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INGREDIENT
INTERACTIONS
Effects on Food Quality
SECOND EDITION
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FOOD SCIENCE AND TECHNOLOGY
A Series of Monographs, Textbooks, and Reference Books
Editorial Advisory Board
Gustavo V. Barbosa-Cánovas Washington State University–Pullman

P. Michael Davidson University of Tennessee–Knoxville
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Lekh R. Juneja Taiyo Kagaku Company, Japan
Marcus Karel Massachusetts Institute of Technology
Ronald G. Labbe University of Massachusetts–Amherst
Daryl B. Lund University of Wisconsin–Madison
David B. Min The Ohio State University
Leo M. L. Nollet Hogeschool Gent, Belgium
Seppo Salminen University of Turku, Finland
John H. Thorngate III Allied Domecq Technical Services, Napa, CA
Pieter Walstra Wageningen University, The Netherlands
John R. Whitaker University of California–Davis
Rickey Y. Yada University of Guelph, Canada
76. Food Chemistry: Third Edition, edited by Owen R. Fennema

77. Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet
78. Computerized Control Systems in the Food Industry, edited by
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79. Techniques for Analyzing Food Aroma, edited by Ray Marsili
80. Food Proteins and Their Applications, edited by Srinivasan Damodaran
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81. Food Emulsions: Third Edition, Revised and Expanded, edited by
Stig E. Friberg and Kåre Larsson
82. Nonthermal Preservation of Foods, Gustavo V. Barbosa-Cánovas,
Usha R. Pothakamury, Enrique Palou, and Barry G. Swanson
83. Milk and Dairy Product Technology, Edgar Spreer
84. Applied Dairy Microbiology, edited by Elmer H. Marth
and James L. Steele
85. Lactic Acid Bacteria: Microbiology and Functional Aspects,
Second Edition, Revised and Expanded, edited by Seppo Salminen
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86. Handbook of Vegetable Science and Technology: Production,
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87. Polysaccharide Association Structures in Food, edited by
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88. Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by
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89. Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa
90. Dairy Technology: Principles of Milk Properties and Processes, P. Walstra,

T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel
91. Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstätter
92. Listeria, Listeriosis, and Food Safety: Second Edition, Revised
and Expanded, edited by Elliot T. Ryser and Elmer H. Marth
93. Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho,
Leon Prosky, and Mark Dreher
94. Handbook of Food Preservation, edited by M. Shafiur Rahman
95. International Food Safety Handbook: Science, International Regulation,
and Control, edited by Kees van der Heijden, Maged Younes,
Lawrence Fishbein, and Sanford Miller
96. Fatty Acids in Foods and Their Health Implications: Second Edition,
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97. Seafood Enzymes: Utilization and Influence on Postharvest Seafood
Quality, edited by Norman F. Haard and Benjamin K. Simpson
98. Safe Handling of Foods, edited by Jeffrey M. Farber and Ewen C. D. Todd
99. Handbook of Cereal Science and Technology: Second Edition, Revised
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100. Food Analysis by HPLC: Second Edition, Revised and Expanded,
edited by Leo M. L. Nollet
101. Surimi and Surimi Seafood, edited by Jae W. Park
102. Drug Residues in Foods: Pharmacology, Food Safety, and Analysis,
Nickos A. Botsoglou and Dimitrios J. Fletouris
103. Seafood and Freshwater Toxins: Pharmacology, Physiology,
and Detection, edited by Luis M. Botana
104. Handbook of Nutrition and Diet, Babasaheb B. Desai
105. Nondestructive Food Evaluation: Techniques to Analyze Properties
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106. Green Tea: Health Benefits and Applications, Yukihiko Hara
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110. Applied Dairy Microbiology: Second Edition, Revised and Expanded,
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112. Alternative Sweeteners: Third Edition, Revised and Expanded,
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113. Handbook of Dietary Fiber, edited by Susan Sungsoo Cho
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114. Control of Foodborne Microorganisms, edited by Vijay K. Juneja
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118. Food Protein Analysis: Quantitative Effects on Processing,

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119. Handbook of Food Toxicology, S. S. Deshpande
120. Food Plant Sanitation, edited by Y. H. Hui, Bernard L. Bruinsma,
J. Richard Gorham, Wai-Kit Nip, Phillip S. Tong, and Phil Ventresca
121. Physical Chemistry of Foods, Pieter Walstra
122. Handbook of Food Enzymology, edited by John R. Whitaker,
Alphons G. J. Voragen, and Dominic W. S. Wong
123. Postharvest Physiology and Pathology of Vegetables: Second Edition,
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124. Characterization of Cereals and Flours: Properties, Analysis, and
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125. International Handbook of Foodborne Pathogens, edited by
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126. Food Process Design, Zacharias B. Maroulis and George D. Saravacos
127. Handbook of Dough Fermentations, edited by Karel Kulp and Klaus Lorenz
128. Extraction Optimization in Food Engineering, edited by Constantina Tzia
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129. Physical Properties of Food Preservation: Second Edition, Revised
and Expanded, Marcus Karel and Daryl B. Lund
130. Handbook of Vegetable Preservation and Processing, edited by Y. H. Hui,
Sue Ghazala, Dee M. Graham, K. D. Murrell, and Wai-Kit Nip
131. Handbook of Flavor Characterization: Sensory Analysis, Chemistry,
and Physiology, edited by Kathryn Deibler and Jeannine Delwiche
132. Food Emulsions: Fourth Edition, Revised and Expanded, edited by
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133. Handbook of Frozen Foods, edited by Y. H. Hui, Paul Cornillon,
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135. Genetic Variation in Taste Sensitivity, edited by John Prescott
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136. Industrialization of Indigenous Fermented Foods: Second Edition,
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137. Vitamin E: Food Chemistry, Composition, and Analysis, Ronald Eitenmiller
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138. Handbook of Food Analysis: Second Edition, Revised and Expanded,
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139. Lactic Acid Bacteria: Microbiological and Functional Aspects: Third Edition,
Revised and Expanded, edited by Seppo Salminen, Atte von Wright,
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140. Fat Crystal Networks, Alejandro G. Marangoni
141. Novel Food Processing Technologies, edited by
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144. Engineering Properties of Foods: Third Edition, edited by M. A. Rao,
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145. Antimicrobials in Food: Third Edition, edited by P. Michael Davidson,
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146. Encapsulated and Powdered Foods, edited by Charles Onwulata
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148. Food Biotechnology, Second Edition, edited by Kalidas Shetty,
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150. Thermal Food Processing: New Technologies and Quality Issues,
edited by Da-Wen Sun
151. Aflatoxin and Food Safety, edited by Hamed K. Abbas
152. Food Packaging: Principles and Practice, Second Edition,
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Packaging, Cook-Chilling and Other Methods, Vazhiyil Venugopal
154. Ingredient Interactions: Effects on Food Quality, Second Edition,
edited by Anilkumar G. Gaonkar and Andrew McPherson
155. Handbook of Frozen Food Processing and Packaging, edited by
Da-Wen Sun
156. Vitamins In Foods: Analysis, Bioavailability, and Stability,
George F. M. Ball
DK3027_title 11/3/05 8:42 AM Page 1
INGREDIENT
INTERACTIONS
Effects on Food Quality
SECOND EDITION
Edited by
Anilkumar G. Gaonkar
Andrew McPherson
Boca Raton London New York
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Dedication
Dedicated to our families, friends, and colleagues

Preface
Formulated food products are complex colloidal systems in which

molecular interactions among different ingredients dictate the struc-
tural, textural, and organoleptic properties of foods. Our understanding
of, and ability to identify and study, the molecular interactions in food
have been greatly enhanced by recent advances in instrumentation and
machine-based computation. Some ingredient interactions are desirable
and help in improving quality, whereas others are undesirable and can
adversely affect food quality. Understanding the interactions among
food ingredients in food is critical to optimizing ingredient performance
and obtaining quality food products. This knowledge provides new and
unique opportunities for food product developers.
The primary aim of the book is to assemble, for convenient reference,
state-of-the-art technical information in the area of ingredient interac-
tions pertaining to foods. The need for such information has become
increasingly apparent to most food scientists and technologists, espe-
cially those who believe that quality improvement is possible through
improved understanding of food systems.
This book contains chapters by scientists actively involved in research,
most of whom have made notable contributions to the advancement of
knowledge in their field of expertise. Real foods as well as model food
systems are considered. Broadly, the book elucidates the interactions of
major food ingredients such as water, carbohydrates, lipids, proteins,
and flavors, with other constituents of food and shows how the ingre-
dient interactions affect the quality of foods. Also included are chapters
on application of tools such as microscopy and rheology in the study
of ingredient interactions.
This book is intended for scientists, engineers, and technologists from
industry, university, and government laboratories involved in food
research, and for undergraduate, graduate, and postgraduate students
and faculty in food science, food engineering, agriculture, and biochem-
istry. Also, it will serve as a reference for food product developers and
scientists in the agricultural and consumer products industries associ-
ated with food ingredients and will prove valuable for in-house training
programs and professional seminars.
We wish to thank all the contributing authors for their dedication,
hard work, and cooperation, and the reviewers for their valuable sug-
gestions. Last, but not least, we thank the management of Kraft Foods,
Inc. and our colleagues, friends, and families for their encouragement.
Editors
Anilkumar G. Gaonkar, Ph.D., is a research principal at Kraft Foods

Global, Inc. in Glenview, Illinois. He has authored or coauthored more
than 50 journal articles, proceedings papers, and abstracts and holds 10
U.S. patents. His research interests include food colloids, emulsions and
foams, controlled delivery of ingredients, and microencapsulation. Dr.
Gaonkar has organized and cochaired several national and international
symposia as well as lectured at numerous conferences and symposia.
He is a member of the Controlled Release Society.
Dr. Gaonkar received his B.S. in chemistry (1973) and M.S. in physical
chemistry (1975) from Karnatak University, Dharwar, India and his
Ph.D. in chemistry (1979) from Poona University, Pune, India. He is the
editor of three books in the area of food science and technology.
Andrew McPherson, Ph.D., is a senior associate principal scientist at

Kraft Foods Global, Inc. in Glenview, Illinois. He has authored or coau-
thored several peer-reviewed articles and holds six U.S. patents. His
research interests focus on the structure–function relationships of car-
bohydrates and their applications in food systems.
Dr. McPherson is an active lecturer on carbohydrates and food chem-
istry. Dr. McPherson received his B.S. in food technology (1992) and
Ph.D. in food science (1999) from Iowa State University, Ames, and M.S.
in cereal chemistry (1995) from Kansas State University, Manhattan.
Contributors
Curtis M. Amundson Dominique Champion

