiological techniques do not allow direct observation of
such processes, calorimetry potentially does. For example, Schffer
et al. (2004) used isothermal calorimetry to monitor the growth of two
cultures commonly found in probiotic dairy products. In addition to
the added probiotic, Prebiolact (a probiotic organism developed by the
Hungarian Dairy Research Institute), the product contains Hansens
CHN-22 mesophilic butter culture to add aroma. It was found that the
growth curves of the two organisms were distinct, the growth curves
showing maxima at 4.5 h and 6.5 h for the Prebiolact and the butter
culture, respectively. When studied in combination, the growth curve
became more complex, but the maxima at 4.5 h and 6.5 h were still
present, indicating the two organisms did not interfere with each other.
Qin et al. (2006) demonstrated the effect of the water solubility
of various chitosans on antimicrobial activity using isothermal calorimetry. The growth curves of Staphyloccus aureus, Escherichia coli,
and Candida albicans were monitored in the presence and absence
of various chitosans (different molecular weights and N-acetylated
derivatives). It was found that water-soluble chitosans had no significant antimicrobial activity and in some cases increased the growth of
C. albicans. Water-insoluble chitosans were found to have antimicrobial activity, however, when in an acidic medium. Chitosans with
molecular weight of approximately 5 104 were found to be most
active.
Riva et al. (2001) used isothermal calorimetry to evaluate the shelf
life of whole eggs, fresh milk, and carrots. The three foodstuffs were
all subject to microbial spoilage, and microbial growth was monitored.
Complex growth curves were recorded, which showed peaks associated with development of the microbial population. With an increase
in temperature, the peaks appeared earlier and were sharper, indicating
faster microbial growth. The authors selected the point at which the
time derivative of the power signal was at a maximum as the stability
245
time. This qualitative outcome was supplemented with data from plate
counting and pH measurements, which confirmed the increasing power
signal derived from an increase in the microbial population.
Galindo et al. (2005) investigated the change in the metabolic
response of foodstuffs pre- and postprocessing by isothermal calorimetry. Here, minimal processing operations were considered, such as
peeling, grating or shredding. The effect of these operations can impact
the quality of the product through several means; changes in respiration
rate, increased biochemical reactions through wounding stress and
microbiological storage. The rate of many of these processes is dependent upon the surface area of the product, and the authors found relationships between surface area and thermal power for several vegetables
(carrots, rutabagas, and potatoes). It was also possible to monitor enzymatic browning of potatoes and the effect of browning inhibitors, such
as citric acid or ascorbic acid.
Quantitative Studies
Quantitative (which may mean simply determining the number and
nature of reaction processes through to recovery of descriptive reaction
parameters, such as rate constants, enthalpies, and activation energies)
interpretation of complexity in isothermal calorimetric data is demanding. Primarily, this is because, as noted, heat is a universal accompaniment to chemical and physical change. Its ubiquitous nature means that
the measured signal is a composite of the powers arising from each of
the individual events occurring (which can involve physical, as well
as chemical, change). Any meaningful analysis thus has the primary
objective of determining the number of processes contributing to the
overall data. This number alone is a useful basis for quantitative interpretation, because it could be indicative of a reaction pathway and
hence could give some indication of mechanism. Once the number of
steps is known, the data must be deconvoluted into their component
parts. Once the individual processes are identified, analysis to recover
quantitative reaction parameters is usually much more straightforward.
Over the past 15 years, a number of methods have been proposed for
quantitative analysis of calorimetric data, from simple model fitting to
model-free chemometric analysis. Here, these approaches are reviewed
and illustrated with examples of application to food products.
246
Empirical Model Fitting

In the complete absence of any knowledge of the processes occurring
in the sample, or in the case where an equation based on a known
mechanism is not available, the simplest approach to modeling calorimetric data is to fit the data to an empirical equation (i.e., an equation
that conveniently fits the data but does not attempt to describe the
reaction processes occurring). A simple example would be to use an
exponential decay model, such as that shown in Equation 10.1:
x
y = y0 A.e t
(10.1)
where x and y are the plotted variables, y0 is the initial value of y, and
A and t are constants. For example, some calorimetric data are represented in Figure 10.2. The exact process that gave rise to these data is
not important, but it shall be assumed that they represent the heat
output of a partially completed reaction. The data can be fitted to
Equation 10.1 by least-squares minimization to determine the equation
parameters that describe them. Once these values have been determined, it is a simple matter to extend the data to the time at which the
power signal falls to zero (shown by the dotted line in Figure 10.2).
The area under the dotted line thus represents the total heat, Q, which
Figure 10.2. The fit of calorimetric data to an exponential model and the subsequent
extrapolation to power = 0.
247
would be generated by the reaction if it went to completion. From this

information, it is easy to determine the percentage of reaction completed at any time, t, by taking fractional areas. Hence, it would be
possible to determine quantitative shelf-life data, even though the reaction processes remain unclear.
Similarly, taking the total heat output of a process (the area under
the power-time data) allows quantification of the amount of material
reacted if the reaction enthalpy is known. This approach can be used
for quite sensitive analysis of components in foodstuffs when an
enzyme is used to degrade a specific ingredient, because the enzyme
is highly specific for a particular substrate and the calorimeter is able
to quantify the reaction in a complex sample without the need for isolation or purification of the reacting components.
A number of groups have reported enzymatic methods for quantification of ingredients in foodstuffs. For instance, Forte et al. (1996)
demonstrated the utility of using lipolytic enzymes for quantification
of fat content in food. Here, pancreatic lipase was used to catalyze the
hydrolysis of triglycerides (lipases) in a number of food products (oils,
milk, and milk derivatives). A calibration curve was prepared with
tributyrin as a model substrate prior to work on the foodstuffs, and it
was shown that the calorimetric response of the enzymatic turnover
reaction was linear up to a substrate concentration of 15 mM. When
analyzing oils, the authors had to dilute the samples 100-fold in buffer
prior to analysis to ensure the experiment was conducted in this linear
region. Two classes of oils were studied: olive oil (extra virgin, olive,
and husk) and seed oil (peanut, soya, and mixed seed). It was found
that it was possible to differentiate oils from different classes, but not
oils within one class, as their heat outputs were the same within experimental error. It was found possible to differentiate milk samples
(whole, semi-skimmed, and skimmed) and also to quantitate fat content
to 0.1 g/l. In milk derivatives (yogurts), again a linear response was
found for fat contents from zero to 3 g/l, although the slope of the line
was observed to be lower than that of the milk samples. The authors
ascribed this to the fact that yogurts have a microbial population also
capable of producing lipase enzymes that would compete with the
assay reaction.
More recently, the same group demonstrated a similar approach for
the quantification of L-malic acid (Antonelli et al. 2008). In this case,
fumarase is used to catalyze the dehydration of L-malic acid. The
248
calibration curve was found to be linear to 2.68 g/l; above this concentration, nonlinearity was found as a result of the inverse reaction also
catalyzed by the fumarase. The authors compared the assay with a
classical spectrophotometric approach and noted the calorimetric technique gave equivalent answers but with no requirement for sample
isolation or cleanup. L-malic acid was successfully quantified in a
range of beverages (red and white wines, soft drinks, and apple juice)
and solid food products (apples, mandarins, and powder for making
carbonated water).
The use of ascorbate oxidase to quantify ascorbic acid (vitamin C)
concentrations has attracted much attention. Antonelli et al. (2002)
found a calibration curve of calorimetric response versus ascorbic acid
to be linear between ascorbic acid concentrations of 3270 mg/l.
Similarly, ONeill (2004) used this approach to investigate the quality
of fresh orange juices and determined the kinetics of ascorbic acid
degradation (discussed further below).
A derivative technique that can offer some useful insight into the
properties of food substances is solution calorimetry. In this experiment, a solid (usually) sample is introduced to a solvent, and the heat
change upon dissolution is recorded (Royall and Gaisford 2005). The
technique is useful because small changes in the physical form of a
material, such as a change in polymorph or percentage of amorphous
content, will result in a change of dissolution enthalpy. Marabi et al.
(2007) have shown that solution calorimetry can be used to study the
dissolution of two food powders, maltodextrin and skimmed milk. As
the moisture content of the powder increased a concomitant decrease
in the exothermic dissolution, heat was seen. The effect was reversible
if the moisture content was reduced unless crystallization occurred in
the sample. The authors used real-time video analysis to follow dissolution kinetics and related these to the calorimetric data.
However, it is possible to interpret the calorimetric data quantitatively to recover dissolution parameters. Conti et al. (2006), in a study
of the dissolution of various hydroxymethylcellulose (HPMC) polymers, demonstrated how it is possible to convert the calorimetric dissolution data into a plot of swelling ratio; this was achieved by assuming
that the total heat output during the experiment, Q (obtained by integration of the power-time data), corresponded to complete swelling, while
the heat output to any time t (qt) corresponded to the fraction of swelling that had occurred to that point. Hence, a plot of qt/Q versus time
249
Figure 10.3. Power-time data for the dissolution of various grades of HPMC into
water. Reprinted from Conti et al. (2006), with permission from Elsevier.
gave a set of data that represented the swelling response; typical plots
of the dissolution profile of the polymer and the swelling ratios are
shown in Figures 10.3 and 10.4. The swelling curves were then analyzed using a power-law model. It was shown that for all polymers
dissolution occurred immediately following hydration of the polymer,
although there was a rate dependence upon both polymer grade and
solution pH.
Modeling Based on Reaction Kinetics
The next step in complexity from the use of empirical models is to fit
data to models based on reaction kinetics. Clearly, for this approach
to be valid, the mechanism of reaction must be known prior to analysis.
This approach is also not appropriate for those processes that involve
physical change (although if the chemical reaction data can be isolated
from any physical change data this approach is legitimate). While the
use of such models has been documented in full elsewhere (Gaisford
and ONeill 2006), it is appropriate to discuss the simplest case, a
single step, A P reaction process here; logical extension of the
analysis can be made to more complex reaction schemes. The rate of
disappearance of reactant A, or the buildup of product P, is given by
250
Figure 10.4. The swelling profiles of various grades of HPMC calculated from the
power-time data shown in Figure 10.1. Reprinted from Conti et al. (2006), with permission from Elsevier.
dA
= kAn
dt
(10.2)
A = A0 x
(10.3)
dx
n
= k ( A0 x )
dt
(10.4)
Since
Then
Where dx/dt is the rate of reaction, k is the rate constant, A0 is the initial
quantity of reactant A that is available for reaction, x is
the quantity of reactant A reacted at time t, and n is the order of reaction. It should be noted that from a mathematical perspective, n may
have any value, integral or nonintegral, but to have meaning as a
kinetic model only integral values are considered. Immediately, therefore, it is possible to check the validity of a model against real data,
for the values of any reaction orders obtained should be integral. For
any given reaction that has gone to completion, the total heat evolved
251
during the course of the reaction, Q, must be equal to the product of

the enthalpy of reaction, H, and the number of moles of material
reacted, A0:
Q = A0 H
(10.5)
q = x H
(10.6)
It follows that
where q is the heat evolved at time t. Substituting q/H for x and Q/H
for A0 in Equation 10.4 and rearranging gives
dq
n
= = k H 1 n (Q q )
dt
(10.7)
where is the calorimetric power (in watts). Assuming n 1, integration of Equation 10.7 gives
1
(Q q ) = [ k t H 1 n (n 1) + Q1 n ]1 n
(10.8)
This expression may be substituted into Equation 10.7 to give

n
= k H 1 n [ k t H 1 n (n 1) + Q1 n ]1 n
(10.9)
Equation 10.9 describes calorimetric data that derive from reactions

that follow a single-step, solution phase process. Calorimetric data
from such a reaction may be entered into a suitable mathematical
package and, by least-squares minimization, the reaction parameters
may be quantified. This process was first described by Bakri (1988)
and was later extended by Willson et al. (1995).
A further consideration of these equations results in methods to
calculate directly the parameters of interest. Knowing these values
reduces the burden on the fitting program and increases confidence in
the values returned. Here, methods to calculate parameters for singlestep reactions that proceed to completion or to equilibrium are discussed. A discussion of the extension of these principles to more
complex reaction schemes can be found in Gaisford and ONeill
(2006).
252
Reactions That Proceed to Completion

Calculation of the Initial Calorimetric Signal 0
In most calorimetric experiments, data over the first few minutes are
lost (because the sample is prepared externally to the instrument and
there will be a heat of friction introduced by loading), which means it
is not possible to measure the value of the power signal at t = 0 (0).
The value must therefore be inferred in some way from the recorded
data set. A convenient strategy is to apply a polynomial series (usually
fourth order is sufficient) to the first 10 h of any experimental data set
and to extrapolate to a value of 0.
Calculation for the Reaction Order
Knowledge of the value of n is vital because it can give some
mechanistic insight to a data set that contains no molecular information. By selecting two power values from the calorimetric data, 1 and
2, it has been shown that the ratio of the two associated times, t1
and t2, for 1 and 2 is dependent only on the order of reaction (Willson
1995). Rearrangement of Equation 10.9 for the two time points
gives
t1 =
k H 1 n
1 n
n
Q1 n
k H 1 n ( n 1)
(10.10)
and
t2 =
k H 1 n
1 n
n
Q1 n
k H 1 n ( n 1)
(10.11)
and therefore,
1 n
t 2 ( 2 ) n
=
t1 ( )1nn
1
(10.12)
253
It follows that the order of reaction may be determined from knowledge of the value of t2/t1. This is most easily achieved through the use
of a suitable mathematical worksheet. The values of 1 and 2 are
converted into percentages of the initial calorimetric signal (0) and,
by using the worksheet, a table of values of t2/t1 calculated from
Equation 10.12, can be constructed as a function of the rate constant
for a particular pair of (1/0) and (2/0) ratios. The experimental t2/t1
constant may then be compared with the table of t2/t1 values, and the
order of reaction can be determined.
Calculation for the Total Heat Released for Complete Reaction

In the unlikely case that the reaction progresses to completion within
the experimental measurement period, Q is simply the area under the
power-time curve. More commonly, this is not the case, and it becomes
necessary to calculate a value for Q from the experimental data.
Recall Equation 10.7:
= k H 1 n (Q q )
(10.7)
If two values of at different points along the calorimetric curve are

taken, and their associated values of q are noted such that
1 = H 1 n k (Q q1 )
2 = H 1 n k (Q q2 )
(10.13)
(10.14)
Then,
n
1 (Q q1 )
=
2 (Q q2 )n
(10.15)
1 n = (Q q1 )
2
(Q q2 )
Setting
(10.16)
254

1
1n
=R
(10.17)
The value for Q can then be derived from Equation 10.18.

Q=
(q1 Rq2 )
(10.18)
1 R
At this point, it should be noted that the values of q1 and q2 must

include the area under the curve for the missing data always encountered at the start of any calorimetric experiment. It is clear that this
value cannot be measured directly, but it can be approximated (with
reasonable accuracy) from the calculated value of 0 and the accompanying polynomial equation used to derive it, described earlier.
Calculation for Reaction Half-Life

The reaction half-life, t1 2, is defined as the time taken for half the
reactable material to be consumed. Assuming n 1, it is easily calculated from
t1 2 =
(2n1 1)
(10.19)
[( n 1) kA0n1 ]
Hence, if k (see below), A0, and n are known, then t1 2 can be

calculated.
Calculation for Rate Constant
Because the reaction order, n, is known, a kinetic equation that describes
the reaction can easily be written. This equation can then be manipulated to reveal the rate constant. Taking a second-order reaction as an
example,
A0
= k H
1 + kA0 t
(10.20)
255
If the ratio of two data points 1 and 2 at times t1 and t2 are taken,
Equation 10.21 is obtained:
1 (1 + kA0 t2 )
=
2 (1 + kA0 t1 )
(10.21)
This equation can be expanded and rearranged to yield a quadratic

expression in terms of k:
k 2 ( RA02 t12 A02 t22 ) + k (2 RA0 t1 2 A0 t2 ) + ( R 1) = 0
(10.22)
This quadratic function can then be solved in the normal way.

Calculation for Reaction Enthalpy
Earlier it was shown that the total heat output, Q, is given by Equation
10.5. The reaction enthalpy is then easily calculated.
Reactions That Proceed to a Point of Equilibrium
It is important to know whether the reaction under study reaches
completion or equilibrium, because knowledge of the number of moles
of material reacted is essential to calculate the correct value for the
reaction enthalpy. Note: In the following treatment, Q represents
the amount of material that will react and is distinct from QT which
is the value of Q if all the sample (i.e., A0) content had reacted.
Test for Complete Reaction
A test for complete reaction is to study the reaction (for identical loads)
over a range of temperatures. Noting that because the equilibrium
constant (if one exists), K, will change as a function of temperature, Q
must also vary as a function of temperature. If the reaction proceeds
to completion, then the value of Q will remain constant across the
temperature range and will be equal to QT for all temperatures.
Determination of K
For the reaction A B, the equilibrium constant is given by
K=
[ A]
[ B]
(10.23)
256
At equilibrium [B] is given by Equation 10.24 and [A] is described by

Equation 10.25.
[ B] =
Q
H
(10.24)
[ A] = AT [ B ]
(10.25)
QT Q
H H
(10.26)
QT Q
K= H H
Q

H
(10.27)
Q
(QT Q )
(10.28)
Hence,
[ A] =
Thus,
And therefore,
K=
Equation 10.28 can be written for studies at different temperatures, Tm

(m = 1, 2, 3, etc.). Note that QT will remain constant for all values of
T providing that the reaction mechanism does not change and that there
is no dependence of change in heat capacity, Cp, over the chosen
temperature range.
Equation 10.28 then permits K to be calculated for any chosen temperature. However, a value for QT is required in order to effect this
calculation. The value of QT is not directly available from the calorimetric data and must be calculated separately.
Calculation of QT
The calculation of QT is undertaken by consideration of the effect of
change in temperature on the equilibrium constant. The vant Hoff
equation states

H
( ln K )
=
1
R

T
257
(10.29)
If it is assumed that H is independent of temperature, then integration

of Equation 10.29 gives
K1
H 1 1
=
K2
R T1 T2
(10.30)
K1
K
will be equal to 2 , for temperatures T2 and T3, if the
K2
K3
temperatures T1, T2, and T3 are such that Equation 10.31 is true.
The ratio
T1T2
T2T3
=
(T2 T1 ) (T3 T2 )
(10.31)
(For instance, if T1 = 298 K and T2 = 303 K, then T3 will be equal to

308.5 K). If this condition is met, then Equation 10.32 can be written.
Q2
Q1
K1 (QT Q1 ) K 2 (QT Q2 )
=
=
=
Q3
Q2
K2
K3
(QT Q2 )
(QT Q3 )
(10.32)
This can be solved for QT, (for temperatures m = 1, 2, and 3):

Q 2Q + Q 2Q 2Q3Q2Q1
QT = 2 1 22 3
Q2 Q3Q1
(10.33)
The enthalpy is now accessible since QT and AT are known:

QT
= H
AT
Once H is known, A can be calculated:
(10.34)
258

Qm
=A
H
(10.35)
Once these parameters are known, it is a simple matter to calculate the

rate constant, k, using the methods described earlier.
Since QT and Q are accessible the equilibrium constant, K, can be
derived from Equation 28 and hence the values for the Gibbs function
and entropy are readily obtained from Equations 10.36 and 10.37
respectively.
G = RT ln K
(H G )
T
= S
(10.36)
(10.37)
The calculated values of K will allow an internal check on the validity of the procedure through the derived value of H. A plot of
ln K versus 1 will yield a straight line with slope H . If the procedure
T
R
is invalid, the mechanism changes with T, or there is a significant
temperature dependency of heat capacity (over the temperature range
used), the vant Hoff plot will not be linear, and/or the value of H
will not match that derived from Equation 10.34.
Since Q and QT are known and if H is accurately recovered, then
it is possible quantitatively to determine the reactable material content
in a heterogeneous sample.
Although there are fewer examples in the literature, kinetic interpretation of calorimetric data for foodstuffs has been reported. Tortoe
et al. (2007) report a kinetic analysis of the osmotic dehydration of
apples, bananas, and potatoes. In osmotic drying, the foodstuff is
placed in a concentrated sugar solution; water is then drawn out of the
foodstuff as a result of osmosis. A side effect of osmotic drying is the
ingress of sugar into the foodstuff, resulting in a dried, sweetened
material. For all apple samples, a three-phase signal was observed. The
initial, large, signal was assumed to be rapid transfer of water and sugar
from the cut surfaces of the food. The second phase represented movement of water from intracellular spaces, and the final phase represented
movement of water from extracellular spaces. For banana and potato
samples, a two-phase signal was observed. In all cases, the processes
259
were first order, which meant that a simple plot of ln (power) versus
time allowed recovery of the rate constants from the gradients of the
respective phases. The rate constants changed in magnitude in the
order k1 > k2 > k3 and increased with temperature. Arrhenius plots of
the data suggested a linear relationship for Golden Delicious
apples and potatoes and nonlinear behavior for Cox apples and
bananas.
As noted, ONeill (2004) studied the kinetics of ascorbic acid in
freshly squeezed orange juice. This is more complex than simple
studies of the oxidation of ascorbic acid because spoilage of fresh
orange juice generally results both from oxidation of ascorbic acid
and from degradation of pectin by pectin methyl esterase. Although it
is still the subject of debate, it is thought that the degradation of
pectin results in protein agglomerates that settle, resulting in clarification of the juice. Clarification is a major cause of spoilage in orange
juice, and strenuous efforts are made to eliminate it. This can be done
in a number of ways. Classically, the juice is pasteurized to denature
the protein as well as destroy the microbial flora. However, heat
treatment adversely affects the flavor and aroma of the juice and is
generally unsatisfactory if the juice is to be marketed as fresh-squeezed.
Alternatively, the juice is frozen during storage and transportation,
reducing the activity of the enzyme and other degradative processes.
This is costly and usually only viable for concentrated juice. An ideal
solution would be some natural additive that does not adversely affect
the desired quality parameters for orange juice but does inhibit pectin
methyl esterase, in quantities that are not harmful, reducing cloud
destabilization. Selection of such a material starts with the ability
quantitatively to monitor the reaction processes directly within orange
juice samples.
Both the oxidation reaction and the enzyme reaction are pH dependent, with the oxidation reaction favoring alkaline conditions and the
enzyme reaction favoring more neutral conditions. By buffering the
orange juice at pH 7.0, ONeill (2004) held the enzyme reaction at
almost optimal conditions, while the oxidation reaction was more
favored compared with the naturally more acidic pH of orange juice.
The calorimetric data revealed two distinct first-order phases (Figure
10.5). The first reaction progressed with a rate constant of 3.9
(1.0) 105 s1, and the second progressed with a rate constant of 2.7
(0.3) 105 s1.
260
Figure 10.5. Observed calorimetric output for buffered orange juice. Reprinted from
ONeill (2004), with permission.
Analysis of the pectin/pectin methyl esterase reaction revealed it to

progress with a rate constant of 3.9 (0.3) 105 s1, identifying this as
the first process in the orange juice sample. A similar analysis of the
oxidation of ascorbic acid in buffer gave a rate constant of 3.0 (0.3)
105 s1, highlighting this as the second process. A further benefit of
the kinetic analysis was the recovery of the enthalpy of oxidation of
ascorbic acid, 155 25 kJ/mol. This information allows the quantification of ascorbic acid content in the orange juice sample. From Figure
10.5, it can be seen that the calorimetric output from approximately
100,000 s onwards can be solely attributed to the oxidation of ascorbic
acid. This being so, then the calorimetric output at t = 0 (0) for the
oxidation reaction can be obtained from the y intercept of the ln
versus t plot. This value is quantitatively proportional to the amount
of ascorbic acid of the sample:
A=
0
kH
where A is the quantity of ascorbic acid.
(10.38)
261
Summary
The study of foods and food ingredients is complicated, largely because
physical form impacts the selection and use of any analytical assay
technique. Because it measures a universal property and is invariant to
physical form, calorimetry offers an exciting opportunity for the investigation of foodstuffs. The full capabilities of calorimetry have not yet
been exploited in this area, although the number of reported applications is increasing. Here, it has been shown that calorimetry can be
used to study a huge variety of samples, from simple ingredients (such
as ascorbic acid) to complex biological processes. Data interpretation
ranges from qualitative to quantitative, the analysis methods being
dependent upon the complexity of the sample. However, a combination
of clever experimental design, sample preparation, and data analysis
mean that quantitative outcomes are increasingly available from calorimetric data matrices, and the application of the technique to foods
and food ingredients can only continue to increase.
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Chapter 11
Use of Thermal Analysis to Design and
Monitor Cereal Processing
Alberto Schiraldi, Dimitrios Fessas, and Marco Signorelli
Introduction
Starch
Proteins
Nonstarch Carbohydrates
Process Applications
Conclusions
References
265
268
272
276
278
285
285
Introduction
The term thermal analysis (TA) means the record of any physical property during a given thermal treatment under strict temperature control.
The main physical property that is monitored in this way is enthalpy
(and the related property, heat capacity); the relevant thermal analysis
is named calorimetry and is performed in a few well-defined conditions, each with a specific name: isothermal calorimetry (IC), differential scanning calorimetry (DSC), temperature-modulated DSC
(TMDSC), and modulated adiabatic scanning calorimetry (MASC).
Another physical property that is traditionally included in the TA
realm is mass; the relevant analysis is named thermogravimetry (TGA)
and is currently used to monitor the mass loss upon heating the sample.
Its trace is often transformed into that of its time derivative (DTG).
The mechanical properties of a given sample, like Youngs modulus,
E, elastic and storage moduli, G and G, respectively, are those
265
266
recorded with the dynamic mechanical analysis (DMA, or DTMA) and

thermomechanical analysis (TMA). Applications related to the dielectric character of the sample go under the name of dielectric analysis
(DEA).
Every type of TA has been used in the study of food stuffs and food
processes. Calorimetry was used to determine heat capacities
(Schwartzberg 1976; Noel and Ring 1992), describe the behavior of
frozen ice-forming food systems (Goff, Montoya, and Sahagian 2002),
and study many phase transitions (Roos 1995), including those that
imply a heat capacity drop with no transition enthalpy, like the socalled glass transition (Slade and Levine 1991). Since most of these
changes imply a substantial modification of the mechanical and rheological properties of the sample, DMA and DEA also were used to
describe these phenomena (Laaksonen and Roos 2000; Vodovotoz,
Hallberg, and Chinachoti 1996). A rather recent improvement of thermogravimetry is the Knudsen TGA that allows determination of the
water activity along the whole dehydration pattern of a given sample
in isothermal conditions (Schiraldi and Fessas 2003).
Because of the intrinsic heterogeneity of many food materials, a
suitable sampling is crucial for the reliability and the reproducibility
of the recorded TA traces, which usually is much poorer than for purified chemicals. Too-small samples may not represent the food investigated; smashed or powdered products may miss some important
feature of the starting material, for example, the surface area/volume
ratio that affects the rate of diffusion-limited processes and the effects
related to the overall texture of sample that can be missed when the
material is ground or finely sliced (Riva, Schiraldi, and Piazza 1994).
The simultaneous occurrence of different changes within a given
sample is another reason for the humping and bumping trend of
the TA traces from food samples. In DSC, baseline shifts due to glass
transitions are often partially overlapped to peak signals of first-order
transitions and chemical, biochemical, and microbe-sustained reactions. This explains why DSC investigations were often devised to
allow just qualitative tests or comparison between samples and why
tentative evaluations of the enthalpy changes, as in the case of starch
gelatinization and retrogradation, are often a major cause of discrepancy between different authors who made different choices for the
baseline trends. As a consequence, DSC has been considered a substantially qualitative analytical tool by many food scientists and
technologists.
Use of Thermal Analysis
267
Fortunately, this is not true, provided that the operators are well
trained and adequately educated in physical chemistry. Suitably performed TA indeed provides a real insight into the processes that occur
within a food system during a thermal treatment, as the relevant change
of the involved physical property is directly related to the laws of
thermodynamics and kinetics. For example, any thermal effect, H,
when coupled with a temperature value, T, is a measure of the stability
of the system, thanks to the relationship,
G = H
T2
T T p
(11.1)
where G is the Gibbs function. Moreover, the rate of heat release or

