Food Chemistry

FOOD SCIENCE AND TECHNOLOGY

FOOD CHEMISTRY

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FOOD SCIENCE AND TECHNOLOGY

FOOD CHEMISTRY

DONGFENG WANG HONG LIN JIANQIAN KAN LINWEI LIU XIAOXING ZENG AND

SHENGRONG SHEN EDITORS

New York

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Food chemistry / editors, Dongfeng Wang ... [et al.]. p. cm. Includes index. ISBN: B 1. Food--Analysis. 2. Food--Composition. I. Wang, Dongfeng. TX531.F555 2011 664'.07--dc23 2011042522

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface

vii

Contributors

ix

About the Editors

xi

Chapter 1

Introduction Dongfeng Wang

1

Chapter 2

Water Jianqian Kan and Guoqing Huang

9

Chapter 3

Carbohydrates Dongfeng Wang, Jipeng Sun, Guoqing Huang, Xiaolin Zhou and Liping Sun

Chapter 4

Lipids Shengrong Shen, Dongfeng Wang and Undurti N. Das

107

Chapter 5

Proteins Hong Lin, Lisha Wu and Shuhui Wang

137

Chapter 6

Vitamins Yibin Zhou, Dongfeng Wang and Ping Dong

191

Chapter 7

Minerals Dongfeng Wang, Lina Yu, Haiyan Li, Bin Zhang, Shuhui Wang and Xingguo Liang

223

Chapter 8

Food Flavors Xiaoxiong Zeng and Guaoqing Huang

247

Chapter 9

Food Additives Linwei Liu and Shiyuan Dong

273

Chapter 10

Toxicants in Foods Wang Dongfeng, Guoqing Huang and Shuhui Wang

305

Index

35

353

PREFACE Foods consist of a large quantity of compounds, of which, some are original from plant or animal materials, some are new ones generated during processing or preservation, some are intentionally added by manufacturers, and some are contaminants produced during processing, preservation or packaging. These compounds undergo various changes during processing and storage and it is hence necessary to understand the effects of processing or storage on these compounds so as to enhance the nutrition, palatability and safety of foods. The purpose of Food Chemistry is to elucidate the structure, physicochemical properties, nutrition and safety of major food constituents and their changes occurred during processing and storage. Due to the extreme importance, Food Chemistry has been accepted as a major fundamental course for food-related majors. Though food chemistry has a history of more than 200 years, it developed into a relatively independent system in the late 1960‘s. Since then, the United States, Japan, Germany and other countries published several authoritative food chemistry textbooks, including Latest Food Chemistry edited by Hayashi Junzo and Kitamura Mitsuo (Japan), Food Chemistry by Sakurai Yoshito (Japan), Food Chemistry by Owen R. Fennema (United States), Food Chemistry by Belitz HD (Germany), Food Chemistry by Zhang Wang (China), and Food Chemistry by Dongfeng Wang (China). Of the works, the publications edited by Fennema and Belitz HD have been widely chosen by university students as textbook. However, the two books contain too many contents and part of them overlaps with those stated in Biochemistry and Organic Chemistry. Besides, the two books are too expensive for readers in developing countries. Hence, there is an urgent demand to publish a simplified Food Chemistry textbook that most university students can afford, which is the case of this book. This book presents the chemistry and properties of the six essential nutrients contained in foods, including water, carbohydrates, lipids, proteins, vitamins and minerals, and their changes occurred during food processing and storage. In addition, this book also deals with the chemistry and properties of flavors, food additives and toxic substances in foods. This book is simplified and cheaper than previously published books without reducing its academic level, and reflects the latest advances in food chemistry. This work can be used as a textbook by university students and especially suitable for students in developing countries and non-English speaking countries for bilingual delivery. The authors would like to thank the postgraduates of the Laboratory of Food Chemistry and Nutrition of Ocean University of China, including Mei Ding, Yan Li, Lu Yu, Xingya Li,

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Dongfeng Wang, Hong Lin, Jianqian Kan et al.

Xiang Gao, Wen Zhou, Zhe Xu, Min Wang, Mengqi Li, and Chunsheng Li, for assistance in literature collection and typesetting, and Ocean University of China for funding the publication.

CONTRIBUTORS Undurti N Das Jawaharlal Nehru Technological University, Kakinada-533 003, India Liping Sun College of Chemistry and Engineering, Kunming University of Science and Technology, Yunnan Province, China Jipeng Sun Third Institute of Oceanography State Oceanic Administration, Xiamen, China Lina Yu Shandong Peanut Research Institute, Qingdao, China Xiaoling Zhou Medical College of Shantou University, Shantou, Guangdong Province, China Bin Zhang School of Food and Pharmacy & Medical School, Zhejiang Ocean University, Zhushang City, Zhejiang Province, China Haiyan Li College of Food Science and Engineering, Ocean University of China, Qingdao, China Banping Wang College of Food Science and Engineering, Ocean University of China, Qingdao, China Xingguo Liang College of Food Science and Engineering, Ocean University of China, Qingdao, China

ABOUT THE EDITORS Dongfeng Wang is a professor of the College of Food Science and Engineering at the Ocean University of China. He has published many books related to food chemistry as editorin-chief, including Food Chemistry (2007), Advanced Food Chemistry (2009), Chemistry of Toxic Substances in Foods (2005), Technology of Experiment & Study of Tea Biochemistry (1997), Experiments on Food Quality & Food Safety (2004) and Technology of Experiments on Food Science and Engineering (2007). He has published over 120 original papers that reflect his research interests in food chemistry, tea biochemistry, carbohydrate chemistry, and preservation. He has received many teaching & academic honors, including The Second Prize for Advanced Science and Technology of China in 2010, The First Prize for Advanced Science and Technology from Ministry of Education of the People‘s Republic of China in 2009, Distinguished Teacher Awards from Shandong Province of China in 2006, and Award for Young Scientists from Anhui Province in 2000. Professor Wang received his BS degree of agriculture in 1982 from Anhui Agricultural College (Anhui, China), the MS degree of tea biochemistry in 1988 from Zhejiang Agricultural University (Zhejiang, China), and the PhD degree of inorganic biochemistry of food in 1999 from University of Science and Technology of China (Hefei, China). Jianquan Kan is a professor of the College of Food Science at Southwest University of China. He is the editor-in-chief of many books related to food chemistry, including The Practical Chemistry of Oil and Fat (1997), Food Chemistry (2002, revised in 2006 and 2008), Advanced Food Chemistry (2011), An Introduction to Food Safety (2009), Food Analysis (2011) and Experimental Methods (2011). He is the author or corresponding author of over 140 original papers covering food chemistry, food analysis and nutrition. He has been honored the Second-Class Prize of Chongqing Science and Technology Advancement (2009) and the Second Chongqing Academic and Technological Leader (2008). Professor Kan received the BS degree of chemistry from Nanchong Normal College (Sichuan, China) in 1986, the MS degree of Product Processing and Storage from Southwest Agricultural University (Chongqing, China) in 1992, and the PhD degree of Product Processing and Storage from Southwest Agricultural University (Chongqing, China) in 2003. Lingwei Liu is a professor of the College of Food Science & Engineering in Northwest A&F University, Yangling, ShaanXi, China. He has done intensive researches related to food chemistry, food analysis, nutrition and food safety. Professor Liu received the BS degree of food science from Northwest Agriculture University (ShaanXi, China) in 1982 and the PhD

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degree of food science from Northwest Agriculture & Forest University (ShaanXi, China) in 1995. Hong Lin received the BS degree in 1984, the MS degree in 1990, and the PhD degree in 1998 in seafood science from the Ocean University of China. Professor Lin is a famous expert in the seafood safety field and his research area covers novel marine organism-derived chemical and biological hazard discovery, quality control during seafood processing, and fast hazard detection method development. Professor Lin has been granted 4 invention patents, published more than 100 original articles, and edited 4 academic books, including Seafood Safety (2010), Aquatic Nutrition and Safety (2007), Effective Use of Aquatic Resources (2007), and Fish Preservation Technologies (2000). Xiaoxiong Zeng received the BS degree from Hunan Agricultural University in 1985, the MS degree from Zhejiang Agricultural University in 1988, and the PhD degree from Shizuoka University (Shizuoka, Japan) in 2000. Dr. Zeng is now a professor of the College of Food Science and Technology, Nanjing Agricultural University, China. He is one of the authors or corresponding author of over 100 original papers related to food chemistry, food biotechnology and glycobiology. Shengrong Shen is a professor of the School of Biosystems Engineering and Food Science of Zhejiang University. Professor Shen was granted the PhD degree by the Department of Biophysics of Zhejiang University in 1997. His research area includes structural analysis of such bioactive compounds as lipids, fatty acids, and polyphenols. He has published more than 100 original papers concerning food chemistry, food safety and applied nutrition in the latest 10 years. Besides, professor Shen has published 5 academic books related to food and health, tea biochemistry and food chemistry. Yibin Zhou is a professor of the Department of Food Science and Engineering at Anhui Agricultural University, Anhui, China. He had edited Food Chemistry (in Chinese) as an assistant, and is the author or corresponding author of over 40 original papers on carbohydrates, food engineering, and biotechnology. Guoqing Huang is a lecturer of the College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, China. Shiyuan Dong is a lecturer of the College of Food Science and Engineering, Ocean University of China, Qingdao, China. Shuhui Wang is a PhD candidate in Biosystems Engineering Department College of Agriculture - Ginn College of Engineering, Aubum University, Auburn, AL 36849-5417, USA

In: Food Chemistry Editors: D.Wang, H. Lin, J. Kan et al.

ISBN: 978-1-61942-125-7 © 2012 Nova Science Publishers, Inc.

Chapter 1

INTRODUCTION Dongfeng Wang College of Food Science and Engineering, Ocean University of China, Qingdao, China

ABSTRACT Food Chemistry is a fundamental discipline for students, engineers, and professionals engaged in the food industry. This chapter provides an overview of this discipline, including its definition, purpose, development, and its role in food science and engineering.

1.1. Food Chemistry and Its History 1.1.1. What Is Food Chemistry Nutrients refer to the indispensable substance that provides nourishment essential for maintenance of life, growth and development of human being. The human body needs a lot of nutrients. Based on chemical structure, the nutrients can be divided into six major categories, including water, carbohydrates, proteins, lipids, vitamins and minerals. A minor difference between terms foodstuff and food should be noted first. Foodstuff refers to materials containing nutrients; while foods are materials that have been processed from foodstuff (ranging from simple cleaning to a modern factory processing) in order to meet people‘s nutritional and sensory requirements. In another word, a food shall be characterized by both nutrition and sensory satisfaction. The nutritional compositions of foods can be determined easily, but sensory satisfaction is a much complex issue and is related to the color, texture, and shape, flavor of foods in addition to the cultural background and dietary habits of consumers. The chemical compositions of foods are very complex (Figure 1-1). Of the components, some are intrinsic in animal or plant materials, some are generated during processing and storage, some are intentionally added by manufacturers, some are contaminants originated from the environment or microorganisms, and some are migrated from packing materials of

2

Dongfeng Wang

food. The purpose of Food Chemistry is to elucidate the structure, physical and chemical properties, nutritional value as well as safety of these components, their changes undergone during storage and processing, and the effects of these changes on food nutrition and palatability. The knowledge is of great importance in improving food quality, developing new food resources, evolving food processing and storage technologies, upgrading food packaging materials, and increasing food safety and quality.

Natural

Water Carbohydrates Proteins Lipids Minerals Vitamins Pigments Hormones Flavor components Toxic substances

Food components Natural additives Food Addtives Synthetic additives Unnatural

From processing Contaminants From environmental pollution

Figure 1-1. Composition of foods.

Food Chemistry is a comprehensive discipline and partially overlaps with chemistry, biochemistry, physical chemistry, botany, zoology, food nutrition, food safety, polymer chemistry, environmental chemistry, toxicology, molecular biology, and many other subjects. Food Chemistry associates the most closely with chemistry and biochemistry and it is the extension of the two subjects to the food area. However, the subjects have different contents and focuses. The chemistry subject deals mainly with the composition, property, and reactions of molecules, biochemistry focuses on the reactions and changes of various components in organisms under suitable or moderately suitable conditions, while food chemistry is interested in the changes of components occurred in such unsuitable conditions as freezing, heating, and drying, their interactions during these processes, and the effects of these changes on the nutrition, safety, and sensory properties (such as color, flavor, taste, and shape) of foods. 1.1.2. History of Food Chemistry It is a short time since Food Chemistry is accepted as an independent subject. However, the researches and reports related to this subject have been started since the last 1700s. Many components were separated from foods by chemists and botanists at that time and Researches on the Chemistry of Food by Justus von Liebig in 1847 is recognized as the first book related to food chemistry.

Introduction

3

As the trading of foods between regions and countries increased, both consumers and manufacturers had urgent needs on the information of water contents and the presence nonfood components in foods. Meanwhile, driven by the rapid development of analysis measures, the desire to understand the natural characteristic of foods also grew. In 1860, German scholars Hanneberg W. and Stohman F. invented a method for the simultaneous determination of water, crude fat, ash, and nitrogen contents. Several years later, diets containing solely proteins, lipids, and carbohydrates were found insufficient for maintaining life. In 1900s, with the advancement of analytical techniques and the biochemistry subject and the rapid development of the food industry, requirements on new food processing technologies and prolonged storage life emerged, which drove the quick development of food chemistry. During this period, a growing number of researches papers were published and the quantity of related journals increased significantly as well, including Archives of Biochemistry and Biophysics (initiated in 1942), Journal of Agricultural and Food Chemistry (initiated in 1953) and Food Chemistry (initiated in 1966). Due to the emergency of increasing deep and systematic publications, Food Chemistry gradually developed into an independent subject. Chinese scholars Yanbin Xia and Ruijin Yang divide the history of Food Chemistry into four stages. Stage one: Many natural components were separated from plants and animals and were identified, including lactic acid, citric acid, malic acid, and tartaric acid. The knowledge was not systematic yet and was reported mainly by chemists. Stage two: In the early 1900s (1820 ~ 1850), food chemistry developed quickly along with the development of agricultural chemistry and gained much importance in Europe. Specialized food chemistry laboratories were established and many professional journals related to food chemistry were issued. Meanwhile, adulteration became a serious issue and the need for impurity determination propelled the development of food chemistry. In this stage, Justus von Liebig invented an optimized method for quantitative analysis of organic substances and published Researches on the Chemistry of Food in 1847. Stage three: In the middle 1900s century, the British scientist Arthur Hill Hassall reported the microscopic images of pure and adulterated foods and food chemistry came into the microanalysis time. In 1871, Jean Baptis M.D.M. proposed that diets containing only proteins, carbohydrates and lipids were insufficient to sustain human‘s life. The interests on the nutritional requirements further accelerated the development of food chemistry. Until the first half of the 20th century, the majority of components in foods were identified and the number of literatures related to chemistry food increased markedly. Food chemistry then turned to be a mature and independent subject in mid-20th century. Stage four: Food chemistry is now in the fourth stage. With the rapid development of society, economy, science and technology, and the improvement of living standards, consumers raise higher requirements on food security, nutrition, palatability, and convenience. Meanwhile, to realize the transformation from traditional to scaled, standardized, and modernized processing of foods, more and more new technologies, materials, and equipment are used, which markedly drive the rapid development of food chemistry. Besides, the advancement of basic chemistry, biochemistry, instrumental analysis and other related subjects guarantee the rapid development of food chemistry. Food chemistry has become a most important subject for food scientists [1, 2].

4

Dongfeng Wang

1.1.3. Food Chemistry Textbooks A series of food chemistry textbooks were published between 1976 to 1985, including Latest Food Chemistry by Hayashi Junzo and Kitamura Mitsuo (Japan), Food Chemistry by Sakurai Yoshito (Japan), Food Chemistry by Owen R. Fennema (United States), and Food Chemistry by Belitz HD (Germany), in which, the works of Fennenma and Belitz HD contributed a lot to the development of food chemistry and has been widely chosen by university students as textbook. Food Chemistry has been chosen as a fundamental course for food related majors.

1.2. The Role of Food Chemistry in Food Science and Engineering Foodstuff undergoes various chemical and biochemical reactions during storage, transport, and processing. These reactions might yield products that are either beneficial to food nutrition and palatability or harmful to consumers. The knowledge of food chemistry is hence of extreme importance, because the purpose of this subject is to elucidate the changes of various food components occurred during storage, transport, and processing and the effects of these changes on food quality. In recent years, the control of composition, property, structure, and interaction of various food components, the chemical nature of the nutrition and palatability of complex food systems, and the exploitation of new food resources constitute the new contents of food chemistry. With the development of science and technologies and the extension of other fundamental subjects to the food industry, more and more toxic and harmful chemicals in foods are identified and food chemistry has turned to be the theoretical foundation for guaranteeing food quality and safety. Food chemistry plays an important role in food science and engineering and is developing quickly. 1.2.1. Role of Food Chemistry in Technology Advancement Nutrition, healthcare, safety, and enjoyment are the four fundamental attributes of foods required by the modern food industry. The theories and application research results of food chemistry are guiding the healthy and sustainable development of the food industry (Table 11). Practice has proved that, no the theoretical guidance of food industry, no the ever growing modern food industry. Table 1-1. Impact of food chemistry on technological advancement of the food industry [3, 4] Food Industry Basic food industry Storage and processing of fruits and vegetables

Application Flour improving; starch modification; new edible materials exploitation; high-fructose syrup; food enzymes; molecular basis of food nutrition; new sweetener and natural additive development; new oligosaccharide production; oil modification; vegetable protein isolates; functional peptides production; microbial polysaccharides and single cell protein development; development and utilization of wild, marine, an edible drug resources, etc. Chemical peeling; color protection; texture control; vitamin retention; deastringency and debittering; coating and waxing; chemical preservation; controlled atmosphere storage; bioactive packaging; enzyme-assisted juicing, filtration and clarification; chemical preservation, etc.

Introduction

5

Table 1-1. (Continued) Food Industry Storage and processing of meats

Beverage industry

Dairy industry Baking industry Edible oils and fats industry Condiments industry Fermented food industry Food safety Food inspection

Application Post-slaughter processing; juice preservation and tenderization; color protection and development; enhancement of the emulsifying capacity, gelling capacity, and viscoelasticity of meat; frozen denaturation of proteins; fresh meat packaging in supermarket; production and application of fumigation agent; artificial meat production; comprehensive utilization of viscera, etc Instant dissolution; ingredient floating and/or sinking inhibition; protein beverage stabilization; water treatment; juice stabilization; juice color protection; flavor enhancement; alcohol degree decrease; beer clarification; beer foamability and bitterness improvement; chemical nature and prevention of beer non-biological stability; off-flavor elimination; juice deastringency; soybean odor elimination, etc. Yoghurt and juice milk stabilization; chymosin substitute development; whey utilization; nutrition fortification of diary products; etc. High-efficiency leavening agent development; crispness improvement; bread color and texture modification; aging and mildewing inhibition; etc. Lipid refinement; lipid modification; development and utilization of DHA, EPA, and MCT; food emulsifier and anti-oxidant development; oil absorption reduction of fried foods; etc. Meat soup production; nucleotide-type flavor enhancers; organic iodinesupplemented salt; etc. Post-processing of fermented foods; flavor changes during postfermentation; comprehensive utilization of biomass and residues; etc. Source identification of exogenous toxicants and their prevention; identification of endogenous toxicants and their elimination; etc Formulation of inspection standards; rapid analysis; biosensor development; fingerprint preparation of products; etc.

Due to the rapid development of food chemistry, some important reactions, including the Millard reaction, caramelization, lipid auto-oxidation, starch gelatinization and aging, polysaccharide hydrolysis and modification, protein hydrolysis and denaturation, pigment discoloration, vitamin degradation, metal-catalyzed reactions, enzyme-catalyzed reactions, fat hydrolysis and transesterification, lipid thermo-oxidative decomposition and polymerization, flavor compound changes, action mechanisms of food additives, generation of harmful ingredients as well as postharvest physiology, are identified in foods. The knowledge on these reactions greatly enhances the development of the food industry. 1.2.2. Role of Food Chemistry in Human Nutrition and Health It has been more than two centuries since proteins, carbohydrates and lipids were identified as the three major nutrients for human. The two most important attributes of foods are to provide consumers with nutrition and sensory satisfaction. One of the objectives of food chemistry is to investigate the nutrition and flavor composition in food materials and processed foods and the interactions of the components occurred during processing and storage and effects of these interactions on food nutrition and palatability. The modern food

6

Dongfeng Wang

chemistry should not only ensure the healthcare and enjoyment attributes of food components, but also guide consumers on rational diet selection. The concept of nutrition has evolved significantly due to social development and the change of the healthy status of consumers. How to reduce the incidences of diet-related diseases, such as cardiocerebrovascular diseases, cancers, and diabetes, has turned to be a new major task of food industry. In addition to the healthcare attribute, foods should also provide desirable flavors so that consumers enjoy the eating process. The emergence of biotechnologies and new food processing technologies guarantees the safety of foods. Contamination of foods by pollutants is currently a worldwide concern due to global environmental deterioration. The analysis and identification of trace and ultramicro substances are of vital importance to the nutrition value and the control of toxicants of foods. The development of food chemistry has been associated with the healthy status and civilization level of human.

1.3. Research Methods of Food Chemistry Each type of food contains a large number of components and is thereby a much complex system. Hence, the research methods of food chemistry are quite different from those of common chemistry subjects. In food chemistry, the knowledge on the chemical composition, physicochemical properties, and changes of food components must be associated with the nutrition, enjoyment, and safety of foods. The experimental design of food chemistry should reveal the complex composition of food systems and the changes of the nutrition value, enjoyment, and safety of foods during processing and storage. The interactions between food components and their changes occurred during storage and processing (such as ultra-high pressure, high temperature, freezing, presence or absence of oxygen) are extremely complex. Hence, many researches are carried out in simplified and stimulated models, which must be then verified in real food systems. The experiments of food chemistry include mainly physicochemical experiments and sensory evaluation experiments. Physicochemical experiments reveal the composition of foods and the structures of the components, including nutrients, toxicants, and flavors; while sensory experiments evaluate the texture, flavor, and color changes of foods through visual inspection. Foods or food materials undergo a series of changes during storage, transport, processing, and sales. The changes include: enzymatic and chemical reactions in raw and fresh materials; changes caused by water activity variance; component decomposition, polymerization, and denaturation under violent conditions (high temperature, high pressure, mechanical actions); oxidation induced by oxygen or other oxidants; photochemical reactions; and migration of packaging materials to foods. Of the changes, non-enzymatic browning, lipid oxidation and hydrolysis, protein hydrolysis and denaturation, protein cross-linking, oligosaccharide and polysaccharide hydrolysis, and change of the presence form of natural pigments and their degradation, are the most important reactions for the food industry. Of the reactions, some are desired, but some are unexpected and must be avoided during processing (Table 1-2). The mechanisms and control of these reactions constitute the key contents of food chemistry.

Introduction

7

Table 1-2. Part reactions occurred during food processing and storage and their influences on foods [3, 4] Reaction Nonenzymic browning Oxidation Hydrolysis

Isomerization Polymerization

Protein denaturation

Examples Color development in bakery foods Oxidation of lipids, vitamins, an phenols Hydrolysis of lipids, proteins, and carbohydrates

Influence on foods Desired or undesired color, smell, taste; loss of nutrition; harmful ingredients. Change color; desired flavor or off-odors and toxicants Increased soluble solids content; texture changes; desired color, flavor, taste, and nutrition; toxicity loss of certain components

cis-trans isomerization of lipids Foam and insoluble brown precipitate forming in frying Egg white coagulation; enzyme inactivation

Discoloration; formation or loss of certain functions Discoloration; loss of nutrition; off-odor development; toxicants formation Improved nutrition; toxicity loss of certain components

The research fruits and methods of food chemistry have been widely absorbed by the food industry and greatly promote the development of the food industry. In the last decades, some new subjects and research areas, such as structural chemistry, free radical chemistry, membrane separation, edible package, microencapsulation, extrusion, superfine comminution, bioactive packaging, supercritical extraction, molecular distillation, membrane catalysis, bioreactor, toxicant chemistry of foods, molecular nutrition, and nutria-genomics, have been established. These new technologies and subjects will undoubtedly facilitate the rapid development of the food industry, which in turn benefits the improvement of the food chemistry subject.

REFERENCES [1] [2] [3] [4]

Wang, DF. Food Chemistry.1st edition. Beijing: Chemistry Industry Press; 2007 Damodaran, S; Parkin, KL; Fennema, OR. Fennema’s Food Chemistry. 4th edition. New York: CRC Press; 2007. Kan, JQ. Food Chemistry. 1st edition. Beijing: China Agricultural University Press; 2002. Wang, Z. Food Chemistry. 1st edition. Beijing: China Light Industry Press; 2005.



Chapter 2

WATER 1

Jianqian Kan1 and Guoqing Huang2

College of Food Science, Southwest University, Chongqing, China College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, China

2

ABSTRACT Water is an important component in many foods. Its content and occurrence status significantly affect the flavor, texture, and stability of foods. This chapter deals with the various physical and chemical properties of water and ice and the interactions with other components in foods. Water occurs in multiple states due to interactions with solutes and the interactions significantly affect the bioavailability of water to chemical reactions and microorganisms. To distinguish the differences between water content and its bioavailability, the term water activity (aw) is proposed and its application in food stability predication are detailed. The relationship between water content and aw can be presented by moisture sorption isotherm (MSI), which is very useful in designing the concentration and dehydration processes of foods. In addition to aw, molecular mobility (Mm) has also been proposed to predict food stability. Its definition and its effect on food stability are also a concern of this chapter. Water is a predominant constituent in many foods (Table 1). Water in proper amount, location, and orientation profoundly influences the structure, appearance, and taste of foods and their susceptibility to spoilage. Because medium water supports chemical reactions and water is a reactant in hydrolytic processes, the removal of water from foods retards many reactions and inhibits the growth of microorganisms, thus improving the shelf lives of a number of foods. Through physical interaction with proteins, polysaccharides, lipids and salts, water contributes significantly to food texture. Water is essential to life: as an important governor of body temperature, as a solvent, as a carrier of nutrients and waste products, as a reactant and reaction medium, as a lubricant and plasticizer, as a stabilizer of biopolymer conformation, as a likely facilitator of the dynamic behavior of macromolecules, including their catalytic (enzymatic) properties, and in other ways yet unknown.

10

Jianqian Kan and Guoqing Huang Table 1. Water contents of some foods [1] Food Pork, raw, composite of lean Beef, raw, retail cuts Chicken, all classes, raw meat without skin

Water content (%) 53~60 50~70

Food

Water content (%) 10~13 20

Fish, muscle proteins

65~81

bananas

75

Berries, cherries, pears Apples, peaches, oranges, grapefruit Rhubarb, strawberries, tomatos Butter, margarine Milk powder

80~85

Cereal flour Honey Avocado, bananas, peas (green) Beets, broccoli, carrots, potatoes Asparagus, beans (green), cabbage, cauliflower, lettuce Bread

85~90

Biscuits

3~8

90~95

Tea

3~7

15 4

Edible oil

0

74

74~80 80~85 90~95 35~45

1. PHYSICAL AND CHEMICAL PROPERTIES OF WATER AND ICE 1.1. The Water Molecule and Its Association 1.1.1. The Water Molecule The water molecule is comprised of two hydrogen atoms interacting with the two sp3 bonding orbitals of oxygen, forming two covalent σ bonds. A schematic orbital model of a water molecule is shown in Figure 1.a and the appropriate van der Waals radii are shown in Figure 1.b.

Figure 1. Schematic model of a single HOH molecule: (a) sp3 configuration, and (b) van der Waals radii for a HOH molecule in the vapor state [1].

Water

11

In the vapor state, the bond angle of an isolated water molecule is 104.5°. The O-H internuclear distance is 0.96 Å and the van der Waals radii for oxygen and hydrogen are 1.40 and 1.2 Å respectively.

1.1.2. Association of Water Molecules Each water molecule has an equal number of hydrogen-bond donors and receptor sites and is able to hydrogen-bond with a maximum of four water molecules. The resulting tetrahedral arrangement is shown in Figure 2. The two unshared electron pairs (n-electrons or sp3 orbitals) of oxygen act as H-bond acceptor sites and the H-O bonding orbitals act as hydrogen bond donor site. The dissociation energy of this hydrogen bond is about 1125kJ/mol. As mentioned above, each water molecule can hydrogen bond with at most four water molecules and the resultant three-dimensional structure is quite stable. This structure is quite different from those formed by other small molecules that also involved in hydrogen bonding (such as NH3 and HF). Ammonia has three hydrogen-bond donors and one hydrogen-bond receptor, while HF has one hydrogen and three receptor sites. Both the two chemicals do not have equal numbers of donor and receptor sites and therefore can form only two dimensional hydrogen-bonded networks. The above mentioned polarization of H-O bonds is transferred via hydrogen bonds and extends over several bonds. Therefore, the dipole moment of a complex consisting of increasing numbers of water molecules is higher as more molecules become associated and is certainly much higher than the dipole moment of a single molecule. Proton transport takes place along the H-bridges. It is actually the jump of a proton from one water molecule to a neighboring water molecule. In this way a hydrate H3O+ ion is formed with an exceptionally strong hydrogen bond (dissociation energy about 100kJ/mol). A similar mechanism is valid in transport of OH- ions, which also occurs along hydrogen bridges (Figure 3).

Figure 2 Hydrogen bonding of water molecules in a tetrahedral configuration. Open circles are oxygen atoms and closed circles are hydrogen atoms. Hydrogen bonds are represented by dashed lines [1].

Figure 3. Proton transport in water [2].

12

Jianqian Kan and Guoqing Huang Table 2. Coordination number and distance between two water molecules [2]

Ice (0°C) Water (1.5°C) Water (83°C)

Coordination number 4.0 4.4 4.9

O-H…O Distance 0.276 nm 0.290 nm 0.305 nm

Table 3. Comparisons of the melting and boiling points of methanol, dimethyl ether, and water Formula H2O CH3OH CH3OCH3

Fp/°C 0.0 -98 -138

Kp/°C 100.0 64.7 -23

1.2. Structures of Water and Ice 1.2.1. The Structure of Water (Liquid) Due to the strong tendency of water molecules to associate through H-bridges, liquid water is highly structured as ice, but not sufficiently established to produce long-range rigidity. The major difference between liquid water and ice lies in the coordination number and the distance between neighboring water molecules (Table 4). The degree of intermolecular hydrogen bonding among water molecules is temperature dependent. Ice at 0°C has a coordination number of 4.0, with nearest neighbors at a distance of 2.76 Å. As the temperature increases, the coordination number increases from 4.0 in ice at 0°C, to 4.4 in water at 1.50°C, then to 4.9 at 83°C. Simultaneously, the distance between nearest neighbors increases from 2.76 Å in ice at 0°C, to 2.9 Å in water at 1.5°C, then to 3.05 Å at 83°C. The increase in the distance between nearest neighbors during ice-water transformation decreases the water density, while the increase in the coordination number increases water density. The maximum water density is observed in 3.98°C and then declines gradually. The hydrogen-bound water structure can be changed in the presence of dissolved salts or molecules with polar and/or hydrophobic groups. For example, in salt solutions the nelectrons occupy the free orbitals of the cations, forming ―aqua complexes‖. Other water molecules then coordinate through H-bridges, forming a hydration shell around the cation and disrupting the natural structure of water. In addition, hydration shells are also formed by polar groups through dipole-dipole interaction or H-bridges, again leading to the disruption of the structure of water. The three-dimensional hydrogen-bound structures of ice and water impart them with unique properties and extra energy is needed for disrupting the structures. Table 3. lists the comparisons of the melting and boiling points between methanol, dimethyl ether, and water.

Water

13

Figure 4. Unit cell of ordinary ice at 0°C. Circles represent oxygen atoms of water molecules. Nearestneighbor internuclear O-O distance is 2.76 Å; θ is 109° [3].

1.2.2. The Structure of Ice Ice is the orderly organized crystal of water molecules. The O-O internuclear distance between nearest neighboring water molecules in ice is 2.76 Å and the O-O-O bond angle is about 109°, which is very close to the perfect tetrahedral angle of 109°28'. As shown in Figure 4, each water molecule is associated with four other water molecules 1, 2, 3, and W'. Because pure water contains H3O+, OH–, and negligible isotope variants (such as those containing 16O, 1H, 17O, 18O, and 2H) in addition to ordinary water molecules, actual ice is not present as the perfect crystal shown in Figure 4. Due to the presence of H3O+, OH– and their dislocation, ice crystals suffer both orientational and ionic defects. Only at temperatures near -180°C or lower will all hydrogen bonds be intact, and as the temperature is raised, the mean number of intact (fixed) hydrogen bonds will decrease gradually. The amount and kind of solutes present in foods influence the quantity, size, structure, location, and orientation of ice crystals. The four major ice structures are hexagonal forms, irregular dendrites, coarse spherulites, and evanescent spherulites. The hexogonal form, which is most highly ordered, is found exclusively in foods, provided extremely rapid freezing is avoided and the solute is of a type and concentration that does not interfere unduly with the mobility of water molecules.

2. STATES OF WATER IN FOODS 2.1. Water-Solute Interactions Mixing of solutes and water results in altered properties of both water and solutes. Hydrophilic solutes change the structure and mobility of adjacent water, and water causes changes in the reactivity, and sometimes structure, of hydrophilic solutes. Hydrophobic groups of added solutes interact only weakly with adjacent water. Interactions between water and specific classes of solutes are considered below.

14

Jianqian Kan and Guoqing Huang

2.1.1. Interaction of Water with Ions and Ionic Groups Ions and ionic groups of organic molecules hinder the mobility of water molecules to a greater degree than do any other types of solutes. The strength of electrostatic water-ion bonds is greater than that of water-water hydrogen bonds, but is much less than that of covalent bonds. The normal structure of pure water (based on a hydrogen-bonded, tetrahedral arrangement) is disrupted by the addition of dissociable solutes. Water and simple inorganic ions undergo dipole-ion interactions. The example in Figure 5. involves hydration of the NaCl ion pair. In a dilute solution of ions in water, second-layer water is believed to exist in a structurally perturbed state because of conflicting structural influences of first-layer water and the more distant, tetrahedrally oriented ―bulk-phase‖ water. In concentrated salt solutions, water structure would be dominated by the ions. The ability of a given ion to alter net structure is related closely to its polarizing power (charge divided by radius) or simply the strength of its electric field. Ions that are small and/or multivalent (mostly positive ions, such as Li+, Na+, H3O+, Ca2+, Ba2+, Mg2+, Al3+, F–, and OH–) have strong electric fields and are net structure formers. These ions strongly interact with the four to six first-layer water molecules, causing them to be less mobile and pack more densely than HOH molecules in pure water. Ions that are large and monovalent (most of the negatively charged ions and large positive ions, such as K+, Rb+, Cs+, Cl–, Br–, I–, NO3– , BrO3–, IO3– and CIO4– have rather weak electric fields and are net structure breakers, although the effect is very slight with K+. These ions disrupt the normal structure of water and fail to impose a compensating amount of new structure. Ions, through their varying abilities to hydrate (compete for water), alter water structure, influence the permittivity of the aqueous medium, and govern the thickness of the electric double layer around colloids, profoundly influence the ―degree of hospitality‖ extended to other nonaqueous solutes and to substances suspended in the medium. Thus, conformation of proteins and stability of colloids (salting-in, salting-out in accord with the Hofmeister or lyotropic series) are greatly influenced by the kinds and amounts of ions present.

Figure 5. Likely arrangement of water molecules adjacent to sodium chloride. Only water molecules in plane of paper are shown [3].

Water

15

2.1.2. Interaction between Water and Neutral Groups Possessing Hydrogen-Bonding Capabilities Interactions between water and nonionic, hydrophilic solutes are weaker than water-ion interactions and about the same strength as those of water-water hydrogen bonds. Therefore, solutes capable of hydrogen bonding might be expected to enhance or at least not disrupt the normal structure of pure water. However, in some instances it is found that the distribution and orientation of the solute's hydrogen-bonding sites are geometrically incompatible with those existing in normal water. Thus, these kinds of solutes, such as urea, frequently have a disruptive influence on the normal structure of water. It should be noted that the total number of hydrogen bonds per mole of water may not be significantly altered by addition of a hydrogen-bonding solute that disrupts the normal structure of water. This is possible since disrupted water-water hydrogen bonds may be replaced by water-solute hydrogen bonds. Hydrogen bonding of water can occur with various potentially eligible groups (e.g., hydroxy1, amino, carbony1, amide, imino groups, etc.). This sometimes results in ―water bridges‖, where one water molecule interacts with two eligible hydrogen-bonding sites on one or more solutes. A schematic depiction of water hydrogen bonding (dashed lines) to two kinds of functional groups found in proteins is shown in Figure 8. A more elaborate example involving a three-HOH bridge between backbone peptide units in papain is shown in Figure 9.

Figure 8. Hydrogen bonding (dotted lines) of water to two kinds of functional groups occurring in proteins [3].

Figure 9. Examples of a three-molecule water bridge in papain; 23, 24, and 25 are water molecules [4].

16


2.1.3. Interaction of Water with Nonpolar Substances The mixing of water and hydrophobic substances, such as hydrocarbons, rare gases, and the apolar groups of fatty acids, amino acids, and proteins, enhances the hydrogen bonding of water molecules in the vicinity of hydrophobic groups due to the repulsion with water. This process has been termed ―hydrophobic hydration‖. Because hydrophobic hydration is thermodynamically unfavorable, water would tend to minimize its association with apolar entities that are present. Thus, if two separated apolar groups are present, the incompatible aqueous environment will encourage them to associate, thereby lessening the water-apolar interfacial area. This process is thermodynamically favorable and is referred to as ―hydrophobic interaction‖. Two aspects of the antagonistic relationship between water and hydrophobic groups are: formation of clathrate hydrates and association of water with hydrophobic groups in proteins. A clathrate hydrate is an ice-like inclusion compound wherein water, the ―host‖ substance, forms a hydrogen-bonded cage-like structure that physically entraps a small apolar molecule known as the ―guest molecule.‖ The guest molecules of clathrate hydrates are characteristically low-molecular-weight compounds with sizes and shapes compatible with the dimensions of host water cages comprised of 20–74 water molecules. Typical guests include low-molecular-weight hydrocarbons and halogenated hydrocarbons; rare gases; short-chain primary, secondary, and tertiary amines; and alkyl ammonium, sulfonium, and phosphonium salts. Interaction between water and guest is slight, usually involving nothing more than weak van der Waals forces. Clathrate hydrates are the extraordinary result of water's attempt to avoid contact with hydrophobic groups. There is evidence that structures similar to crystalline clathrate hydrates may exist naturally in biological matter, and if so, these structures would be of far greater importance than crystalline hydrates since they would likely influence the conformation, reactivity, and stability of molecules such as proteins.

Figure 10. Schematic depiction of a globular protein undergoing hydrophobic interaction. Open circles are hydrophobic groups, ―L-shaped‖ entities around circles are water molecules oriented in accordance with a hydrophobic surface, and dots represent water molecules associated with polar groups [2].

Water

17

Because exposure of protein nonpolar groups to water is thermodynamically unfavorable, association of hydrophobic groups or ―hydrophobic interaction‖ is encouraged, and this occurrence is depicted schematically in Figure 10. Hydrophobic interaction provides a major driving force for protein folding, causing many hydrophobic residues to assume positions in the protein interior. Hydrophobic interactions also are regarded as being of primary importance in maintaining the tertiary structure of most proteins. It is therefore of considerable importance that a reduction in temperature causes hydrophobic interactions to become weaker and hydrogen bounds to become stronger.

2.1.4. Interaction of Water with Amphiphilic Substances Water functions as the dispersion medium of amphiphilic compounds, such as fatty acid salts, lipoproteins, glycolipids, polar lipids, and nucleic acids, in some foods. Water associates with the hydrophilic entities (COO–, OH, PO4–, –C=O, or those containing the nitrogen atom) and dissolves the compounds. Amphiphilic compounds occur as micelles in water and each micelle contains hundreds or thousands of the molecules. The apolar groups are directed to the interior of the micelles, while polar groups are distributed in the water environment.

2.2. Water in Foods Foods are composed of proteins, polysaccharides, minerals, pigments, and many other constituents in addition to water. These constituents interact with water and significantly affect the properties and status of water. Generally, the water in foods can be classed as ―bulk water‖ and ―bound water‖.

2.2.1. Bound Water ―Bound water‖ is water that exists in the vicinity of solutes and other nonaqueous constituents and binds to other solutes through covalent bonds. According to the binding strength, bound water is further divided into the following three types: Constitutional water: Water of this type is a constituent of other compounds and binds the most tightly. Water in hydrates belongs to this type. Monolayer water: Water of this type is the first layer water bound to the hydrophilic groups of solutes. The forces involved include mainly water-ion or water-polar association, followed by hydrogen bonding between water and solutes. Multilayer water: Water of this type refers to water distributed in multiple layers around nonaqueous components. The forces involved are water-water and water-solute hydrogen bonding. Multilayer water binds tightly to nonaqueous components, but the strength is lower than that of monolayer water. Besides, multilayer water has changed properties compared with ordinary water. 2.2.2. Bulk Water Bulk water or free water is not chemically bound to nonaqueous compounds and mainly includes water that is physically entrapped. Based on the physical interaction, bulk water is further divided into two types:

18


Entrapped water: Water of this type is entrapped by microstructures or ultrastructures and cannot flow freely as pure water. Capillary water: Water of this type is restricted in the gaps between cells or the capillaries of food structures. Capillary water has similar reduced fluidity and vapor pressure as entrapped water. As mentioned above, the states of water in foods depend on the composition of foods and the physical status of the components. Water states and contents significantly influence the structure, processing properties, and stability of foods. The differences between bulk water and bound water are shown in Table 4. According to Table 4, bound water and bulk water differ in the following: 1. Bound water associates with nonaqueous constituents more tightly and its vapor pressure is much lower than bulk water. More energy is required for removing bound water than bulk water and the removal of bound water might irreversibly degrade the flavor, texture, and other properties of foods. 2. Bound water freezes in much lower temperature than bulk water. This explains why plant seeds and microbial spores can survive low temperatures. In contrast, juicy fruits and vegetables have much higher water contents and their tissues are susceptible to damage by ice crystals in low temperatures. 3. Bound water cannot dissolve solutes. 4. Bulk water can be utilized by microorganisms, while bound water cannot. Table 4. Comparisons of bulk water and bound water Item General description Freezing point Solute solubilization capability Molecular movement compared with pure water Enthalpy of vaporization compared with pure water Percentage among total water in high-moisture (90%) foods

Bound water Occurs in vicinity of solutes and other nonaqueous constituents and includes constitution water, monolayer water, and multilayer water Not frozen even at temperatures lower than -40°C

Bulk water Locates far away from solutes and occurs as water-water hydrogen bonding Slightly lower than that of pure water

None

Yes

Markedly reduced or none

Changed slightly

Increased

Nearly not changed

Less than 0.03%

ca. 96%

3. WATER ACTIVITY Intensive researches have indicated that no relationship can be established between the water content of a food with its physiochemical properties or stability. It has also been

19

Water

observed that various types of foods with the same water content differ significantly in perishability. Thus, water content alone is not a reliable indicator of perishability. This situation is attributable, in part, to differences in the intensity with which water associates with nonaqueous constituents. The term ―water activity‖ (aw) was developed to account for the intensity with which water associates with various nonaqueous constituents. Experience shows that food stability, safety, and other properties can be predicted far more reliably from aw than from water content.

3.1. Definition and Measurement of aw The water activity (aw) is defined as follows: aw

P P0

RVP

ERH 100

(1)

where, RVP is the relative vapor pressure; P is the partial vapor pressure of food moisture at temperature T; P0 is the saturation vapor pressure of pure water at temperature T, and ERH is the equilibrium relative humidity at temperature T. Equation (1) applies only to ideal solutions and thermodynamically equilibrium systems and the values obtained are only approximate for food systems. The RVP of a food can be determined by placing it in a closed chamber for a time sufficient to achieve apparent equilibrium (constant weight) and then measuring either pressure or relative humidity in the chamber. The vapor pressures of the aqueous solution of solutes are generally lower than that of pure water and aw hence falls in the range 0~1.

3.2. Temperature Dependence of aw aw is temperature dependent, and the modified Clausius-Clapeyron equation (3) can be used to precisely present its relationship with the absolute temperature: d ln aw d (1/ T )

H R

(3)

where, T is the absolute temperature, R is the gas constant, and ⊿H is the isosteric net heat of sorption at the water content of the sample. By rearrangement, equation (2-4) can be obtained: Inaw

k

H R

1 T

(4)

where, R and T have the same meaning as those in Equation (2-3), ⊿H is the latent heat of vaporization of pure water (40.5372kJ/mol), and k is calculated from the following formula:

20 k

Jianqian Kan and Guoqing Huang Absolute temperature of the sample - Absolute temperature of pure water in vapor pressure p Absolute temperature of pure water in vapor pressure p

Plots of Inaw versus 1/T are not always linear over broad temperature ranges, and they generally exhibit sharp breaks with the onset of ice formation. Figure 11. is a plot of logaw versus 1/T, illustrating that (a) the relationship is linear at subfreezing temperatures, (b) the influence of temperature on RVP is typically far greater at subfreezing temperatures than at above-freezing temperatures, and (c) a sharp break occurs in the plot at the freezing point of the sample. Below freezing temperatures, the water activity (aw) can be calculated as follow:

aw

p ff

pice

p0 ( SCW )

p0 ( SCW )

(5)

where, pff is the partial pressure of water in partially frozen food, p0(SCW) is the vapor pressure of pure supercooled water, and pice is the vapor pressure of pure ice. Two important distinctions should be noted when comparing aw values at above- and below-freezing temperatures. First, at above-freezing temperatures, aw is a function of sample composition and temperature, with the former factor predominating. At subfreezing temperatures, aw becomes independent of sample composition and depends solely on temperature; that is, in the presence of an ice phase aw values are not influenced by the kind or ratio of solutes present. Hence, the knowledge of aw at a subfreezing temperature cannot be used to predict aw at an above-freezing temperature. Second, as the temperature is changed sufficiently to form or melt ice, the meaning of aw, in terms of food stability, also changes. For example, in a product at -15°C (aw =0.86), microorganisms will not grow and chemical reactions will occur slowly. However, at 20°C and aw 0.86, some chemical reactions will occur rapidly and some microorganisms will grow at moderate rates.

Figure 11. Relationship between relative vapor pressure and temperature for a complex food above and below freezing [5].

Water

21

4. MOISTURE SORPTION ISOTHERM 4.1. Definition and Zones of Moisture Sorption Isotherm A plot of water content (expressed as mass of water per unit mass of dry material) of a food versus aw at constant temperature is known as a moisture sorption isotherm (MSI). The MSI of a food system is of great importance for the following reasons: 1. The ease of dehydration during concentration or drying is aw dependent; 2. The migration of water between materials during blending must be avoided; 3. It determines whether the determination of the moisture barrier properties of packaging materials is necessary; 4. It can be used to predict the water content that inhibits microbial growth; 5. It can be used to predicate food stability. Shown in Figure 12. is a schematic MSI for a high-moisture food plotted to include the full range of water content from normal to dry. Omission of the high-moisture region and expansion of the low-moisture region, as is usually done, yields an MSI that is much more useful (Figure 13). Several substances that have MSIs of markedly different shapes are shown in Figure 14. Isotherms with an S shape are characteristic of most foods. Foods such as fruits, confections, and coffee extract that contain large amounts of sugar and other small soluble molecules, and are not rich in polymeric materials exhibit a J-type isotherm shown as curve 1 in Figure 14.