Kraft Foods Global, Inc. Research Group in Molecular and
Madison, Wisconsin Sensory Engineering of Foods
and Pharmaceuticals
Marc Anton ENSBANA
INRA Université de Bourgogne
Biopolymers Interactions Mazan, France
Assemblies Laboratory
Interfaces and Dispersed Systems Milena Corredig
Team Department of Food Science
Nantes, France University of Guelph
Ontario, Canada
Tarek S. Awad
Centre for Food and Soft Merete Faergemand
Materials Science Danisco
Department of Food Science Brabrand, Denmark
University of Guelph
Ontario, Canada Kathy Groves
Leatherhead Food International
Genevieve Blond Surrey, United Kingdom
Research Group in Molecular and
Sensory Engineering of Foods Hans Peter Heldt-Hansen
and Pharmaceuticals Novozymes
ENSBANA Bagsvaerd, Denmark
Université de Bourgogne
Mazan, France Scott Helstad
Cargill Sweeteners North
Sumana Chakrabarti America
General Mills Technology Minneapolis, Minnesota
Riverside
Minneapolis, Minnesota
Nazlin K. Howell John J. Mitolo
School of Biomedical and National Starch Food Innovation
Molecular Sciences Bridgewater, New Jersey
Division of Nutrition and Food
Safety Martin Preininger
University of Surrey Kraft Foods Global, Inc.
Guildford, United Kingdom Glenview, Illinois
Arun Kilara Gaelle Roudaut

Arun Kilara Worldwide Research Group in Molecular and
Concord, New Hampshire Sensory Engineering of Foods
and Pharmaceuticals
Niels Krog ENSBANA
Danisco Université de Bourgogne
Brabrand, Denmark Mazan, France
Martine Le Meste Denise Simatos

Research Group in Molecular and Research Group in Molecular and
Sensory Engineering of Foods Sensory Engineering of Foods
and Pharmaceuticals and Pharmaceuticals
ENSBANA ENSBANA
Université de Bourgogne Université de Bourgogne
Mazan, France Mazan, France
Alejandro G. Marangoni Rodrigo Tarté

Centre for Food and Soft Kraft Foods Global, Inc.
Materials Science Madison, Wisconsin
Department of Food Science
Ontario, Canada
Yoshinori Mine
Department of Food Science
Ontario, Canada
Contents
1 Interactions of Ingredients in Food Systems: An

Introduction ............................................................................1
Arun Kilara
2 Microscopy: A Tool to Study Ingredient Interactions

in Foods ................................................................................ 21
Kathy Groves
3 Probing Ingredient Functionalities in Food Systems

Using Rheological Methods ............................................... 49
Sumana Chakrabarti
4 Interaction of Water with Food Components ................... 87

Martine Le Meste, Gaelle Roudaut, Dominique Champion,
Genevieve Blond, and Denise Simatos
5 Starch Selection and Interaction in Foods ..................... 139

John J. Mitolo
6 Ingredient Interactions: Sweeteners ................................ 167

Scott Helstad
7 Protein Interactions in Muscle Foods ............................. 195

Rodrigo Tarté and Curtis M. Amundson
8 Protein-Protein Interactions in Food ............................... 283

Milena Corredig
9 Interaction of Proteins with Selected Small

Molecules ............................................................................ 309
Nazlin K. Howell
10 Ingredient Interactions: Phospholipid-Protein
Interactions in Food Systems ........................................... 343
Yoshinori Mine and Marc Anton
11 Macromolecular Interactions in Enzyme Applications

for Food Products .............................................................. 363
Hans Peter Heldt-Hansen
12 Interactions of Emulsifiers with Other Components

in Foods .............................................................................. 389
Merete Faergemand and Niels Krog
13 Ingredient Interactions Affecting Texture and

Microstructure of Confectionery Chocolate ................... 423
Tarek S. Awad and Alejandro G. Marangoni
14 Interactions of Flavor Components in Foods ................. 477

Martin Preininger
Index ............................................................................................. 543

1
Interactions of Ingredients in Food Systems:
An Introduction
Arun Kilara
CONTENTS
Introduction ......................................................................................... 2
Example Interactions............................................................................ 4
Protein-Water Interactions ........................................................ 4
Protein-Protein Interactions ..................................................... 7
Protein-Lipid Interactions ....................................................... 10
Protein-Carbohydrate Interactions ......................................... 12
Protein-Microcomponent Interactions .................................... 14
Summary ............................................................................................ 16
References .......................................................................................... 17
2 Ingredient Interactions: Effects on Food Quality, Second Edition
Introduction
In an agrarian society, the problem of storing seasonal foods for consump-
tion at a later date was recognized. Many methods of preserving perish-
able commodities were developed, including pickling, brining,
fermenting, drying, and smoking. Each of these processes produced a
food that had different attributes from the original raw material. This
eventually led to the processes of canning and later freezing as means of
preserving foods in a condition as close to the fresh material as possible.
The food industry has evolved from preserving foods in a safe manner
to providing safe, nutritious, and convenient foods for all segments of the
population on demand. This implies the shift from mere safety to other
aspects that appropriately address consumer needs. Of course, safety of
any food is an essential prerequisite, and, therefore, its importance should
never be minimized. As a result of globalization of economies, palates of
consumers are becoming internationalized as well. Many restaurants
today do not cook food from scratch but merely assemble and present
precooked ready-to-serve items.
Consumers demand variety in their foods in addition to safety, nutri-
tion, convenience, fair price, uniform quality, and taste. Food processing
today consists of assembly of ingredients, processing, packaging, ship-
ping, storage, and distribution functions. Consumers purchase the food
at the point of sale, spending a minimum amount of time cooking the
purchased food and consuming the prepared food. Due to shopping hab-
its, consumers purchase foods weekly and sometimes less often. Condi-
tions of storage and distribution and in-home storage vary widely and
are often not controlled as well as they can be. The net result is that
carefully processed, safe, nutritious, good-tasting food can lose some of
these desirable characteristics by the time the consumer finally puts this
food in his or her mouth.
These are the changes in consumer demands that are driving the food
industry in the 21st century. As the population becomes older, the drivers
of change include the demands for functional foods. These are foods that
contain biologically active (bioactive) molecules that deliver palliative
properties for various disease states. Maintenance of biological activity
necessitates minimizing interactions of the bioactives with other food
components. Similarly, there will be other new demands arising out of
demographic and lifestyle changes in the coming years.
The word interaction is used in a nonspecific sense, but its use seems
to convey some specific meaning. Interactions can be physical, chemical,
or physicochemical. The physical interactions involving ingredients or the
manufactured food are adsorption, absorption, evaporation, drying, and
Interactions of Ingredients in Food Systems: An Introduction 3
particle size reduction. Chemical interactions can occur among classes of
constituent molecules of the food (e.g., proteins, lipids, carbohydrates,
minerals, and vitamins) or can occur between the food and the packaging
materials in which the food is contained. Given that there are a number
of different food proteins with differing amino acid compositions and
concentrations, and similarly the other constituents are also different
chemical entities, the exact permutations and combinations of reactions
can indeed be mind-boggling. Physicochemical interactions include the
formation of foams, emulsions, gels, etc.
The main components of foods can be classified as macrocomponents
(water, proteins, lipids, carbohydrates) and microcomponents (minerals,
vitamins, enzymes, nutraceuticals). Interactions can take place between
macrocomponents, e.g., protein-protein, protein-lipid, or protein-carbo-
hydrate. Interactions can also take place between macro- and microcom-
ponents, e.g., protein-enzyme, lipid-enzyme, or carbohydrate-enzyme. In
some instances, vitamins can play a role as cofactors for these enzyme
interactions. Similarly, some enzymes require minerals to maintain their
conformational state that leads to catalysis.
Physical conditions of temperature, moisture, pH, and time affect the
intensity of interactions. Secondary interactions can then ensue between
the reaction products from the primary reaction and the food constituents.
The value judgments of whether these reactions are desirable or undesir-
able depend upon the substrates and the intended use of the end product.
For example, the carbonyl-amine reaction commonly referred to as Mail-
lard browning is undesirable in dried egg whites used as food ingredients,
but is preferred in certain baked goods for generating color and aroma.
Therefore, the number of interactions and their desirability is a variable.
Conceptually, the interactions in a processed food can be diagrammed
(Figure 1.1).
A few examples of interactions using proteins as a substrate are dis-
cussed in this introduction. This chapter is by no means a definitive
treatise on the subject. The book, however, is a comprehensive compilation
of the complex subject of ingredient interactions and their effects on food
quality. Chapters 2 and 3, respectively, describe how microscopy and
rheology can be used to study ingredient interactions in food systems.
Chapter 4 describes the interactions involving water. Chapters 5 and 6
deal with the interactions involving carbohydrates, whereas Chapters 7
to 10 deal with interactions pertaining to proteins. Interactions of
enzymes, emulsifiers, lipids, and flavor components are the subject matter
of Chapters 11 to 14, respectively.
Nutrition Type
Type Breed
Cultivars Genetics
Management Sex
Environment Management
Nutrition
Plants Ingredients Animals
Assembly
Foods
Processing
Processed Foods
Packaging, storage, distribution
Consumer
In-home storage, preparation
Consumed Food
FIGURE 1.1
Conceptual diagram of the interaction among food components.
Example Interactions
Proteins are an important component of the diet and supply essential
nutrients (amino acids) to sustain life. Using this class of ingredients as
an example, the interactions of proteins with the other macro- and micro-
components of foods are discussed. This overview may provide insight
into the complexities of interactions and their effects on the quality of
foods.
Protein-Water Interactions
Chapter 4 in this book delves deeply into the interactions of water with
food components. Water has numerous functions in foods. It is abundant
in many foods, especially fruits, vegetables, meat, fish, eggs, and other
muscle foods. Water aids in dispersion and solublization of many constit-
uents in foods. Some constituents, once solubilized, affect the interactions
between other molecules. The case of solubilizing salts and their impact
on proteins has been well characterized. Similarly, the presence of water
affects the confirmation and molecular organization of proteins (Le Meste
and Davidou, 1995).
Functional properties of proteins are those physicochemical attributes
that make these macromolecules useful in foods. A number of the impor-
tant functional properties are a result of the interaction of proteins with
water. Solubility, water absorption, water binding, viscosity, gelation,
cohesion, adhesion, elasticity, emulsification, and foaming are all depen-
dent on the interaction of proteins with water.
Solubility is an important attribute for the use of proteins in beverages.
The mechanism involves hydrophilicity, and solubility is influenced by
the presence of ions. Both the type of ions and their concentrations affect
solubility. This important attribute of proteins also is a prerequisite for
other functionalities such as viscosity, foaming, emulsification, and gela-
tion. Since the mechanism for solubility involves hydrophilicity, the amino
acid composition of a protein plays an integral role in determining the
interaction of this type of macromolecule with water.
Protein solubility is pH dependent. For most proteins the point of min-
imum solubility occurs near its isoelectric point. pH values above and
below the isoelectric point alter the charge on the molecule and, therefore,
its interaction potential with water. Some proteins, such as whey proteins,
retain their solubility at their isoelectric point.
Increasing temperature leads to disorder in the structure of the protein
and also a loss in solubility. The effects of heat on proteins have been
reviewed (Kilara and Sharkasi, 1986). Thermal properties of proteins can
be measured using calorimetry. There is a direct correlation between these
thermal properties measured using a differential scanning calorimeter and
the solubility of the protein. As was reported by Kilara and Mangino
(1991) with whey protein concentrates, as the enthalpy of denaturation of
a protein increased so did solubility.
The desirable sensory properties of such foods as gravies, soups, and
beverages are derived from their viscosity. Viscosity is highly dependent
on the solute type and concentration. Macromolecules increase viscosity
at low concentrations depending on the size, shape, flexibility, and hydra-
tion of the protein in question (Damodaran, 1997). The viscosity of protein
solutions increases exponentially with concentration. Increase in hydro-
dynamic radius caused by heating proteins also tends to increase viscosity.
Environmental factors such as pH, ionic strength, and temperature also
affect viscosity of protein solutions. Viscosity of protein solutions
decreases as the isoelectric point is approached. The effect of heat on the
viscosity of whey protein solution at two concentrations was discussed
by Zhu and Damodaran (1994) (Table 1.1). Similarly, under comparable
protein concentrations, heat denaturation of whey protein concentrate at
an acidic pH caused a tenfold increase in apparent viscosity when com-
pared to unheated whey protein concentrate (Modler and Emmons, 1977).
TABLE 1.1
Effects of Heat on Specific Viscosity of Whey Protein Isolate Solutions at Two
Concentrations at pH 7 and 25°C
Heating Time (min) Specific Viscosity
70°C and 5% 90°C and 9%
0 0.21 0.21
5 0.28 1.87
20 0.43 2.25
40 2.33
60 2.38
Source: Reprinted with permission from Zhu, H. and Damodaran, S. J. Agric. Food
Chem., 42: 846–852, 1994. Copyright 1994 American Chemical Society.
It is also observed that the replacement of sodium ions with calcium ions
increases the viscosity of whey protein concentrate solutions.
Gelation is a phenomenon in which protein solutions transform into a
solid structure that is capable of holding large amounts of water. Proteins
can form either coagula or transparent gels. Amino acid composition and
the environmental conditions dictate the nature of the structure formed
(Damodaran, 1997). Proteins with a high frequency of nonpolar amino
acid residues tend to form coagula (Shimada and Matsushita, 1980). When
the sum of Val, Pro, Leu, Ile, Phe, and Trp residues in the protein is above
31.5 mol%, coagulum-type gels are likely to form. Translucent gels hold
more water than coagulum-type gels.
Gelation of proteins can be induced by heating, altering the pH, adding
salts to protein solutions, and by the action of enzymes. The general
principle is to alter the structure of the protein by the addition of one of
the above-mentioned agents. The interaction of the altered protein with
water and with other protein molecules results in gelation. In all instances
concentration of the reactive species is an important variable. Gelation is
a process that not only involves protein-water interactions, but also pro-
tein-protein interactions. Tofu, cheese, puddings, sausages, and cakes are
also example foods in which gelation plays a key role.
Interfacial properties of proteins are key in foaming and emulsification.
Foams are formed at the air-water interface, whereas emulsions form at
oil-water interfaces. Whipped toppings, ice cream, cakes, and desserts are
all examples of foods in which foams are involved. Foods in which emul-
sification plays an important role are salad dressings, mayonnaise, bolo-
gna, soups, gravies, and cakes. A prerequisite for foaming or
emulsification is for the protein to be soluble in water. The soluble protein
can then rearrange itself at the interface, thus lowering the surface and/
or interfacial tension and facilitating the formation of foams or emulsions.
As far as the molecular nature of the protein-water interactions are
concerned, these involve both polar and nonpolar amino acid residues
on the protein molecules. The interaction itself may be mediated via
dipole-dipole, charge-dipole, and dipole-induced dipole interactions.
Charged amino acids can bind up to 6 mol of water per residue, whereas
uncharged polar residues and nonpolar residues bind as much as 2 and
1 mol of water per residue, respectively. Therefore, the interaction of
water with a protein is governed by the amino acid composition of the
protein. Normally, the majority of nonpolar residues and a significant
number of polar residues are buried in the interior of the protein and
thus are unavailable for interactions until the structure is perturbed. Such
perturbations can occur by changing the environmental conditions (pH,
ionic strength, and temperature) as frequently happens in the normal
course of food processing.
Protein-Protein Interactions
Chapter 8 is an in-depth discussion of protein-protein interactions, and
other examples of interactions of proteins are covered in Chapters 7, 9,
and 10. The reader is referred to these for greater details on this aspect.
Here, a cursory treatment of the effects of these interactions on the quality
of foods is accorded.
The molecular forces involved in protein-protein interactions are similar
to those that are involved in the formation of secondary and tertiary
structure of the individual protein molecules. Quaternary structure of
protein is one example of protein-protein interaction. These forces are van
der Waals interactions, hydrophobic interactions, electrostatic interac-
tions, and hydrogen bonds. Kinetic or other constraints may prevent
interactions from occurring (Dalgleish and Hunt, 1995). Confirmation of
the molecules involved is also important for the interactions to occur.
Nonnative confirmations favor protein-protein interactions and perturba-
tion in confirmation can be accomplished by heat, pH, ionic strength, and
processing conditions. Alterations in confirmations can be subtle, and
denaturation is not a prerequisite for protein-protein interactions.
Blood plasma proteins have potential uses for functional and economic
reasons in many foods. This potential was examined in a model cake
batter system containing 6% protein, and 45% sucrose in distilled water
at pH 8.0. The proteins consisted of egg albumin and blood plasma pro-
teins (Howell and Lawrie, 1987). The solution was heated from 30 to 79°C
for 30 min and then cooled to 20°C, during which time the viscosity was
monitored by a plate and cone viscometer. Mixtures of these proteins had
lower viscosities than the additive value calculated for each component
in the mixture, indicating a negative interaction. However, if the temper-
ature of heating was between 73 and 79°C, synergistic interactions
occurred between these proteins. The forces involved in the observed