adsorption, Q = dQ/dt (where Q and t stand for heat and time, respectively), is directly related to the rate of the underlying process,
d
Q = H ,
dt
(11.2)
similar expressions holding for the change rate of practically every

physical property recorded during a temperature scan.
These laws are of great help in recognizing the true trend of a
baseline (Roduit 2000) and the true transition points, because they
provide means to make reliable predictions. The problem that cannot
be easily overcome concerns the number of partially overlapped phenomena that take place during a given heating or cooling run, each
relevant to a single component of the system, as in the case of cerealbased food products. A deconvolution of the relevant trace therefore
becomes a necessary step of the data treatment (Schiraldi 2003).
This chapter reports a short critical review of the applications of TA
to the study of cereal-based food and related processing. The main
concern of these products deals with the transitions of starch in the
presence of other components of the system, such as proteins and
nonstarch carbohydrates, which compete for the available moisture
and/or affect its partition between the different regions or phases of
a given food system, and lipids, which are responsible for specific
interactions that produce peculiar DSC signals. Since water is ubiquitous in most food systems, it is expedient to look at the changes that
take place within a given sample by focusing on water and directly
detecting changes in the state of water that are concomitant with
268
changes relevant to the other components of the system. Particular

emphasis therefore is given in this chapter to approaches based on the
choice of water as a probe compound.
Starch
Starch is a supramolecular substance that nature assembles within the
seeds of cereals and pseudocereals, legumes, and tubers. Starch may not,
therefore, be referred to as a chemical compound: it has neither a definite molecular mass nor a fusion point, and it does not react with other
substances until its granular structure is rotten and its glucose polymers, amylose and amylopectin, are exposed to the surrounding environment. Starch chemistry starts with surface processes that take place
at the pores and defects of the granule structure, which remains practically unaffected at temperatures below 45 C, even with excess water.
An aqueous suspension of starch granules is easy to prepare and
investigate with TA techniques. A superficial wetting takes place when
starch granules are dispersed in excess water. The process is exothermic and can be detected with isothermal calorimetry (IC) and suitable
mixing cells (Riva, Piazza, and Schiraldi 1991).
DSC (and related variations) equipment (Liu and Shi 2006) is instead
enough to monitor changes that take place when the starch suspension
is heated (Figure 11.1).
Across a 30 C range, starting from an onset temperature that depends
on the vegetal origin of the starch investigated (e.g., 45 , 50 , and 65 C,
for potato, wheat and rice, respectively), water enters the granule and
disaggregates the internal crystal regions that are mainly formed by the
side branches of amylopectin molecules. The whole starch granule is
transformed in a swollen jelly ghost of the original hard and birefringent
body. A gentle stirring turns the starting suspension into a dispersion of
amylopectin gel and amorphous insoluble amylose. The two glucose
polymers are mutually incompatible (Kalichevsky and Ring 1987),
which means that they may not stay in the same phase, being competitors
for the available moisture.
Because of this, the system is rather heterogeneous and unstable. On
further heating, the amylopectin gel turns into a sol, while around
90 C, nucleation of amylose crystals can take place. The whole process,
currently dubbed starch gelatinization, is therefore a multistep, fully
269
Figure 11.1. DSC trace of a suspension of starch (Merck product) granules in excess
(72.5%, w/w) water, recorded on heating (upper curve) and cooling (lower curve) at
5 C min1 scanning rate (authors unpublished data).
irreversible (Figure 11.1) transformation of the starting suspension of

starch granules. The corresponding DSC trace presents a wide endothermic signal, with maximum at a temperature that depends on the
moisture content of the system: the lower the moisture, the higher the
temperature of the maximum (Figure 11.2).
Moreover, mainly because of the different sizes of the starch granules, samples with low moisture content (in any case, never below
70%) show a high T shoulder, which has been attributed to the delayed
degradation of larger granules (Biliaderis, Page, Maurice, and Juliano
1986), although the reliability of this interpretation has yet to be convincingly demonstrated. A further endothermic signal (in the 90
115 C range) can often be observed (Figures 11.1 and 11.2) in the
DSC of aqueous suspensions of starches extracted from some flours
that contain lipids: the signal corresponds to the fusion of amyloselipid complexes formed in the course of the starch gelatinization
(Eliasson 1994; Bulpin, Welsh, and Morris 1982; Le Bail 1999).
Because amylopectin molecules can carry phosphate groups (the
number of which once again depends on the vegetal origin of the
starch), the properties of the gel obtained are affected by the ionic
strength and the pH of the surrounding medium. In particular, when
270
Figure 11.2. DSC traces of suspensions of starch (Merck product) granules at various
water contents (72.5%, 61.1%, and 42.5%, w/w) recorded on heating at 5 C min1
scanning rate (authors unpublished data).
the system is cooled to room temperature (and below), the amylopectin

gel becomes the matrix of growing crystals that entrap water and cause
the system to harden. Although the structure of the original granules
is by no means restored, the term starch retrogradation is currently
used to indicate this process. It can be easily monitored with DSC
investigations (Riva, Fessas, and Schiraldi 2000). It must be understood that the crystal phases formed are sufficiently extended to allow
X-ray detection (Zobel 1988; Yuryev et al. 2004), but they should be
envisioned as islets of ordered arrays of chains with disordered moieties dangling out of their boundaries, rather than as well-shaped
microcrystals. Crystal islets of different extension are present throughout a given amylopectin gel, and various types of crystal structures
may be formed, named according to the respective X-ray diffraction
patterns A, B, C, etc. (Zobel 1988; Yuryev et al. 2004). For these
reasons, the progress of the crystal growth can imply the coexistence
of different polymorphs that have different thermal stability, and when
the retrograded system is warmed, the fusion process therefore
encompasses a temperature range between 30 and 90 C (Figure 11.3)
(Riva, Fessas, and Schiraldi 2000).
If the system undergoes a suitable annealing treatment, formation
of amylose crystals becomes possible (Wasserman et al. 2007). Their
fusion takes place in the 125 140 C range and can be easily detected
with a DSC investigation (Wasserman et al. 2007). Figure 11.4 shows
271
Figure 11.3. Endothermic effect related to the fusion of amylopectin crystals formed
within the starch gel phases of a bread dough (data are given as excess heat capacity,
namely heat flux, divided by the scanning rate sample mass product). Dashed curve
describes the trend of the of the relevant enthalpy versus the storage time. The records
were obtained at 2 C min1 heating rate. Modified from Riva et al. (Riva, Fessas, and
Schiraldi 2000).
two DSC traces obtained from the same aqueous starch suspension:
the dashed profile refers to the first heating (2 C min1) run, during
which starch gelatinization and fusion of amylose-lipid complexes
occurred, whereas the full line profile refers to the heating run performed after a suitable annealing treatment (Wasserman et al. 2007)
that favors the formation of amylose crystals. The latter shows the
endothermic effect related to the fusion of amylopectin crystals, while
a second endothermic peak with onset at a much higher temperature
is related to the fusion of amylose crystals. The growth of these crystal
phases prevents the formation of amylose-lipid complexes (no signal
appears in the expected temperature range).
These amylose crystals are resistant to the amylase assisted hydrolysis: the hosting system has therefore received the misleading name
of resistant starch, which meets industrial and commercial needs
rather than the chemical truth.
A starch gel heated in an open crucible releases its moisture with a
diffusion-limited mechanism (Fessas and Schiraldi 2005). This means
that solvation water can be easily exchanged with water engaged in
the structure.
272
Figure 11.4. DSC traces obtained from an aqueous starch suspension (heating rate
2 C min1; data are transformed in apparent heat capacity, dividing the heat flux by
sample mass heating rate). The dashed line refers to the first heating. The heavy
line corresponds to the record of the reheating run performed after a suitable annealing
treatment (Wasserman et al. 2007). Notice that the fusion of the amylopectin crystals
(formed during the annealing) occurs at lower temperature, while no signal relevant
to amylase-lipid complexes appears and that a new endotherm occurs at much higher
T corresponding to the fusion of amylose crystals (formed during the annealing).
In short, all these starch transformations take place in a cereal flour

dough in the presence of many other substances that compete for the
available moisture or may directly interact with starch carbohydrates.
The relevant DSC traces should therefore be interpreted by taking into
account the possible interactions between different dough components.
Blends of flours from cereals and pseudocereals allow dough preparations in which many interactions are expected (Fessas et al. 2008).
These complications can be addressed by considering the potential
role of the other nonstarch main components of a dough, namely,
proteins, nonstarch carbohydrates, and lipids.
Proteins
In most cereals, both globular and networking proteins are present. The
former, dubbed albumins and globulins after Osborne (Osborne 1924),
can be either enzymes or carriers, are soluble in aqueous media, and
273
are therefore easily extractable. The latter tend to form wide threedimensional meshes that entrap aqueous phases and separated bodies,
like starch granules. Gluten is the most important representative of this
family: it is not soluble in water and therefore can be separated by
washing a dough loaf with hot water to wash away starch carbohydrates and globular proteins. Minor amounts of nonsoluble compounds
remain entrapped in the gluten meshes and represent the unavoidable
contaminants of any gluten preparation. Details about the chemistry
of the cereal proteins are beyond the scopes of this chapter; suffice it
to say that when a dough is prepared from a cereal flour, globular
proteins play mainly a surfactant role that is crucial in stabilizing the
air bubbles formed in a leavening loaf, whereas gluten is responsible
for the overall rheological behavior of the system.
Both globular proteins and gluten fix water molecules, although in
rather different ways. The former are normally solvated at the surface
polar groups and modify their own solvation shell when unfolding and
denaturation take place. Gluten instead uses water molecules as bridges
between the next neighboring chains (Belton 1999) and develops an
extended network, due to a large number of hydrogen bonds (Figure
11.5).
Because of this, gluten can entrap large amounts of interstitial water
within its meshes. Some disulphide bonds provide more robust interand intrachain links and affect the overall extensibility of the network.
Any elongation strain squeezes the interstitial water out of the meshes
so that the next neighboring chains become closer to one another; when
the strain is allowed to relax, water can reoccupy the interstitial regions
and make the meshes swell back to the starting size. This view has
been recently perfected (Belton 2005; Kontogiorgos and Goff 2006),
and it is still adequate to suggest general guidelines for our understanding of the competition of various flour components for the available
water that undergoes displacement between the coexisting aqueous
phases of a cereal flour dough from its early mixing, to proofing and
baking, or freezing and storing.
This a fundamental issue to be considered. Because of the thermodynamic incompatibility (Tolstoguzov 2003) between proteins and
carbohydrates, as well as between different carbohydrates (Liu and Shi
2006), a flour dough is indeed a dispersed system in which several
aqueous phases coexist that can exchange the solvent between one
another.
274
Figure 11.5. Naive picture of the water bridges and direct hydrogen bonds between
hydro-compatible biopolymer chains (which can be referred to as carbohydrates or
gluten proteins).
This partition of water can be detected with thermogravimetry investigations (Fessas and Schiraldi 2005): the related DTG trace shows a
broad signal that can be deconvoluted in two or more components,
each relevant to a given water fraction (Figure 11.6).
The water fraction that sustains the evaporation at mild temperatures
mainly belongs to the imbibing moisture (in the earliest steps) and by
the separated (because of the thermodynamic incompatibility of their
solutes, such as carbohydrates and globular proteins) aqueous phases:
the solvent that evaporates from one aqueous phase is quickly replaced
by the water migrating from any neighboring aqueous phase. As a
result, the dehydration of the samples looks like a single process governed by the core-to-surface diffusion of moisture (Fessas and Schiraldi
2005). The rest of the water (about 15% of the starting overall dough
moisture) tends to remain close to the network forming polymers (mainly
gluten) and can be stripped away only at temperatures above 100 C.
Starch gelatinization is mainly sustained by the former water fraction when the dough is being baked. Because the starch gelatinization
275
Figure 11.6. Deconvolution of the DTG trace obtained from a wheat dough sample.
The high-temperature peak can be related to the water fraction tightly trapped within
the gluten meshes, while the lowest broad peak is related to the imbibing water that
is released through a Fickian diffusion mechanism. The other minor peaks should be
referred to as contributions from moderately bound water fractions.
rate is strongly dependent on the starch/water mass ratio, the loss of

water reduces the degree of gelatinization attained (Fessas and Schiraldi
2000) (Figure 11.7).
The effect is obviously different in the various regions of a dough
loaf, being more severe at the surface where dehydration is faster and
the crust is being formed.
Two main situations can be envisaged: namely, before and after the
onset of the starch gelatinization. Wetted starch granules fix a relatively small amount of water, while most of the solvent is engaged by
salts, globular proteins, water-soluble nonstarch carbohydrates, and
gluten. The hydration shell water is the poorly available fraction of the
overall dough moisture, whereas the rest of the solvent, including water
molecules trapped in the gluten meshes and in the nonstarch carbohydrate-entangled coils, can be driven toward the starch granules once
the gelatinization onset is trespassed. This onset in turn depends on the
available moisture: addition of small molecular mass solutes that can
fix water produces a delay of the starch gelatinization, while addition
of hydratable polymers, such as water-soluble arabinoxylans, has no
significant effect. The reason for this difference is due to the different
effect on the relative humidity (RH) of the system: for a given dry
matter content, an aqueous solution of simple sugars or salts shows a
276
Figure 11.7. Deconvolution of the DTG trace of a manually mixed dough with 42%
moisture content (30 mg sample, 2 C/min heating rate) and starch gelatinization of
a dough undergoing heating at the same heating rate in a open DSC pan. Modified
from Fessas and Sachiraldi (Fessas and Schiraldi 2000).
much lower RH than an aqueous solution of arabinoxylans (Fessas and

Schiraldi 1998).
Combinations of TA with other techniques, such as NMR relaxometry (Vittadini et al. 2003; Lopes-Da-Silva et al. 2007), MRI (Hills
1998), and NIR (Huang et al. 2003), provide the experimental evidence
of these changes, even in a very complex system such as staling bread
(Morgan, Fourneaux, and Stanley 1992; Schiraldi, Piazza, and Riva
1996). They can, however, be better understood once the principle of
thermodynamic incompatibility is put at work.
Nonstarch Carbohydrates
Cereal and pseudo-cereal flours contain some nonstarch carbohydrates
coming from various regions of the seed. A fraction of them is not
water extractable and therefore is segregated from the aqueous phases
of the dough: the role of this fraction has little relevance to the physical
behavior of the other dough components (safe for some sensorial properties of the final products).
277
Figure 11.8. DTG traces of dough prepared with buckwheat (a), wheat (b), and 50%
(w/w) buckwheat/wheat flour (c) (authors unpublished data).
The water-extractable fraction conversely plays a crucial role on the

overall viscosity of the dough and to the partition and displacement of
water (Fessas and Schiraldi 2001a; Courtin and Delcour 1998; Fessas
and Schiraldi 2001b).
Although only one water molecule per single sugar monomer can
be engaged to solvate these polymers, much larger amounts of moisture are trapped within their entanglements and can be easily displaced
under the effect of chemical potential gradients.
Because a flour dough is a quite viscous environment, only shortrange displacements are indeed allowed. This explains why when the
dough is being dehydrated, as in the course of a TGA run, the water
displacements through the samples are rapidly hindered by the concurrent increase of the viscosity.
The flour of some gluten-free cereals and pseudo-cereals, such as
buckwheat, soy, and amaranth, can trap water because of different
proteins but cannot form a stable dough because the polymer chains
do not arrange themselves in a web. This water fraction is therefore
much more mobile than the moisture trapped within gluten meshes.
The relevant DTG trace therefore shows a single diffusional (see
above) peak (Figure 11.8). When one of these flours is mixed with
wheat flour to form a blend, then the dough can be formed upon kneading. The relevant DTG trace (Figure 11.8) shows two peaks: the high
T signal is once again attributed to the moisture fixed by the gluten
278
meshes, although the temperature gap between the two maxima, which
is related to the looseness of the gluten network, is smaller than in the
trace of a wheat flour dough. This effect is mainly related to nonstarch
and nongluten proteins, which form separate phases (droplets) that do
not allow gluten to attain an extended and tight reticulation (Fessas
and Schiraldi 1998).
Process Applications
The information collectable with TA investigations can be directly
used to improve food formulations and process conditions. Of great
interest are the use of the so-called state diagrams that highlight the
role of the glass transition temperature as a border between high- and
low-molecular-mobility regions, which have been collected in Rooss
book (Roos 1995). Few other examples are helpful to the reader.
Any given thermal treatment, such as cooking, baking, frying, etc.,
corresponds to a thermal history experienced by the system. A direct
reproduction of such history can be difficult to plan when using TA
equipment. Nonetheless, one can inscribe any thermal history in a TTT
(time temperature transformation) diagram that can be defined on the
experimental basis of TA evidence of a given transformation responsible for a specific signal in the TA record. Starch gelatinization monitored with DSC is a good example. The relevant signal is an endothermic
shouldered peak, the area of which, once divided by heatingrate starch-mass product, corresponds to the specific enthalpy change,
H (joules per gram of starch units), accompanying the gelatinization.
The peak area swept at any given T within the (Tonset, Tend) range is
related to the progress degree, , achieved at that T:
(T ) =
partial peak area

.
total peak area
(11.3)
To account for the effect of the moisture content, the reference to be

chosen is the thermal effect recorded in excess water, , which is
larger than those observed at low water contents since only a fraction
of the overall starch undergoes gelatinization (or experience a complete transformation) when moisture is less than 40% (w/w). The total
peak area must accordingly be scaled with respect to the area of the
279
peak recorded in excess moisture. To clarify with an example, if the

recorded H is 0.75 H, then
(T ) =
partial peak area

0.75
total peak area
(11.4)
Since starch gelatinization is an irreversible transformation, the relevant DSC signal reflects the process kinetics and is affected by the
heating rate, = dT/dt. The higher the heating rate, the higher the end
temperature of the signal (there is also a shift of the apparent onset
temperature that is of minor concern for the present discussion,
however). One therefore has to define the temperature range where
starch gelatinization takes place for each and the progress degree
within that range:
d
d dT
d
Q = H = H = H
dt
dT dt
dT
(11.5)
where Q is the recorded heat flux per unit mass of starch and H is
the related enthalpy change.
Once the DSC runs at the selected heating rates are performed, the
increase of on sweeping the related peaks can be determined: more
precisely, one has to detect the temperatures at which attains some
given levels (say = 0.1, 0.2, 0.3, etc.) for each considered (Figure
11.9).
The TTT diagram is a T-versus-t plot, where the straight lines corresponding to the various heating rates considered in the experimental
design of the DSC investigations have to be drawn first.
The selected iso- temperatures (see above) have to be marked
along the corresponding straight line in the TTT diagram. Eventually,
a map of iso- points is obtained that can be used to draw iso- curves
(Figure 11.10).
Remember that the maximum attainable does not depend on ,
being mainly related to the available moisture. This is a third fundamental parameter to be accounted for. The experimental design must
therefore include DSC runs performed with samples of a different
moisture content, and a third axis can be added to the TTT diagram to
account for it.
In the three-dimensional TTT diagram, the iso- loci are surfaces.
For a more practical approach, samples can be partially dehydrated by
Figure 11.9. Extent of starch gelatinization, , on increasing temperature, according

to the DSC data recorded at various heating rates, . Iso- temperatures have to be
reported in the TTT diagram.
0.5
140
120
0.5
T / C
100
0.8
0.6
80
0.4
60
0.1
0.2
40
20
0
0
50
100
150
200
250
t / min
Figure 11.10. Iso-moisture (excess water) TTT plane relevant to starch gelatinization
in bread dough samples. The dotted straight lines correspond to different heating
temperatures (0.5 , 1 , 2 , 5 C/min), while the data points are the drawn from the
-versus-T plots to evidence the attainment of a given level (values reported at the
right-hand side). The tie lines connecting these points are the iso- curves. Modified
from Fessas and Schiraldi (Fessas and Schiraldi 2000).
280
281
Figure 11.11. Bread dough samples: starch gelatinization extent achieved in DSC
open pans. Modified from Fessas and Schiraldi (Fessas and Schiraldi 2000).
heating them in open DSC pans within the furnace of the instrument
at a given heating rate. The run has to be stopped at a temperature, Ti,
that can represent a rough average of the real thermal history experienced by the product. The DSC sample pan is then quickly cooled to
room temperature, sealed, and again heated at the same rate to evaluate
the residual starch gelatinization. The relevant moisture content of the
sample at Ti (and in the second DSC run with sealed pans) can be
eventually determined as the mass loss after an overnight rest at 105 C
after piercing the cover of the sample pan with a needle (Figure 11.11).
This experiment allows determination of the fraction of starch gelatinization that occurred during the first (open pans at variable moisture
content) and the second phase (sealed pans at constant moisture content)
(Fessas and Schiraldi 2000). An ideal experiment should in principle
be performed with a TGA-DSC combined instrument. Unfortunately,
this cannot be a realistic choice, since the vaporization enthalpy of
water (2.3 kJg1) is 2 orders of magnitude larger than the enthalpy of
starch gelatinization. A superposition of separate investigations can
nonetheless be performed (see Figure 11.7).
The corresponding TTT diagram (Figure 11.12) is indeed a curved
section of the three-dimensional plot. On the same diagram, any
thermal history actually experienced by the system, as well as the
282

120
T / C
100
0.7
0.6
0.5
0.4
80
0.3
0.2
0.1
60
a
40
20
thermal histroy
10
15
t / min
20
25
30
Figure 11.12. TTT diagram corresponding to the data reported in Figure 11.11. The
figure is a projection of a surface of the three-dimensional TTT (moisture-vs.temperature-vs.-time) diagram.
moisture loss that occurred in the real process, can be represented with
a process path. The corresponding curve crosses the iso- surfaces
(Figure 11.13).
The highest attained may not regress and can indeed be referred
to as the maximum progress of starch gelatinization achievable at the
end of the thermal history considered. Obviously, one must take into
account that real systems have a much larger mass than a DSC sample
and therefore experience temperature and moisture gradients on
cooking or baking. For a more detailed simulation of the process, one
therefore must first record the thermal history and the moisture changes
in each region of the real system and then draw the relevant information about the extent of the starch gelatinization achieved.
Another practical use of the information drawn from TA investigations concerns the effects of mechanical treatment, such as kneading,
and layering, that affect the tightness of the gluten network of wheatbased products. Mechanical stresses squeeze some water out of the
gluten phase, thus allowing protein moieties to come closer to one
another and, possibly, to form direct links (mainly hydrogen bonds
[Belton 1999; Belton 2005]). If the product is baked just after the
experience of such mechanical stresses, more water evaporates on
baking, and the final product is harder and brittle. This occurs in bis-
283
Figure 11.13. Sketched representation of the real thermal history experienced by the
product (sigmoid curve) that crosses the iso- surfaces in the TTT diagram. The figure
is a projection of a surface of the three-dimensional TTT (moisture-vs.-temperaturevs.-time) diagram. Modified from Fessas and Schiraldi (Fessas and Schiraldi 2000).
cuits (Piazza and Schiraldi 1997). The experimental evidence related

to this effect is provided by the DTG trace of the dough. The high T
gluten peak of a stressed (overmixed) dough is smaller and closer
to the large low T peak (Fessas and Schiraldi 2005). This indeed
means that some water migrates from the gluten phase toward the
neighboring starch-rich regions (these are short-range displacements),
where it can evaporate more easily. A couple of hours rest is, however,
enough to restore the previous water partition (Fessas and Schiraldi
2005). The relaxed dough leads to a softer baked product (Piazza
and Schiraldi 1997).
The DTG trace can be of help also in predicting the effect of extra
ingredients on the final properties of a given product. The effect of the
nonstarch carbohydrates acting as hydrocolloid sinks of moisture
appears in the DTG traces with a downward shift of the high T peak.
The main difference from the effects related to mechanical stresses is
that this peak corresponds to more than 20% of the overall dough
moisture. This means that the water fractions bound to hydrocolloids
and gluten, respectively, have similar fugacities, both being less free
284
A

B
Figure 11.14. Pictures of the alveolar crumb structure of bread from wheat (A) and
a wheat-buckwheat blend added with soluble polysaccharides (B).
than the other water fractions of the dough. Some visual evidence of
this picture is provided by light microscopy investigations (Autio and
Salmenkallio-Marttila 2001; Autio and Laurikainen 1997) that show
islets of nonstarch hydrocolloids dispersed within the gluten meshes,
apparently hindering the contacts between them and preventing the
formation of a tight network. If this situation is not perturbed by
mechanical stresses, the water loss during baking is smaller than for a
dough with no extra hydrocolloids. The result is a product with broader
crumb alveoli (Fessas and Schiraldi 1998). The nonstarch hydrocolloid
water sinks keep the final product softer for a longer period, thus
acting as antistaling ingredients (Fessas and Schiraldi 2001a). One may
define an optimal shift of the DTG peak that corresponds to the
desired tightness of the final network by adjusting the amounts of extra
hydrocolloids added to the recipe.
The case of bread prepared from a blend of wheat and buckwheat
flours can be a suitable example (Fessas et al. 2008), where the nonstarch carbohydrates were those water-extracted from the buckwheat
hull in a separate process and added as an aqueous solution to the
dough. The bread prepared from a blend of wheat and dehulled buckwheat had a crumb with alveolar distribution quite similar to that of
the crumb of the bread obtained from wheat flour (Figure 11.14),
although with a 45% greater density.
In this modified dough, the effects of soluble nonstarch polysaccharides has been tuned through the counterbalancing action of
285
globular proteins that play the role of surfactants that stabilize the
dough matrix/air interface (Fessas and Schiraldi 1998).
Conclusions
Thermal analysis is very suitable for monitoring the transformations
that take place in cereal-based food products. It requires, however,
specific training for operators because the records obtained from a food
sample usually have to be mathematically treated to unveil the single
contributions coming from largely overlapped events relevant to different components or phases of the system.
Combination with other experimental techniques is nonetheless of
great help, especially when water partition and displacements are
involved in the changes induced by the process.
Specific applications to simulate thermal treatments and adjust the
recipe of a given product can be drawn from either DSC or TGA data.
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Chapter 12
Importance of Calorimetry in Understanding
Food Dehydration and Stability
Yrj H. Roos
Introduction
Phase and State Transitions of Food Components
Calorimetric Glass Transition Measurement
Dielectric and Mechanical Relaxations
Thermal Analysis in Characterization of Food
Systems
The Frozen State of Foods Systems
State Diagrams and Dehydration
Spray-Drying
Freeze-Drying
Glass Transition and Stability of Dehydrated Materials
Conclusions
References
289
292
293
297
298
301
303
305
306
307
308
309
Introduction
Dehydration involves removal of solvent water from dissolved and
hydrated food components. The process requires heat for evaporation
or sublimation of water and concentration of food solids at high levels.
This results in water removal to an almost anhydrous state of food
components. Traditional dehydration processes are based on empirical
knowledge of food material properties and processing needs to achieve
desired product characteristics. Advanced dehydration processes, such
289
290
as spray-drying and freeze-drying require more a fundamental

understanding of water or ice properties and their removal as well as
knowledge of physicochemical properties of the dehydrated solids
(Roos 1995, 2002a, 2004). Thermal analytical and calorimetric
measurements can often be used to characterize phase transitions of
food solids and water (2002b). These measurements provide data that
can be used to adjust dehydration conditions and temperatures to
improve dehydration processes and product characteristics, such as
flavor retention, storage stability, and flowability of powders (Roos
1995, 2004).
Various phase and state transitions occur in food dehydration and
storage of dehydrated foods. Phase transitions typically include
evaporation of water and crystallization of food components (precrystallization before dehydration, crystallization during dehydration, and
crystallization during storage). Most dehydrated materials, however,
contain noncrystalline, amorphous solids. These can exist as
solid glasses or viscous, supercooled fluids (White and Cakebread
1966; Slade and Levine 1995; Roos 1995, 2004). The glassy state
of materials refers to the nonequilibrium, solid state, which is universal
of all glass-forming materials, such as inorganic glasses, and synthetic
noncrystalline polymers, sugars, and proteins as the main amorphous
food components. Typical characteristics of the glassy state include
transparency, solid appearance, and brittleness (White and Cakebread
1966; Sperling 1992). In noncrystalline, amorphous systems, molecules have no ordered structure, and the volume of the system is
larger than that of the equilibrium crystalline systems with the
same composition. Amorphous, noncrystalline systems can exist
as glassy solids or supercooled liquids (rubber, leather, syrup) (Slade
and Levine 1991; Roos 1995, 2004; Slade and Levine 1995), depending on their physical state, that is, apparent solid or liquid-like
properties.
The transition in which a solid, glassy material undergoes a change
to a supercooled liquid is a change in state of the material rather than
a change in phase (Sperling 1992; Roos 1995). This state transition is
universally referred to as glass transition. Glass transition involves a
change in heat capacity, which can be measured by calorimetric
methods. As the glass transition is a change in the state of the system,
it also results in a dramatic change in mechanical properties (Roos
1995). Changes in state and flow properties in food systems greatly
Importance of Calorimetry in Understanding Food
291
affect their behavior in dehydration equipment and storage stability at

low water contents. Glass transitions of amorphous components in
foods are often monitored by differential scanning calorimetry and
recorded from a step change in heat capacity (Roos 2002b).
Understanding the physical state of food materials requires that properties of individual food components and their interactions with
each other are well characterized.
The first studies referring to glass formation by food components
were those of dairy powders and glucose. It was recognized that sugars
formed solid, noncrystalline structures (glasses) and that the properties
of noncrystalline lactose in dairy powders and ice cream were often
responsible for dramatic changes in product quality (White and
Cakebread 1966). Slade and Levine (1991, 1995), Karel et al. (1994),
and Roos (1995, 2004) emphasized that solid, dehydrated food systems,
as well as frozen food systems, contain noncrystalline (amorphous)
components and that the physical state of the components controls food
properties and stability. For example, water can be removed from milk
by dehydration or freezing. These processes remove solvent water, and
the solute molecules often remain in a disordered, dissolved or dispersed amorphous state. Macroscopic observations of such systems
have suggested that dehydration may result in glass formation.
Calorimetric and other systematic studies are then required for full
characterization of the glass-forming components and their properties
in the dehydrated food systems.
Carbohydrates and some proteins are the most typical hydrophilic
components of food solids. These components may form amorphous,
noncrystalline structures at low water contents (White and Cakebread
1966; Slade et al. 1991; Roos 1995, 2004). The most typical food
processes resulting in glass formation by amorphous or partially
amorphous food components include baking, extrusion, dehydration,
and freezing (Roos 1995). Noncrystalline food solids are extremely
sensitive to water and may show various time-dependent changes that
result in a dramatic decrease in food quality. The most important
quality-controlling parameter of amorphous food solids is their glass
transition. Structural relaxations associated with increased molecular
mobility in the vicinity of the glass transition are observed from rapid
changes in food properties above the glass transition. The glass transition describes a temperature range over which a change of a solid glass
to a softened material takes place, with the concomitant appearance
292
of vibrational and translational mobility of component molecules