Figure 12. Schematic moisture sorption isotherm encompassing a broad range of moisture contents [3].

22


Figure 13. Generalized moisture sorption isotherm for the low-moisture segment of a food (20°C) [3].

Figure 14. Resorption isotherms for various foods and biological substances. Temperature 20°C, except for number 1, which is 40°C: (1) confection (main component powdered sucrose), (2) spray-dried chicory extract, (3) roasted Columbian coffee, (4) pig pancreas extract powder, (5) native rice starch [6].

The MSI can be prepared in two ways. For high-moisture foods, the desorption isotherm can be obtained by plotting the water content versus aw during dehydration. For low-moisture foods, the resorption isotherm can be determined by plotting water content versus aw during addition of water to the foods. The shape and position of the isotherm are determined by several factors including sample composition, physical structure of the sample (e.g., crystalline or amorphous), sample pretreatments, temperature, and methodology To deeply understanding the meaning and usefulness of sorption isotherms it is sometimes appropriate to divide them into three zones as indicated in Figure 13.

Water

23

(1) Water present in Zone I of the isotherm is most strongly absorbed and least mobile. This water associates with accessible polar sites by water-ion or water-dipole interactions, is unfreezable at -40°C, has no ability to dissolve solutes, and is not present in sufficient amount to have a plasticizing effect on the solid. It behaves simply as part of the solid. The high-moisture end of Zone I (boundary of Zones I and II) corresponds to the ―Brunauer-Emmett-Teller (BET) monolayer‖ moisture content of the food. Zone I water constitutes a tiny fraction of the total water in a high-moisture food material. (2) Water added in Zone II occupies first-layer sites that are still available. This water associates with neighboring water molecules and solute molecules primarily by hydrogen bonding, is slightly less mobile than bulk water, and most of it is unfreezable at -40°C, it exerts a significant plasticizing action on solutes, lowers their glass transition temperatures, and causes incipient swelling of the solid matrix, leads to acceleration in the rate of most reactions. Water in Zones I and Zone II usually constitutes less than 5% of the water in a high-moisture food material. (3) Further addition of water (Zone III) causes a glass-rubber transition in samples containing glassy regions, a very large decrease in viscosity, a very large increase in molecular mobility, and commensurate increases in the rates of many reactions. This water is freezable, available as a solvent, and readily supports the growth of microorganism. Zone III water is referred to as bulk-phase water. The bulk-phase water of Zone III, either entrapped or free, usually constitutes more than 95% of the total water in a high-moisture food.

4.2. Hysteresis of MSI An MSI prepared by addition of water (resorption) to a dry sample will not necessarily be superimposable on an isotherm prepared by desorption. This lack of superimposability is referred to as ―hysteresis,‖ and a schematic example is shown in Figure 15. Typically, at any given p/p0, the water content of the sample will be greater during desorption than during resorption. MSIs of polymers, glasses of low molecular-weight compounds, and many foods exhibit hysteresis. The following explanations have been proposed for the occurrence of hysteresis: 1. Some moisture cannot be released during desorption due to the interaction with nonaqueous components. 2. Different vapor pressures are needed for evacuating or filling the moisture entrapped by capillaries. 3. The tissues of foods are changed during desorption. As a result, moisture cannot bind the same tightly to the tissues during resorption and higher aw is resulted in the same water content. 4. The magnitude of hysteresis, the shape of the curves, and the inception and termination points of the hysteresis loop can vary considerably depending on factors such as nature of the food, the physical changes it undergoes when water is removed or added, temperature, the rate of desorption, and the degree of water removal during desorption.

24

Jianqian Kan and Guoqing Huang Table 6. Water activity and growth of microorganisms in foods [7]

Range of aw

Inhibited Microorganisms

1.00–0.95

Pseudomonas, Escherichia Proteus, Shigella, Klebsiella, Bacillus, Clostridium perfringens, some yeasts

0.95–0.91

Salmonella, Vibrio parahaemolyticus, C. botulinum, Serratia, Lactobacillus, some molds, yeasts (Rhodotorula, Pichia)

0.91–0.87

Many yeasts (Candida, Torulopsis, Hansenula, Micrococcus)

0.87–0.80

Most molds (mycotoxigenic penicillia), Staphylococcus aureus, most Saccharomyces (bailii) spp., Debaryomyces

0.80–0.75 0.75–0.65

0.65–0.60

Most halophilic bacteria, mycotoxigenic aspergilli Xerophilic molds (Aspergillus chevalieri, A. candidus, Wallemia sebi),Saccharomyces bisporus Osmophilic yeasts (Saccharomyces rouxii), few molds (Aspergillus echinulatus, Monascus bisporus)

0.50

No microbial proliferation

0.40


0.30


0.20


Foods generally within this range Highly perishable (fresh) foods and canned fruits, vegetables, meat, fish, and milk; cooked sausages and breads; foods containing up to approximately 40% (w/w) sucrose or 7% sodium chloride Some cheeses (Cheddar, Swiss, Muenster, Provolone),cured meat (ham), some fruit juice concentrates; foods containing up to 55% (w/w) sucrose or 12% sodium chloride Fermented sausage (salami), sponge cakes, dry cheeses, margarine; foods containing up to 65% (w/w)sucrose (saturated) or 15% sodium chloride Most fruit juice concentrates, sweetened condensed milk, chocolate syrup, maple and fruit syrups; flour, rice, pulses containing 15–17% moisture; fruit cake; country-style ham, fondants, high-ratio cakes Jam, marmalade, marzipan, glacé fruits, some marshmallows Rolled oats containing approximately 10% moisture; grained nougats, fudge, marshmallows, jelly, molasses, raw cane sugar, some dried fruits, nuts Dried fruits containing 15–20% moisture; some toffees and caramels; honey Pasta containing approximately 12% moisture; spices containing approximately 10% moisture Whole egg powder containing approximately 5% moisture Cookies, crackers, bread crusts, etc. containing 3–5% moisture Whole milk powder containing 2–3% moisture; dried vegetables containing approximately 5% moisture; corn flakes containing approximately 5% moisture; country style cookies, crackers

Water

25

Figure 15. Hysteresis of moisture sorption isotherm [5].

Figure 16. Relationships among relative water vapor pressure, food stability and sorption isotherms. (A) Microbial growth versus p/p0. (B) Enzymatic hydrolysis versus p/p0. (C) Oxidation (nonenzymatic) versus p/p0. (D) Maillard browning versus p/p0. (E) Miscellaneous reaction rates versus p/p0. (F) Water content versus p/p0. All ordinates are ―relative rate‖ except for F [3].

26


5. WATER ACTIVITY AND FOOD STABILITY It has been widely recognized that aw is a much better indicator of food stability than water content. The data in Figure 16 and Table 6. provide examples of these relationships.

5.1. Water Activity (aw) and Growth of Microorganisms in Foods Shown in Table 6. are various common microorganisms and the range of aw permitting their growth. Most bacterial growth is affected above water activity 0.90 and most yeast and molds, however, can grow above water activity 0.80. No microorganisms survive in water activity lower than 0.5.

5.2. Water Activity (aw) and Chemical and Enzymatic Reactions in Foods The relationship between water activity and the rates of chemical and enzymatic reactions is very complex. First, water is a reactant of many chemical and enzymatic reactions and its content significantly influences the balance of the reactions. Second, water can bind to polar or ionic groups through hydration and significantly affects their contact with other reactants. Third, many biomolecules swell in the presence of water and more reaction sites are exposed, leading to accelerated reaction. However, high water contents dilute solutes and retard the proceeding of reactions. As shown in Figure 16, all chemical and enzymatic reactions, except oxidation reactions, have the lowest reaction rates at the boundary of Zone I and Zone II, corresponding aw 0.2~0.3.

5.3. Water Activity (aw) and Lipid Oxidation Figure 6.(c) indicates the relationship between lipid oxidation rate and aw. Within a specific aw range, the rate of lipid oxidation decreases with the increase of aw. When aw further increases to the boundary of Zone II and Zone III, the lipid oxidation rate starts to rise. Generally, the lowest lipid oxidation rate is found in aw 0.35. The moisture in food might either enhance or suppress lipid oxidation. When aw is lower than 0.35, lipid oxidation is suppressed for the following reasons. Firstly, water covers the oxidizable sites and prevents their contact with oxygen. Secondly, water hydrates ions and eliminates the oxidation reactions initiated by ions. Thirdly, water hydrogen bonds with hydroperoxides and retards the oxidation induced by them. Finally, water facilitates the binding between free radicals and disrupts the chain reactions involving the free radicals. When aw is greater than 0.35, water enhances lipid oxidation though two ways. Water dissolves solutes and facilities their movement. Meanwhile, biomolecules swell in high water content and more accessible sites are exposed.

27

Water

5.4. Water Activity (aw) and Maillard Reaction Figure 16(c) illustrates the influence of aw on Maillard reaction. It could be seen that Maillard reaction occurs mainly in aw range 0.3~0.7. In low aw, the water-solute hydrogen bonding and the association of water with neighboring molecules retard the movement of solutes and subsequently suppress the Maillard reaction. As the aw increases gradually, reactants and products move more easily and the rate of Maillard reaction increase as a result. When aw exceeds a specific value, solutes are diluted and the Maillard reaction is consequently retarded.

5.5. Calculation of BET Monolayer Value As shown in Figure 16, all the chemical enzymatic reactions, except oxidation, occur the slowest in the boundary of Zone I and Zone II (corresponding to aw 0.2~0.3) and further decrease of the water content does not change the minimum rates. The water content at the first-encountered rate minimum is the ―BET monolayer‖ water content. The BET theory is a theory proposed by Brunauer, Emett, and Teller in 1938 and the theory was named after them. The BET monolayer value of a food provides a good first estimate of the water content providing maximum stability of a dry product. One can use the BET equation to compute the monolayer value:

aw m(1 - aw )

1 m1c

c 1 m1c

(10)

where, aw is water activity, m is water content (in g H2O/g dry matter), m1 is the BET monolayer value, and c is a constant. From this equation, it is apparent that a plot of versus aw, known as a BET plot, should yield a straight line. An example for native potato starch, with aw replaced by p/p0, is shown in Figure 17. The linear relationship, as is generally acknowledged, begins to deteriorate at p/p0 values greater than about 0.35. The BET monolayer value can be calculated as follows:

Monolyaer value (m1 )

1 (y intercept) (slope)

(11)

From Figure 17, the y intercept is 0.6. Calculation of the slope from Figure 17 yields a value of 10.7. Thus,

m1

1 0.6 10.7

0.088g H2O/g dry matter

In this particular instance, the BET monolayer value corresponds to a aw, of 0.2.

28


Figure 17. BET plot for native potato starch (resorption data, 20°C) [8].

6. FREEZING AND FOOD STABILITY Although freezing is regarded as the best method of long-term preservation for most kinds of foods, the benefits of this preservation technique derive primarily from low temperature as such, not from ice formation. The formation of ice in cellular foods and food gels has two important adverse consequences: (1) All water converted to ice increases 9% in volume. Consequently, ice crystals formed in a disperse system can cause locally increased pressures, which can in turn cause mechanical damage. Hence water will leave the cells, which will shrink considerably; enzymes will release into solution, whereby they become active; this may result in a product of poor quality. (2) Nonaqueous constituents become concentrated in the unfrozen phase, the unfrozen phase changes significantly in properties such as pH, titratable acidity, ionic strength, viscosity, freezing point (and all other colligative properties), surface and interfacial tension, and oxidation reduction potential. In addition, solutes sometimes crystallize, supersaturated oxygen and carbon dioxide may be expelled from solution, water structure and water-solute interactions may be drastically altered, and macromolecules will be forced closer together, making interactions more probable. These changes in concentration-related properties often favor increases in reaction rates. Thus, freezing can have two opposing effects on reaction rate: lowering temperature, as such, will always decrease reaction rates, and freeze-concentration, as such, will sometimes increase reaction rates.

7. MOLECULAR MOBILITY AND FOOD STABILITY 7.1. Molecular Mobility In addition to water activity, molecular mobility (Mm) has also been used to predicate and control the stability of foods. Molecular mobility involves all the movements of food

Water

29

components during storage that are related to the stability and processability and includes: molecular movement or deformation caused by liquid movement or mechanical stretch; Brownian movements or atomic rotation caused by molecular diffusion; relative movement of materials in containers or pipelines. Some properties and behavioral characteristics of food that are dependent on Mm are shown in Table 7. Mm is mainly influenced by hydration and temperature. The water content and the interaction between water and nonaqueous components determine the fluidity of the liquid phase. As temperature is increased, the translational and rotational motion (Mm) becomes easier, while upon cooling to Tg, translational motion of polymer segments stop.

7.2. State Diagrams It is necessary to introduce the concept of state diagram before the discussion of the relationship between Mm and the stability of dried, partially dried, or frozen foods, State diagrams are supplemented phase diagrams, and contain equilibrium information as well as information on conditions of nonequilibrium and metastable equilibrium ―states‖, and are appropriate because foods that are dried, partially dried, or frozen do not exist in a state of thermodynamic equilibrium. A simplified temperature-composition state diagram for a binary system is shown in Figure 19. Table 7. Some properties and behavioral characteristics of foods that are governed by molecular mobility (diffusion-limited changes in products containing amorphous regions) [9] Dry or semidry foods Flow properties and stickiness Crystallization and recrystallization Sugar bloom in chocolate Cracking of foods during drying Texture of dry and intermediate moisture foods Collapses of structure during secondary (desorption) phase of freeze-drying Escape of volatile encapsulated in a solid, amorphous matrix Enzymatic activity Maillard reaction Gelatinization of starch Staling of bakery products caused by retrogradation of starch Cracking of bakery goods during cooling Thermal inactivation of microbial spores

Frozen foods Moisture migration (ice crystallization, formation of in-package ice) Lactose crystallization (―sandiness‖ in frozen desserts) Enzymatic activity Structural collapse of amorphous phase during sublimation phase of freeze-drying Shrinkage (partial collapse of foam-like frozen desserts)

30


Figure 19. State diagram of a binary system. Assumptions: maximal freeze concentration, no solute crystallization, constant pressure, no time dependence. Tml is the melting point curve, TE is the eutectic point, Tms is the solubility curve, Tg is the glass transition curve, and Tg‘is the solute-specific glass transition temperature of a maximally freeze concentrated solution. Heavy dashed lines represent conditions of metastable equilibrium. All other lines represent conditions of equilibrium [1].

Most foods are so complex that they cannot be accurately or easily represented on a state diagram. Differential scanning calorimetry (DSC) can successfully determine the glass transition temperature (Tg) of simple polymer systems, but is inapplicable to complex food systems. The Tg of complex food systems is often determined by using dynamic mechanical analysis (DMA) or dynamic mechanical thermal analysis (DMTA). Glass transition temperature (Tg) is the temperature at which a supersaturated solution (amorphous liquid) converts to a glass, and is dependent on solute type and water content. Tg‘ is a special Tg that applies only to samples containing ice, and only when ice has been formed so maximum freeze-concentration occurs (very slow cooling). Below Tg or Tg‘ of a complex sample, all but small molecules lose their translational mobility while retaining limited rotational and vibrational mobility. In the glassy state, the food will have greater stability (shelf life). As long as the temperature remain below Tg‘, the composition of the system is virtually fixed. This implies physical stability: crystallization, for instance, will not occur. But some chemical reactions may still proceed, albeit very slowly because of the high viscosity and the low temperature.

7.3. Molecular Mobility, State Diagram, and Food Properties 7.3.1. Reaction Rates and Molecular Mobility Mm is causally related to diffusion-limited properties of foods that contain, besides water, substantial amounts of amorphous, primarily hydrophilic molecules, ranging in size from monomers to polymers. Foods of this type include starch-containing foods, such as pasta, boiled confections, protein-based foods, intermediate-moisture foods, and dried, frozen, or freeze-dried foods. The utility of the Mm approach for predicting many kinds of physical changes has been reasonably well established. However, situations do exist where the Mm

Water

31

approach is of questionable value or is clearly unsuitable. Some examples are (1) chemical reactions whose rates are not strongly influenced by diffusion, (2) desirable or undesirable effects achieved through the action of specific chemicals (e.g., alteration of pH or oxygen tension), (3) situations in which sample Mm is estimated on the basis of a polymeric component (Tg of polymer) and where Mm of small molecules that can penetrate the polymer matrix is a primary determinant of the product attribute of interest, and (4) growth of vegetative cells of microorganisms (p/p0 is a more reliable estimator than Mm). Examples of diffusion-limited reactions are proton transfer reactions, radical recombination reactions, acid-base reactions involving transport of H+ and OH-, many enzyme-catalyzed reactions, protein folding reactions, polymer chain growth, and oxygenation/deoxygenation of hemoglobin and myoglobin. At constant temperature and pressure, three primary factors govern the rate at which a chemical reaction will occur: a diffusion factor, D (to sustain a reaction, reactants must first encounter each other), a frequency-of-collision factor, A (number of collisions per unit time following an encounter), and a chemical activation-energy factor, Ea (once a collision occurs between properly oriented reactants the energy available must be sufficient to cause a reaction, that is, the activation energy for the reaction must be exceeded). For a reaction to be diffusion-limited, it is clear that factors A and Ea must not be rate-limiting. Diffusion-limited reactions typically have low activation energies (8–25 kJ/mol). When a food is cooled and/or reduced in moisture content so that all or part of it is converted to a glassy state, Mm is greatly reduced and diffusion-limited properties become stable.

7.3.2. Free Volume and Molecular Mobility The free volume of a food system decreases as the temperature decreases and the translational and rotational motion become more difficult, which affects the motion of the segments and local viscosity of polymers. When the temperature decreases to below Tg, the free volume decreases significantly and the translational motion of polymer segments stops. Hence, foods have stable diffusionlimited properties in temperatures below Tg. The increase of free volume, which is often unexpected, can be achieved by adding small molecular-weight solutes such as water or by increasing the temperature. Both the two practices improve the translational motion of solutes and are not beneficial for food stability. However, this relationship is applicable only to certain food systems and free volume cannot be used as a quantitative indicator of food stability to present. 7.3.3. Moisture Content and Tg The moisture content has special effect on the Tg of food systems. The Tg of water is as low as -135°C and water is a strong plasticizer. First, water has a much smaller size and moves more easily than other solutes such as polysaccharides, proteins, and lipids. The ease of motion provides space required for the movement of segments. Second, water interacts with the polar groups on other components and replaces partial inter-molecular or intra-molecular hydrogen bonds, which decreases the rigidity of the components and consequently reduces the Tg. Generally, the increase of moisture content by 1% decreases the Tg by 5~10°C.

32


It should be noted that the plasticizing effect is valid only when water gets entrapped in the amorphous region of the components. In the absence of environmental effects, moisture content is the predominant factor that affects Tg, especially in low-moisture foods. Table 8. Relationship between the Tg and moisture content of pre-gelatinized starch and wheat starch Pre-gelatinized starch Moisture content Tg/°C 0.153 62 0.166 53 0.181 40 0.222 28 0.247 25

Native wheat starch Moisture content Tg/°C 0.151 90 0.164 67 0.178 59 0.221 40 0.256 33

For example, the Tg of the anhydrous mixture of 50% starch and 50% sucrose is about 60°C; when the moisture content increases to 2%, the Tg decreases to 20°C; when the moisture content further increases to 6%, Tg falls to as low as 10°C. Table 8. lists the relationships between the Tg and moisture content of native wheat starch and pre-gelatinized starch. It could be seen that the Tg of both the materials increases along the decrease of moisture content.

7.3.4. Carbohydrates, Proteins, and Tg Carbohydrates and proteins are major constituents in many foods and their presence and contents markedly influence the Tg of foods. Besides, the sizes of the components also affect the Tg. Generally, carbohydrates or proteins with higher average molecular weight have more compact structure, higher viscosity, lower free volume, and consequently higher Tg. Table 9. lists the Tg of maltodextrin with different DE and concentration. It is seen that Tg decreases as DE increases in the case of same moisture content. Generally, Tg is dependent on solute type and water content, while Tg‘ is solely solute type dependent. For glycosides and polyols with molecular weight less than 1200, Tg or Tg‘ increases as molecular weight rises. When the average molecular weight exceeds 3000 (DE of 6 or more for starch hydrolysis products), g becomes independent of MW, as shown in Figure 25. An exception occurs when biomolecules are present in the entanglement networks form. In this case, g continues to rise with increasing MW. Most biomolecules, including starch, maltodextrin, cellulose, hemicellulose, carboxymethyl cellulose, glucan, xanthan gum, gluten, glutenin, gliadin, zein, collagen, elastin, keratin, albumin, globulin, casein, and gelatin, have similar glass transition curve and Tg‘, which approaches -10°C.

7.3. Mm, State Diagram and Food Stability Knowledge on the relationship between Tg or Tg‘ and food components is of great importance for the processing and storage of the foods.

33

Water Table 9. Tg of maltodextrin with different Des DE5 Moisture content 0.00 0.02 0.04 0.11 0.18

Tg/°C 188 135 102 44 23

DE10 Moisture content 0.00 0.02 0.05 0.10 0.19

Tg/°C 160 103 84 30 -6

DE15 Moisture content 0.00 0.02 0.05 0.11 0.20

Tg/°C 99 83 65 8 -13

Figure 25. Relationship between average molecular weight and dextrose equivalent (DE) of commercial starch hydrolysis products with Tg‘ [10].

7.3.1. Temperature, Mm, and Food Stability Good correlation exists among temperature and Mm or viscosity for foods containing amorphous regions in temperature range 10~100°C. Most molecules are present in the glassy or rubbery state in temperatures below Tg or Tg‘. In this case, the motion of food components is restricted and food stability is increased. 7.3.2. Food Stability Predication Based on State Diagram The approximate stability of foods can be predicated according to the state diagram. When foods are stored in temperatures lower than Tg or Tg‘, the diffusion of molecules is restricted and the shelf life is markedly prolonged. In contrast, the foods are susceptible to spoilage in temperatures higher than Tg or Tg‘. Hence, temperatures below or approaching Tg or Tg‘ should be preferred during food storage. Generally, Mm is more effective in predicating diffusion-limited properties, such as the physiochemical properties of frozen foods and optimum freeze-drying conditions. However, aw is more effective in predicating non-diffusion-limited properties and microbial growth of

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ice-free foods. Because aw can be measured conveniently and quickly, it is still the major indicator for judging food stability.

REFERENCES Fennema, OR. Food Chemistry. 3rd edition. New York: Marcel Dekker; 1996. Damodaran, S; Parkin, KL; Fennema, OR. Fennema’s Food Chemistry. 4th edition. New York: CRC Press; 2007. [3] Belitz, HD; Gorsch, W. Food Chemistry. 2nd edition. Berlin: Springer-Verlag, 1999. [4] Berendsen, HJC. Specific interactions of water with biopolymers. In: Franks, F. Water A Comprehensive Treatise. New York: Plenum Press, 1975; 293-349. [5] Fennema, OR. Enzyme kinetics at low temperature and reduced water activity. In: Crowe, JH; Clegg, JS. Dry Biological Systems. New York: Academic Press, 1978; 297322. [6] Ferry, JD. The evaluation of water activity in aqueous solutions from freezing point depression. International Journal of Food Technology, 1980, 16, 21-30. [7] Beuchat, LR. Microbial stability as affected by water activity. Cereal Foods World, 1981, 26, 345-349. [8] Van den Berg, C. Vapour Sorption Equilibria and Other Water-Starch Interactions; A Physico-Chemical Approach, PhD thesis, Wageningen: Wageningen Agricultural University, 1981. [9] Slade, L; Levine, H. Beyond water activity: Recent advances based on an alternate approach to the assessment of food quality and safety. Critical Reviews in Food Science and Nutrition. 1991, 30, 115–360. [10] Levine, H., and L. Slade (1986). A polymer physico-chemical approach to the study of commercial starch hydrolysis products (SHPs). Carbohydrate Polymers, 6, 213–244. [1] [2]



Chapter 3

CARBOHYDRATES

1

Dongfeng Wang1, Jipeng Sun2, Guoqing Huang1,3, Xiaolin Zhou4 and Liping Sun5 College of Food Science and Engineering, Ocean Universityof China, Qingdao, China 2 Third Institute of Oceanography, State Oceanic Administration, Xiamen, China 3 College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, China 4 Department of Biology, Shantou University Medical College, Shantou, China 5 College of Chemical Engineering, Kunming University of Science and Technology, Kunming, China

ABSTRACT Carbohydrates account for 3/4 of the dry weight of terrestrial plants and algae and can be found in all the plants, animals and microorganisms that human can eat. Carbohydrates are one of the major components in foods. The compounds not only provide human beings with energy, but also impart foods with desired textures and tastes. Carbohydrates undergo various changes during food processing and storage and yield substantial compounds that affect the flavor, quality and safety of foods. This chapter deals with the classification of carbohydrates and their most important functional properties, in which, special attention is paid to the non-enzymatic browning reaction, its influences on food quality, and its control. The structures, proportions, and applications of most important polysaccharides and oligosaccharides in foods are then detailed one by one. Dietary fiber as an important healthy diet component is also introduced at the end of this chapter.

36

Dongfeng Wang, Jipeng Sun, Guoqing Huang et al.

INTRODUCTION Carbohydrates Classification Carbohydrates are natural organic compounds converted from carbon dioxide and water through photosynthesis of plants. According to the number of monosaccharide units, carbohydrates are divided into monosaccharides, oligosaccharides and polysaccharides. Monosaccharides are the simplest sugars in structure and can no longer be hydrolyzed. A monosaccharide often contains three, four, five, or six carbon atoms and its functional group might be the aldehyde or keto group. An oligosaccharide generally consists of 2 to 20 monosaccharide units and can be hydrolyzed to simple sugars. Oligosaccharides are often found in glycoproteins or lipopolysaccharides. According to monosaccharide composition, oligosaccharides are further divided into homo-oligosaccharides and hetero-oligosaccharides. A homo-oligosaccharide is composed of only one type of monosaccharide, such as maltose and dextrin with degree of polymerization less than 20. In contrast, a hetero-oligosaccharide consists of two or more types of monosaccharide units. Polysaccharides, with degree of polymerization greater than 20, are formed by dehydration of multiple monosaccharide units and consist of homo-polysaccharides, such as cellulose and starch, and hetero-polysaccharide such as seaweed and tea polysaccharides. Polysaccharides can also be classified into plant, animal and microbial polysaccharides according to their origins or storage and functional polysaccharides according to their biological functions. Polysaccharides contain multiple hydroxyl groups and can covalently attach to the side chains of proteins or peptides to form glycoproteins or protein polysaccharides. Polysaccharides also react with carboxyl-containing molecules to form esters such as lipopolysaccharide (LPS) and sulfate polysaccharides. Besides, polysaccharides can complex with transition metals due to the presence of hydroxyl groups. These polysaccharide derivatives are generally referred to as polysaccharide complexes.

Carbohydrates in Foods Starch is one of the most abundant carbohydrates in plant-derived foods and is the most abundant in seeds, roots and tubers. Glycogen is found in animal-derived foods especially in muscle and liver, and it is structurally similar to amylopectin. Starch is insoluble in aqueous solutions and does not contribute to the sweetness of foods, unless it was hydrolyzed into oligosaccharides or glucose. The majority of plant-derived foods contain only a small amount of free sugars and most sugars are present as starch. For example, maize contains only 0.2%~0.5% D-glucose, 0.1%~0.4% D-fructose and 1%~2% sucrose. Free sugars not only provide the sweet taste for foods, but also participate in flavor and color formation during thermal processing. An increase in free sugar content during processing improves food quality. For example, to increase the sweetness of sweet corn, sweet corn must be harvested before sugars are converted to starch. Many fruits are often harvested before they are fully mature for two reasons. Firstly, the high rigidity of immature fruits facilitates their transport and storage. Secondly, starch is

Carbohydrates

37

converted to sucrose or other sweet sugars during transport and storage. This change makes the fruits sweet and soft. The post-harvest ripening is the reverse of the starch synthesis process in the grain, tuber and root of plants. The contents of water-soluble sugars in processed foods are generally higher than the corresponding materials, because sugars are intentionally added by manufacturers to meet the requirements of consumers on flavor and color.

Carbohydrates and Food Quality Carbohydrates are the main constitutes in foods and are closely related to the nutrition, color, taste, texture and functionality of foods. Carbohydrates are essential nutrients for human body and 70% of the energy needed by human body is provided by carbohydrates. Reducing sugars with free aldehyde or ketone contribute to the formation of colors and flavors in food thermal processing and thus affects food quality. Besides, many free sugars are sweet and positively affect the mouth feel of foods. Some carbohydrates, such as guar gum and carrageenan, have special viscoelastic properties and can give pleasant textures for foods. Cellulose, pectin, and many other macromolecules can provide desired food texture and adjust the intestine flora as dietary fibers. In addition, some polysaccharides or oligosaccharides, such as lentinan and tea polysaccharides, have specific physiological functions and can be directly added to foods as functional ingredients.

PHYSICOCHEMICAL PROPERTIES AND FUNCTIONS OF CARBOHYDRATES Carbohydrate Structure Monosaccharide Monosaccharide generally contains 5 or 6 carbon atoms with the general formula Cn(H2O)n. Monosaccharides are asymmetric and optically active. Taking glyceraldehyde as an example, the C atom in position 2 is chiral and glyceraldehyde therefore has two enantiomers. D-glyceraldehyde is dextrorotatory and is distinguished by the prefix ―+‖ or ―d‖ and Lglyceraldehyde is levorotatory and is labeled with prefix ―-‖ or ―l‖. Monosaccharides derived from D-glyceraldehyde are termed D-ketones and those from L-glyceraldehyde are termed Lketones. The carboxyl group of a monosaccharide ring can react with a free hydroxyl group in the same molecule to yield more stable 5- or 6-membered hemiacetal or hemiketal, called lactol. Lactol formation provides a new chiral center. Thus, there are two additional diastereomers for each pyranose or furanose. These isomers are called anomers and are denoted as α- or βforms. Most naturally occurring simple sugars are present in the D form. Hence, it is sometimes unnecessary to indicate the configuration of these sugars. Monosaccharides can produce various biologically important derivatives after chemical modification. Monosaccharide derivates that have been identified in foods include

38


monosaccharide phosphates, deoxy monosaccharides, glucosamines, aldonic acids, uronic acids, saccharic acids, ascorbic acid, sugar alcohols, myo-inositol, glycosides, etc.

Sugar Alcohol, Inositol and Glycoside Sugar alcohols are the hydrogenated products of monosaccharides and are also known as polyols. Most sugar alcohols are the reducing products of their corresponding monosaccharides except mannose, which occurs naturally in algae with high contents. Most sugar alcohols are white crystals and soluble in water. Table 3-1. Isomers of inositol

39

Carbohydrates

Sugar alcohols have lower calorific values and are sweeter than their monosaccharide precursors. Sugar alcohols do not undergo the typical reactions of sugars and are stable against heating and pH variations. Sugar alcohols share the same chemical properties as common alcohols and are not involved in the Maillard reaction. Inositol is a cyclic hexatomic alcohol and has 9 stereoisomers (Table 3-1), of which, 7 are mesomeric and 2 are optically active. Among the isomers, only the myo isomer is biologically active. Inositol is often present as free form in the muscle, heart, liver, and lung of animals. The hydroxyl groups in inositol can react with phosphate acid to produce phosphoinositides. In higher plants, all the six hydroxyl groups in inositol are phosphated to inositol hexaphosphate. Phosphoinositides can complex with Ca2+ and Mg2+, forming calcium and magnesium salts of phytic acid. Glycosides are the condensation products of monosaccharides and non-saccharide ligand through the hemiacetal hydroxyl of monosaccharide. The bond between sugar and ligand is referred to as the glycosidic linkage. Glycosides contain one furanose or pyranose ring and the new chiral center might be present in the alpha or beta form. Most glycosides occur in the beta form in nature.

Oligosaccharides Oligosaccharides are water-soluble and occur widely in nature. Generally, naturally occurring oligosaccharides contain less than six monosaccharide units, of which, most are disaccharides and trisaccharides. For example, sucrose and maltose are disaccharides, and affinose is a trisaccharide. Some high-molecular weight oligosaccharides, such as cyclodextrins (or schardinger dextrin), have gained wide applications in the food industry. Cyclodextrins consist of 6~8 Dglucopyranose units, corresponding to α-, β-, and γ- cyclodextrins respectively. In addition to molecular weight, the three cyclodextrins differ in their water solubility and cavity size, as shown in Table 3-2. X-ray diffraction reveals that α-cyclodextrin is a highly symmetric cylinder. Six C6 hydroxyl groups are located in the bottom of the cylinder and 12 C2 and C3 hydroxyl groups are arranged on the cylinder top. The inner wall of the cylinder is covered with C-H groups and hence more hydrophobic than the external surface. Cyclodextrins are used to stabilize hydrophobic substances by entrapping them in the cavity during food processing. Table 3-2. Chemical and physical properties of cyclodextrins Item Number of glucose residues Molecular weight Solubility in water at 25°C (g/l) Optical rotation Inner diameter of cavity (nm) Cavity height (nm)

α-cyclodextrin 6 972 145 +150.5 0.57 0.67

β-cyclodextrin 7 1135 18.5 +162.5 0.78 0.70

γ-cyclodextrin 8 1297 232 +174.4 0.95 0.70

40


Polysaccharides Structure The degrees of polymerization (DP) of polysaccharides range from 21 to several thousands. Polysaccharides consist of one type of structural unit (homoglycans) or multiple types of structure units (heteroglycans) and the structural units can be linked in a linear (such as cellulose and amylose) or branched pattern (such as amylopectin and glycogen). The monosaccharide units generally occur periodically in polysaccharides and each periodic repeat contains one or more alternative structural units. An example of the exceptions is the carbohydrate components in glycoproteins, in which the monosaccharide compositions are nonperiodic all along the chain. The DPs of polysaccharides are heterogeneous, that is, polysaccharides have no fixed molecular weight (MW) and display the Gauss distribution. The heterogeneity of polysaccharide molecular weight is associated with the metabolic status of organisms. For example, the MW of glycogen is closely dependent on the blood glucose level of animals. When the blood glucose level is low, the liver glycogen is hydrolyzed and glycogen is cleaved to small segments. In contrast, when the blood glucose level increases, glycogen is synthesized in the liver and the glycogen MW is increased. In addition, many polysaccharides are present as complexes, such as glycoproteins, glycopeptides and glycolipids. In this case, the MWs of polysaccharides are determined by much more factors than polysaccharide alone. Conformation Polysaccharides are either straight or branched molecules, but they have much more complex conformations. Some typical conformations are elucidated in the following by taking glucans and some other polysaccharides as examples. Extended or stretched ribbon-type conformation This conformation is typical for 1,4-linked β-D-glucopyranosyl residues (Figure 3-1), for instance that in cellulose fibers. This formula shows that the stretched chain conformation is due to the zigzag geometry of monomer linkages involving oxygen bridging. The chain may be shortened or compressed to enable formation of H-bonds between adjacent resides and thus contribute to conformational stabilization. In this type of conformation, if the number of monomers in turn is denoted as n and the pitch (advancement) in the axial direction per monomer unit is denoted as h, n ranges from 2 to ±4 and h equals the length of a monomer unit. Thus, the chain given in Figure 3-2(a) has n value of -2.55 and h value of 5.13 Å. A strongly plated ribbon-type conformation might also occur, as shown by a segment of a pectin chain (1,4-linked α-D-galactopyranosyl-uronate units) and an alginate chain (1,4linked α-L-gulopyranosyluronate units), as shown in Figure 3-3.

Figure 3-1. Conformation of 1, 4-linked β-D-glucopyranosyl residue.

41

Carbohydrates

Figure 3-2. Conformations of some β-D-glucans. Linkages: a 1→4, b 1→3, c 1→2.

(a) Peetin

(b) Alginate

Figure 3-3. Plated ribbon-type conformation of pectin and alginate.

Since alginate contains multiple oxygen atoms, it can complex with many transition metals. As shown in Figure 3-3(b), Ca2+ stabilizes the conformation of alginate. In this case, two alginate chains are assembled in a conformation which resembles an egg box, which is referred as the egg box type of conformation (Figure 3-4). Hollow helix-type conformation This conformation is typical for 1,3-linked β-D-glucopyranose units and occurs in the polysaccharide lichenin, for example, as shown in Figure 3-5 (a). The formula shows that the helical conformation of the chain is imposed by a U-form geometry of the monomer linkages. Amylose (1,4-linked α-D-glucopyranosyl residues) also has such a geometry, and hence a helical conformation (Figure 3-5 (b)).

Figure 3-4. Sketch map of egg box type of conformation.

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(a) Lichenin.

(b) Amylose

Figure 3-5. Conformation of lichenin and amylase.

a

b

c

Figure 3-6. Stabilization of helical conformations. a, clathrate compounds; b, coiled double or triple helices; c, nesting.

The number(n) of monomers per turn and the pitch in the axial direction per residue (h) might differ significantly in this conformation. The value of n varies from 2 to ±10, while h can be near its limit value of 0. The conformation of a β(1-3)-glucan, with n value of 5.64 and h value of 3.16 Å, is shown in Figure 3-2(b). The helical conformation can be stabilized in various ways. When the helix diameter is large, inclusion (clathrate) compounds can be formed (Figure 3-6(a)). More extended or stretched chains, with smaller helix diameter, can form double or triple stranded helices (Figure 3.6(b)), while strongly-stretched chains, in order to stabilize the conformation, have a zigzag, plated association and not stranded (Figure 3-6(c)). Crumpled-type conformation This conformation occurs with, for example, 1,2-linked β-D-glucopyranosyl residues (Figure 3-2 (c)). This is due to the wrinkled geometry of the monomer O-bridge linkages. Here, the n value varies from 4 up to −2 and h is 2–3 Å. The conformation reproduced in Figure 3-2 (c), the n = 2.62 and h = 2.79 Å. The likelihood of such a disorderly form associating into more orderly conformations is low. Polysaccharides of this conformational type play only a negligible role in nature. Loosely-jointed conformation This is typical for glycans with 1,6-linked β-D-glucopyranosyl units, because they exhibit a particularly great variability in conformation. The great flexibility of this glycan-type conformation is based on the nature of the connecting bridge between the monomers. The bridge has three free rotational bonds and, furthermore, the sugar residues are further apart.

Carbohydrates

43

Figure 3-7. Conformations of β-D-galactopyranosyl-4-sulfate and 3,7-anhydro-α-D-galactopyranosyl-2sulfate residues in ι-carrageenan.

Figure 3-8. Biosynthesis of ι-carrageenan.

Conformations of heteroglycans The examples considered so far have demonstrated that a prediction is possible for a homoglycan conformation based on the geometry of the bonds of the monomer units which maintain the oxygen bridges. It is more difficult to predict the conformation of a heteroglycan with a periodic sequence of several monomers, which implies different types of conformations. Such a case is shown by ι-carrageenan, in which the β-D-galactopyranosy l-4sulfate units have a U-form geometry, while the 3,6-anhydro-α-Dgalactopyranosyl-2-sulfate residues have a zigzag geometry (Figure 3-7). Calculations have shown that conformational possibilities vary from a shortened, compressed ribbon band type to a stretched helix type. X-ray diffraction analyses have proved that a stretched helix exists, but as a double stranded helix in order to stabilize the conformation. Interchain interactions As mentioned above, the periodically arranged monosaccharide sequence in a polysaccharide can be interrupted by nonperiodic segments. Such sequence interferences result in conformational disorders. This will be explained in more detail with ι-carrageenan.

44


Initially, a periodic sequence of altering units of β-D-galactopyranose-4-sulfate (Figure 3-8, I) and α-D-galactopyranose-2,6-disulfate (Figure 3-8, II) is built up in carrageenan biosynthesis: When the biosynthesis of the chain is complete, an enzyme-catalyzed reaction eliminates sulfate from most of α-D-galactopyranose-2,6-disulfate (Figure 3-7, II), transforming the unit to 3,6-anhydro-α-D-galactopyranose-2-sulfate (Figure 3-7,III). This transformation is associated with a change in linkage geometry. Some II-residues remain in the sequence, acting as interference sites. While the undisturbed, ordered segment of one chain can associate with the same segment of another chain, forming a double helix, the nonperiodic or disordered segments cannot participate in such associations (Figure 3-9). In this way, a gel is formed with a three dimensional network in which the solvent is immobilized. The gel properties, e. g., its strength, are influenced by the number and distribution of α-D-galactopyranosyl-2,6-disulfate residues, i.e. by a structural property regulated during polysaccharide biosynthesis. The example of the ι-carrageenan gel-building mechanism, involving a chain–chain interaction of sequence segments of orderly conformation, interrupted by randomly-coiled segments corresponding to a disorderly chain sequence, can be applied generally to gels of other macromolecules. Besides a sufficient chain length, the structural prerequisite for gel-setting ability is interruption of a periodic sequence and its orderly conformation. The interruption is achieved by insertion into the chain of a sugar residue of a different linkage geometry (carrageenans, alginates, pectin), by a suitable distribution of free and esterified carboxyl groups (glycuronans) or by insertion of side chains. The inter-chain associations during gelling (network formation), which involve segments of orderly conformation, can then occur in the form of a double helix (Figure 3-10(a)); a multiple bundle of double helices (Figure 3-10(b)); an association between stretched ribbon-type conformations, such as an egg box model (Figure 3-10(c)); some other similar associations (Figure 3-10(d)); or, lastly, forms consisting of double helix and ribbon-type combinations (Figure 3-10(e)).

Figure 3-9. Schematic representation of a gel setting process.

Carbohydrates

45

Figure 3-10. Interchain aggregation between regular conformations. a Double helix. b double helix bundle. c egg-box. d ribbon–ribbon. e double helix, ribbon interaction.

Physiochemical Properties Solubility Most monosaccharides, such as sugar alcohols, glycosides, and oligosaccharides, are water soluble. At 20°C, up to 195 g of sucrose can dissolve in 100 g of water. The solubility of sugar alcohols varies significantly with species. For example, sorbitol has a higher solubility than sucrose and reaches up 220 g per 100g water, while that of mannitol, erythritol and isomaltitolto is only 17, 50, and 100 g per 100g water, respectively. Sugar alcohols absorb much more heat than sugars when dissolution and thus produce cooling sensation in the mouth. Sugar alcohols are added to candies and chewing gums to provide the cooling sensation. The solubility of glycosides is closely correlated with their ligands. Generally, glycosides are more water soluble than corresponding ligands. For example, flavonoids are usually insoluble, but the corresponding glycosides are soluble. Flavonoids provide foods with different colors and tastes in the soluble glucoside form. Each sugar unit in polysaccharides contains an average of three hydroxyl groups and each hydroxyl group can hydrogen bond with one or more water molecules. In addition, the oxygen atoms in the sugar ring and glycosidic bond can also form hydrogen bonds with water. Therefore, monosaccharide units in polysaccharides can be completely solvated and most polysaccharides have strong water-holding capabilities and are highly hydrophilic. Polysaccharides in foods affect the movement of water and significantly influence the functional properties of foods. The presence of polysaccharides does not increase the penetrability or significantly reduce the freezing point of water, although polysaccharides can be solvated by water. Therefore, polysaccharides are good frozen stabilizers. Taking starch solution as an example, when a starch solution is frozen, a two-phase system is formed, of which, one phase is composed of crystal water and another phase is in the glass state consisting of 70% starch and 30% unfrozen water. Due to the extremely high polysaccharide concentration in the glassstate phase, the viscosity is high and the movement of unfrozen water in the glass-state phase is restricted. In addition, polysaccharides are concentrated in low temperatures. In this case,

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the mobility of water is further restricted and water molecules can no longer adsorb to crystal nucleus or the active sites for crystal growth. Both high and low molecular weight polysaccharides can effectively protect food texture and structure from being damaged during frozen storage. Some polysaccharides occur in a highly ordered form. In these molecules, the chains are tightly bound to form crystals. Because the number of exposed hydroxyl groups is significantly reduced, these polysaccharides are insoluble in water unless the inter-chain hydrogen bonds are broken. Taking cellulose as an example, the structural unit β-Dglucopyranosyl residues are arranged orderly and linearly along the chain and form interchain hydrogen bonds with parallel chains. The crystal regions of cellulose are not water soluble and are very stable. The majority of carbohydrates is not present as crystals and readily dissolves or swells in water. Water-soluble polysaccharides and their derivates used in the food industry are often termed gums or hydrophilic colloids. The solutions of most macromolecular polysaccharides are viscous and the viscosity depends on the molecular size, shape, net charge and conformation in solution. The thickening and gelling properties of polysaccharides significantly affect the quality of foods.

Hydrolysis Hydrolysis of Glycosides Though glycosides occur in low contents in foods, they impart important physiological effects and functionality to foods. For example, nature saponins are strong foaming agent and stabilizer, flavonoids produce bitterness and color for foods. In addition to a small amount of sweet glycosides such as stevioside and osladin, most glycosides taste bitter or astringent, particularly when the ligand is larger than methyl. When glycosides are hydrolyzed, their solubility is reduced and the bitterness or astringency is alleviated. O-glycoside bonds are stable in neutral and weak alkaline solutions, but easily hydrolyzed in acidic conditions. Most glycosides in foods (except for strong acidic foods) are stable. In the enzymatic hydrolysis of glycosides, the sugar component is transformed to highly active half-chair conformation and the glycosidic bond is weakened. Then, a proton is transferred from the enzyme to an oxygen atom in glycoside. When the oxygen atom is separated from the carbon atom, a positively-charged carbon ion is generated. This carbon ion then reacts with the negatively-charged -COO- group in the enzyme and is temporarily stabilized until it is completely hydrolyzed by reacting with the -OH- group in solvent. N-glycosidic bond is not as stable as O-glycosidic bonds and is susceptible to hydrolysis in water. For example, glycosylamines are unstable in water and can be hydrolyzed to colored products through a series of reactions. These reactions are the main reason of Maillard reaction initiation (to be discussed in Section 3.2.4). Thioglycosides, which contain S-glycosidic bonds, occur naturally in mustard and horseradish and are very stable and water-soluble. Thioglycosides can be hydrolyzed by thioglucosidase to produce isothiocyanates, as shown in Figure 3-11. Cyanogenic glycosides are another category of glycosides that significantly affect food safety. Cyanogenic glycosides are widely present in apricot, cassava, sorghum, bamboo, and lima beans and can yield toxic hydrocyanic acid upon hydrolysis. Amygdalin and

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mandelonitrile are the most important cyanogenic glycosides. The complete hydrolysis of amygdalin generates D-glucose, benzaldehyde, and hydrocyanic acid (Figure 3-12). Table 3-3 lists the main thio-glycosides and their hydrolysates. Excessive intake of cyanogenic glycosides leads to cyanide poisoning. In addition to enzyme activity and environmental acidity, the hydrolysis of glycosides is also affected by glycodic bond conformation, substitution of the sugar ring, and sugar ring size. Generally, glycosides with β glycodic bond are hydrolyzed faster than those with α glycodic bond. Substitution on the sugar ring reduces the hydrolysis rate and furanosides are hydrolyzed faster than corresponding pyranosides (Table 3-4). The hydrolysis rate of glycosides increases rapidly with elevated temperature, as shown in Table 3-4. Hydrolysis of oligosaccharides and polysaccharides Similar to glycosides, oligosaccharides are hydrolyzed easily by acid and enzymes and are stable in alkaline solutions. Sucrose can be hydrolyzed by acid to yield equamolar mixture of glucose and fructose that is called invert sugar.