synergy were speculated to be similar to those encountered in gelation.
When solutions of globular proteins are heated, conformational changes
occur. These changes can be followed by changes in viscosity, circular
dichroism, surface hydrophobicity, and absorption of ultraviolet radia-
tion. Differential scanning calorimetry is a valuable tool in monitoring
thermally induced changes in protein molecules. Conformational changes
expose reactive groups, and covalent and noncovalent interactions within
the same molecules and between different molecules can occur. If the
protein concentration is appropriate, gelation may even occur.
Whey proteins consist mainly of β-lactoglobulin, α-lactalbumin, bovine
serum albumin, and protease peptones. β-Lactoglobulin has a molecular
weight of 18,000 and exists as a dimer of molecular weight 36,000. There
are two variants of this protein termed variant A and variant B. Variant
A is the more abundant form of β-lactoglobulin. Both variants are stabi-
lized by two disulfide linkages, and these proteins are susceptible to heat
denaturation around 72°C under the environmental conditions encoun-
tered in milk. The dimer can be dissociated by lowering the pH to below
3.5 or by raising the temperature to above 40°C (Dalgleish and Hunt,
1995). In contrast, β-lactoglobulin can form octameric aggregates at pH
values close to its isoelectric point. The state of aggregation affects the
nature and strength of gels formed by β-lactoglobulin.
Morr and Josephson (1968) identified two whey protein-casein aggre-
gates formed by heating skim milk. One species was due to thiol-disulfide
interchange reactions, whereas the second was an agglomeration of aggre-
gates involving calcium bridges. These investigators pointed out that the
large aggregate was probably held in colloidal suspension in skim milk
by nonspecific calcium-mediated linkages with the casein micelle. It can
be inferred from this study that the functional properties of heated milk
can be affected if such interactions occur during the thermal process.
Soy proteins contain 7S, 11S, 15S, and 2S proteins. These proteins possess
quaternary structure. The method of isolation affects the percentage of
proteins in the isolate. The quaternary structure is influenced by environ-
mental conditions used during the isolation steps. Heating 7% solutions
of soy protein isolates increases their viscosity and may result in gelation
in 10 to 20 min at 70 to 100°C (Circle et al., 1964). Disulfide cleaving agents,
such as cysteine and sodium sulfite, inhibit protein gelation. The same
agents promote the solubility of heated and unheated soy proteins with
an accompanying lowering of viscosity. Dilute soy protein solutions can
be heated and concentrated and then reheated to form gels (Aoki and
Sakurai, 1969). In these experiments, soy meal extracts were heated to
90°C with steam prior to precipitation of proteins with hydrochloric acid
or calcium chloride. Gels resulted when these acid-precipitated proteins
were heated at 90 or 95°C after dilution to 20%. Here, too, Aoki and
Sakurai (1969) observed that mercaptoethanol and sodium bisulfite inhibit
gelation, which confirmed earlier observations of Circle et al. (1964).
Therefore, it has been suggested that sulfhydryl-disulfide interchange
during heating results in intermolecular cross-links, which stabilize the
gel network. Alternately, intramolecular disulfide linkages may maintain
protein conformations that favor other interactions necessary for gelation.
Soy protein gels are important for tofu manufacture and in the preparation
of meat analogues.
Catsimpoolas and Meyer (1970) studied protein-protein interactions
involved in gelation of soybean globulins. They pointed out that protein
gels were formed by intermolecular interactions that resulted in a contin-
uous three-dimensional protein gel network exhibiting structural rigidity.
The mechanism of gel formation seemed to differ among various proteins
and included cross-linking involving multiple hydrogen bonds, sulfhy-
dryl-disulfide interchange, and formation of peptide groups. A critical
parameter observed in gelation was protein concentration. Below a min-
imum concentration (<8%), aggregation and increase in viscosity were
observed without any attendant gel formation. It was suggested that an
effective overlapping of the functional groups between adjacent protein
molecules or dissociated subunits was necessary for network formation.
At concentration between 8 and 14%, gelation of soybean globulins was
accomplished by heating and subsequent cooling of the protein disper-
sions. Catsimpoolas and Meyer (1970) suggested the overall schematic for
the gelation of soybean globulins (Figure 1.2).
The nature of the bonding forces involved in the various states shown
in Figure 1.2 was also discussed by Catsimpoolas and Meyer (1970). Since
the Gel-Progel transition was reversible and dependent on protein con-
centration, noncovalent bonds were hypothesized to be predominant.
Both pH and temperature had a pronounced effect on the formation of
the Progel. The gel strength and the maximum temperature of Progel
formation were affected by pH. The gel formed by cooling was thought
to be dependent on hydrogen and electrostatic bond formations, which
are favored by lowering temperatures. The lowest gel strengths were
SOL PROGEL GEL
METASOL
FIGURE 1.2
Scheme for soy protein gelation. (From Catsimpoolas, N. and Meyer, E.W., Cereal Chem., 47:
559–570, 1970. With permission.)
obtained at extreme acidic (pH 1 to 2) and extreme alkaline (pH >10)