(Sperling 1992).
There are several glass transition-related changes in foods that affect
their properties and stability. These include stickiness and caking of
powders and sugar-containing products; collapse in freeze-drying and
collapse of dehydrated structures; crispness of snack foods and breakfast cereals; crystallization of amorphous sugars; recrystallization of
gelatinized starch; ice formation and recrystallization in frozen foods;
and to some extent, nonenzymatic browning and enzymatic reactions
(Roos 1995; Slade and Levine 1995; Roudaut et al. 2004). The objective of the present review is to highlight important properties of food
components associated with their thermal behavior and the use of calorimetry and other thermal analytical techniques in the characterization
of food systems, particularly with regard to their dehydration properties and stability control of dehydrated food systems.
Phase and State Transitions of Food Components

Phase transitions indicate changes in the equilibrium state of materials,
and they can be classified according to changes in thermodynamic
properties (Roos 1995). The main requirement for any phase to coexist
with another phase is that the Gibbs free energy of two or more phases
at the transition pressure and temperature is the same. The equilibrium
state is always that with the lowest Gibbs free energy. According to
the thermodynamic classification of phase transitions, first-order phase
transitions are those at which the first derivatives of the thermodynamic
functions exhibit discontinuity; that is, at a first-order transition temperature there is a discontinuity in heat capacity and thermal expansion
coefficient (Roos 1995). Such discontinuity occurs in melting/crystallization and boiling/condensation temperatures. A second-order phase
transition shows a step change in heat capacity and thermal expansion
coefficient.
Glass transitions occur in thermodynamically nonequilibrium
systems, and therefore they do not involve a thermodynamic change
in phase. A glass transition may be considered as a state transition
of an amorphous material with some of the thermodynamic characteristics of a second-order phase transition. The amorphous state, however,
is a nonequilibrium state, and its properties are time-dependent. For
293
Equilibrium
Liquid
(Pressure)
C
oo
lin
g
R
ap
id
Cooling
Crystallization
ow
Sl
tin
g
g
in
ol
Co
GLASS
ea
Heating
g
in
at
He
Equilibrium
Liquid
Cooling
RUBBER
Nonequilibrium
Solid
Heating
CRYSTAL
a
Pl
Calorimetric
Measurements
eh
yd
ra
tio
n
a
iz
tic
n
tio
a
t
ur
at
io
n
Equilibrium
Solid
So
lu
bi
liz
at
io
n
SOLUTION
MELT
Figure 12.1. Equilibrium and nonequilibrium states of materials. Materials suffer

state and phase transitions in various food-processing and storage conditions, which
include equilibrium phase transitions such as crystallization and melting and nonequilibrium state transitions as a result of changes in temperature, water content, or both.
example, molecules in an amorphous material can have an infinite

number of intermolecular arrangements; that is, amorphous materials
have similar molecular disorder to that of liquids and gases, and any
observed characteristics may be specific to the material only at the time
of observation. Changes in amorphous materials may be followed as
a function of time, and the rates of changes are likely to depend on
rates of molecular relaxations and diffusion within the amorphous
state. In food dehydration processes, depending on the rate of solvent
(water) removal or cooling of the amorphous food solids into the solid
glassy structures, different characteristics of the glassy state of the
same material can be obtained. Various states of materials, their phase
and state transitions, and glass formation in dehydration processes are
described in Figure 12.1.
Calorimetric Glass Transition Measurement

The glass transition is a change in state associated with a considerable
change in molecular mobility. Molecular mobility is time-dependent
and no exact glass transition temperatures can be measured or defined.

Exothermal Heat Flow
294
Exotherm
Onset
Midpoint
Time-dependent
changes
C p
Endset
Endotherm
T
Figure 12.2. Schematic representation of typical DSC curves obtained for amorphous materials in heating over their glass transition temperature range. The glass
transition temperature, Tg, is often taken from the onset temperature of the glass
transition or as the midpoint value corresponding to 50% change in heat capacity
occurring over the glass transition. Glass transition may involve an exotherm or an
endotherm corresponding to differences in glass formation (heating/cooling rates;
solvent removal/sorption rates).
Observed changes in heat capacity and characteristic changes around

the glass transition occur over a temperature range. The glass transition
temperature is often the onset temperature of the glass transition temperature range (onset Tg) or the temperature corresponding to a 50%
heat capacity change over the transition (midpoint Tg) as measured by
differential scanning calorimetry (DSC; Figure 12.2).
Glass transitions have been reported for a wide range of food components, including sugars and other carbohydrates as well as proteins
(Slade and Levine 1995; Roos 1995). Pure food components often
show a single, clear glass transition in DSC thermograms about 100
to 150 C below their equilibrium melting temperature, Tm. Calorimetric
techniques measure the change in heat capacity associated with glass
transition. This can be observed in heating or cooling of the material,
as the glass transition is reversible. An increase in heat capacity occurs
when a supercooled liquid is heated over its glass transition. The temperature range of the glass transitions is highly dependent on food
composition and molecular weight of components. Low-molecularweight components, for example, water and simple sugars, show glass
295
transition over a relatively narrow temperature range followed by

rapidly increasing flow above the glass transition (Roos 1993). Highmolecular-weight food components, such as proteins and starch, as
well as heterogeneous food systems often show glass transition over a
wide temperature range (Hoseney et al. 1986; Roos 1995; Slade and
Levine 1995; Ronda and Roos 2008).
The glass transition of systems with two or more components is
dependent on component properties and their miscibility. Solvents,
such as water in foods, often have a low molecular weight, and they
are fully miscible with their glass-forming solutes. The glass transition
temperature of a solute is highly dependent on the presence and concentration of solvents. Even very small amounts of solvent may substantially decrease the observed glass transition temperature. It has
been found that an increasing amount of water decreases both the glass
transition temperature and its temperature range, but it also increases
the change in heat capacity of the transition (Roos and Karel 1991a,
1991b). Other mixtures of miscible components, for example, sugars,
also show composition-dependent glass transition temperatures, and
they are often substantially affected by the lower-molecular-weight
components (Roos 1995). This can also be observed in edible films
plasticized by other plasticizers, such as glycerol and sorbitol (Talja
et al. 2007). In food systems, several components (e.g., starch, proteins) can exist in partially amorphous states, and many of them exhibit
only partial miscibility or remain immiscible, forming single or several
phases within food microstructure (Kalichevsky and Blanshard 1993;
Vega et al. 2005).
In food processing and storage, the glass transition occurs in both
cooling and heating over the glass transition temperature range, and
glass transition of food solids often takes place during removal of water
in freezing and dehydration processes (Figure 12.1). The materials
show the reversible characteristics of the glass transition and also a
tendency to transform toward the equilibrium state. As described by
Figure 12.1, the glassy state is a nonequilibrium amorphous state and
the glass transition is a time-dependent property. It may occur at
varying temperatures at different experimental time scales (e.g., frequency), as shown in Figures 12.3 and 12.4. Furthermore, depending
on the rate of glass formation and possible changes occurring with time
in the glassy state (aging), various relaxations may be observed over
the glass transition (Figures 12.2, 12.3, and 12.4).
V
H
S
Anomalous Changes
In Thermodynamic
Properties Depending
on Glass Properties
Non-equilibrium
State
Equilibrium
State
Non-equilibrium
State
Liquid
Supercooled
liquid
Hm
Crystal
Glass
100-150C
Tg
Tm
MECHANICAL OR DIELECTRIC PROPERTY
Figure 12.3. Thermodynamic states of materials. The equilibrium liquid state occurs
at and above the equilibrium melting temperature, Tm. At lower temperatures, the
crystalline solid state may exist at equilibrium. Noncrystalline systems can be supercooled liquids or they may become solidlike supercooled materials (glasses) below
the glass transition temperature, Tg. However, the glassy state is a nonequilibrium and
time-dependent state. It may have various molecular arrangements with different
enthalpy, H, entropy, S, and volume, V, states. Melting of crystals involves heat of
melting, Hm, but the glass transition involves no latent heat of the transition.
Storage modulus or
dielectric constant
Increasing frequency
Mechanical and
dielectric relaxations
Loss modulus or
dielectric loss
and relaxations
Tg
relaxation
TEMPERATURE
Figure 12.4. Mechanical and dielectric relaxations of materials in the glassy state
and around the glass transition.
296
297
Dielectric and Mechanical Relaxations

Dielectric analysis (DEA/DETA) and dynamic mechanical analysis
(DMA/DMTA) are thermal analytical methods that allow complementary characterization of amorphous food systems (Kalichevsky et al.
1992; Talja and Roos 2001; Roudaut et al. 2004). These techniques
detect relaxations in dielectric and mechanical properties of the materials. In general, amorphous structures are fairly stable in the solid,
glassy state (Sperling 1992; Slade and Levine 1995), and the relaxation
times extend to several years or decades. At temperatures around and
above the transition, the solid state is rapidly transformed to a supercooled liquid state (viscous fluid) with more rapid time-dependent flow
(White and Cakebread 1966; Roos 1995; Roudaut et al. 2004). The
change in mechanical properties is observed by DMA and detected as
a change in the complex moduli of the material. These changes are
referred to as relaxations, which also appear as analogous changes
in dielectric properties in a DEA analysis (Figure 12.4). For example,
dehydrated, glassy foods have a solid and brittle behavior, whereas the
materials may flow as syrups or become soggy above the glass transition (e.g., freeze-dried foods). This change is associated with a decreasing modulus appearing in a DMA analysis as well as a decrease of the
dielectric constant.
Mechanical and dielectric properties detect glass transition in
foods by their sensitivity to relaxations and changes in modulus and
dielectric properties (Kalichevsky et al. 1992; Sperling 1992; Talja and
Roos 2001). Glass transition as such cannot be measured by DEA or
DMA, but these techniques detect relaxations associated with the
change in heat capacity (Figure 12.4). Several relaxations may appear
at lower temperatures ( and relaxations), and they appear as changes
in storage modulus, E or G; loss modulus, E or G; dielectric constant, ; dielectric loss constant, ; and mechanical and dielectric loss,
tan , below the glass transition (Figure 12.4). The relaxation is the
main relaxation associated with the glass transition. The observed
relaxation temperatures are highly dependent on the frequency, f, of
the applied stress or dielectric disturbance, which clearly indicate the
time-dependent characteristics of noncrystalline materials under disturbance. Many researchers have used frequencies around 1 Hz in
DMA and DEA measurements. However, it seems that frequencies
indicating relaxations at the calorimetric glass transition onset
298
temperature occur at much lower frequencies (Talja and Roos 2001).

These frequencies may be in the range of 0.010.1 Hz, and they indicate the onset of molecular mobility or viscous flow within the glassy
material; that is, the solid characteristics are changing to liquid-like
material properties.
Thermal Analysis in Characterization of Food Systems

DSC is the most universal thermal analytical technique used to detect
phase and state transitions of food systems. DSC requires minimal
sample preparation, and the materials studied can be hermetically
sealed in sample pans for the analysis at known water contents.
Amorphous or partially amorphous structures in foods are formed in
food processing, particularly as the result of water removal and dehydration. Loss of water causes the concentration of solids to increase,
for example in baking, dehydration, freezing, and extrusion, as
described by Figure 12.1. Depending on the rate of solvent removal or
cooling into the solid state, glasses with different properties can be
obtained (Figure 12.2 and 12.3). These food processes form concentrated, supercooled, amorphous, nonequilibrium materials that exhibit
time-dependent changes. The materials exhibit a thermodynamic
driving force toward an equilibrium state, for example, the crystalline
state. This is typically observed in a DSC scan of pure food components, such as lactose and sucrose, which show a glass transition followed by a crystallization exotherm. Crystallization is time-dependent,
but pure substances often show instant crystallization at a scanning
rate-dependent temperature (Figure 12.5). The glass transition occurs
over a temperature range, although it is often referred to with a single
temperature value (Figure 12.3). Glass transition may be present in
either low-moisture and dehydrated foods or frozen foods in which a
concentrated solute phase is formed because water is separated as a
crystalline ice phase within the material. Several real food systems and
components may exist as only partially amorphous materials, and
many food components are only partially miscible or immiscible
forming single or several phases within food microstructure; for
example, carbohydrate, protein or lipid-rich regions or phases.
Glass transition occurs in both cooling and heating and also in
removal or sorption of a plasticizer or a solvent or both. The main
299
Glass transition
ENDOTHERMAL HEAT FLOW
Tg
Melting
T cr
Tm
Crystallization
TEMPERATURE
Figure 12.5. Schematic representation of DSC curve typical of amorphous sugars

with glass transition, Tg, and crystallization, Tcr, of the amorphous phase and melting
of the crystals at Tm.
plasticizer of amorphous food solids is water. Water is a plasticizer to

most carbohydrates and proteins. Lipids exist in separate, hydrophobic
phases and show little interactions with hydrophilic components and
functional groups of food polymers (Roos 1995). Water softens food
solids by decreasing their glass transition temperature toward that of
water, at around 135 C. The presence and interaction of water molecules with food solids results in changes in the amorphous structure.
The glass transition of dehydrated food solids decreases as a result of
water sorption (water uptake from surroundings), and their properties
may change from those of the glassy solid to viscous liquids or syrup
(sugar systems) or leathery material (protein systems) in an isothermal
water sorption process.
The glass transition of amorphous sugars occurs over a temperature
range of 10 20 C, whereas the glass transition of food polymers may
extend over a temperature range of more than 50 C (Hoseney et al.
1986; Roos 1995; Roudaut 2004). The change in heat capacity (Cp)
of sugar glasses around their glass transition is around 0.51.0 J/g C,
and the transition occurs over a temperature range of about 10 20 C
(Roos 1993). The Cp of proteins and starch is often quite small, and
the glass transition may occur over a broad temperature range. The
magnitude of the glass transition often increases with increasing water
content, and the transition occurs over a more narrow temperature
300
range. The glass transition of proteins and polysaccharides may only

be measurable by DSC with a relatively large level of water plasticization (Roos 1995). Food systems also may exhibit numerous glass
transitions depending on composition and extent of phase separation
(miscibility). Some anhydrous food components, for example several
monosaccharides and polyols, have their anhydrous glass transitions
below room temperature, and they cannot be dehydrated to solid materials (Roos 1993, 1995). Many carbohydrates and proteins as well as
other polymeric food components have glass transitions in dry states
above 200 C, which approaches their decomposition temperature. A
common problem in observing glass transitions in food systems at
intermediate water content is that liquid-crystalline transitions of lipids
often overlap the glass transition of hydrophilic food solids.
The effect of water on the glass transition can be predicted, for
example, using the Gordon-Taylor equation (Gordon and Taylor 1952).
We have combined the water sorption data and glass transition data to
establish diagrams showing critical values for water content and water
activity that result in glass transition at the storage temperature (Figure
12.6). Such diagrams can be established using, for example, the
Gordon-Taylor equation to model water plasticization and the
120
60
0.6
Glass Transition Region

(Temperature-dependent
critical storage parameters)
40
20
0
Tg
-20
-40
Critical Water
Activity (25C)
Critical Water
Content (25C)
-60
0
10
20
30
0.4
0.2
WATER ACTIVITY
TEMPERATURE (C)
80
-80
0.8
Extrapolated
GAB Sorption
Isotherm
Time-dependent
crystallization
100
0
40
WATER CONTENT (g/100g dry solids)
Figure 12.6. Glass transition and water sorption behavior of lactose. Water sorption
results in lactose crystallization as the glass transition at the observation temperature
is exceeded, and the water activity and water content become higher than the critical
values.
301
Guggenheim-Anderson-De Boer (GAB) equation to model water sorption (Roos 1995).
The Frozen State of Foods Systems

Freezing of water in food systems occurs at temperatures below the
equilibrium melting temperature of water, Tm. The equilibrium melting
temperature refers to the temperature at which the last ice crystals melt
during heating of a frozen material. The equilibrium melting temperature is dependent on dissolved food components and their concentration. Below Tm, freezing of water may continue until an equilibrium
amount of ice appears at the freezing temperature or a kinetically
limited maximum amount of ice has formed at a lower temperature
(Roos and Karel 1991c, Roos 1995). A maximally freeze-concentrated
system shows an initial solute concentration-independent glass transition temperature, Tg, of solutes plasticized by the unfrozen water. The
unfrozen water is a continuous phase, with dispersed ice crystals that
exhibit an onset of ice melting during heating at an initial concentration-independent temperature, Tm. This behavior has been well established for many common sugars and carbohydrates (Goff 1995; Roos
1995; Slade and Levine 1995; Singh and Roos 2005). Typically, the
solute concentration of a glassy unfrozen solute phase dispersing the
maximum amount of ice formed in a frozen system is around 80%
(w/w) (Slade and Levine 1991; Roos 1993; Talja and Roos 2001; Singh
and Roos 2005). These transitions can be described by DSC curves of
nonannealed and annealed systems (Figure 12.7). The kinetic limitations for ice formation and time-dependent characteristics of maximum
freeze concentration can be related to the limited diffusion, high viscosity, and longer relaxation times as the glassy state of the unfrozen
solids-unfrozen water phase is approached (Figure 12.8).
Phase and state transitions of maximally freeze-concentrated materials are complex and show the nonequilibrium nature of ice formation
in calorimetric and thermal analytical studies. However, the same
thermal analytical techniques, as well as electron spin resonance (ESR)
and nuclear magnetic resonance (NMR) techniques, provide information about transitions of freeze-concentrated solids and ice melting
(Slade and Levine 1995; Roos 1995; Laaksonen et al. 2002; Roudaut
et al. 2004). Low-molecular-weight components, such as sugars, often
302

ENDOTHERMAL HEAT FLOW
Initial Tg
Unfrozen state
Annealing time
t0
Partial freeze-concentration
Tm
t1
t2
T'g
Maximal freeze-concentration
T'm
t3
Annealing temperature
TEMPERATURE
Figure 12.7. A schematic representation of time-dependent ice formation in freezeconcentrated food systems. A rapidly cooled system shows a glass transition corresponding to the solute-water ratio of the solution when no ice is formed. Ice formation
may be achieved by annealing (isothermal holding) at a temperature below Tm. This
can be detected from an increasing glass transition temperature, Tg, for the freezeconcentrated unfrozen solute phase and an increasing size of the ice-melting endotherm. At maximum ice formation, more ice cannot be formed, and the glass transition
occurs at initial water content-independent temperature, Tg, and is followed by onset
of ice melting at Tm.
exhibit clearly observable and separate but time-dependent transitions

(Roos and Karel 1991b,c). In DSC measurements, the glass transition
temperature of the maximally freeze-concentrated solute, Tg, must be
taken from the onset temperature of the transition temperature range.
The midpoint or endpoint of the transition cannot be defined because
the glass transition is often not complete prior to the ice-melting transition (Roos 1995). Hence, the Cp of the glass transition may remain
unknown or a value that is too low may be obtained because the glass
transition may not be complete when the first ice crystals in the frozen
system melt. Melting of ice gives a relatively sharp endothermic peak,
and its onset temperature can be taken as the onset of ice melting, Tm,
for ice within the maximally freeze-concentrated system (Roos and
Karel 1991c). As described in Figure 12.7, freezing to this maximally
freeze-concentrated state may require an isothermal treatment (anneal-

TT g
Relative
Relaxation Time
(C)
40
2.4x10 8
TEMPERATURE
Tm
20
1.3x10 5
1.0x10
303
Supercooled
liquid
Tm
low
sf
u
co
olid
Vis
ys
Tg
ation
ion
-relax
ansit
r
t
s
Glas
0
ss
Gl a
Thermal plasticization
Maximum Ice Formation
Water plasticization
WEIGHT FRACTION OF SOLIDS
1.0
Figure 12.8. Schematic state diagram showing the decrease in glass transition with
increasing water content and decreasing relaxation times at increasing levels of
thermal or water plasticization. Maximum ice formation takes place time dependently
over the temperature range from the glass transition temperature of the maximally
freeze-concentrated unfrozen phase, Tg, and onset of ice melting in the maximally
freeze-concentrated unfrozen phase, Tm. Equilibrium ice melting occurs according to
the equilibrium ice-melting temperature, Tm, curve.
ing) at a temperature favoring maximum ice formation (Roos and Karel

1991c; Singh and Roos 2005).
State Diagrams and Dehydration

Calorimetric transition temperatures can be shown in state diagrams
(Figure 12.8 and 12.9). State diagrams are often used to show phase
and state transition data to describe material states at various temperatures and levels of water plasticization (Roos and Karel 1991a; Roos
1995; Slade and Levine 1991, 1995). A typical state diagram shows
the glass transition temperature against water content with Tg, Tm, and
Tm data as shown for lactose in Figure 12.9. The effect of water on the
glass transition can be predicted, for example, using the Gordon-Taylor
equation (Roos 1995), which uses the solids, water glass transition
50
-50
-100
Glass transition
Solubility
range
(equilibrium mixture
Time-dependent
of - and
crystallization
-lactose)
Supercooled
Calorimetric
transition
Tg
liquid
temperatures
T'm
Equilibrium freezing zone
T'g
Temperature range for maximum

ice formation
Temperature (C)
100
Tg
Glass
-150
0.0
Gla
s
304
0.2
0.4
0.6
0.8
Weight Fraction of Lactose
C'g
1.0
Figure 12.9. State diagram of lactose with the glass transition temperature, Tg, curve,
and transition temperatures for maximally freeze-concentrated lactose solutions
(Tg is the glass transition temperature of a maximally freeze-concentrated solution,
and Tm is onset temperature of ice melting in a maximally freeze-concentrated
solution).
temperatures, and their weight fractions to predict the glass transition

temperatures at various water contents. We also have used combined
water sorption and glass transition data to establish diagrams showing
critical values for water content and water activity. The critical water
activity and water content have been defined as those corresponding
to the glass transition occurring at a processing or storage temperature
for which water sorption isotherm is shown (Figure 12.6). Such diagrams can be obtained, for example, by using the Gordon-Taylor equation to model water plasticization and the GAB relationship to model
water sorption (Roos 1995). The critical water content and water activity diagrams, together with state diagrams, are important tools in
explaining changes in time-dependent mechanical and flow properties
that are related to glass transition and water plasticization (Slade et al.
1991; Kokini et al. 1994; Roos 1995; Roos et al. 1996; Rahman 2006).
Isoviscous states or relaxation time curves can be shown in state diagrams to describe rapid changes of time-dependent characteristics of
food systems above the glass transition.
305
Spray-Drying
Spray-drying is an efficient dehydration method for a large variety of
liquid materials and slurries, which can be converted to small liquid
droplets using rotating disks or pressure nozzle atomizers. The tiny
droplets can be dehydrated in hot air within seconds, which allows
continuous production of free-flowing powders. Although the principle
of the process is relatively simple, phase and state transitions of food
solids have a significant impact on whether the materials can be spraydried successfully or whether the powders have free-flowing properties
in handling, packaging stages, storage, and use.
It has been suggested that formation of the glassy state from solids
in spray-drying, particularly the glass-forming properties of carbohydrates, have a correlation with spray-drying behavior of fruit juices and
materials rich in sugars (Bhandari and Howes 1999). These materials
are often extremely difficult to dehydrate because the solids tend to
stick on drier surfaces and cake inside dehydration- and powderhandling equipment. Stickiness is probably the most important property in establishing criteria for the suitability of food materials to
spray-drying. Studies of glass transitions of dehydrated sugars and
high-sugar products have confirmed that stickiness is related to the
glass transition of amorphous powders (Roos and Karel 1990). Based
on the knowledge of phase and state transitions in dehydration of
liquids with dissolved substances, it may be assumed that the rapid
removal of water causes vitrification of the liquid droplets (i.e., solids
in the droplets dehydrate and form glasslike structures) within a short
time and formation of a solid particle surface (Roos 2004). Glass transition of the solids at the surface layer of a drying droplet is a key
parameter in defining stickiness behavior of the particles and formation
of liquid bridges occurring in subsequent caking as a result of rapid
decrease in surface viscosity above the glass transition. Therefore,
materials with an anhydrous glass transition below room temperature
cannot be spray-dried, as they cannot be converted to a solid state at
room temperature.
The viscosity changes resulting from the glass transition also can be
used to control agglomeration of fine particles and in the manufacturing of instant powders (Roos 1995). In such processes, it is essential
to allow controlled stickiness on particle surfaces and adhesion of
particles to form clusters. The formation of clusters is followed by
306
Freeze-concentrated
unfrozen solute phase
Freeze-dried
glass solute
membranes
Ice
Pores
Freeze-drying
below T m
id
Sol
Flo
w
Freeze-drying
above T m
Collapsed
liquid
Figure 12.10. The role of onset temperature of ice melting, Tm, in successful freezedrying and liquid flow resulting in collapse as ice temperature exceeds Tm in a freezedrying process.
removal of water and cooling to solidify the surfaces into the glassy
state. The final product will have larger particles and remain free
flowing and stable at appropriate storage conditions.
Freeze-Drying
The Tg and Tm temperatures of biological materials are extremely
important determinants of appropriate operation parameters in freezedrying (Roos 2004). Freeze-drying requires that dissolved substances
are freeze-concentrated to an almost solid state and that the highly
viscous state is retained throughout the dehydration process; that is,
the material should consist of solid ice and a freeze-concentrated, solid,
glassy, unfrozen phase (Figure 12.10). Ice melting above Tm has a
dramatic plasticization effect in a freeze-concentrated system and
results in liquid flow. The effect of state transitions and ice melting
above Tm are described for freeze-drying in Figure 12.10. Accordingly,
the highest allowable pressure (or ice temperature) in freeze-drying is
defined by the initial melting temperature of ice in the system. At
conditions allowing melting, flow may occur, and some dehydration
occurs from the liquid state; the process no longer can be referred to
as freeze-drying (Figure 12.10).
307
Loss of structure known as collapse in freeze-drying occurs above

a critical temperature that allows the viscous flow of freeze-concentrated amorphous solutes (Bellows and King 1973) as they are plasticized by unfrozen water (Roos 2004). The onset temperature of ice
melting, Tm, determines a temperature at which a maximally freezeconcentrated system becomes plasticized by dissolving ice crystals;
therefore, Tm can be used as a critical reference temperature for production of properly freeze-dried materials (the ice vapor pressure in
freeze-drying must be kept below the ice vapor pressure at Tm by the
control of drying pressure and heat supply for sublimation of ice).
Collapse can be avoided by the use of ice temperatures below Tm in
freeze-drying, and the Tm values agree well with collapse temperatures
reported for freeze-drying of carbohydrate systems (Bellows and King
1973; To and Flink 1978; Roos 1995).
Glass Transition and Stability of Dehydrated Materials