Figure 3-11. Hydrolysis of thioglycosides by thio-glucosidase.

Figure 3-12. Hydrolysis of amygdalin.

Table 3-3. Main thioglycosides found in foods and their hydrolysates Glycoside Amygdalin and prunasin Linamarin Vicianoside Linarin

Occurrence Almond kernel and dried alpinia japonica Lima bean, linseed (flax), cassava Tare Sieve (black bean) and Chickpea horsebean

Hydrolysates D-Glucose + hydrocyanic acid + benzaldehyde D-Glucose + hydrocyanic acid + acetone Vicianose + hydrocyanic acid + benzaldehyde D-Glucose + hydrocyanic acid + acetone (to be confirmed)

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Dongfeng Wang, Jipeng Sun, Guoqing Huang et al. Table 3-3. (Continued) Glycoside Lotaustralin Dhurrin Sinigrin Glocoside

Occurrence Arabicus of lotus Broomcorn and corn Black mustard Brassicaceae

Hydrolysates D-Glucose + hydrocyanic acid + lotoflavin D-Glucose + hydrocyanic acid + salicylide D- Glucose + propyl isorhodanide + KHSO4 D-Glucose+ 5-ethenyl-2-oxazolidinethione, or goitrogen+ KHSO4

Table 3-4. Effect of temperature on the hydrolysis rate of glucosidesa Ka 70°C Methyl-α-D-pyran-glycoside 2.82 Methyl-β-D-fructofuranoside 6.01 a Note: First order reaction rate constant, ×106 sec-1. Glycosides (in 0.5mol/L H2SO4)

80°C 13.8 15.4

93°C 76.1 141.0

Polysaccharides are also prone to acidic or enzymatic hydrolysis, accompanying reduced viscosity and increased sweetness. In the food industry, α-amylase and glucose glucoamylase have been widely used in corn starch hydrolysis to produce D-glucose. The hydrolysate is further treated by D-glucose isomerization to yield a balanced mixture of 54% D-glucose and 42% D-fructose, which is known as fructose syrup. This mixture has replaced sucrose as a low-cost sweetener in many foods.

Oxidation Carbohydrates with reducing free aldehydes or ketoses that can be converted to aldehyde groups can be oxidized into aldonic acid in the presence of weak oxidants in alkaline conditions. When strong oxidants are present, both the aldehyde and primary hydroxyl of aldoses are oxidized to carboxyl and aldaric acids are produced as a result [3, 18].

Figure 3-13. Oxidation of D-glucose by glucose oxidase.

Some enzymes catalyze the oxidation of aldoses. For example, dehydrogenases oxidize the primary hydroxyl of some aldoses to produce uronic acids. D-glucuronic acid, Dgalacturonic acid, and D-manuronic acid are the components of many heteropolysaccharides. D-glucose can be oxidized to D-gluconic acid by glucose oxidase. Figure 3-13 illustrates the preparation of D-gluconic acid and gluconolactones. D-gluconic acid-δ-lactone can be transformed to γ-lactone and both can be hydrolyzed into D-gluconic acid at room

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temperature. As the hydrolysis proceeds, the pH value decreases gradually. Hence, gluconolactones can be used as a mild acidifier. Gluconolactones have gained applications in meat, dairy and soy products and especially baked goods as a leavening agent.

Reduction The carbonyl groups of monosaccharides can be reduced to the hydroxyl group. Reduction of a ketose yields to two sugar alcohols isomers due to the formation of a new chiral carbon atom. Figure 3-14 shows sugar alcohols produced by the reduction of glucose and fructose. Esterification and Etherification Due to the presence of hydroxyl groups, sugars can be esterified by organic acids or some inorganic acids, such as D-glucose-6-phosphate and D-fructose-1,6-diphosphate (Figure 315). The ester derivatives of starch, such as starch succinate, have found wide applications in the food industry. Another typical example is sucrose fatty acid ester, which is a commonly used an emulsifier.

Figure 3-14. Reduction of D-glucose and D-fructose.

Figure 3-15. Esterification of glucose and fructose by phosphorus acid (left: D-glucose; right: Dfructose).

Sugars can also be etherified, but naturally occurring sugar ethers are not as diverse as sugar esters. Etherification of polysaccharides can significantly improve their functional properties. Carboxymethyl cellulose (CMC) is an important ester of cellulose and is soluble in water. CMC has been widely used in the food industry as thickening agent or emulsion stabilizer.

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Properties of Carbohydrates Hydroscopicity Carbohydrates contain abundant hydrophilic hydroxyls and can bind water through hydrogen bonding. For example, when monosaccharides or oligosaccharides are placed in environments of different relative humidity (RH), the compounds can absorb moisture from air (Table 3-5). Table 3-5. Moisture adsorption of different sugars (%) Sugar D-glucose D-fructose Sucrose Anhydrous maltose Hydrous maltose Anhydrous lactose Anhydrous lactose

20°C in different RHs 60%, 1h 60%, 9d 0.07 0.07 0.28 0.63 0.04 0.03 0.80 7.0 5.05 5.1 0.54 1.2 5.05 5.1

100%, 25d 14.5 73.4 18.4 18.4 Not measured 1.4 Not measured

Figure 3-16. Hydroscopicity and water holding capacity of tea polysaccharide in different RHs (left: RH=81%; middle: RH=43%; right: RH=43%).

All sugar alcohols, except mannitol and isomaltulose, exhibit hygroscopicity especially in high RH environments. The hygroscopicity of sugar alcohols varies with their purity and lowpurity compounds have relatively higher hygroscopicity. Sugar alcohols are used as moisturizing agent in cream foods and soft pastries. Polysaccharides also absorb moisture from air and hence have good water holding capacity (Figure 3-16). The hydroscopicity of carbohydrates, which is often referred to as moisture retention capacity, is the most important attribute and determines their applications in foods. For example, to overcome the sticky problem of icing sugar powders after packaging, sugars with low hydroscopicity, such as lactose and maltose, are preferred in such products.

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Viscosity and Gelling Property Definition of Viscosity Viscosity is a measure of the resistance of a fluid which is being deformed by either shear stress or tensile stress. Viscosity can be measured with various types of viscometers and rheometers, such as capillary viscometer, rotational viscometer, falling ball viscometer and vibration-type viscometer. The aqueous solutions of monosaccharides, sugar alcohols, oligosaccharides and soluble macromolecular polysaccharides are viscous. Many factors affect the viscosity of carbohydrates. Internal factors include the average molecular weight size and shape of molecular chains and external factors are carbohydrates concentration, environmental temperature, etc. Viscosity of Polysaccharide Solutions Polysaccharides (gums, hydrocolloids) are primarily used to thicken and/or gel aqueous solutions and otherwise to modify and/or control the flow properties and textures of liquid foods and beverages and the deformation properties of semisolid foods. They are generally used in concentrations 0.25–0.50%, indicating their great ability to produce viscosity and to form gels. The viscosity of a polysaccharide solution is related with the molecular size, shape, net charge and conformation of the polysaccharide in solution. Polysaccharides are often present as random coils in solutions (Figure 3-17) and the specific conformations are closely related to their compositions and connection modes. Straight-chain and branched-chain polysaccharides of the same degree of polymerization (DP) differ markedly in viscosity in aqueous solutions. Compared with their linear counterparts of equal molecular weights and equal concentrations, solutions of branched polysaccharides have a lower viscosity. It is assumed that the viscosity reflects the ―effective volume‖ of the macromolecule. The ―effective volume‖ is the volume of a sphere with diameter determined by the longest linear extension of the molecule. Molecules with larger effective volumes undergo collision more frequently than those with smaller effective volumes. Hence, linear polysaccharides can provide high viscosity even in low concentrations.

Figure 3-17. Random coiled structure of polysaccharides.

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Figure 3-18. Effective volume of straight-chain and branched-chain polysaccharides with equal molecular weights in solution.

Branched polysaccharides (amylopectin, glycogen) are more soluble in water than their perfectly linear counterparts since the chain–chain interaction is less pronounced and there is a greater extent of solvation of the molecules. This is especially the case for highly branched polysaccharides, because they have much less effective volumes than their linear counterparts, as shown in Figure 3-18. In addition to the DP, unfolding degree, and rigidity, the viscosity of polysaccharides is also affected by the shape and flexibility of polysaccharides after solvaiton. The charge carried by solvated polysaccharides significantly influences the viscosity of the solution. For example, straight-chain polysaccharides containing carboxyl, sulfate hemiester or phosphate groups are often negatively charged. The electrostatic repulsion between the molecules unfolds the molecules and increases their effective volume, leading to increased viscosity. Hence, pH has significant impact on the viscosity of polysaccharide solutions, since it is closely related to the charge statues of the molecules. The viscosity of polysaccharide solutions decreases with the increase of temperature, except xanthan solution, whose viscosity remains nearly unchanged in the temperature range 0~100°C. Besides, the viscosity of xanthan gum solutions decreases with shear rate increase. Hence, xanthan gum solutions are pseudoplastic. A practical use would be in salad dressing: xanthan gum makes it thick enough at rest in the bottle to keep the mixture fairly homogeneous, but the shear forces generated by shaking and pouring thins it, so it can be easily poured. When it exits the bottle, the shear forces are removed and it thickens back and clings to the salad.

Gelation Gelation is another important property of polysaccharides. In food processing, polysaccharides or proteins and other macromolecules can form a sponge-like threedimensional network gel structure through hydrogen bonding, hydrophobic interaction, Van der Waals attraction, ionic cross bridges, entanglement or covalent bonding (Figure 3-19). Liquids containing small-sized solutes and polymers fill the holes of the gel network.

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Gels possess the properties of both solids and liquids. They are not as flowable as continuous liquids or rigid as orderly organized solids. Instead, gels are viscoelastic semisolids and can retain their shape even in the presence of external stresses. Polysaccharide gels generally contain only about 1% polymer—that is, they may contain as much as 99% water. Examples of polysaccharide gels are dessert gels, aspics, structured fruit pieces, structured onion rings, meat-analog pet foods, and icings. The firmness of a gel depends on the extent of junction zone formation. If the conjunction zone area is not long enough, polysaccharide chains cannot bind tightly. In this case, the chains can be separated by pressure or high temperature due to increased mobility of the chains. Such gels are easily damaged and are heat-labile. If the conjunction area contains long chain segments, the interaction between chains is strong enough to withstand applied pressure or thermal stimulation. Gels of this type are hard and stable. Therefore, gels of different hardness and strength are available by controlling the length of the conjunction zone. Branched-chain polysaccharides or heteropolysaccharides cannot bind to each other well as linear molecules and large enough conjunction areas cannot be formed between these molecules. Hence, gels cannot be formed by branched-chain or heteropolysaccharides. This is also the case for charged molecules, such as those containing carboxyl groups, because the Coulomb repulsion between chains hinders the formation of conjunction zone.

Figure 3-19. A diagrammatic representation of the type of three-dimensional network structure found in gels.

The selection of a specific polysaccharide for a particular application depends on the viscosity or gel strength desired, required rheology, pH of the system, temperatures during processing, interactions with other ingredients, desired texture, and cost.

Flavor Retention Aroma release from food matrix and the subsequent delivery of flavor to the olfactory and gustatory receptors is greatly dependent on the type of food ingredients and on the physicochemical properties of the aroma compound. It has been shown that macromolecules, such as polysaccharides, are involved in the retention of volatile compounds. Polysaccharides influence the volatility of aroma compounds and their partitioning between different phases [1].

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The use of carbohydrates may induce a significant decrease in flavor perception and/or release. Some factors affecting the retention and release of volatile flavor compounds by carbohydrates are depending on physicochemical properties of flavor compounds, type of carbohydrates and their concentrations. Firstly, high molecular weight flavor compounds tend to retain in carbohydrate than low molecular weight flavor compounds. Additionally, long linear chain length molecules will be retained in polysaccharide matrix higher than short chain molecules or aromatic one. Among the volatile flavor compounds such as alcohol, aldehyde, ester and ketone, alcohol are usually the best retained in carbohydrates. The retention of polar (hydrophilic) volatiles flavor compounds is expected to be very low in carbohydrates complex. The second factor is depending on type of carbohydrates. Each type of carbohydrate resents different structure that influence on the interaction between flavor compounds and its structure and also the retention and release. Thirdly, the concentration of carbohydrates generally shows that an increase in the concentration of sugar is proportional of the release of flavor compounds due to the salting out effect. On the other hand, an increase in polysaccharide concentration leads to a decrease the release of flavor compounds due to complexation and viscosity effect of that polysaccharide themselves. This knowledge can be used to optimize product quality in term of flavour retention during preparation or processing and its release during eating [2].

Browning and Food Flavor The non-enzymatic browning reaction of carbohydrates yields abundant volatile substances in addition to melanoidin and imparts peanut, coffee bean, and bakery foods with special flavors. Some compounds, such as maltol (3-hydroxy-2-methyl-4H-pyran-4-one) and ethyl maltol (3-hydroxy-2-ethyl-4H-pyran-4-one), not only exhibit special flavors on their own, but also enhance the flavors of other components in foods. Maltol lowers the threshold of sucrose by a factor of two. Ethyl maltol is more effective as a sweetness enhancer than maltol. Maltol enhances the sweet taste of foods, especially the sweetness produced by sugars, and is able to mask the bitter flavor of hops and cola. Ethyl maltol enhances the same sensation but is 4- to 6-times more powerful than maltol. In contrast to maltol, ethyl maltol is not a natural constituent in foods. Table 3-6. Relative sweetness of sugars and sugar alcohols to sucrose in 10% aqueous solution Sugar/sugar alcohol Sucrose

Relative sweetness 100

Sugar/sugar alcohol D-Mannitol

Relative sweetness 69

Galactitol D-Fructose D-Galactose D-Glucose Invert sugar Lactose Maltose

41 114 63 69 95 39 46

D-Mannose Raffinose D-Rhamnose D-Sorbitol Xylitol D-Xylose

59 22 33 51 102 67

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Sweetness Sweetness is a value relative to that of sucrose under the same conditions, which is often referred to as 100. All sugars, sugar alcohols and oligosaccharides are sweet (Table 3-6) with varying intensity of sweetness and some glycosides and polysaccharide complexes are excellent sweeteners. For example, the sweetness of honey and most fruits is contributed by sucrose, D-fructose or D-glucose. The sweetness perceived by consumers varies with the composition, conformation, and physical aspects of sugars. The intensities of sweetness of sugar alcohols differ markedly from those of corresponding sugars. For example, sorbitol is sweeter than glucose and that of xylitol is greater than xylose. Generally, all sugar alcohols, except xylitol, are less sweet than sucrose. Sugar alcohols are usually incompletely absorbed into the blood stream from the small intestines which generally results in a smaller change in blood glucose than sucrose. This property makes them popular sweeteners among diabetics and people on low-carbohydrate diets. As an exception, erythritol is actually absorbed in the small intestine and excreted unchanged through urine, so it has no side effects at typical levels of consumption.

Nonenzymatic Browning Reactions Nonenzymatic browning, or oxidative browning, is a chemical process that produces a brown color in foods without the participation of enzymes. The two main forms of nonenzymatic browning are caramelization and Maillard reaction. Ascorbic acid is also involved in nonenzymatic browning under certain conditions. Because phenols are important components in some foods and these compounds readily undergo autooxidation to yield brown color, the nonenzymatic browning of phenols is also discussed in this section.

Types and Process of Nonenzymatic Browning Reactions Maillard Reaction and Its Process The Maillard reaction is a form of nonenzymatic browning and involves the complex reactions between reducing sugars and amino acids/proteins. French chemist Louis-Camille Maillard described the reactions for the first time in 1912. John Hodge then named the reaction after Maillard and summarized the reaction process for the first time in 1953, as shown in Figure 3-20. The process of the Maillard reaction is often divided into three stages. The initial stage of the Maillard reaction involves the condensation of a carbonyl group, for example from a reducing sugar such as glucose, with a free amino group, typically the epsilon amino group of lysine residues within proteins. This glycation reaction results in the formation of an unstable Schiff base (aldimine) that spontaneously rearranges to the more stable 1-amino-1-deoxy-2-ketose (ketoamine), which is also known as the Amadori product after the Italian scientist Mario Amadori. When the initial sugar is glucose, the Amadori product is commonly known as fructoselysine (FL) (Figure 3-21).

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Figure 3-20. Maillard reaction scheme recommended by John Hodge [3].

Amadori products are degraded via various pathways in the intermediate stage, leading to the formation of furfurals, reductones and fragmentation products (carbonyl and hydroxycarbonyl compounds). Furfural or hydromethylfurfural (HMF) formation (Figure 322) is favored under acidic conditions, while alkaline media favor the production of reductones (Figure 3-23). The products of 1-amino-1-deoxy-2-ketose fragmentation in alkaline media, such acetol, diacetyl, pyruvaldehyde, etc, are able to react with amino acids via the Strecker degradation (named after the German chemist Adolph Strecker, Figure 3-24) to give Strecker aldehydes of the amino acids and aminoketones; the latter subsequently condense to form pyrazines. Strecker aldehydes and pyrazines contribute to aroma formation in heated foods.

Carbohydrates

Figure 3-21. Scheme of the initial stage of the Maillard reaction.

Figure 3-22. Formation of hydromethylfurfural from 1-amino-1-deoxy-D-fructose.

Figure 3-23. Formation of reductones from 1-amino-1-deoxy-D-fructose.

Figure 3-24. Scheme of the Strecker degradation.

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Figure 3-25. Scheme of the aldolisation of aldehydes and ketones.

The majority of the compounds displaying color are formed in the final stage of the Maillard reaction. Furfurals, reductones, aldehydes either undergo aldol condensation without the intervention of amino compounds or react with amino compounds as other intermediates, leading to the ultimate reaction products known as melanoidins. Hodge defined melanoidins as ‗brown, nitrogenous polymers and copolymers‘. The structures of melanoidins remain unknown till now. The pigment generated in the Maillard reaction is soluble in the early stage of polymerization and exhibits no characteristic absorption pick in the visible range. Infrared spectrum and chemical composition analysis indicate that melanoidins contain unsaturated bonds, heterocycles, and complete amino acid residues. The reaction mechanism of the Maillard reaction proposed by John Hodge is largely unchanged after 60 years and is still widely cited. However, the hypothesis has some flaws. Firstly, the mechanism presents only the general process of the Maillard reaction and the details on the reaction are not described. Secondly, some new reactions have been identified by other researchers. For example, the work of Japanese scientists Namiki and co-workers found that the carbonyl fission products can also be formed directly from N-substituted glycosylamine through a free radical-mediated pathway, which is known as the Namiki pathway [4].

Mechanism of Caramelization Heating of carbohydrates without the presence of nitrogen-containing compounds causes a complex group of reactions termed caramelization. Unlike the Maillard reaction, caramelization of carbohydrates is thermolysis as compared to the reaction with amino acids. Mild heating or thermolysis in the early stage leads to anomeric shifts, ring size change, glycosidic bond breakdown, and new glycosidic bond formation. Mostly, thermolysis causes dehydration of the sugar molecule with introduction of double bonds or formation of anhydro rings. Caramelization yields hundreds of flavor compounds and these compounds impart foods with pleasant color and flavor. The caramels derived from different sugars have similar compositions. Generally, the products of caramelization include caramel, which is the polymerization product of sugar dehydration, and aldehydes, ketones, phenols, etc. The caramelization process is divided into two stages. Caramel formation Figure 3-26 illustrates the formation of caramel by taking sucrose as the example. It can be seen that caramelization is the removal of water from a sugar, proceeding to isomerization and polymerization of the sugars into various high-molecular weight compounds.

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The aqueous solutions of caramels are colloidal and the isoelectric points range from 3.0~9.0, with some exceptions with pI lower than 3. The presence of acids or certain salts facilitates the reaction. The caramel prepared by heating sucrose solution in the presence of acid or acidic ammonium salts has been widely used in food coloration. The caramelization of sucrose consists of three steps: Step 1: Caramelization of sucrose starts with the melting of the sugar at high temperatures followed by foaming (boiling). At this stage sucrose decomposes into glucose and fructose. This is followed by a condensation step, in which the individual sugars lose water and react with each other to form the isosaccharosan (Figure 3-27). Isosaccharosan loses the sweetness of sugar and tastes bitter instead.

Figure 3-26. General mechanism for thermal promoted caramelization of sucrose.

Figure 3-27. Structure of isosaccharosan.

Step 2: This step involves further dehydration reactions. Isosaccharosan dehydrates and condensates to caramelan. Caramelan is one of the three main products of sucrose caramelization. Caramelan are lightly brown and tastes bitter with formula of C24H36O18. Caramelan melts at 138°C and is soluble in water and ethanol.

Step 3: This step includes both fragmentation reactions (flavor production) and polymerization reactions (color production). With respect to color production, caramelan is further dehydrated to form caramelen. If the mixture is further heated, polymerization reaction occurs and the insoluble caramelin is generated. The caramelan melts at 154°C, tastes bitter, and is soluble in water with formula of C36H50O25. Caramelin appears dark brown and are not soluble in water with formula of C125H188O80.

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The presence of iron enhances the color of caramels. Phosphates, inorganic salts, alkalines, citric acid, ammonia, and ammonium sulfate catalyze sugar caramelization. Ammonia and ammonium sulfate increase the yield of caramel. However, the addition of ammonia and ammonium sulfate cause the generation of 4-methylimidazole at high temperatures, which is an anticonvulsants and can cause nervous system damage after longterm consumption. Ammonia and ammonium sulfate have been prohibited in the production of caramels. Caramel flavors from thermal fragmentation a. Formation of aldehydes in acid media. In acid media, aldoses or ketones undergo enolization to yield hexose-1,2-enediol when heated, followed by a series of dehydration reactions, as shown in Figure 3-28. b. Formation of aldehydes in alkaline media. In alkaline conditions, the intermediate 1,2-enol hexose, such as fructose, is produced via the tautomery of reducing sugars and is then fragmented when heated, as shown in Figure 329.

Figure 3-28. Formation of aldehydes from sugars in acid media and thermal condition.

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Figure 3-29. Thermal fragmentation of 1, 2-enediol-hexose in alkaline media.

Mechanism of Nonenzymatic Browning of Ascorbic Acid Ascorbic acid is a well-known natural antioxidant. It can be readily oxidized in two ways. In the presence of oxygen, ascorbic acid is oxidized to dehydroascorbic acid, which is then dehydrated to yield 2,3-diketogulomic acid (DKG). DKG is further decarboxylated to yield xylosone and subsequently reductones, which participate in the reactions in the intermediate and final stages of the Maillard reaction. Ascorbic acid degrades very quickly in the presence of oxygen and the specific rate is related to the level of dissolved oxygen and extraneous gas. If the food matrix contains components with reduction potential higher than that of ascorbic acid, ascorbic can be oxidized to dehydroascorbic acid even in the absence of oxygen. In both cases, dehydroascorbic acid is then transformed to DKG in the presence of water. DKG further undergoes decarboxylation and dehydration to yield aldofuranoses or reductions. These products can participate in the Maillard reaction, leading to the formation of brown pigments (Figure 3-21). Ascorbic acid is slowly oxidized to dehydroascorbic acid in acidic solutions with pH < 5 and this process is reversible. Mechanism of Polyphenols Browning Some plant-derived foods contain high levels of phenolic compounds. For example, the content of polyphenols in green tea is up to 30%. The phenolic hydroxyl group of polyphenols is very susceptible to oxidation, especially in alkaline conditions. Polyphenols undergo auto-oxidation at high temperature and high moisture conditions and the oxidation products exhibit different colors. Polyphenols are an important cause of food browning and the browning mechanism is elucidated by taking catechin as an example.

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Figure 3-30. Mechanism of ascorbic acid browning.

Structure of catechin Catechin possesses two benzene rings (called the A- and B-rings) and a dihydropyran heterocycle (the C-ring) with a hydroxyl group on carbon 3, as shown below:

Catechin contains multiple phenolic hydroxyl groups and is very susceptible to oxidation, polymerization, and condensation. Catechin is white crystal and is oxidized to yellowishbrown compounds in the air. Catechin is soluble in water, ethanol, methanol, acetone, and acetic anhydride, partially soluble in ethyl acetate and acetic acid, and insoluble in chloroform and anhydrous ether.

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Mechanism of oxidation The hydroxyl groups in catechin have different activities with respect to auto-oxidation. Hydroxyl groups in adjacent and vicinal positions are oxidized easily, while the –OH in carbon 3 on ring C cannot be oxidized. The mechanism of the nonenzymatic auto-oxidation of catechin is very complex and not well understood to the present. It is generally accepted that the process involves two reactions. The first is the formation of quinine. Quinine is unstable and rapidly undergoes condensation. The condensation product in the early stage is soluble, light yellow and tastes bitter. As the reaction proceeds, the intermediates are further condensed to brown nitrogenous polymer in the presence of amino compounds. The general process is illustrated in Figure 331.

Effects of Nonenzymatic Browning on Foods Nonenzymatic browning reaction is one of the most important reactions during the storage and processing of foods. A large number of food components, such as carbohydrates, amino acids, phenolic compounds, and ascorbic acid, are involved in the reaction. The reaction yields multiple volatiles and nonvolatile compounds, which significantly influence the color, aroma, taste, nutrition, and safety of foods.

Figure 3-31. Mechanism of nonenzymatic browning of catechin.

Effects on Color The composition of nonenzymatic browning reaction products is very complex. Many components affect the color of foods and their molecular weights range from several hundreds to up to more than 100000 Dolton. Several coloring compounds have been isolated from different model systems.

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Dongfeng Wang, Jipeng Sun, Guoqing Huang et al. Xylose – Lys model system

A yellow compound is isolated from the Maillard reaction product of the xylose – Lys model system and MS and NMR reveals that its possible structure is one of the following two [5]:

(1) Furan-2-carboxaldehyde – L-alanine model system Two red products are isolated and identified from the Maillard reaction product and their structures are shown below [6]:

(2) Xylose – L-alanie model system A white compound is isolated:

(3) Glucose – propylamine model system in ethanol solution. A yellow product is identified:

(4) Xylose – alanie model system in the presence of carbonyl compounds. Two yellow compounds are isolated:

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(5) One red compound is isolated:

(6) Starting materials, as well as reaction conditions, markedly affect the elemental composition and structure of melanoidins. Thus far, the structures of the melanoidins have not been elucidated, although some structural insights have been gained from model reactions. In a glucose/amino acid Maillard reaction system, the carbonyl compound reacts mainly via the Amadori product to form several deoxyosones which are able to react with each other in an aldol-type condensation to form a basic melanoidin skeleton of amino-branched sugar degradation products. Figure 3-32 shows the possible structure of a melanoidin formed from 3-deoxyhexosuloses in this way.

Figure 3-32. Part of possible melanoidin structure formed from 3-deoxyhexosulose involving amino compounds [7].

Figure 3-33. Strecker degradation of L-Lys with dicarbonyl compound.

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Figure 3-34. pH changes of various sugar-amino acids solutions heated at 100°C. From up to down: glucose, lactose, lactose + alanine, glucose +alanine, lactose + glycine, glucose + glycine, glucose + lysine, lactose+ lysine.

Effects on Food Aroma and Taste The intermediate and final products of nonenzymatic browning reactions markedly influence the aroma and taste of foods. Under high temperatures, carbohydrates undergo dehydration, fragmentation, isomerization, and oxidation/reduction, giving rise to formic acid, acetic acid, lactic acid, acetol, acetoin, diacetyl, pyruvic acid, and many other products. The dicarbonyl compounds generated in non-enzymatic browning can initiate the changes of other food components. For example, amino acids are deaminated and decarbonylated to yield abundant aldehydes, as shown in Figure 3-33: Non-enzymatic browning can produce both desirable and undesirable flavors. Maltol and isomaltol in breads gives the caramel-like flavor for breads and 4-hydroxy-5-methyl-3(2H)furanone delivers the flavor of roasted meat and is used as flavor and sweetness enhancer, while some pyrazine and aldehyde compounds are the source of undesired burned taste in some foods. CO2 is a product of the Strecker degradation and its volume released is proportional to the moiety of dicarbonyl compounds. Besides, the reducing ketones and aldehydes produced can be readily oxidized to acidic compounds. Therefore, non-enzymatic browning reaction reduces the acidity of foods. Figure 3-34 shows the changes of pH in different model systems. Antioxidation The antioxidant activity of Maillard reaction products (MRPs) was firstly reported by Franzke and Iwainsky in 1954. The two researchers found that the thermal reaction product of glycine and glucose improved the stability of margarine against oxidation [8]. However, this research did not attract much attention until 1980s. The antioxidant components in various MRPs are not identified yet and the antioxidant activity of MRPs is studied mainly by using various model systems.

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Elizalde et al. found that the volatile compounds of the MRP of the glucose-glycine system showed a significant antioxidant activity and prolonged the induction period of soybean oil thermoxidation by up to 3 times in relation to the control [9]. Bedinghaus and Ockerman compared the lipid oxidation inhibitor activities of fifteen MRPs prepared by heating one of three sugars (glucose, xylose, and dihydroxyacetone) with one of five amino acids (arginine, histidine, leucine, lysine, and tryptophan) by using cooked ground pork patties as the model food. It was found that the most effective MRPs were xylose-lysine, xylose-tryptophan, dihydroxyacetone-histidine, and dihydroxy-acetone-tryptophan when compared to controls [10]. Yamaguchi et al. obtained a fraction from the MRP of the xylose – glycine system through multiple chromatographic steps and found that it is more effective in preventing linoleic acid oxidation than BHA and propyl gallate [11]. Yoshimura et al. investigated the MRP of the glucose-glycine system on the inhibition toward active oxygen and fount that it inhibited more than ca. 90% of active oxygen species existing in the form of hydroxyl radicals (•OH) [12]. Morales and Jiménez-Pérez investigated the DPPH· scavenging activities of MRPs produced by heating glucose or lactose with lysine, alanine or glycine. It was found that all the MRPs were effective DPPH· scavenging agents. Browning was not directly related to the free radical scavenging properties of MRPs formed at prolonged heating conditions and fluorescence measurement is more effective than browning to follow the formation of MRPs with free radical scavenging activities, as shown in Figure 3-35. Although the antioxidant effects of MRPs have been well recognized, their application as effective antioxidants in foods is still limited due to insufficient knowledge on the structure and antioxidant mechanism of MRPs. Early researches indicated that antioxidant capability of MRPs is contributed by the intermediate reductones of the Maillard reaction and the metal chelating ability of MRPs. Latest researches indicate that MRPs also have strong active oxygen scavenging capabilities and can reduce peroxides.

Note: A: up to down: glucose+alanine, glucose+glycine, glucose+lysine. B: up to down: lactose+alanine, lactose+glycine, lactose+lysine. Figure 3-35. Free radical scavenging activity in sugar/amino acid mixtures heated at 100°C up to 24 h [13].

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Effects on Nutrition One of the most obvious negative consequences of the Maillard reaction in food is the loss of nutritive value of proteins due to decreased digestibility, destruction and/or biological inactivation of amino acids, including essential amino acids like lysine and tryptophan, inhibition of proteolytic and glycolitic enzymes, and interaction with metal ions. Lysine is the most liable to loss in non-enzymatic browning, followed by alkaline amino acids, including L-Arg and L-His. In addition to sugar-amino acid reactions, Strecker degradation is also involved in amino acid loss. The formation of complex macromolecules reduces the solubility of proteins and reduces their nutritional value. The effect of the Maillard reaction on amino acid availability has been investigated by using rainbow trout (Salmo gairdneri) as the model. It was found that arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37°C and the apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein [14]. Vitamin C is also involved in browning and therefore suffers loss during the process. The products of non-enzymatic browning reduce the bioavailability of minerals. Whitelaw et al. employed the dialysis procedure to determine the effect of the Maillard reaction on apparent 65Zn availability. In the presence of 65ZnCl2, the amino acids glycine, Dleucine, L-proline, L-lysine and L-glutamic acid were combined with D-glucose and autoclaved (110°-120°C, 15 atm, 10 min) to produce high molecular weight 65Zn binding compounds that were not dialyzable (6-8KD). Experiments in stimulated gastrointestinal digestive conditions revealed that the Maillard reactions significantly reduced the bioavailability of Zn [15]. Harmful Compounds The safety of MRPs has attracted wide attentions in recent years. With the development of new instrumental analysis measures, more and more harmful compounds have been isolated and identified. For example, mutagenic compounds have been found in instant and caffeine-free coffee and the compounds consist of dicarbonyl compounds, methylglyoxal, diacetyl and glyoxal, among which the methylglyoxal presented highest mutagenic activity [16]. Also in both fried and grilled meat and fish, mutagenic compounds were identified, mainly stemming from heterocyclic amines [17]. The reaction mechanism seems to have a major influence on the mutagenicity of the reaction products. For instance, ketose sugars showed a higher mutagenic activity than the corresponding aldose sugars [18]. However, due to the complexity of non-enzymatic browning reactions and the poor stability of intermediates, only very few harmful compounds have been well elucidated, of which, acrylamide is the most intensively studied. Acrylamide is a well-known cancerogen and can cause neurologic damage. Acrylamide has been detected in nearly all the foods, but fried and roasted foods that are processed at high temperatures have much higher acrylamide contents, as presented in Table 3-7.

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Carbohydrates Table 3-7. Content of acrylamide in some foods Foods Potato chips French fries Biscuit Fried bread American breakfast Cornflakes Bread

Acrylamide contents (μg/kg) Median Minimum ~ maximum 1,200 330-2,300 450 300-1,100 410 <30-650 140 <30-1,900 160 <30-1,400 150 120-180 50 <30-160

Number of samples 14 9 14 21 15 3 20

Factors Influencing Nonenzymatic Browning Type of Sugar and Amino Acid The structures of sugars and amino acids significantly affect the rates of the nonenzymatic browning reactions between them. Among common reducing sugars, pentoses are 10 times more susceptible to non-enzymatic browning than hexoses. Under the same conditions, the reactivity of some pentoses follow the sequence ribose > arabinose > xylose and that of some hexoses are galactose > mannose > glucose. Among carbonyl compounds, -hexenal has the highest browning rate, followed by dicarbonyl compounds and ketones in sequence. Alkaline amino acids undergo browning more readily than acidic and neutral amino acids and those with the amino group locating at the end or ε- position are more susceptible to browning than those with amino group at the α- position. Non-enzymatic browning also occurs between proteins and carbonyl compounds, but the reaction is slower than that of peptides. Temperature and Time Reaction temperature significantly affects browning. When the temperature increases by 10 °C, the browning rate can be 3~5 times higher. Generally, non-enzymatic browning occurs rapidly at temperatures higher than 30 °C. The degree of browning is also affected by reaction time. When the mixture of disaccharide or monosaccharide and amino acid is heated at 100°C, the absorbance of the solutions at 420nm increases with heating time, indicating that the formation of the brown color is positively correlative with the reaction time (Figure 3-36). In addition to the color and flavor of foods, reaction temperature and time influence the generation of harmful compounds as well. For example, when equimolar amount of asparagine and glucose is heated, the formation of acrylamide is detected in 120 °C and the content continues increasing with temperature elevation until 170 °C. The formation of acrylamide generated in the mixtures of glucose and glutamine, methionine, or asparagine heated at 180 °C as a function of time (5~60 min) reveals that the contents of acrylamide varies with amino acids. The highest acrylamide content is found in the glucose/asparagine system and the highest content appears in the 5th minute. When the reaction proceeds, the level of acrylamide keeps decreasing. In the glucose/glutamine system, the highest

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acrylamide formation is detected within 10 min and the content remains unchanged thereafter. While in the glucose/methionine system, the formation of acrylamide keeps increasing in the first 30 min and then remains unchanged in remaining time.

pH Medium pH affects multiple reactions of non-enzymatic browning. Aqueous solutions of sugar (xylose or glucose) and amino acid (glycine or lysine monohydrochloride), are heated without pH control for up to 120 min and the total reaction products are analyzed by HPLC and compared with those of the corresponding model systems maintained at pH 5 throughout heating. For xylose-lysine, seven of these peaks are common to the systems heated both with and without pH control for 15 min, while no peak is common to both glucose-lysine systems heated for 120min, indicating different products are formed under the conditions [19]. Generally, the browning rate between sugars and amino acids increases with pH when the medium pH is greater than 3.5 and is reversely proportional to pH values in the range 2.0~3.5. Hence, browning can be suppressed by reducing pH. This is why browning does not occur readily in acidic foods such as pickled vegetables. Sulfites inhibit nonenzymatic browning by reacting with carbonyl intermediates, thereby preventing their further reaction to form brown pigments [20].

Water Content and Metals The rate of non-enzymatic browning is a function a water activity. Generally, nonenzymatic browning occurs readily in the water activity range 10%~15% and is suppressed in water activity less than 3%. Moderate water content facilitates the mobility of solutes, but too high water contents lead to solute dilution and consequently reduced browning. Metal ions are also involved in the process of non-enzymatic browning, as evidenced by the fact that the browning of grapefruit juice is inhibited by the addition of a chelating agent EDTA [21]. Cu(I), Cu(II), Fe(II), and Fe(III) speed up the browning of ascorbic acid and phenols, but other metals, such as Pb, Zn, and Sn, seem to have little effect.

Figure 3-36. The time course of the browning develop in heated sugars -amines solutions at 100°CUp to down: glucose+lysine, lactose +lysine, glucose+glycine, lactose+ glycine, glucose+alanine, lactose +alanine, lactose, and glucose.

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High Pressure High pressure (100-1000 MPa), which is gaining increasing importance as a foodprocessing technology particularly in combination with moderate temperatures (30-60 °C), may influence the Maillard reaction and thereby affects the flavor, color, and nutritional value of foods. It has been proposed that high pressure exerts its influence on non-enzymatic browning by changing the medium pH. The effect of high pressure on Maillard reaction has been investigated by using the glucose-lysine model system over a range of pH values (5-10) at 60 °C either under atmospheric pressure or at 400 MPa. The results obtained showed that high pressure affected in different ways the different stages of the Maillard reaction and that such effects were strongly influenced by pressure-induced changes in the pH of the systems. In unbuffered media, at an initial pH 8.0, the formation of Amadori rearrangement products (ARP) was not considerably affected by pressure, whereas the intermediate and advanced stages of the Maillard reaction were suppressed, suggesting a retardation of the degradation of the ARP. In buffered media, at pH values 8.0, pressure slowed the Maillard reaction from the initial stages. These effects are attributed to the pH drop caused by the pressure-induced dissociation of the acid groups [22]. Control of Non-Enzymatic Browning The non-enzymatic browning has both beneficial and adverse impacts on food qualities. Researchers have developed rational approaches to minimize adverse consequences of browning reactions and optimize beneficial ones. Non-enzymatic browning can be prevented by chemical or biochemical methods [23]: Sulfhydryl compounds Sulfur-containing amino acids such as cysteine, N-acetylcysteine, and the tripeptide glutathione play key roles in the biotransformation of toxic compounds by actively participating in their detoxification. These antioxidant and antitoxic effects are due to a multiplicity of mechanisms including their ability to act as (a) reducing agents, (b) scavengers of reactive oxygen (free radical species), (c) destroyers of fatty acid hydroperoxides, (d) strong nucleophiles that can trap electrophilic compounds and intermediates, (e) precursors for intracellular reduced glutathione, and (f) inducers of cellular detoxification. Under certain conditions, SH-containing compounds may be as effective as sodium sulfite in preventing nonenzymatic browning of both apples and potatoes. Acetylation of amino groups Modifications of amino groups prevent them from participating in browning reactions. For example, treatment of foods with the enzyme transglutaminase will transform lysine amino groups to amide groups. The former initiate browning, whereas the latter do not. Antioxidants Oxygen seems to be required for some nonenzymatic browning reactions. Hence, antioxidants could suppress browning in some foods. Besides, antioxidants can trap or

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prevent the formation of intermediates in the Maillard and related reactions, thus prevent the formation of undesirable compounds during food processing. Deglycation An extract from soil microorganisms catalyzed the deglycation of α- and εfructosyllysines to lysine. This finding suggests that these purified enzymes could be used to prevent or reverse Maillard reactions in foods and in vivo provided they are safe in other regards. Non-enzymatic browning can also be prevented by controlling processing conditions: Low temperature Non-enzymatic browning occurs slowly in low temperatures. Storage at low temperatures can delay non-enzymatic browning in foods. Sulfurous acid The condensation product of carbonyl group and sulfurous acid can react with R-NH2 and the resultant product cannot be transformed to Schiff‘s base. Hence, SO2 and sulfites suppress non-enzymatic browning. pH Non-enzymatic browning does not occur readily in acidic conditions as in alkaline conditions. Product concentration Products with lower concentrations suffer lighter browning. For example, because lemon juice is more susceptible to browning than orange juice, the concentration factor of lemon juice is 4:1, which is lower than 6:1 of orange juice. Insensitive sugar The presence of free carbonyl groups is essential for non-enzymatic browning. Replacement of reducing sugar with sucrose can prevent browning. Removal of sugar Some foods contain only trace amount of sugars and these sugars can be removed to prevent non-enzymatic browning. For example, glucose oxidase and catalase have been used to remove the trace glucose in dried egg yolk and dehydrated meat. Calcium salts Calcium can complex with amino acids to form precipitates.

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IMPORTANT OLIGOSACCHARIDES AND POLYSACCHARIDE IN FOODS Oligosaccharides and polysaccharides occur widely in the nature and some of them contribute largely to the quality and nutrition of foods. Oligosaccharides and polysaccharides have gained important applications in the food industry as thickening agent, gelling agent, crystallization inhibitors, clarifying agent, stabilizing agent, film forming agent, flocculating agent, controlled-release agent, expansive agent and encapsulation agent. Some carbohydrates posses important physiological functions and many functional foods have been developed based on them.

Oligosaccharides Oligosaccharides occur naturally in a variety of foods, especially plant-derived foods, such as vegetables, grains, legumes, and algae. Oligosaccharides have also been identified in animal-derived foods, including milk, honey and insects. Sucrose, maltose, lactose and cyclodextrin are the most important oligosaccharides for the food industry. Some oligosaccharides, such as fructo-oligosaccharides, xylo-oligosaccharides, and Konjac oligosaccharides, have obviouse physiological functions and can be used as the effective ingredients of functional foods. Cellobiose, maltose, isomaltose, gentiobiose and trehalose are common disaccharides. All these disaccharides, except trehalose, contain a free semi-acetal group and hence are reducing sugars. Sucrose, lactose, lactulose, and melibiose are hybrid-oligosaccharides and each contains a reducing group except sucrose. Of the sugars, lactose deserves special attention. Lactose is found notably in milk and non-fermented dairy products and accounts for 2~8% of milk by weight. In small intestine, lactose is hydrolyzed by lactase to D-glucose and D-galactose, which are then absorbed by the human body. However, some consumers lack the lactase and cannot digest or metabolize lactose. These consumers might suffer abdominal pain, bloating, flatulence, diarrhea, nausea, and acid reflux and this is called lactose intolerance or lactase deficiency. Some natural foods contain functional oligosaccharides. These compounds cannot be absorbed by human body and provide very few energy, but can enhance the proliferation of intestinal bifidobacteria and prevent dental caries and colon. Some important functional oligosaccharides are described below. Soybean oligosaccharide Soybean oligosaccharide has once been regarded as an antinutritional factor and has been reported to increase the incidence of diarrhea in rats. However, recent researches reveal that soybean oligosaccharide has the potential as new functional ingredients in functional foods. Soybean oligosaccharide has recently been found to be ―probiotic material‖ and have been approved by the Food and Drug Administration as generally recognized as safe material in the USA. Composition analysis indicates that soybean oligosaccharide was composed of

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galactose (65.3%), mannose (15.6%), fructose (7.8%) and glucose (8.7%) [24]. Intake of 3~5 g soybean oligosaccharide per day is sufficient to enhance the proliferation of bifidobacteria. Fructo-oligosaccharide Fructo-oligosaccharides (FOS) are fructose oligosaccharides containing a single glucose moiety. FOS are mainly composed of 1-kestose (GF2), nystose (GF3), and 1-β-fructofuranosyl nystose (GF4), in which fructosyl units (F) are bound at the β(2 → 1) position of sucrose molecule (GF). FOS occur naturally in various fruits and vegetables, such as bananas, onions, chicory root, garlic, asparagus, barley, wheat, jícama, tomatoes, and leeks, but commercial FOS are often prepared by transfructosylation from sucrose and chemical or enzymatic hydrolysis of inulin. FOS are about 0.4 and 0.6 times as sweet as sucrose and have been used in the pharmaceutical and food industries as functional sweeteners. FOS with low polymeric grade have better therapeutic properties than those with a high polymeric degree. They present properties such as low caloric values, non-cariogenic properties, decreased levels of phospholipids, triglycerides and cholesterol, help gut calcium and magnesium absorption, and are used as prebiotics to stimulate the bifidobacteria growth in the human colon [25]. Figure 3-37 illustrates the structure of several FOS. Xylo-oligosaccharide Xylo-oligosaccharides (XOS) consist of 2~7 xylose residues connected through by the β (1→4) glycosidic bond. XOS are about 40 times as sweet as sucrose. XOS have good thermal stability and are not decomposed in acidic conditions (pH2.5~7) when heated. XOS can be used in yogurt, lactobacillus drinks, carbonated drinks and other acidic beverages. Commercial XOS consists mainly of xylose, xylobiose, and less amount of polymers with DP greater than 3. Xylobiose is the main constituent of XOS and its content determines the quality of XOS products. XOS can be prepared by the enzymatic (xylanase) hydrolysis of xylan-rich materials, such as corncob, bagasse, cottonseed hull, and bran. Many fungi and bacteria can produce xylanase, of which, the endoxylanase produced by Chaetomiu globosum has been used in the industrial production of XOS. XOS cannot be digested but can selectively activate bacterial reproduction within intestines and hence are prebiological substances. It can obviously improve intestinal microecological balance, proliferate bifid bacteria and gastric function. Chito-oligosaccharides Chito-oligosaccharides (COS) are oligomers of N-acetyl-D-glucosamine and Dglucosamine connected through the β-1, 4 glycosidic bond. COS are water soluble in contrast to chitosan and chitin. COS carry positive charge, which allows COS to bind to negatively charged cell surface strongly. This property contributes to many physiological functions of COS, such as antitumor, immunostimulatory, and anti-inflammatory. Besides, COS stimulate the proliferation of Bifidobacteria bifidium and Lactobacillus sp. [26].

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Figure 3-37. Structure of FOS.

Other oligosaccharides Palatinose (6-O-α-D-glucopyranosyl-D-fructose), also referred as isomaltulose, is a reducing disaccharide identified during the processing of sugar from sugar cane. It is completely digested and provides the same caloric value as sucrose, but it is non-cariogenic and digested much slower, leading not only to a low glycemic response but also to a prolonged glucose supply, indicating its potential as a parenteral nutrient acceptable to both diabetics and non-diabetics. Its ingestion selectively promotes the growth of beneficial bifidobacteria amongst the human intestinal micro flora. It is more stable than sucrose, which facilitates the maintenance of its sweetness and taste in fermented foods and beverages. It has been suggested as a non-cariogenic alternative to sucrose, and as such is currently widely used as a sugar substitute in foods. This disaccharide has a sweet taste and very similar physical and sensory properties to sucrose [27]. Lactulose (4-O-β-D-galactopyranosyl-D-fructose) is a synthetic disaccharide and is also termed isomerized lactose, because it can be prepared from the isomerization of lactose. It is a Bifidus factor in nutrition and is a very important humanizing factor in infant formula and is added to commercial infant formula products and various milk products. This sugar has greater sweetness and solubility than lactose and if produced economically, it could widely be used in baking and confectionery applications [28].