ranges. In the very acidic range, carboxylic groups showed minimum
ionization, whereas when pH exceeded 10, the ionization of tyrosine was
increased and that of the lysine decreased. Therefore, Catsimpoolas and
Meyer (1970) suggested that at strongly basic pH values, interactions of
carboxylate-phenolic groups and carboxylate-protonated amino groups
were inhibited. However, at pH 1 to 2 the homologous bond between
protonated carboxyl groups can still form. At pH 10, the bond between a
carboxylate group and arginine is possibly formed. At pH 12 where amino
groups of arginine are easily titrated, there is no gel formation. The lower
gel viscosities obtained in solutions of high ionic strength also suggested
participation of ionic bonds in the gelation phenomenon of soybean glob-
ulins. It was also cautioned that soybean globulins may be more stable to
heat treatment in the presence of high concentrations of NaCl, and this
stability can best be visualized as an inhibition of dissociation of these
proteins into subunits.
Protein-Lipid Interactions
Chapter 10 covers details of this important topic. Protein-lipid interactions
are important in biology, as they are the constituents of the cell mem-
branes. They are also indispensable in foods. One aspect of such interac-
tions mentioned briefly is interfacial behavior that leads to the formation
of emulsions. Cheese, cream, milk, mayonnaise, dough, and meat prod-
ucts (e.g., sausages) all involve lipid-protein interactions.
Colicin A is a protein that kills Escherichia coli by forming channels in
the membrane. This protein binds spontaneously to negatively charged
surfaces of the bacterial membrane and assumes an acidic “molten glob-
ule” state prior to fully inserting itself into the membrane. Electrostatic
interactions play an important role in both processes of attachment and
embedding (Chobert and Haertle, 1997). The mechanism of incorporation
of the ion channel forming C-terminal fragment of colicin A into nega-
tively charged lipid vesicles is an example of the insertion of a soluble
protein into a lipid bilayers. The N-terminal portion of colicin A forms a
well-defined complex with dimyristylphosphatidyl glycerol.
In emulsions, proteins adsorb at the oil-water interface and then
undergo unfolding and rearrangement, allowing the hydrophobic parts
of the protein to interact with the oil phase and the hydrophilic portion
of the same molecule to anchor in the aqueous phase. Typically food
emulsions are produced using combinations of small molecule surfactants
and macromolecular surfactants. Although competitive adsorption may
occur, there also can be interactions between proteins and the low-molec-
ular-weight surfactants. Cooperative interactions lead to emulsion stabil-
ity, and competitive or negative interactions result in macromolecular
desorption. One common method of improving the stability of dairy
emulsions is to add mono- and diglycerides to the system. These com-
pounds adsorb onto the fat droplet surface and impart stability toward
coalescence (Morr, 1981).
β-Lactoglobulin has been observed to bind to dipalmitoylphosphatidyl-
choline through lysyl or asparagyl residues interacting with the negatively
charged lipid (Brown et al., 1988; Cornell and Patterson, 1989). This pro-
tein can also bind fatty acids and small hydrophobic ligands like retinol,
alkanones, Tween®, and protoporphyrin. β-Lactoglobulin may belong to
a class of proteins that transport hydrophobic molecules (lipocalicins).
Food processing facilitates protein-lipid interactions. Processed cheese
can be manufactured using either a batch process or an extrusion cooker.
Blond et al. (1988) studied the effects of recipe and process variations on
the protein-lipid interactions in processed cheese. The addition of emul-
sifying salts or premelted cheese increased protein-lipid interactions, but
the proportion of bound lipid did not increase with increasing fat content.
During the extrusion cooking, cooling with slow mixing increased lipid
binding, but a similar effect was not observed in the batch process. Extru-
sion cooking also resulted in increased proteolysis when compared to the
batch method of processed cheese manufacture. In the extrusion cooking
process, increasing temperature and screw speed (rotation rate) resulted
in decreased bound lipid and increased proteolysis. These researchers
proposed the use of a ratio of lipid fraction bound to nonsedimentable
nitrogen as a tool for assessing protein-lipid interactions.
The lipid-protein interactions in concentrated infant formula were stud-
ied by adding radiolabeled κ-casein or β-lactoglobulin to skim milk used
in the manufacture of the formula (Rowley and Richardson, 1985). When
the formula was subjected to ultracentrifugation three fractions resulted,
namely, a lipid phase with some protein, free casein micelles, and a fluid
phase. The radiolabeled protein was distributed in a diverse manner, and
the protein content of these fractions also varied widely. Addition of KOH
prior to sterilization decreased the amount of protein in the lipid layer.
They concluded that the physical properties and stability of the milk-
based lipid-rich products were related to the protein-lipid interactions
and to the interactions with calcium phosphate.
Extrusion is a processing step that involves high pressure and temper-
ature. These conditions may promote protein-lipid interactions. Corn
snacks made with zein and corn oil by extrusion at temperatures of 120
and 165°C were tested for protein-lipid interactions (Izzo and Ho, 1989).
The variables tested in these experiments were screw speed, temperature
of extrusion, and moisture content. Extractable lipids decreased post
extrusion and were mainly affected by moisture content. Low moisture
and high extrusion temperatures resulted in maximal interactions,
whereas high moisture and high temperature decreased the amount of