Stickiness and caking are common problems in handling of powders
containing amorphous carbohydrates. Stickiness and caking appear as
the viscosity of the amorphous components decreases and powder
particles adhere as they gain liquid-like flow properties at conditions
resulting in glass transition. Glass transition of lactose may occur as a
result of water plasticization in dairy powders. Such plasticization is
often the cause of time-dependent lactose crystallization. An instant
crystallization may be observed at a high level of rapid thermal and
water plasticization (Jouppila et al. 1997; Haque and Roos 2005). A
schematic representation of glass transition-related flow and its effect
on food material behavior, including development of stickiness, caking,
and crystallization, is shown in Figure 12.11. The crystallization of
lactose has been found to be highly time-dependent following the
typical crystallization rate behavior of amorphous solids (Roos and
Karel 1991a, Jouppila et al. 1997). The time-dependent lactose crystallization in dairy powders is often observed in water sorption studies
(Haque and Roos 2005). These have shown that above a critical storage
relative humidity, there is a loss of sorbed water (Figure 12.6). The
loss of sorbed water in dairy powders corresponds to the difference in
water sorption by amorphous and crystalline lactose. However, it
should be noted that the loss of sorbed water is time-dependent, and
Stability Zone
Solid
Glass Transition
Fermis Model
(M. Peleg)
CRITICAL ZONE
VISCOUS FLOW
Increasing Diffusion
Structural Transformations
SOLID
Crispness
RELAXATION TIME
Months
Days
Hours
Minutes
Seconds
Glassy State
Flow
LIQUID
Critical Zone
Mobility Zone
Highly time-dependent
Instant changes
EXTENT OF CHANGE IN PROPERTY
Years
Hardening, Cracking
308
TEMPERATURE, WATER ACTIVITY OR WATER CONTENT
Figure 12.11 Changes in relaxation times as a result of thermal or water plasticization in food systems. Around and above the glass transition rapidly appearing liquidlike properties of the materials result in dramatic changes in mechanical properties
and diffusion. The changes in mechanical properties around glass transition may be
modeled using the Fermi relationship (Peleg 1993).
the crystalline form of lactose produced is dependent on the crystallization conditions (Jouppila et al. 1997; Haque and Roos 2005). Most
crystals formed are anhydrous, but at the higher storage humilities
increasing amounts of -lactose monohydrate is formed. Crystallization
of amorphous lactose in sealed packages and in bulk storage also
results in an increase in water activity and acceleration of most deteriorative changes, such as browning reactions and oxidation.
Conclusions
Thermal and calorimetric properties of food and biological materials
at various water contents are extremely important determinants of
their dehydration and stability characteristics. Calorimetric measurements provide data for selection of appropriate dehydration parameters
and manipulation of solids composition to enhance dehydration and
improve storage stability. Several dehydrated materials, particularly
spray-dried and freeze-dried, exist as amorphous, glassy solids.
Formation of a solid structure contributes to the success of dehydration
processes and the quality characteristics of dehydrated materials.
Knowledge of glass transitions and ice-melting properties of sensitive
309
materials is the basis of successful freeze-drying. Freeze-drying may

only take place below the onset temperature of ice melting in a frozen
system, as higher temperatures allow flow of freeze-concentrated
matrices as well as collapse and loss of quality. Relationships between
flavor retention and encapsulation of volatiles and dispersed components, and formation of a glassy, continuous hydrophilic phase in
dehydration processes, are important in stabilization of such components. Solids crystallization, lipid oxidation, nonenzymatic browning,
and enzymatic changes are often interrelated and controlled by the
glass transition and water.
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Bhandari B.R. and Howes T. 1999. Implication of glass transition for the drying and
stability of dried foods. J Food Eng, 40:7179.
Goff H.D. 1995. The use of thermal analysis in the development of a better understanding of frozen food stability. Pure Appl Chem, 67:18011808.
Gordon M. and Taylor J.S. 1952. Ideal copolymers and the second-order transitions
of synthetic rubbers. I. Non-crystalline copolymers. J Appl Chem, 2:493500.
Haque M.K. and Roos Y.H. 2005. Crystallization and x-ray diffraction of spray-dried
and freeze-dried amorphous lactose. Carbohydr Res, 340:293301.
Hoseney R.C., Zeleznak K., and Lai C.S. 1986. Wheat gluten: A glassy polymer.
Jouppila K., Kansikas J., and Roos Y.H. 1997. Glass transition, water plasticization,
and lactose crystallization in skim milk powder. J Dairy Sci, 80:31523160.
Kalichevsky M.T. and Blanshard J.M.V. 1993. The effect of fructose and water on
the glass transition of amylopectin. Carbohydr Polym, 20:107113.
Kalichevsky M.T., Jaroszkiewicz E.M., Ablett S., Blanshard J.M.V., and Lillford P.J.
1992. The glass transition of amylopectin measured by DSC, DMTA, and NMR.
Carbohydr Polym, 18:7788.
Karel, M., Anglea, S., Buera, P., Karmas, R., Levi, G., and Roos, Y. 1994. Stabilityrelated transitions of amorphous foods. Thermochim Acta, 246:249269.
Kokini J.L., Cocero A.M., Madeka H., and de Graaf E. 1994. The development of
state diagrams for cereal proteins. Trends Food Sci Technol, 5:281288.
Laaksonen T.J., Kuuva T., Jouppila K., and Roos Y.H. 2002. Effects of arabinoxylans
on thermal behavior of frozen wheat doughs as measured by DSC, DMA, and
DEA. J Food Sci, 67:223230.
Peleg, M. 1993. Mapping the stiffness-temperature-moisture relationship of solid
biomaterials at and around their glass transition. Rheol Acta, 32:575580.
Rahman M.S. 2006. State diagram of foods: Its potential use in food processing and
product stability. Trends Food Sci Technol, 17:129141.
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Ronda F. and Roos Y.H. 2008. Gelatinization and freeze-concentration effects on

recrystallization in corn and potato starch gels. Carbohydr Res, 343:903911.
Roos Y. 1993. Melting and glass transitions of low molecular weight carbohydrates.
Carbohydr Res, 238:3948.
Roos Y.H. 1995. Phase Transitions in Foods. Academic Press: San Diego.
Roos Y.H. 2002a. Importance of glass transition and water activity to spray drying
and stability of dairy powders. Le Lait, 82:475484.
Roos Y.H. 2002b. Thermal analysis, state transitions, and food quality. J Therm Anal
Calorim, 71:197203.
Roos Y.H. 2004. Phase and state transitions in dehydration of biomaterials and foods.
In: Dehydration of Products of Biological Origin, A.S. Mujumdar, editor, pp. 322.
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Roos Y. and Karel M. 1990. Differential scanning calorimetry study of phase transitions affecting the quality of dehydrated materials. Biotechnol Progr,
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Roos Y. and Karel M. 1991a. Applying state diagrams to food processing and development. Food Technol, 45, 66, 6871, 107.
Roos Y. and Karel M. 1991b. Nonequilibrium ice formation in carbohydrate solutions. Cryo-Letters, 12:367376.
Roos Y. and Karel M. 1991c. Amorphous state and delayed ice formation in sucrose
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Roos Y.H., Karel M., and Kokini J.L. 1996. Glass transitions in low moisture and
frozen foods: Effects on shelf life and quality. Food Technol, 50(11):95108.
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food products. J Food Technol, 1:7382.
Chapter 13
High-Pressure Calorimetry and
Transitiometry*
Stanislaw L. Randzio and Alain Le Bail
Introduction
High-Pressure Calorimetry
Scanning Transitiometry
Applications
Water in Pork Muscle
Frozen Water Ratio in Gelatine Gels
Pressure Shift Freezing
Gelatinization of Starch
Phase Stability of Systems Containing Lipids
Conclusions
References
311
313
317
324
324
326
329
330
336
337
338
Introduction
The discovery by Bridgman in 1914 (Bridgman 1914a) of a pressureinduced coagulation of egg white enhanced research on the influence
of pressure on biological and food systems. From the results obtained
over the years, it became evident that the use of pressure can lead to
developments of food products with new properties, such as highpressure-processed jams prepared by nonheating food processing,
which entered the market in Japan in 1992 (Hayashi 2002). Other
important technological applications are concerned with high-pressure
processing of food, especially freezing-thawing and crystallization
*M. Malecki is acknowledged for technical assistance in the preparation of this
chapter.
311
312
processes (Knorr 1999) and the use of extruders. An understanding of

the effects of processing on the physical properties of food materials
should allow prediction of the formulation of raw materials and processing conditions so as to achieve desired end product properties. To
achieve this goal, systematic studies must be done to develop a database on the physical properties of food materials as a function of
variables relevant to processing (Kaletun and Breslauer 1996).
Calorimetry is a powerful tool for determination of thermophysical
properties of matter and processes over wide ranges of external conditions (Randzio 1998; Randzio 2002), and thus it is a suitable tool for
creating such databases. However, the calorimetric techniques, mainly
differential scanning calorimetry (DSC), to date have been mostly used
only to evaluate the effects of high-pressure processing, the calorimetric measurements being performed after processing under atmospheric
pressure. For example, Stute et al. (Stute et al. 1996) compressed
aqueous suspensions of wheat starch at 293 K at various pressures up
to 500 MPa, and then after compression up to a selected pressure, the
sample was decompressed, removed from the autoclave, and analyzed
with a classical DSC to verify the degree of pressure-induced gelatinization at 293 K. Douzals et al. (Douzals et al. 2001) have placed the
autoclave in a thermostat and were able to perform similar pressure
measurements over the temperature interval from 253 K to 373 K.
However, the degree of gelatinization caused by the processing at
various pressures and temperatures also was determined after processing by using classical DSC under atmospheric pressure. However, one
must note that such a use of DSC can give indicate the effect of pressure only for irreversible phenomena. To be able to understand the real
role of pressure in food processing one must know the phase diagrams
of its constituents and the mechanisms of transitions between the
phases or states. Phase diagrams of biomacromolecules and biopolymers can be extremely complicated (Smeller 2002), and an interplay
between temperature and pressure is sometimes difficult to interpret.
For this reason, it is important to make calorimetric measurements
under typical processing conditions of high hydrostatic pressure and
temperature under well-determined pressure and temperature conditions and to determine the thermodynamic and thermomechanic parameters of the transitions. This chapter describes such direct high-pressure
calorimetric techniques and reviews their applications in investigation
of selected systems important for food science and technology.
313
High-Pressure Calorimetry
Figure 13.1 presents a schematic diagram of a high-pressure calorimetric system developed by LeBail et al. (Le Bail et al. 2001; Zhu et al.
2004). The high-pressure calorimetric system is composed of a differential calorimetric detector made from 220 thermocouples interconnected between the two calorimetric vessels, high-pressure pump,
hydraulic fluid reservoir, pressure detector (Asco Instruments, France),
and a circulating liquid thermostat. The pressure in the system was
controlled by proportional-integral-derivative computer software
through a stepping motor and a gear box of the high-pressure pump
(Nova Swiss, Switzerland). The processing of the calorimetric signal
was performed with computer software. The investigated substance
was always placed in a small flexible plastic pouch. A detailed view
of the calorimetric system elements can be seen in Figure 13.2. The
high-pressure vessels connected to the high-pressure hydraulic system
by flexible stainless steel capillary tubing can be easily introduced into
the cavities of the differential calorimetric detector, placed in the calorimetric block, which in turn is surrounded with a copper coil in which
Figure 13.1. Schematic diagram of a differential high-pressure calorimeter: (1) differential calorimetric detector, (2, 3) calorimetric vessels, (4) high-pressure pump, (5)
hydraulic fluid reservoir, (6) pressure detector, (7) circulating liquid thermostat, (8),
pressure control, (9) calorimetric signal processing, (10) investigated substance in a
flexible plastic pouch.
314
Figure 13.2. Experimental setup of a high-pressure calorimeter: (1) high-pressure

vessels, (2) flexible stainless steel capillary tubing, (3) cavities of the differential
calorimetric detector, (4) calorimetric block, (5) copper cooling coil.
a temperature-controlled liquid circulates, ensuring a good temperature

stability. The whole system is placed in a stainless steel flask filled
with heat-insulating material. Figure 13.3 presents a detailed view of
a high-pressure calorimetric vessel made from stainless steel. The
investigated substance was sealed under vacuum in a polyethylene
pouch. The high-pressure closing of the vessels was done from one
side with a nitrile O-ring placed on a plug, with a threaded plug holding
it in place. From the other side, the calorimetric vessels were connected
to the hydraulic high-pressure system through stainless tubing (3.2 mm
outside diameter) and standard Harwood connections. The internal
volume of the calorimetric vessels was 4.6 cm3.
The pressure sensor was calibrated against a Bourdon reference
pressure gauge. The calorimeter temperature was calibrated against a
K-type thermocouple (Omega, USA) placed in the calorimetric vessel
at selected temperatures between 253 K and 293 K. The calibration of
the calorimetric detector was carried out by joule effect using a 100ohm resistance settled in the high-pressure vessel.
Processes investigated in this calorimeter can be induced either by
pressure variations at constant temperature or by temperature variations at constant pressure. Figure 13.4 presents an example of melting
of ice at 265.9 K induced by pressure variations at a rate of 16.7 kPa/s
(1 MPa/min). Integration of the calorimetric trace gave the value of the
Figure 13.3. High-pressure calorimetric experimental vessel: (1) body of the vessel
made from stainless steel, (2) polyethylene pouch containing the investigated substance, (3) nitrile O-ring placed on a plug, (4) threaded plug holding the closing in
place.
Figure 13.4. Calorimetric trace of melting of ice at 265.9 K induced by pressure

variations at a rate of 16.7 kPa/s (1 MPa/min).
315
316
latent heat of fusion equal to the value derived from Bridgmans highpressure volumetric data (Bridgman 1912) within a 3% agreement,
which is a good validation of the instrument correctness. It is worth
noting that the Calvet-type calorimeters are also suitable instruments
for measurements performed under typical processing conditions of
high hydrostatic pressure and temperature of various processes under
well-determined pressure and temperature conditions that are important for food science and technology. Such instruments have been used
either as a single calorimetric detector (Chourot, LeBail, and Chevalier
2000) or as a differential calorimetric device (Randzio, Grolier, and
Quint 1994). In differential mounting, a Setaram C80 calorimeter was
used in an upside-down position. This permitted using differential
calorimetric vessels fixed on a laboratory table and connected to the
high-pressure pump and pressure detectors with rigid stainless steel
tubing, allowing performance of direct calorimetric measurements up
to 400 MPa. This was the first pressure-controlled scanning calorimeter
with linear pressure variations at rates from 0.5 kPa/s (30 kPa/min) to
0.2 MPa/s (12 MPa/min).
When performing high-pressure calorimetric measurements, one
should realize that the significance of the calorimetric signal depends
on the use of the pressure-transmitting fluid. If the pressure is transmitted to the calorimetric vessel through the substance under investigation
(liquid, liquid suspension, liquid emulsion, or even a paste), the heat
developed as a result of pressure variation is proportional to the coefficient of thermal expansion of the substance under investigation
(Randzio 1985). This is because the mass of the substance contained
in the calorimetric vessel varies, m = VE/V, where VE is the internal
volume of the calorimetric vessel and V is the molar or specific volume
of the substance under investigation, the latter one being pressuredependent. This is a kind of open mass vessel (quasi-constant volume),
in which the substance under investigation entirely fills the calorimetric vessel and at least a part of the external tubing connecting to the
pressure generator. In investigating solid samples, the pressure must
be transmitted by a fluid. Thus, the thermal effect developed due to a
pressure variation is composed of two contributions. The first is proportional to (dV/dT )p of the solid sample under investigation and the
second to the thermal expansion coefficient of the pressure-transmitting fluid. However, an exact separation of the two contributions
requires careful treatment of the data (Rodier-Renaud et al. 1996).
317
Scanning Transitiometry
Figure 13.5 presents schematically a relatively new technique called
scanning transitiometry (Randzio 1996). The function of scanning
transitiometry consists of scanning one of the three variables ( p, V, or
T ) when the second is kept strictly constant. During the scanning, the
variations of the dependent variables and the associated calorimetric
signal are simultaneously recorded. From these two quantities and the
scanned variable, two thermodynamic derivatives, thermal and mechanical, are simultaneously determined for the system under study. Figure
13.6 presents four thermodynamic situations covered by scanning transitiometry, where from the state variables and the heat effect one can
determine four pairs of thermodynamic derivatives (Randzio 1997).
Each of the situations has specific applications, which prove its particular utilities. In studying food systems, the most useful is the use of
temperature as a scanned variable at constant pressure and the use of
pressure as a scanned variable at constant temperature. In the former
case, the output variables are heat capacity at constant pressure and
thermal expansion. After proper integration, one obtains enthalpy and
volume variations caused by the applied temperature change. In the
latter case, the output variables are pressure derivative of entropy and
compressibility. After proper integration, one obtains heat of transition
and associated volume change. Sometimes, especially for transitions
volume 15 cm3
5 +106 5 +103 cm3/s
temperature
pressure
heat flux
223673K
0.15mK/s
COMPUTER
0.1400MPa
0.0010.05MPa/s
107101W
Figure 13.5. Scheme of basic principles of scanning transitiometry.
318

INPUTS
OUTPUTS
T = const
P = f(t)
PVT
Thermal
Mechanical
T = const
V = f(t)
PVT
Thermal
Mechanical
P = const
T = f(t)
PVT
Thermal
Mechanical
V = const
T = f(t)
PVT
Thermal
Mechanical
(S/P)T = (V/T)P
(V/P)T
(S/P)T = (P/T)V
(V/P)T
(H/T)P
(V/T)P
(U/T)V
(P/T)V
Figure 13.6. Thermodynamic scheme of scanning transitiometry.
with a negative slope of the equilibrium line, the use of temperature

as scanned variable at constant volume is advantageous. As it is shown
in Figure 13.5 during the transitiometric experiment, the experimenter
can see all thermodynamic variables of the process under investigation.
The screen in Figure 13.5 exhibits an isobaric investigation of thermal
gelatinization of a 50% water suspension of wheat starch by scanning
temperature at a rate of 2.5 mKs1 under 90 MPa of pressure. The fundamental advantage of the scanning transitiometry with respect to
calorimetry is that the former technique gives simultaneously two
contributions to a thermodynamic potential change, thermal and
mechanical, thus permitting description of a transition in a single
experiment; with calorimetry, such a description requires at least a few
measurements performed at various pressures, and it is not as precise.
Figure 13.7 presents a schematic diagram of a scanning transitiometer, which was used in investigation of pressure influence on the phase
transformation occurring during thermal gelatinization of aqueous
wheat starch suspensions (Randzio and Orlowska 2005). It consists of
a calorimeter equipped with high-pressure vessels, a pressure-volumetemperature system, and a LabView-based virtual instrument (VI) software. Two calorimetric detectors made from 622 thermocouples each
are mounted differentially and connected to a nanovolt amplifier. The
calorimetric detectors are placed in a calorimetric metallic block, the
temperature of which is directly controlled with an entirely digital
feedback loop of 22-bit resolution (104 K), which is part of the transitiometer software. The calorimetric block is surrounded by a heatingcooling shield. The temperature difference between the block and the
319
calorimetric detector
upper entries
heat.-cool. shield
air
cone plug
TDIFF
calorimetric
signal
TSECURITY
80W
300W
ampoule
temp.
control
calorimetric detector
investigated
substance
computer
spring
data acquisition
&
process control
high pressure
tubing
heat insulation
calorimetric
block
dry air flow
cooling fluid
pump
Pt 100
measuring vessel
step motor
control
pressure
detector
reference vessel
Figure 13.7. Schematic diagram of a scanning transitiometer.
heating-cooling shield is set as constant (5, 10, 20, or 30 K) and using

an additional controller. The temperature measurements, both absolute
and differential, are performed with calibrated Pt 100 sensors. The
heaters are homogeneously embedded on the outer surfaces of both the
calorimetric block and the heating-cooling shield. The whole assembly
is placed in a thermal insulation embedded in a stainless steel body
and placed on a stand that permits moving the calorimeter up and down
over the calorimetric vessels. When performing measurements near
273 K or below, dry air is pumped through the apparatus.
The calorimetric vessels are made from 0.8-cm internal diameter
316 SS tubing and are fixed on a mounting table attached to the mobile
stand. A flexible ampoule containing the sample is placed in the measuring vessel on the top of a spring, ensuring placement of the sample
in the center of the calorimetric detector. Another technique is to use
mercury as the hydraulic fluid and place a sample of prepared material
directly on the mercury. Mercury offers a great advantage because its
compressibility is very low, which is extremely advantageous for measurements of both quantities of volume variations and heat flux. Only
the measuring vessel is connected to the PV line. The reference vessel
acts only as a thermal reference; a stainless steel bar of appropriate
dimensions is placed in it to balance the baseline of the differential
320
calorimetric signal. The tubing of both measuring and reference vessels

are connected to reducers placed inside the calorimeter when it is in
the lowered (measuring) position. The connections from the reducers
to the manifold are made with thin stainless steel capillaries to reduce
heat losses to the environment. The vessels are closed, with a cone
plug fixed in place by an internally threaded cover, which also acts as
a heat exchanger between the calorimetric vessel tubing and the calorimetric detector. Two sleeves also are fixed on the calorimetric vessel
tubing below the cover to help control the heat exchange between the
calorimetric vessel tubing and both the calorimeter block and the
shield.
The piston pump (9 cm3 of total displaced volume) is driven by a
stepping motor controlled by the transitiometer software (manual
control is possible during preparatory operations). The pressure detector is a Viatran 245 transducer, 100 MPa full range with a precision of
0.15% full scale deflection (fsd).
The pressure detector, the output of the calorimetric amplifier, and
the stepping motor are connected to a NI PCI-MIO-16XE-50 multifunction board through a NI SCB-68 shielded connector block. The
temperature measurements and digital control of the calorimetric block
are performed through a serial port. The software, elaborated with the
use of LabView language, performs as a virtual instrument (VI). It
consists of 90 subVI, each responsible for a particular function: pressure measurement, temperature measurement, counting the motor steps
for recording the volume variations, measuring the calorimetric signal,
etc., and each performs independently. However, all the subVIs form
a hierarchical structure with a top window, where the experimenter can
see simultaneously all four variables (pressure, P, volume variations,
V, temperature, T, and heat flux, q) associated with the process under
investigation and the current status of the temperature and pressure
control loops.
The experiments are performed by starting thermal and mechanical
stabilization for at least 5000 s, then the temperature scanning starts,
which is accompanied by automatic volume compensation to keep the
pressure constant. At the end of the scan, the temperature is kept constant for at least 5000 s. Any static baseline shift of the calorimetric
signal between the low and high temperature stabilizations is corrected.
No corrections are made for the calorimetric signal recorded during
the temperature scan.
321
Figure 13.8. Transitiometric vessels: (a) standard high-pressure vessels; (b) the
vessels kept in a holder to facilitate reproducible closing and opening of the vessel
by a dynamometric wrench; (c) a sample of a gelatinized sample of 50% aqueous
suspension of wheat starch pushed out after a transitiometric isobaric experiment
under 90 MPa.
The method presented here is rather simple and safe in practice. The
total volume of the liquid phase under pressure is only about 20 ml; the
energy accumulated in it is rather small and not dangerous. The mercury
used as a hydraulic fluid is always contained in a closed space. In case of
a leak, the mercury is collected on a special protecting plate. The calorimetric vessels are conveniently and reproducibly closed and opened
with a torque wrench with the vessels placed in a specially designed
holder. Figure 13.8ac presents a view of transitiometric vessels: standard high-pressure vessels, the vessels kept in a holder to facilitate
reproducible closing and opening of the vessel by a dynamometric
wrench, and a sample of a gelatinized sample of a 50% aqueous suspension of wheat starch pushed out after a transitiometric experiment.
Because of the high sensitivity of the instrument, some precautions
must be taken to ensure valid measurements. In the case of the calorimetric signal, the main precaution is to carefully compensate the
thermal balance of the differential calorimetric vessels. It is also important to keep the initial mercury level always in the same position, just
above the entry to the calorimetric detector zone. Adjustment of the
mercury level is easily done with the motorized pump. With respect to
the volumetric component, it is very important that displacement
322
of the mercury during calibration experiments be sufficiently slow to

avoid overpressure in the flow lines, and differentiation of the piston
displacement must be carefully done to avoid excessive noise on the
one hand and excessive damping on the other.
The temperature and energy scales of the differential calorimetric
detector were calibrated under atmospheric pressure with the fusion of
gallium, Tm = 302.91 K, Hm = 5.59 kJ mol1; p-bromochloro-benzene,
Tm = 337.73 K, Hm = 18.760 kJ mol1; p-di-bromobenzene,
Tm = 360.45 K, Hm = 20.530 kJ mol1; benzoic acid, Tm = 395.55 K,
Hm = 18.062 kJ mol1; and indium, Tm = 429.75 K, Hm = 3.28 kJ mol1.
The calibration experiments were done by enclosing a calibration substance in a 75-mm-long thin glass tube placed in the center of the
calorimetric vessel. In order for the internal heat exchange to resemble
that occurring in the real experiment, but to avoid thermal effects of
gelatinization, the remaining inner space of the calorimetric vessel was
filled with dried starch. The precision of the temperature scale is
0.2 K. The energetic calibration constant kc of the calorimetric detector depends on temperature and is described by Equation 13.1:
kc (WV 1 ) = 3.423 10 3 + 9.993 10 6 T K
(13.1)
The mean deviation between Equation 13.1 and the calibration data is
1.4%. The reproducible resolution of the calorimetric detector varies
from 1.3 107 W at 303 K to 1.6 107 W at 430 K. As reported previously, for properly designed experimental vessels, the energetic
calibration constant of the calorimetric detector does not depend on
pressure (Randzio, Grolier, and Quint 1994).
The volumetric calibration of the high pressure pump was performed
by weighing 11 mercury samples displaced by known numbers of
motor steps. Each motor step corresponded to a displacement of
(5.22 0.03) 106 cm3.
The volumetric calibration of the high pressure pump and both
energetic and temperature calibrations of the calorimetric detector
were verified by test measurements using isobaric fusion of benzene,
for which both enthalpy and volume of the transition are exactly known
(Bridgman 1914b). Figure 13.9 presents an example of such measurements performed at a scanning rate of 2.5 mK s1 at 100 MPa. The mean
results from eight independent measurements gave the following:
fusV(100 MPa) = 0.1038 0.0028 cm3g1 [the respective literature
323
120
10
Endo
100
80
50
60
70
40
90
110
130
299
20
b
301
303
305
Temperature (K)
dV/dT (mm3 K1 g1)
Heat flux (mWg1)
30
307
0
309
Figure 13.9. Example of simultaneous transitiometric traces (heat flux and volume
variations) of isobaric melting at 100 MPa of 0.3858 g of benzene used as a verification test for thermal and volumetric calibrations of the transitiometer used in the
present study: (a) heat flux, (b) dV/dT.
value is fusV(100 MPa) = 0.1026 cm3g1]; fusH(100 MPa) = 131.1