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Starch and Glycogen Starch Starch is the major constituent of many foods and is the most important carbohydrate source for human nutrition. Corn, wheat, potato, sweet potato, and rice are the most important materials for starch production. Table 3-8 lists the contents of starch in some crops. Starch can be divided into two fractions: amylose and amylopectin. Natural starches are the mixture of amylose (10~20%) and amylopectin (80~90%). The percentages of amylose and amylopectin in starches derived from different sources are shown in Table 3-9. Gelatinization does not occur easily in starches with high amylose contents and the gelatinization temperature can reach up to 100 °C. However, gelatinized amylose is unstable and is prone to aging in contrast to the high stability of gelatinized amylopectin. Due to the unique physicochemical properties and nutritional functions, starches are of incomparable importance to human beings. Starch and its derivatives have been widely used as thickening agent, bonding agent and stabilizing agent and as the materials of pudding, soup, sauce, vermicelli, infant foods, pie, and mayonnaise. Chemistry structure Amylose is a linear polymer of α-D-glucopyranosyl residues connected through the 1→4 glycosidic bond. The number of repeated glucose subunits is usually in the range from 300 to 3000, but can be up to many thousands. For example, the polymerization degree of wheat starch is in the range 500~6000, while in potato it can rise up to 4500. Amylose also contain few α(1→6) bonds, accounting for 0.3%~0.5% of total glycosidic bonds. Based on X-ray diffraction diagrams, native starches can be divided into types A, B, and C. An additional form, called the V-type, occurs in swollen granules. While types A and B are real crystalline modifications, the C-type is a mixed form. The A-type is largely present in cereal starches, and the B-type in potatoes, amylomaize, and in retrograded starches. The Ctype is not only observed in mixtures of corn and potato starches, but is also found in various legume starches [29]. Table 3-8. Contents of starch in some materials (%) Species Unpolished rice Corn Barley Kidney bean Sweet potato (fresh)

Content 73 70 40 49 19

Variety Potato Wheat Sorghum Buckwheat noodles Pea

Content 16 66 60 72 58

Table 3-9. Percentages of amylose and amylopectin in some starches (%) Starch High amylose corn starch Corn starch Waxy corn starch

Amylose 50~ 85 26 1

Amylopectin 15~50 74 99

Starch Potato starch Cassava starch Wheat starch

Amylose 21 17 28

Amylopectin 79 83 72

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Type B amylose shows the left-hand double helices structure, which are packed in a parallel arrangement. Hydroxyl groups are located outside of the chains and the double helix is stabilized by the hydrogen bridges between amylose molecules. The internal channel of the helix is hydrophobic and hence can enclose only hydrophobic compounds, such as lipids. The A-type is very similar to the B-type, except that the central channel is occupied by another double helix, as shown in Figure 3-38. Amylopectin is the highly branched polymer of glucose. The backbone chain is composed of glucose units linked in a linear way with α(1→4) glycosidic bonds and branching takes place through α(1→6) bonds. In amylopectin, α(1→6) bonds account for 4%~5% of total glycosidic bonds.

Figure 3-38. Unit cells and arrangement of double helices (cross section) in type-A amylose (left) and type-B amylose (right) [29].

Due to the presence of multiple linear branches, amylopectin has multiple reducing ends and are hydrolyzed by enzymes faster than amylose. The branches of amylopectin are arranged in parallel or double helix, as shown in Figure 3-39. Amylopectin contributes to the crystalline structure of starch granules. The molecular weight of amylopectin ranges from 5×107~5×108. The ratio of amylopectin in starches generally exceeds 75% and the value can reach up to 99% in some species, such as waxy corn starch, as shown in Table 3-9. Starches derived from potato also contains a high phosphorus content (0.06~0.1%). Hence, potato starches are slightly negatively charged and swell more rapidly in warm water due to coulombic repulsion. Gelatinization Starch granule: Starches are present as granules in plant cells. Starch granules can be round, oval, and polygonal and their sizes range from 0.001~0.15 mm, depending on the plant

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species, of which, potato starch granules have the largest size and cereal starch granules have the smallest size, as shown in Figure 3-40. Polarization microscopy observation and X-ray diffraction find double refraction and X-ray diffraction in starch granules, indicating the presence of crystalline structure alternate layers of crystal region and amorphous region (Figure 3-41 I). About 70% of the mass of a starch granule is regarded as amorphous and about 30% as crystalline. The amorphous regions contain mainly amylose and the crystalline regions consist primarily of amylopectin. Amylose can enclose fatty acids and hydrocarbons due to the presence of the hydrophobic internal channel and the complexes are termed inclusion complexes. Amylose occur as double helices as in starch granules. Amylose and amylopectin are arranged radially in starch granules, as shown in Figure 3-41 II.

Figure 3-39. Structural models (I, II) for amylopectin with parallel double helices. III is an enlarged segment of I or II.

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Figure 3-40. Shapes of starch grains in electron microscope (×1200).A, Green bean starch (mean grain size: 0.016nm); B, Potato starch (mean grain size: 0.049nm; C, Common corn starch (mean grain size: 0.013nm); D, Sweet potato starch (mean grain size: 0.017nm).

Figure 3-41. Sketch of crystallization section and amorphous section of starches (I), and radial shape of amylose and amylopectin in starches (I, II).

Gelatinization: Due to the inter-molecular hydrogen bonding, starch is not soluble in cold water in spite of the abundance of hydroxyl groups. When the suspension is heated, the vibration of starch molecules increases and the hydrogen bonding between molecules is disrupted. As a result, more hydroxyl groups are exposed and starch-water hydrogen bonding is formed instead. With the diffusion of water into starch granules, many long chains are separated and the confusion degree increases markedly. Meanwhile, the number and size of

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6ISCOSITY"RABENDERUNITSX

crystal regions reduce significantly. When the suspension is further heated, the swelling becomes reversible. In this case, random coils are observed in amylopectin due to hydration and the ordered structure of starch is destroyed. This process is termed gelatinization and the temperature at which irreversible changes occur is called the gelatinization temperature. Starch gelatinization can be divided into three phases. In phase I, water diffuses into granules and absorbs to the polar groups in amorphous regions below the gelatinization temperature. The starch can restore to their original form if dehydrated. In phase II, when the gelatinization temperature is reached, bulk water enters starch granules and the granules swell significantly. Due to the increase of volume, the fraction between swollen starch increases and the suspension becomes viscous, which can be followed by Brabender amylograph. In this phase, water molecules enter the microcrystalline areas and the original arrangement is disrupted. When the temperature further increases, the viscosity of the suspension increases accordingly. In phase III, the swollen starch granules are disintegrated and the suspension viscosity drops sharply, as shown in Figure 3-42. It could be seen that the shape of the curve varies greatly for different starches. The gelatinization of starch is affected by the following factors: Water activity: Gelatinization occurs readily in medium with high water activity. The presence of high concentration of sugars significantly suppresses gelatinization, because the molecules can compete for water with starch.

4IMEMIN

Figure 3-42. Gelatinization properties of various starches. Brabender viscoamylograph. 40 g starch/460 ml water, temperature programming: start at 50 °C, heated to 95 °C at a rate of 1.5°C/min. Held at 95°C for 30 min – potato, - - - waxy corn, −−− normal corn, and ••• amylomaize starch.

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Helix amylose

Fatty acid Figure 3-43. Scheme of the lipid-starch inclusion complex.

Starch structure: Starches with higher ratio of amylose is less susceptible to gelatinization and their gelatinization temperature might reach up to over 100 °C. Salts: Salts in high concentrations suppress the gelatinization of starch, but those in low concentrations have no the inhibitory effect, except potato starch, because salts affect the charge carried on phosphorus groups. Lipids: Lipids can be enclosed inside the helix of starch (Figure 3-43). The enclosed lipids cannot be easily removed from the helix and can prevent the diffusion of water into starch granules. Hence, any lipids that can complex with starch can prevent starch swelling and gelatinization. The addition of C16~18 monoacylglycerol increases the gelatinization temperature, reduces the gelation temperature, and weakens the gel strength. pH: When the medium pH is lower than 4, starch is hydrolyzed to dextrins, which reduces the suspension viscosity. Hence, crosslinked starch instead of natural starch should be preferred as thickening agent in acidic foods. Starch gelatinization is not affected in the pH range 4~7. Starch gelatinization occurs rapidly in medium with pH greater than 10, but such high pH is not found in foods. Amylase: In the initial phase of gelatinization, starch granules start to absorb water and swell and amylase is not inactivated yet. Amylase can hydrolyze starch and accelerate gelatinization. This is why new rice is gelatinized more easily than old rice, because the former contains higher amylase activity. Retrogradation When a hot starch paste is cooled down, the suspension generally transforms to a viscoelastic gel and starch becomes insoluble. The process of soluble starch becoming insoluble is called retrogradation. The presence of conjunction zones in the gel indicates that retrogradation is actually the recrystallization of starch molecules. The qualities of many foods are deteriorated during retrogradation. For example, the staling of breads and the loss of viscosity of soups and sauces are partially caused by starch retrogradation. The retrogradation of starch is influenced by the following factors: Amylose to amylopectin ratio: Amylose is more susceptible to retrogradation due to its linear structure, of which, amylose with DP of 100~200 is the most resistant to retrogradation. Starch concentration: Retrogradation occurs readily in high starch concentrations due to frequent molecular collision, but water content of less than 10% hinders the process. The highest retrogradation rate is generally found in the water content of 30~60%. Presence of inorganic salts: Inorganic salts hinder the repositioning of starch molecules. The hindering effects of some common ions are SCN-, PO43-, CO3-, I-, NO1-, Br-, CI-, Ba2+, Ca2+, K+, and Na+ in the decreasing order.

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Medium pH: Starch undergoes retrogradation readily in the pH range 5~7 and the process is inhibited in alkaline or acidic solutions due to charge repulsion. Temperature: The optimum temperature for starch retrogradation is 2°C~4°C and retrogradation does not occur in temperatures above 60°C or below -20°C. Cooling speed: When starch paste is cooled slowly, starch molecules have sufficient time to align and retrogradation occurs readily. If the paste is cooled rapidly, the water in the paste crystallizes quickly, which prevents the approaching of starch molecules. Hence, retrogradation occurs slowly in rapid cooling. Presence of other ingredients: Lipids and emulsifying agents prevent retrogradation. Compounds such as glyceryl monopalmitate (GMP), other monoglycerides and their derivatives, and sodium stearoyl 2-lactylate (SSL) are often added into doughs of bread and other baked goods to prevent starch retrogradation and increase shelf life. Hydrolysis of starch Starches can be randomly hydrolyzed by acids and enzymes. Under mild conditions, starch is only partially hydrolyzed by acids. This process is called thinning and the products are termed acid-modified or thin-boiling starch. Acid-modified starch has increased gel transparency and strength and is less susceptible to retrogradation. Acid-modified starch has wide applications and can be used as film forming agent and adhesive agent in products such as pan-coated roasted nuts and candy. Besides, they are also used as encapsulating agents and flavor carriers. Enzymatic hydrolysis has been used in the production of commercially important syrups. In the production of high-fructose corn syrup, corn is firstly hydrolyzed by α-amylase and glucoamylase to obtain high purity D-glucose. D-glucose isomerase is then added and Dglucose is converted to D-fructose. The final product is the mixture of 58% D-glucose and 42% D-fructose. High-fructose corn syrup is commonly used as sweetener in soft drinks. The degree of hydrolysis of starch is measured by dextrose equivalency (DE), which is defined as the percentage of reducing sugar in the syrup. DE is related to the degree of polymerization and is calculated in the following equation:

(1) Glucose polymers with DE less than 20 are defined as maltodextrins and those with DE in the range 20~60 are termed corn syrup. Table 3-14 lists the functional properties of some hydrolysis products of starch. Modified starch Physical, chemical and biochemical modifications have been implemented to enhance the functional properties of starches and modified starches have gained wide applications in the food, pharmaceuticals, and chemical industry. Important modified starches that are widely used in the food industry are described below. Low viscosity starch: Starch of this type is also termed acid-modified starch. When starch is exposed to acid below the gelatinization temperature, hydrolysis occurs only in the

Carbohydrates

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amorphous region and the crystal region nearly remains intact. The modified starch produced under such conditions is not soluble in cold water but is readily soluble in boiling water. Compared with native starch, low viscosity starch has decreased viscosity and gel strength of hot paste and elevated gelatinization temperature. Low viscosity starch can be used as thickening and film forming agents. Pre-gelatinized starch: This product is obtained by heating starch suspension above the gelatinization temperature and then dried with drum drying, spray drying or extrusion. Pregelatinized starch can dissolve and form gel in cold water. Pre-gelatinized starch can be used in elder and infant foods, surimi products, ham, sausage, and bakery foods. Besides, pregelatinized starch has gained applications in cooking-free instant foods due to its solubility in cold water. Table 3-14. The functional properties of hydrolysis products from starch Properties enhanced by greater Properties enhanced in products of less hydrolysis A conversion B Sweetness Viscosity production Hygroscopicity and humectancy Body formation Freezing point depression Foam stabilization Flavor enhancement Sugar crystallization prevention Fermentability Ice crystal growth prevention Browning reaction Note: A high DE syrups; B low DE syrups and maltodextrins.

Etherified starch: All the three free hydroxyl groups in each D-glucopyranose unit can be etherified. Hydroxyethyl starches of low degree of substitution (DS) have reduced gelatinization temperature, increased swelling rate, and lowered tendency of pastes and gels to retrogradation. Hydroxyalkyl starches, such as hydroxypropyl starch, can be used in salad dressings, pie fillings, and other foods as thickening agent. Esterified Starch: Starch can be esterified by acidic orthophosphates, acid pyrophosphates, and tripolyphosphates to produce various starch esters. Compared with native starch, monostarch phosphates gelatinize in lower temperatures and those with DS greater than 0.07 can swell in cold water and have increased paste viscosity and transparency, and decreased retrogradation. These properties are quite similar to those of potato starch, which has high phosphorus content. Because monostarch phosphates have good freezing-thawing stability, they are preferred in frozen foods, such as frozen broth and frozen cream pie, as thickening agent to unmodified starch. Starch can also be esterified by various organic acids, such as acetic acid, long-chain fatty acids (C6~C26), succinic acid, adipic acid, and citric acid. These esters are superior in thickening and paste transparency and stability to native starch and can be used in bakery products, soup powder, sauce, pudding and frozen foods as thickening agent and stabilizer, as well as in dehydrated fruit as protective agent and in flavor processing as encapsulating material. Crosslinked starch: Starch can react with sodium trimetaphosphate, phosphorus oxychloride, epichlorohydrin, and acid anhydride to yield crosslinked derivates. Crosslinking

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prevents starch granule swelling and increases the stability against heating. Crosslinking by phosphorus increases the stability of swollen starch granules, but the paste of distarch phosphates is opaque in contrast to that of monostarch phosphate. Table 3-15. Comparisons of the properties of native and modified starches Starch

Amylose/Amylopectin ratio 1:3

Gelatinization temperature range (°C) 62~72

Properties

0:1

63~70

High-amylose starch Acidmodified starch Hydroxyethyl starch Monostarch phosphate Crosslinked starch

3:2-4:1

66~92

Variable

69~79

Low tendency of paste to retrogradation Less birefraction of granule than common starch granules Reduced viscosity of hot paste

Variable

58~68 (DS0.04)

Variable

56~66

Variable

Acetylated starch

Variable

Higher than unmodified starch, depending on the degree of crosslinking. 55~65

Common starch Waxy starch

Poor freezing-thawing stability

Increased paste transparency and decreased retrogradation Reduced gelatinization temperature and retrogradation. Reduced peak viscosity and increased paste stability Increased paste transparency and stability

Starches with high degree of crosslinking are very stable against high temperature, low pH and mechanical vibration and their gelatinization temperature is proportional to the degree of crosslinking. Some starches with high degree of crosslinking do not swell even in boiling water. Crosslinked starches are mainly used in infant foods, salad dressing, fruit pie filling and cream-style corn as thickeners and stabilizers. They also provide resistance to gelling and retrogradation, show good freeze-thaw stability, and do not undergo syneresis or weep on standing. Oxidized starch: Starch is hydrolyzed and oxidized when its aqueous suspension is incubated with sodium hypochlorite below the gelatinization temperature and the carboxyl group occurs once every 25 to 50 glucose residues in the resulting oxidation products. Oxidized starch has relative low viscosity even in high concentrations and is used as thickening agent in salad dressing and mayonnaise. Compared with low viscosity starch, oxidized starch is less susceptible to retrogradation or forming opaque gel. Table 3-15 lists the nature of a variety of starch before and after modification.

Glycogen Glycogen is also called animal starch and is the major storage carbohydrate in muscle and liver tissue of animals. Because glycogen accounts for only 0.02%~0.1% of fresh animal

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tissues, it is less important for the food industry than starch. Glycogen is much branched than starch and is similar to amylopectin in structure.

Cellulose and Hemicellulose Cellulose Cellulose is the major component of plant cell walls and often associates with hemicellulose, pectin, and lignin. The mode and degree of their association significantly affect the texture of plant-derived foods. Cellulose cannot be digested by the enzymes in human digestive tract and hence is a good source of dietary fiber. Cellulose is a linear homopolysaccharde consisting of D-glucopyranose units that are connected through β-D-1,4-glycodic bonds. Native cellulose contains both crystal and amorphous zones, of which, amorphous zone is more susceptible to solvent and chemical reagent action. This difference has been used in the preparation of microcrystalline cellulose, in which the amorphous zone is hydrolyzed by acids and the acid-resistance crystal zone is remained. The molecular weight of microcrystalline cellulose ranges from 30~50kDal and its tradename is avieol. Microcrystalline cellulose is insoluble in water and is often added to lowcalorie foods as fillings and rheology control agents. The degree of polymerization (DP) of cellulose varies with the plant origin and ranges from 1000~14000. Due to the large molecular weight and the presence of crystal structures, cellulose is insoluble in water and its swelling power or the ability of absorb water is poor or negligible. Carboxymethyl cellulose (CMC) CMC is the most widely used derivative of cellulose. It is obtained by treating alkaline cellulose with chloroacetic acid. The DS of commercially important CMC ranges from 0.3~0.9 and DP from 500 to 2000. CMC has long and rigid chains and carries positive charge. CMC solution is viscous and stable due to the electrostatic impulsion. However, these properties depend on the DS and DP of the product. Low-DS (≤ 0.3) CMC is insoluble in water but soluble in alkaline solutions, while high-DS (> 0.4) are water soluble. Besides, the solubility and viscosity are also affected by medium pH. CMC with DS of 0.7~1.0 is used to increase the viscosity of foods. Their aqueous solutions exhibit the characteristics of non-Newtonian fluid and the viscosity decreases with temperature elevation. CMC solution is soluble in pH range 5~10 and is most stable in pH 7~9. Monovalent cations form soluble complex with CMC, divalent cations reduces CMC solubility, whereas trivalent cations can cause gelling or precipitation of CMC. CMC can be used to improve the solubility of such food proteins as gelatin, casein, and soybean protein by complexing with these molecules. CMC can maintain the stability of the dispersion system of proteins even in their isoelectric points. Due to the excellent rheologic properties, safety, and indigestibility, CMC is added to jellies, paste fillings, spreadable process cheeses, salad dressings, and cake fillings as binding and thickening agent. Meanwhile, because CMC has strong water binding capacity, it is

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widely used in ice creams and other foods to prevent ice crystal formation. CMC also increases the volume and elongate shelf life of cakes and other bakery foods, improves the mouthfeel of sucrose and prevent CO2 escape from low-calorie carbonated beverage. Methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) MC is the etherified derivative of cellulose and is prepared by treating alkaline CMC with chloroform. The DS of commercially important MC lies in the range 1.1~2.2. MC is characterized by its gelling properties. When a MC solution is heated, the initial viscosity drops with rising temperature and then increases sharply. This can be explained by the breakdown of hydration layer around MC molecules upon heating, which increases the hydrophobic interactions between MC molecules. Electrolytes such as NaCl and nonelectrolytes such as sucrose and sorbierite reduce the gelling temperature of MC by competing for water molecules. MC cannot be digested by human body and is a calorie-free polysaccharide. HPMC is prepared by incubating cellulose with methyl chloride and cyclopropane in alkaline solutions. The DS of commercial HPMC ranges from 0.002 to 0.3. The initial viscosity of HPMC solution decreases with temperature elevation and the formation of gel is reversible at specific temperature, which is similar to that of MC. The gelling temperature and gel strength are related to the type of substation groups, DS and concentration of the soluble gel. The hydroxypropyl group stabilizes the hydration layer of HPMC and thereby increases the gelling temperature. Changing the proportion of methyl to hydroxypropyl substituents can vary the jelling temperature within a wide range. MC and HPMC increase the water retention and absorption capabilities of foods and prevent excessive oil absorption in fried foods. MC can be added to functional foods as fillings and dehydration and shrinkage inhibitors. The two derivatives are added to salad dressings as thickening and stabilizing agents and to various foods as edible coatings and fat substitutes.

Hemicellulose Hemicelluloses occur along with cellulose in the cell walls of plant cells and consist of multiple monomers in contrast to cellulose, such as xylose, mannose, galactose, rhamnose, and arabinose in addition to glucose. The compositions of hemicelluloses vary with the plant origin and tissue. For example, wheat and rye contain mainly arabinoxylans, while β-glucans predominate in barley and oats. In the food industry, hemicellulose is mainly added to bakery foods to increase the water binding capability of flour, improve the kneading quality of dough by reducing the energy required by kneading, and increase bread volume. Breads containing hemicellulose have delayed hardening time compared with those without hemicellulose. Hemicellulose is an important source of dietary fiber.

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Pectin Pectins are polymers consisting of D-galactopyranosyluronic acids connected through the α-1,4-glycodic linkage. In addition to galacturonic acid, pectins might also contain rhamnose, galatose, arabinose, and other sugars. Pectins are present in the cells and intercellular layers of plants. Pectins originated from various sources differ mainly in their contents of methoxyl groups or degrees of esterification (DE). The DE of pectins is defined as the percentage of the number of esterified D-galacturonic acid residues among all D-galacturonic acid residues. Pectins with DE greater than 50% are termed high-methoxyl pectin (HM) and those with DE less than 50% are called low-methoxyl pectin (LM). Protopectins are the highly methyl esterified and insoluble form of pectins and are found in immature fruits and vegetables. Pectins with low DS are called pectinic acids and can be converted from protopectins by the hydrolysis of protopectinase and pectin methyl esterase. Pectinic acids are either colloidal or soluble in water depending on the degree of polymerization and degree of methyl esterification, of which, water soluble pectinic acids are also referred as low-methoxyl pectin. Pectinic acids can be completely hydrolyzed by pectin methyl esterase to pectic acids. Various pectinases participate in the post-harvesting ripening of plants. During this process, protopectinase converts protopectins to colloidal or soluble pectinic acids. Pectinesterase removes methoxyl groups from pectins to produce poly-D-galacturonic acid or pectic acid, which is then hydrolyzed by polygalacturonase to yield D-galacturonic acid units. These enzymes work together in the ripening of fruits and are important for the texture formation of fruits and vegetables. Pectin is an important polysaccharide with applications in foods, pharmaceuticals, and a number of other industries. Its importance in the food sector lies in its ability to form gel in the presence of divalent ions such as Ca2+ or a solute at low pH. In the case of HM, gels are formed only in low pH and high sugar concentrations. Generally, HM concentration 1%, pH 2.8~3.3 and sucrose concentration 58%~75% facilitate the gelation of HM. In HM, the crosslinking of pectin molecules involves a combination of hydrogen bonds and hydrophobic interactions between the molecules. Low pH suppresses the dissociation of carboxylic groups and the loss of charges minimizes the electrostatic impulsion between HM molecules. Meanwhile, sugars compete for water molecules and reduce the solvation of HM chains, facilitating the hydrogen bonding between HM molecules and consequently their gelation. HM gels can maintain their original properties even when heated at 100 °C. The strength of pectin gels is positively proportional to the molecule weights and intermolecular association of pectins. Generally, the gelation time increases as the degree of methyl esterification increases from 30%~50%, because the raise of carbomethoxy groups increases the steric hindrance for hydrogen bonding between pectin molecules. Pectins with degree of methyl esterification in the range 50%~70% have enhanced hydrophobic interactions and therefore gel in shorter time. The gelling characteristics of pectins are the function of degree of methyl esterification, as shown in Table 3-16. In the case of LM, the presence of divalent ions, such as Ca2+, is a prerequisite for their gelation and the ions function as the bridge between LM molecules. Increasing the concentration of Ca2+, which is the only divalent cation allowed in the food industry, increases the gelling temperature and gel strength of LM, which is similar to the role of Ca2+ in the formation of the egg-box structure in alginate gels. LM is not as sensitive to pH as HM and can gel in a wider pH range 2.5~6.5. Though sucrose is not a prerequisite for LM

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gelation, the addition of 10%~20% sucrose markedly improves the texture of gels. LM gels are less rigid or elastic than common pectin gels, but sugars or plasticizing agents improve the properties. LM pectin, since it does not require sugar for gelation, is used to make dietetic jams, jellies, and marmalades.

Agar Agar is a gelatinous polysaccharide extracted from some species of Rhodophyceae by a hot water extraction process.

Structure and Properties Agar is a linear heteropolysaccharide consisting of alternate β-D-galactopyranose and 3,6-anhydro-α-L-galactopyranose that are connected through 1→4 and 1→3 linkages respectively. Table 3-16. Effects of degree of methyl esterification on the gelling properties of pectin Degree of methyl esterification >70 50~70 <50

Gelling conditions pH Sugar concentration (%) 2.8~3.4 65% 2.8~3.4 65% 2.5~2.6 None

Presence of divalent ions No No Yes

Rate of gelation High Low High

The hydroxyl groups on the chains are more or less esterified by methyl, sulfuric acid, and pyruvic acid. Commercial agar is colorless or light yellow strips or powder. Agar is insoluble in cold water, but dissolves slowly in hot water. The β-1,4-glycodic bonds can be specifically hydrolyzed by the agarlytic enzymes derived from soil and marine organisms to yield agar oligosaccharide. Figure 3-44 illustrates the structure of agarose.

Figure 3-44. Structure of agarose.

Carbohydrates

Figure 3-45. Molecular structures of the seven carrageenan classes.

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Application Agar is a most potent gelling agent in the food industry and is widely used in candies, puddings, ice cream, gelly, and many other foods as gelling agent. Besides, agar is indigestible and is a good source of dietary fiber. In the fermentation industry, agar has been widely used to encapsulate enzymes and microbes.

Carrageenan Carrageenans are mixtures of several related galactans having sulfate half-ester groups attached to the sugar units and are extracted from the red seaweeds (Rhodophyceae). Carrageenans are important commercial hydrophilic gels. Carrageenans are linear chains of D-galactopyranosyl units joined with alternating (1,3)D- and (1,4)-D-glycosidic linkages, with most sugar units having one or two sulfate groups esterified to a hydroxyl group at carbon atoms C-2 or C-6. The molecular weights (MWs) of carrageenan range from (1~5)×105 and the average MW of food-grade carrageenans is 2×105. According to the positions of sulfate half-ester groups, carrageenans are divided into κ-, ι-, λ-, μ-, υ-, θ-, and δ- classes. Figure 3-45 presents the structures of the 7 carrageenan classes. Table 3-17. Properties of κ-, ι-, and λ- carrageenans Item Solubility

Condition Hot water

κ-carrageenan Soluble above 70°C

Cold water

Sodium salt soluble Na+ salt soluble, From limited to high swelling of K+, Ca2+ and NH4 salt Soluble Insoluble

Hot milk Cold milk

Gelling properties

Miscibility

ι-carrageenan Soluble above 70°C Ca2+ salt gives thixotropic dispersions

λ-carrageenan Soluble

Soluble Insoluble

All salts soluble

Cold milk (Tetrasodium pyrophosphate) Concentrated sugar solutions Concentrated salt solutions Organic solvents Cations

Thickens or gels

Thickens or gels

Soluble hot

Difficulty soluble

soluble Disperses with thickening Increased thickening or gelling Soluble hot

Insoluble cold and hot

Soluble hot

Soluble hot

Insoluble Hard gel with K+

Insoluble Non-gelatinous

Gel type

Rigid with syneresis

Insoluble Strong gel with Ca2+ Elastic without syneresis Non-synergetic

Non-gelatinous

Effect of locust bean Synergetic Non-synergetic gum Stability against Stable Stable Stable neutral and alkaline conditions Stability in pH3.5 Hydrolysis, accelerated Stable hydrolysis solutions upon heating; gel state Miscible with nonionic and anion surfactants, immiscible with cation surfactants

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Carbohydrates

Structure and Properties Commercial carrageenans are odorless, colorless or light yellow powder. Carrageenans have strong gel forming ability and the gel is heat reversible, that is, formed gels can be transformed to solutions by heating or vice versa upon cooling. All the carrageenans are soluble in hot water or hot milk, but their potassium and calcium salts only absorb water in cold water without dissolution. Carrageenans are not soluble in methanol, ethanol, propanol, isopropanol, or acetone. Of the seven classes, κ-, ι-, and λ- carrageenans are commercially important for the food industry and their properties are summarized in Table 3-17. Applications in the Food Industry Carrageenans have been used as nature food additives for hundreds of years. Carrageenan utilization in food processing is based on the ability of the polymer to gel, to increase solution viscosity and to stabilize emulsions and various dispersions. A level as low as 0.03% in chocolate milk prevents fat droplet separation and stabilizes the suspension of cocoa particles. Carrageenans prevent syneresis in fresh cheese and improve dough properties and enable a higher amount of milk powder incorporation in baking. The gelling property in the presence of K+ salt is utilized in desserts and canned meat. Protein fiber texture is also improved. Protein sedimentation in condensed milk is prevented by carrageenans which, like κ-casein, prevent milk protein coagulation by calcium ions. Carrageenans are also used to stabilize ice cream and clarify beverages. Besides, because carrageenans are cheaper than agar, they have replaced agar in some foods. The applications are summarized in Table 3-18. Table 3-18. Applications of carrageenans in the food industry Food Ice cream Chocolate milk Milk Processed cheese Infant formulations Milk pudding Milk shake Acidic dietary products

Function Ice cream formation and syneresis inhibition Coca suspension Smoothing and texture modifier Syneresis inhibition, shape retention, and curd formation reduction Fat and protein stabilization Gelling agent and texture modification Ingredients suspending and texture modification Texture modifier and

Food Dessert gels

Function Gelling

Fruit drinks Bread

Fruit particles suspension and gelling Water retention increasing, staling delaying Texture modification

Pie

Seasonings Canned foods

Ingredient suspension and shape retention Gelling and fat stabilization

Meats

Syneresis prevention and bonding

Beer

Clarification and stabilization

Alginate Alginate, also termed algin or alginic acid, is an anionic polysaccharide obtained from brown seaweeds. Commercial alginate is often available as sodium salt.

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Structure and Properties Alginates are linear copolymers composed of -D-mannopyranosyluronic acid and -Lgulopyranosyluronic acid units, which are joined by 1→4 linkages. The ratio of the two sugars (mannuronic/guluronic acids, M/G) depends on the source and is generally 1.5. Pure alginates are colorless and amorphous and are insoluble in pure water, ethanol, carbon tetrachloride or other organic solvents. Alginates can absorb much water and swell in pH 5.8~7.5 to produce transparent and viscous solution. Dissolved alginates can be precipitated by acid or calcium salts, which are the methods for the industrial production of alginates. The presence of proteins, sugars, salts, glycerol, and trace amount of starch and phosphates has no effect on the solubility of sodium alginate. Alginate solutions are very viscous and the specific viscosity depends on alginate molecular weight. Generally, the higher the molecular weight, the higher the viscosity of alginate solutions. The viscosity of alginate solutions increases markedly with the increase of alginate concentration. When alginate concentration exceeds 3%, solutions lose the fluidity. High temperature reduces alginate viscosity. When the temperature increases by 1°C, the viscosity decreases by about 3%. When the temperature exceeds 80°C, decarboxylation reaction occurs and the viscosity drops dramatically. Alginates are liable to decreases of degree of polymerization and viscosity due to the effects of temperature, light, metal ions, and microbes during storage. Alginates are soluble in solutions with pH greater than 5.8. When the pH decreases to below this value, the solubility decreases and gel is formed. When a calcium salt is added to alginate sodium solution, the calcium ion replaces the sodium ion and gelling occurs. Hence, alginate gels can be formed by adding either Ca2+ or acids. Applications in the Food Industry Alginate is a powerful thickening, stabilizing and gel-forming agent. It has gained wide applications in the food industry, as listed in Table 3-19. Table 3-19. Properties of alginate and its major application in foods Application Cold drinks and snacks (ice cream) Jams, mayonnaise, tomato paste, seasoning dressing Noodles, instant noodle, macaroni, bread Jelly foods (meat jell, fish jell) Wines (beer, white wine, fruit wine) Candies (cerealose, chocolate)

Property utilized Thickening, hydratability, reaction with Ca2+ Gelling, thickening, and emulsifying Gelling, thickening, acid resistance Gelling Foam stabilizing, coagulating, clarifying Thickening, bonding

Meat emulsion, fish emulsion

Stabilizing, bonding

1

Note: PGA, propyleneglycol alginate.

Alginate type Sodium alginate, calcium alginate, PGA1 Sodium alginate, calcium alginate, PGA PGA, sodium alginate PGA, sodium alginate PGA, sodium alginate Sodium alginate, calcium alginate, PGA Sodium alginate, PGA

Carbohydrates

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Chitin and Chitosan Chitin is the second most abundant natural polysaccharide in the world and its annual product is 1×1011 tones, which is only behind that of cellulose. It is also the most abundant naturally occurring polysaccharide that contains amino sugars. Chitosan is the deacetylated products of chitin and has improved solubility and functional properties. Their abundance, combined with their specific properties and functions, impart them with wide applications in the food industry.

Structure and Properties Chitin is a linear polymer consisting of N-acetylglucosamine connected through β-1,4 linkage. The general formula of chitin is (C8H13NO5)n and the molecular weight is of the magnitude 106. Chitin is white or gray amorphous powder and decomposes at 270°C. Chitin is insoluble in water, ethanol, diluted acid or diluted alkaline solutions and is soluble in only several solvents, including hexafluoroacetone, trichloroacetic acid-dichloropropane, ketopyrrolidine-lithium chloride, and some chlorohydrins. Chitin can be hydrolyzed by concentrated inorganic acids. Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked Dglucosamine and N-acetylglucosamine (acetylated unit). It is obtained by the deacetylation of chitin. Commercial chitosan is white or gray amphorous powder and decomposes at 185°C. Chitosan is insoluble in water, diluted alkaline solutions or some diluted acid solutions, for example, sulfuric acid, nitric acid, and phosphorous acid, but soluble in diluted organic acids and some inorganic acids such as hydrochloric acid. Chitosan carries positive charges and is hence a polar polymer. Applications of Chitosan in the Food Industry Antibacterial agent Chitosan and its derivates have strong antibacterial activities. Chitosan carries positive charges and can adhere to negatively charged membranes of bacteria, leading to the leakage of intracellular proteins and other components. Fruit preservative Chitosan can prevent the spoilage and decay of peach, kiwi fruit, cucumber, pepper, and strawberry and markedly increase their shelf lives. This is because chitosan has strong film forming capability. The film formed hinders the diffusion of oxygen to fruits and the escape of CO2 generated by respiration, but allows the release of ethylene. Antioxidants It has been reported that the addition of chitosan at 1% resulted in a 70% decrease in the 2-thiobarbituric acid (TBA) values of meat after 3 days storage at 4 °C. The mechanism by which this inhibition takes place is believed to be related to the chelation of free iron released

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from the hemoproteins of meat during heat processing or storage. This would in turn inhibit the catalytic activity of iron ions [30]. Functional ingredients After chitosan is absorbed, it can form indigestible complexes with triglycerides, fatty acids, bile acid, and cholesterol. To complement for the bile acid that is associated with chitosan, the liver increases the secretion of bile acid. Since bile acid is converted from cholesterol in the liver, the content of cholesterol in liver and blood is deceased. Hence, chitosan is an effective ingredient of functional foods. Juice clarificant Fruit juices become turbid after long-time storage due to the presence of negatively charged pectin, cellulose, tannin, and pentosan. The positively charged chitosan can coagulate these components by electrostatic attraction and hence is widely used in fruit juice clarification. Chitosan has high affinity to polyphenolic compounds, such as catechin and cinnamic acid. Chitosan has been added to grape wines to remove these components. Chitosan-treated grape wines are dark golden yellow compared to the light yellow of untreated products and hence have improved quality. Water clarificant Chitosan is more effective in removing polychlorobiphenyl (PCB) than active carbon. Chitosan in combination with bentonite can effectively remove particles, color, and odor of drinking water and that in combination with polysilicate, polyaluminosilicate, and ferric chloride markedly reduces the COD and turbidity of water.

Applications of Chitooligosaccharides in Food Industry Chitooligosaccharide (COS) is the partial hydrolysis products of chitin and chitosan and its molecular weight is generally less than 104. COS has much better solubility than chitin and chitosan and has been found to posses various physiological functions. COS has great potential applications in the food industry. Micro-ecological regulator COS is a Bifidobacterium proliferative factor and can selectively enhance the growth of intestine probiotics and inhibit the proliferation of harmful bacteria and formation of spoilage substances. Functional sweetener Chitobiose and chitotriose have pleasant sweetness and are superior to sucrose in thermostability. The two compounds cannot be digested by human body and are ideal functional sweeteners for the diets of diabetic and fat patients.

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Antibacterial agent The antibacterial activity of COS is related to its molecular weight and medium pH. It is generally recognized that COS of molecular weight 1500 has the highest antibacterial activity. Medium pH affects the dissociation of amino groups and therefore influences the antibacterial activity. COS is more effective in inhibiting microbial growth in acidic medium than in neutral or alkaline medium. Short-chain COS, such as those with seven or more monomers, can enter inside microbial cells and inhibit the biosynthesis of mRNA and proteins by binding to DNA, thus killing the microbes. COS is more effective in inhibiting the growth of molds in soy sauce than benzoic acid and sodium benzoate without affecting the taste and color of soy sauce. Preservative COS has abundant free amino and hydroxyl groups and therefore has strong water retention capability. The mixture of COS and high-molecular weight chitosan with strong film forming ability has been used as fruit and vegetable coatings to prevent water evaporation and Vc loss and elongate the shelf life. COS also significantly reduces the spoilage rate of fruits and vegetables as chitosan. Calcium absorption enhancer COS of DP 3~7 reduces the excretion of fecal calcium and increases the fracture force of rat thighbone in contrast to chitosan, which hinders the absorption of calcium.

Guar Gum and Locust Bean Gum Guar gum and locust bean gum are important thickening polysaccharides and are widely used in the food and other industries. Guar gum produces the highest viscosity of all natural and commercial gums. Guar gum is also known as guaran and occurs in the endosperm of the seeds of guar bean (Cyamopsis tetragonolobus). It is composed of galactose and mannose and the backbone is a linear chain of β-1,4-linked mannose residues to which galactose residues are 1,6-linked at every second mannose, forming short side-branches. The structure of guar gum is shown in Figure 3-46. Guar gum readily absorbs water and is rapidly hydrated in cold water to produce highly viscous and thixotropic fluids. The viscosity of guar gum solutions is affected by temperature and the presence of other components. Heating accelerates the dissolution of guar gum, but it can be decomposed in high temperatures. Because guar gum yields very high viscosity even in low concentration, the addition of guar gum in foods is generally less than 1%. The aqueous solution of guar gum is neutral and its viscosity is not affected by pH. Hence, guar gum is compatible with most food components. Ionic strength has little effects on the viscosity of guar gum solutions, but the presence of high concentrations of sucrose

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reduces the viscosity and delays the arrival of the peak viscosity. Wheat starch synergistically improves the viscosity of guar gum solutions. Guar gum prevents ice crystal growth and produces desired consistency, chewiness, and heat stimulation resistance in ice cream. In cheese, guar gum is added to avoid syneresis. In bakery foods, guar gum elongates the shelf life and reduces the water absorption of sucrose in the glace of pastries. Locust bean gum, also called carob gum, occurs in the seeds of carob tree (Ceratonia siliqua). It structure is similar to that of guar gum, except that the galactose residues are distributed randomly along the β-1,4-linked mannose backbone. The ratio of mannose/galactose residues of locust bean gum ranges from 3:1 to 6:1 and its molecular weight is about 31000. Due to the irregular distribution of galactose residues, some mannose residues are exposed or naked without attached galactose residues. Due to this difference, locust bean gum has different properties compared to guar gum. For example, the viscosity of locust bean gum solutions is lower than that of guar gum solutions. Locust bean gum is used as a thickener, binder and stabilizer in canned meats, salad dressings, sausages, soft cheeses, and ice creams. It also improves the water binding capacity of dough especially in the case of low-gluten flour. Locust bean gum can improve the viscosity of other gelatinous polysaccharides. For example, the viscosity of the solution containing 0.5% agar and 0.1% locust bean gum is 5 times higher than that of agar alone.

Gum Tragacanth Gum tragacanth is a plant exudate collected from the plants of the Astragalus genus. The gum has been used for over 2000 years as Arabic gum and is mainly produced in Iran, Syria, and Turkey. Gum tragacanth has a very complex structure and composition. When gum tragacanth is suspended in water, one fraction is dissolved and this part is called tragacanthic acid. Tragacanthic acid accounts for 60%~70% of gum tragacanth and consists of 43% Dgalacturonic acid, 10% fucose, 4% D-galactose, and 40% D-xylose and L-arabinose, of which, the galacturonic acid residues form the backbone and the other residues are attached as side chains. The molecular weight of tragacanthic acid is about 800000. The insoluble fraction is termed bassorin, which contains 75% L-arabinose, 12% D-galactose, and 3% Dgalacturonase and L-rhamnose with molecular weight of 84000.

Figure 3-46. Structure of guar gum.

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Gum tragacanth produces high viscosity and hence is used as thickening agents. Gum tragacanth is used as a thickening agent and a stabilizer in salad dressings (0.4–1.2%) and in fillings and icings in bakery goods. As an additive in ice creams (0.5%), it provides a soft texture.

Microbial Polysaccharides Microorganisms can produce multiple polysaccharides, of which, dextran and xanthan are the most important for the food industry. Dextran is an extracelluar polysaccharide produced by Leuconostoc mesenteroides, Streptobacterium dextranicum, Streptococcus mutans and other species. Dextran is composed of glucose units, of which, the monomers in the straight chain are connected through α-1,6 linkages, while branches begin mainly from α-1,3 linkages (Figure 3-47), with part 1,4- and 1,2- linkages. The type and number of the glycosidic linkages vary with the source. The dextran produced by Leuconostoc mesenteroides NRRL B512 contains 95% 1,6- linkages and the remaining 5% are 1,3- and 1,4- linkages. Dextran is used in confections to improve viscosity and water retention and inhibit sugar crystallization. In chewing gum and soft sweets, dextran is added as gelling agent. Dextran inhibits ice crystal formation in ice creams and provides desired consistency and mouthfeel for puddings.

Figure 3-47. Structure of dextran.

Figure 3-48. Structure of xanthan.

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Xanthan is secreted by multiple species of the Flavobacterium genus and the commercial xanthan is produced by Xanthomonas campestris. Xanthan can be considered as derivative of cellulose. On an average, every second glucose residue bears in the 3-position a trisaccharide of the structure β-D-Manp- (1→4)-β-D-GlcpA(1→2)-α-D-Manp as the side chain. The mannose bound to the main chain is acetylated in position 6 and ca. 50% of the terminal mannose residues occur ketalized with pyruvate as 4,6-O-(1-carboxyethylidene)-D-mannopyranose (Figure 3-48). The molecular weight of xanthan is greater than 2×106 Dal. Xanthan is readily soluble in both cold and hot water and provides high viscosity even in low concentrations, but the jellification is weakened in high xanthan concentrations. Xanthan solutions are pseudoplasitc fluids and exhibit obvious shear thinning. Xanthan is compatible with most edible salts and edible acids and can coexist with them in foods. Xanthan interacts with guar gum to give rise to a synergistic increase in solution viscosity and with locust bean gum to produce a heatreversible gel. Xanthan is widely used to stabilize aqueous dispersions, suspensions, and emulsions. Xanthan is added to juices and canned foods as suspending and stabilizing agents. In starch gels, the addition of xanthan gum substantially improves their freeze-thaw stability and reduces syneresis. Due to the high stability of xanthan gels, xanthan can be used in seasonings with high salt contents or acidity.

Glucomannan Glucomannan is slightly branched polymer consisting of β-(1→4)-linked D-mannose and D-glucose in a ratio of 1.6:1. Short side chains of 11-16 monosaccharides occur at intervals of 50-60 units of the main chain attached by β1→3 linkages. Some native glucomannan are partially acetylated at C-2 and C-3 of certain of mannose residues [31]. The weight-average molecular weights range from 105~106 and depend on source. Glucomannan is soluble in water to produce highly viscous pseudoplastic fluids. In alkaline conditions, glucomannan is deaceylated and aggregates to form the threedimensional network and strong thermo-irreversible gel [32]. However, glucomannan can interact with xanthan to yield thermo-reversible gel. Glucomannan has strong hydrophilicity, gelation capacity, and film-forming ability. Glucomannan is added to jellies, jams, confections, dietary products, ice creams, meat products, and breads as thickening and stabilizing agents.

Gum Arabic Gum Arabic is extracted from the exudate of various Acacia species, primarily Acacia senegal. Gum Arabic is a proteoglycan and its molecular weight lies in the range 260000~1160000. The glycan fraction accounts for 70% of the total gum and consists of Larabinose, D-galactose and D-glucuronic acid in the molar ratio of 3.5:1.1:2.9:1.6. The backbone of gum Arabic consists of β-D-galactopyranosyl residues linked by 1→3 bonds and side chains are attached at position 6 (Figure 3-49). Gum Arabic is present as acidic or neutral salts and the counter ions are Ca2+, Mg2+, and K+. The protein fraction accounts for 2% of the

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total gum, but the value can reach up to 25% in some species. The glycan fraction is covalently connected to the hydroxyproline and serine residues of the protein fraction. Gum Arabic is readily soluble in water and provides low viscosity, but the viscosity increases dramatically in high gum Arabic concentrations. This property is different from other polysaccharides, which can provide high viscosity even in low concentrations. The solubility of gum Arabic reaches up to 50% by weight. Arabic gum solutions with concentration less than 40% are Newtonian fluids, but those with higher concentrations exhibits the pseudoplastic behavior. In the presence of ions, the viscosity of gum Arabic solutions varies with pH and the highest viscosity is obtained at pH 6~8. The viscosity reduces proportionally when the valence and concentration of ions increases. Gum Arabic is compatible with most gums, except gelatin and sodium alginate.

Figure 3-49. Structure of the glycan fraction in gum Arabic.

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Gum Arabic is used as emulsifier and stabilizer. It retards sugar crystallization and fat separation in confectioneries and large ice crystal formation in ice creams, and can be used as a foam stabilizer in beverages. Gum Arabic is also applied as a flavor fixative in the production of encapsulated, powdered aroma concentrates. For example, essential oils are emulsified with gum Arabic solution and then spray-dried. In this process, the polysaccharide forms a film surrounding the oil droplet, which protects the oil against oxidation and other changes.