interaction.
Baked goods are a category of foods where protein-lipid interactions
have been studied. Two different sources of lipids are involved: first, the
lipids present in wheat flour (endogenous lipids) and second, the lipids
added during the manufacturing process (exogenous lipids). The endog-
enous lipids are associated with the starch granule, and this complexation
with amylose may help prevent retrogradation, thus preventing staling
of starch-based foods. Endogenous lipids not bound to starch can form
organized structures in the presence of water. These structures are called
liposomes, bilayers, and vesicles. Generally, triglycerides do not partici-
pate in these structure formations and also do not have desirable effects
on the properties of the dough. Polar lipids, such as glycolipids and
phospholipids, are effective in improving the quality of bread, and they
also form lamellar liquid crystals in water (Le Meste and Davidou, 1995).
Bound lipids in dough are predominantly polar lipids and form organized
structures in the gluten network of the dough.
Protein-Carbohydrate Interactions
Chapters 5 and 6 in this book will deal more with interaction of starches
and sweeteners, respectively. Once more, an attempt here is to merely cite
some representative examples of these interactions.
The most studied and common family of reactions involve those
between the reducing groups of sugars and the amino groups of proteins
known as the Maillard reaction. More recently, the reactivity between the
amino group of asparagine and the reducing group of sugars has been
implicated in the formation of acrylamide in starch-based foods subjected
to high temperatures (Mottram et al., 2002). Although the presence of
acrylamide in foods has been irrevocably established, its public health
significance remains unclear. In most proteins, the amino groups are
involved in the peptide linkage, and, therefore, the only free amino groups
available for reactions are those from glutamine, asparagine, lysine, and
the N-terminal amino group. There are, however, free amino acids and
peptides also present in foods. The peptide and free amino acid content
of foods increase in the event of proteolysis. Proteolysis can be caused by
endogenous proteases in fresh foods, exogenous proteases added to
achieve a desirable property, or subjection of the food substrate to fer-
mentation. A Maillard reaction occurs during processing and storage of
foods. Its effects can be desirable in producing flavors and imparting color,
or it may be undesirable in certain instances when nutritional properties
are adversely affected or the color and flavor may be undesirable. A
Maillard reaction also leads to flavor generation, cross-linking of protein
chains (in advanced stages), loss of nutritional value due to loss of lysine,
formation of a variety of colors, antioxidative effects, and even mutagenic
and carcinogenic effects. These have been described in detail by Ames
(1992). While the Maillard reaction has been extensively studied and
numerous papers and symposia published on this subject, it is not the
only carbohydrate-protein interaction of interest.
Protein and starch constitute the major components in many food sys-
tems. Cereals, legumes, and tubers, generally consumed after cooking, are
examples of foods composed mainly of starch and protein. In products
such as bakery foods, pasta, snack foods, breakfast cereals, and the like,
starch and protein are components that are shaped and flavored in a
variety of ways. The characteristic flavor and texture of these foods is due
in part to the protein-starch interactions. The breakdown of starch by
enzymes to yield high fructose corn syrups, maltodextrins, corn syrup
solids, etc. are also specific instances of protein-starch interactions because
enzymes are catalytic proteins.
In the absence of heat, the mechanism of the protein-starch interaction
is a charge-charge interaction. Because of this it is highly pH dependent
and on the isoelectric point of the protein (Takeuchi, 1969; Dahle, 1971).
Heating increases the complexity of the reactions between starch and
protein. Thermal changes in protein lead to denaturation (Kilara and
Sharkasi, 1986). This denaturation is accelerated in the presence of mois-
ture. Denaturation of cereal proteins leads to disulfide-sulfhydryl inter-
change reactions resulting in the extensive cross-linking of proteins, i.e.,
protein-protein interactions. Starch undergoes a loss of crystallinity, swell-
ing of granules, and leaching of amylose out of the granules leaving
mostly amylopectin behind. The granules collapse and the matrix of amy-
lose forms a part of the gel network (Remson and Clark, 1978). When
protein and starch are in contact, a stable protein-starch matrix involving
hydrogen bonding, covalent bonding, and ionic linkages may form
(Moore and Carter, 1974).
In certain foods like pasta, snacks, and breakfast cereals, heating takes
place under restricted moisture conditions. In these instances, higher tem-
peratures are required for denaturation of the protein and for the gelati-
nization of the starch. Often extruders are used for the conversion of raw
materials to food products. Extruders involve not only high temperature
and low moisture, but also use pressure and shear for the conversion of
raw materials to finished products. Changes that take place in protein
molecules under these conditions are poorly understood. Changes taking
place in the starch granule have been reviewed (Lai and Kokini, 1991).
Marshall and Chrastil, (1992) also provide a good review of protein-starch
interactions.
Hydrocolloids or large-molecular-weight polysaccharides are used as
stabilizers in many processed foods. Some hydrocolloids interact with
proteins, and these interactions are considered in the next section. Proteins
and hydrocolloids can also interact to form desirable structures in foods.
This is a topic of considerable interest to the food industry. When protein
and polysaccharide solutions are mixed, four different results can occur.
In two cases, stable solution mixtures are formed. In the other two cases,
phase-separated mixtures are formed. The protein-polysaccharide mix-
tures can be soluble or insoluble. Interpolymer complex formation occurs
at pH values below the isoelectric point of the protein and at low ionic
strengths (<0.3; Tolstoguzov, 1997). Proteins are positively charged below
their isoelectric point and negatively charged above their isoelectric point.
Carboxyl-containing polysaccharides at acidic to neutral pH range carry
a net positive charge. Therefore, electrostatic attraction between a protein
and a polysaccharide is possible, depending on the pH and the isoelectric
point of the protein. Such a reaction is reversible by changing either the
ionic strength or pH values of the solutions. Sulfated polysaccharides form
soluble complexes with proteins at pH values above the isoelectric point
of the protein. Irreversible complexation between polysaccharides and
proteins is nonelectrostatic, and the functional properties of the complex
are different from those of either reactants.
Limited compatibility of biopolymers leads to single-phase and biphasic
mixtures and usually takes place at high ionic strength or pH values above
the isoelectric point of the protein. Such conditions are commonly encoun-
tered in food systems. Understanding the phase behavior can lead to new
food processes and new food products (Tolstoguzov, 1997).
Protein-Microcomponent Interactions
Microcomponents are molecules that are added at low levels to achieve
their intended effects. Examples of microcomponents are stabilizers, emul-
sifiers, and flavors. Other microcomponents can be colors, buffering salts,
vitamins, antioxidants, etc. Chapters 12 and 14 deal with the interactions
of some of the microcomponents with other components in food.
The interaction of stabilizers with proteins has been alluded to in the
preceding section. The intent was to discuss the use of stabilizers to create
new structures and new food products. In this section, however, the more
frequently used purposes of the stabilizer are discussed along with the
flavor-protein interactions.
The general nature of the electrostatic interactions between charged
polysaccharides and proteins has been described previously. pH is an
important factor, as is ionic strength in such interactions. In fact, such
interactions have been used to recover proteins from dilute waste streams,
e.g., cheese whey (Hidalgo and Hansen, 1971). The complexes contain up
to 50% polysaccharide on a dry weight basis and require alkali to solubi-
lize them. The alkali-treated complex yields a viscous solution with excel-
lent whipping properties (Hansen and Black, 1972).
Addition of hydrocolloids to milk at pH 6.7 changes the aggregation of
casein micelles. Ionic polysaccharides like carrageenan and carboxyme-
thyl cellulose have the greatest impact (Hansen, 1968).
Experiments in model systems have demonstrated that κ- and ι-carra-
geenan and to a lesser extent λ-carrageenan can stabilize αs1- and β-casein
against precipitation by calcium ions (Hansen, 1968; Lin and Hansen,
1970). Carrageenan mimics the action of κ-casein in behaving as a protec-
tive colloid. Further, the weight ratio of carrageenan required to achieve
this action is similar to weight ratio of κ-casein. In the absence of calcium,
κ-carrageenan does not interact with αs-casein.
Smith et al. (1982) investigated the effects of polysaccharides on the
extrusion of soy grits. The polysaccharides tested were carrageenan, car-
boxymethyl cellulose, sodium pectate, and sodium alginate. When these
hydrocolloids were present at the 1% level, sodium alginate was found
to be the most influential in altering the properties of the extrudate.
Presence of sodium alginate reduced the dough viscosity and temperature
at the die, and the torque and extrudate expansion ratio was also dimin-
ished. For optimum texture and expansion, higher extruder temperatures
and lower feed moisture content were required in the presence of the
alginate.
Milk protein-stabilizer interactions impact the quality of frozen dairy
desserts. Protein instability during processing and storage of the mix is
undesirable. Stabilizers such as carboxymethyl cellulose, guar gum, and
locust bean gum may cause protein destabilization, but carrageenan has
an opposite effect (Phillips and Williams, 1995). Both κ- and λ-carrag-
eenans react with casein, but not with whey proteins, in imparting
stability to these proteins. This reaction is influenced by the presence of
calcium ions.
Hydrocolloids have been used to replace fat in the formulation of fat-
free and low-fat foods. In such applications, the interactions of hydrocol-
loids with proteins have been significant in developing texture attributes.
Trailblazer®, a Kraft Foods’ invention, was one such application in which
xanthan gum-whey protein interactions resulted in a fat mimetic (Chen
and Soucie, 1985). This complex formation results from electrostatic inter-
actions when anionic polysaccharides interact with proteins below their
isoelectric point. Flavor-impact molecules are also bound by proteins and
may lead to off-flavor generation in foods containing soy protein. Similar
binding of flavor molecules may also attenuate the flavor impact in fat-
free food formulations. Both of these interactions are undesirable and ones
that food scientists have to contend with.
Using an equilibrium dialysis method, Damodaran and Kinsella
(1981a,b) studied the interactions of various aliphatic carbonyls and soy
protein. Under aqueous conditions, β-conglycinin or 7S fraction of soy

protein binds carbonyls that may cause the off-flavor in soy protein prod-
ucts. It was further reported that on an equivalent weight basis, soy
protein, β-conglycinin, and gylcinin (11S) had 5, 2, and 3 binding sites per
100,000 molecular weight, respectively (O’Neill and Kinsella, 1987). The
affinity of β-conglycinin for 2-nonanone was fivefold higher than those
of gylcinin or the whole soy protein.
The lipid-binding properties of β-lactoglobulin have been briefly men-
tioned elsewhere. It has been suggested that β-lactoglobulin belongs to a
family of proteins that have strong interactions with small hydrophobic
molecules such as retinol, pyrazines, biliverdines, etc. (Papiz et al., 1986;
Godovac-Zimmerman, 1988; Pervaiz and Brew, 1985). Whey protein con-
centrate binds model flavor compounds like 2-nonanone and nonanal,
and this protein can be used as a flavor carrier in food systems (Jasinski
and Kilara, 1985). At concentrations as low as 0.5%, bovine serum albumin
binds diacetyl.
Phenols present in a wide variety of foods, such as coffee, beer, and
fruits, bind with protein, and consequently, the astringency of these foods
is minimized (Clifford, 1968). When tea-containing milk is cooled, a white
insoluble precipitate forms that is called tea cream. Tea cream consists of
protein, phenolics, theaflavine, and caffeine. Tea cream formation is
thought to reduce the flavor of teas (Sheshadri and Dhanraj, 1988).
The binding of 2-nonanone to partially denatured whey is reversible,
whereas heptenal binding was partially irreversible and binding was
dependent on temperature and pH value (Bakker, 1995). Irreversible bind-
ing was increased as either the temperature increased or the pH increased.
It is suggested that heptenal reacts with the ε-amino group of lysine in
proteins, whereas ketone binding may be facilitated by hydrophobic inter-
actions.
Summary
The interactions of ingredients are a complex and evolving area of food
chemistry. The examples provided above are illustrative of some of the
known effects of such interactions. The consequences of such interactions
may be desirable in some instances and undesirable in others, but their
impact on food quality is pronounced. Desirable interactions increase the
acceptability of foods, and the undesirable interactions decrease the
acceptability of the foods by consumers. A thorough understanding of
this area is important to maximize the desirable interactions while pre-
venting the undesirable ones.
Processing of foods changes the nature of the proteins and, hence, their
reactivity. Additionally, increases in temperatures increase the rates of
reactions. Changes in pH affect the charge status of molecules and, hence,
their ability to interact through electrostatic interactions.
Recent trends point to enhancing nutraceutical qualities of foods
through the addition of bioactive molecules. Such molecules may be inher-
ently present in foods or may be added to enhance a perceived physiologic
attribute. The interaction of these molecules with other molecules present
in foods and their bioavailabilty as a consequence of such interactions is
not understood.
Packaged foods, when exposed to light, may undergo interactions that
reduce the nutrient content of the food and also generate undesirable off-
flavors. In this example, the food is properly prepared and packaged with
minimal effects on its quality. However, the defects and interactions may
occur when the food is exposed to light during storage and handling.
Milk is a good example of such a process. Potentially, all organic molecules
in milk are subject to reaction with visible and ultraviolet light as an
energy source. There may be an interaction between proteins, protein and
riboflavin, and between riboflavin and free amino acids. These reactions
result in hydrolysis and aggregation of milk proteins, and the photo-
induced reactions result in a significant loss of riboflavin (Dimick and
Kilara, 1983). No evidence is available to indicate that there is a significant
loss in essential amino acid content of the proteins. Dimick and Kilara
(1983) observed that subsequent reactions following photooxidation of
proteins might result from the generation of free radicals that are impor-
tant in lipid oxidation reactions. Photooxidative reactions promote pro-
tein-protein interactions and protein-lipid interactions that may
ultimately influence the functionality of the components. Undoubtedly,
one of the most significant factors relative to nutrient intake is the flavor
acceptability by the consumer; and, thus, in fluid milk the light-induced
protein degradation leading to off-flavors is an important and relevant
problem.
It is hoped that this book with its authoritative chapters on interactions
between ingredients and their effects on food quality will encourage fur-
ther research in this important area of food chemistry.
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2
Microscopy: A Tool to Study Ingredient
Interactions in Foods
Kathy Groves
CONTENTS
Introduction ....................................................................................... 21
Light Microscopy .............................................................................. 24
Electron Microscopy ......................................................................... 33
Concluding Remarks .......................................................................... 42
Acknowledgments ............................................................................. 45
References .......................................................................................... 45
Introduction
The use of microscopy to study foods has gained increasing popularity
over recent years, as it has become recognized as an essential technique
that can provide a link in understanding why food products have the
properties they do. This chapter aims to provide an overview of the main
microscopy techniques with an emphasis on how they can be used to look

at ingredient interactions in food products. A brief consideration of more
recent techniques is also included. The content is aimed at those in food
product development who have little experience with microscopy, and
attempts to put forward the considerations and concepts involved, with
references to more detailed reviews or research where appropriate.
Generally the use of microscopy in food applications can be divided
into three main areas:
1. To identify foreign bodies. This deals with looking at the foreign