2.1 J g1 [the respective literature value is fusH(100 MPa) = 126.3 Jg1];
Tfus,onset(100 MPa) = 304.2 0.3 K and Tfus,peak(100 MPa) = 305.7
0.5 K [the literature value is Tfus(100 MPa) = 305.6 K given without any
specification]. The agreement with volumetric data is very good. Small
differences with the thermal data probably are caused by internal heat
exchange conditions in the calorimetric vessel. In test measurements,
only 0.4 g of benzene was floating on the mercury, whereas in the calibration experiments, the calibration substances were placed in the
center of the calorimetric vessel and were surrounded by a dry starch
powder.
Figure 13.10 presents results of another test performed on the temperature and pressure dependence of the thermal expansion of the
mercury used as hydraulic liquid. The hydraulic liquid was displaced
to the top of the empty calorimetric vessel and temperature-scanning
measurements performed at a rate of 2.5 mK s1 at various pressures.
The thermal expansion of the hydraulic fluid is almost constant at a
given pressure and only very slightly decreases with temperature. The
mean values are as follows: 0.949 0.033 mm3 K1 at 10 MPa,
0.895 0.038 mm3 K1 at 60 MPa, and 0.888 0.050 mm3 K1 at
100 MPa. This test was important for analysis of transitions observed
324
Figure 13.10. Volume variations during isobaric temperature scans at various pressures with only hydraulic liquid (mercury) present in the system. The data at 10 MPa
and 100 MPa are shifted by +0.3 mm3K1 and 0.3 mm3K1, respectively, to avoid
overlapping.
in food systems as a function of temperature under isobaric conditions,

which are discussed later.
Applications
Water in Pork Muscle
High pressure has a dramatic effect on the phase transition behavior
of water, encompassing several forms of ice crystals, depending on the
pressure and temperature (Bridgman 1912). This phase transition phenomenon offers several potential applications in food processing, such
as pressure-shift freezing, high-pressure freezing, high-pressure
thawing, etc. (Cheftel, Thiebaud, and Dumay 2002; Kalichevsky,
Knorr, and Lillford 1995). Water is a major component of most foods,
especially fresh products (e.g., meat, fish, vegetables, fruits), and properties of such foods are strongly related to the properties and content
of water. Such foods containing water are, with respect to phase transition phenomena, somewhat similar to water-ice phase transitions when
subjected to high-pressure and low-temperature processing (Le Bail et
al. 2003).
Water in foods can exist either in a free and thus freezable state or
in a bound and thus nonfreezable state. The content of freezable water
325
traditionally has been considered to depend on freezing temperature

(Pham 1987). The presence of solutes in the aqueous phase shifts the
phase diagram of water, lowering the freezing point or ice-melting
temperature at atmospheric pressure. This effect is enhanced with
increasing solute concentrations (Fennema 1973; Cheftel, Levy, and
Dumay 2000). Therefore, phase transition processing of water in foods
during high-pressure treatment is more complex than that of pure
water. In this respect, an example of high-pressure calorimetric applications in the investigation of real foods is an evaluation of the phasetransition behavior of water in pork muscle (Zhu, Ramaswamy, and
Le Bail 2004), described below.
Small samples of fresh pork muscle specimen (0.620.72 g) were
prepared and vacuum-packaged in polyethylene bags (80-m-thick
multiplayer film). While awaiting calorimetric experiments, the packaged samples were stored at 277 K. To make measurements, the investigated sample was placed in the calorimetric vessel and either isothermal
pressure scanning or isobaric temperature scanning was performed.
After calorimetric experiments, moisture content in each investigated
sample was determined by drying in an oven at 376 K for 24 h.
For isothermal pressure-scanning measurements, the calorimetric
temperature was set at a selected value (268, 263, 258, and 253 K). Once
the calorimetric signal showed a stable baseline, the pressure was
increased linearly at a rate of 5 kPa/s (0.3 MPa/min), and the heat flow
was recorded every 5 s. When the pressure reached the corresponding
phase-change temperature, the frozen sample started to melt, resulting
in a peak of heat flow. Figure 13.11 presents a comparison of calorimetric heat flow signals of thawing of pure ice and of ice in a frozen pork
muscle at 263 K induced by a linear pressure scans at a rate of 5 kPa/s
(0.3 MPa/min). Figure 13.12 presents similar isothermal heat flow
signals of pressure-induced thawing of ice in frozen pork muscle
recorded at various temperatures. It is worth noting that this is a heat
flow calorimetric detector; the temperature increase is extremely small,
thus it ensures quasi-isothermal conditions for the measurements
performed.
The temperature-scanning measurements were carried out at various
constant pressures by increasing (for thawing) or decreasing (for freezing) the temperature at a prescribed rate. Figure 13.13 presents results
of a typical temperature-scanning measurement of freezing of ice in a
pork muscle performed at a rate of 2.5 mKs1 (0.15 K/min) under pres-
326

0
Heat flux (mW/g)
20
40
60
80
Pork
100
0.1
Water
50
100
Pressure (MPa)
150
200
Figure 13.11. Comparison of thawing heat flux of pure ice and frozen pork muscle
induced by pressure scans at a rate of 5 kPa/s (0.3 MPa/min) at 263.1 K.
Heat flux (mW/g)
10
20
30
268.0K
40
0.1
25
50
263.1K 258.0K 253.1K

75
100 125 150

Pressure (MPa)
175
200
250
Figure 13.12. Thawing heat flux of frozen pork muscles induced by a pressure scan
at a rate of 5 kPa/s (0.3 MPa/min) at various temperatures.
sure of 111.7 MPa. At the transition, the temperature scan was perturbed
because of a rapid release of heat during fast crystallization of ice.
Frozen Water Ratio in Gelatine Gels
The same experimental procedure as that described above for pure
water and water in pork muscle has also been used with gelatine gels
containing 2% and 10% of dry gelatine (Chevalier-Lucia et al. 2003).

2.5
327
259
258
1.5
Temperature
1.0
0.5
257
Temperature (K)
Heat flux (mV)
2.0
Heat flux
0.0
0.5
256
0
1200
3600
Time (s)
6000
Figure 13.13. Heat flux of crystallization of water in pork muscle induced by cooling
under pressure of 111.7 MPa.
Dried powdered gelatine (Merck, Darmstadt, Germany) was dissolved

in distilled water and placed in an hermetic flask. The solution maintained at 293 K was mixed for 1 h at 100 rpm with a magnetic stirrer.
The mixture was then heated for 30 min at 323 K at the same stirring
rate. The formed gelatine gel was then stored for 12 h at 277 K to allow
maturation. Three samples of 5 g of gel were dried at 375 K for 24 h to
check the final water content in the gels before the calorimetric measurements. The pressure-controlled scanning calorimetric measurements were then performed with gels containing 2% and 10% of dry
matter at three temperatures: 268, 263, and 258 K. For each temperature, the onset pressure, the peak pressure, and the latent heat were
measured, always for three different samples. It was observed that
whatever the concentration in dry matter, the latent heat of gelatine
gels decreased with the melting temperature as observed previously
for pure water. As illustrated in Figure 13.14, whatever the concentration in dry matter, the latent heat of the gelatine gel decreased with the
melting temperature as observed for water. The latent heat versus
temperature evolution was fitted by a second-order polynomial expression given by Equations 13.2 and 13.3 for 2% and 10% gelatine gels,
respectively.
L2% = 0.0921T 2 + 6.668 T + 318.8 R 2 = 0.991
(13.2)
L10% = 0.0884 T 2 + 8.922 T + 296.9 R 2 = 0.999
(13.3)
328
Latent heat (J/g)
400
300
200
100
253
258
263
268
273
Temperature (K)
Figure 13.14. Evolution of the latent heat of pure water (square, experimental data;
dash, Bridgmans data) and of gelatine gels (circle, 2%; diamond, 10% in dry matter)
according to the melting temperatures.
Figure 13.15. Evolution of the ratio of latent heat of melting gelatine gels and latent
heat of melting water as a function of melting temperature (or respective pressure):
circle, 2% gelatine gels; diamond, 10% gelatine gels.
The dry matter content appeared to have an influence on the latent

heat under pressure. The higher the dry matter, the lower was the latent
heat. This phenomenon observed under atmospheric pressure was also
valid under high pressures. Figure 13.15 presents the ratio of the latent
heat observed for gelatine gels and that of pure water. Under atmospheric pressure (273 K), this ratio for the 2% and 10% gelatine gels
was 0.98 and 0.9, respectively. The difference between these results
and the water content of the gels is probably due to the bound water
fraction. The bound water fraction, which does not freeze, corresponds
to some water molecules fixed on polar groups of components such as
proteins. It can be seen in Figure 13.15 that when the melting temperature decreases (the phase-change pressure increases), this ratio
329
decreases. Thus, it can be concluded that the amount of the bound

water in gelatine gels increases with pressure.
Pressure Shift Freezing
Pressure shift freezing (PSF) is increasingly receiving attention in
recent years because of its potential benefits for improving the quality
of frozen food. Generally, the PSF process consists of three successive
steps: (1) cooling the product under pressure to a low temperature (e.g.,
253 K at 200 MPa) without involving phase change; (2) a quick depressurization (adiabatic expansion) to create supercooling for instantaneous, uniform, and partial initiation of the freezing process (resulting
largely in ice nucleation); and (3) completion of the freezing process
(ice crystal growth) under atmospheric pressure. It has been demonstrated that the PSF process produces fine and uniform ice crystals
throughout the food samples (Chevalier, Le Bail, and Ghoul 2002),
thus reducing ice crystal-related textural damage to frozen products
(Chevalier et al. 2001). In the PSF process, after the pressure release,
a portion of the liquid water is frozen, and the resulting crystals are
usually very small in size (like ice nuclei in conventional freezing) and
will then grow when the freezing is completed under atmospheric pressure. Evaluation of the amount of ice nuclei formed instantaneously
by depressurization is important for a better understanding of PSF
process. The high-pressure calorimetry can be very helpful in this
respect (Zhu, Ramaswamy, and Le Bail 2005). Figures 13.16 and 13.17
Pressurization
Nucleation
Supercooling
Water
Cooling
F
Cooling
Temperature (K)
273
Ice-I
C
G
D
Depressurization
252
0.1
Pnuc
210 Pressure (MPa)
Figure 13.16. Basic procedure of pressure-shift freezing based on phase transition

between water and ice I under pressure; Pnuc is the pressure under which ice nucleation
starts.
330

Temperature
300
253.1
200
Heat flux
100
0
252.1
0
Pressure
100
0
Temperature (K)
Heat flux (mW/g)

or Pressure (MPa)
400
1200
2400
Time (s)
3600
251.1
4800
Figure 13.17. Typical profiles of pressure, heat flux, and temperature during pressure-shift freezing of pork muscle under pressure of 199 MPa at 253.1 K.
present procedures of a PSF experiment performed in a high-pressure

calorimeter using pork muscle under pressure of 199 MPa at 253.1 K.
After placing the sample into the experimental vessel, the calorimeter
was rapidly pressurized to target level (AB in Figure 13.16). Then the
sample was cooled to the preset temperature under constant pressure
(BC in Figure 13.16). When the baseline of the calorimetric signal
became stable (AB in Figure 13.17), pressure was released to initiate
the nucleation process (CDEF in Figure 13.15 and BC in Figure 13.17).
Because the depressurization was carried out rapidly (within a matter
of 1 or 2 s), water was instantaneously supercooled in the liquid state
(a metastable one), even after the complete release of pressure (i.e.,
Pnuc = 0.1 MPa and EF in Figure 13.16), and then ice nucleation
occurred. After ice nucleation, sample temperature increased to the
freezing point (EF in Figure 13.16) due to the latent heat of crystallization. Finally, the sample was allowed to complete freezing under atmospheric pressure (FG in Figure 13.16 and CD in Figure 13.16). Figure
13.18 shows the ratio of ice crystal to the whole mass of the sample
formed during depressurization of the pork, determined from the highpressure calorimetric results described above.
Gelatinization of Starch
Starch is one of the most important natural macromolecules. Its importance stems from the fact that the starch granule is an almost universal

293
Temperature (K)
40
Ice ratio and regression curve
273
30
20
253
10
50
100
150
Pressure (MPa)
200
0
250
Ratio of ice to sample mass (%)
50
Phase-change curve
0.1
331
Figure 13.18. Ratios of ice crystal to sample mass instantaneously formed after
depressurization during pressure-shift freezing of pork muscle (74.2% moisture
content) at various initial pressures, with temperatures slightly higher than the corresponding phase-change points of water.
form for packaging and storing carbohydrate in green plants. It is also

one of the main components of food materials, especially those submitted to elevated pressure extruder processing.
The process of preparing a homogeneous sol phase from a mixture
of native starch and water is called gelatinization. Starch gelatinization
is a combined process consisting of hydration of amorphous regions
and subsequent melting of crystalline arrays. It was demonstrated
recently (Hayert et al. 2003) that all the transformations occurring
during starch gelatinization can be observed with a high-sensitivity
DSC done at a low rate of temperature scanning under atmospheric
pressure. Typical results are shown in Figure 13.19 for pastes or emulsions of wheat starch with various total water contents. The main
endothermic transition occurring from 319 K to 333 K independently
of the water content is likely associated with melting of the crystalline
part of the starch granules followed by a helix-coil transformation in
amylopectin, the main component of starch. This endothermic transition is followed by a water-dependent, slow, exothermic transformation, which is probably related to reassociation of the unwound helices
of amylopectin with parts of amylopectin molecules other than their
original helix-duplex partner, forming physical junctions and creating
more general hydrogen-bonded associations. The high-temperature
endothermic transition occurring at water contents around 50 wt % and
332

26
Endo
46.8
Heat flux difference (mW/g)
28
30
56.0
32
60.3
34
64.8
36
38
40
300
310
320
330
340
350
360
370
380
Temperature (K)
Figure 13.19. DSC traces obtained at atmospheric pressure at a rate of 16.67 mK s1

for aqueous native wheat starch emulsions at selected concentrations of water (total
water contents in wt %). M, main endothermic transition, occurring from 319 K to
333 K independently of the water content; A, water-dependent, slow, exothermic
transformation; N, high-temperature endothermic transition occurring at water contents around 50 wt % and higher.
higher is associated with destruction of amylose-lipid complexes or

with a nematic-isotropic transition, which ends the formation of the
isotropic colloidal SOL phase. The pressure influence on those transitions has been investigated under the process conditions of pressure
and temperature, using a scanning transtitiometer described above. A
mixture of native wheat starch with 50 wt % of added water (56.0 wt
% total water content) has been selected for such transitiometric highpressure studies (Randzio and Orlowska 2005).
Figure 13.20 presents results obtained in isobaric experiments by
scanning temperature at a low rate of 2.5 mK s1 (0.15 K min1) under
pressures of 10, 60, and 100 MPa. Results at each pressure present two
output signals recorded simultaneously as a function of temperature,
the heat flux and dV/dT (thermal expansion), both quantities expressed
per gram of dry starch. The most important observation is that all
the transitions recorded previously under atmospheric pressure at
a temperature-scanning rate of 16.7 mK s1 (1 K min1) with a highsensitivity DSC (see the respective trace at 56.0 wt % of total water
Figure 13.20. Transitiometric traces (heat flux and dV/dT per gram of dry starch)
obtained simultaneously and under the process conditions of pressure and temperature
by scanning temperature at a rate of 2.5 mK s1 at various pressures for a starch-water
emulsion (56.0 wt % total water content). A, exothermic transformation; N, endothermic transition.
333
334
content in Figure 13.19) also are observed in the transitiometric traces

in Figure 13.20 performed under elevated pressures at a much lower
temperature-scanning rate of 2.5 mK s1 (0.15 K min1). The transitiometric method also measures simultaneously the volume changes at
those transitions. In Figure 13.19, the right ordinate presents the dV/
dT of the sample; the dV/dT from the hydraulic liquid (see Figure
13.10) have been subtracted from the experimental data. The dV/dT of
the sample at the main endothermic transition (M) decreases over the
pressure range under investigation (10100 MPa). Also note that the
changes of dV/dT at the particular transitions are rather small, while
the general tendency is for dV/dT to rise considerably with temperature
over the whole temperature range.
The last phenomenon is associated with swelling of starch granules
during gelatinization, even under elevated pressures. This allows a
more detailed analysis of the main transition (M). For several degrees
prior to and after the transition, dV/dT increases linearly with temperature with the same or very similar slope. Assuming this, dV/dT during
the transition can be divided into two contributions, one due to the
assumed linear swelling and one due to the phase transition itself.
Figure 13.21 presents an example of such a division of the results
obtained at 10 MPa. Once the division is made, the two contributions
can be integrated separately to give volume changes occurring in the
transition, a positive change for the swelling and a negative change for
the transition itself.
Figure 13.21. Division of dV/dT for the main endothermic transition into a positive
dV/dT due to swelling and a negative dV/dT due to the transition itself.
335
Integrated volumetric data, together with thermal data, all obtained

for each pressure from at least four independent experiments and performed each time on a freshly prepared sample, are given in Table
13.1. The errors are the standard deviations from the mean values of
all experiments performed at each pressure. Table 13.1 also contains
data at 0.1 MPa obtained previously (Randzio, Flis-Kabulska, and
Grolier 2002) with a DSC. From linear approximations of pressure
dependence of the parameters presented in Table 13.1, the following
slopes could be obtained: dHtrans/dp = (9.85 2.25) mJ MPa1 g1,
dVtrans/dp = 2.27 0.37 103 mm3 g1 MPa1, dVswelling/dp = (9.28 2.05)
103 mm3 g1 MPa1 and dTtrans/dp = (24.6 6.9) mK MPa1. Assuming
the main transition (M) is an equilibrium first-order transition, the last
slope also can be obtained from the Clapeyron equation and data from
Table 13.1, from 10 MPa to 100 MPa (dTtrans/dp)Clapeyron = (78.3 2.5)
mK MPa1. Although the slopes are both negative, the agreement is
poor, implying that the mechanism of the transition is more complicated than the first-order transition assumed by the Clapeyron equation.
Also, the pressure dependence may not be linear, especially at low
pressure. Future studies will focus on that problem.
Despite a large number of pressure studies on starch gelatinization,
only the results of Rubens and Heremans (Rubens and Heremans 2000)
were obtained from under the process conditions of pressure and temperature studies and are in agreement with the present results. In Figure
Table 13.1. Thermodynamic data for the main transition (M) in an aqueous
emulsion of wheat starch (56 wt % total water) expressed per gram of
completely dry starch.
Pressure (MPa)
Quantity
transH
(Jg1)
transV
(mm3g1)
swellingV
(mm3g1)
Ttrans,onset (K)
0.1
10
60
100
3.52 0.07
3.12 0.12
2.65 0.07
2.45 0. 7
0.788 0.038
0.647 0.040
0.586 0.035
1.53 0.10
1.22 0.05
0.683 0.036
320.5 0.5
319.5 0.5
318.1 1.0
317.9 0.6
Source: Data at 0.1 MPa are from Randzio et al. (Randzio, Flis-Kabulska, and Grolier 2002).
336
4 of their study, performed using infrared spectroscopy and a diamond

anvil cell, Rubens and Heremans (Rubens and Heremans 2000) show
that the temperature of the transition is lowered by pressure increase.
In opposition to the pressure effects on the main endothermic transition
(M), the pressure influence on both the exothermic transformation (A)
and the high-temperature endothermic transition (N) is positive. At
10 MPa, the exothermic transformation starts at 348.6 0.6 K and is
shifted by pressure to higher values at a mean rate of 38.9 9.9 mK
MPa1. Also at 10 MPa, the high-temperature endothermic transition
starts at 382.7 0.2 K and is shifted by pressure to higher values at a
mean rate of 96.1 3.4 mK MPa1. These observations are in agreement
with a general thermodynamic approach to these transitions.
In Figure 13.20, in transition (A), the exothermic effect is always
associated with a decrease of thermal expansion, which is most probably caused by a negative volume change at that transition, similar to
the observation made on the main transition (M). In transition (N), the
endothermic effect is associated with a small increase of thermal
expansion, which is most probably caused by a positive volume change
at that transformation. Thus, the Clapeyron equation in both cases
also would give positive dT/dP slopes.
The uncertainty limits given for the above results contain both a
purely instrumental contribution, 15%, and a contribution from the
preparation of the emulsion, which can be several percent.
Phase Stability of Systems Containing Lipids
Lipids are components of various food products, especially oils. The
content and the nature of lipids influence the phase stability of such
products. Figure 13.22 presents a pressure-temperature phase diagram
(a) and pressure dependence of latent heat of fusion (b) for cocoa
butter, palm oil, copra oil, and for comparison, water. All the data were
obtained from high-pressure calorimetric measurements (Hayert et al.
2003). It can be seen that the cocoa butter and copra oil, which do have
almost no high unsaturated fatty acids, solidify at rather low pressures
below 60 MPa. In contrast, the palm oil, which contains more high
unsaturated fatty acids, solidifies under much higher pressure of
122 MPa. All the transitions in the investigated systems containing
lipids have positive slopes and their latent heats do not depend on
pressure, which can suggest that the volume variations of those transi-
337
Figure 13.22. (a) PT phase diagram of lipids (cocoa butter, palm oil, copra oil) and
water determined from high-pressure calorimetric measurements. (b) Pressure dependence of latent heat of fusion of lipids (cocoa butter, palm oil, copra oil) and water
determined from high-pressure calorimetric measurements.
tions are positive. These features are in opposition to the respective

properties of water, which should be taken into consideration in highpressure processing of food products containing those components.
Conclusions
Presented in this chapter are a review and a description of highpressure calorimetric and transitiometric techniques, which allow
338
investigation under typical processing conditions of high hydrostatic

pressure and temperature of various transitions and processes occurring in model and real food systems under well-determined pressure
and temperature conditions. The thermodynamic and thermomechanic
parameters of the transitions under investigation determined with the
described techniques permit understanding of the effects of high-pressure processing on the physical properties of food materials and thus
should allow prediction of the formulation of raw materials and processing conditions so as to achieve desired end-product properties. A
detailed description of selected applications of the presented techniques in the analysis of high-pressure processing of selected systems
important for food science and technology should also stimulate further
development of such applications in other food systems.
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Chapter 14
Calorimetric Analysis of Starch Gelatinization
by High-Pressure Processing
Kelley Lowe and Gnl Kaletun
Introduction
Gelatinization of Starch by Heat
Gelatinization of Starch by High Hydrostatic Pressure
High-Pressure Processing of Wheat Starch Suspensions
Storage of Gelatinized Starch
Conclusions
References
341
342
344
344
347
348
349
Introduction
Starches are used in many food products to increase viscosity or to
form gels. Because starch is insoluble in water, a mixture of starch
with water forms a suspension. Starch granules in suspension swell
with heat, and the viscosity of the suspension increases, depending on
starch concentration. Thermal processing changes the physicochemical
properties of starch, such as increased water solubility and development of viscoelastic behavior (Fennema 1996; Jobling, 2004).
Because starch affects the texture of food products, characterization
of aqueous starch suspension behavior and its interaction with other
food additives under conditions relevant to food processing and storage
is important for assessment of food stability. This chapter provides
a review of starch characterization studies by calorimetry relevant
341
342
to thermal processing, high hydrostatic pressure (HHP) processing,

and retrogradation of processed starch during storage.
Gelatinization of Starch by Heat

Aqueous starch suspension undergoes a phase transition between 60 C
and 70 C. During the phase transition, starch granules swell and optical
properties of starch granules, such as light polarization or iodine coloration, change. Starch gelatinization is a water-assisted melting process
that exhibits an endothermic transition and is monitored by differential
scanning calorimetry (DSC). Figure 14.1 shows the gelatinization endotherms of 15% (w/w) for wheat and corn starches heated in a DSC. The
peaks observed are characterized by two parameters, namely, the peak
temperature, thermal stability of the starch phase; and the peak area
under the curve, enthalpy of gelatinization (H). It is apparent that the
gelatinization temperature of corn starch is higher than that of wheat
starch in terms of onset (66 C vs. 58 C) and peak temperatures (70 C
vs. 64 C) of the gelatinization endotherm. Corn starch also requires a
larger heat energy for gelatinization per gram of dry starch (11.2 Jgds1
vs. 16.1 Jgds1). Similar results are reported in the literature for starches
of different origins (Roos 1995). Because thermally induced gelatinization requires higher temperatures and exhibits larger enthalpy change
for corn starch than wheat starch, corn starch is considered to have a
more thermally stable crystalline structure.
Starch exists in foods together with other ingredients. Gelatinization
characteristics of starch are also affected by the presence of other food
ingredients, such as sugars and proteins (Figure 14.2). Curve C in
Figure 14.2 is a thermogram of a mixture of starch, sugar, and milk
protein, which is a representative composition of milk pudding. Figure
14.2 shows that when the total dry matter is kept constant, replacing
some of the starch with sugar only (curve B) and sugar and proteins
(curve C) shifts the thermal stability of the corresponding mixture to
higher temperatures up to approximately 5 C in comparison with the
thermal stability of the starch-water mixture (curve A). Similarly, the
heat energy required for gelatinization changes with the composition
of the mixture. Therefore, the parameters obtained by calorimetry are
directly applicable to processing protocols and should be taken into
account when process conditions are selected.
0.32
65.72C
2.416J/g
Heat Flow (W/g)
0.34
0.36
70.33C
58.30C
1.673J/g
0.38
0.40
63.63C
0.42
45
Exo Up
50
55
60
65
70
75
80
85
90
Universal V2.6D TA Instruments
Temperature (C)
Figure 14.1. DSC thermograms of 15% (w/w) native corn and wheat starch suspensions. Corn starch (solid line) and wheat starch (dashed line).
30
40
50
60
70
80
90
100
Temperature (C)
Figure 14.2. DSC thermograms of wheat starch (A), wheat starch-sugar (B), and
wheat starch-sugar-milk protein (C). Total solids: 30% for all cases.
343
344
Gelatinization of Starch by High Hydrostatic Pressure

HHP has been shown to affect high-molecular-weight polymers
causing denaturation of proteins (Messens et al. 1997; Famelart et al.
1998) and gelatinization of starch (Douzals et al. 1996, 1998, 2001;
Zuo et al. 1999). Ezaki and Hayashi (1992), based on a study including
20 starches, reported that A-type starches (cereals) are more susceptible to high pressure than B-type starches (tubers), and C-type
starches (pea, tapioca) show intermediate behavior. A similar study
conducted by Stute et al. (1996) on two starches of various crystalline
structures stated that A- and C-type starches are susceptible to
gelatinization at intermediate pressure levels, and B-type starches
are the most pressure resistant. Among different starches, potato
starch appears to be the most resistant starch to high-pressure
gelatinization. Studies in the literature show that starch can be
gelatinized by pressure partially depending on the level of pressure
applied (Ezaki and Hayashi 1992; Stute et al. 1996; Douzals et al.
1998).
High-Pressure Processing of Wheat Starch Suspensions