DIETARY FIBER Dietary fibers (DF) were once considered as nonnutritive crude fibers and unimportant for human diets. However, with the rapid improvement of social economy and people's living standards, people‘ dietary structure changes greatly. The proportion of plant-derived foods in diets decreases obviously and that of animal-derived foods containing high calorie, high protein content and high fat contents increases significantly instead, which impairs the nutrition balance of the diets. Due to the imbalance, the incidences of adiposity, hypertension, diabetes, cancer, and cardiovascular diseases keep rising. The emergency of these diseases has been contributed to the decreased intake of DF and DF has been widely accepted as the seventh essential nutrient.

Structure and Properties Definition Various definitions of dietary fibers have been proposed. In 1987, FDA and WHO defines dietary fiber as ―the material isolated by AOAC method 985.29‖. In 1998, dietary fiber was defined by a committee of dietary fiber experts of the American Association of Cereal Chemists (AACC) as ―the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary fiber includes polysaccharides, oligosaccharides, lignin, and associated plants substances. Dietary fibers promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood glucose attenuation‖. The definition proposed by AACC has been widely accepted in the world. Classification By Solubility Based o solubility, DF is subdivided into soluble DF (SDF) and insoluble DF (IDF). SDF is soluble in warm and hot water and absorb water to gel. Dissolved SDF can be precipitated by 4 volumes of ethanol. SDF mainly includes pectins, gums (gum Arabic, guar gum, locust bean gum, and agar), mucilages, and some hemicelluloses. IDF is insoluble in hot water and is the important component of plant cell wall, including cellulose, hemicellulose, ligin, protopectin, vegetable wax and some animal-derived compounds, such as chitin and chitosan.

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SDF and IDF have different physiological and health functions. IDF cannot be metabolized by human body, but can absorb water as it moves through the digestive system, thus easing defecation. SDF can be partially fermented by the microorganisms inhabited in the colon and produce active byproducts. SDF protects against cholesterol gallstone [33], increase heavy metal evacuation, reduce cholesterol levels in serum and liver, inhibit postprandial blood glucose increase, and prevent hypertension and heart disease. IDF increases defecation and prevents obesity, laxation, and intestinal cancers. The ratio of IDF to SDF markedly affects the physiological functions of DF.

By Source DF can be obtained by extraction from plants (including algae) and animals or by synthesis, of which, plants are the major source at present. Chitin and chitosan are typical representatives of animal-derived DF. Dextran is a water soluble DF. In addition to microorganism, it is also synthesized from dextrin by transglucosylation of dextrin dextranase (EC 2.4.1.2) [34]. Physichemical Properties Solubility and viscosity DF molecules with ordered and less branched structures have strong inter-molecular bonding capacities and thereby poor solubility. For example, the linear cellulose is insoluble, but pectins with irregular structure are readily water soluble. Insoluble DF can be converted to soluble DF by high temperature, high pressure, or shearing force. Conversion of IDF to SDF is an important measure to improve DF quality. SDF are viscous upon ingestion and can complex with heavy metals, fats, and cholesterol. Water retention Most DFs contain abundant hydrophilic groups and can absorb much water. The water retention capacity of DFs varies with the source, composition, structure, preparation method, and particle size. Generally, cellulose-based DFs have lower water retention capacity compared with other DFs. Too low particle sizes reduce the index. Besides, high temperature treatment, cooking, and enzymatic hydrolysis could change the physical properties of DFs and thereby influence the water retention capacity. Organic compounds absorption The abundant active groups on the surface of DF can absorb bile acid, cholesterol, and mutagens in the intestines of human body and therefore affects their metabolism and adsorption. Cation binding and ion exchange

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Some DFs contains carboxyl groups, hydroxyl groups, amino acids, and some other active groups. Various cations, such as Ca2+, Fe2+, Zn2+, Cu2+, and Pb2+, can bind to these groups or exchange with the ions attached to these groups. The binding or exchange is reversible and changes the pH, osmotic pressure, and redox potential of the intestine and provides a buffering environment that is beneficial for digestion and absorption. Cations with high polarizability, such as Pb2+, are preferred in absorption by DF. Hence, DF has the function of removing toxic metals. The ions bound to DF can exchange with Na+ and K+. The exchange reduces the Na+/K+ ratio in the blood and consequently lowers the blood pressure. It should be noted that DF also reduces the adsorption of some beneficial elements. Hence, it is necessary to supplement some minerals in DF-containing foods to avoid mineral imbalance. Prebiotics DFs are only partially fermented in the colon. DFs are the nutrition source of the microbes in the colon and support the growth of multiple bacteria, of which, most are beneficial for human body. Besides, DFs can induce the substantial propagation of aerobes and thereby reduces the incidence of some cancers. Bulking effect DFs have markedly increased volume after swelling and can cause satiety. Besides, the presence of DF affects the digestion and absorption of other food components and delays the feeling of hunger. Hence, DFs are effective in preventing adiposity.

Metabolism As mentioned in the definition, DFs cannot be digested in the mouth, stomach, or small intestine and only partially fermented by the microbes in the large intestine. The degree and rate of decomposition are associated with the solubility, chemical structure, particle size, and ingestion mode of DF. Soluble DFs, such as pectin and seaweed gums, are readily digested in the large intestine, but insoluble cellulose cannot be easily utilized by intestinal microorganisms. Some of the metabolites, such as fatty acids, can be absorbed by human body as energy source and some metabolites have important physiological functions.

Safety Aspects Although DFs have multiple beneficial effects on human body, excessive intake of DFs can cause discomfort and affect the absorption of fats, proteins, and essential elements. It should be mentioned that these effects vary with DF composition and individuals. Some IDFs, such as guar gum, can be fermented by intestinal microorganisms to produce volatile fatty acids, carbon dioxide, and methane, which can cause abdominal distension. The filling effect of DFs reduces the adsorption of proteins, fatty acids, and carbohydrates and the increased viscosity of gastrointestinal fluids prevents the contact between these substrates

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with digestive enzymes. Hence, DFs reduce their bioavailability. DFs can bind metal ions and can reduce their absorption. Some DFs interfere with the absorption of vitamins. It is reported that konjac mannan reduces fat-soluble vitamin absorption by removing bile acids, but does not reduce fatinsoluble vitamin absorption in the intestine [35]. No uniform standards have been formulated for the daily intake of DFs due to the complexity of DFs and individual differences. The daily intake recommended by FDA is 20~35g for adults and that in Japanese is 20 g or more. The Chinese Nutrition Association enacted the Chinese dietary intake references in 2000 and suggests that the daily dietary fiber intake of adults is 30.2 g.

REFERENCES [1]

Terta, MG; Blekas, G; Paraskevopoulou, A. Retention of selected aroma compounds by polysaccharide solutions: A thermodynamic and kinetic approach. Food Hydrocolloids, 2006, 20, 863-871. [2] Naknean, P; Meenune M. Factors affecting retention and release of flavour compounds in food carbohydrates. International Food Research Journal, 2010, 17, 23-34. [3] Hodge, JE. Dehydrated Foods, Chemistry of Browning Reactions in Model Systems. Journal of Agricultural and Food Chemistry, 1953, 1, 928-943. [4] Namiki, M; Hayashi, T. Development of novel free radicals during the amino-carbonyl reaction of sugars with amino acids. Journal of Agricultural and Food Chemistry, 1975, 23, 487-491. [5] Arnoldi, A., et al., New Colored Compounds from the Maillard Reaction between Xylose and Lysine. Journal of Agricultural and Food Chemistry, 1997, 45, 650-655. [6] Hofmann, T. Characterization of the Chemical Structure of Novel Colored Maillard Reaction Products from Furan-2-carboxaldehyde and Amino Acids. Journal of Agricultural and Food Chemistry, 1998, 46, 932-940. [7] Cämmerer, B; Jalyschko, W; Kroh LW. Intact Carbohydrate Structures as Part of the Melanoidin Skeleton. Journal of Agricultural and Food Chemistry, 2002, 50, 20832087. [8] Franzke, C; Iwainsky, H. Antioxidant activity of melanoidins. Dtsch. Lebensm. Rundsch., 1954, 50, 251-254. [9] Elizalde, B; Rosa, M; Lerici, C. Effect of maillard reaction volatile products on lipid oxidation. Journal of the American Oil Chemists' Society, 1991, 68, 758-762. [10] Bedinghaus, AJ; Ockerman, HW. Antioxidative Maillard Reaction Products from Reducing Sugars and Free Amino Acids in Cooked Ground Pork Patties. Journal of Food Science, 1995. 60, 992-995. [11] Yamaguchi, N. Antioxidant activity of the oxidation products prepared from melanoidins. Developments in Food Science, 1986, 291-299. [12] Yoshimura, Y., et al., Antioxidative Effect of Maillard Reaction Products Using Glucos- lycine Model System. Journal of Agricultural and Food Chemistry, 1997, 45, 4106-4109.

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[13] Morales, FJ; Jiménez-Pérez, S. Free radical scavenging capacity of Maillard reaction products as related to colour and fluorescence. Food Chemistry, 2001, 72, 119-125. [14] Plakas, SM; et al., Effect of Maillard Browning Reaction on Protein Utilization and Plasma Amino Acid Response by Rainbow Trout (Salmo gairdneri). The Journal of Nutrition, 1985, 115, 1589-1599. [15] Whitelaw, ML; Weaver, CM. Maillard Browning Effects on In Vitro Availability of Zinc. Journal of Food Science, 1988, 53, 1508-1510. [16] Nagao, M., et al., Mutagens in coffee and tea. Mutat Res, 1979, 68, 101-106. [17] Martins, SIFS; Jongen, WMF; van Boekel, MAJS. A review of Maillard reaction in food and implications to kinetic modelling. Trends in Food Science and Technology, 2000, 11, 364-373. [18] Alink, GM; van Boekel, MAJS; Jongen, WMF. Mutagenicity of Heated Sugar-Casein Systems: Effect of the Maillard Reaction. Journal of Agricultural and Food Chemistry, 2000. 48, 2271-2275. [19] Monti, SM; Bailey, RG; Ames, JM. The influence of pH on the non-volatile reaction products of aqueous Maillard model systems by HPLC with diode array detection. Food Chemistry, 1998, 62, 369-375. [20] Wedzicha, BL; Adamu, DJM. Reactivity of vegetables towards sulphur dioxide. Food Chemistry, 1987, 26, 159-173. [21] Kanner, J; Shapira, N. Oxygen- and Metal-Ion-Dependent Nonenzymatic Browning of Grapefruit Juice, in Quality Factors of Fruits and Vegetables. American Chemical Society, 1989, 55-64. [22] Moreno, F.J., et al., High-Pressure Effects on Maillard Reaction between Glucose and Lysine. Journal of Agricultural and Food Chemistry, 2002, 51, 394-400. [23] Friedman, M., Food Browning and Its Prevention: An Overview. AU - Friedman, Mendel. Journal of Agricultural and Food Chemistry, 1996, 44, 631-653. [24] Chen, H., et al., Chemical composition analysis of soybean oligosaccharides and its effect on ATPase activities in hyperlipidemic rats. International Journal of Biological Macromolecules, 2010, 46, 229-231. [25] Caicedo, L; Silva, E; Sánchez, O. Semibatch and continuous fructooligosaccharides production by Aspergillus sp. N74 in a mechanically agitated airlift reactor. Journal of Chemical Technology and Biotechnology, 2009, 84, 650-656. [26] Lee, HW, et al., Chitosan oligosaccharides, dp 2-8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp. Anaerobe, 2002, 8, 319-324. [27] Krastanov, A; Yoshida, T. Production of palatinose using Serratia plymuthica cells immobilized in chitosan. Journal of Industrial Microbiology and Biotechnology, 2003, 30, 593-598. [28] T., M., et al., Lactulose as a sugar with physiological significance. Bulletin 212, International Dairy Federation, Brussels, 1987, 69-76. [29] Galliard, T., Starch: Properties and Potential. 1st edition. Chichester: John Wiley and Sons; 1987. [30] Kamil, JYVA; Jeon, YJ; Shahidi,F. Antioxidative activity of chitosans of different viscosity in cooked comminuted flesh of herring (Clupea harengus). Food Chemistry, 2002, 79, 69-77. [31] Teleman, A., et al., Isolation and characterization of O-acetylated glucomannans from aspen and birch wood. Carbohydrate Research, 2003, 338, 525-534.

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[32] Huang, L., et al., Gelation Behavior of Native and Acetylated Konjac Glucomannan. Biomacromolecules, 2002, 3, 1296-1303. [33] Schwesinger, W.H., et al., Soluble dietary fiber protects against cholesterol gallstone formation. The American Journal of Surgery, 1999, 177, 307-310. [34] Yamamoto, K.;Yasuda, J; Too, K. Arrhythmias in newborn Thoroughbred foals. Equine Veterinary Journal, 1992, 24, 169-173. [35] Doi, K., et al., Influence of dietary fiber (konjac mannan) on absorption of vitamin B12 and vitamin E. Tohoku J Exp Med, 1983, 141, S677-S681.



Chapter 4

LIPIDS 1

Shengrong Shen1, Dongfeng Wang2 and Undurti N. Das3 College of Biosystem Engineering and Food Science, Zhejiang University, China 2 College of Food Science and Engineering, Ocean University of China, China 3 UND Life Science, USA Jawaharlal Nehru Technological University, Kakinada, India

ABSTRACT Lipids are a broad group of naturally-occurring molecules that are soluble in organic solvents but insoluble or only sparingly soluble in water. Triacylglycerols account for 95% of total lipids in foods. Lipids are major energy source for human body and protect human body from mechanical damage and heat loss. Lipids are also the carriers of fatsoluble vitamins and the precursors of many bioactive molecules such as prostaglandin, sex hormone, and epinephrine. This chapter deals with the nomenclature, classification, and fatty acid distribution theories of lipids and their functional properties, including polymorphism, plasticity, and emulsification. Lipids undergo multiple chemical reactions during processing and storage, such as hydrolysis, oxidation, and thermal decomposition. The effects of these reactions, especially oxidation, on food flavor and safety are elucidated in detail. Lipid processing techniques, including refinement, hydrogenation and interesterification, are also concerned in this chapter.

1. INTRODUCTION It should be understood that though the term lipid is used majority of the times as a synonym for fats, in fact, fats are a subgroup of lipids called triglycerides. Lipids encompass molecules such as fatty acids and their derivatives including tri-, di-, and monoglycerides and phospholipids, as well as other sterol-containing metabolites such as cholesterol. Humans and other mammals have a variety of pathways to synthesize and metabolize lipids. Some lipids cannot be synthesized by mammals and therefore must be ingested from the diet. Such lipids are termed essential fatty acids (EFAs).

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All plants and animals eaten by humans contain lipids. Various vegetables and most fruits contain very little amounts of lipids, generally about 0.3%, except avocado, whose edible portion contains about 20% lipids. The lipid content in the muscle tissue of lean beef, fish, white poultry, and shell fish is about 2%, about 3.7% in cow‘s milk, about 2-4% in grains, about 30% in fatty pork, 32% in an egg yolk, and about 35% in fillets of fatty fish. Oilbearing nuts and seeds contain from 20% fat in soybeans to 65% fat in walnuts.

1.1. Nomenclature 1.1.1. Fatty Acids Fatty acids refer to any aliphatic monocarboxylic acids that are released by the hydrolysis of naturally occurring lipids. More than 800 natural fatty acids have been identified, of which, most contain a linear chain and even number of carbon atoms. Based on the presence or absence of double bonds, lipids are divided into saturated and unsaturated fatty acids. Palmitic acid and stearic acid are examples of saturated fatty acids. Unsaturated fatty acids contain two or more double bonds. For instance, oleic acid has only one double bond, linoleic acid has two double bonds, linolenic acid contains three double bonds, and arachidonic acid contains up to four double bonds. Fatty acids can be named by using one of the following four nomenclature systems. Trivial nomenclature Lipids are named according to their sources, such as palmitic acid, lauric acid, butyric acid, stearic acid, and oleic acid. Systematic nomenclature Carbon atoms are counted from the carboxylic acid end. Symbol nomenclature Fatty acids can be represented by a simple numerical expression consisting of two terms separated by a colon, with the first term depicting the number of carbon atoms and the second the number of double bonds. For example, hexadecanoic acid, CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2COOH can be named as 16:0 and linoleic acid (9, 12-octadecadienoic acid) can be presented as 18:2. This notation can be ambiguous, because some different fatty acids might have the same numbers. Omega-x or n-x nomenclature

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Unsaturated fatty acids can be named in the n-x or ω-x system. In this system, the terminal methyl carbon is designated as ω or n and x represents the location of the first double bond. Carbon atoms are counted from the terminal methyl carbon (namely ω or n) to the carbonyl carbon. Because all adjacent double bonds in all natural polyenoic acids (containing 2 to 6 double bonds) are spaced by a methylene, the locations of all other bonds can be determined if the first double bond is specified. For example, linoleic acid is presented as 18:2(ω-9). Because the first double locates in the 9th carbon, the second double occurs between C12 and C13. The geometric configuration of double bonds is usually designated by the use of cis and trans, indicating whether the alkyl groups are on the same or opposite sides of the molecule. Unsaturated fatty acids occur naturally in the cis form, but the trans form is more thermodynamically stable. If the four groups attached to the two carbon atoms of a double bond are different, the configuration can be designated according to the Cahn-Ingold-Prelog rules. In this system, each group is assigned a priority. If the two groups with higher priority (greater atom number) locate in the same side of the double bond, the letter Z is used to designate the configuration. If the two groups are on the opposite sides, the letter E is used. Table 4-1 lists the systematic and common names of some fatty acids in naturallyoccurring lipids. Table 4-1. Nomenclature of some common fatty acids Abbreviation 4:0 6:0 8:0 10:0 12:0 14:0 16:0 16:1(n-7) 18:0 18:1(n-9) 18:2(n-6) 18:3(n-3) 20:0 20:4(n-6) 20:5(n-3) 22:1(n-9) 22:5(n-3) 22:6 (n-6)

Systematic name Butanoic Hexanoic Octanoic Decanoic Dodecanoic Tetradecanoic Hexadecanoic 9-Hexadecanoic Octadecanoic 9-Octadecanoic 9,12-Octadecanoic 9,12,15- Octadecanoic Arachidic 5,8,11,14-Eicosatetraenoic 5,8,11,14,17-Eicosatetraenoic 13-Docosenoic 7,10,13,16,19-Docosapentaenoic 4,7,10,13,16,19-Docosahexaenoic

Common name Butyric Caproic Caprylic Capric Lauric Myristic Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Eicosanoic Arachidonic EPA Erucic

Symbol B H Oc D La M P Po Sta O L Ln Ad An E

DHA

Note: aSome authors use S for stearic, but this can be confusing, since S is also used for ―saturated‖ whenever triaclycerol composition is expressed in terms of saturated (S) and unsaturated (U) fatty acids. For example, S3 or SSS=all three fatty acids saturated, SU2 or SUU = diunsaturated monosaturated, and so on.

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Figure 4-1. Structure of acylglycerols. R denotes fatty acid; there may be fatty acid, two fatty acids or three fatty acids in the acylglycerols, which is mono-acyglycerol, di-acyglycerol or triacylglycerol respectively; R1=R2=R3, the triacylglycerol is defined as simple triacylglycerol, otherwise mixed triacylglycerol.

1.1.2. Acylglycerols Lipids are the mixtures of mono-, di-, and triesters of glycerol with fatty acids, which are termed monoacylglycerols, diacylglycerols, and triacylglycerols, respectively. Natural fats are mainly present as triacylglycerols. In order to designate the configuration of glycerol derivatives, the stereospecific numbering (Sn) system is used in acylglycerol nomenclature. In this system, the carbon atom that appears on top in that Fischer projection that shows a vertical carbon chain with the hydroxyl group at carbon-2 to the left is designated as C-1 and the prefix 'sn' (for stereospecifically numbered) is printed immediately preceding the glycerol term. An example is shown in Figure 4-1. If R1 denotes stearic acid (St), R2 oleic acid (O), and R3 linoleic acid (L) in the structure, this triacylglycerol is named Sn-glycerol-1-stearic acid-2-oleic acid-3linoleic acid, Sn-18:0-8:1-18:2, or Sn-StOL. If the product is the racemic mixture of two antipodes, the prefix 'rac-' (for racemo) can be used preceding the full name, such as in rac-StOM. In this case, the middle acid (O, standing for the oleic acid) is in position 2 and the remaining two fatty acids (St and M, standing for stearic and myristic acids respectively) are equally distributed in positions 1 and 3. If the prefix β is used before the name, such as β-StOM, it indicates that the middle acid is in position 2, but the distribution of the other two acids is unknown. In the case of simple triacylglycerols or those with unknown fatty acids distribution, the prefix can be omitted. For example, StOM can be used to express any mixture of Sn-StOM, Sn-MOSt, Sn-OStM, Sn-MStO, Sn-StMO, and Sn-OMSt.

1.1.3. Phospholipids Phospholipids refer to any lipids that contain phosphoric acid in the structures as monoor diester. According to the type of hydroxyl donors, phospholipids are divided into phosphoglycerides and sphingolipids (sphingophospholipids), in the former structure the alcohol is glycerol and the later sphingosine.

Figure 4-2. Structure of lecithin.

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In phospholipids, amino alcohols, such as choline, ethanolamine, serine, and inositol, attach to phosphoric acid via an ester linkage, which are known as phosphatidylcholine, phosphatidyethanolamine, phosphatidyserine and phosphatidylinositol respectively. Phosphoglycerides are named as the derivatives of phosphatidic acids or similar to the Sn system. For example, lecithin, whose structure is shown in Figure 4-2, is named as 3-snphosphatidylcholine or sn-glycerol-1-stearic acid-2-linoleic acid-3-phosphocholine. Sphingophospholipids adopt sphingosine as their backbone. The amino group in carbon 2 is connected to a long-chain fatty acid through the amido linkage to form ceramide and the hydroxyl in carbon 3 is esterified by phosphoric acid. Choline or neovaricaine then attaches to the phosphoric acid to produce various sphingomyelins.

1.2. Classification Based on chemical structure and composition, lipids can be divided into simple lipids, compound lipids, and derived lipids. Simple lipids are compounds consisting of fatty acids and alcohols and include fats, oils, and waxes. Compound lipids contain many other groups in addition to fatty acids and alcohols. Glycerophosphonolipids, sphingomyelins, cerebroside, and ganglioside are the examples of compound lipids. Derived lipids are the derivatives of simple and compound lipids and have the common properties of lipids. Steroids, hydrocarbons, carotenoid, and fat-soluble vitamins are examples of this group. Most lipids in foods are acylglycerols. According to composition, lipids in animal and plant-derived foods are divided into the following: Milkfat Milkfat exists in the latex of mammals. Milkfat consists mainly of palmitic acid, oleic acid, and stearic acid. Compared with other animal-derived lipids, milkfat contain abundant C4~C12 short-chain fatty acids, small amounts of odd- and branch-chain fatty acids, and trans double bonds. Laurates Laurates are mainly found in palme plants such as coconut palm and babassu. Laurates are featured by their high lauric acid contents, which reach up to 40%~50%. Laurates have medium contents of C6, C8, and C10 fatty acids, low unsaturated fatty acid contents, and low melting points. Plant butter Plant butter originates from the seeds of tropical plants and is characterized by narrow melting point range. Although plant butter has a higher content of saturated fatty acids than

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unsaturated fatty acids, trisaturated glycerides have not been identified in plant butter. Plant butter is widely used in candies and cocoa butter is the most important plant butter. Oleate-linoleate Lipids of this category are the most abundant in nature and are found only in plants. The lipids have high contents of oleic acid and linoleic acid and the contents of saturated fatty acids are less than 20%. Peanut oil, corn oil, olive oil, palm oil, sesame oil, cottonseed oil, and sunflower oil are important members of this category. Linolenates Linolenates contain large amount of linolenic acid and soybean oil, wheat germ oil, and perilla oil belong to this category. Animal fats Lipids of this group, such as lard and tallow, are the storage fat of domestic animals. Animal fats contain large amount of C16 and C18 fatty acids and medium contents of unsaturated fatty acids, mostly oleic and linoleic acids, and small amount of odd-numbered fatty acids. Animal fats contain appreciable amount of fully saturated triacylglycerols and thus exhibit high melting points. Marine oils Marine oils contain abundant long-chain unsaturated fatty acids and are rich in vitamins A and D. Eicose pentaenois acid (EPA) and docosahexenoic acid (DHA) are typical examples of this category. Due to high unsaturation degree, marine oils are susceptible to oxidation.

1.3. Distribution of Fatty Acids in Nature Lipids 1.3.1. Theories of Triacylglycerol Distribution Pattern In addition to fatty acid species and content, the properties of lipids are also affected by the distribution of the fatty acids. Many theories on the fatty acid distribution of triacyglycerols have been proposed and the most important are illustrated below: Even or widest distribution theory This theory was proposed by Hilditch and Williams in 1964 [1]. According to this theory, the fatty acids in triacylglycerols tend to be distributed as broadly as possible. In this case, when an acid, S, accounts for less than one-third of the total fatty acids present, it should not appear more than once in any triacylglycerol. If X refers to the other fatty acids present, only XXX and SXX species should be found. If a fatty acid accounts for between one-third and two-thirds of the total fatty acids, it occurs at least once but never three times in any one

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molecule; that is, only SXX and SSX should be present. If a fatty acid forms more than twothirds of the total acids, it should occur at least twice in every molecule; that is, only SSX and SSS should be present. However, analysis of many natural fats, especially those of animal origin, reveals marked deviation from this theory. Trisaturated acylglycerols have been found in fats containing less than 67% saturated fatty acids. This theory is applicable only to triacyglycerols that consist of two components and does not take positional isomers into consideration. This theory is no longer considered valid. Random (1, 2, 3-Random) distribution theory According to this theory, fatty acids are distributed randomly both within each triacylglycerol molecule and among all the triacylglycerols. Thus, the fatty acid composition of all the three positions of glycerol should be the same and also equivalent to the fatty acid composition of the total fat. The proportion of any given fatty acid species can be calculated according to the following equation: %sn-XYZ = (mol% X in total fat) × mol% Y in total fat) × mol% Z in total fat) × 10-4. For example, if a fat contains four fatty acids, including 8% palmitic acid (P), 2% stearic acid (St), 30% oleic acid (O), and 60% linoleic acid (L), 64 possible triacylglycerol species would exist. The percentages of any species can be calculated by taking the following equations as example: Sn-OOO(%) = 30%×30% ×30% ×10-4 = 2.7. Sn-PLSt(%) = 8×60×2×10-4 = 0.096. Sn-LOL (%) = 60×30×60×10-4 = 10.8. The fatty acid distribution of most lipids does not conform to the random distribution theory. In natural lipids, the fatty acids in the Sn-2 position are different from those in positions Sn-1 and Sn-3. Hence, great deviation exists between the predicted and actual distribution of fatty acids in natural lipids. However, this theory can be used to predict the fatty acid distribution in randomly interesterified fats. Restricted random distribution theory This theory was initiated by Kartha in 1953 [2]. According to this theory, saturated and unsaturated fatty acids in animal fats are distributed randomly. Fully saturated triacylglycerols (SSS) can be present, but only to the extent that the fat can remain fluid in vivo. Excess SSS, according to this theory, can exchange with UUS and UUU to generate SSU and SUU. Kartha's calculations do not account for positional isomers or positioning of individual acids. 1, 3-Random-2-Random distribution

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This theory was proposed by Vander al, Coleman, and Fulton between 1960 and 1961 based on their researches of directed hydrolysis of Sn-1 and Sn-3 fatty acids by steapsin. According to this theory, the fatty acid composition at position 2 is different and independent from that of positions 1 and 3 and fatty acids are distributed randomly in Sn-2 and Sn-1,3. Thus, the composition at positions 1 and 3 will, presumably, be identical. According to this theory, the content of any given triacylglycerol can be calculated with the following equation: Sn-XYZ(%) = (mol%X in positions 1 and 3)×(mol%Y in position 2)×(mol%Z in positions 1 and 3)×10-4. This theory is very useful in the exact predication of plant-derived lipids. 1-Random-2-Random-3-Random distribution theory This theory was proposed by Tsuda in 1962. According to this theory, fatty acids are separately but randomly distributed at each of the three positions of the triacylglycerol molecules of a natural fat. Thus, the possibility of a given fatty acid appearing in each Sn position would likely be different. The content of a given triacylglycerol species can be calculated as: %sn-XYZ = (mol% X at sn-1)×(mol% Y at sn-2)×(mol% Z at sn-3)×10-4. This theory is applicable to the predication of common animal fat, milkfat, and seed fats.

1.3.2. Positional Distribution of Fatty Acids in Natural Fats Generally, the Sn-2 position is preferred by unsaturated fatty acids, especially linoleic acid, in seed-derived fats and saturated fatty acids are distributed at Sn1- and Sn-3 positions. In most cases, the distributions of both saturated and unsaturated fatty acids at positions Sn-1 and Sn-3 are equivalent. Animal-derived fats have higher saturated fatty acid contents at position Sn-1 than plantderived fats and their fatty acid compositions at positions Sn-1 and Sn-3 also differ markedly. In most animal fats, palmitic acid is esterified at position Sn-1 with priority, and myristic acid is esterified at position Sn-2. Table 4-2. Positional distribution of fatty acids in triacylglycerols of some natural fats [3] Source Coconut

Peanut

Soybean

Position 1 2 3 1 2 3 1 2 3

14:0

16:0 34 2 37 14 2 11 14 1 13

18:0 50 2 53 5 5 6 22 6

18:1 12 87 9 59 59 57 23 70 28

18:2 1 9 19 39 10 48 7 45

18:3

9 8

20:0

20:1

1 4

1 3

20:2

20:0

6

1 0.5 3

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Lipids Table 4-2. (Continued) Source Beef

Pig

Position 1 2 3 1 2 3

14:0 4 9 1 1 4 -

16:0 41 17 22 10 72 -

18:0 17 9 24 30 2 7

18:1 20 41 37 51 13 73

18:2 4 5 5 6 3 18

18:3 1 1 1

20:0

20:1

20:2

20:0

Lard has different fatty acid distribution patterns compared with other animal-derived fats. For example, palmitic acid is mainly found in position Sn-2, stearic acid is distributed in Sn-1, linoleic in Sn-3, and oleic acid in Sn-3 and Sn-1. In milkfat, short-chain fatty acids attach specifically to position Sn-3, while the longchain polyunsaturated fatty acids are preferably esterified at position Sn-2 of marine oils. The fatty acid distributions of some natural fats are listed in Table 4-2.

2. PHYSICAL PROPERTIES OF LIPIDS Pure oils and fats are odorless and colorless, but processed fats and oils often display yellow-green color due to the incomplete decoloration of such pigments as carotenoid and chlorophyll. The odor of fats and oils is often attributed to the presence of non-lipid impurities and lipid oxidation.

2.1. General Physical Properties 2.1.1. Density The density of fatty acids and glycerides reduces with the increase of carbon chain length and the decrease of degree of unsaturation. Generally, fatty acids and glycerides with high hydroxyl and carbonyl contents have the highest density. Natural lipids are the mixture of multiple acylglycerols and thus the relationship between density and composition is rather complex. The densities of lipids are smaller than that of water at room temperature and tend to decrease in higher temperatures. It is estimated that, when a triacylglyceride is melted, its density decreases by about 10%. 2.1.2. Refractive Index The refractive index of lipids rises with the increase of degree of unsaturation and chain length. The refractive index of a monoacylglycerol is generally higher than that of the corresponding triacylglycerol. The refractive index is sometimes used in lipid identification to determine whether adulteration occurs.

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2.1.3. Melting Point The melting point of saturated fatty acids is closely related to their chain length. Generally, the index increases as the chain length rises. However, the melting points of odornumbered fatty acids are lower than that of the exactly adjacent even-numbered fatty acids and the differences decrease as the chain length increases. The melting points of unsaturated fatty acids are lower than their saturated forms and are affected by the number, location, and configuration of double bonds. Fatty acids with more double bonds have lower melting points and those with double bonds close to the two ends exhibit higher melting points. In the case of fatty acids with the same carbon number, branched ones are melted in lower temperatures than those of straight chain ones and hydroxyl-containing ones have higher melting points due to hydrogen bonding. For acylglycerols, monoacylglycerols have the highest melting point, followed by diacylglycerols and triacylglycerols in sequence. The melting point of triacylglycerols is positively correlated with the content and chain length of fatty acids due to the increase of inter-molecular action. The presence of trans fatty acids increases the melting point compared with cis fatty acids, because the interaction between fatty acids is hindered in the cis configuration. Similarly, because conjugation enhances the interaction between fatty acids, lipids containing conjugated double bonds are melted at higher temperatures than those without conjugated double bonds. Natural lipids are the mixture of multiple acylglycerols and hence no specified melting points can be given. The melting points of natural lipids are defined as a range of temperature from lipid melting initiation to completion. 2.1.4. Smoke Point, Flash Point and Fire Point The smoke point refers to the temperature at which a lipid begins to produce smoke, which indicates the decomposition of the lipid. The smoke point of lipids is normally in the range of 200–230 °C. The flash point of a volatile liquid is the lowest temperature at which it can vaporize to form an ignitable mixture in air. The fire point, a higher temperature, is defined as the temperature at which the vapor continues to burn for over 5 seconds after being ignited. Smoke point, flash point, and fire point are three important properties that determine the quality of lipids and can be used to determine the presence of impurities.

Figure 4-3. Crystalline forms of triacylglycerols.

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For example, the smoking point of refined edible oil is generally higher than 240 °C, but the presence of free fatty acids dramatically decreases the index. The flash point of plantderived edible oil is generally over 225-240 °C and the fire point is usually 20-60 °C higher than the flash point.

2.2. Polymorphism of Lipid 2.2.1. Crystallization and Polymorphism Polymorphic forms are crystalline phases of the same chemical composition but differ in structure and yield identical liquid phases upon melting. The polymorphic forms have different stabilities and the less stable forms can automatically and unidirectionally transform to the more stable forms without melting. If all the polymorphic forms are in their stable states, the transformation is multidirectional and is decided by the temperature. When lipids are placed in a temperature below freezing point, multiple crystal forms can be identified. Polymorphism is an important property of lipids. X-ray diffraction and infrared spectrum analysis indicate the existence of three crystal forms, namely α, β’, and β in triacylglycerols, as shown in Figure 4-3. In the α form, the fatty acid chains are arranged randomly. Hence, crystals in this form have lower melting point, density and stability. Crystals in the β’ and β forms are arranged more orderly, especially for the β form, in which the fatty acids are packed in the same direction. Hence, β’ and β form crystals exhibit higher melting point, density, and stability. The polymorphic behavior of natural lipids is affected by fatty acid composition and their distribution in triacylglycerols. Generally, lipids consisting of relatively few closely related triacylglycerol species tend to transform rapidly to the stable β form, while those consisting of more triacylglycerol species are transformed slowly to the β' form. Soybean oil, peanut oil, corn oil, olive oil, coconut oil, safflower oil, cocoa butter and lard are present mainly in the β form, but cottonseed oil, palm oil, rapeseed oil, and milkfat are found mainly in the β' form. 2.2.2. Effects of Polymorphism on Food Processing The different crystal forms in polymorphism have different physicochemical and sensory properties. Conditions must be well controlled in food processing to yield desirable crystal forms. One example is the production of salad oil from cottonseed oil. Cottonseed oil must be winterized to remove high-melting-point fats before further processing. During winterization, the temperature must be decreased slowly to ensure sufficient time for bulk crystallization in the β form, which is beneficial for subsequent filtration. If the temperature is reduced too fast, fine crystals are formed and the filtration process becomes difficult. Crystals in the β' form can impart desired coating performance and mouthful for margarine. In margarine production, oils are firstly rapidly cooled to produce α form crystals and then cooled in a slightly higher temperature to enable the transformation from α form to the more stable β' form. Chocolate should melt at 35 °C in the mouth without causing the greasy feeling. Besides, chocolate must has smooth surface and the crystal particles cannot be too rough. For this purpose, conditions must be controlled exactly to yield the desired β form. Cocoa butter is

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heated to above 55 °C until completely melts and is then allowed to cool slowly until 29 °C. The crystals are reheated to 32 °C to melt other crystal forms than the β form. The later process is repeated until cocoa butter is completely transformed to the β form.

2.3. Plasticity Lard and butter that are solids in room temperature are actually the mixtures of liquid oils and solid fats. The lipids are completely solidified only in very low temperatures. Such homogeneous mixtures of liquid oils and solid fats are defined as the plastic fat. Plastic fat can maintain their shape even in exposure to external forces. The plasticity of fats is affected by the following factors: Fat crystal form Fat crystals in the β' form have the best plasticity, because substantial amount of small bubbles are trapped in the β' crystals. In contrast, only a small amount of air bubbles are trapped in β crystals and the air bubbles are large in volume. Hence, fats in the β form have less plasticity. Melting point range Fats with wider melting point ranges exhibit better plasticity. Solid to liquid ratio The fat/oil ratio significantly affects lipid plasticity. If the percentage of the solid phase is too high, rigid crosslinking occurs and the fat exhibits high hardness and poor plasticity. If the percentage of liquid phase is too high, the fat is flowable, soft, and susceptible to deformation.

Figure 4-4. Heat content (H) or dilatometric (D) melting curve of a glyceride mixture [3].

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The solid phase to liquid phase ratio is also termed as the solid fat index (SFI). SFI is the most important index that determines the plasticity of lipids and can be determined by measuring the expansion of the fat, i.e. the volume increase of the fat during its transition from solid to liquid. The volumes of solid fat and liquid oil increase when heated. The expansion without phase transformation is defined as thermal expansion. When solid fat is transformed to the liquid state in high temperatures, the volume increase is called melting dilation. The meltexpansion curve of plastic fats can be followed by measuring the specific volume change of liquid oil and solid fat in different temperatures. As shown in Figure 4-4, solid fat starts to melt at point X and is gradually melted until to point Y, at which the fat is completely liquefied. The fat in point b is a mixture of solid and liquid. In this point, the percentage of solid fat is ab/ac and that of liquid oil is bc/ac. Hence, the SFI in point b is ab/bc. If a lipid melts in a very narrow temperature range, the slope between points X and Y is high. Otherwise, the lipid exhibits a wide plastic range. Hence, the plastic range of lipids can be modified by adding lipids with different melting points. SFI can be measured exactly by using a dilatometer. However, measurement with dilatometer is time consuming and is applicable only to fats with SFI less than 50%. Pulsed nuclear magnetic resonance is the most common method for SFI determination. Because ultrasonic travels faster in solid fat than in liquid oil, ultrasonic methods have been developed for SFI measurement.

2.4. Lipid Emulsification and Emulsifier 2.4.1. Emulsion Emulsions are dispersed system composed of two immiscible liquids. In an emulsion system, one phase is dispersed in another phase in the form of liquid crystal or droplets with diameter ranging from 0.1 μm to 50 μm and is thus designated as the inner phase or dispersed phase and is surrounded by the outer phase or continuous phase. According to the natures of the two phases, two emulsion systems, including oil-in-water (O/W) and water-in-oil emulsions (W/O), have been identified. Milk is an example of the O/W emulsion and cream is an example of the W/O emulsion. The formation of small dispersed droplets increases the area of the interface between the two phases and the interface area increases exponentially when the droplet diameter is decreased. Due to the increased interface area, high energy is required to maintain positive free energy for the interfaces. Hence, emulsions are unstable thermodynamic systems and demulsification might occur under specific conditions. Emulsions can be destabilized in one of the following ways: Creaming When the two phases differ in density, one phase migrates to the top or bottom of the emulsion due to gravity. Large droplet size of the dispersed phase and density difference accelerate the creaming process.

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Shengrong Shen, Dongfeng Wang and Undurti N. Das Flocculation

When the amount of static charge on the surface of dispersed droplets turns insufficient, the repulsion between droplets is reduced. As a result, the droplets attach to each other and flocculation occurs. The membranes of droplets remain intact after flocculation. Coalescence The membranes of dispersed droplets are disrupted and the drops move to each other, leading to coalescence. Coalescence reduces the interface area of the dispersed phase and planar interface might occur when coalescence is serious. Coalescence can be prevented by adding emulsifiers. Emulsifiers are surfactants containing both hydrophilic and lipophilic groups and are located in the oil/water interface. Emulsifiers decrease the interfacial tension and thus reduce the energy required for emulsion formation. Although surfactants reduce the interfacial tension, the interfacial free energy is still positive and emulsions are still thermodynamically unstable.

2.4.2. Emulsifier Emulsifiers are divided into anionic, cationic and nonionic types according to structure and property. Based on origin, emulsifiers can be divided into natural and synthetic ones. Based on functions, emulsifiers are divided into surfactants, viscosity reinforcing agents and solid absorption agents. According to polarity, emulsifiers are divided into hydrophilic and lipophilic ones. Fatty monoglycerides and their derivatives (including glycerol monostearate and stearic acid glyceride), sucrose fatty acid ester, sorbitan fatty acid ester and their derivatives (such as sorbitan monooleate (Span 80) and polyoxyethylene sorbitan monostearate (Span 60)), and phospholipids (such as modified soybean phospholipid), are widely used in the food industry as emulsifiers.

3. CHEMICAL PROPERTIES OF LIPIDS 3.1. Hydrolysis Lipids can be hydrolyzed by acid, alkali, heat, and enzymes to release free fatty acids in the presence of water. Triacylglycerols are firstly hydrolyzed to diacylglycerols, followed by monoacylglycerols and finally glycerol in sequence. Free fatty acids are not found in living animals, but can be liberated by enzymatic hydrolysis after animals are slaughtered. When mature oil seeds are harvested, lipid hydrolysis occurs. Free fatty acids are less stable than the corresponding glycerides and are more susceptible to oxidative rancidity. Hence, oils are neutralized by alkali in refinement to reduce free fatty acid content and improve the quality and shelf life.

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When foods are fried, water might migrate into hot lipids, leading to lipid hydrolysis and fatty acid release. Frying decreases the smoke point of lipids and deteriorate the quality of foods. Lipid hydrolysis is not preferred in most cases. However, moderate lipid hydrolysis can create special flavors for some foods. For example, in cheese production, microbes and milk lipases are added to produce desired flavor. In breads and yogurts, lipid hydrolysis also contributes to flavor formation.

3.2. Oxidation Lipid oxidation is a major cause of quality deterioration of lipid-containing foods. Lipid oxidation generates various off-flavors and leads to rancid (oxidative rancidity), which render these foods less acceptable. In addition, oxidation decreases the nutritional quality of foods and produces potential toxicants. The prevention of lipid oxidation has been recognized as an important issue of lipid chemistry.

3.2.1. Autoxidation The autoxidation of lipids is a typical free radical chain reaction and the autoxidation rate is markedly inhibited by chemicals which can interfere with free radical reactions. Light and free radical-producing substances catalyze the reaction and the quantum yields exceed unity when the oxidation reaction is initiated by light. Lipid autoxidation yields high levels of hydroperoxides (ROOH) and the induction periods of pure substrates are longer than mixture substrates. The lipid autoxidation process is comprised of three steps: initiation, propagation, and termination. Initiation: Propagation: Termination:

RH→R• +H• R• + O2 → ROO• ROO• + RH → ROOH + R• R• + R• → R-R R• + ROO• → R-O-O-R ROO• + ROO• → R-O-O-R + O2

In initiation, the hydrogen atom on the α-carbon atom of the double bonds of unsaturated fatty acids or their triacylglycerols (designated as RH) is detached by metal catalysis or exposure to light or heat and a fatty acid radical known as the alkyl radical (R•) is produced. R• then reacts with oxygen to produce peroxy radicals ROO•, which grabbles the hydrogen atom from α-carbon atom of another molecules (RH) to yield hydroperoxides (ROOH) and new free radicals R•. The reactions are repeated and lead to the propagation of lipid autoxidation. Once these free radicals are combined to produce stable non-free radical products, the chain reaction is terminated. Initiation needs high activation energy and is therefore the limiting step of lipid autoxidation.

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3.2.2. Formation of Hydroperoxides Hydroperoxides are the main product in the early phase of lipid autoxidation and their structures vary with substrates (unsaturated fatty acids). After the hydrogen carbon is detached from the α-methylene group neighboring a double bond, oxygen attacks the α carbon atom connected the double bond, resulting in the formation of hydroperoxides. The reaction is usually accompanied by a shift in position of double bonds. Oleate In autoxidation, the hydrogen atoms in C8 and C11 of oleates are detached and two allylic radical intermediates are generated. Oxygen then attacks the end carbons of each radical intermediates and an isomeric mixture of 8-, 9-, 10-, and 11-allylic hydroperoxides are produced, as shown in Figure 4-5. The percentages of 8- and 11-hydroperoxides are slightly higher than those of the 9- and 10-isomers in the mixture. When autoxidation occurs at 25 °C, the product mixture contains equalmolar cis and trans 8- and 11-hydroperoxides and predominant percentages of trans 9and 10-isomers. Linoleate The hydrogen atoms attached to position 11 is highly active. Hence, only one radical intermediate is produced upon autoxidation and an equalmolar mixture of conjugated 9- and 13-diene hydroperoxides is generated. The two hydroperoxide products undergo geometric isomerization and hence are found in both the cis, trans and the trans, trans forms, as shown in Figure 4-6. Linolenate Linolenate contains two 1, 4-pentadiene structures and their autoxidation products are a mixture of 9-, 12-, 13-, and 16-hydroperoxides (Figure 4-7).

Figure 4-5. Autoxidation of oleate [4].

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Lipids

Figure 4-6. Autoxidation of linoleates [5].

Figure 4-7. Autoxidation of linolenate [4]. CHO

OOH R

H2 C

C H

C H

CHO

R

C H

C H

C H

CH2

CHO

CHO

Figure 4-8. MDA formation by unsaturated aldehydes. C5H11

O

C5H11

3C5H11CHO O

O

C5H11

Figure 4-9. Tripentyltrioxane formation by polymerization of three hexanals.

RCHO

124


Each of the four hydroperoxides has multiple geometric isomers with the conjugated diene system in the cis, trans or the trans, trans configuration, but the unconjugated double bond is always cis. Linolenate autoxidation results in more 9- and 16-hydroperoxides than the 12- and 13-isomers, because oxygen prefers C9 and C16 and the 12- and 13-hydroperoxides are decomposed rapidly. The 12- and 13-hydroperoxides can form six-membered peroxide hydroperoxide via 1,4-cyclization or prostaglandin-like endoperoxides via 1,3-cyclization.