material found by customers in food products or in the ingredi-
ents used to make the products. By identifying these, their origin
can be deduced, and possible problems in the processing line or
at the retail stage remedied. These contaminants are varied and
include intrinsic material that might naturally be found in the
food ingredients, such as strawberry hulls or stalks in jam or fruit-
containing products. They also include extrinsic material such as
glass, plastic, and metals, and it is mainly these that need to be
identified so that the company can trace the source wherever
possible.
2. To control and understand existing products or processes. Micros-
copy can play an important part in assessing ingredient quality
or batch variability. Perhaps, more importantly, by seeing how
the different ingredients interact to form the structure of a good
product, it then becomes easier to understand why things go
wrong. In this way by looking at the relationships between struc-
ture, processing, ingredient interactions, and texture, the sensory
properties and stability of a product can be more closely con-
trolled. This knowledge will lead to improved product quality,
shelf life, or process control.
3. To develop new products or new processing technology. By
understanding how food structure relates to functional properties
and the involvement of different ingredients, or how the manu-
facturing process puts the ingredients together, novel products or
improvements in processes can be developed.
In using microscopy as a tool to study ingredient interactions in foods,

the main application has been in the last area, namely that of understand-
ing the relationship between structure and functionality.
There is a pattern to the way that research has progressed using micros-
copy that can be applied to other techniques as well, because it appears
to be a natural progression. Initial research in food science tends to take
methods or techniques that have been developed for other disciplines
Microscopy: A Tool to Study Ingredient Interactions in Foods 23
(mostly medical or biological research) and use them in the study of foods.
As the methods progress, they often have to be adapted to cope with the
differences in the nature of foods, particularly between manufactured
products and "natural" foods. This can involve the modification of, or
invention of, new preparation methods and the use of stains or probes
not normally associated with food systems.
Given a new instrument or technique, the starting point often is to look
at the "natural" foods first and establish their basic structure. Hence, early
food microscopy studies looked at the structures of fruits, vegetables,
grains, herbs, and meat products. Following this, researchers have looked
at the effects of processing or storage on these structures and at how these
effects relate to changes in the texture or other properties of the foods.
The next step in the progression of microscopy has been to look at the
structures of manufactured or processed food products. This is often more
difficult than the "natural" foods since they are frequently high in sugar
or fat, both of which cause problems for the microscopist in preparation.
In addition, the removal of sugar and fat in preparation for some micros-
copy techniques can produce a collapse of the manufactured structure.
This collapse is not all bad news as it can be used to provide information
on the original structure of the product and so is often deliberately used
to determine how the ingredients contribute to the structure. Frequently,
the removal of one or more ingredients in the product is necessary to see
more clearly the structure of the others. This approach is not one used
often by microscopists in the fields of biological or medical sciences, but
is an extremely useful one for food studies.
Following this in the study of the manufactured food product, the effects
of changing the ingredients were then examined. By looking at how the
food product properties alter when ingredient amounts or type are
changed, some information on ingredient interactions can be obtained. In
doing this there is a need to know the structure, appearance, and prop-
erties of the ingredients. Hence, there are studies on the individual ingre-
dients commonly used in foods, such as starches, proteins, and
polysaccharides. These include ways to stain or identify them as well as
following changes in their structure and properties as they are cooked.
In the study of ingredient interactions, the majority of the research has
been to look at the effects of removing ingredients or adding different
ones on the properties of the model system or real food product. Much
has been reported on the use of microscopy to look at the functional
properties of ingredients, such as gelling, whipping, and emulsifying, and
on how these change with altered environment. To look at the interactions
between ingredients is not so easy and usually must be achieved in an
indirect way.
Light microscopy does not have the ability to resolve molecular struc-
ture; it is more macrotechnique than molecular. It has a theoretical reso-
lution of about 200 nm, but in practice it is unlikely to show much below
about 1 μ. However, the light microscope can be used to look at the
location of ingredients in the food and also changes that take place when
ingredients interact. These changes might be in visual appearance (a factor
important to the consumer) or might be in differences in distribution of
ingredients or overall structure of the product.
A simple example would be to consider the effects of addition of an
emulsifier to an oil-in-water system. The addition of a good emulsifier
will produce small stable oil droplets, but a poor emulsifier will give large
droplets that are likely to coalesce. The molecular structure of the interface
of the oil droplet is different, but this is not seen directly in the light
microscope; only the indirect effects on the droplet size are visible. These
indirect effects are of course vitally important for the success of the food
product as they affect the quality and sensory aspects of the emulsion.
The development of the confocal laser scanning microscope for food
research has allowed the ability of the light microscope to be enhanced.
The resolution is slightly better than that of a conventional light micro-
scope, but the optical sectioning ability gives a much-improved practical
resolution, allowing changes in molecular aggregation to be seen more
clearly.
To see the molecular interactions between ingredients directly, the res-
olution of the transmission electron microscope is necessary. This can be
used to image strands of gel network, the interfacial structure of oil or
water emulsions, and crystal formation. Further resolution can be
obtained by scanning probe techniques such as atomic force microscopy.
The next section describes typical approaches and preparation methods
for light, confocal, and electron microscopy, together with a brief review
of the literature and some examples of their use.
Light Microscopy
A good starting point in the microscopy of foods and ingredient interac-
tions is to use a light microscope. There are different types of light micro-
scopes, and each has its own advantages.
A stereolight microscope is a simple relatively cheap instrument that
magnifies the food product or ingredient. An advantage for the non-
microscopist is that the image is clearly identifiable and gives very good
depth of field. Although the magnification is not large, it is surprising
how much information can be obtained. Little preparation, if any, is
needed, and therefore the problem with artifacts is avoided. A cold light
source (fiber optic) is recommended as it avoids the heat damage encoun-
tered with conventional lights. Examples of uses include looking at pow-
dered foods to see if they are a mixture of ingredients, or have been
codried. Some powdered samples are hollow and some solid. This will
affect their dispersibility and can be seen by simply fracturing or grinding
the powder and looking at it under the stereolight microscope. In studies
on the effect of antibloom agents on the development of chocolate bloom,
Subramaniam et al. (1999) and Subramaniam and Groves (2003) used the
stereomicroscope to determine when the bloom had formed, and a mac-
rolens to photograph the appearance of the chocolate. This microscope is
more usually associated with the identification of contaminants rather
than ingredient interactions, but is still worth including in any study.
The more common light microscope in laboratories is the compound
transmitted microscope. This relies on light passing through the sample.
Liquids or soft foods such as sauces and emulsions can be placed directly
on the microscope slide, and powders can be dispersed in a suitable
mountant, so very little preparation is needed. However, for most foods
it is necessary to get the sample thinner and therefore transparent. The
easiest is to gently squash the sample; the next is to cut a fairly thin section
by hand using a razor blade or similar tool. Squashing the sample can
produce shear effects and distort the structure, but should always be
considered as it can give information on the behavior of the sample to
that shear, and how the sample breaks up reveals the nature of the under-
lying structure. Recently the use of specially designed shear stages on the
microscope allowed the observation of controlled shear effects on the
behavior of the food (Mackley et al., 1999).
Cutting a section by hand is often successful enough to reveal much of
the structure, especially if the sample is cooled or frozen. Specialized
instruments (microtomes) are available for cutting thinner sections. These
generally involve holding the sample rigidly while the blade cuts to a
predefined thickness. Many foods are compressible or soft, and so a micro-
tome that is held in a freezing chamber (cryostat) is useful. With this, the
sample is rapidly frozen, usually in liquid nitrogen, then held in the
chamber frozen, generally in the range –20 to –40°C. The sections are cut
frozen and then warmed to room temperature. If the sample needs to be
kept frozen, such as ice cream, the frozen sections can be placed in a cooled
stage and examined frozen. If a cryostat is not available the sample can
be embedded in wax or resin, but this usually involves fixation and
dehydration (described below).
All samples or sections usually need to be mounted in a liquid to get
good images without distortion. If this liquid mountant is not a stain
(dye), then it needs to be suitable for the individual food. For example, if
the product is essentially fat or oil based, water would be a good mountant
as it would not disperse or dissolve the main ingredients. If the sample
is water-soluble, liquid paraffin would be a good choice. However, most
products contain both fat and water-soluble ingredients, and some

dissolution of ingredients will probably take place. This can be used to
advantage as it can give useful information on the location of the ingre-
dients and how they are incorporated into the product. For example, a
fat-continuous matrix will not dissolve in water but will in liquid paraffin,
and so the continuous nature can be identified by how the sample interacts
with the mountant. In addition, how the sample breaks up in the moun-
tant can give information on the makeup of the product.
Once the sample is on the microscope, optical contrast techniques can
be used to get useful information on the structure. These operate on the
principle of using the optical properties of the sample to give detailed
images. Phase contrast is often available on microscopes in routine labo-
ratories as it is a technique frequently used by microbiologists. The images
of inclusions in foods are often improved by phase contrast, but the most
useful simple optical additions to a light microscope for the study of foods
or ingredients are the polarizing filters. With two filters, a polarizer below
the sample and an analyzer above, information on the birefringence or
ordering of molecules within a sample can be obtained. Crystalline sugar
or fat appears white (birefringent) under crossed polarizing filters (the
polarizer is rotated so that it is at 90° to the analyzer). Ungelatinized starch
grains have a characteristic "Maltese cross" appearance that is lost when
they gelatinize, so information on the extent that they have swollen and
dispersed can be seen with polarized light. The size and shape of the
starch grain can be used to identify the type of starch when considering
the ingredients. Plant cell walls and muscle fibers also have some bire-
fringency, especially when in the native state. Typical food processes such
as heating affect the ordering of molecules, and this is reflected in a
reduced birefringency or brightness under crossed polarizing filters.
An example of the use of polarized light and also deliberate dispersion
of one ingredient to examine another in a product is shown in Figures 2.1
and 2.2. These show the distribution of fat and sugar in a sugar-based
chewy confectionery product.
In Figure 2.1 a thick section of the product is mounted dry so that no
ingredient has dissolved. Any crystalline sugar and the crystalline fat both
appear white. It is not possible from this image to see how the fat is held
in the sample, that is, whether it is in droplet form or in large areas.
In Figure 2.2 the sample is mounted in water so the sugar has dissolved,
leaving the fat component visible as white. The way the fat is formed in
the product is only visible in the light microscope by dissolving the sugar.
This technique is useful for comparison of different fats or processes.
The state of the fat could be seen by using a fluorescent fat stain and
imaging a thick sample by confocal laser scanning microscopy. However,
as this instrument is not always available, this example shows a way to
achieve the information using conventional light microscopy.
FIGURE 2.1
Polarized light image of a confectionery product. Fat and sugar crystals appear white.
(Courtesy of Leatherhead Food International, U.K.)
FIGURE 2.2
Polarized light image of a confectionery product mounted in water. The sugar has dissolved,
leaving the crystalline fat visible as white. (Courtesy of Leatherhead Food International, U.K.)
Generally, polarized light microscopy is useful in looking at crystalli-

zation of fat or sugar as well as following changes in starch gelatinization.
Examples of published research using polarized light to consider ingre-
dient interactions include: following the effects on fat graininess in
margarine with different triacylglycerols (Heertje, 1993); imaging the crys-