The impact of HHP on gelatinization of starch is investigated by
applying a pressure treatment between 0.1 and 700 MPa to a 30%
wheat starch suspension. The high-pressure-processed starch is
then characterized by performing DSC studies. A preliminary experiment at various starch concentrations showed that it requires at
least 12% wheat starch concentration to form a gel by using HHP
processing. Although the DSC results exhibited a complete gelatinization, a characteristic gel texture cannot be obtained below 12%
wheat starch concentration. A similar observation was reported
by Stolt et al. (2001) for barley starch: while the increase of viscosity
was slight for 10% suspension after pressure treatment at 550 MPa,
a strong paste with creamy texture was obtained with 25% suspension.
Stute et al. (1996) also stated that HHP processed starches exhibited
low viscosity values at the concentrations at which gel formation
typically occurs by heat gelatinization, but they formed smooth pastes
or rigid gels within the concentration range from 15% to 30% dry
matter.
345
Sample preparation
A Hydrostatic High Pressure Unit (Quintas QFP-6; ABB Autoclave
Systems, Columbus, OH) with a maximum pressure of 900 MPa was
used to gelatinize the starch suspensions at ambient temperature. A
water-ethylene glycol (1 : 1 vol/vol) mixture was used as the pressuretransmitting fluid. The rate of pressurization was 400 MPa/min, with a
pressure release time of less than 20 s. During pressurization, the pressure, temperature, and time were kept constant using an automatic
device and recorded throughout the cycle using a data logger. Fifty
milliliters of 30% starch suspension was placed in a sterile, polyethylene bag and sealed under vacuum. The sample bags were placed inside
the Hydrostatic High Pressure Unit. HHP processing was performed
at 25 C for 15 min at a range of pressures from 100 MPa to 700 MPa.
The pressure-treated samples were analyzed using a DSC (model
2090; TA Instruments, New Castle, DE) to determine the degree of
gelatinization. Samples (5055 mg) of HHP-treated starch were placed
in a high-volume stainless steel crucible, and the thermograms were
recorded from 1 C to 100 C at a heating rate of 5 C/min. Each thermogram was analyzed to calculate the onset and the peak temperatures
and the enthalpy of the endothermic transition corresponding to the
melting of ungelatinized starch.
Results
The degree of gelatinization of a 30% starch solution is directly related
to the level of pressure applied at 25 C. Figure 14.3 shows that the
peak area corresponding to melting of ungelatinized starch decreases
progressively as the level of applied pressure is increased. In addition,
as the pressure level increases, the thermal stability of the ungelatinized starch phase decreases, indicating changes in the crystal structure
of starch, although some light microscopy studies show intact starch
granules after pressure treatment (Douzals et al. 1998; Stolt et al.
2001). However, some microscopic observations showed some swelling of starch granules (Douzals et al. 1996), which could lead to the
observation of reduced thermal stability.
The data in Figure 14.3 were analyzed further to calculate the percentage of gelatinized starch as a function of applied pressure (Figure
14.4). Percent starch gelatinization data from Douzals et al. (1996) and
Stute et al. (1996) for wheat starch also are included in Figure 14.4.
Although the initial starch concentrations are different, 16% (Douzals
0.30
600
500
0.32
Heat Flow (W/g)
400
0.34
300
200
0.36
100
0.38
0.40
50
Exo Up
55
60
65
70
75
80
Temperature (C)
Figure 14.3. Gelatinization endotherm of 30% wheat starch suspension after HHP
processing at various pressure levels.
100
Percent gelatinization
80
60
40
20
0
0
100
200
300
400
500
600
Pressure (MPa)
Figure 14.4. Percent gelatinization of wheat starch as a function of HHP. Open

square, 30% starch (this study); filled circle, 16% wheat starch (data taken from
Douzals et al. 1996); filled diamond, 25% wheat starch (data taken from Stute et al.
1996).
346
347
et al. 1996), 25% (Stute et al. 1996), and 30% (w/w) dry solids (this
work), the trend seems to be similar. A fairly sharp increase in the
extent of gelatinization starts at 300 MPa for all starch data, and wheat
starch suspensions are completely gelatinized after a treatment at 500
600 MPa. The results suggest that starch can be partially or completely
gelatinized by increasing pressure at constant temperature.
Douzals et al. (2001) further characterized the behavior of the wheat
starch-water suspensions at 5% dry starch concentration over a pressure range of 0.1 and 600 MPa and over the temperature range of 20
and 96 C. The microscopic measurements of the loss of birefringence
of the granules calibrated by DSC was used to determine the extent of
gelatinization. The data were used to develop the pressure-temperature
(P-T) gelatinization diagram. The P-T gelatinization diagram is similar
to the P-T diagram of denaturation of proteins. The P-T diagram indicates that starch can be gelatinized under various pressure-temperature
combinations; however, the gelatinized starch can have different properties based on the gelatinization conditions.
For corn starch, Zuo et al. (1999) report the presence of native starch
after a treatment at 700 MPa for 2 min, but complete gelatinization after
5 min, which indicates that starch gelatinization by pressure is a kinetically controlled process. This issue becomes significant for investigation of starch kinetics at HHPs. The starch gelatinization kinetics
should be decoupled from the rate of increase of pressure with
time. In fact, Stolt et al. (2001) state that treatment of barley starch
suspension at 550 MPa with a zero-minute holding time exhibited
almost complete gelatinization, indicating pressurization at a rate of
20 MPamin1 was sufficiently slow for complete gelatinization.
Storage of Gelatinized Starch

Gelatinized starch recrystallizes during storage, affecting the texture
and shelf life of food products. This phenomenon is known as retrogradation. Retrogradation contributes to the quality defects in foods, such
as loss of viscosity, syneresis, and precipitation. Jouppila et al. (1998)
reported that water content, storage temperature, and the temperature
difference between storage temperature and glass transition temperature were important factors in retrogradation of thermally treated corn
starch. Furthermore, the formation of bonds between the macromole-
348
cules during retrogradation depends on the gelatinization conditions,

including concentration and degree of gelatinization (Stute et al. 1996).
Douzals et al. (1998) reported that crystallization of the HHPgelatinized wheat starch (30% dry matter) during storage approached
an asymptotic value after 6 days, and the extent of retrogradation was
higher for starch gelatinized by heat than starch gelatinized by pressure
at 600 MPa. However, Stolt et al. (2001) reported that the retrogradation of heat-treated (90 C, 30 min) and pressure-treated (550 MPa,
30 C, 10 min) 25% (w/w) barley starch stored at 4 C was similar and
did not approach an asymptotic value after 7 days of storage. The
results may suggest that retrogradation depends on the botanical source
of the starch as well as gelatinization conditions, storage temperature,
and starch concentration.
Conclusions
An understanding of the effect of high pressure on the properties of
starch-based food systems under conditions relevant to food storage
are necessary to predict storage stability of such systems so that HHPprocessing protocols can be optimized for successful development of
HHP-processed commercial food products.
Studies on the retrogradation properties of starch in the presence of
common food ingredients are also essential for optimization of processing conditions so as to improve the physical stability and textural
characteristics of HHP-processed food products. Because DSC instruments operating under conditions relevant to HHP-processing conditions are not commercially available, starch samples have to be treated
before performing DSC on the samples. The thermodynamic basis of
starch gelatinization involves initial and final states that can be experimentally defined and energetic and/or structural differences that can
be measured using calorimetry. The comparison of various final states
as a function of exposure to various levels of pressure, starting from
the same initial state, makes it possible to predict the effectiveness of
HHP to produce gelatinized starch. However, development of DSC
equipment working at high pressures will allow one to perform pressure scans to determine the pressure dependence of the gelatinization
event, to investigate the kinetics of starch gelatinization at constant
pressure, and to separate the irreversible and reversible events.
349
References
Douzals J.P., Marechal P.A., Coquille J.C., and Gervais P. 1996. Microscopic study
of starch gelatinization under high hydrostatic pressure. J Agric Food Chem,
44(5):14051409.
Douzals J.P., Perrier-Cornet J.M., Gervais P., and Coqulle J.C. 1998. High-pressure
gelatinization of wheat starch and properties of pressure-induced gels. J Agric
Food Chem, 46:48244829.
Douzals J.P., Perrier-Cornet J.M., Coqulle J.C., and Gervais P. 2001. Pressuretemperature phase transition diagram for wheat starch. J Agric Food Chem,
49:873876.
Ezaki S. and Hayashi R. 1992. High-pressure effects on starch: Structural changes
and retrogradation. In: High Pressure and Biotechnology, Vol. 224, Balny C.,
Hayashi R., Heremans M.P., editors, pp. 163165. Colloque INSERM/John Libbey
Eurotext: Montrouge, France.
Famelart M.H., Chapron L., Piot M., Brule G., and Durier C. 1998. High pressureinduced gel formation of milk and whey concentrates. J Food Eng, 36:149164.
Fennema O.R. 1996. Food Chemistry, 3rd edition, pp. 201204. Marcel Dekker: New
York.
Jobling S. 2004. Improving starch for food and industrial application. Cur Opin Plant
Biol, 7:210218.
Jouppila K., Kansikas J., and Roos Y.H. 1998. Factors affecting crystallization and
crystallization kinetics in amorphous corn starch. Carbohyd Polym, 36:143149.
Messens W., Van Camp J., and Huyghebaret A. 1997. The use of high pressure to
modify the functionality of food proteins. Trends Food Sci Technol, 8:107112.
Roos Y.H. 1995. Phase Transitions in Food. Academic Press: San Diego, CA.
Stolt M., Oinonen S., and Autio K. 2001. Effect of high pressure on the physical
properties of barley starch. Innov Food Sci Emerg Technol, 1:167175.
Stute R., Heilbronn R.W., Klingler R.W., Boguslawski S., Eshtiaghi M.N., and Knorr
D. 1996. Effects of high pressures treatment on starches. Starch/Staeke,
48:399408.
Zuo C., Ma, C., and Zhang S. 1999. Effects of high hydrostatic on gelatinization of
corn starch. In: Proceedings of the International Conference on Agricultural
Engineering (ICAE), Vol. 4, pp. 9193 Beijing, China.
Chapter 15
Use of Calorimetry to Evaluate Safety
of Processing
Hans Fierz
Scope
Concepts
Severity: Adiabatic Temperature Rise
Probability: Time to Maximum Rate
Critical Conditions
Autocatalysis
Screening
Comparison of Open and Closed Measurement Methods
Estimation of q(T )
Isoconversional Methods
High-Sensitivity Calorimetry
Adiabatic Measuring Methods
Dewar Vessels
Accelerating Rate Calorimeter
Reactions with Oxygen
Screening Test
Determination of Self-Ignition Temperature
Applications
Formation of Hot Spots in Dryers
Storage and Hot Discharge
Prevention of Molasses Incidents
Transport Safety
Conclusion
References
351
352
352
353
353
354
356
356
356
357
357
360
361
361
361
362
363
363
364
364
364
364
365
365
366
366
352
Scope
Food, as does every other chemical, shows chemical reactivity, and if
handled in bulk can be dangerous. Numerous incidents involving
wheat, milk powder, coffee, or molasses are known, due to, for example,
self-heating, self-ignition of hot spots, or dust explosions.
This chapter focuses on the methodology for characterizing the
thermal consequences of exothermic decompositions in bulk and the
correspondent safety risks. Of course, there are desired synthetic exothermic reactions in bulk, with food presenting a thermal risk, as for
example the hydrogenation of fats. These cases are rather rare and may
not justify a treatment from a specialized point of view such as food
chemistry.
However, the methodology of how to treat and quantify the risk of
both desired reactions and decompositions is not specific to food chemistry, but was developed for process chemistry in general. A thorough
discussion of this topic can be found in books about process safety
(Stoessel 2008).
One of the major risks in food production is dust explosions in mills
or dryers, for example, in sugar refining. Because this chapter deals
with applications of calorimetry, we consider dust explosions only
insofar as hot spots serve as ignition sources. Further literature about
this topic can be found in Bartknecht (1981).
Concepts
The risk of an exothermic decomposition can be defined as the product
of its severity and its probability, both of which can be discussed within
the framework of the so-called adiabatic scenario.
Adiabatic means that there is no heat exchange at all between the
system and the surroundings. A situation with no or negligible heat
exchange could occur in various cases, including failure of heat transfer (breakdown of stirrer or cooling system) during storage of a liquid
or storage of reactive solids in bulk.
The latter situation can occur either intentionally, for example, when
a solid is stored in drums or containers at elevated temperature, or
unintentionally in a dryer or a mill when after a technical incident the
product is no longer agitated and the product cannot be discharged.
353
Severity: Adiabatic Temperature Rise

In an ideal adiabatic environment any heat generated by a decomposition will be accumulated and converted to a temperature rise, Tad,
Tad =
Qr
(15.1)
cp
where Qr is the specific heat of reaction and cp the specific heat

capacity.
The adiabatic temperature rise is a measure of the severity of an
incident. An adiabatic temperature rise of 50 K can hardly be considered critical, but one of 400 K could lead to formation of gases, to an
explosive rupture of the vessel, and finally to a fire due to self-ignition
of the dispersed material.
Probability: Time to Maximum Rate
The rate of an exothermic reaction is proportional to its heat release
rate and if no heat can be removed, to its temperature increase rate:
Q
dT q
=
= r r
dt c p
cp
(15.2)
where q is the specific heat production in watts per kilogram and r is

the specific reaction rate in kilograms per second.
As the reaction rate increases with the temperature (law of Arrhenius),
temperatures increase quickly resulting finally in a so-called thermal
explosion. This can be characterized by an adiabatic temperature rise
and a time until the explosion sets in, the so-called time to maximum
rate under adiabatic conditions (TMRad).
For reactions of nth order, which generate a large amount of heat,
the time to maximum rate can be described by the following
equation:
TMR ad (T ) =
RT 2
Ea
cp
q (T )
(15.3)
where R is the universal gas constant and Ea the activation energy.
354

80
70
Temperature [C]
60
50
t/8
40
t/4
30
t/2
20
t
10
0
0
10
20
30
40
50
60
70
Time [min]
Figure 15.1. Temperature as function of time using the assumption that a 10

increase in temperature doubles the reaction rate. Under adiabatic conditions, this
produces a thermal explosion after a defined time, as described in the section
Probability: Time to Maximum Rate.
The time until the system explodes is a function of the initial heat
release rate q and thus of the temperature (Figure 15.1). Note that in
the beginning, the temperature increase rate is only moderate. The
TMRad is therefore related to the probability of an incident. At very
long times, countermeasures may be taken, for example, discharging
the container or filling it with water. A very short time, however, does
not allow any action to be taken, and the thermal explosion cannot be
avoided.
Critical Conditions
Critical heat release rate
The adiabatic case is the worst-case assumption. Any real system loses
heat to its surroundings either by convection, in the case of liquids, or
by conduction, in solids. A steady temperature will be obtained when
the heat release rate of the reaction equals this heat loss rate, which is
the definition of the critical heat release rate qcrit. Any heat release rate
higher than this will lead to heat accumulation, to a temperature
increase, and finally to a thermal explosion.
355
Critical layer thickness

In solids, the dominant heat transport mechanism is conduction. For
each heat release rate, there is a critical layer thickness or critical radius
rcrit of the bulk, above which the decomposition heat can no longer be
dissipated (Equation 15.4). For a given shape of the bulk, the radius
will define the volume.
r crit =
RT 2
Ea
q(T )
(15.4)
Critical temperature
As the rate is related to the temperature via Arrhenius law, there is also
a critical temperature for a given bulk volume. Details can be found
in Gray and Lee (1967). The critical temperature, Tcrit, is therefore a
function of the layer thickness, r, and its physical properties (density
, thermal conductivity , geometry ), as well as of the macrokinetics
of the decomposition characterized by (q(T), Ea). Figure 15.2 shows
a typical dependency of the critical radius of the temperature.
Many methods originally developed by trial and error use, in fact,
the concept of critical temperatures (see below).
Critical radius [m]
1.00E+00
1.00E-01
unstable, thermal explosion
1.00E-02
stable, no thermal explosion
1.00E-03
0
50
100
Temperature [C]
150
200
Figure 15.2. Dependence of the critical radius r of the temperature T for a typical
reaction as described in the text. Combinations of T and r in the overcritical region
will lead to a thermal explosion; in the undercritical region, the system will be stable.
356
Autocatalysis
The concepts described above rely on the assumption that the heat
release rate does not depend on the conversion. There are, however, many cases in which the heat release rate initially increases with
increasing conversion, reaches a maximum, and then decreases
again. This behavior is called formal autocatalysis or, in short,
autocatalysis.
For such cases, the equations previously discussed for the TMRad
and the critical layer thickness can be applied only with caution. It is
therefore important to identify this type of formal reaction. However,
using temperature-programmed thermoanalytical measurements for
this is not obvious and requires experience: Bou-Diab and Fierz (2002)
describe an identification approach.
Differential scanning calorimetry (DSC) is often used either as a
screening tool or to establish the thermal kinetics of a decomposition.
Here, the change in heat flow as function of the oven temperature is
recorded.
Becasue starting materials and products may be volatile, correct
results are obtained only by using closed and pressure-tight crucibles.
Measurements in which the samples are allowed to lose mass can show
quite different behavior from those made in closed crucibles. An
example describing this situation is given for saccharose in the
Comparison of Open and Closed Measurement Methods section below.
Typically, pressure-resistant gold-coated 40 l crucibles can be used,
which can withstand 400 C/220 bars.
Screening
From one temperature-programmed run, both the reaction and decomposition energy and its temperature range can be deduced (ASTM
E537-98).
As often is the case in calorimetry, the determination of the baseline
is difficult because, for reactions, the recorded signals usually cover a
broad temperature range. Interpolation of the baseline over such a
broad range and therefore determination of the decomposition potential
2.5
2
1.5
1
0.5
0
-0.5
-1
-1.5
120
Mass loss in %
100
80
TGA
SDTA
60
357
40
20
0
-20
DeltaT [K]
0
50
0
45
0
40
0
35
0
30
0
25
0
20
0
15
0
10
50
Temperature [C]
Figure 15.3. Saccharose measured in an open crucible at 4 K/min (thermogravimetric

analysis) as an illustration of the difference between using either open or closed
crucible. Shown is the thermogravimetry TGA signal (mass loss in percentage as
function of temperature) and the qualitative DTA signal (SDTA: T sample, oven).
can be quite difficult. To cool down the sample after a run and to
perform a second scan without removing the sample crucible from the
sensor helps establish the baseline.
Comparison of Open and Closed Measurement Methods
Figure 15.3 shows a typical thermogravimetric trace of sugar (saccharose). Thermogravimetric analysis is based on mass loss of the sample;
the sample crucible is open to the atmosphere and thus the method is
called an open method. At 220 C, there is a sudden decrease in mass
followed by a gradual mass loss until, at 450 C, all the sample has
evaporated. At the same time, the instrument used records a qualitative
heat flow signal, which indicates a peak heat release rate at 350 C.
The same substance measured in DSC in a closed crucible shows a
quite high decomposition potential at lower temperatures (240 C)
(Figure 15.4).
Estimation of q(T)
The detection limit of a modern DSC apparatus is about 1 W/kg. If this
value is set equal to qcrit and used to calculate the critical radius rcrit in
Equation 15.4, a value of about 0.1 m results, which is equivalent to a
cubic container of approximately 8 L, depending on the assumptions
358

Signal [W/g]
1
0.5
0
-0.5
-1
0
50
100
150
200
250
300
350
400
Temperature [C]
Figure 15.4. DSC measurement of saccharose at 4 K/min in a closed pressureresistant crucible. Note that the decomposition occurs at lower temperatures than in
Figure 15.3.
made. In other words, lower heat release rates that may lead to a
thermal explosion in a bigger volume may remain undetected.
Extrapolation of heat release rates to lower temperatures is thus
often necessary. A conservative estimate (that is an estimate giving
high values) for q at lower temperatures can be obtained as follows,
Once a baseline is drawn, the deviation of the signal from the baseline at any temperature T is proportional to the heat release rate q(T ).
If this is determined at the beginning of the signal, the influence of the
conversion can be neglected, and q(T ) can be used as reference value
(Figure 15.5). In practice, a value of 20 W/kg for q(T ) is a good compromise between a value too small to be measured and one so high
that there is already noticeable conversion.
To estimate heat release rates at other temperatures, a value for the
activation energy Ea is needed. This is generally not known a priori.
To overcome this problem, a very low value of the activation energy,
for example 50 kJ/mol, can be used, which in the case of real decompositions is practically never encountered.
This can now be used to calculate a too-short and thus conservative
value of the TMRad (Figure 15.6) or of the critical radius rcrit. If acceptable TMRad values or critical volumes are then obtained at the desired
temperature, no further action has to be taken. If not, more refined
kinetic methods are needed.
Thus, from one measurement both the severity Tad and the probability of a runaway TMRad can be estimated. This approach is in
practical use in many laboratories and has been verified theoretically
(Keller et al. 1997). Also, by comparing actual adiabatic experiments
to the results of the described method, it has been shown that the results
140
Heat release rate [Watt/kg]
120
100
80
60
20 Watt/kg
40
20
0
0
50
100
150
200
250
300
Temperature [C]
Figure 15.5. Estimation of q(T) values from a DSC measurement. Shown is a schematic DSC thermogram and the determination of the heat release rate at small
conversion.
Figure 15.6. Influence of different activation energies on the safety margin of the
extrapolated TMRad. A measured heat release rate (15 W/kg at 160 C, upper right)
can be used to extrapolate the heat release rate corresponding to a TMRad of 24 h using
a high activation energy (lower curve) or a low activation energy (upper curve).
Extrapolated temperatures of 100 C and 50 C, respectively, result.
359
360
so obtained in all studied cases were on the conservative side (Pastr

et al. 2000).
Some words of caution are necessary, however:
The point of the curve where 20 W/kg is reached should correspond
to a conversion of less than 10%.
The method should not be used for extrapolations to higher
temperatures.
It works best when the detectable beginning of the signal lies below
250 C.
It depends on a good definition of the baseline. Especially strongly
curved baselines will make the determination of the heat release rate
unreliable at small conversion.
One should not extrapolate across a melting point, because decomposition mechanisms in the solid state can be very different from
those in the liquid state.
Isoconversional Methods
Isoconversional methods deliver formal kinetic information by using
a set of measurements where the heat release rate is measured at different temperatures and always at the same conversion. Once the
kinetic information is known, it can be used to calculate the heat
release rate and the conversion at different conditions, such as at fixed
temperatures or under adiabatic conditions.
Such an isoconversional set can consist of measurements at different but constant heating rates. Whereas simple methods are easy to
use but limited to nth-order reactions (ASTM E698), the more advanced
methods depend on sophisticated computer algorithms (Opfermann
and Hdrich 1995; Roduit 2000), which are outside the scope of this
chapter.
Although the isoconversional method can be considered to be state
of the art, it is not always possible to apply it. For example, in cases
where the product melts and decomposes in the liquid state, it is often
not possible to determine the decomposition kinetics in the solid state.
Alternatively, one can use a set of measurements at different but
constant temperatures, where the signal is recorded as a function of
time. This approach works best with kinetics of nth-order, where the
highest heat release rate occurs at the beginning of the isothermal
361
phase. An Arrhenius graph of the maxima of the heat release rate q is

then plotted as a function of the temperatures T, and from this reference
the values for the heat release rate and the activation energy can be
obtained. It is assumed that at the maximum of the heat release rate
there is not yet any significant conversion. This method is easy to
understand and to use; however, the temperature range and therefore
the number of points in the Arrhenius graph is limited: (1) For isothermal measurements at high temperatures, there is some conversion
during heatup, so the assumption of zero conversion therefore may not
be true; and (2) at low temperatures, measurement time can be very
long, as the baseline is known only after complete conversion.
High-Sensitivity Calorimetry
High-sensitivity calorimetry (Suurkuus and Wads 1982) can be used
to avoid the cumbersome extrapolation of heat release rates to lower
temperatures. In these instruments, heat release rates as low as 0.001
0.03 W/kg can be measured directly. The method works at constant
temperatures between 30 and 80 C, and measurements usually take
several days to perform.
Adiabatic Measuring Methods

In an adiabatic calorimeter, there is no heat exchanged with the surroundings. Any heat produced in a system will therefore remain inside
the system and produce a temperature rise. This can be measured, and
once the heat capacity of the system is known, the heat of reaction can
be determined. So at least in principle, one could put the decomposing
substance in such a calorimeter and obtain both the adiabatic temperature rise and the TMRad.
Dewar Vessels
The use of adiabatic calorimeters seems to be an attractive choice, and
the type most commonly used is the Dewar vessel (Rogers 1989).
These are quite well insulated, but a 500-ml vessel filled with a liquid
still has a heat loss of typically 0.04 W/kg/K. Heat release rates of less
than 0.20.4 W/kg will in this case not lead to self-heating.
362
Dewar vessels are usually made of glass and are not pressureresistant. Gases and vapors must be allowed to escape from the system,
and because this is normally an endothermic process, it also will
contribute to the heat loss. The sensitivity of such an arrangement
can be improved (1) by minimizing the temperature difference between
Dewar and oven by adapting the temperature of the oven to that of
the Dewar and (2) by placing the Dewar vessel in an autoclave to suppress the evaporation of gases and vapors (Grewer 1994). The use of
containment is also advisable for reasons of safety and industrial
hygiene.
This improvement requires the use of rather sophisticated experimental facilities, and especially in the case of autoclaves, a room with
concrete walls and with remote control should be used.
Accelerating Rate Calorimeter

The accelerating rate calorimeter was first described by Townsend
(Townsend and Tou 1980). In this instrument, it is possible to measure
both the TMRad and the adiabatic temperature rise with a relatively
small sample of a few grams. A small pressure-resistant container is
filled with the sample and placed in an oven at a temperature closely
matches that of the sample container. There is therefore no heat
exchange between sample and oven, and heat generated by a reaction
will produce a proportional temperature rise. Despite the small sample
size, the instrument is quite sensitive. It can detect approximately
0.3 W/kg, which corresponds to a heat rate of approximately 0.01 K/
min or 0.6 K/h, and its sensitivity corresponds to that of a 400-ml
Dewar vessel.
The proportionality between the specific heat of reaction Qr4 and
Tad or the specific heat production q and the rate of temperature rise
dT/dt is the specific heat capacity of the system. The heat produced by
the sample will heat not only the sample itself but also the sample
container. To obtain TMRad and Tad values of the substance itself, the
measured values TMRmeas and Tmeas must be corrected with a factor
as follows:
TMRadcorr =
meas
TMR ad
(15.5)
363
and
Tadcorr = Tadmeas
(15.6)
where
= 1+
mb c p,b
ms c p , s
(15.7)
and m is mass, cp is specific heat capacity, and subscript b and s

mean sample container and sample, respectively. The correction for
the TMRad is valid for reactions of 0th order. At small conversion, nthorder reactions can be treated as being of 0th order, as conversion can
be neglected.
Reactions with Oxygen

Tests for the determination of the self-ignition temperature simulate a
dryer, where the powder sample is in close contact with hot air.
Different test arrangements are in use, which can be classified according to how oxygen is delivered to the sample: by forced convection or
diffusion (Grewer 1971). In all these tests, the temperature of the hot
air is either held constant or increased continuously, and the temperature difference between the sample and either an inert reference or the
surroundings is recorded. With methods using relatively big sample
volumes, temperatures in the center of the sample can deviate considerably from the temperature of the surrounding air and can reach overcritical values. These instruments will therefore not give quantitative
heat release rates, but will be more suited for the detection of critical
temperatures.
Screening Test
Currently, a widely used screening test apparatus, which is described
by Grewer (1971), uses preheated air that flows through both a sample
and a reference channel containing the respective samples in small wire
baskets. An inert substance such as graphite is used as reference.
364
Determination of Self-Ignition Temperature