3.2.3. Decomposition of Hydroperoxides Hydroperoxides are extremely unstable and are decomposed immediately after they are formed. In the initiation of autoxidation, the rate of hydroperoxides formation is higher than decomposition, but the trend is reversed in subsequent steps. The decomposition of hydroperoxide involves the production of alkoxy radical and its further breakdown, with the products including aldehydes, ketones, alcohols, acids, epoxy compounds, and hydrocarbons. The aldehydes and ketones include nonanal, 2-isodecenoic aldehyde, 2-ecenal, hexanal, cis-4-heptenal 2,3-pentanedione, 2,4- glutaconaldehyde, 2,4decadiene, and 2,4,7-decatrienal. The resultant epoxy compounds are mainly the homologues of furan. Unsaturated aldehydes can be further oxidized to malonaldehyde (MDA) (Figure 48), which not only deteriorate the flavor of foods, but also impose safety concerns. Saturated aldehyde products can be easily oxidized to corresponding acids or generate new products through polymerization and condensation. For example, tripentyltrioxane can be produced through the polymerization of three hexanal molecules (Figure 4-9). The reactions involved in lipid autoxidation and decomposition are given in Figure 4-10.. 3.2.4. Factors Influencing Lipid Autoxidation The following factors influence the autoxidation of lipids: Fatty acid composition The quantity, location, and geometrical configuration of double bonds in fatty acid components significantly affect lipid autoxidation. Lipids containing more double bonds are oxidized faster than those with less double bonds. For example, the relative oxidation rates of arachidonic acid, linolenic acid, linoleic acid, and oleic acid are 40, 20, 10, and 1 respectively. The cis fatty acids are more susceptible to oxidation than the trans isomers. Conjugated double bonds are oxidized more easily than unconjugated double bonds. Saturated fatty acids are much more resistant to oxidation than unsaturated fatty acids. Compared with triacylglycerols, free fatty acids are oxidized more rapidly and the autoxidation rate of lipids increases when the free fatty acid content exceeds 0.5%. Random distribution of fatty acids in lipids decreases the autoxidation rate. Temperature High temperature accelerates lipids autoxidation, though high temperature decreases the solubility of oxygen. This is because the release of free fatty acids and the decomposition of hydroperoxides are enhanced in high temperatures.

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Figure 4-10. Schemes of lipid autoxidation and decomposition [6].

Oxygen concentration The autoxidation of lipids is not affected by oxygen level in the case of sufficient partial pressure of oxygen. However, when the partial pressure of oxygen is low, the lipid autoxidation rate is proportional to the partial pressure of oxygen. Surface area The lipid autoxidation rate is positively correlated with the area of the interface in contact with air. In the case of a large surface area to volume ratio, the decrease of oxygen partial pressure exerts no significant effect on oxidation rate. In O/W emulsions, the autoxidation rate is determined by the diffusion of oxygen to the oil phase.

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Shengrong Shen, Dongfeng Wang and Undurti N. Das Water activity

Lipids are oxidized very rapidly in dried foods with aw less than 0.1. When aw is increased, lipid autoxidation rate decreases dramatically. When the aw reaches 0.3, lipids are oxidized in the minimum rate. This is because water reduces the catalytic activity of metals, quenches free radicals, enhances non-enzymatic browning (producing antioxidant compounds), and prevents the contact between foods and oxygen. When aw increases in the range 0.3~0.7, the water solubility of oxygen and the fluidity of catalysts increase and more reaction sites on substrates are exposed, leading to accelerated oxidation. When the aw further increases, the lipid oxidation rate is reduced again due to diluted oxygen and catalyst concentrations. Auxiliary oxidants Some divalent or polyvalent transit metals with appropriate redox potentials, such as Co, Cu, Fe, Mn, and Ni, are effective auxiliary oxidants. These metals can shorten the initiation period and accelerate lipid oxidation in concentrations as low as 0.1 mg/kg. Light and radiation Visible light, UV light and high-energy radiation can initiate the formation of free radicals and decompose hydroperoxides, thus stimulating lipid oxidation. Hence, lipids or lipid-containing foods should be stored in dark place or packed with opaque materials. Special attention should be paid to this issue in the radiation sterilization of foods. Antioxidants Antioxidants can delay and retard the autoxidation of lipids. The effects of antioxidants on lipid oxidation are detailed in Section 3.2.7.

3.2.5. Photosensitized Oxidation Photosensitized oxidation, also referred to as photoxidation, is the reaction between the double bonds of unsaturated fatty acids and singlet oxygen. Photosensitized oxidation can initiate the autoxidation of lipids. Pigments in foods are natural photosencitizers. After exposure to light, photosencitizers are excited to reactive molecules. Photosensitized oxidation occurs in one of two ways. When photosencitizers are excited, they directly react with lipids to form free radicals and initiate the autoxidation of lipids; or react with triplet oxygen (3O2) to produce active excited singlet oxygen. The resultant excited oxygen directly attacks the atoms on double bonds of unsaturated fatty acids and leads to the formation of trans hydroperoxides. The number of hydroperoxide species is twice of the number of double bonds. Figure 4-11 is the mechanism of the photosensitized oxidation of linoleic acid. Because singlet oxygen is extremely active, the rate of photosensitized oxidation is 1000 times faster than the autoxidation of lipids. Besides, the hydroperoxide products can be decomposed to free radicals to initiate autoxidation.

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Singlet oxygen does not show obvious specificity to substrate structure and can react with any double bonds in unsaturated fatty acids. The rate of photosensitized oxidation is proportional to the number of double bonds and is not affected by their locations. This property is evidenced by the rate constants of the reactions of singlet oxygen with oleic acid, linoleic acid, linolenic acid, and arachidonic acid, which are 0.74 × 105 mol-1 · S-1 · L, 1.3 × 105 mol-1 · S-1 · L, 1.9 × 105 mol-1 · S-1 · L, and 2.4 × 105 mol-1 · S-1 · L respectively. In the case of triplet oxygen, it is much less active and the rate constant of the reaction with linoleic acid is only 89 mol-1 · S-1 · L, which is 1450 times lower than that of singlet oxygen. Hence, the oxidation initiated by singlet oxygen predominates in the oxidation of unsaturated fatty acids.

3.2.6. Enzymatic Oxidation Lipoxidases catalyze the oxidation of lipids. Lipoxidases are widely present in organisms, especially plants. Lipoxidases act specifically and attacks only polyunsaturated fatty acids that contain the cis,cis-1,4-pentadiene structure, such as that in oleic acid, linoleic acid, linolenic acid, and arachidonic acid. The mechanism of lipoxidase-catalyzed oxidation of lipids is elucidated by taking linoleic acid as an example. After the hydrogen of the ω-8 methylene is detached, a free radical is formed and then isomerized to the trans configuration. As a result, the ω-6 and ω-10 hydroperoxides with conjugated double bonds are produced, as shown in Figure 4-12. 13

HOO

13

12

OOH

9

HOO

10

Figure 4-11. Mechanism of the photosensitized oxidation of linoleic acid.

Figure 4-12. Mechanism of the enzymatic oxidation of linoleic acid.

9

OOH

128


In animals, lipoxidases specifically oxidize arachidonic acid to produce prostaglandin, prothrombin, and other active compounds. The generation of beany flavor of soybean products has been attributed to the lipid oxidation catalyzed by lipoxidase.

3.2.7. Lipid Antioxidation and Antioxidants Oxidation reduces the quality of lipids and deteriorates the quality of lipid-containing foods. The prevention of lipid oxidation is a major concern of oil chemistry. The oxidation of lipids can be delayed or retarded by both chemical and physical methods, but antioxidants are the most effective and economic methods. Antioxidant mechanism of antioxidants Based on action mechanism, antioxidants are divided into free radical scavengers, singlet oxygen quenchers, hydroperoxide decomposers, enzyme inhibitors, and antioxidant synergists. Free radical scavengers are either hydrogen or electron donors. Hydrogen donors, such as phenolic antioxidants, can convert free radicals to their more stable forms by supplying an H· radical. Because the resultant free radical cannot initiate a new free radical reaction, the chain reaction is terminated as a result. Antioxidants of the electron donor type exert their functions in the similar mechanism. Singlet oxygen quenchers such as vitamin E react with singlet oxygen and convert it to less active ground state oxygen and the quenchers themselves are transformed to the excited state, which release the energy and return to the ground state immediately. Hydroperoxide decomposers inhibit lipid oxidation by converting hydroperoxides generated in chain reactions to non-active compounds. Superoxide dismutases catalyze the formation of ground state oxygen and peroxide from superoxides. The produced peroxide is then hydrolyzed to water and ground state oxygen by catalase. Antioxidant synergists are used together with antioxidants and can enhance the antioxidant capability of antioxidants. Citric acid, fumaric acid, amino acids, vitamin C and its esters, tartaric acid, and phospholipids are common antioxidant synergists. Synergists reduce the catalytic activities of metal ions as well by complexing with them. Besides, some synergists act by reducing the intermediate free radicals generated by antioxidants. Common antioxidants Antioxidants can be natural or synthetic compounds according to their origins. The antioxidants that are approved in China include tocopherol, tea polyphenol, bamboo leaf flavone, propyl gallate, vitamin C, butylated hydroxyarisol (BHA), 2,6-butylated hydroxytoluence (BHT), and tertiary butylhydroquinone (TBHQ).

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3.3. Chemical Reactions in High Temperatures Lipids might undergo oxidation, degradation, polymerization and condensation and produce lower fatty acids, hydroxyl acids, esters, aldehydes, dimmers, and trimers in high temperatures. These reactions deteriorate the quality of foods and lead to darkening, viscosity increase, iodine value decrease, smoke point reduction, acid value elevation, and even irritant odor. The changes occurred during frying are associated with fatty acid composition, food composition, frying temperature, frying duration, and presence of metal ions. The thermal decomposition and polymerization reactions of lipids are illustrated in Figure 4-13.

Figure 4-13. Thermal degradation and polymerization of lipids.

Figure 4-14. Non-oxidative thermal degradation of saturated fatty acids.

3.3.1. Thermal Degradation Both saturated and unsaturated fatty acids undergo oxidative or non-oxidative thermal degradation in high temperatures. For saturated fatty acids, non-oxidative thermal degradation occurs only in very high temperatures, as shown in Figure 4-14: In the presence of oxygen, saturated fatty acids undergo oxidative-thermal degradation. All the methylenes of fatty acids can be attacked by oxygen, but those at α, β, and γ carbons are attached with priority. The degradation products, namely hydroperoxides, are further decomposed to aldehydes, ketones, and low molecular weight hydrocarbons. The non-oxidative thermal degradation of unsaturated fatty acids yields various dimmers and some low-molecular weight compounds. The oxidative thermal decomposition of unsaturated fatty acids occurs in the same pathway of lipid autoxidation in low temperatures, but in much higher rates.

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3.3.2. Thermal Polymerization The thermal polymerization of lipids might be oxidative or non-oxidative reactions. Nonoxidative thermal polymerization follows the Diels-Alder reaction. That is, a conjugated diene reacts with a double bond to produce a cyclohexene. R1

R1 R3

R3

＋ R4

R4 R2

R2

Figure 4-15. Inter-molecular non-oxidative thermal polymerization of lipids.

Figure 4-16. Intra-molecular non-oxidative thermal polymerization of lipids.

Thermal polymerization occurs either between different molecules (Figure 4-15) or inside the same molecule (Figure 4-16). In oxidative thermal polymerization, homolysis occurs in the α-carbon of a double bond and the resultant free radicals react with each other to produce non-cyclic dimmers or add to a double bond to yield cyclic or non-cyclic compounds. The thermal polymerization of lipids deteriorates the quality of foods and adversely affects food nutrition and safety. However, these reactions are desired in some cases. For example, thermal polymerization contributes to the formation of special flavors in fried foods.

3.4. Lipid Processing 3.4.1. Refining Oils extracted from the tissues of plants and animals by compression, organic solvent extraction, and cooking contain free fatty acids, phospholipids, sugars, proteins, water, pigments, and many other impurities. These impurities adversely affect the color, flavor, and stability of lipids and some compounds, such as aflatoxin and gossypol, threaten the health of consumers. Hence, the refinement process is required to remove these impurities. Degumming Phospholipids are removed in this step. Phospholipids are soluble in oils with low water content. When the water content increases, phospholipids are hydrated and precipitated from

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oil. In degumming, oils are added with 2%-3% water or vapor and then agitated. The water phase is then separated by settling or centrifugation. Carbohydrates and proteins are also removed in this step. Neutralization To remove free fatty acids and part phospholipids and pigments, NaOH solutions of appropriate concentrations are added to heated oils. The resultant foots or soapstock is separated and used for making soap. Gossypol and aflatoxin are also removed or destroyed in neutralization. Bleaching Chlorophyll, lutein, carotene, and other pigments affect the appearance of oil and decrease the stability against oxidation. These coloring materials can be removed by absorbents, such as floridin, emathlite, and active carbon. Other impurities, such as phospholipids, soaps, and some oxidation products are also absorbed by the absorbents. The absorbents are then separated by filtration. Deodorization The off-flavors of lipids are caused by the volatile compounds generated in lipid oxidation. These compounds can be removed by distillation in reduced pressure. Some nonvolatile off-flavor compounds are also destroyed by water steam and are then distilled away. Table 4-3. Content changes of harmful substances in rapeseed oil during refining(μg/kg) Compounds Anthracene Phenanthrene 1,2-benzanthracene 3,4-benzopyrene

Crude oil 10.1 100 14 2.5

Neutralization 5.8 68 7.8 1.8

Bleaching 4.0 42 5.0 1.0

Deodorization 0.4 15 3.1 0.9

The quality of oils is obviously improved after refining. Table 4-3 lists the removal of various harmful substances in rapeseed oil during refining. It should be noted that some useful components, such as fat-soluble vitamins and natural antioxidants are lost during refining. Hence, additional antioxidants are often added to refined oils to improve the antioxidant capacity.

3.4.2. Hydrogenation Hydrogenation is the addition of hydrogen to the double bonds of unsaturated fatty acids. Due to the insufficient supply of natural solid fat, liquid oils are hydrogenated to produce semisolid fats. Hydrogenated fats have gained wide applications in the production of margarine and shortening oil.

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Oils can be either partially or completely hydrogenated. The products of fully hydrogenated are used in soap production, and partially hydrogenated products are mainly used in the food industry. Iodine values in the range 60~80 are preferred for partially hydrogenated products, because the products exhibit desired melting point and solidity and good thermal and oxidation stability. The hydrogenation of oils is catalyzed mainly by nickel. In partial hydrogenation, the reaction occurs in pressure 1.5~2.5 atm and temperature 125-190 °C. The degree of hydrogenation can be measured according to the change of the refractive index. When the desired hydrogenation degree is reached, the reaction can be stopped by cooling and centrifugation. Oils must be refined before hydrogenation and the hydrogen used must be anhydrous and free of sulfur, SO2, ammonia, or other impurities. In addition to nickel, platinum, copper, and copper-chromium mixture are also used in oil hydrogenation as catalysts. However, platinum catalyst is expensive and copper can cause toxicity and its separation is difficult. Nickel is the most widely used catalyst. The hydrogenation reaction comprises three steps. Firstly, a carbon-metal complex is formed at an end of an olefin bond. This intermediate then reacts with the hydrogen attached to catalyst to form an unstable half-hydrogenated state, in which only one olefin bond is connected with the catalyst and thus can rotate freely. The half-hydrogenated compound reacts with another hydrogen atom to yield a saturated product and separates from the catalyst. Non-naturally occurring fatty acids may be generated during hydrogenation. Besides, some double bonds are translocated and isomerized during the process. Hence, partially hydrogenated oils are a complex mixture of multiple fatty acids. The products of linolenic acid hydrogenation are shown in Figure 4-17.

Figure 4-17. Hydrogenation products of linolenic acid [3].

Table 4-4. Effects of operating conditions on hydrogenation selectivity and rate Operating parameter High temperature High temperature High catalyst concentration High-intensity agitation

SR High Low High High

Trans fatty acids High Low High High

Rate High High High High

The selectivity of unsaturated fatty acid hydrogenation can be controlled by selecting different hydrogenation conditions and catalysts. The selectivity of hydrogenation is measured by the selectivity ratio (SR). SR is defined by Albright and is the ratio (rate of hydrogenation of linoleic to oleic)/(rate of hydrogenation of oleic to stearic).

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Reaction rate constants can be calculated from the starting and ending fatty acid compositions and the hydrogenation time. Selective hydrogenation reduces the formation of fully saturated glycerides, avoids excessive hardening of fats, and increases fat stability by decreasing the content of linoleic acid. However, selective hydrogenation yields undesired amount of trans isomers that cannot be absorbed by human body. As shown in Table 4-4, both catalyst species and operating conditions affect the hydrogenation selectivity.

3.4.3. Interesterification The distribution pattern of fatty acids in lipids markedly affects the properties of lipids. Because these properties affect the application of lipids in industries, interesterification has been performed to improve lipid performance. Interesterification changes the distribution pattern of fatty acid in triacylglycerols and is thus of industrial importance. Both intra(Figure 4-18) and inter-molecular interesterification (Figure 4-19) can occur, depending on the composition and structure of triglycerides. Interesterification can be accomplished by heating lipids in high temperatures for a long period. In the presence of catalyst, interesterification occurs at lower temperatures in a short period (50°C, 30min). Alkali metals and alkylated alkali metal are effective low-temperature catalysts and sodium methoxide is the most common one. The dosage of catalyst is generally about 0.1% of lipids. R1

R2

R1 R1

R2

R3 R2

R3

R3 Figure 4-18. Intra-molecular interesterification.

R1 R1

R1

R2 R2

＋ R2 R1

R2

R2 ＋

R1

R1

R2

R1

R2

R1

＋ R2

R2 R1

Figure 4-19. Intermolecular interesterification.

Excessive catalyst leads to more lipid loss due to the formation of soap and methyl esters. Impurities, such as free fatty acids and peroxides, can react with sodium methoxide and these impurities must be present in low levels in interesterification. Besides, sodium methoxide can react with water and lost the catalytic activity. Hence, interesterification reactions can be terminated by adding water.

134


Figure 4-20. Effect of interesterification on solid content index of lard [7].

Interesterification occurs either in the directed or the randomized mode. If the temperature is higher than the melting point of lipids, randomized interesterification occurs. In this mode, fatty acids are randomly connected to glycerol and the proportions of various glycerides depend on the contents of fatty acid contents in original lipids, which can be calculated according to the 1, 2, 3-random distribution theory mentioned previously. If the temperature is lower than the melting point, directed interesterification occurs. In this case, triglycerides with melting points higher than the reaction temperature crystallize and separate from the reaction mixture and the reaction proceeds until most of the fatty acids are precipitated. In directed interesterification, the proportions of trisaturated acylglycerols S3 (as crystals) and triunsatured acylglycerols U3 (liquid) increase simultaneously. Interesterification has found wide application in the manufacture of shortenings. Lard, due to its high proportion of disaturated triacylglycerols with palmitic acid in the 2 position, forms relatively large and coarse crystals, even when rapidly solidified in commercial chilling machines. Randomized interesterification of lard improves its plastic range and makes it a better shortening. Directed interesterification, however, produces a product with a higher solid content at high temperatures and thus extends its plastic range, as shown in Figure 4-20. Enzymatic interesterification utilizes lipase as catalyst. Compared with chemical interesterification, enzymatic interesterification occurs in mild conditions with high specificity, less byproducts, and low energy consumption. Hence, enzymatic interesterification is a promising method for lipid processing. Enzymatic interesterification has been used in the production of high-quality products such as cocoa butter equivalent and breast milk fat substitutes.

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REFERENCES [1] [2]

[3] [4]

[5]

[6] [7]

Hilditch, TP; Williams, PN. The Chemical Constitution of Natural Fats, 4th edition. London: Chapman and Hall, 1964. Kartha, ARS. The glyceride structure of natural fats. II. The rule of glyceride type distribution of natural fats. Journal of the American Oil Chemists' Society, 1953, 30, 326–329. Fennema, OR. Food Chemistry. 3rd edition. New York: Marcel Dekker; 1996. Chan, HW; Levett, G. Autoxidation of methyl linoleate: Analysis of methyl hydroxylinoleate isomers by high performance liquid chromatography. Lipids, 1977, 12, 837-840. Chan, HW; Levett, G. Autoxidation of methyl linoleate: Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates. Lipids, 1977, 12: 99-104. Frankel, EN. Autoxidation in Fatty Acids. Journal of the American Oil Chemists' Society, 1979, 353-390. Sreenivasan, B. Interesterification of fats. Journal of the American Oil Chemists' Society, 1978, 796–805.



Chapter 5

PROTEINS 1

Hong Lin1, Lisha Wu1 and Shuhui Wang2

College of Food Science and Engineering, Ocean University of China, Qingdao, China 2 Biosystems Engineering Department, College of Agriculture – Ginn College of Engineering, Aubum University, Auburn, AL, US

ABSTRACT Protein is one of the most important components in foods. Most proteins are constituted with 20 kinds of amino acids that are linked through peptide linkages. The combination of these amino acids leads to the occurrence of substantial amount of proteins with different structures and functions. Theoretically, all proteins produced by organisms can be utilized by human body. However, only proteins that are readily digestible, non-toxic and nutritious with specific functions are used in diets. This chapter briefly describes the properties and structures of amino acid components and proteins and highlights the properties of important dietary protein sources. Denaturation is an important change in food processing. The mechanism of denaturation, properties of denatured proteins and factors causing denaturation are also included in this chapter. In addition to the nutrition attribute, some functional properties, including emulsifying, foaming, gelling, and viscosity, also contribute to its wide applications in the food industry. These functional properties are described in detail in this chapter. Proteins undergo various changes during processing. These changes might greatly affect the quality of foods. This chapter also emphasize on the changes of proteins occurred in various processing techniques.

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Hong Lin, Lisha Wu and Shuhui Wang

1. PHYSICAL AND CHEMICAL PROPERTIES OF AMINO ACIDS AND PROTEINS 1.1. Amino Acids 1.1.1. Acid-Base Properties Amino acids are amphiphilic molecules that contain an acidic carboxyl group and one alkaline amine group. The dissociation constants of the amine and carboxyl groups in amino acids are lower than common carboxyl and amino groups. For example, the Ka value of glycine is 1.6×10-10, which is much lower than 10-5 of most carboxylic acids. Hence, the two groups do not occur as free forms in most cases. At around neutral pH, amino acids are present as dipolar ions or zwitterions. Amino acids can react with both acids and bases. Carboxylic acid groups (–COOH) can be deprotonated to become negative carboxylates (–COO－), and α-amino groups (–NH2－) can be protonated to become positive α-ammonium groups (+NH3－), depending on the pH of the environment. At pH values greater than the pKa of the carboxylic acid group, the negative carboxylate ion predominates. At the pH values lower than the pKa of the αammonium group, the nitrogen is predominantly protonated as a positively charged αammonium group. Thus, at pH between the pKa of carboxylic and amine groups, amino acids have net zero charge and this state is known as zwitterion. Below the pKa of carboxylic acid, the predominant form will have a neutral carboxylic acid group and a positive α-ammonium ion, and above the pKa of the amine group, most amino acids have a neutral amine group and a negative carboxylate. 1.1.2. Isoelectric Point At pH values between the two pKa values, the zwitterion predominates, but coexists in dynamic equilibrium with small amounts of net negative and net positive ions. At the exact midpoint between the two pKa values, the trace amount of net negative and trace of net positive ions exactly balance, so that average net charge of all forms present is zero. This pH is known as the isoelectric point pI, so pI = ½(pKa1 + pKa2). Amino acids have zero mobility in electrophoresis and minimum solubility at their isoelectric points. Hence, amino acids can be isolated by precipitation from water by adjusting the pH to their isoelectric points. The pI ranges of neutral, acidic, and alkaline amino acids are 5.5~6.3, 2.8~3.2, and 7.6~10.6 respectively. Generally, amino acids with more amine groups have higher pI values and those with more carboxylic groups have lower pI values. 1.1.3. Chemical Reactions Amino acids show the usual reactions of both carboxylic acids and amines and these reactions might occur during food processing. (1) Acylation of amine group Activated acid derivatives, such as acid halogenides and anhydrides, can react with the amine group of amine acids as acylating agents. The following is the reaction between carbobenzoxy chloride and amino acids

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The acylated products are more stable against various reagents and can be removed by many methods. Hence, carbobenzoxy chloride has been widely used in protein synthesis for amine group protection. (2) Alkylation of amine group Amino acids can react with 1-fluoro-2, 4-dinitrobenzene (FDNB) to yield N-2,4dinitrophenyl amino acids (DNP-amino acids), which are yellow compounds and crystallize readily. This reaction is very important in N-terminal acid labeling for protein sequencing in Sanger‘s method.

(3) Reaction with nitrous acid All α-amino acids, except proline, can react with nitrous acid to yield nitrogen and hydroxyl acid. The liberated nitrogen gas is contributed by equal molar amine group and nitrous group. Hence, the contents of free amine groups in amino acids can be determined by measuring the volume of nitrogen gas formed by reaction with nitrous acid. This is the principle of the Van Slyke method.

(4) Reaction with ninhydrin All α-amino acids, except proline, can react with ninhydrin in alkaline solutions to produce purple products. This reaction has been widely used in the identification and colorimetric quantification of amino acids. (5) Reactions of carboxylic groups. The carboxylic groups of amino acids can undergo various chemical reactions. Azide reaction is a typical reaction on the carboxylic group. Amino acid ester reacts with hydrazine to yield hydrazide and the product then reacts with nitrous acid to produce azide:

The azide compound can react with another amino acid ester by condensation to produce a dipeptide:

140


Peptides synthesized in this method are optically pure. When heated, the carboxylic group can react with the amine group of in the same molecule to produce diketopiperazine. This compound contributes to the special flavor of foods.

1.3. Sensory Properties 1.3.1. Bitterness Amino acids contain multiple functional groups and can act with various taste receptors to exhibit different tastes. Sweet amino acids are primarily found among amino acids of the D form, while the tastes of L-series amino acids are significantly affected by their side chains. When the side chain is small, the sweet taste predominates. For example, glycine (Gly), alanine (Ala), proline (Pro), hydroxyproline (Hpr), homocysteine (Hcys), serine (Ser), threonine (Thr), asparagine (Asn), and glutamic acid methyl ester (Glu-OMe) contain small side chains and taste sweet. When the side chain contains more than three carbons, such as leucine (Leu), isoleucine (Ile), norleucine (Nle), phenylalanine (Phe), tyrosine (Tyr), tryptophane (Trp), histone (His), lysine (Lys), arginine (Arg), the bitter taste predominates. If the side chain is of moderate size, the amino acids taste both sweet and sweet, such as valine (Val) and ornithine (Orn). The sensory property of amino acids also depends on the hydrophobicity of side chains. If the side chain is only slightly hydrophobic, the amino acid tastes slightly sweet without bitterness, such as glutamin (Gln), cysteine (Cys), and methionine. If the side chain is acidic, the amino acids taste sour, such as aspartic acid (Asp) and glutamic acid (Glu). Table 5-1 lists the relationship between the sensory properties and side chain structure of amino acids. Table 5-1. Relationship between the side chain structure and taste of amino acids Category І II III IV V

Amino acids Glu, Asp, Gln, Asn Thr, Ser, Ala, Gly, Met, Cys Hpr, Pro

Side chain property Acidic Short

Taste Sour Sweet

Pyrrole

Val, Leu, Ile, Phe, Tyr, Trp His, Lys, Arg

Large and long

Sweet, with slight bitterness Bitter

Alkaline

Sweet, with slight bitterness

The sensory properties of oligopeptides, especially dipeptides, are decided by the original tastes of the amino acid components. The rules are as follows:

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(1) Neutral peptides consisting of type I and V amino acids and those containing only type II amino acids exhibit light tastes. (2) The sodium salts of oligopeptides consisting of only type I amino acids or type I and type II amino acids taste fresh, such as Glu-Glu, Glu-Asp, Glu-Ser, and Glu-Thr. (3) The combination of type III amino acids with type I amino acids remove the bitter taste, but remain the sour taste. (4) Peptides composed of type III, IV, or V amino acids only or their combinations are bitter. (5) Peptide formation, carboxyl group esterification, or diketopiperazine formation by coupling of type IV and V amino acids enhances the bitter taste. (6) Peptides with type IV and V amino acids locating at the C terminal are 3~5 times bitterer than those with the amino acids locating at the N terminal or the middle of the peptides. (7) Type II amino acids (especially Gly) locating at either terminals or cyclized increase the bitterness. All peptides contain AH polar groups. However, because peptides differ markedly in molecular weight and nature of hydrophobic groups, they have different bitterness receptor binding capabilities. The bitterness intensity of peptides can be predicated by calculating the average hydrophobicity of side chains, which is related to the total hydrophobicity of nonpolar amino acids. Table 5-2 lists the hydrophobicity of various amino acids. The average hydrophobicity of a peptide can be calculated by using the following formula:

In which, ⊿G is the free energy change of the side chains of amino acids and n is the number of resides in the peptide. Peptides with Q greater than 6.85kJ/mol are bitter and those with Q less than 5.43kJ/mol are not bitter. No rules have been found for the taste of peptides with Q in the range 5.43~6.85kJ/mol. However, exceptions have been found. For example, all peptides with molecular weight more than 6000 taste light despite of the Q value. If Gly is attached to one or both ends of a peptide, the peptide tastes bitter even though Q is less than 5.43kJ/mol. This is possible due to the less steric hindrance of Gly that facilitates the contact with bitterness receptors. For this reason, it has been proposed to exclude Gly from Q calculation. Arg-Arg, Lys-Ala, Val-Ah, Arg-Gly-Pro, and Ser-Lys-Gly-Leu, are also exceptions to the rule. The Q values of these peptides are less than 5.43kJ/mol, but they taste bitter. Besides, the bitterness intensity of peptides with similar Q values might differ significantly. The bitterness intensity of Gly-Gly-Gly-Leu is greater than its three positional isomers and that of Leu polymers are Leu-Leu-Leu-Leu, Leu-Leu-Leu, Leu-Leu, and Leu in decreasing order. In the case of bitter peptides in cheese, the bitter taste disappears after the Gln at the N terminal is cyclized, though the Q value is not changed. These inconsistencies indicate that the bitterness intensity of peptides is also affected by their molecular weights and higher structures in addition to Q values.

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Hong Lin, Lisha Wu and Shuhui Wang Table 5-2. Hydrophobicity of the side chain of amino acids (transfer from ethanol to water)

Amino acid Ala Arg Asn Asp Cys Gln Glu Gly His Ile

⊿G0(side chain)/(kj/mol) 2.09 2.1 0 2.09 4.18 -0.42 2.09 0 2.09 12.54

Amino acid Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

⊿G0(side chain)/(kj/mol) 9.61 6.25 5.43 10.45 10.87 -1.25 1.67 14.21 9.61 6.27

1.3.2. Delicious Flavor The delicious flavor is a complex taste and is generally contributed by various flavor enhancers, including some amino acids, as shown in Table 5-3. Table 5-3. Main delicious flavor-contributing components in some foods Material

MSG

Animal meat Fish meat Shrimp, crab Shellfish Octopus, squid Kelp Vegetable Mushroom Soy sauce

+ + + +++ ++ +++

Amino acid amine and peptides ++ ++ + +++ +++ ++ ++

5‘-IMP

5‘-GMP

Sodium succinate

+++ +++ ++ +++

+++ +++

+++

Note: MSG, monosodium glutamate; 5‘-IMP, 5‘-inosine monophosphate; 5‘-GMP, 5‘-guanosine monophosphate.

(1) Common properties of flavor enhancers. Flavor enhancers are compounds that enhance the aroma of a food, though they themselves have no distinct odor or taste in concentrations used. Many compounds have been found to have the flavor enhancing capability. Only dissociated glutamic acid salts can enhance flavor, of which, monosodium glutamate (MSG) has the purest taste. Glutamic acid derivates that cannot dissociate have no the flavor enhancing capability. 5‘-Inosine monophosphate (5‘-IMP), 5‘-guanosine monophosphate (5‘-GMP), and 5‘-xanthine monophosphate (5‘-XMP) are good flavor enhancers, but adenylate has no the flavor enhancing ability. L-Cys, sodium salt of thiosulfonic acid, homocysteine, L-Asp, L-aminoadipic acid, and succinic acid, display flavor enhancing abilities similar to L-MSG.

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Many fruit acids, such as malic acid, tartaric acid, and citric acid, enhance the taste of foods. The combination of anyone of the acids with lactic acid can improve the odor of soybean product. Besides, citric acid juice enhances the taste of strawberry. Fumaric acid and maleic acid can suppress the unpleasant odor of garlic. Glutathione enhances the taste of meat broth. Polyphosphates improves the taste of chicken meat and cheese. The diamine salts of dicarboxylic acids with carbon atom number ranging from 3 to 10 can be used as the substitutes of table salt. It is proposed that all flavor enhancers show a common structure O-(C)n-O, in which, n ranges from 3 to 9. In another word, the compounds must contain an alkyl chain with 3~9 carbons and the two ends must be negative charged. Compounds with 4~6 carbons have the highest enhancing ability. The alkyl chain can be straight, branched, or cyclic and the carbon atoms can be replaced by O, N, S, and P. The negative charges on the two ends of the molecule are crucial for the enhancing ability. If the carboxylic acid group is esterified, amidated, or converted to lactone or lactam upon heating, the enhancing ability is obviously reduced. The negative charge of an end can be replaced by a negative dipole. For example, the enhancing abilities of tricholomic acid and ibotenic acid are 5~30 times higher than that of L-MSG.

For economic and safety reasons, only Glu- and nucleotide-type flavor enhancers have gained commercial applications. (2) Glu-type flavor enhancers. Flavor enhancers containing Glu are fatty compounds and have specific spatial configuration for their functions. The negatively-charged groups at the two ends, such as – COOH, –SO3H, and –SH, determine the flavor enhancing capability and hydrophilic groups, such as –L-NH2OH, assist in the capability. Dipeptides or tripeptides with hydrophilic amino acids attached to the carboxylic end of Glu have the flavor enhancing ability, but those with hydrophobic amino acids attached are bitter, as shown in Table 5-4. It is noteworthy that all the amino acids have more than one taste. Table 5-5 lists the tastes of three amino acids. The flavor enhancing capability of MSG is affected by the environmental pH. MSG has the highest enhancing ability in pH6.0. Besides, temperature also affects the ability. When MSG is heated for a long time or at 120°C, inter-molecular dehydration occurs and pyroglutamic acid (shown below) is generated. This product losses the flavor enhancing ability and is toxic to human body.

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Table 5-4. Relative flavor enhancing abilities of MSG-containing flavor enhancers (with L-MSG as 1) Flavor enhancer L-MSG sodium salt of L-Asp Sodium salt of L-α- aminoadipic acid Sodium L- tricholomic acid sodium salt of L-cysteine thiosulfonic acid Glu-γ-NH2 Glu-γ-Ome Glu-α-NH2 Glu-Asp Glu-Ser Thr-Glu Asp-Glu-Ser Ser-Glu-Glu Glu-Glu-Glu Glu-Gly-Ser Glu-Glu-Ser Glu-α,γ-(NH2)2 (Erythrose)-HO2CCH2CHOHCHNH2CO2Na (Stachyose)- HO2CCH2CHOHCHNH2CO2Na (R,S)-HO2CCHMeCH2CHNH2CO2Na (S,S)-HO2CCHMeCH2CHNH2CO2Na L-HO2CCHOHCHNH2CO2Na L-NaO3SCH2CHNH2CO2Na L-NaO3S(CH2)2CHNH2CO2Na L-NaO3S(CH2)3CHNH2CO2Na Ac-Glu-α,γ-(OMe)2

Relatively ability 1.00 0.08 0.10 5~30 0.10 Delicious Sweet 0 0.15 0.10 0.10 0.10 0.15 0.20 2.00 2.00 Bitter 0.1 1.8 0.1 0.1 Nearly 0 0.1 1.0 0.1 0

Table 5-5. Taste proportions of three amino acids Amino acid L-Glu L-MSG L-Try

Delicious taste 21.5% 71.4% 1.2%

Sour 64.2% 3.4% 5.6%

Salty 2.2% 13.5% 0.6%

Sweet 0.8% 9.8% 1.4%

Bitter 5.0% 1.7% 87.6%

In addition, MSG is racemized under alkaline solutions when heated and loses flavor enhancing ability. Hence, MSG should be added after dishes or soups are cooked. (3) Nucleotide-type flavor enhancers. Flavor enhancers of this type are aromatic heterocyclic compounds. The hydrophilic ribose phosphate determines the flavor enhancing ability and e hydrophobic substituents attached to the aromatic heterocycle assist in the ability.

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2. STRUCTURE OF PROTEIN 2.1. Primary Structure Primary structure is the sequence of amino acid residues in proteins and is decided by the genetic codes in genes. The primary structure decides the higher structures of proteins. All proteins are composed of 20 amino acids and the combination of these components leads to the occurrence of tremendous number of proteins with different spatial structures and biological functions.

2.2. Higher Structures The polypeptide chains of proteins occur as folded and twisted conformation under suitable conditions and these conformations determine the biological and physicochemical properties of the molecules in addition to their amino acid sequences.

2.2.1. Secondary Structure Secondary structure is the three-dimensional form of local segments of polypeptides. This term does not involve the conformation of the side chains of amino acid residues. Two forms of periodic secondary structures have been found: α-helices and β-sheet. The α-helical structure is characterized by the following: (1) Multiple peptide bond planes rotate through the α-carbon atom spin, twisting each other into a tight solid right-handed helix. (2) Each helical rotation involves 3.6 amino acid residues. Each residue extends the axial length by 1.5 Å and the angle of rotation per residue is 100°. (3) α-Helices are stabilized by hydrogen bonding. In this structure, each backbone N-H group is hydrogen bonded to the C=O group of the fourth preceding residue in the same chain. (4) The side chains of amino acid residues are located on the outer of the helix and their shape, size, and charge affect helix formation. α-Helix is often not found in areas with abundant acidic or basic amino acids due to charge repulsion or areas consisting more amino acids with large side chains, such as tryptophan and isoleucin. Proline is generally not

146


involved in α-helix formation, because it cannot rotate or hydrogen bond with other amino acids readily. The presence of glycine affects helix stability, because its side chain (H) occupies the minimum space. The β-sheet structure is characterized by the following: (1) The β-sheet structure is an extended structure. Amino acid residues involved are arranged in a zigzag pattern and their residues are located on the upside or downside of the pleat plane. The angle between adjacent peptide bond planes is 110°. (2) The conformation is stabilized by hydrogen bonding of C=O and –NH between adjacent strands of a peptide. (3) When adjacent strands run in the same direction, the peptide chains are parallel. When the chains run in opposite directions, a planar, antiparallel sheet structure is stabilized. Both parallel and antiparallel sheets can occur in one peptide. (4) In parallel β-sheet, the distance between any two adjacent residues is 0.65 nm and that in anti-parallel β-sheet is 0.7 nm.

2.2.2. Tertiary Structure The secondary structures of a polypeptide chain can further fold to form a compact three-dimensional structure termed as tertiary structure. The tertiary structure of proteins are stabilized mainly by secondary bonds, including hydrogen bond, hydrophobic interaction, salt linkage, and Vander Wasls force, as shown in Figure 5-1. These secondary bonds might occur between the side chains of amino acid residues that are quite far away in primary structure. Secondary bonds are non covalent bonds and are affected by the pH, temperature, and ionic strength of the environment and are hence subject to change. Disulfide bond is not a secondary bond, but it can connect two segments of a peptide that are far away in a peptide chain. Hence, disulfide bonds are very important for stabilizing tertiary structures. Proteins can be divided into two large groups on the basis of conformation: fibrous and globular proteins. Fibrous proteins, such as fibroinare, the slender molecules with axial ratio are greater than 10. Plasma albumin, globulin, and myoglobin, are examples of globular proteins. In globular proteins, hydrophobic groups are located on the interior of a protein and the hydrophilic groups are distributed on the surface. Hence, globular proteins are hydrophilic molecules and most enzymes are globular proteins. The distribution of the side chains leads to the formation of special areas with special biological functions, such as the active center of enzymes. Figure 5-2 shows the folding patterns of two proteins. 2.2.3. Quaternary Structure Multiple folded peptide chains can combine with each other through secondary bonding to form a more complex structure. This structure is called the quaternary structure of proteins. Each peptide chain with specific tertiary structures is a subunit of the protein. Hence, quaternary structure is also defined as the arrangement and interaction of subunits. The secondary bonds involved in subunits interaction are loose than those in secondary and tertiary structures and proteins with quaternary structure can dissociate to separate subunits with their conformation unchanged under certain conditions.

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a

electrovalent bond; hydrogen bond; c hydrophobic bond; d Vander Wasls force; e disulfide bond. b

Figure 5-1. Secondary bonds involved in tertiary structure stabilization [1].

Figure 5-3 shows the combination pattern of the subunits of hemoglobin. The structures of subunits in a protein may be the same or different. For example, the coat protein of tobacco streak virus consists of 2200 identical subunits and normal hemoglobin A is a tetramer containing two α subunits and two β subunits. The smallest subset of different subunits of a protein is defined as the protomer. For example, the aspartate carbamyl transferase contains six protomers and each protomer consists of one catalytic subunit and one regulatory subunit. Some proteins can further aggregate into polymers and each repeating unit is called a monomer. According to the number of monomers, protein polymers can be divided into dimer, trimer, oligomer and multimer. For example, insulin might occur as dimer and hexamer in human body.

Figure 5-2. Tertiary structures of myoglobin and triosephosphate isomerase [2].

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Figure 5-3. Binding pattern of hemoglobin subunits [3].

2.3. Structure-Function Relationships 2.3.1. Relationship between Primary Structure, Conformation and Function Primary structure determines the spatial arrangement of peptide chains and the conformation is mainly maintained by various secondary bonds of side chains in amino acid residues. Once a polypeptide chain is synthesized in vivo, the protein folds automatically to the correct conformation. Proteins with similar primary structure have similar conformations and functions. For instance, proteins of same functions isolated from different organisms only differ slightly in primary structure and the difference is much smaller between organisms with closer evolutionary relationships. 2.3.2. Relationship between Spatial Conformation and Function The diverse functions of proteins are closely related to their spatial conformations. Once the conformations changed, the functional activities vary accordingly. The relationship between conformation and function of proteins can be evidenced by protein denaturation and renaturation, which will be detailed later.

3. CLASSIFICATION OF PROTEINS 3.1. Classification by Amino Acid Composition and Quantity According to the composition of amino acids and their contents in proteins, proteins are divided into complete, partially complete, and incomplete proteins.

3.1.1. Complete Protein A complete protein (or whole protein) contains an adequate proportion of all nine of the essential amino acids necessary for the dietary needs of humans or other animals. Many animal products, such as milk, eggs, fish, and meat, contain complete proteins.

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3.1.2. Partially Incomplete Protein A partially incomplete protein contains all the amino acid species, but their proportions are insufficient for human demand. Partially incomplete proteins can maintain life, but cannot enhance growth and development. For example, gliadin in wheat contains a low proportion of Lys and is thus designated as a partially incomplete protein. Any essential amino acid that is present in insufficient quantity for body protein synthesis is defined as a limiting amino acid. Corn proteins contain low levels of Lys and thus Lys is the limiting amino acid. 3.1.3. Incomplete Protein Incomplete proteins lack one or more essential amino acids in correct proportions as necessary for good nutrition and health. Organism cannot grow, develop, or maintain their lives only on incomplete proteins. Collagen in pork skin is an example of incomplete protein.

3.2. Classification by Solubility Based on solubility, proteins are divided into seven categories.

3.2.1. Albumin Albumin refers to any protein that is soluble in water and moderately soluble in concentrated salt concentrations. Albumin can be precipitated by saturated ammonium sulphate. Albumin occurs widely in nature. Leucosin in wheat seeds, blood plasma proteinand ovalbumin in eggs are examples of albumin. 3.2.2. Globulin Globulins are insoluble in water but soluble in diluted salt, acid, or alkaline solutions and can be precipitated by half-saturated ammonium sulphate. Globulins have important biological functions. Legumin in soybean seeds, seroglobulin in blood, myoglobulin in muscle, and immunoglobulins are of this category. 3.2.3. Histone Histones are highly alkaline proteins and are soluble in water or diluted acid solutions. Histones contain rich quantity of Arg and Lys and are the structural components of chromosomes. 3.2.4. Protamine Protamines are small, arginine-rich proteins and are soluble in water and diluted acid solutions. Protamines contain abundant alkaline amino acids and lack Trp and Tyr. The proteins are found in mature sperm cells and replace histones late in chromosome the haploid phase of spermatogenesis. 3.2.5. Prolamin Prolamins are a group of plant storage proteins with high proline contents and found in the seeds of cereal grains. They are characterized by a high glutamine and proline content and are generally soluble only in 70%~80% ethanol solutions.

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3.2.6. Gluten Glutens are contained in plant seeds, such as barley and rye. Proteins of this category are insoluble in water or diluted salt solutions but soluble in diluted acid and alkaline solutions. 3.2.7. Albuminoid Proteins of this category are not soluble in water or salt, diluted acid and diluted alkaline solutions and occur mainly in skins, hairs, and nails. The roles of such proteins include protection and support, forming connective tissue, tendons, bone matrices, and muscle fiber. Keratin, collagen, elastin, and fibroin are all albuminoids.

3.3. Classification by Chemical Composition 3.3.1. Nucleoprotein A nucleoprotein is any protein which is structurally associated with nucleic acid. Deoxyribonucleoprotein, ribosome, and tobacco mosaic virus are examples of nucleoprotein. 3.3.2. Lipoprotein A lipoprotein is a complex molecule consisting of both protein and lipid. The lipid might be phospholipids, sterols, or neutral fats. Many transporters in blood and lipovitellin are lipoproteins. 3.3.3. Glycoprotein And Mucoprotein In glycoproteins, proteins are covalently attached to oligosaccharides and the glycans can be galactose, mannose, hexosamine, hexuronic acid, or many other sugars. Mucoprotein is a typical example of glycoprotein and it is primarily composed of mucopolysaccharides. 3.3.4. Phosphoprotein In phosphoproteins, phosphoric acid is connected to the Ser or Thr resides of proteins through ester bonds. Casein and pepsin belong to this category. 3.3.5. Metalloprotein Metalloproteins refer to any proteins that contain metal ion cofactors. Many important enzymes, transport proteins and storage proteins are metalloproteins. For example, hemoglobin contains iron, chlorophyl contains magnesium, and hemocyanin contains copper. 3.3.6. Flavoprotein Flavoproteins are proteins that contain a nucleic acid derivate of riboflavin. Succinic acid dehydrogenase and d-amino acid oxidase are examples of flavoprotein.

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4. PROTEIN DENATURATION 4.1. Definition of Denaturation When a protein is exposed to such physical stresses as heating and ultraviolet irradiation or such chemical stresses as strong acid and base, its properties are changed, such as decrease of solubility and loss of activity, but the primary structure remains unaltered. This process is termed denaturation. Denaturation is an extremely important property of proteins and markedly affects food processing.

4.2. Mechanism of Denaturation The denaturation theory was first proposed by a Chinese biochemist in 1931. It was believed that denaturation is the transfer of natural proteins molecules from ordered and compact states to disordered and loose states because of environmental stresses. The compact structure of natural proteins is maintained by various secondary bonds. These secondary bonds are easily interrupted by physical and chemical factors, which induce the destruction or alternation of protein conformation. Hence, protein denaturation is actually caused by the changes of secondary, tertiary, and quaternary structures of proteins. The destruction of spatial conformation of proteins leads to reduced solubility, aggregation, irreversible gelation, increased sensitivity to protease hydrolysis, and loss of physiological activities. A protein denatured in mild conditions is present in a relaxed conformation. After the denaturation factor is removed, the protein can restore to its natural conformation. Hence, this denaturation process is reversible. For example, when ribonuclease is treated with 8 mol/L urea and mercaptoethanol, the disulfide bonds in the molecule is reduced and the enzyme loses its activity due to changed conformation. However, after the regents are removed by dialysis, the denatured protein is automatically oxidized and returns to its natural conformation (Figure 5-4).

Figure 5-4. Denaturation and renaturation of ribonuclease [4].