tallization process in cocoa butter and blends of cocoa butter, milk fat,
and canola oil (Singh et al., 2004); and the study of chocolate bloom
(Hartel, 1999). The last two studies considered the interaction between
milk fat and cocoa butter and the effects on the polymorphism of the
cocoa butter.
Polarized light has also been used extensively to follow starch gelatini-
zation in many studies. Seetharaman et al. (2004) found that the state of
dough development and starch gelatinization was linked to the final
product quality. In addition, the interaction between starch and other
ingredients is now very topical as the phenomenon of phase separation
caused by interaction between hydrocolloids is a means of producing
encapsulated structures or products. Light microscopy is used to follow
this phase separation. Abeysekera and Robards (1995) describe the inter-
action between gelatin and starch as seen by light and electron microscopy.
There are other techniques such as differential interference contrast
(DIC) or the use of fluorescence. As this latter technique is normally used
with stains, it is discussed below. DIC is a similar technique to polarized
light microscopy in principle but gives a more three-dimensional view of
the sample.
These contrast techniques are useful in highlighting the structural fea-
tures of ingredients without any further processing or manipulation of
the sample and are generally used as a first step in understanding the
basic nature of the sample.
Further information or contrast to distinguish components and their
part in the nature of the food requires the use of stains or dyes to highlight
particular ingredients. Most stains are general rather than specific but are
very useful in identifying ingredients such as protein, starch, polysaccha-
ride, and fat. Commonly used stains include iodine in potassium iodide
for starch. This shows amylose as blue-black and certain modified starches
or amylopectin as red-brown. It is worth using the stain at both the usual
concentration (2%) and also diluted about 1 in 10, as the diluted version
can distinguish the amylopectin component more easily.
Light green, eosin, or toluidine blue are commonly used to stain pro-
teins, although iodine also stains proteins yellow. The stains are usually
prepared in water, although eosin can be used in alcohol. Toluidine blue
is a metachromatic stain, which means it will stain different colors
depending on the ionic environment and nature of the material. It can
distinguish between polysaccharides (pink) and proteins (blue-green) if
used carefully.
Fats can be stained, often slowly, using stains such as one of the Sudan
series or Nile blue. The solvent for the stain needs to be considered
carefully as, if correctly chosen, it will allow the stain to pass through
water regions into fat or oil. It should be noted that sometimes stains soak
into regions in the sample rather than binding specifically; for example,
iodine will go into oil, coloring it yellow, so care needs to be taken in the
interpretation of results. Even so, stains are extremely useful. A number
of detailed stains for food ingredients, including different gums and
starches, are discussed by Lewis (1978), Flint (1994), and Kalab et al. (1995).
The use of several stains in sequence is useful to show the distribution
of more than one ingredient in a product. For example, in a sausage the
meat can be stained with light green, the fat with osmium or Sudan IV,
and the rusk with periodic acid-Schiff’s.
The advantage in using different stains for each ingredient is that
changes to the distribution and interaction between ingredients can be
seen readily. A good example of a sequential staining procedure for con-
nective tissue and other ingredients in meat products is described by Flint
and Pickering (1984). In this procedure, the combination of specific stain-
ing and polarized light microscopy is used to demonstrate the identifica-
tion of ingredients in meat products and to assess their heat treatment.
Light microscopy and staining was used to follow interactions between
starch and emulsifiers in pastilles and yogurts (Titoria et al., 2004a). The
addition of certain emulsifiers in yogurt reduced the swelling of the starch
granules.
The effects of addition of calcium and pretreatment on the texture and
properties of selected fruits and vegetables were examined and light
microscopy used to follow structural changes in the cell walls (Burke et
al., 1993; and Clegg and Groves, 2002). In these studies, the changes in
the cell wall structure during processing as depicted by light microscopy
could be reduced by pretreatment with calcium.
Fluorescence microscopy is a subject for a book chapter in itself and
will only be referred to briefly here. It is commonly used with incident
light (epiillumination). This means that the light reflects back from the
sample into the eyepiece of the microscope, or camera, rather than passing
through the sample.
Incident light microscopy without fluorescence can be used to look at
the surface of products or packaging but has not been used widely in
ingredient or food product evaluation, except when combined with fluo-
rescence or confocal microscopy. Fluorescence microscopy requires the
sample to be autofluorescent, or if it is not, requires the use of specific
stains that bind to ingredients.
Plant cells have been extensively studied using fluorescence. A useful
review of the technique for identification of components in cereals is given
by Fulcher (1982).
Confocal laser scanning microscopy (CLSM) also uses fluorescence but
has an added advantage that the design of the optics allows some optical
sectioning of the sample. In practice this means that a thick or unprepared
sample can be examined directly, and a good, clear image of a thin optical
plane can be seen (in conventional light microscopy, thick samples pro-
duce blurred images as several planes of focus are visible). The image
from the confocal microscope is stored directly on computer, and other
images above and below that plane can be produced simply by moving
the stage up or down. This allows the microscopist to see the internal
three-dimensional structure of the sample while acquiring the images.
Using special software these vertical images can be added together and
viewed as a sum of the optical sections (projection) or as red-green ste-
reopairs. Additionally, the sections can be added together at different
angles so that a rotating three-dimensional view of the sample is obtained.
This means that the information seen in three dimensions by the micros-
copist, as the sample is moved up and down on the microscope, can be
communicated to the nonmicroscopist more easily.
This technique is limited by the depth that the laser can penetrate, but
depending on the sample, a depth of about 100 μ into the product can
reveal very useful information. The technique is also limited by the laser
wavelengths available and by the stains and penetration of these into the
product. As a result of the increasing popularity of this technique for food
research, developments in lasers and applications of stains are removing
these limitations.
The main initial application of the confocal techniques to foods was in
the study of fats and fat products. Reviews of the technique with some
examples are given by Brooker (1995) and by Heertje et al (1987).
The change in low-fat spread properties and structure was followed by
confocal microscopy (see also Bavington et al., 1992.) Changes in the
microstructure were related to differences in aqueous phase stabilizers,
and these affected the sensory characteristics. A typical image of a spread
using Nile red to stain the fat is shown in Figure 2.3.
This shows a water-in-oil (or -fat) emulsion. A change in the level of fat
or in the manufacturing process can produce an oil-in-water emulsion, or
a complex, biphasic structure, where the water forms large continuous
regions and contains dispersed fat, and the fat forms a continuous matrix
with dispersed water. The structure of these products is difficult to image
clearly, particularly as they are very shear-sensitive. CLSM is ideally
suited to studies on these products as very little shear is needed to produce
good images of the biphasic nature. Figure 2.4 shows a projection of
several optical sections of such a spread. The change in structure is
reflected in very different sensory properties.
An interaction between protein and sugar in chocolate has been shown
to be present by CLSM (Subramaniam et al., 1994). In this study strong
heat resistance in one commercially available chocolate was due to an
interaction between the sugar and the protein to form a network in the
chocolate, restricting the fat and helping to prevent melting. However,
this network contributed also to grittiness in the chocolate.
FIGURE 2.3
Confocal image of a low-fat spread showing a water-in-oil structure. Stained with Nile red
to show fat as white. (Courtesy of Leatherhead Food International, U.K.)
FIGURE 2.4
Confocal image of a low-fat spread showing a biphasic structure. Stained with Nile red to
show fat as white. (Courtesy of Leatherhead Food International, U.K.)
In terms of ingredient interactions, confocal microscopy allows multiple

labeling or staining as in light microscopy. The different signals produced
by two or more stains are obtained from different channels set to the
correct wavelength for each stain. Usually the signal from confocal micros-
copy is white; however the images from each channel can be combined
and each signal given a different color. For example protein stained by
Texas red, Fast Green FCF, or Rhodamine B can be assigned one color,
and fat stained in Nile red or Nile blue can be assigned another, and the
distribution of fat and protein within the product can be seen in three
dimensions. Auty et al. (2001) show differences in the structure develop-
ment of mozzarella cheese using this technique. In addition, differences
in viscosities of chocolate made with different milk powders were shown
to be due to a change in the distribution of fat in the product when it was
double-labeled for fat and protein.
Loren et al. (1999) combined CSLM with image analysis to determine
the microstructure of gelatin-maltodextrin systems. Image analysis is a
technique used to quantify aspects of microscopy images. It can be used
for relatively simple measurements, such as area, volume, or size of com-
ponents. It can also be used more sophisticatedly to assess boundary
interactions or interfacial areas.
Loren et al. (1999) used image analysis in phase-separated gelatin-mal-
todextrin systems to assess the area fraction or space each phase took in
the microstructure. They found that cooling time and holding temperature
affected the area fraction of the maltodextrin. By understanding precisely
how processing parameters affect the interaction between the two hydro-
colloids in terms of phase volume, it should be possible to control the
formation of specific microstructures.
In another aspect of the use of CSLM, Hermansson et al. (2000) used
the technique to examine the interaction between an emulsifier and β-
lactoglobulin (β-lg) during gelation. They presented dynamic studies of
the formation of gels with and without emulsifier.
The effects of emulsifiers on the gelation and microstructure of different
proteins were examined by Titoria et al. (2004b). These effects depended
on the type of emulsifier. The images in Figures 2.5 and 2.6 demonstrate
20 μ
FIGURE 2.5
Confocal image of whey protein isolate gel alone. Stained with Rhodamine B. Protein appears
white. (Courtesy of Leatherhead Food International, U.K.)
20 μ
FIGURE 2.6
Confocal image of whey protein isolate gel containing stearoyl lactylate. Stained with
Rhodamine B. Protein appears white. (Courtesy of Leatherhead Food International, U.K.)
the clear changes in aggregation and association of whey protein isolate

molecules when the emulsifier stearoyl lactylate is present. These changes
are represented on a macroscale by changes in gel strength and appear-
ance, as the gel made with emulsifier was softer and more translucent.
Phase separation can also occur in commercial products. In an exami-
nation of the microstructure of commercial confectionery gels, Groves
(2003) used conventional and stereolight light microscopy to show differ-
ences in ingredient interaction between the gelatin and modified starch
in products. In some products gelatin was present both as a continuous
gel and as phase-separated spheres. These spheres could be seen dispers-
ing into the surrounding water-based stain by confocal microscopy (Fig-
ure 2.7).
Electron Microscopy
Electron microscopy has been used many times in food research and to
investigate ingredient interactions. These studies include Heertje (1993)
for looking at fats, Angold (1979) for cereals, Jewell (1979) for fruits and
vegetables, Lewis (1979) for meat products, and Kalab (1981) for dairy
products. A good review of the techniques in electron microscopy is given
by Heertje and Paques (1995); this details the preparation methods and
advantages of the different techniques and gives examples.
Simplistically, electron microscopy is similar to light microscopy but
uses electrons to produce the image instead of light or photons. The
FIGURE 2.7
Confocal image of commercial confectionery gel. Stained in Rhodamine B. The spheres are
gelatin separating as the sample disperses in the stain. (Courtesy of Leatherhead Food Inter-
national, U.K.)
electrons have a shorter wavelength to allow a greater magnification of