In the European Union, a testing method of the diffusion type is in
official use (method A16 of the European Commission Joint Research
Centre). Here, the sample is placed in a wire basket surrounded by hot
air. The temperature of the oven in which the sample reaches 400 C
due to self-ignition is called the self-ignition temperature.
Applications
Formation of Hot Spots in Dryers
Many solid food products are produced by spray-drying of water-based
solutions. Such a solution is sprayed into a stream of hot air. The water
is evaporated, and the solute is converted to a powder. Examples are
the production of powdered milk or coffee whitener.
Reactions with oxygen can lead either to product deterioration or to
smoldering if there are deposits. Deposits not only occur in the dryer
itself but also in equipment downstream, for example, in filters. As
discussed previously, if the heat release rate due to the oxidation in
such a product layer is overcritical, the formation of hot spots that can
trigger a dust explosion is unavoidable. In general, the self-ignition
temperature should therefore be determined.
Storage and Hot Discharge
Sometimes the product is discharged from a dryer to drums or containers while still hot. In a dryer, heat produced by an ongoing decomposition reaction can be removed by convection because the product is
agitated. In a container, however, this agitation is lacking, and heat is
removed by conduction only. This is a much less efficient mechanism
of heat transfer. The product can therefore self-heat, the self-ignition
temperature can be reached, and a fire can occur.
If the decomposition kinetics are known, it is possible to calculate
the critical temperature for a given container size. Alternatively, one
can establish a temperature limit using a series of isothermal Dewar
experiments by starting at a relatively high temperature where a signal
is observed and then lowering the temperature in steps of 10 K until
no signal is observed. From this temperature a safety margin, for
365
example 50 K, is deduced. This gives a conservative estimate for the

allowed discharge temperature.
Prevention of Molasses Incidents
Several incidents where big molasses reservoirs burst are recorded, for
example, the 1919 Great Molasses Disaster in Boston, Massachusetts,
in which a large tank burst, flooding the streets with molasses and
killing several people. The bursting could have been caused by various
reasons. One reason is certainly the exothermic decomposition in combination with the formation of carbon dioxide (Strecker degradation).
The DSC thermogram of the molasses studied in our laboratories
shows a decomposition signal between 120 C and 250 C, with an
energy of several hundred kilojoules per kilogram. Isothermal measurements in the thermal activity monitor showed a heat release rate
of 0.004 W/kg at 50 C and 0.06 W/kg at 75 C, which gave an activation energy of about 100 kJ/Mol. The critical radius at 30 C calculated
by Equation 15.4 and using the above-determined kinetics is fairly low,
approximately 3.5 m, which is equivalent to a capacity of 100,000 l and
therefore comparable with the size of actual reservoirs. These findings
are confirmed in the literature (Platje, Wittenberg, and Timmermans
2006). Heating of molasses in the reservoir to reduce the viscosity and
to facilitate pumping should therefore be avoided.
Transport Safety
The UN recommendations for the transport of dangerous goods
(UNECE 2003) require a chemical to be tested for self-reactivity (Class
4.1) if it releases more than 300 kJ/kg. It is considered to be selfreactive if a 50-kg package has a self-accelerating decomposition
temperature (SADT) less than 75 C. The SADT is defined as
T = Ti Ta 6 K, where Ti is the temperature of the package and Ta
is the temperature of the environment.
According to the recommendations, a 50-kg package can be substituted by a Dewar vessel with a specific heat loss of 0.08 W/kg/K.
For screening purposes, the specific heat release rate can thus be
estimated at 81 C using the method outlined in the Estimation of q(T)
section. If the specific heat release rate is higher than 0.48 W/kg, a full
determination of the SADT must then be made using either the heat
366
accumulation storage test (HAST) H.4, based on a 500-ml Dewar

vessel, or the isothermal storage test (IST) H.3, based on isothermal
measurements. Depending on the outcome of these tests, transport
restrictions apply; for example, packages have to be cooled during
transport or air transport is not allowed. Typical curves of food products, which currently would probably have to be classified as class 4.1,
have been published previously (Raemy and Lambelet 1982).
Conclusion
Food, especially carbohydrate- and protein-based food, can undergo
highly exothermal decompositions. Unlike other chemicals, food itself
is by definition not toxic; nevertheless, the consequences of an accident
involving bulk food can be devastating.
It is possible, however, to assess the risk of such decompositions
using the same principles as for process chemistry. If these principles
are properly applied and the stability data correctly determined, safe
conditions for handling can be established and accidents in the food
industry prevented.
References
ASTM Standard E 698. 2005. Test Methods for Arrhenius Kinetic Constants for
Thermally Unstable Material. ASTM International: West Conshohocken, PA.
Retrieved from: www.astm.org.
ASTM Standard E 537-98. 2007. Standard Test Method for Assessing the Thermal
Stability of Chemicals by Methods of Thermal Analysis. ASTM International: West
Conshohocken, PA. Retrieved from: www.astm.org.
Bartknecht, W. 1981. Explosions. Course Prevention Protection. Springer: Berlin.
Bou-Diab, L. and Fierz, H. 2002. Autocatalytic decomposition reactions, hazards, and
detection. J Hazard Mater, 93(1):137146.
European Commission Joint Research Centre, Institute for Health and Consumer
Protection. Directive 67/548/EEC, Annex V, Method A 16. Retrieved from: http://
ecb.jrc.it/testing-methods.
Gray, P., Lee, P.R. editors. 1967. Thermal Explosion Theory, Oxidation and
Combustion Reviews, 2nd edition. Elsevier: Amsterdam.
Grewer, T. 1971. Zur Selbstentzndung von abgelagertem Staub. Staub-Reinhaltung
der Luft, 31(3):97101.
Grewer, T. 1994. Thermal Hazards of Chemical Reactions, Industrial Safety Series,
Vol. 4. Elsevier: Amsterdam.
367
Keller, A., Stark, D., Fierz, H., Heinzle, E., and Hungerbhler, K. 1997. Estimation
of the time to maximum rate using dynamic DSC experiments. J Loss Prevent
Process Ind, 10:3141.
Opfermann, J. and Hdrich, W. 1995. Prediction of the thermal response of hazardous
materials during storage using an improved technique. Thermochim Acta,
263:2950.
Pastr, J., Wrsdrfer, U., Keller, A. and Hungerbhler, K. 2000. Comparison of
different methods for estimating TMRad from dynamic DSC measurements with
ADT 24 values obtained from adiabatic Dewar experiments. J Loss Prevent
Process Ind, 13:717.
Platje, T., Wittenberg, A., and Timmermans, A. 2006. Study of the runaway behaviour of technical sucrose solutions. Zuckerindustrie, 131(4):231238.
Raemy, A. and Lambelet, P. 1982. A calorimetric study of self heating in coffee and
chicory. J Food Technol, 17:451460.
Roduit, B. 2000. Computational aspects of kinetic analysis. Part E: The ICTAC
Kinetics Projectnumerical techniques and kinetics of solid state processes.
Rogers, R.R. 1989. The advantages and limitations of adiabatic Dewar calorimetry
in chemical hazard testing. Plant Operations Progress, 8:109.
Stoessel, F. 2008. Thermal Safety of Chemical Processes: Risk Assessment and
Process Design. Wiley-VCH: Weinheim.
Suurkuus, J. and Wads, I. 1982. A multichannel microcalorimetry system. Chem
Scripta, 20:155163.
Townsend, D.I. and Tou, J.C. 1980. Thermal hazard evaluation by an accelerating
rate calorimeter. Thermochim Acta, 37:130.
UNECE Transport Division. 2003. International Recommendations on the Transport
of Dangerous Goods, Manual of Test and Criteria, 4th edition. UNECE: New
York.
Index
Accelerating rate calorimeter,

36263
Activation enthalpy, protein heatinduced transformations
with, 129
Adiabatic measurement methods
accelerating rate calorimeter,
36263
Dewar vessels, 36162
food-processing safety with,
36163
Adiabatic temperature rise, 353
ADSC. See Alternating DSC
Aggregation
DSC technique v. vessel/heating
mode with, 28t
globular proteins in bulk phase
system, 12429, 126f128f
heat effects of, 88
heating mode with, 3637, 37f
protein postdenaturation, 11012
Alcohols, protein denaturation
affected by, 99100, 101f
11S globulin, 99100, 101f
Setschenow equation for, 100
Alfalfa, 93
Alginate, denaturation temperature
of -lactoglobulin with,
110
Alternating DSC (ADSC), foodprocessing design with, 204

AMF. See Anhydrous milk fat
Ampoule mixing vessel, mixing and
reaction heat flux
microcalorimeter with, 30,
31t
Anhydrous milk fat (AMF)
Avrami plots from, 139f
calorimetric parameters observed
with, 136t
heat-induced transformations
with, 13341, 135f, 136t,
137f, 139f, 139t, 140f,
18487, 186f, 187f
heating/cooling curves, 135f
isothermal curves, 137f
protein-stabilized, 135f
surfactant added, 135f, 136t, 137f
Antibiotics, 15355, 153f, 155f
Apple, heat capacity for, 36t
Arabic gum, denaturation
temperature of 11S globulin
with, 106t
Autocatalysis, 356
Autoclave, 54, 54f, 55f, 57
Avrami equation, oil-in-water
emulsions with, 13839,
139f, 139t
369
370
Index
Bacillus megaterium, DSC analysis

of, 149
Bacteria
Bacillus megaterium DSC
analysis of, 149
calorimetry with growth of, 43
Citrobacter freundii DSC
analysis of, 149
Clostridium perfringens DSC
analysis of, 14953, 151f
results for, 15052, 151f
sample preparations for,
14950
DSC analysis of foodborne,
14764, 151f, 153f, 155f,
157f, 160f, 162t
antibiotics effect on, 15355,
153f, 155f
cold shocking, 148
heat shocking, 148, 151
mode with, 28t
endothermic/exothermic effects
of, 19t
Escherichia coli DSC analysis of,
148, 15558, 157f
erythromycin treatment of,
15455, 155f
heat inactivation parameters of,
15960, 160f
nonthermal treatment of,
16263, 162t
food-processings effect on,
1011
food-processing treatment
evaluation by DSC for,
15864, 160f, 162f
antimicrobials in, 16364
heat inactivation parameters of
bacteria in, 15861, 160f,
162f
HHP in, 161
nonthermal treatment of
bacteria in, 16163, 162f
hydrostatic pressure resistance of,
4445, 44f
inactivation of, 10
Lactobacillus plantarum DSC
Listeria monocytogenes DSC
153f
159
14950
Mycoplasma laidlawii DSC
analysis of, 149
Staphylococcus aureus
nonthermal treatment,
16263, 162t
Batch high-pressure vessel, mixing
and reaction heat flux
31t
Batch mixing vessel, high
sensitivity heat flux
calorimeter with, 27, 28t
Batch standard vessel, mixing and
reaction heat flux
31t
Benzene, transitiometry verification
test using, 322, 323f
Binding
data quantifies high-affinity,
7577
mixing and reaction calorimetry
with, 4142
processes, protein in dilute
solution with, 78, 79, 80f
Blank test heat flow equation, 32
Index
Bovine -lactoglobulin, 9495
Bovine serum albumin (BSA),
protein denaturation affected
by, 104
Broad beans, 11S globulin from,
99100, 101f, 105
BSA. See Bovine serum albumin
Bulk phase system, denaturationaggregation of globular
proteins in, 12429,
126f128f
Butter, heat capacity for, 36t
Butyric acid, melting point of,
171f
Cabbage, heat capacity for, 36t
Calibration, 2325, 24f, 25f
Calvet type calorimeter, 23,
206
food-processing design, 206
heat calibration procedure for
HP-DSC, 60f, 6163, 62f
high pressure calorimetry, 314
HP-DSC, 5763, 60f, 62f
Joule effect in, 2324, 24f, 25f
MICROCALIX, 177
necessity for HP-DSC, 57
parameters for, 58
heating rate, 58
pan type, 58
sample mass, 58
temperature, 58
reference substances for, 58
indium, 58, 60, 61
lead, 58
tin, 58, 59
zinc, 58
scanning transitiometry, 32223
table of corrections for, 57
temperature calibration procedure
for HP-DSC, 5861
uncertainty with HP-DSC, 58
371
Calorimeter
accelerating rate, 36263
symmetrical, two-chamber,
1718
Calorimetry. See also Calvet type
calorimetry; Differential
scanning calorimetry; Heat
flux calorimeters; Heat flux
microcalorimetry; High
pressure calorimetry; High
pressure differential
scanning calorimetry;
High-sensitivity calorimetry;
High sensitivity heat flux
calorimeter; Isothermal
calorimetry; Isothermal
solution calorimetry;
Isothermal titration
calorimetry;
Microcalorimetry; Mixing
and reaction calorimetry;
Mixing and reaction heat
flux microcalorimeter;
Pressure calorimetry
advantages for using, 7
applications of, 1545
under controlled relative
humidity, 45
food dehydration understood
with, 289309
calorimetric glass transition
measurement for, 29396,
294f, 296f
dielectric and mechanical
relaxations with, 296f,
29798
freeze-drying for, 290, 3067
freezing in, 3013, 302f, 303f
glass transition and stability of,
3078, 308f
phase and state transitions of,
290, 29293, 293f
372
Index
Calorimetry (continued)
spray-drying for, 290, 3056,
306f
state diagrams with, 3037,
304f
thermal analysis in, 298301,
299f, 300f
food industry interest in, 226
food-processing design in, 2026
alternating DSC, 204
Calvet type calorimetry, 203,
206
differential scanning
calorimetry, 2026
differential thermal analysis,
203
dynamical mechanical analysis,
225
dynamical mechanical thermal
analysis, 225
methods, 2056
modulated DSC, 204
samples, 206
techniques, 2035, 204f
food-processing safety evaluated
with, 35166, 354f, 355f,
357f359f
adiabatic measurement
methods for, 36163
applications for, 36466
concepts for, 35256, 354f,
355f
critical conditions in, 35456,
355f
critical heat release rate in,
35455
critical temperature, 35556,
355f
estimation of q(T) in, 35760,
359f
formation of hot spots in
dryers, 364
high-sensitivity calorimetry in,

361
isoconversional methods in,
36061
open v. closed measurement
methods in, 357, 357f, 358f
prevention of molasses
incidents, 365
reactions with oxygen in,
36364
screening in, 35657
storage and hot discharge,
36465
transport safety, 36566
isothermal, 38
isothermal performance of, 19
isothermal solution, 220
methods with food using, 513
parameters of, 8
interpretation of overlapping
peaks, 8
magnitude of heat flow, 8
moisture loss, 8
time scale, 8
pressure, 4345, 44f
scanning mode, 3536, 44
solution, 218
step heating in, 40, 40f
suitability for food of, 52
ultrasensitive to proteins, 8
Calvet principle, 2223, 22f, 23f, 26
Calvet type calorimetry, 2223, 22f,
23f, 203
calibration of, 23, 206
food-processing design with, 203,
206
Capric acid, melting point of, 171f
Caproic acid, melting point of, 171f,
172f
Caprylic acid, melting point of,
171f
Carbohydrates
Index
C80 technique v. vessel/heating
mode with, 31t
cereal with, 12
of, 19t
gelatinization of starch-water
systems, 207
glass transition with, 294
hydrophilic component of,
291
thermal analysis of cereal
nonstarch, 27678, 277f
thermal behavior of food
constituents in, 2068, 207f,
208f
Carboxymethylcellulose,
denaturation temperature of
11S globulin with, 106t
Carp, heat capacity for, 36t
-Carrageenan
denaturation temperature of 11S
globulin with, 106t
-lactoglobulin with, 110
-Carrageenan
globulin with, 106t
Carrots, isothermal traces at
temperatures for, 39f
Caseins, 12223
CB. See Cocoa butter
Cellobiose, calorimetric curves of,
208f
Centre National de la Recherche
Scientifique (CNRS), 176
Cereal, 12
mode with, 31t
Cereal processing, thermal analysis
to design/monitor, 26585
373
nonstarch carbohydrates, 27678,

277f
process applications, 27885,
280f284f
proteins, 27276, 274f, 275f
starch, 26872, 289f272f
Chaotropic salts (Salting-in salts),
95
Chocolate
mode with, 31t
mode with, 28t
Citrobacter freundii, DSC analysis
of, 149
Closed measurement method, 357,
357f, 358f
Clostridium perfringens
DSC analysis of, 14953, 151f
sample preparations for, 14950
CNRS. See Centre National de la
Recherche Scientifique
Cocoa butter (CB)
DSC and XRD with, 17984,
180f, 183f
MICROCALIX for, 18284, 183f
polymorphism, 18182f
polymorphism of 1,2-dipalmitoyl3-oleoylglycerol, 18284,
183f
Coffee, C80 technique v. vessel/
heating mode with, 31t
Cold denaturation, protein in dilute
solution with, 75
Cold shocking, 148
Complete reaction, test for, 255
Cream, heat capacity for, 36t
Critical conditions, food-processing
safety with, 35456, 355f
heat release rate, 35455
temperature, 35556, 355f
374
Index
Crystallization
mode with, 28t
heating mode with, 36
isothermal, 39
lard, 190, 191f, 191t
lipids, 21011
milk fat, 18990
oil-in-water emulsions, 13236,
135f, 136t
water in pork muscle with, 327f
CSC, 21
Dairy, heat capacity for, 36t
Debye-Hckel approximation, 98,
99t
Dehydration. See Food dehydration
Denaturation
cold, protein in dilute solution
with, 75
defined, 122
mode with, 28t
globular proteins in bulk phase
system, 12429, 126f128f
11S globulin, 8992, 91f
alcohols effect on, 99100,
101f
different pH values in, 91f, 92
polysaccharides effect on,
1056, 106t, 110
salts effect on, 9698, 97f, 99t
two-state model to analyze, 89
heat effects of, 88
Kunitz inhibitor, polysaccharides
effect on, 10710, 108f
-lactoglobulin, 125
methodological approaches to
study, 89
of protein, 87113, 91f, 97f, 99t,
101f, 103f, 106t, 108f
effects of alcohols on, 99100,

101f
effects of odorants on, 1024,
103f
effects of pH on, 8995, 91f
effects of polysaccharides on,
10410, 106t, 108f
effects of salts on, 9599, 97f,
99t
reversibility of, 123
two-state model of, 123
Dextran, denaturation temperature
of 11S globulin with, 106t,
108f
Dextran sulfate, 106t, 108f
Differential scanning calorimetry
(DSC), 67, 9, 16, 265
Bacillus megaterium analysis by,
149
calibration of, 2325, 24f,
25f
Calvet principle with, 2223, 22f,
23f, 26
Calvet type of, 2223, 22f, 23f,
203, 206
calibration of, 23, 206
efficiency ratio of, 23f
schematic of, 22f
Citrobacter freundii analysis by,
149
Clostridium perfringens analysis
by, 14953, 151f
14950
cold denaturation with, 75
data quantifies high-affinity
binding with, 7577
assumptions of, 75
concentration in, 77
equilibrium in, 7677
Index
hydrogen ion buffer selection
in, 77
purity in, 77
two-state, reversible transitions
in, 7677
efficiency ratio of flat-shaped,
21f
Escherichia coli analysis by, 148,
15558, 157f
15455, 155f
15960, 160f
nonthermal treatment of,
16263, 162t
foodborne bacteria analysis by,
14764, 151f, 153f, 155f,
157f, 160f, 162t
153f, 155f
cold shocking, 148
heat shocking, 148, 151
methods, 2056
samples, 206
with XRD, 225
food-processing safety with, 355f,
35661, 357f359f
estimation of q(T) for,
35760, 359f
high-sensitivity calorimetry for,
361
isoconversional methods for,
36061
methods for, 357, 357f, 358f
screening for, 35657
food-processing treatment
evaluation by, 15864, 160f,
162f
antimicrobials in, 16364
375
heat inactivation parameters of
bacteria in, 15861, 160f,
162f
HHP in, 161
nonthermal treatment of
bacteria in, 16163, 162f
glass transition with, 294f
heat flux type of, 2021, 2630,
27f, 28t
heatings role in, 88
high pressure, 5164, 54f56f,
60f, 62f
applications of, 63
calibration of, 5763, 60f, 62f
construction of, 5357,
54f56f
Lactobacillus plantarum analysis
by, 15558, 157f
Listeria monocytogenes analysis
by, 14953, 151f
153f
159
14950
microcalorimetry v., 16, 1925,
20f25f
heat flux microcalorimetry,
1925, 20f25f
Mycoplasma laidlawii analysis
by, 149
power compensated type of,
2022
protein in dilute solution with,
6877, 71f
equations, 7074, 71f
heat capacity change origins
for, 74
information content, 6869
instrumentation, 6970
376
Index
Differential scanning calorimetry

(DSC) (continued)
simulated DSC thermogram of,
70, 71f
vant Hoff enthalpy change,
7274
schematic of plate-shaped sensor
for, 20f
schematic representation of, 299f
sensor plate thickness in
efficiency of, 21f
nonthermal treatment with,
16263, 162t
starch analysis with, 26872,
289f272f
starch gelatinization by heat
monitored with, 342, 343f
two types of, 20
X-ray diffraction with, 16994,
171t, 172t, 173f, 177f, 180f,
183f, 186f, 187f, 189f, 191f,
192f
applications for, 17993, 180f,
183f, 186f, 187f, 189f, 191f,
192f
cocoa butter in, 17984, 180f,
183f
lard in, 19093, 191f, 192f
MICROCALIX using, 170,
17679, 177f, 180f, 183f,
186f, 187f, 189f, 191f, 192f
milk fat in, 18490, 186f, 187f,
189f
results using, 17993, 180f,
183f, 186f, 187f, 189f, 191f,
192f
triacylglycerols in, 16976,
171t, 172t, 173f
Differential thermal analysis (DTA),
53, 173
food-processing design with, 203
Dilute solution, calorimetry of

protein in, 6784, 71f, 80f,
83f
Dilute systems, 10
Dissolution, mixing and reaction
calorimetry with, 41
DMA. See Dynamical mechanical
analysis
DMTA. See Dynamical mechanical
thermal analysis
Drying
freeze-, 290, 3067
hot spots with, 13
spray-, 290, 3056, 306f
DSC. See Differential scanning
calorimetry
DTA. See Differential thermal
analysis
Dynamical mechanical analysis
(DMA)
food dehydration with, 296f,
29798
glass transition detected with,
296f, 297
thermal analysis with, 266
Dynamical mechanical thermal
analysis (DMTA)
food dehydration with, 296f,
29798
glass transition detected with,
296f, 297
thermal analysis with, 266
Eggs, isothermal traces at
temperatures for, 39f
Electromotive force (Emf), 23, 25
Electron spin resonance (ESR),
301
Emulsions
lipids, 214
Index
oil-in-water, 13241, 135f, 136t,
137f, 139f, 139t, 140f
anhydrous milk fat, 13341,
135f, 136t, 137f, 139f, 139t,
140f, 18487, 186f, 187f
Avrami equation for, 13839,
139f, 139t
crystallization in, 13236,
135f, 136t
fat crystal growth in, 138
Gompertz model for, 13940,
139t, 140f
ice cream, 133
kinetics of, 13641, 137f, 139f,
139t, 140f
melting of fat droplets in,
13236, 135f, 136t
triacylglycerols, 133
whipped cream, 133
proteins role in, 10
Enthalpy, 265
activation, 129
estimate of apparent denaturation,
112
reaction, 255
vant Hoff enthalpy change,
7274
Entropy, protein heat-induced
transformations with, 129
Enzymatic reactions, mixing and
reaction calorimetry with,
42, 42f, 43f
Enzyme
mode with, 31t
mode with, 28t
of, 19t
Erythromycin, Escherichia coli
treatment with, 15455, 155f
Escherichia coli
377
DSC analysis of, 148, 15558,

157f
15455, 155f
15960, 160f
4445
nonthermal treatment of, 16263,
162t
ESR. See Electron spin resonance
Exothermic decomposition, 13
Fat. See also Lipids
mode with, 31t
mode with, 28t
of, 19t
oxidative stability of, 21112
Fatty acids, 17073, 171t, 172t,
173f
crystallographic/energetic
properties of, 172f
hexagonal, 172, 172f, 173f
melting point of, 171f, 172f
orthorhombic perpendicular, 172,
172f, 173f
triclinic parallel, 172, 172f,
173f
Fermentation, mixing and reaction
Fish, heat capacity for, 36t
Fluid mixing vessel, high sensitivity
heat flux calorimeter with,
27, 28t
Foams, proteins role in, 10
Food dehydration, 289309
calorimetric glass transition
measurement for, 29396,
294f, 296f
378
Index
Food dehydration (continued)

dielectric and mechanical
relaxations with, 296f,
29798
freezing in, 3013, 302f, 303f
glass transition and stability of,
3078, 308f
phase and state transitions of,
290, 29293, 293f
state diagrams with, 3037, 304f
freeze-drying, 290, 3067
spray-drying, 290, 3056, 306f
thermal analysis in, 298301,
299f, 300f
Food flavorings, 89
odorants with, 102
Food-processing design, 20127
food industry interest in
calorimetry for, 226
related techniques for, 225
DMA, 225
DMTA, 225
DSC combined with XRD,
225
safety aspects for, 21718
thermal analysis/calorimetry on,
2036, 204f
methods, 2056
samples, 206
constituents in, 20617,
207f, 208f, 210f, 213f, 215f,
216f
carbohydrates, 2068, 207f,
208f
lipids, 20814, 210f, 213f
proteins, 21416, 215f, 216f
sugars, 2068, 207f, 208f
water, 21617
thermal behavior of raw/
reconstituted food in, 217
thermodynamic parameters for,

21825, 221f223f
heat of combustion, 225
heat of solution, 21824,
221f223f
specific heat, 22425
Food-processing safety
adiabatic measurement methods
with, 36163
accelerating rate calorimeter,
36263
applications for, 36466
formation of hot spots in
dryers, 364
prevention of molasses
incidents, 365
storage and hot discharge,
36465
transport safety, 36566
calorimetry for, 35166, 354f,
355f, 357f359f
concepts with, 35256, 354f,
355f
adiabatic temperature rise, 353
autocatalysis, 356
probability, 35354, 354f
severity, 353
time to maximum rate, 35354,
354f
critical conditions with, 35456,
355f
heat release rate, 35455
temperature, 35556, 355f
differential scanning calorimetry
for, 355f, 35661, 357f359f
estimation of q(T) with,
35760, 359f
high-sensitivity calorimetry
with, 361
isoconversional methods with,
36061
Index
methods with, 357, 357f,
358f
screening with, 35657
reactions with oxygen in, 36364
determination of self-ignition
temperature for, 364
screening test for, 363
Formal autocatalysis, 356
Fourier transform infrared
spectroscopy, 11
Free protein, denaturation
with, 106t
Freeze-drying, 290, 3067
Fruit, heat capacity for, 36t
Galactose, calorimetric curves of,
208f
Gas-flow vessel, mixing and
reaction heat flux
31t
Gelatin, endothermic/exothermic
effects of, 19t
Gelatin gels, frozen water ratio in,
32629, 328f
Gelatinization
mode with, 28t
starch, 12, 274, 27879, 280f
calorimetric analysis by HPP
of, 34149, 343f, 346f
heat in, 342, 343f
high pressure calorimetry on,
33036, 332f334f, 335t
storage of, 34748
thermodynamic data for, 335t
wheat, 34447, 346f
starch-water systems, 207
Gelatin molecules, 122
379
Gelation, heating mode with, 3738