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Trypsin and hemoglobin also undergo reversible denaturation. After trypsin is denatured by heating at 70~100°C for a short time in an acidic solution, the enzyme loses its catalytic activity. When the solution is cooled down in a proper rate, trypsin can recover all its properties. Haemoglobin can be denatured by low-concentration sodium salicylate. After sodium salicylate is removed, the protein can restore to its natural conformation. However, the denaturation of most proteins is irreversible. For example, egg protein is gelated when cooking and this process is irreversible; soybean protein is irreversibly denatured in tofu production. The reversibility of protein denaturation is related to the denaturation agent species, protein property and the degree of protein conformation destruction. Generally, in reversible denaturation, the tertiary and quaternary structures of proteins are destroyed, but secondary structures remain intact. In contrast, in the irreversible denaturation, the secondary, tertiary and quaternary structures are all interrupted.

4.3. Properties of Denatured Protein The physical, chemical and biological properties of proteins undergo obvious changes after denaturation. (1) Hydrophobic groups originally located inside molecules get exposed on molecule surface and the hydration layer is destroyed. Hence, the solubility of denatured proteins decreases significantly. (2) Denatured proteins cannot crystallize as original ones. (3) The spatial structure of denatured proteins is in random shape. The friction between molecules increases and the fluidity decreases. Hence, denatured proteins have reduced diffusion coefficient and their solutions have lower fluidity. (4) The optical activity of denatured protein changes and the isoelectric point increases slightly. (5) Denatured proteins are more susceptible to enzymatic hydrolysis. For example, natural haemoglobin cannot be hydrolyzed by trypsin, but denatured haemoglobin can be hydrolyzed easily. This is why cooked foods are digested and absorbed more easily. (6) Denatured proteins have more exposed side chains, such as –SH and –OH. Hence, the degree of protein denaturation can be estimated by the reaction between these groups and specific reagents. (7) The bioactivities of denatured proteins are completely or partially destroyed. For example, denatured enzymes lose their catalytic activities; denatured hemoglobin can no longer transport oxygen; and denatured antigens lose their immunological competences.

4.4. Denaturation Agents and Their Mechanisms 4.4.1. Heat Heating is the most common physical factor of protein denaturation. The temperature required for detectable denaturation varies with the nature of protein and is also affected by

Proteins

153

protein purity and pH. The denaturation of most proteins can be detected in the temperature range 45~50°C and the process is accelerated when the temperature is elevated to 55°C. Denaturation occurring at lower temperatures involves only the disruption of non-covalent bonds and proteins become unfolded. For example, natural serum albumin is of the oval shape with the axial ratio of 3:1, while the axial ratio of denatured serum albumin increases to 5:5. Such denaturation induced by short-time exposure to low temperatures is reversible. When the temperature increases to 70~80°C, disulfide bonds are broken. Hence, protein denaturation occurring in long-time high-temperature treatment is irreversible. Heat treatment leads to protein denaturation by disrupting hydrogen bonding and many other secondary bonds that maintain protein conformation. It is estimated that the rate of denaturation increases by up to 600 folds when the temperature increases by 10°C in the denaturation temperature range. The sensitivity of a protein to thermal denaturation depends on multiple factors, such as protein property, protein concentration, water activity, pH, ionic strength and ion species. Proteins, enzymes, and microbes are more resistant to thermal denaturation in watercontaining conditions than in dry conditions. Besides, high concentration of denatured protein hinders the renaturation process. The temperature used in the thermal processing of foods is generally around 100°C and the central temperature of materials often exceeds 70°C. Hence, most proteins are denatured and pathogens are inactivated during thermal processing of foods.

4.4.2. Radiation The impact of radiation on proteins depends on the energy and wavelength of the rays used. Ultraviolet radiation can be absorbed by aromatic amino acid residues (tryptophan, tyrosine and phenylalanine) and can change the conformation of proteins. Radiation by highenergy rays disrupts disulfide bonds. γ radiation and other ionizing radiation can also make conformation changes, amino acid oxidization, covalent bond disruption, ionization, proteins free radical formation as well as polymerization. 4.4.3. Denaturation at Interfaces The denaturation of proteins attached to water-air, aqueous-nonaqueous solution, and water-solid interfaces is generally irreversible. Proteins are surfactants and tend to migrate to the interface and be adsorbed. The adsorption rate of proteins is controlled by diffusion rate of natural proteins to the interface and the absorption stops when the interface is saturated by denatured proteins (about 2mg/m2). Figure 5-5 indicates that the migration and absorption of globular proteins in the aqueous-nonaqueous interface. During the migration to interfaces, proteins interact with high-energy water molecules (compared with the water molecules far away from the interface) at the interface. The hydrogen bonding between protein molecules is then disrupted and the protein conformation becomes slightly unfolded (Figure 5-5b). Because more hydrophobic groups are in contact with water, the partially unfolded protein is hydrated and activated and transforms to an unstable state. The protein further extends and unfolds at the interface and the hydrophobic and hydrophilic residues are positioned in the water and non-aqueous phases respectively, leading to protein denaturation at the interface. Proteins that are mainly stabilized by disulfide bonds are not readily absorbed at the interface. Hence, these proteins are less susceptible to denaturation at interface.

154

Hong Lin, Lisha Wu and Shuhui Wang a)

b)

c)

Figure 5-5. Schematic diagram of protein interface denaturation [5].

The interfacial properties of proteins are important for their applications in various food systems. For example, proteins absorbed at interfaces are beneficial for the formation and stabilization of emulsions and foams. To keep the natural conformation and functional properties of proteins, dispersion systems, such as foams and emulsions, should be avoided during food processing and separation.

4.4.4. pH It is irreversible that protein denaturation occurs in mild and extreme pH conditions. Extreme pH changes the ionization of some groups in polypeptide chains and disrupts the hydrogen bonding required for maintaining conformation and the electrostatic interaction between differently charged groups. If the pH is lowered far below the isoelectric point (pI) or elevated above the pI, the protein will contain only positive or negative charges. The charges will repel each other and prevent the protein from aggregation. In areas with large charge density, the intramolecular repulsion may be great enough to cause unfolding of the protein. In some cases the unfolding may be extensive enough to expose hydrophobic groups and

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cause irreversible aggregation. Yogurt is a typical example of acid-induced protein denaturation.

4.4.5. Heavy Metals Heavy metal salts containing Hg2+, Pb2+, Ag+, T+, Cd2+ and other metals with high atomic weights can disrupt the salt bridges and disulfide bonds in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal-protein salt. This reaction is used for its disinfectant properties in external applications. For example AgNO3 is used to prevent gonorrhea infections in the eyes of new born infants. Silver nitrate is also used in the treatment of nose and throat infections, as well as to cauterize wounds.

Figure 5-6. Denaturation of prion protein by alcohol.

Figure 5-7. Denaturation of prion protein by reducing agents.

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This same reaction is used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have swallowed a significant quantity of a heavy metal salt. As an antidote, a protein such as milk or egg whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce vomiting so that the precipitated metal protein is discharged from the body.

4.4.6. Chemical Reagents Most organic solvents can cause protein denaturation. Organic solvents disrupt the hydrogen bonding between water and proteins, which leads to change the dielectric constant of medium. Non-polar organic solvents can enter the hydrophobic area of proteins and denature proteins by destroying the hydrophobic interaction. A 70% alcohol solution is often used as a disinfectant on the skin. Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds are formed instead between alcohol and protein side chains. In the prion protein, Tyr 128 is hydrogen bonded to Asp 178, which cause one part of the chain to be bonding with a part some distance away. After denaturation, the graphic show substantial structural changes, as shown in Figure 5-6. Urea denatures protein also by disrupting the hydrogen bonding, because it forms new hydrogen bonds with proteins. Guanidine hydrochloride denatures proteins in the similar mechanism as urea. Different protein chains or loops within a single chain are often held together by the strong covalent disulfide bonds. Reducing agents, such as dithiothreitol, can split disulfide bonds apart and cause protein denaturation. Figure 5-7 shows the denaturation of proteins by reducing agents.

4.5. Effects of Combined High-Pressure and Thermal Treatment on Beef Protein Denaturation Thermal treatment in combination with high pressure improves the tenderness of beef and enhances the sterilization effect. Besides, the combined treatment changes the composition of beef muscles and affects its functional properties, such as color, texture, fat oxidation and flavor, which are related to protein denaturation. Differential scanning calorimetry(DSC) is an effective tool for studying the structural and thermodynamic properties of natural polymers. DSC has been used in the investigation of denaturation and resultant texture changes of muscle proteins. Figure 5-7(a) Figure 3 shows the thermograms of intact beef muscle after treatment at pressures from 0.1 to 800MPa at 20°C for 20min. The three major endothermic transitions seen have been attributed to myosin, collagen and actin and the peak maxima in this study are seen at 54.6°C (myosin), 67.1°C (collagen) and 77.3°C (actin). Treatment at 200MPa decreased the myosin and actin peaks and at 400MPa and higher, the actin peak is not seen. Figure 5-7(b) shows the thermograms when the samples are treated at 0.1-800MPa at 40°C for 20min. At ambient pressure about 43% of the myosin, is denatured but there is no obvious effect on collagen and actin. At 200MPa the myosin and collagen are merged into one broad peak with an onset temperature of 50.1°C. At 400MPa, the actin peak is not seen. When the pressure is increased to 600 and 800MPa, the new myosin peak and that of collagen are still discernible.

157

Proteins a)

Peak 1

Peak 3 Peak 2

0.2 mw

0.1 MP2 Peak N

Heat Flow (mw)

200 MP2 400 MP2

600 MP2 800 MP2 Rescan

50

55

60

65

70

75

80

85

Temperature (˚C)

b)

40

45

Figure 5-7. Thermograms of beef muscle, heated at 10 ˚C min−1 after treatment at different pressures in air for 20min at 20°C and 40°C [7].

Although the combination of high pressure and thermal treatment has complex effect on protein properties, it has wide applications in food processing: Improving texture and tenderizing by causing protein unwinding and aggregation; Extending shelf life by inactivating enzymes, microbes, and toxins activity; Increasing digestibility; Increasing the ability of maintaining flavors, pigments and vitamins by increasing the surface hydrophobicity due to unwinding; Improving product flavor and overall acceptability.

4.6. Effects of Freezing on Aquatic Protein Denaturation Frozen fish alter in characters during storage at sub-zero temperatures, becoming progressively tougher to eat, and extruding much fluid or drip on thawing. The deteriorative changes in texture as a consequence of long-term storage are considered to be due to protein denaturation during frozen storage and the change proceeds more slowly in low temperature. Most of these observed changes have been attributed to the changes of proteins in low temperatures.

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5. FUNCTIONAL PROPERTIES OF PROTEINS The functional properties important for the food processor are attributes, which at the proper concentration of the respective components or additives and at appropriate conditions, provide for the desirable characteristics of the product. These properties of proteins are displayed in interactions with the surrounding solvent, ions, other proteins, saccharides, lipids, and numerous other components, as well as in surface phenomena (chemical and functional properties of food components). Table 5-6 lists the functional properties of proteins involved in different food systems. Table 5-6. Functions of proteins in different food systems [8] Functionality Solubility Viscosity Water holding Gelation

Food Systems Beverage Soup, sauce, salad dressing, dessert Sausage, cake, bread Meat, gell, bakery product, cheese

Conglutination

Meat, sausage, noodle, bakery product

Elasticity

Meat, bread

Emulsification

Sausage, bologna sausage, soup, cake, dessert Ice cream, cake, dessert Low-fat bakery product, doughnut

Foamability Fat and flavor binding

Proteins involved Lactalbumin Gelatin Muscle protein, egg protein Musscle protein, egg protein, milk protein Muscle protein, egg protein, lactalbumin Musscle protein, cereal protein, milk protein Muscle protein, egg protein, lactalbumin Egg protein, lactalbumin Milk protein, egg protein, cereal protein

The sensory properties of foods result from the complex interaction between various food ingredients. For example, the flavor, texture, color and shape of a cake is contributed by the combination of the thermal gelation capacity, foaming capacity, water holding capacity, emulsification, elasticity and browning reaction of the materials used. Hence, proteins as food materials must have multiple functional properties (Table 5-7). Animal proteins, such as those derived from milk, egg and meat, are mixture of multiple protein components and thus have diverse physical and chemical properties. For example, egg white has good water holding, gelling, conglutination, emulsification, foaming, and thermal coagulation capabilities and these properties are contributed by ovalbumin, conalbumin, ovomucin, lysozyme and other albumins and their interactions. Plant proteins, such as those from soybean and oilseeds, are also mixtures of multiple protein components. However, these proteins have only limited functional properties and are only used in common foods in less quantity.

5.1. Interfacial Properties of Proteins Bakery products, desserts, beer, milk, ice cream, butter, chopped meat, and other foods containing emulsions or foams are dispersion systems and amphiphilic components must be present to stabilize the systems. Proteins are amphiphilic molecules and can spontaneously

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migrate to the air-water or oil-water interface and form highly viscoelastic film at the interface. The interface system of proteins is more stable than that formed by surfactants. Table 5-7. Functional properties of proteins required for various food [9] Food Beverage, soup, sauce Formed bakery products Dairy products The substitute for egg Meat products Meat extender Food film Dessert

Functionality required Solubility in solutions of different pH, thermal stability, viscosity, emulsifying and water holding capacity Casting and viscoelastic film forming, coherence, thermal denaturation, gelation, swelling, emulsification, foaming, browning reaction Emulsification, fat retention, viscosity, foaming, gelation, coagulation Foaming, gelation Emulsifying, gelation, coherence, absorption and retention of water and fat Absorption and maintain of water and fat, insolubility, hardness, chewiness, coherence, thermal denaturation Coherence, adhesion Dispersity, emulsification

In addition to the intrinsic properties, including amino acid composition, structure, conformation, distribution and ratio of hydrophobic and hydrophilic groups, number of disulfide bonds, and shape, size, and flexibility, the interfacial properties of proteins are also affected by environmental conditions, such as pH, temperature, ionic strength, salt species, interface composition, protein concentration, presence of saccharides and low-molecular weight surfactants, and energy input as well as container and operation sequence. A protein as an ideal surfactant should has the following three attributes: moving rapidly to the interface; extending and directing rapidly once reaching the interface; and forming films of strong cohesion and viscoelasticity with neighboring molecules. Hence, the distribution of hydrophobic and hydropholic groups in proteins and protein shape markedly affect the interfacial properties. Supposing the surface of a protein is highly hydrophilic and no discernable hydrophobic domain is found on the surface, the protein has a positive adsorption free energy. That is, the free energy of the protein in the aqueous phase is lower than in the interface or non-aqueous phase and the adsorption dose not occur. In contrast, if the protein contains multiple hydrophobic domains on the surface, the protein can be absorbed on the interface spontaneously, as shown in Figure 5-8. In another word, absorption to the interface occurs only when the number of hydrophobic areas on the surface is sufficient for providing the energy for hydrophobic domain-interface interaction. The hydrophobic and hydrophilic groups of a protein must be positioned correctly at the interface for the formation and stabilization of foams and emulsions. Generally, most proteins are distributed in the bulk phase and only a small proportion is absorbed at the interface. High flexibility facilitates the adsorption and positioning of proteins at the interface. For example, casein has very good flexibility. Once casein is absorbed at the interface, conformation change occurs rapidly and more peptide segments are absorbed at the interface. Rigid globular proteins, such as lysosome and soybean protein, do not undergo marked conformation transition at the interface. Flexible polypeptide chains might exhibit three typical conformations at the interface: train, tail, and loop, as shown in Figure 5-9. A flexible protein can be present as one or more of these conformations at the interface. Generally, peptide segments in directly contact with

160


the interface occur in the train conformation, the segments suspended in the aqueous phase are arranged as loops, while the peptide chains with the N- and C- terminal in the aqueous phase are present mainly in the tail conformation. Of the three conformations, the train conformation occurs in higher probability than the other two conformations and exhibits lower interfacial intension. )NTERFACE

-ODERATEPROBABILITY OFADSORPTION

.OADSORPTION

(YDROPHILIC SURFACE

3TRONGPROBABILITY OFADSORPTION

.ONPOLARSURFACE

(YDROPHOBICCORE

!QUEOUSPHASE

Figure 5-8. Probability of protein adsorption to the interface as a function of hydrophobic domain number [11].

1IBTF USBJO *OUFSGBDF

UBJM

MPPQ

1IBTF Figure 5-9. Configuration of flexibility polypeptide on interphase [12].

The stability of a protein at the interface determines the mechanical strength of the film formed at the interface and the film strength is also affected by the electrostatic attraction, and hydrogen bonding, and hydrophobic interaction between molecules. Disulfide bonds increase the film viscoelasticity. When the concentration of protein in the interfacial film reaches about 20%~25% (W/V), gelation occurs. To maintain the stability viscoelasticity of the gelled film, the secondary interactions involved must be in balance. If the hydrophobic interaction is too strong, the protein flocculates, aggregates, and even precipitates. If the electrostatic repulsion far exceeds electrostatic attraction, a thick cohesive membrane is not formed readily.

5.1.1. Emulsifying Properties Proteins play important role in the stabilization of many food emulsion systems, such as milk, butterfat, ice cream, and soy milk. Proteins are less capable of stabilizing W/O emulsions than O/W emulsions, because most proteins are highly hydrophilic and most absorbed proteins are located in the aqueous phase.

161

Proteins Table 5-8. Emulsifying capability index (EAI) of some proteins [12] EAI (m2/g) pH6.5 pH8.0 197 149 166 153 119 142

Protein BSA Casein ache sodium β-lactoglobulin Powder form lactalbumin

Protein hemoglobin Yeast protein lysozyme Ovalbumin

EAI (m2/g) pH6.5 pH8.0 75 8 59 50 49

Note: The data were obtained when proteins are dispersed in 0.5% PBS buffer of ionic strength of 0.1.

The emulsifying capability of a protein can be evaluated by droplet size and distribution, emulsifying activity, emulsion capacity, and emulsion stability. Emulsifying activity refers to the total interfacial area of an emulsion. The emusifying activity of a protein is often presented as the emulsifying activity index (EAI), namely the interfacial area created per unit mass of protein. The EAI of a protein can be determined easily in the turbidimetric method. The turbidity of an emulsion can be calculated by the following equation:

A is the absorbance of the solution and L is the path length. According to the Mie theory of light scattering, the interfacial area of an emulsion is two and C is the weight of protein per unit volume of the aqueous phase, then the EAI of the protein can be calculated by:

Emulsion capacity (EC) is the volume (ml) of oil that can be emulsified by per gram of protein before phase inversion (a change from oil-in-water emulsion to water-in-oil) occurs. In the determination of TC, oil or melted fat are added at a constant rate and temperature to an aqueous protein solution that is continuously agitated. Phase inversion is detected by an abrupt change in viscosity or color (usually a dye is added to the oil). EMULSIFYINGACTIVITYINDEX%!)-G

$(

Figure 5-10. Effect of molecular weight on EAI (DH is the degree of hydrolysis) [13].

162


Note: ○ - 0.1mol/L NaCl; ● – 0.2 mol/L NaCl; ▲ – 0.5mol/L NaCl; □ – 1.0mol /L NaCl. Figure5-11. Effect of pH and NaCl concentration on the emulsion volume of peanut protein isolate [14].

The emulsion stability (ES) is measured as the final volume of the emulsion after centrifuging the initial volume or standing for several hours at specified conditions. It may also be determined as the quantity of oil or cream separated from the emulsion, or the time required for the emulsion to release a specified quantity of oil.

The properties of protein-stabilized emulsions are influenced by many factors. Intrinsic factors are pH, ionic strength, temperature, presence of low-molecular-weight surfactants, sugars, oil-phase volume, type of protein, and the melting point of the oil used. Extrinisic factors include type of equipment, rate of energy input, and rate of shear. For example, the emulsion capacity or emulsion stability of proteins is generally positively correlative with protein concentration in the range 25~80%. Molecular weight also affects the EAI, as shown in Figure5-10. The addition of sodium chloride increases the emulsion volume of meat stuffing (Figure 5-11). Proteins exhibit different emulsifying activities at their isoelectric points (pI). Proteins have minimum solubility at pI and hence have decreased emulsifying activities. On the other hand, proteins are present in a highly viscoelastic structure at their pI. This structure hinders the unfolding or adsorption at the interface, which is not beneficial for emulsion formation, but can stabilize the film that is formed by already absorbed proteins. Hence, proteins might have increased emulsifying activity under certain conditions. Collagen, serum protein and ovalbumin have the maximum emulsifying activities at their pI, but soybean protein, peanut protein, casein, lactalbumin, bovine serum albumin and troponin have the maximum emusifying activites at other pH than their pI.

5.1.2. Foaming Properties Food foams are dispersions of gas bubbles in a continuous liquid or semisolid phase. Foaming is responsible for the desirable rheological properties of many foods, such as the texture of bread, cakes, whipped cream, ice cream, and beer froth. In most of these products, proteins are the main surface-active agents that help in the formation and stabilization of the dispersed gas phase. Thus foam stability may be an important food quality criterion. However, foams are often a nuisance for the food processor, such as in the production of potato starch or sugar.

163

Proteins

In most cases, the disperse phase is air or carbon dioxide and the continuous phase is composed of two films of proteins adsorbed on the interface between a pair of air bubbles, with a thin layer of liquid between them. The diameters of bubbles might range from 1μm to several centimeters, depending on the surface tension and viscosity of the aqueous phase and energy input. Evenly distributed small bubbles can impart foods with desired consistency, exquisite texture, and softness and improve the dispersivity and flavor. The formation of bubbles is shown in Figure 5-12.

#

&

% $

" GASLATEX

LIQUID

BUBBLE

Figure 5-12. Formation of bubbles [15].

Bubbles can also be formed by whipping or violently shaking a protein-containing solution in the presence of gas. Whipping is the most common measure used to produce bubbles. Compared with the first method mentioned above, whipping can produce stronger mechanical stress and shearing force and hence gases are dispersed more uniformly. However, too strong mechanical stress might affect the aggregation and formation of bubbles by hindering the absorption of protein at the interface. Besides, the amount of proteins required for stabilizing bubbles is also increased by 1%~40% (w/v). The volumes of materials can increase by 300%~2000% after whipping. The third method of producing bubbles is to suddenly release the pressure of a pressured solution. This method has been used in the production of whipped butter. The foaming power of proteins and the stability of bubbles foams are affected by many factors, such as pH, salts, saccharides, lipids, and protein concentration. Table5-9 lists the foaming power of some proteins. Table 5-9. Foaming capability of protein [16] Protein type Bovine serum albumin Whey protein isolate Ovalbumin Egg albumen

Foaming power at 0.5% protein concentration (W/V) 280

Protein type β-Lactoglobulin

Foaming power at 0.5% protein concentration (W/V) 480

600

Fibrinogen

360

40

Soybean protein (enzyme hydrolyzed) Gelatin (acid-processed pig skin produce)

500

240

760

The pH is an important factor that affects the foaming ability and foam stability of proteins. Generally, high solubility is a prerequisite for good foaming ability and foam stability. However, insoluble protein granules, such as fibrillin in its isoelectric point, may

164


impart good foam stability. Some proteins give rise to small to foam volume in their isoelectric points, but the produced foams have excellent stability, such as globulins in pH 5~5, glutelin in pH 6.5~7.5, and lactalbumin in pH 4~5. Some protein-stabilized foams are more stable in extreme pH values, possible due to increased viscosity of involved proteins. The foams in most foods are formed in other pH than the isoelectric points of contained proteins. Sugars suppress the expansion of protein-stabilized foams, but increase the foam stability. This is because sugars can increase the viscosity of the bulk phase and reduce the drainage of the lamella fluid. Sugars can stabilize the conformation of proteins and hinder their adsorption and unfolding on the interface. Hence, proteins cannot produce large interfacial area and foam volume in the presence of sugars. This is why sugars are often added after the foam expansion has already been completed in the manufacture of meringue and other sugar-containing foods. Lipids in low concentrations (about 0.1%) seriously impair the foam ability of proteins. This is because lipids are more surface-active than proteins. Lipids readily adsorb at the airwater interface and inhibit adsorption of proteins during foam formation. Besides, lipid films lack the cohesive and viscoelastic properties necessary to withstand the internal pressure of the foam bubbles, the bubbles rapidly expand, then collapse during whipping. Thus, lipid-free whey protein concentrates and isolates, soy proteins, and egg proteins without egg yolk display better foaming properties than do lipid-contaminated preparations. Partially denatured proteins have improved foaming abilities than their nature forms. Moderate heat treatment can enhance the foaming ability of soybean protein (70~80°C) and lactalbumin (40~60°C). Although moderate heat treatment increases the expansion capacity, it decreases the foam stabilization. Excessive heat treatment might impair the foaming ability of proteins. To obtain enough quantity of foam, the whipping operation must last long enough in suitable strength for sufficient unfolding and adsorption. However, excessive whipping can reduce the expansion volume and foam stability. For example, ovalbumin is very sensitive to excessive whipping. Whipping of ovalbumin for 6~8min can lead to coagulation-flocculation in the air-water interface. These insoluble proteins could not be completely absorbed on the interface and the viscosity of the formed liquid sheet cannot meet the requirement of high stability of foams. Most proteins contain more than one subunit. Hence, the foaming ability of a protein is also affected by the interaction between the subunits absorbed on the interface. For example, the excellent foaming ability of egg whit has been attributed to its protein composition. Acidic proteins can get improved foaming ability when complexing with an alkaline protein. No close correlation between emulsifying ability and foaming ability has been found for protein. Salts affect the foaming of proteins by influencing the solubility, viscosity, unfolding and aggregation of proteins. The foaming ability and foam stability of most globular proteins, such as bovine serum albumin, egg albumin, gluten and soy proteins increase with increasing concentration of NaCl. This behavior is usually attributed to neutralization of charges by salt ions. However, some proteins, such as whey proteins, exhibit the opposite effect: Both foam ability and foam stability decrease with increasing concentration of NaCl (Table 5-10). Bivalent cation ions, such as Ca2+ and Mg2+, in concentration 0.02~0.04mol/L could bridge

Proteins

165

with the carboxyl group of proteins to form protein films with good viscoelasticity. Hence, the presence of these ions can increase the foam stability.

5.2. Viscoelasticity Viscoelasticity is the property of materials that exhibit both viscous and elastic characteristics when undergoing deformation. Table 5-10. Effect of NaCl on the foaming ability and foam stability of whey protein isolate [17] Concentration of NaCl(mol/L) 0.00 0.02 0.04 0.06 0.08 0.10 0.15

Total interfacial area (cm2/mL of foam) 333 317 308 307 305 287 281

Times of 50% collapse of initial area (second) 510 324 288 180 165 120 120

Wheat protein is very unique among food proteins because it can produce dough with both viscous and elastic properties. When a mixture of wheat flour and water (about 3:1 ratio) is kneaded, a dough with viscoelastic properties forms that is suitable for making bread and other bakery products. These unusual dough characteristics are mainly attributable to the proteins in wheat flour. Wheat flour contains 20% soluble and 80% insoluble protein fractions. The soluble proteins are primarily albumin, globulin-type enzymes and minor glycoproteins. These proteins do not contribute to the dough-forming properties of wheat flour. The major storage protein of wheat is gluten. Gluten is a heterogeneous mixture of proteins, mainly gliadins (soluble in 70%~90% ethanol) and glutenins (insoluble in water or ethanol, but soluble in acidic and alkaline solutions). Gluten can entrap gas during fermentation to produce the viscoelastic property. Besides, the starch granules, pentosan, polar and non-polar lipids, and soluble proteins in wheat flour facilitate the formation of the viscoelastic protein network and dough texture. Gluten is rich in Gln (more than 33% of the amino acid residues) and hydroxylcontaining amino acids. Hence, the amino acids can form hydrogen bonding readily and this property contributes to the water adsorption and cohesion-adhesion property of gluten. About 30% of gluten's amino acid residues are hydrophobic, and the residues contribute greatly to its ability to form protein aggregates by hydrophobic interactions and to bind lipids and other nonpolar substances. Cysteine and cystine residues account for 2~3% of gluten's total amino acid residues. During formation of the dough, these residues undergo sulfhydryl-disulfide interchange reactions resulting in extensive polymerization of gluten proteins. Several physical and chemical transformations occur during mixing and kneading of a mixture of wheat flour and water. Under the applied shear and tensile forces, gluten proteins absorb water and partially unfold. The partial unfolding of protein molecules facilitates hydrophobic interactions and sulfhydryl-disulfide interchange reactions, which result in

166


formation of thread-like polymers. These linear polymers in turn are believed to interact with each other, presumably via hydrogen bonding, hydrophobic associations, and disulfide crosslinking, to form a sheet-like film capable of entrapping gas. Because of these transformations in gluten, the resistance of the dough increases with time until a maximum is reached, and this is followed by a decrease in resistance, indicative of a breakdown in the network structure. The breakdown involves alignment of polymers in the direction of shear and some scission of disulfide cross-links, which reduces the polymer size. The time it takes to reach maximum dough strength during kneading is used as a measure of wheat quality for bread making—a longer time indicating better quality. The development of the viscoelastic dough is thought to be related to the extent of sulfhydryl-disulfide interchange reactions. This is supported by the fact that when reductants, such as cysteine, or sulfhydryl blocking agents, such as N-ethylmaleimide are added to dough, viscoelasticity decreases greatly. On the other hand, addition of oxidizing agents, such as iodate and bromate, increase the elasticity of the dough. This implies that wheat varieties that are rich in SH and S-S groups might possess superior bread-making qualities, but this relationship is unreliable. Thus, the role of sulfhydryl-disulfide interchange reactions in the development of viscoelastic dough is poorly understood. The strength of dough is related to the contents of glutenin and completely insoluble residue proteins. Experiments on wheat flour supplemented with glutenins and gliadins of different proportions indicate that glutenins determine the elasticity, cohesiveness, mixture tolerance, extensibility, the expansion of dough and the mobility of gliadins. Suitable proportions of the two categories of proteins are important for bread making. High glutenins contents can increase the cohesiveness of dough and suppress the expansion of entrapped CO2, while high gliadins contents increase the elongation and make the gluten film fragile and permeable, leading to poor CO2 retention and bread collapse. The different effects of glutenins and gliadins on bread-making may be related to the differences in their structure and composition. In gluten these exist as single polypeptides with molecular weight ranging from 30,000 to 80,000. Although gliadins contain about 2~3% half-cystine residues, they apparently do not undergo extensive polymerization via sulfhydryl-disulfide interchange reactions. The disulfide bonds appear to remain as intramolecular disulfides during dough making. Dough made from isolated gliadins and starch is viscous but not viscoelastic. Glutenins are heterogeneous polypeptides with molecular weight ranging from 12,000 to 130,000. These are further classified into high-molecular-weight (M.W. >90,000, HMW) and low-molecular-weight (M.W. <90,000, LMW) glutenins. In gluten, glutenin polypeptides are present as polymers joined by disulfide cross-links, with molecular weights ranging into the millions. Because of their ability to polymerize extensively via sulfhydryl-disulfide interchange reactions, glutenins contribute greatly to the elasticity of dough. Therefore, an optimum ratio of gliadins and glutenins seems to be necessary to form viscoelastic dough. Some studies have shown a significant positive correlation between HMW glutenin content and bread quality in some wheat varieties, but not in others. Available information indicates that a specific pattern of disulfide crosslink association among LMW and HMW glutenins in gluten structure may be far more important to bread quality than the amount of this protein. For example, association/polymerization among LMW glutenins which have the similar structure form the HMW gliadin. This type of structure contributes to viscosity of the dough, but not to elasticity. In contrast, if LMW glutenins link to HMW glutenins via disulfide cross-

Proteins

167

links (in gluten), then this is believed to contribute to dough elasticity. It is possible that in good-quality wheat varieties, more of the LMW glutenins may polymerize with HMW, whereas in poor-quality wheat varieties, most of the LMW glutenins may polymerize among themselves. These differences in associated states of glutenins in gluten of various wheat varieties may be related to differences in their conformational properties, such as surface hydrophobicity, and reactivity of sulfhydryl and disulfide groups. Baking does not lead to the re-denaturation of gluten, because glutenins and gliadins occur naturally in partially unfolded form and are further unfolded during kneading. When heated in 70~80°C, gluten loses water and the water is absorbed by gelatinized starch granules. Hence, gluten can maintain the softness and water content (40%~50%) of bread in baking. Soluble protein fractions, including albumin- and globulin-type proteins, are denatured and coagulated during baking. This partial gelling is beneficial for the cohesion of crumbs. Hence, supplementation of external proteins is good for bakery foods quality, such as nutrition fortification. However, not all proteins are beneficial for gluten network formation. For example, supplementation of wheat flour with albumin- and globulin-type proteins, such as whey proteins and soy proteins, adversely affects the viscoelastic properties and baking quality of the dough. These proteins decrease bread volume by interfering with formation of the gluten network. Addition of phospholipids or other surfactants to dough counters the adverse effects of foreign proteins on loaf volume. In this case, the surfactant/protein film compensates for the impaired gluten film. Although this approach results in acceptable loaf volume, the textural and sensory qualities of the bread are less desirable than normal.

5.3. Gelation Gelation is the process of aggregation and ordered network formation of denatured proteins. The gelation of proteins is very important for some foods, including various dietary products, jelly, gelatin gel, soybean protein gel, and textured vegetable proteins. Tofu, which is a favorite food in China, is a typical food prepared by protein gelation. Heating is necessary for the formation of most protein gels. The addition of salts, especially those containing Ca2+, improves the gelation rate and gel strength. Proteins can also interact with polysaccharides to form gels. For example, the positively-charged gelatin can bind to negatively-charged sodium alginate through nonspecific ionic interaction to form gel with a high melting point (80°C). Many protein gels are highly hydrated and each gram of protein can hold up to more than 10 grams of water. Many food components can be trapped in the network of protein gels. The water contents of some protein gels might reach up to 98%. Although the trapped water has properties similar to the water in dilute solutions, the water cannot be squeezed out easily.

5.4. Hydration The conformation of each protein in a solution is affected by their interactions with water. The interaction between proteins and water also occurs in solid foods. In addition, dry concentrated proteins or protein isolates must be hydrated before use. Hence, the hydration

168


and rehydration properties of proteins markedly affect their applications in the food industry. Figure 5-13 illustrates the sequence of interactions occurred between dry protein and water.

Dry protein

Water molecules are absorbed by binding to polar groups

Multilayer water is formed

Liquid water condenses

Solution is formed

Protein starts swelling

Protein disperses in water

Figure 5-13. Sequence of the interactions between dry protein and water [10].

The protein-protein and protein-water interactions are affected by environmental conditions, including protein concentration, pH, temperature, time, ionic strength and the presence of other components. The pH alternation affects the ionization and net charge of proteins and thus changes the attraction and repulsion between proteins and their water binding capacity. The water binding capability of proteins decreases with the increase of temperature. High temperature decreases the bonding force between hydrogen bonds and leads to protein denaturation and aggregation. Protein denaturation or aggregation reduces the surface area of the protein and consequently the hydration of polar amino acids. When proteins with compact strength are heated, the proteins are unfolded and the peptide chains and polar chains that are originally located inside the structure are exposed to water and the hydration capability is increased as a result. The gelation of some proteins, such as lactoalbumin, is irreversible. If the gels of these proteins are dried, the gel network is transformed to the capillary of the protein powder. The dried proteins thus have significantly increased hydration capability. However, the rate and degree of hydration are also affected by the size and the holes on the surface and inner of proteins. Ions might compete for the side chains of amino acids with water and subsequently influence the hydration capability, swelling property, and solubility of proteins. Salts in low concentrations enhance the hydration capability of proteins, but salts in high concentrations can lead to dehydration due to the excessive water-salt interaction over the water-protein interaction.

5.5. Solubility It is a long process for proteins to reach their equilibrium solubility and the protein solubility varies with the pH, ionic strength, and temperature of the environment and protein concentration. The solubility of most proteins decreases irreversibly when heated. However, heat treatment is unavoidable during food processing and protein denaturation might occur during extraction and purification even in mild conditions. Hence, the nitrogen solubility index (NSI) of soybean protein powder, concentrated soybean protein, and soybean protein isolate vary in the range 10%~90%.

Proteins

169

The solubility of proteins is essential for determining the optimum conditions of protein extraction, purification and separation and is an important indicator of its application range. The degree of insolubility can be used as a measure to evaluate the denaturation and agglutination of proteins. It is reported that proteins with greater initial solubility can disperse quickly and yield well dispersed hydrocolloid system with macrostructure and smooth texture. In addition, high initial solubility facilitates the diffusion between of proteins to the gas/water and oil/water interface for enhanced their surface activity.

5.4. Viscosity The viscosity or consistency of a liquid reflects its resistance to flow under an applied force or shear stress and can be represented in viscosity coefficient η, which is defined as the ratio of shear stress to shear rate (or flow rate). The viscosity of protein fluids is largely related to the apparent diameter of dispersed protein molecules or protein particles. The apparent diameter of proteins is decided by the following parameters: (1) the inherent nature of protein molecules, such as molar concentration, molecular size, molecular volume, molecular structure and charge; (2) protein-solvent interaction, which affects the swelling and solubility of proteins and the hydrokinetics of the fluid surrounding protein molecules; (3) protein-protein interaction. The interaction determines the size of protein aggregates and the interaction opportunity increases in high protein concentrations. The viscosity coefficient of protein-containing solutions, such as homogenates, emulsions, pastes and gels, might decrease with the increase of shearing stress. This behavior is known as shear thinning. During shearing, protein molecules are redirected to the flow direction and the friction drag is reduced as a result. Besides, the break of hydrogen bonds and other weak bonds can cause the dissociation of protein aggregates and network. The break of weak bonds is a slow process so that the shear stress and apparent viscosity decreases with the proceeding of shearing before the protein fluid reaches steady flow. When the shearing force is no longer applied, the original aggregates or network can or cannot be reformed. If the aggregates or networks can be completely or partially reformed, the change of viscosity coefficient is irreversible. The viscosity or consistency of protein system is an important property for liquid and semisolid-type foods such as beverages, broth, sauces and cream. The fluid property of protein dispersion systems is of practical significance in determining the optimum operation conditions. For example, during transfer, blending, heating, cooling, and spray drying, the fluidity of protein dispersion systems significantly affects the transfer of both mass and heat.

6. PROPERTIES OF FOOD PROTEIN MATERIALS AND

NEW PROTEIN DEVELOPMENT

6.1. Muscle Protein Muscle derived from mammals, birds and fish is the main source of meat. The skeletal muscle of these animals contains 16%~22% proteins. Muscle protein can be divided into myofibrillar protein, sarcoplasmic protein and matrix protein. Proteins of these three

170


categories differ markedly in their solubility. Sarcoplasmic proteins can be extracted by water or buffers with low ionic strength (0.15mol/L or lower), myofibrillar protein can be extracted only in high salt concentrations, and matrix proteins are insoluble. Sarcoplasmic proteins contain large amounts of glycolytic enzymes and many other enzymes in addition to myoglobin and haemoglobin, which affect the color of meat. Myofibrillar proteins account for about half of animal skeletal muscle proteins. These proteins are intensively charged hydrated and are insoluble under physiological conditions. Matrix proteins participate in the formation of connective tissues. Collagen, reticulum and elastin are examples of matrix proteins. Collagen is fibrous and occurs in all muscle tissues. Reticulin refers to a type of fiber in connective tissues composed of type III collagen. The proteins crosslink and form a fine mesh network. This network acts as a supporting mesh in soft tissues such as liver, bone marrow, and the tissues and organs of the lymphatic system. Elastic fiber is composed of the protein fibrillin and elastin made of simple amino acids such as glycine, valine, alanine, and proline. Elastin helps skin to return to its original position when it is poked or pinched. Elastin is also an important load-bearing tissue in the bodies of vertebrates and used in places where mechanical energy is required to be stored. These matrix proteins are less water soluble than sarcoplasmic and myofibrillar proteins.

6.2. Casein Milk contains multiple types of proteins and the proteins exhibit different properties (Table 5-11). In skimmed milk, casein accounts for about 80% of total protein. Casein is a mixture of phospho-contaning proteins and precipitate out from skimmed milk at pH4.6 and 20°C. Casein is extremely hydrophobic and occurs as micelles in milk. Casein is composed of αs1-casein, casein, β-casein, αs2-casein and κ-casein. The diameters of casein micelles range from 50nm to 300nm with molecular weight range of 107~3×1010 Daltons. Casein micelles are aggregated by sub-micelles, which has a diameter range from 10 to 20nm and molecular weight range from 3×105 to 6×105. Sub-micelles consist of αs1-casein, β-casein, αs2-casein and κ-casein and the fractions aggregate through hydrophobic interaction. The core of sub-micelles is hydrophobic in contrast to the hydrophilic surface. The carbohydrate-rich κ-casein is restricted on a region of the surface through self-association. Therefore, the surface of sub-micelles contains one carbohydrate-rich regions and one phosphate-rich region that is formed by other caseins. Casein micelles are the aggregation of sub-micelles. The electrostatic interaction contributed by colloidal calcium phosphate facilitates the aggregation of sub-micelles. The aggregation occurs between the negatively charged serine residue on casein and the positively-charged colloidal calcium phosphate, which occurs in Ca9 (PO4)6 clusters. Because κ-casein contains no phosphate groups, the electrostatic interaction occurs only between αcasein and β-casein. During casein micelle formation, sub-micelles with low content of κ-casein or without κcasein are located in the interior of casein micelle. When the surface of the micelle is completely covered by κ-casein, the micelles stop growing up.

171

Proteins

6.3. Whey Protein The main constitutients of whey proteins are β-lactoglobulin, α-lactoglobulin and serum albumin in decreasing order in content, as shown in Table 5-9. These proteins are soluble at pH 4.6 in their natural or denatured forms. In the manufacture of caseinate and country cheese, these protein fractions remain in the whey. Hence, whey protein is a by-product of cheese making. Whey protein is soluble in a wide range of pH, temperature and ionic strength, pH4~5, which is near the isoelectric point. Solubility is most important physicochemical and functional property of natural whey protein. In addition, whey protein produces stable gel when heated. This property together with its surface properties imparts whey protein with important application in the food industry. Table 5-11. Proteins in milk [21] Protein Casein αs1-casein αs2-casein β- casein κ- casein Whey protein β-whey protein α-whey protein albumin Immunoglobulin IgG1 IgG2 IgA IgM Secretory component

Content in skimmed milk (g/L)

Relative molecular weight

12~15 3~4 9~11 2~4

22068~23724 25230 23944~24092 19007~19039

2~4 0.6~1.7 0.4

18205~18363 14147~14175 66267

0.3~0.6 0.05~0.1 0.05~0.15 0.05~0.1 0.02~0.1

153000~163000 146000~154000 385000~417000 1000000 79000

6.4. Wheat Protein According to solubility, wheat proteins are divided into albumins (water soluble), globulins (soluble in 10% NaCl but insoluble in water), gliadin (soluble in 70%~90% ethanol), and glutenin (insoluble in water or ethanol but soluble in acid or alkali solutions). Albumin and globulin account for about 10% to 15% of wheat endosperm proteins. The two fractions contain free sulfhydryl and higher proportion of alkaline and charged amino acids. Albumins are relative smaller molecules with molecular weight ranging from 12000 to 26000 compared with that of globulin of up to 100000. Gliadin and glutenin are the major constituents of gluten and account for about 85% of flour protein. Both gliadin and glutenin are complex mixtures of multiple protein components and the two fractions share similar contents in wheat flour. Amino acid analysis indicates that gliadin and glutenin have very high contents of glutamine and praline and very low contents of lysine and ionized amino acids. Gliadin and glutenin are among the least charged proteins. Although the sulfurcontaning amino acids are low, these amino acids play an important role in gluten structure and dough formation.

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The compact spherical structure of gliadin is related to the intramolecular disulfide bond. Glutenins associate with each other through inter-molecuar disulfide bonding. Hence, glutenins are found as large and stretched associated molecules. Glutenin is insoluble due to intermolecular disulfide bonds, but treatment with reducing agents increases its solubility. The formation of gluten is the result of both covalent and non-covalent interactions of multiple protein constituents. Glutenins contribute to the formation of the network structure, while gliadin and other proteins are trapped within the structure. Wheat flour contains 12% protein, 70% starch and 2% fat. During kneading, these components are incorporated into the gluten network structure and a starch-protein-fat composite matrix is formed. The reduction of disulfide bonds during kneading the re-oxidation of sulfhydryl during dough storage occur mainly in cross-linked glutenin. The reduction of disulfide bonds facilitates the gluten relaxing and enhances the non-covalent interactions between fat, starch and other additives, leading to the formation of a continuous starch-protein-fat complex.

6.5. Soybean Protein Soybean contains 42% protein, 20% oil and 35% carbohydrates on dry basis. Soybean protein is very sensitive to physical and chemical treatment. For example, heating under moisture conditions and alteration of pH can cause significant changes in the physical properties of soybean protein, such as solubility, viscosity and molecular weight. Globulins are the most important constituents of soybean protein. Globulins are insoluble in their isoelectric points, but the addition of NaCl or CaCl2 dissolves the proteins. Globulins are soluble in water in the absence of NaCl or CaCl2 in other pHs than their isoelectric points. Soybean proteins are least soluble in the pH range of 3.75~5.25, but are most soluble in pH1.5~2.5 and that higher than 6.3. At pH6.5, about 85% nitrogen of defatted soybean protein can be extracted by water and the yield can be further increased by 5%~10% by increasing the pH. According to the sedimentation coefficient, the water-extractable components of soybean protein are divided into four fractions: 2S, 7S, 11S, and 15S, of which, 7S and 11S protein are the most important. 7S protein accounts for 37% of the total protein, while the 11S fraction accounts for 3l%. Soybean protein concentrates and soybean protein isolates are the most important commercial products of soybean protein and their contents are higher than 70% and 90% respectively. Soy protein concentrate can be prepared from defatted soybean meal in one of three methods, namely by extraction with 60%~80% ethanol, with pH 4.5 aqueous solution, or with water after heat denaturation. In these methods, extraction with the solvents remove soluble carbohydrates from the material and the nitrogen content in the residue is increased as a result. Removal of soluble carbohydrates markedly improves the flavor of soy protein concentrate and flatulence factors are removed at the same time. Soy protein concentrates are often used as the material of thermoplastic extrusion. In the preparation of soybean protein isolate, proteins in defatted soybean meal are firstly extracted with water at neutral or higher pH. Then, the proteins are precipitated by adjusting the pH to the isoelectric point. The protein precipitate is collected and redissolved by changing the pH and finally is applied to spray dryer. Soy protein isolate can be modified by chemical, heat or enzymatic treatments to improve its functional properties.

Proteins

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6.6. New Protein Resources Development Due to the increase of world population and people‘s living standard, the demand on protein keeps increasing and the deficiency of protein resources has turned to be a worldwide issue. Improving the utilization efficiency of existing protein resources and exploiting new protein resources are effective measures to solve the protein resource deficiency problem.