the structure than light. The electrons hit a photosensitive screen, produc-
ing light that can be seen.
There are two main types of electron microscopy commonly used for
food: scanning electron microscopy (SEM) and transmission electron
microscopy (TEM). In SEM, the electron beam scans the surface of the
sample, giving a higher magnification of the type of image seen in the
stereolight microscope. This results in images that are more easily iden-
tifiable to a nonmicroscopist. Although the beam scans the surface of the
sample, the internal appearance can be studied by fracturing the sample
before it is placed in the microscope.
For TEM, the sample is usually fixed, dehydrated, and then embedded
in a resin so that very thin sections can be cut.
The technique for both SEM and TEM normally uses instruments set at
high vacuum to get the best image quality from the easily deflected
electron beam. The need for a high vacuum imposes some restrictions on
the sample, mainly in that the sample must be very dry and not contain
any ingredient that might evaporate under the intense energy of the
electron beam. Two ways of achieving this are chemically treating the
sample to preserve the structure, or freezing the sample and looking at it
while it is still frozen. The chemicals used to prevent the components from
changing during the removal of the water or fat, or under the vacuum,
are called fixatives. Typically glutaraldehyde or formaldehyde are used
to cross-link proteins and osmium to fix and keep membranes and liquid
fat present, although fat is often removed. Fixatives for starches and
polysaccharides include periodic acid, thiocarbohydrazide and osmium,
or uranyl acetate.
Once fixed, water is removed from the sample, usually using ethanol.
For SEM the sample is dried, using critical-point drying equipment, or
air dried using a suitable solvent. The aim is always to minimize the
drying effects on the structure. The sample is then attached to a carrier
stub and coated with a very thin electrically conducting element, usually
gold, palladium, or carbon, before inserting it into the microscope.
Many studies involve using SEM of chemically fixed samples to look
at gel structures, dairy products, meat products, and fruits and vegetables.
Groves et al. (1996) combined the results from stereolight microscopy and
low-voltage SEM to understand the relationship between sorbitol struc-
ture and the properties of chewing gum.
An alternative to conventional SEM, with its need for fixation and
drying, is the use of a cold stage in the instrument. This technique involves
freezing the sample, usually in liquid nitrogen slush, coating it in a con-
ductive material, and examining it in the microscope at high vacuum
while still frozen at about –180°C. In addition, the sample can be fractured
after freezing and the ice removed by sublimation. This removes any frost,
but also allows the identification of the regions of water or ice more clearly.
The technique is very useful for foods as they often contain large
amounts of water or fat, and these can be seen in situ. In addition, the
artifacts produced by chemical fixation and removal of water and other
components are not present. There are some disadvantages to this tech-
nique. The freezing process can cause some damaging changes, and it can
be difficult to see the structural details of an individual ingredient when
all the components are present, particularly in low-moisture foods. How-
ever, it is a very valuable method for food products.
The technique of cold-stage SEM is useful for high-fat products and has
been applied to the study of chocolate for investigating interactions
between ingredients on the stability of chocolate to bloom (Subramaniam
et al., 1994). In this study, differences in fat crystal appearance were noted
after storage under accelerated conditions, when antibloom fats were
added to the chocolate. Examples of the appearance of some of these
systems are shown in Figures 2.8 to 2.10. In some conditions, long needle-
like fat crystals are produced rather than the wider conventional bloom.
In TEM, once the samples have been fixed, dehydrated in ethanol, and
embedded in resin, sections are cut. This requires a specialized microtome
as the sections are typically 100 nm or less in thickness and have to be
floated on water. The sections are picked up on grids having a very fine
mesh so that the sections are supported but are also held over gaps for
the electron beam to pass through. After this, the sections are usually
stained with a heavy metal salt such as uranyl acetate or lead citrate or
FIGURE 2.8
Cold-stage scanning electron microscopy image of stored control chocolate with no visible
bloom. (Courtesy of Leatherhead Food International, U.K.)
FIGURE 2.9
Cold-stage scanning electron microscopy image of stored control chocolate showing typical
bloom. (Courtesy of Leatherhead Food International, U.K.)
both. These heavy metal salts bind to the proteins or other structural
elements and make them opaque to the electrons.
Alternatives to the preparation of resin sections for TEM are the less
common techniques of replica manufacture and negative staining. In the
first technique, the food sample is usually frozen and fractured in spe-
cialist equipment under vacuum. A very thin replica of the fractured
surface is made (by evaporating a metal such as platinum) from an angle
FIGURE 2.10
Cold-stage scanning electron microscopy image of stored chocolate containing a commercial
antibloom agent. The bloom level is reduced but also slightly different in appearance. (Cour-
tesy of Leatherhead Food International, U.K.)
to highlight the contours, followed by an overhead evaporation of carbon

to give support. The food sample is dissolved away and the replica exam-
ined in the microscope. This technique was used early in the application
of electron microscopy to study food microstructure. It was applied to the
examination of fats used in baking and showed the importance of the fat
crystal structure at the air bubble interface. The technique was also applied
to investigate the properties of chocolate and cocoa butter, revealing the
role of polymorphism in the formation of chocolate bloom. Later work
on toffee used the replica technique to show the change in protein distri-
bution in the emulsion when the milk protein was varied. Good examples
of some of this early work are given by Lewis (1981).
In negative staining, the sample is dried onto a coated grid, and an
electron dense stain is then allowed to dry around the edges of the sample.
Thus, the sample, which is transparent to the electrons, is seen as clear
with a black outline of stain. This technique is more commonly used with
bacteria or viruses but has been used to look at the behavior of gelatin
and collagen.
A less commonly used method as an alternative to the above prepara-
tions for TEM, the technique of cryosectioning or cutting very thin frozen
sections and looking at these in the electron microscope, has been
described for foods by Heertje and Paques (1995). This method has usually
been applied when precise information on the distribution of elements or
ingredients at the resolution of the transmission electron microscope is
required.
Electron microscopy, especially TEM, is very suitable for the study of

ingredient interactions at the molecular level. The gelation of proteins and
polysaccharides and the effects of the variation of pH and addition of
salts, emulsifiers, or other ingredients have been extensively reported
(Lewis, 1981; 1986; Olsson et al., 2003; Walkenstrom et al., 2003). Meat
products and the dispersion of meat proteins with ingredients have been
studied by Lewis et al. (1986), and confectionery products by Eeles et al.
(2002a,b).
The aggregation of molecules into networks is affected by ingredients
and environment as well as processing or holding conditions. Different
ingredients can interact to produce networks with different properties. In
a study in confectionery gels (gums), Eeles et al. (2002b) used TEM to
compare microstructures of pilot-scale products made with different hydro-
colloids. Sensory attributes changed on storage, and these changes were
dependent on the microstructure of the gel network. Gelatin gels had a
dense network and were firm initially, whereas starch gels were softer and
had a larger pore structure. Mixtures of gelatin and starch produced a
phase-separated system, with starch as the continuous network.
Products stored under humid conditions developed sugar graining, and
the changes in the network could be seen using TEM. Examples of typical
networks are given in Figures 2.11 to 2.13. Sensory evaluation of the
gelatin-based gums revealed that flavor release was less marked than with
the starch-based samples. This suggests that flavor release can be related
to network structure.
FIGURE 2.11
Transmission electron microscopy image of a thin section of a gelatin-based gum showing a
dense network structure. (Courtesy of Leatherhead Food International, U.K.)

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DK3027_half 11/2/05 2:40 PM Page 1

FOOD SCIENCE AND TECHNOLOGY

A Series of Monographs, Textbooks, and Reference Books

Editorial Advisory Board

Gustavo V. Barbosa-Cánovas Washington State University–Pullman

76. Food Chemistry: Third Edition, edited by Owen R. Fennema

90. Dairy Technology: Principles of Milk Properties and Processes, P. Walstra,

118. Food Protein Analysis: Quantitative Effects on Processing,

147. Dairy Science and Technology: Second Edition, Pieter Walstra,

Boca Raton London New York

© 2005 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4200-2813-3 (eBook - PDF)

and the CRC Press Web site at

Dedicated to our families, friends, and colleagues

Formulated food products are complex colloidal systems in which

Anilkumar G. Gaonkar, Ph.D., is a research principal at Kraft Foods

Andrew McPherson, Ph.D., is a senior associate principal scientist at

Curtis M. Amundson Dominique Champion

Arun Kilara Gaelle Roudaut

Martine Le Meste Denise Simatos

Alejandro G. Marangoni Rodrigo Tarté

1 Interactions of Ingredients in Food Systems: An

2 Microscopy: A Tool to Study Ingredient Interactions

3 Probing Ingredient Functionalities in Food Systems

4 Interaction of Water with Food Components ................... 87

5 Starch Selection and Interaction in Foods ..................... 139

6 Ingredient Interactions: Sweeteners ................................ 167

7 Protein Interactions in Muscle Foods ............................. 195

8 Protein-Protein Interactions in Food ............................... 283

9 Interaction of Proteins with Selected Small

11 Macromolecular Interactions in Enzyme Applications

12 Interactions of Emulsifiers with Other Components

13 Ingredient Interactions Affecting Texture and

14 Interactions of Flavor Components in Foods ................. 477

Index ............................................................................................. 543

Packaging, storage, distribution

In-home storage, preparation

occurred between these proteins. The forces involved in the observed

SOL PROGEL GEL

obtained at extreme acidic (pH 1 to 2) and extreme alkaline (pH >10)

whereas high moisture and high temperature decreased the amount of

protein. Under aqueous conditions, β-conglycinin or 7S fraction of soy

Rowley, B.O. and Richardson, T. Protein-lipid interactions in concentrated infant

microscopy techniques with an emphasis on how they can be used to look

1. To identify foreign bodies. This deals with looking at the foreign

In using microscopy as a tool to study ingredient interactions in foods,

products contain both fat and water-soluble ingredients, and some

Generally, polarized light microscopy is useful in looking at crystalli-

margarine with different triacylglycerols (Heertje, 1993); imaging the crys-

In terms of ingredient interactions, confocal microscopy allows multiple

the clear changes in aggregation and association of whey protein isolate

electrons have a shorter wavelength to allow a greater magnification of

to highlight the contours, followed by an overhead evaporation of carbon

Electron microscopy, especially TEM, is very suitable for the study of

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