Gibbs function, 267
Glass transition, 29092
behavior of lactose with, 300f
calorimetric measurement for,
29396, 294f, 296f
cooling/heating with, 295
mode with, 28t
DSC with, 294f
Gordon-Taylor equation with,
300
mechanical/dielectric relaxations
in, 296f, 297
stability of dehydrated materials
with, 3078, 308f
studies referring to, 291
sugar/carbohydrates with, 294
two or more components with,
295
Globulin, 7S, different pH values in
denaturation of, 93
Globulin, 11S
alcohols effect on protein
denaturation using, 99100,
101f
broad beans, 99100, 101f, 105
denaturation of, 8992, 91f
different pH values in, 91f, 92
two-state model to analyze, 89
polysaccharides effect on protein
106t, 110
-glucosidase, ITC of binding
inhibitors to, 80f
Gompertz model, oil-in-water
emulsions with, 13940,
139t, 140f
Gordon-Taylor equation, 300, 304
Grapefruit, heat capacity for, 36t
Guar gum, denaturation temperature
of -lactoglobulin with, 110
380
Index
Heat calibration procedure,

HP-DSC, 60f, 6163, 62f
Heat capacity
constant pressure processes with,
30
defined as ratio, 30
determination, 3035, 33f, 34f,
36t
foods in, 35, 36t
liquids in, 3435, 34f
temperature-scanning mode in,
3133, 33f
temperature step mode in,
3334
DSC with, 6
formula for heat flux with, 17
Heat flow
blank test heat flow equation, 32
magnitude of, 8
Heat flux
electrical signals correlation
with, 24
pork muscle crystallization with,
327f
pork muscle thawing with, 326f
power dissipations correlation
with, 24
pressure shift freezing with, 330f
ratio of measured to total, 2021,
21f
Heat flux calorimeters, 2021,
2630, 27f, 28t
high sensitivity, 2627, 27f, 28t
batch mixing vessel for, 27,
28t
fluid mixing vessel for, 27,
28t
mixing vessels for, 27, 27f
temperature control in, 26
thermal conductive block of,
26
mixing and reaction, 2930, 31t
ampoule mixing vessel for, 30,

31t
batch high-pressure vessel for,
29, 31t
batch standard vessel for, 29,
31t
gas-flow vessel for, 29, 31t
membrane mixing vessel for,
2930, 31t
mixing vessel for, 29, 31t
Heat flux calorimetric principle,
1719, 18f, 19t
parts of, 17
temperature equivalent formula
for, 17
thermal contribution due to heat
capacity formula for, 17
Heat flux microcalorimetry, DSC v.,
1925, 20f25f
calibration in, 2325, 24f,
25f
Calvet principle in, 2223, 22f,
23f, 26
Heat-induced transformations
oil-in-water emulsions with,
13241, 135f, 136t, 137f,
139f, 139t, 140f
anhydrous milk fat, 13341,
135f, 136t, 137f, 139f, 139t,
140f, 18487, 186f, 187f
139f, 139t
crystallization in, 13236,
135f, 136t
139t, 140f
ice cream, 133
kinetics of, 13641, 137f, 139f,
139t, 140f
13236, 135f, 136t
Index
whipped cream, 133
peak temperatures and heat of
reaction in, 128f
protein solutions with, 11932,
126f128f, 130f, 131t, 132f,
141
activation enthalpy of, 129
denaturation-aggregation of
globular proteins in, 12429,
126f128f
entropy of, 129
kinetics of, 12932, 130f, 131t,
132f
Lumry-Eyring model for, 129,
130f, 131t
protein structures in, 12123
thermodynamics of, 12324,
12932, 130f, 131t, 132f
whey protein isolate in, 125,
126f, 128f, 132f
Heating mode, 3540, 37f, 39f, 40f
aggregation, 3637, 37f
crystallization, 36
denaturation, 3637, 37f
gelatinization, 38
gelation, 3738
isothermal calorimetry, 38
isothermal crystallization, 39
oxidative stability, 38
retrogradation, 28t, 38
scanning calorimetry, 3536
shelf life, 38, 39f
step heating in calorimetry, 40,
40f
Heat of combustion, parameters for
food-processing design of,
225
Heat of solution, parameters for
food-processing design of,
21824, 221f223f
Heat release rate, critical, 35455
381
Heat shocking, 148, 151

HHP. See High hydrostatic pressure
processing
High hydrostatic pressure
processing (HHP), 9
food-processing treatment DSC
evaluation with, 161
starch gelatinization by, 34149,
343f, 346f
starch gelatinization with, 12
wheat starch suspensions by,
34447, 346f
results with, 34547, 346f
sample preparation for, 345
High pressure calorimetry, 31138,
313f315f
frozen water ratio in gelatin
gels, 32629, 328f
gelatinization of starch,
33036, 332f334f, 335t
phase stability of lipid
containing systems, 33637,
337f
pressure shift freezing, 32930,
329f331f
water in pork muscle, 32426,
326f, 327f
calibration of, 314
calorimetric signal processing in,
313f
calorimetric vessels in, 313f
differential calorimetric detector
in, 313f
experimental setup of, 314f
experimental vessel for, 315f
high-pressure pump in, 313f
hydraulic fluid reservoir in, 313f
pressure detector in, 313f
pressure-transmitting fluid with,
316
schematic diagram of, 313f
382
Index
High pressure differential scanning

calorimetry (HP-DSC),
5164, 54f56f, 60f, 62f
advantages of, 6364
applications of, 63
autoclave of, 54, 54f, 55f, 57
availability of, 63
calibration of, 5763, 60f, 62f
heat calibration procedure for,
60f, 6163, 62f
necessity for, 57
parameters for, 58
reference substances for, 58
table of corrections for, 57
temperature calibration
procedure for, 5861
uncertainty with, 58
ceramic housing of, 54, 55f
construction of, 5357, 54f56f
danger with, 54
differential thermal analysis with,
53
disadvantages of, 64
fluid medium as limit to, 52
furnace of, 54, 56, 56f
operating temperature range of,
56
power compensation principle
with, 53
setup of, 54f
spindle pump of, 54, 54f
High-sensitivity calorimetry,
food-processing safety with,
361
High sensitivity heat flux
calorimeter, 2627, 27f, 28t
batch mixing vessel for, 27, 28t
fluid mixing vessel for, 27, 28t
mixing vessels for, 27, 27f
temperature control in, 26
thermal conductive block of, 26
Hot spots
dryers with, 364

drying and, 13
HP-DSC. See High pressure
differential scanning
calorimetry
Hydrocolloid
mode with, 31t
mode with, 28t
of, 19t
IC. See Isothermal calorimetry
Ice cream
heat capacity for, 36t
with, 133
ICTAC. See International
Confederation for Thermal
Analysis and Calorimetry
Indium, HP-DSC calibration using,
58, 60, 61
Initial calorimetric signal 0,
calculation of, 252
International Confederation for
Thermal Analysis and
Calorimetry (ICTAC), 20
Interpolyelectrolyte complex
formation, 107
Isoconversional methods, foodprocessing safety with,
36061
Isothermal calorimetry (IC), 265
shelf life analysis with, 23761
calculation for rate constant,
25455
calculation for reaction
enthalpy, 255
calculation for reaction halflife, 254
Index
calculation for reaction order,
25253
calculation for total heat
released, 25354
calculation of initial
calorimetric signal 0, 252
calculation of QT, 25660, 260f
determination of K, 25556
empirical model fitting for,
24649, 246f, 249f, 250f
qualitative studies on, 23945,
241f
quantitative studies on, 245
reaction kinetics based model
of, 24951
reactions that proceed to
completion for, 25255
equilibrium for, 25560,
260f
test for complete reaction, 255
Isothermal crystallization, heating
mode with, 39
Isothermal solution calorimetry, 220
Isothermal titration calorimetry
(ITC), 910
dependence of model with, 82
heat of interaction measured with,
84
protein in dilute solution with,
68, 7784, 80f, 83f
binding processes in, 78, 79,
80f
data analysis for, 7982
information content, 7778
instrumentation, 7879, 80f
power compensation design in,
78
range of applicability with,
8283, 83f
shape of titration curve with,
8283, 83f
383
ITC. See Isothermal titration

calorimetry
Joule effect, 2324, 24f, 25f, 32
K, determination of, 25556
KCl, salt-protein interaction with,
98, 99t
KI. See Kunitz inhibitor
Kirchhoff s law, 93, 96, 100
Kosmotropic salts (Salting-out
salts), 95
Kunitz inhibitor (KI), 94
interpolyelectrolyte complex
formations effects on, 107
polysaccharides effect on protein
denaturation with, 10710,
108f
dextran sulfate with, 108f
soybean seeds with, 107
Lactobacillus plantarum, DSC
-lactoglobulin
-carrageenan with, 110
-carrageenan with, 110
denaturation of, 125
milk protein with, 9495
porcine, 95
salts effect on protein
97f, 99t
Lactose
glass transition behavior of, 300f
state diagrams of, 304f
Lard
crystallization in, 190, 191f, 191t
191f, 192f
DSC curves of, 191f
SAXS of, 191t, 192f
WAXS of, 191t, 192f
384
Index
Lauric acid
melting point of, 171f
MICROCALIX calibration with,
177
Lead, HP-DSC calibration using, 58
Linoleic acid, melting point of, 171f
Linolenic acid, melting point of,
171f, 172f
Lipids, 169. See also
Triacylglycerols
antioxidant efficacy of, 212
crystallization kinetics of, 21011
emulsifier-water systems with,
21214
emulsions, 214
melting profile of, 209
polymorphism of, 20910, 210f
quality control for, 211
210f, 213f
Liquids, heat capacity determination
for, 3435, 34f
Listeria monocytogenes
153f
DSC analysis of, 14953, 151f
159
sample preparations for, 14950
Lumry-Eyring model, 104
protein heat-induced
transformations with, 129,
130f, 131t
Lyophilization, DSC technique v.
vessel/heating mode with,
28t
Maltodextrin (MD), moisture
content of, 22224, 223f
MASC. See Modulated adiabatic

scanning calorimetry
MD. See Maltodextrin
MDSC. See Modulated DSC
Meat, heat capacity for, 36t
Membrane mixing vessel, mixing
and reaction heat flux
microcalorimeter with,
2930, 31t
Methyl cellulose, denaturation
with, 106t
MicroCal, 21
MICROCALIX, 170, 17679, 177f,
180f, 186f, 187f, 189f, 191f,
192f
cocoa butter in, 18284, 183f
experimental setup of, 177f
experiments using, 179
laboratory with conventional
x-ray source and, 17778
lauric acid for calibration of,
177
synchrotron radiation XRD bench
with, 17879
temperature-controlled cryostat
for, 177f
temperature controller for, 177f
Microcalorimetry
DSC v., 16, 1925, 20f25f
calibration in, 2325, 24f, 25f
Calvet principle in, 2223, 22f,
23f, 26
heat flux, 16, 1925, 20f25f
methods of, 3045, 33f, 34f, 36t,
37f44f
controlled relative humidity in,
45
heat capacity determination,
3035, 33f, 34f, 36t
heating mode in, 3540, 37f,
39f, 40f
Index
mixing and reaction
calorimetry, 4043, 41f43f
pressure calorimetry, 4345,
44f
Milk
bovine -lactoglobulin of, 9495
mode with, 28t
of, 19t
isothermal traces at temperatures
for, 39f
protein, 9495, 131
skim milk powder, 22224, 223f
Milk fat
anhydrous, 13341, 135f, 136t,
137f, 139f, 139t, 140f,
18487, 186f, 187f
crystallization properties of,
18990
186f, 187f, 189f
globules, 18889, 189f
Mixing and reaction calorimetry,
4043, 41f43f
batch mixing in, 40
binding, 4142
dissolution, 41
enzymatic reactions, 42, 42f, 43f
fermentation, 43
flow mixing in, 41
neutralization, 41, 41f
solubility, 41
Mixing and reaction heat flux
microcalorimeter, 2930, 31t
ampoule mixing vessel for, 30,
31t
batch high-pressure vessel for,
29, 31t
batch standard vessel for, 29, 31t
gas-flow vessel for, 29, 31t
385
membrane mixing vessel for,

2930, 31t
mixing vessel for, 29, 31t
Mixing vessel, mixing and reaction
heat flux microcalorimeter
with, 29, 31t
Modulated adiabatic scanning
calorimetry (MASC), 265
Modulated DSC (MDSC), foodprocessing design with, 204
Moisture content
maltodextrin, 22224, 223f
skim milk powder, 22224, 223f
thermodynamic response with,
21920
Moisture loss, calorimetry with, 8
Molasses incident, 365
Mycoplasma laidlawii, DSC
analysis of, 149
Myristic acid, melting point of, 171f
NaCl
globulin with, 106t
salt-protein interaction with, 98,
99t
Neutralization, mixing and reaction
calorimetry with, 41, 41f
(NH4)2SO4, salt-protein interaction
with, 98, 99t
NMR. See Nuclear magnetic
resonance
Nuclear magnetic resonance
(NMR), 301
Odorants
food flavorings with, 102
protein denaturation effected by,
1024, 103f
BSA in, 104
Lumry-Eyring model in, 104
ovalbumin in, 1024, 103f
386
Index
Oersted law, 25
Oil. See also Lipids
mode with, 31t
mode with, 28t
of, 19t
Oil-in-water emulsions, 13241,
135f, 136t, 137f, 139f, 139t,
140f
anhydrous milk fat, 13341, 135f,
136t, 137f, 139f, 139t, 140f,
18487, 186f, 187f
139f, 139t
crystallization in, 13236, 135f,
136t
139t, 140f
ice cream, 133
kinetics of, 13641, 137f, 139f,
139t, 140f
13236, 135f, 136t
whipped cream, 133
Oleic acid, melting point of, 171f
One-cell calorimetric principle,
18f
Open measurement method, 357,
357f, 358f
Orange juice, heat capacity for,
36t
Ovalbumin, protein denaturation
effected by, 1024, 103f
Overlapping peaks, interpretation
of, 8
Oxidative stability, heating mode
with, 38
Oxygen, reactions with, 36364

determination of self-ignition
temperature for, 364
screening test for, 363
Palmitic acid, melting point of,
171f, 172f
Parameter Be, salt-protein
interaction with, 98, 99t
Pectin, denaturation temperature of
11S globulin with, 106t
pH
globulin with, 106t
7S globulin denaturation with
different values of, 93
11S globulin denaturation with
different values of, 91f, 92
protein denaturation affected by,
8995, 91f
RBPC denaturation with different
values of, 92
Phaseolin, 93
Phase transitions, food dehydration
in, 290, 29293, 293f
Polypeptide chains, 121
Polysaccharides
globulin with, 106t
10410, 106t, 108f
11S globulin in, 1056, 106t,
110
Kunitz inhibitor in, 10710,
108f
protein thermodynamic
incompatibility with, 10910
Pork
muscle
heat flux of crystallization for,
327f
Index
thawing heat flux of, 326f
water in, 32426, 326f, 327f
Postdenaturation aggregation
aggregation rate determined by
denaturation rate with, 111
estimate of apparent denaturation
enthalpy with, 112
irreversible, 110
kinetic parameters of, 111
of protein, 11012
reversible, 110
Potato, heat capacity for, 36t
Power compensation principle, 53
Pressure calorimetry, 4345, 44f
Pressure shift freezing (PSF)
basic procedure of, 329f
heat flux during, 330f
high pressure calorimetry with,
32930, 329f331f
ice crystal/sample mass ratio
formed during, 331f
pressure during, 330f
temperature during, 330f
Propylene glycol, denaturation
temperature of
Protein
20 amino acids constituting, 120
behavior upon heating of, 89
bovine -lactoglobulin of milk,
9495
calorimetry of dilute solution of,
6784, 71f, 80f, 83f
cold denaturation with, 75
DSC data quantifies highaffinity binding with, 7577
DSC for, 6877, 71f
ITC for, 68, 7784, 80f, 83f
cereal with, 12
conformation stability of, 121
mode with, 28t
387
emulsions/foams, role in, 10
free, 106t
heat-induced transformations in
solutions of, 11932,
126f128f, 130f, 131t, 132f,
141
denaturation-aggregation of
globular proteins with,
12429, 126f128f
kinetics of, 12932, 130f, 131t,
132f
protein structures with, 12123
thermodynamics of, 12324,
12932, 130f, 131t, 132f
hydrophilic component of, 291
milk, 9495, 131
polysaccharides thermodynamic
incompatibility with, 10910
postdenaturation aggregation of,
11012
structures, 12123
caseins, 12223
gelatin molecules, 122
polypeptide chains, 121
secondary structures, 121
tertiary structures, 12122
processing with, 27276,
274f, 275f
gluten fix of water molecules
for, 273
soluble in aqueous media for,
273
starch gelatinization with, 274
215f, 216f
thermal denaturation of, 87113,
91f, 97f, 99t, 101f, 103f,
106t, 108f
effects of alcohols on, 99100,
101f
388
Index
Protein (continued)
effects of odorants on, 1024,
103f
effects of pH on, 8995, 91f
effects of polysaccharides on,
10410, 106t, 108f
effects of salts on, 9599, 97f,
99t
reversibility of, 123
two-state model of, 123
thermodynamic compatibility of
denatured/native, 89
ultrasensitive calorimetry to,
8
Protein-protein interactions
(Exothermic reaction),
12728
PSF. See Pressure shift freezing
QT, calculation of, 25660, 260f
Rate constant, calculation for,
25455
RBPC. See Ribulose 1,5
biphosphate carboxylase
Reaction enthalpy, calculation for,
255
Reaction half-life, calculation for,
254
Reaction kinetics, model of based
on, 24951
Reaction order, calculation for,
25253
Retrogradation
mode with, 28t
heating mode with, 28t, 38
starch, 270
Ribulose 1,5 biphosphate
carboxylase (RBPC),
different pH values in
denaturation of, 93
Saccharides, dissolution behavior

of, 219
Safety. See Food-processing safety;
Transport safety
Salmon, heat capacity for, 36t
Salting-in salts. See Chaotropic salts
Salting-out salts. See Kosmotropic
salts
Salts
mode with, 31t
chaotropic, 95
kosmotropic, 95
9599, 97f, 99t
Debye-Hckel approximation
for, 98
-lactoglobulin, 9698, 97f,
99t
Scanning mode, 3536, 44. See also
Temperature-scanning mode
heating curves for different rates
of, 127f
Scanning transitiometry, 31138,
317f319f, 321f, 323f, 324f
frozen water ratio in gelatin
gels, 32629, 328f
gelatinization of starch,
33036, 332f334f, 335t
phase stability of lipid
containing systems, 33637,
337f
pressure shift freezing, 32930,
329f331f
water in pork muscle, 32426,
326f, 327f
benzene as verification test for,
322, 323f
calibration of, 32223
calorimetric vessels in, 31920
piston pump in, 320
Index
precautions for, 32122
pressure detector in, 320
schematic diagram of, 319f
scheme of basic principles of,
317f
temperature and energy scales of,
322
thermodynamic scheme of, 318f
transitiometric vessels for, 321f
Self-ignition temperature, 364
Setschenow equation, 100
SFC. See Solid fat content
Shelf life, 23761
empirical model fitting for
analysis of, 24649, 246f,
249f, 250f
qualitative studies on, 23945,
241f
quantitative studies on, 245
reaction kinetics based model of,
24951
completion for analysis of,
25255
calculation for rate constant,
25455
calculation for reaction
enthalpy, 255
calculation for reaction halflife, 254
calculation for reaction order,
25253
calculation for total heat
released, 25354
calculation of initial
calorimetric signal 0, 252
equilibrium in analysis of,
25560, 260f
calculation of QT, 25660, 260f
determination of K, 25556
389
test for complete reaction, 255

Skim milk powder (SMP), moisture
content of, 22224, 223f
Small-angle X-ray diffraction
(SXRD), 176
lard in, 191t, 192f
SMP. See Skim milk powder
Sodium alginate, denaturation
with, 106t
Solid fat content (SFC), 20910
Solubility, mixing and reaction
Solution calorimetry, 218
Specific heat
moisture contents effect on
thermodynamic response
with, 21920
parameters for food-processing
design of, 22425
pharmaceutical substances, 219
solution calorimetry with, 218
Spindle pump, HP-DSC, 54f
Spray-drying, 290, 3056, 306f
44, 44f
nonthermal treatment of, 16263,
162t
Starch
mode with, 31t
cereal with, 12
mode with, 28t
of, 19t
gelatinization, 12, 274, 27879,
280f
calorimetric analysis by HPP
of, 34149, 343f, 346f
heat in, 342, 343f
390
Index
Starch (continued)
high pressure calorimetry on,
33036, 332f334f, 335t
storage of, 34748
thermodynamic data for, 335t
wheat, 34447, 346f
HHPs effects on, 12
retrogradation, 270
processing with, 26872,
289f272f
aqueous suspension of starch
granules for, 268
DSC for, 26872, 289f272f
State diagrams
food dehydration in, 3037, 304f
freeze-drying, 290, 3067
lactose, 304f
spray-drying, 290, 3056, 306f
State transitions, food dehydration
in, 290, 29293, 293f
Stearic acid, melting point of, 171f,
172f
Step heating, calorimetry with, 40,
40f
Sucrose, calorimetric curves of,
207f, 208f
Sugar
mode with, 31t
calorimetric curves of sucrose,
207f, 208f
mode with, 28t
glass transition with, 294
constituents in, 2068, 207f,
208f
SXRD. See Small-angle X-ray
diffraction
TA. See Thermal analysis
TCC. See Temperature-controlled

cryostat
Temperature, critical, 35556, 355f
Temperature calibration procedure,
HP-DSC, 5861
Temperature-controlled cryostat
(TCC), 177f
Temperature modulated DSC
(TMDSC), 265
Temperature-scanning mode
blank test heat flow equation,
32
heat capacity determination using,
3133, 33f
Temperature step mode, heat
capacity determination
using, 3334
TG. See Triacylglycerols
TGA. See Thermogravimetry
Thermal analysis (TA). See also
Differential thermal analysis;
Dynamical mechanical
thermal analysis
design/monitor of cereal
processing, 26585
nonstarch carbohydrates,
27678, 277f
process applications, 27885,
280f284f
proteins, 27276, 274f, 275f
starch, 26872, 289f272f
food dehydration understood
with, 298301, 299f, 300f
food-processing design with,
2036, 204f
methods, 2056
samples, 206
Thermogravimetry (TGA), 265
Time scale, calorimetry with, 8
Tin, HP-DSC calibration using, 58,
59
Index
TMDSC. See Temperature
modulated DSC
Total heat released, calculation for,
25354
Transitiometry scanning technique,
12
Transition state theory, 111
Transport safety, 36566
Triacylglycerols (TG)
composition of, 169
DSC and XRD in study of,
16976, 171t, 172t, 173f
fatty acids, 17073, 171t, 172t,
173f
crystallographic/energetic
properties of, 172f
hexagonal, 172, 172f, 173f
melting point of, 171f, 172f
orthorhombic perpendicular,
172, 172f, 173f
triclinic parallel, 172, 172f,
173f
with, 133
main types of, 173f
melting profile of, 209
polymorphism of, 17073, 171t,
172t, 173f, 20910, 210f
Trypsin inhibitor, 94
Vanillin, 102
Vant Hoff enthalpy change, DSC
measured, 7274
Vegetable, heat capacity for, 36t
Water. See also Oil-in-water
emulsions
emulsifier-water systems with
lipids, 21214
frozen water ratio in gelatin gels,
32629, 328f
gluten fix with molecules of, 273
391
pork muscle with, 32426, 326f,

327f
starch-water systems, 207
constituents in, 21617
Wheat, starch gelatinization by
HHP for, 34447, 346f
results with, 34547, 346f
sample preparation for, 345
Whey protein isolate, heat-induced
transformations with, 125,
126f, 128f, 132f
Whipped cream, heat-induced
transformations with,
133
Wide-angle X-ray diffraction
(WXRD), 175
lard in, 191t, 192f
WXRD. See Wide-angle X-ray
diffraction
Xanthan, denaturation temperature
of -lactoglobulin with,
110
X-ray diffraction (XRD), 11
applications with DSC and,
17993, 180f, 183f, 186f,
187f, 189f, 191f, 192f
cocoa butter in DSC and, 17984,
180f, 183f
DSC with, 16994, 171t, 172t,
173f, 177f, 180f, 183f,
186f, 187f, 189f, 191f,
192f
food-processing design with DSC
and, 225
lard in DSC and, 19093, 191f,
192f
MICROCALIX using DSC and,
170, 17679, 177f
milk fat in DSC and, 18490,
186f, 187f, 189f
392
Index
X-ray diffraction (XRD) (continued)

results using DSC and, 17993,
180f, 183f, 186f, 187f, 189f,
191f, 192f
triacylglycerols in DSC and,
16976, 171t, 172t, 173f
X-ray diffraction with temperature
function (XRDT), 11, 176
X-ray diffraction with time function
(XRDt), 11, 176
XRD. See X-ray diffraction
XRDT. See X-ray diffraction with
temperature function
XRDt. See X-ray diffraction with
time function
Yeast
mode with, 31t
mode with, 28t
of, 19t
Yogurt processing, DSC technique
v. vessel/heating mode with,
28t
Youngs modulus E, 265
Zinc, HP-DSC calibration using,
58

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Calorimetry in

The IFT Press series reflects the mission

scientists and related agriculture professionals

IFT Book Communications Committee

IFT Press Editorial Advisory Board

A John Wiley & Sons, Inc., Publication

A John Wiley & Sons, Inc., Publication

Edition first published 2009

Titles in the IFT Press series

For my parents, my son, and my husband for their patience and

This book is also dedicated to the memory of the late Professor

Part 1 Analysis of Food and Biological Materials by

Calorimetric Methods as Applied to Food: An

Methods and Applications of Microcalorimetry

High-Pressure Differential Scanning

Calorimetry of Proteins in Dilute Solution

Chapter 5 Thermal Analysis of Denaturation and

Heat-Induced Phase Transformations of Protein

Calorimetry as a Tool for Process Design

Overview of Calorimetry as a Tool for

High-Pressure Calorimetry and Transitiometry

Calorimetric Analysis of Starch Gelatinization by

Use of Calorimetry to Evaluate Safety of

The global food industry is very large, producing sales worldwide on

understanding of the mechanism of processing-induced changes. The

deconvolute the contributions from different components of the system.

Bayles, Darrell O. (Chapter 7)

Hhne, Gnther W.H. (Chapter 3)

Michel Ollivon (Chapter 8, published posthumously)

Calorimetry in Food Processing

understanding of the impact of processing and storage conditions. The

Calorimetric Methods as Applied to Food: An Overview

Calorimetry in Food Processing

An Overview of the Book

Calorimetric Methods as Applied to Food: An Overview

neers with knowledge about the potential uses of calorimetry as a tool

Calorimetry in Food Processing

design and execution of ITC experiments are described with emphasis

Calorimetric Methods as Applied to Food: An Overview

processing variables other than heat on bacteria. Chapter 7 describes

Calorimetry in Food Processing

examples are discussed, from simple ingredients to complex biological

Calorimetric Methods as Applied to Food: An Overview

Calorimetry in Food Processing

crystallization, gelation, gelatinization, denaturation, and oxidation.

Methods and Applications of Microcalorimetry in Food

The Heat Flux Calorimetric Principle

where dh/dt is heat flux produced by the transformation of the sample

Calorimetry in Food Processing

Figure 2.1. One-cell calorimetric principle.

Here, Cr is heat capacity of the reference, including the container, and

By combining Equations 2.4 and 2.5, the characteristic equation for

Methods and Applications of Microcalorimetry in Food

Melting, lipidic transition

If dh/dt corresponds to an endothermic transformation or reaction,

Calorimetry in Food Processing

International Confederation for Thermal Analysis and Calorimetry

Figure 2.2. Schematic of a plate-shaped DSC sensor.

Methods and Applications of Microcalorimetry in Food

Calorimetry in Food Processing

Figure 2.4. Schematic of the Calvet type calorimeter.

Methods and Applications of Microcalorimetry in Food

Ratio of energy (%)