6.6.1. Proteins From Oil Crops The byproducts of the oil processing industry are often used as feed or fertilizer. These byproducts have high protein content and are good protein resources for human beings. Soybean protein is a good example. Soybean contains about 40% protein and defatted soy protein contains up to 50% protein. The contents of all the essential amino acids, except Met and Cys, are close to the recommendations of FAO and their nutritional values are similar to that of animal proteins. Hence, soybean protein is good vegetable protein. At present, soybean protein is the most extensively used oil crop protein. Soybean protein isolate has gained wide applications in noodles, bakery foods and many other foods and the demand keeps increasing in recent years. It is expected that the yield and consumption of soy protein powder will exceed those of soybean protein isolate after its production and deodorization technologies are mature. Soy protein can significantly improve the quality of flour-based foods. For example, it can whiten flour and replace existing chemical brighteners; be added into noodles or dumplings to improve their tenacity and reduce the dissolution rates of starch during cooking; be added to bakery foods to improve biscuit crisp, bread tenacity, and cake softness; be added to buns, steamed buns and other steaming food to yield smooth surface; and be added to instant noodles and other fried foods to reduce oil consumption and the greasy mouth feel of fried foods. 6.6.2. Single Cell Protein Single-cell protein refers to such micro-organisms as yeast, lactic acid bacteria and mold, etc. which are cultivated by industrial means. This biomass is rich in protein, which can be used as human food or animal feed. Single-cell proteins are concentrated protein products and their crude protein contents range from 50% to 85%. Single cell proteins have complete amino acid composition and their bioavailabilities are very high. In addition to proteins, the biomass also contains vitamins, inorganic salts, fats and sugars. Hence, single cell proteins have higher nutritive values than fish powder and soybean powder. Besides, single cell proteins have the following advantages in protein resource development. 1) A wide range of materials can be used for single cell protein production. For example, fiber resources such as straw, wood chips and bagasse can be used as the fermentation substrate. 2) The investment for single cell protein production is low and the biomass grows very quickly. The generation time of microorganisms is about only tens of minutes. It is estimated that a 500 kg cow produces only 0.4kg protein, while yeast of the same weight can produce at least 5000 kg proteins per day. 3) The industrial production of single cell protein does not need large area of arable lands and are not affected by seasons and climate conditions.

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The development of single cell protein resources has bright prospect. However, the high content of nucleic acid in most single-cell proteins restricts their direct consumption by consumers. Heat and alkaline treatment can improve the digestibility and amino acid availability of single cell protein and reduce nucleic acids contents. Long-term toxicological study indicates that heat- or alkaline-treated single cell protein is nontoxic.

6.6.3. Insect Protein Insects have the largest biotic population on the earth and are characterized by high food conversion rate, rapid propagation and high protein contents. Insects have been recognized as the largest and most potential animal protein resource in the world. The protein contents of many insects exceed 50% on dry basis. For instance, the protein contents of maggot butterfly, crickets, cicadas and ants are 61%, 71%, 75%, 75%, 72% and 67% respectively. In some species, the protein contents are even greater than 80%. The protein contents of these insects are far greater than those of chicken, fish, pork and eggs, and is similar to that of beef liver. Meanwhile, the distribution and ratio of essential amino acids in insect protein is close to the ratios recommended by FAO. Therefore, insects can provide high-quality proteins. 6.6.4. Leaf Protein Leaf proteins are extracted from the green stems and leaves of plants. After the materials are squeezed, the juice is collected and the proteins are then separated, concentrated, and dried. Leaf protein products contain 55%~72% protein and 18 amino acids, including 8 essential amino acids. The amino acid composition is close to the recommendations of FAO for adults and the Lys content is high. Hence, leaf protein is potential for consumers of the Third World countries in which cereals are consumed as the staple food. Leaf proteins have high contents of Ca, P, Mg, Fe and Zn, which are 5~8 times higher than seeds. Besides, the contents of carotenes and lutein are 20~30 and 4~5 times higher than those of leaves respectively. Lead proteins do not contain cholesterol as in animal proteins and have multiple physiological functions. Leaf protein has been recognized by FAO as a high-quality protein source and is a potential protein resource.

7. NUTRITION AND SAFETY ASPECTS OF PROTEIN The nutritive values of proteins vary with their sources and the contents of essential amino acids and their digestibility are the main factors that determine proteins‘ nutritive value. Therefore, the daily protein requirement depends on the quality and quantity of proteins in foods.

7.1. Quality of Protein The quality of proteins is mainly decided by the composition of essential amino acids and their digestibility. High-quality proteins should contain all the essential amino acid and their contents should be higher than the reference levels recommended by FAO/WHO/UNU. Besides, the digestibility of high-quality proteins should be comparable or greater than that of albumen and milk protein.

Table 5-12. Essential amino acid contents and nutritive values of proteins derived from several foods (mg/g protein) [18, 19] Item

His Ile Leu Lys Met+Cys Phe+Tyr Thr Trp Val Total Protein content (%) Chemical score /% (by FAO/WHO pattern) PER BV(on rat) NPU

Egg

Milk

Beef

Fish

Wheat

Rice

Corn

Source Barley

22 54 86 70 57 93 47 17 66 512 12 100

27 47 95 78 33 102 44 14 64 504 3.5 100

34 48 81 89 40 80 46 12 50 480 18 100

35 48 77 91 40 76 46 11 61 485 19 100

21 34 69 23(1) 36 77 28 10 38 336 12 40

21 40 77 34(1) 49 94 34 11 54 414 7.5 59

27 34 127 25(1) 41 85 32(2) 6(2) 45 422 / 43

3.9 94 94

3.1 84 82

3.0 74 67

3.5 76 79

1.5 65 40

2.0 73 70

/ / /

Soybean

Broad bean

Pea

Peanut

Kidney bean

20 35 67 32(1) 37 79 29(2) 11 46 356 / 55

30 51 82 68 33 95 41 14 52 466 40 100

26 41 71 63 22(2) 69 33 8(1) 46 379 32 73

26 41 70 71 24(2) 76 36 9(1) 41 394 28 82

27 40 74 39(1) 32 100 29(2) 11 48 400 30 67

30 45 78 65 26 83 40 11 52 430 30 /

/ / /

2.3 73 61

/ / /

2.65 / /

/ / /

/ / /

Note: Chemical score is defined as the proportion of the content of a limiting amino acid in the material to that of the reference protein.

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Hong Lin, Lisha Wu and Shuhui Wang Table 5-13. Recommended patterns of essential amino Acids in foods (mg/g protein) [19]

Amino acid His Ile Leu Lys Met+Cys

Recommended pattern Infant Preschool (2~5) children 26 19 46 28 93 44 66 44 42 22

Amino acid Adults 16 13 19 16 17

Phe+Tyr Thr Try Val Total

Recommended pattern Infant Preschool (2~5) children 72 22 43 28 17 9 55 25 460 241

adults 19 9 5 13 127

The proteins derived from major cereals and beans are deficient in at least one essential amino acid. For example, the proteins of rice, wheat, barley and oat, are deficient in Lys but rich in Met and this is reversed for the proteins of beans and seeds of oil crops. The proteins of some seed plant seeds, such as peanuts, are deficient in both Met and Lys. Essential amino acids with contents lower than that in reference protein are defined as limiting amino acids. Adults that intake proteins only from crops and beans cannot maintain health and children that intake only one of the two kinds of protein mentioned above cannot grow in the normal rate. Table 5-12 lists the contents of essential amino acid in some proteins. Generally, the proteins derived from plants and animals contain enough His, Ile, Leu, Phe, Trp and Val and these amino acids are often not limiting amino acids in common foods. However, Lys, Thr, Trp and sulfur-containing amino acid are often present in insufficient quantity and are regarded as limiting amino acids. Mixing a protein that deficient in an essential amino acid with another protein that is rich in the essential amino acid can increase the nutrition value of the protein. For example, the mixture of cereal protein and bean protein can provide complete and balanced essential amino acids. The nutritive value of proteins can be improved also by supplementing the corresponding limiting amino acids. For instance, supplementation of Met and Lys improves the nutritive value of cereal and bean proteins. If the protein or protein mixture contains all the essential amino acids and the contents and proportions of the amino acids can maintain the optimum growth rate and health conditions for consumers, the protein or protein mixture is though to have an ideal nutritive quality. Table 5-13 lists the ideal amino acid distribution patterns for adults and children.

7.2. Digestibility Protein digestibility is defined as the proportion of absorbed nitrogen to ingested nitrogen. The contents of essential amino acids in proteins are important indicators of protein quality. However, the utilization of these amino acids determines the real quality of proteins. Table 5-14 lists the digestibility of various foods proteins in human body. It could be seen that plant proteins have higher digestibility than animal proteins. The digestibility of proteins is affected by the following factors: (1) Structure: The structure of proteins markedly affects their hydrolysis by enzymes. Generally, native proteins are more resistant to enzymatic hydrolysis than their denatured forms and the hydrolysis of insoluble fibrous proteins is more difficult than that of thoroughly denatured globular proteins.

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Proteins Table 5-14. Digestibility of some food proteins in human body [19] Protein source Egg Milk, cheese Meat, fish Corn Refined rice Whole wheat Refined flour Gluten Oat

Digestibility (%) 97 95 94 85 88 86 96 99 86

Protein source Pea Peanut Soybean meal Soybean protein isolate Kidney bean Corn products Wheat products Rice products Wheat

Digestibility (%) 88 94 86 95 78 70 77 75 79

(2) Antinutrients: Most vegetable proteins isolated and protein concentrates contain trypsin inhibitors, chymotrypsin inhibitor, and exogenous lectins. The inhibitors prevent the complete hydrolysis of proteins derived from legumes and oil seeds by trypsin. Lectins hinder the absorption of amino acids in intestines. Heat treatment can destroy these antinutrients and make proteins more susceptible to digestion. In addition to the antinutrients mentioned above, proteins derived from plants also contain phytic acid, tannin and other anti-nutrients. (3) Association with other components: Association with polysaccharides and dietary fiber reduces the hydrolysis speed and thoroughness of proteins. (4) Processing: Heat and alkaline treatment can cause chemical reactions in proteins, such as Lys residue generation. These changes reduce the digestibility of proteins. Besides, Millard reactions might occur between reducing sugars and proteins. These reactions reduce the digestibility of Lys residues.

7.3. Toxic Proteins Toxic proteins have been found in many kinds of plants. Chemically, the toxic proteins can be divided into two categories: lectins and enzyme inhibitors. Lectins occur widely in leguminous plants, such as soybeans, peas, kidney beans, sword bean and castor seed. These toxins can agglutinate red blood cells. Hence, the intake of uncooked or undercooked seeds of these plants can cause poisoning. The main symptoms of lectin poisoning include nausea, vomiting, and even death. Enzyme inhibitors are found in beans, potatoes, taros, wheat and immature bananas. These proteins inhibit the activities of trypsin or amylase and affect the digestion and absorption of nutrients by human body.

7.3.1. Toxic Peptides Toxic peptides are found in the most quantity in poisonous weeds. Amanita mushroom and phallus mushroom contain the highest levels of poisonous peptides. Intake of as low as 50 grams of these mushrooms could lead to death. 7.3.2. Toxic Amino Acids Some legumes contain toxic amino acids. For example, grass vetch contain α,γ-2-amino butyric acid and kidney bean contains β-cyano-alanine. Canavanine in canavalia hampers arginine metabolism in the body and L-3, 4-dihydroxyphenylalanine in kidney bean can cause acute hemolytic anemia. Consumption of excessive green kidney bean can lead to poisoning no matter whether the whether the beans are cooked or peeled.

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7.3.3. Allergic Proteins In most cases, food-induced irritability is caused by the proteins in foods. For example, the parvalbumins in myogen of fish are sensitinogens. Parvalbumins are small acidic glycoproteins with molecular weight of 11-12kD. The isoelectric point of these proteins are around 4.75 and two Ca2+ ions are bound to the Asp-Asp-Ser-Glu-Glu-Phe and Asp-Asp-AspGlu-Lys regions. Allergic proteins in crustacean are mainly tropomyosins, of which, an acidic glycoprotein with molecular weight of 36kD, isoelectric point of 4.5, and sugar content of 4.0%, has been identified as the major sensitinogen. With the development of technologies, more and more allergic proteins will be identified from crustacean. At least 13 allergic proteins have been identified in crustacean. The molecular weights of these proteins are in the range 16~166kD and most are in the molecular weight 36kD. These proteins do not contain Trp and have low Tyr and Phe contents.

8. CHANGES OF PROTEINS DURING FOOD PROCESSING AND STORAGE 8.1. Effects of Processing Methods on Protein Quality During food processing, temperature, moisture, pH, presence of other components and pressure are main factors that lead to protein changes. Among these factors, moisture and temperature have the largest effect on proteins. Hence, only the effects of dry and moist heat treatments on protein are detailed.

8.1.1. Boiling Boiling is a common processing method in the food industry. Boiling does not reduce the quantity of proteins or essential amino acids of fish and meat. Boiling of milk neither affects the amino acid content nor reduces the bioavailability. The egg white and yolk have different sensitivity of heat treatment. The digestibility of raw egg white is only 50%, but raw yolk can be digested and absorbed completely. Moderate cooking of eggs can yield of nearly 100% of digestibility and biological value due to heat denaturation of proteins. Moist heat treatment is desired for most vegetable proteins, especially for proteins derived from beans. Moist heat treatment increases the nutritive value of bean proteins due to protein denaturation, inactivation of protease inhibitors and toxic substances, and disrupt of tissues and starch granules. Enzymes, such as lipase, lipoxygenase, protease, and polyphenol oxidase, can be inactivated during blanching or steaming. Enzyme inactivation can prevent the formation of undesired colors and flavors and the loss of vitamins. Heat treatment of rape seeds inactivates myrosinase and avoids the formation the goitrogenic 5-vinyl-2-sulfur-oxazolidinone. 8.1.2. Pasteurization It is generally believed that either the tank pasteurization method or the high-temperature short time pasteurization has only slight effects on milk proteins, though whey proteins are partially denatured. Extensive studies on infants, school-age children, adults and experimental animals indicate that the nutritional value of milk is not affected by pasteurization. Sealed container must be heated thoroughly until the contents are sterile. The required intensity of heat treatment depends largely on the physical state of the contents. In

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pasteurization, the heat transferred from the vessel wall to the center in semi-solid foods is much less than in foods containing liquids as the heat transfer medium. High-frequency oscillatory electron heating and aseptic canning can overcome these problems, but their applications are still very limited. Pasteurization reduces the biological value of liquid milk by 6%, lysine content by 10% and cystine content by 13%. The decrease in biological value may be due to the loss of cystine. Pasteurization has much more significant effects on concentrated milk. Hence, pasteurization can significantly reduce the bioavailability of proteins with limited lysine contents.

8.1.3. Can Sterilization Meat cans need more intensive sterilization conditions than dietary products, because the heat transfer in solids or semi-soldiers are more difficult than in liquids. The heat sterilization method used in the current meat industry does not lead to significant loss of amino acids, except cysteine. However, canned beef has reduced digestibility and biological value than unprocessed beef. Besides, the bioavailability of Lys and Met in canned meats is slightly lower than that of fresh meat. Similar trends have also been found in canned fish. Sterilization reduces the biological values of most animal proteins. In contrast, the biological values of many vegetable proteins are increased after suitable sterilization. This is because sterilization not only improves the digestibility, but also destroys natural antinutrients. In the heat treatment of soybean products, both the loss of nutrients and inactivation of toxic components (such as typsin inhibitors) must be taken into consideration. The optimum sterilization conditions should minimize nutrient loss and maximize toxicant inactivation. Researches on moist heat treatment-caused Lys loss indicate a first-order reaction with reaction rate constant of 0.166/h and activation energy of 125.5kJ/mol. 8.1.4. Dehydration Milk and dairy products can be dehydrated by spray drying or drum drying. The effects of these methods on the nutritional value of milk proteins have attracted wide attentions. In drum drying, milk is spread on the drum and the nutrient loss varies with the conditions applied. Dehydration might lead to the loss of Lys. However, since Lys is not the limiting amino acid of milk, moderate dehydration dose not impair the nutritive value of milk powder. At present, dehydration has not been widely used in the industrial processing of meats, because dehydration cannot impart meats with competitive price, quality, and consumer acceptability compared with other products, through freeze-dried meats are more nutritional than heat-treated or irradiated products. Nevertheless, the quality of freeze-dried products cannot last long and critical changes might occur during storage. Fish meal prepared from fish by dehydration is widely used as feed and only very few are used in human foods. Fish can be dehydrated in many technologies, including flame drying, steam pipe drying, and solvent drying. Therefore, the changes of protein quality vary with the technology used. 8.1.5. Freeze-Drying Most foods are dried in conventional methods. However, freeze-drying has potential applications in the drying of unstable and gourmet foods. Studies have confirmed that freezedrying under proper conditions does not impair the quality of proteins.

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8.1.6. Baking and Puffing Cereal foods are often dried by dry heating. During dry heating, the moisture contents in materials decrease rapidly and proteins might be lost seriously. Baking causes a marked loss of the nutritional value of proteins on material surface. Hence, cookies and biscuits suffer serious protein loss during processing and the losses depend on the thickness of the foods. Generally, the loss of bioavailable amino acids in biscuits are is the function of thickness, heat intensity and heating time. The presence of sucrose increases the loss of bioavailable Lys. Breads lose 10%~15% of lysine during baking, and further 5% of lysine is lost when breads become hard. Intensive baking or puffing can lose proteins nutritive value in instant cereal foods. Though the content of Lys is only slightly affected, its bioavailability is significantly reduced after baking or puffing. 8.1.7. Other Processing The application of various protein isolates in the food industry keeps increasing in recent years. In the production of these isolates, the oilseed protein and fish protein are extracted with alkali; then, the protein in the supernatant is precipitated by acid or heat treatment to various yield protein isolates. These products can be used directly in foods or after medication by spinning. Treatment of casein, peanut flour and soybean protein with strong alkaline reduces the quality of the proteins and generate new amino acids, such as lysinoalanine, which is the condensation compound of lysine and alanine.

8.2. Mechanisms of Protein Loss in Food Processing 8.2.1. Effects of Heating Heating affects the spatial arrangement of food proteins and enhances the thermal oscillation of the molecules. These changes weaken the inter-molecule interaction, unfold the proteins, and disrupt the disulfide bridges. This process is termed heat denaturation. Heat denaturation only changes the structures of proteins and cannot alter amino acid sequences. The denaturation induced by moderate heat treatment is reversible. However, the denaturation of most proteins occurred during food processing is irreversible. Denatured proteins have different solubility and functional properties compared with their natural forms. Hence, protein denaturation plays important role in food processing. However, the nutrition aspects of protein denaturation have attracted much less attention. It is generally believed that denatured proteins are more susceptible to enzyme hydrolysis and thus have improved nutritive values. In digestion, proteins are firstly denatured and hydrolyzed by the combination of hydrochloride acid and pepsin in the stomach. Heat denaturation facilitates this process and is hence believed to be beneficial from the point of nutrition view. However, this is not always the case. If a protein plays a special function in the digestive tract, it is crucial to maintain its structure after ingested. For example, it has been proved that feeding of denatured whey protein is not good for the growth of calves. This truth has also been found in newborn infants. Proteins in breast milk are only partially digested and these proteins are more resistant to trypsin hydrolysis compared with milk proteins. It is therefore believed that some proteins have specific functions in the digestive tract. Some of such proteins have been identified. For example, lactoferrin is involved in iron delivery; immunoglobulin imparts

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Infants with passive immunity; lysozyme destroys microorganisms; and streptogenin promotes the growth of lactic acid bacteria.

8.2.2. Effects of other Components (1) Protein-carbohydrate interactions Moderate reactions with reducing sugars (Maillard reactions at early stage) Maillard reactions, also termed non-enzymatic browning reactions, occur between reducing sugars and the free amino groups of peptides, proteins, and amino acids. Carbonyl compounds, such as aldehydes and ketones, also undergo Maillard reaction with reducing sugars. Maillard reaction occurs readily during the processing and storage of foods, especially in dietary products. The rates of Maillard reactions depend on the temperature and moisture of foods. The first step of Maillard reactions includes the condensation between free amino groups and carbonyl groups. The condensation products are converted to Schiff's bases, which are subsequently isomerized to N-substituted glycosylamines. N-substituted glycosylamines derived from amine compounds are relative stable, and those derived from amino acids are unstable and immediately undergo Amado‘s rearrangement to produce 1-amino-l-deoxy-2ketones, which are the major products of Maillard reaction in milk after mild heating. The ketosamines are subject to further reactions and the products include reductones, furfural and unsaturated carbonyl compounds. These compounds then react with each other to produce soluble polymers and finally insoluble melanoidins in sequence. All the Lys residues and amino acids at the N-terminal are completely destroyed in these reactions. In addition to nutritional value loss of proteins, Maillard reactions can produce potential harmful compounds. It has been proved that the brown soluble compounds (pre-melanoidins) generated by the thermal reaction between free amino acids and glucose have particular physiological and anti-nutritional properties. However, no physiological functions have been found in pre-melanoidins formed by proteins and sugars, because the addition of severely heated protein-glucose mixture to a well balanced diet dose not affect the growth function of experimental animals. Violent reaction with carbonyl groups (Maillard reactions at later stage) Many researchers have confirmed that reactions of proteins with glucose at high temperature and pressure significantly reduce the nutritive value of proteins, even when the reaction time is short. Under these conditions, carbonyl compounds cause extensive crosslinking of proteins through Strecker degradation and aldol condensation. In this case, the value of any protein as the amino acid source is very limited. Reaction with sucrose As mentioned above, reducing sugars can react with proteins and cause amino acid loss. However, it has also been found that sucrose is also involved in the reaction with proteins. The initial reaction between lysine and sucrose is the 'aminolysis' of the 1, 2 glycosidic linkage between the glucose and fructose residues by the ε-amino group of lysine. Breakdown

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of the glycosidic linkage yields reducing groups, which undergo typical Maillard reaction with another molecule of lysine. It has been reported that sucrose at relatively high levels in oil seeds may be largely responsible for the damage to protein quality observed when they are severely processed [22]. (2) Protein-lipoid interactions The reactions between oxidized lipids and proteins have been extensively reported. However, no general conclusions can be drawn on the reactions, because lipids are oxidized in multiple modes. During oxidation, unsaturated fatty acids firstly bind to catalysts to produce free radicals. Free radicals then react with oxygen to form hydrogen peroxides, which can undergo reactions in a variety of ways. Some of the products are carbonyl compounds that can participate in Maillard reactions. These products are subject to either degradation or polymerization. When oxidized lipids and proteins are incubated, cross-linking occurs between lipid free radicals and proteins, which reduces the protein solubility, increases the resistance to digestive enzymes, and destroy most amino acids. Then, sulfur-containing amino acids are oxidized by hydroperoxides and methionine is converted to methionine sulfoxide. Free methionine sulfoxide as a methionine source is the same as methionine. However, methionine sulfoxide in peptides or proteins is less bioavailable than methionine, because it is resistant to enzymatic hydrolysis. Cystine is quickly degraded and cysteic acid is one of the degradation products. Cysteic acid is not a source of cystine for human body. The newly formed active carbonyl compounds can react with the amino group of Lys resides in a way similar to browning reactions. These reactions make Lys physiologically unavailable. In electrostatic fields, oxidized lipids can react with amino group-free proteins to yield simple compounds. In this case, the nutritive loss of proteins is very small. (3) Protein-aldehyde interactions. In addition to the interactions between carbonyl and amino groups mentioned above, two special reactions of proteins with aldehydes have also been identified during food processing. One is the reaction with gossypol during cottonseed processing. The other is the reaction with formaldehyde during meat fumigation. Both the two reactions decrease the bioavailability of lysine.

8.3. Effects of Processing on the Nutritional Value of Proteins Proteins as food materials are exposed to physical or chemical treatments during blending and application. These treatments affect the functional and nutritional properties of proteins. Besides, purposive modifications have been applied to improve the existing properties of protein or create new functions.

8.3.1. Heat Treatment Heat treatment can cause protein structure change, peptide bond hydrolysis, amino acid side chain modification and condensation with other molecules. The later two reactions are harmful to the nutritive value of proteins. Structural changes and limited peptide bond hydrolysis caused by mild heat treatment do not affect the nutritive quality of proteins, but

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significantly affect the functional properties of proteins. The degree and result (conformational change and aggregation) of thermal denaturation depend largely on the nature of proteins and environmental conditions. Mammalian collagens start to unfold, dissociate, and dissolve when heated to 65°C in the presence of large quantity of water. In contrast, actins undergo contraction, aggregation and water holding capacity reduction under same conditions. Caseinate monomers are very resistant to heat treatment. Vigorous heat treatment dose not affect the properties of caseinate. Mild heat treatment dose not lead to the destruction or generation of covalent bonds or serious changes of higher structures in proteins. Hence, from the nutritional point of view, the changes induced by mild heat treatment are beneficial. Heat treatment can eliminate the toxic effects of lectins in legumes, because the lectin is a thermal unstable protein which can bind polysaccharides. It can reduce the nutritional value of natural plant proteins in the diet. The presence of lectins can form compounds by combining intestinal brush border membrane polysaccharide, which can diminish the abilities of shifting and digestion in amino acid, produce toxicity to human and animal; heat treatment can eliminate their harmful effects. Protease inhibitors in foods, including trypsin inhibitors and chymotrypsin inhibitors, are inactivated during heat treatment. High temperature treatments, such as high-pressure sterilization, puffing, sterilization, cooking and baking, can destroy all antinutrients factors (Figure 5-14). Therefore, moderate heat treatment can markedly improve the nutritional value of vegetable proteins. Many proteins, such as soybean globulin, collagen and egg albumin, are more digestible after moderate heat treatment.

Figure 5-14. Effect of steaming on trypsin inhibitor activity and protein efficiency ratio of soybean meal feed [20]

After heat treatment, the proteins are more susceptible to enzymatic hydrolyze due to unfolding and amino acid residues exposure. Besides, heat treatment can remove off-odor compounds in soybean protein and avoid the undesired flavor caused by lipid oxidase.

8.3.2. Dehydration When the water in a protein solution is removed, aggregation occurs to the proteins. This is the case especially when water is removed by evaporation at high temperatures, because the

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solubility and surface activity decline sharply. Hence, the effects of drying on the functional properties of protein should not be ignored. Drying changes the size and internal and surface porosity of particles and consequently alters the wettability, absorption, diapersion and dissolution of food proteins. When water is quickly removed as steam, particles shrink in the minimum level and salts and carbohydrates migrate to the dried surface, yielding porous powder particles. This change can be observed in freeze-drying and spray-drying. Introduction of bubbles to protein solution before drying or the implementation of controlled aggregation during drying can be applied to increase the porosity of particles.

8.3.3. Irradiation Irritation causes inter- and intra-molecular cross-linking of proteins or peptide bond breakdown in foods with low moisture content. In the presence of catalase, Tyr is oxidized and condensed to dityrosine. 8.3.4. Oxidation Oxidants are important additives in the food industry. For example, hydrogen peroxide is sterilization and bleaching agent which can be used as a low-temperature fungicide in package. It can also been used to improve the color of fish protein concentrates, flours and oil seed protein isolates. Benzoyl peroxide can oxidize cysteine in flour to cystine so as to enhance gluten strength and bleach flour. In some cases, benzoyl peroxide can also be used in whey powder bleaching. Peroxides are also found in foods without artificially added oxidants and these peroxides are often the reason for the degradation of proteins present in the foods. Light oxidation, irradiation, trace metals, oxygen, hot air drying and aeration during fermentation can all induce oxidative denaturation of amino acids in proteins. Trp is the most sensitive amino acids to oxidation, followed by tyrosine and histidine in sequence. The oxidation of sulfur-containing amino acids occurs mainly in the sulfurcontaining side chains and that of aromatic-containing amino acids mainly occurs in the side chains containing aromatic rings. 8.3.5. Maillard Reaction Foods containing proteins and reducing carbohydrates or carbonyl compounds (such as aldehydes and ketones produced by lipid oxidation) are subject to Maillard reactions during processing and storage. These reactions yield brown or black melanoidins, which impart breads and bakery products with the brown color. The formation of melanoidins is accompanied by the cross-linking of a small amount of proteins. The cross-linking significantly reduces the digestibility of these proteins. Studies on some protein-carbohydrate model systems reveal that melanoidins are mutagenic and the capacity depends on the degree of Maillard reactions. Melanoidins are insoluble in water and are only weakly absorbed on intestinal wall. Hence, melanoidins are only slightly physiologically toxic to human. However, the low molecular weight precursors of melanoidin are absorbed easily. 8.3.6. Enzyme Treatment Enzymatic hydrolysis can improve the quality of food proteins. For example, papain can tenderize meat and chymotrypsin can coagulate casein. Papain, bromelain, pepsin and protease produced by microorganisms can partially hydrolyze thermal denatured or solvent denatured proteins and improve their solubility. The hydrolytes can be used in acidic or hot-

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processed drinks. The hydrolytes can also be used as alternative protein sources for patients allergic to milk and gluten or those with digestive tract diseases. Partial hydrolysis can improve the emulsifying and foaming abilities of thermally denatured proteins, because the increased solubility of proteins facilitates their diffusion to the oil/water and gas/water interfaces. However, when the degree of hydrolysis exceeds 3%~5%, the viscosity and thickness of protein layer absorbed on the interface are insufficient to stabilize emulsion and foam. Hydrolysis reduces the volume of proteins, which is unfavorable for the gelling ability and viscoelasticity of proteins, but limited hydrolysis of gluten can enhance the expansion degree of dough in bread making and improve the flakiness of biscuits. In addition, proteases can connect low molecular weight peptides through peptide transferring to produce new larger peptides, namely proteinoids.

8.3.7. Functional Peptides from Protein Hydrolysates Proteins often undergo hydrolysis during processing. Hydrolysis not only improves the nutritional value of foods, but also yields functional peptides, such as those with antihypertensive, immunomodulatory, neuroactive, antimicrobial and antioxidative activities. Hydrolysis using proteases has been preferred as the main method for producing functional peptides from food proteins. Fish and other seafood proteins have gained particular interest as potential functional peptide sources among various food protein resources due to their abundance as byproducts. For example, antioxidative peptide could be prepared from Alaska pollack (Theragra chalcogramma) and yellowfin sole (Limanda aspera) frame proteins by using a crude enzyme mixture from mackerel intestine [23, 24]. The pepsin hydrolysate, which exhibits a strong DPPH scavenging activity at a concentration of 6.80μg/mL, could be obtained from a marine fish half-fin anchovy [25] and its antibacterial activity is higher than the hydrolysates produced by other proteases [26]. Functional peptides can be generated by protease hydrolysis and/or fermention. Use of exogenous enzymes is preferred in most cases over autolysis. Various food-grade proteases, such as alcalase, flavourzyme and protamex from microorganisms, papain from plant, and pepsin and trypsin from animal sources, have been widely used in producing functional peptides in large scale. Some functional peptides can be generated during fermentation or food processing. In fermented foods, antioxidative peptides can be produced by the action of microbes and endogenous proteolytic enzymes. For example, the free radical scavenging peptides could be produced from fermented mussel sauce [27]. Milk whey fermented by lactic acid bacteria has been reported to contain antioxidative peptides [28]. The peptide cyclo (His-Pro) is an endogenous cyclic dipeptide with antioxidant activity. It has been isolated from processed foods such as dried shrimp and fish sauce [29].

9. CONTRIBUTION OF PROTEINS TO THE COLOR, FLAVOR, AND TASTE OF FOODS 9.1. Bitterness Some amino acids are bitter and the partial hydrolysis of proteins can also yield bitter peptides, such as Leu-Leu and Arg-Pro. The bitterness of protein hydrolyzates and mature cheese is contributed by short peptides and amino acids.

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Peptides with molecular weight greater than 6kD cannot the action sites of taste receptors and are hence tasteless. Whether a peptide with molecular weight less than 6kD is bitter and its bitter intensity is related to its hydrophobicity. For details, see Section 5.1.3.

9.2. Off-Odors Some protein products must undergo the deodorizaton process to remove off-odor compounds bound to proteins. Ketones, aldehydes, alcohols, phenols, and lipid oxidation products can produce beany flavor, rancidity, bitterness, and harsh taste. These compounds can bind to proteins. When the proteins are cooked or chewed, these compounds are released and the undesired tastes or odors are delivered to consumers, which markedly reduce the acceptability of foods. In addition to off-odors, proteins can also serve as the carrier of flavors. For example, textured vegetable proteins can impart the meat flavor. Ideally, the desired volatile flavor components should bind to carriers during storage and processing and be released rapidly and completely in the oral cavity upon ingestion. Any factors that change the conformation of proteins affect the binding to volatile compounds to proteins. In the presence of water facilitates the binding to polar compounds to proteins, but shows no effect on that of nonpolar compounds. Volatile compounds have only limited diffusion in dried protein components. However, when the water activity is slightly improved, volatile compounds diffuse to their binding sites rapidly. Casein can bind more volatile carbonyl compounds, alcohols and esters in neutral and alkaline conditions than in acidic solutions. Oxidates and sulfates in high concentrations can cause protein unfolding and thus improve the binding of proteins to carbonyl compounds. Agents that can dissociate proteins or interrupt disulfide bonds improve the binding of proteins to volatile compounds. When oligomers are dissociated to subunits, the hydrophobic areas between subunits are masked during conformation change of the subunits. Hence, the binding to nonvolatile compounds is reduced. Thorough hydrolysis of proteins reduces their ability of binding volatile compounds. For example, one kilogram of soybean protein can bind 6.7 mg caproaldehyde. After the protein is hydrolyzed by acidic protease, the binding capability is reduced to 1 mg/kg protein. Hence, the beany flavor of soybean protein can be alleviated by hydrolysis. Thermally denatured proteins generally have improved volatile binding capabilities. For example, when 10% soybean protein isolate is heated at 90°C for 1~24h in the presence of caproaldehyde and is then free dried, the amount of caproaldehyde bound is 3~6 times more than that of unheated protein. Dehydration process, such as free drying, can release more than 50% of bound volative compounds. However, if a volatile compound has a low vapour pressure and is present in low content, it can be well retained after the treatments. It should be noted that the presence of lipids is beneficial for the binding and retention of volative compounds, including those generated by lipid oxidation.

9.3. Sweetness of Protein Derivates Development of safe and strong sweeteners by the modification of non-sugar and unsweet natural compounds is an emerging field since 1960s. Such sweeteners that have been put into industrial production are divided into tow categories: amino acids and their dipeptide

Proteins

187

derivates, and dihydrochalcone and their derivates. The potential of perilla aldehyde and its derivates as sweetener has also attracted much attention. Some naturally occurring amino acids have been found to be sweet, including Gly, DAla, L-Ala, D-Ser, L-Ser, D-Thr, L-Thr, D-Trp, D-Pro, D-hydroxyproline, and D-Glu. Some amino acid derivates are also found to be sweet. For example, the specific saccharinity of 6methyl-D-Trp reaches 1000 and is a potential novel sweetener. In 1969, it was found that many dipeptide derivates of aspartic acid are sweet and phenylalanine methyl ester (aspartame) has been approved by FDA for use in foods as a sweetener. Aspartame is the sweetest compound among all sweeteners identified and is 2.7 times sweeter than sucrose. These dipeptide sweeteners are non-sugar compounds. Their components are natural molecules and can be metabolized by human body. Besides, their sweetness curves fit that of sucrose very well and the sweetness tastes very well. The shortcoming is that they are unstable in high temperatures. Sweet dipeptide derivatives have the following structural characteristics: The two amino acids are of the L form. The amino acid in the N terminal is Asp and its –COOH and –NH2 groups are free. Another amino acid is neutral and one of the ends must be esterified. The smaller the ester group, the sweeter the dipeptide. The sweet intensity decreases with the increase of molecular weight.

9.4. Flavor Binding The proteins of food have little flavor of their own, but are known to bind trap aroma compounds. The most extensively studied protein is the milk protein β-lactoglobulin. The protein can interact with many flavour compounds, such as aldehydes and ketones, ionones and esters. Both reversible binding and irreversible binding can occur between proteins and flavor compounds. The type of interaction between a protein and a flavor compound depends on the nature of both the protein and the aroma compound. As most aroma compounds are hydrophobic in nature, hydrophobic and reversible binding is predominant. On the other hand, certain flavor compounds, such as aldehydes, can form covalent bonds with proteins and such interactions are irreversible. Proteins can be used as flavor carriers in the food industry. It is suggested that βlactoglobulin, the major whey protein, could be engineered to bind and protect a wide range of volatile and unstable flavors during food manufacturing or to release them in more or less controlled ways by chemical or physical modifications. Processing significantly affects the interactions between flavors and proteins. Studies on the interactions between whey protein isolate and flavor compounds (2-nonanone, 1-nonanal, and trans-2-nonenal). It was found that hydrophobic interactions are weakened upon heating or high pressure denaturation, whereas covalent interactions were enhanced [30]. Conditions of high hydrostatic pressure treatment (HHP) and flavor species significantly affect flavor retention. HHP treatments did not result in significant changes in the flavor retention for benzaldehyde in WPC solutions, but significant decreases of nonanone retention were only observed at 100 ppm when WPC solutions were HHP-treated for 10 min. While use of HHP treatment of WPC has potential in real food systems, these findings demonstrate the

188


importance of careful selection of HHP treatment times and flavor concentrations for desired outcomes in food application [31]. Milk protein-flavor interactions are very dependent on the conformational state of a protein. Therefore, factors such as pH, temperature, and high pressure that influence protein conformation can markedly change flavor binding characteristics of proteins. Interactions of vanillin with soy, casein, and whey proteins Bindings of vanillin with casein and whey protein were enthalpy driven, while the interaction of vanillin with soy protein was highly entropy driven. The researchers inferred that conformational changes of soy protein might be important in binding of vanillin [32].

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

[17]

[18]

[19]

Guang B, Lin H, Wang GC. Food Protein Chemistry. Beijing: Chemical industry press, 2005. Murray RK, Granner DK, Rodwell VW, Mayes PA. Harper's Biochemistry. 25th edition. McGraw-Hill Companies, 1999. http://www.hemaquest.com/indications/hemoglobin.asp. Xie, BJ. Food chemistry. 1st edition. Beijing: Science Press, 2004: 255. Damodaran S. Influence of protein conformation on its adaptability under chaotropic conditions. International Journal of Biological Macromolecules, 1989, 11:2-8. Han JM, Ledward DA. High pressure/thermal treatment effects on the texture of beef muscle. Meat Science, 2004, 68: 347-355. Wang DF. Food Chemistry. 1st edition. Beijing: Chemistry Industry Press. 2007: 118. Xie BJ. Food chemistry. 1st edition. Beijing: Science Press, 2004: 259. Xie BJ. Food chemistry. 1st edition. Beijing: Science Press, 2004: 260. Hu WW, Xie BJ. Food Chemistry. 1st edition. Beijing: Science Press, 1992: 165. Damodaran S. Interfaces, protein films, and foams. Advances in Food and Nutrition Research. 1990, 34: 1-79. Xie BJ. Food chemistry. 1st edition. Beijing: Science Press. 2004: 280. Wang DF. Food Chemistry. 1st edition. Beijing: Chemistry Industry Press. 2007: 118. Xie BJ. Food chemistry. 1st edition. Beijing: Science Press. 2004: 278. Xie BJ. Food chemistry. 1st edition. Beijing: Science Press. 2004: 281. Pusztai A, Clarke EMW, Grant G, King TP. The toxicity of Phaseolus vulgaris lectins. Nitrogen balance and immunochemical studies. Journal of the Science of Food and Agriculture. 1981, 35: 701-711. Zhu H, Damodaran S. Effects of calcium and magnesium ions on aggregation of whey protein isolate and its effect on foaming properties. Journal of Agricultural and Food Chemistry. 1994, 42: 856-862. Eggum BO, Beames RM. The nutritive value of seed proteins. In: Gottschalk W, Müller PH. Seed Proteins: Biochemistry, Genetics, Nutritive value. Berlin: Springer. 1983: 499-531. FAO/WHO/UNU. Energy and Protein Requirements, Report of a Joint FAO/WHO/UNU Expert Consultation. World Health Organization Technical Report Series. 1985: 724.

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[20] Lund D. Effect of commercial processing on nutrients. In: Karmas E, Harris RS. Nutritional Evaluation of Food Processing. New York: Van Nostrand Reihhold. 1988: 319-354. [21] Wang DF. Food Chemistry. 1st edition. Beijing: Chemistry Industry Press. 2007: 124. [22] Hurrell RF, Carpenter KJ. Mechanisms of heat damage in proteins. The role of sucrose in the susceptibility of protein foods to heat damage. British Journal of Nutrition. 1977, 38: 285-297. [23] Je JY, Park PJ, Kim, SK. Antioxidant activity of a peptide isolated from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. Food Research International. 2005, 38: 45-50. [24] Kim SY, Je JY, Kim SK. Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. The Journal of Nutritional Biochemistry. 2007, 18: 31-38. [25] Song R, Wei RB, Zhang B, Yang ZS, Wang DF. Antioxidant and Antiproliferative Activities of Heated Sterilized Pepsin Hydrolysate Derived from Half-Fin Anchovy (Setipinna taty). Marine Drugs. 2011, 9: 1142-1156. [26] Song R, Wei RB, Zhang B, Yang ZS, Wang, DF. Optimization of the Antibacterial Activity of Half-Fin Anchovy (Setipinna taty) Hydrolysates. Food and Bioprocess Technology. 2011: 1-11. [27] Rajapakse N, Mendis E, Jung WK, Je JY, Kim SK. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Research International. 2005, 38: 175-182. [28] Virtanen T, Pihlanto A, Akkanen S, Korhonen H. Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria. Journal of Applied Microbiology. 2007, 102: 106-115. [29] Minelli A, Bellezza I, Grottelli S, Galli F. Focus on cyclo(His-Pro): history and perspectives as antioxidant peptide. Amino acids. 2008, 35: 283-289. [30] Kühn J, Considine T, Singh H. Binding of Flavor Compounds and Whey Protein Isolate as Affected by Heat and High Pressure Treatments. Journal of Agricultural and Food Chemistry. 2008, 56: 10218-10224. [31] Liu XM, Powers JR, Swanson BG, Hill HH, Clark S. High Hydrostatic Pressure Affects Flavor-binding Properties of Whey Protein Concentrate. Journal of Food Science. 2005, 70: C581-C585. [32] Li Z, Grün IU, Fernando, LN. Interaction of Vanillin with Soy and Dairy Proteins in Aqueous Model Systems: A Thermodynamic Study. Journal of Food Science. 2000, 65: 997-1001.



Chapter 6

VITAMINS 1

Yibin Zhou1, Dongfeng Wang2 and Ping Dong2 College of Tea and Food Technology, Anhui Agriculture University, China 2 College of Food Science and Engineering, Ocean University of China, China

ABSTRACT Vitamin is one important group of nutrients that determines the nutritional value of foods. These compounds play extremely important role in many metabolisms in organism as cofactors of various enzymes and long-term vitamin deficiency can cause various diseases and even death. Besides, some vitamins, such as Vc and sulfur-containing compounds, also markedly affect the color and flavor of foods. This chapter introduces the structures, properties, physiological functions and deficiency symptoms, and stability of the vitamins that have been identified one by one. Then, the factors that influence vitamin contents in foods and the changes of vitamins during processing and storage are discussed.

1. CLASSIFICATION OF VITAMINS Vitamins are classified by their biological and chemical activities, as well as the chronological order of being found, but not their structures. Basically each "vitamin" has a multiple chemical substances (called vitamers) that show similar biological activity, rather than having a fixed chemical structure. For example, Vitamin A includes retinol, retinal, and four known carotenoids. The classification and some functions of vitamins are summarized in Table 6-1. Vitamins are classified into two water-soluble and fat-soluble ones according to solubility. Among the 13 vitamins identified, the eight members of the B family and vitamin C are water soluble, while vitamins A, D, E, and K are fat soluble.

192

Yibin Zhou, Dongfeng Wang and Ping Dong Table 6-1. Classification of vitamins and their functions

Category Watersoluble

Name VB1

Synonyms Thiamine, Antineuritic factor

Physiological functions Antineuritic Prevent beriberi

VB2

Riboflavin

VB3,VPP

Nicotinic acid, Niacin Nicotinamide Antipellagra factor Pyridoxine, pyridoxal, pridoxal Folic acid, folinic acid

Prevent inflammation of lip and tongue, Facilitate growth Prevent pellagra Form Coenzyme I or II

VB6 VB9 VB12

Fat-soluble

VB7,VH

Cyanocobalamin, hydroxycobalamin, methylcobalamin Biotin

V B5

Pantothenic acid

VC

Ascorbic acid

VA1,2

Antixerophthalmic vitamin, carotenoids, retionol, retinal

VD1,3

Cholecalciferol, Lexacalcitol, Resistant rickets

VE VK1,2,3

Tocopherol, Aantisterility vitamin Phytomenadione, phylloquinone

Play a role in amino acid metabolism Prevent pernicious anemia Prevent pernicious anemia

Prevent skin disease Facilitate lipid metabolism Prevent Paresthesia Prevent or cure scurvy Facilitate the growth of intercellular substance Work as a light-sensitive substance in a visiual cell, prevent epidermal cell keratinization, facilitate growth, prevent and cure xerophthalmia Regulate metabolism of calcium and phosphate, prevent rachitis and gibbosity Prevent barrenness Accelerate blood coagulation

Because water-soluble vitamins can dissolve in body fluid and can not be stored readily, consistent daily intake is required. On the other hand, fat-soluble vitamins, absorbed through the intestinal tract with the help of lipids (fats), can be easily accumulated in the body. Watersoluble vitamins are readily excreted from the body so their consumption can be simply checked by the degree of urinary output.

2. VITAMINS IN FOODS As described above, vitamins are a class of natural organic compounds that maintains the normal physiological functions of human body. Although, the amount required for daily diet is only several micrograms or milligrams, they are essential for human health. Microorganisms in intestines can synthesize some vitamins, but the quantity far from the need of human body. Therefore, vitamins have to be largely provided by foods.

2.1. Vitamin A Vitamin A, including vitamin A1 (retinol) and vitamin A2 (3-dehydroretinol), is also called antixerophthalmic vitamin, and its deficiency can cause night-blindness,

193

Vitamins

hyperkeratosis, and keratomalacia. Vitamin A is needed by the retina of the eye as the lightabsorbing molecule of retinal, without which the vision will be lost. An oxidized form of vitamin A – retinoic acid, is a hormone-like growth factor for epithelial and other cells. Vitamin A was discovered in 1917 by Elmer McCollum (University of Wisconsin–Madison), Lafayette Mendel (Yale University) and Thomas Burr Osborne (Yale University). In 1919, Steenbock (University of Wisconsin) proposed that yellow plant pigments (beta-carotene) might be vitamin A, which was named as ―fat-soluble factor A". Vitamin A exists in animal tissues, plant tissues and fungi. (1) Structure. Structures of vitamin A and its precursors are shown in Table 2.

Table 6-2. Carotenoids structure and activity of vitamin A [1] Compounds

Structure

Relative activity

β-carotene

50

α-carotene

25

β-Apo-8´carotenal

25-30

Zeaxanthin

0

Canthaxanthin

0

Astacin

0

Lycopene 0

Both vitamin A1 (retinol) and vitamin A2 (3-dehydroretinol) have the unsaturated monohydric alcohol (an isoprenoid chain, also called a retinyl group) with the beta-ionine ring shown as follows: Both the retinyl group and the beta-ionine ring are essential for the activities of vitamin A. The difference between vitamin A1 and vitamin A2 lies in the bond between C3 and C4 in

194

Yibin Zhou, Dongfeng Wang and Ping Dong

the beta-ionine ring. Usually vitamin A refers to vitamin A1, retinol. The biological effect of vitamin A2 is about 40% of that of vitamin A1. In foods of animal origin, the major form of vitamin A is an ester, primarily retinyl palmitate, which is converted to retinol in small intestine. Retinol can be converted to its visually active aldehyde form, retinal. The associated acid (retinoic acid), a metabolite that can be irreversibly degraded from vitamin A, has only partial vitamin A activity, and does not function in the retina for the visual cycle. Food carotenoids, mainly include beta-carotene and lutein (Table 6-2), are also precursors of vitamin A. Usually they have two connected retinyl groups, which are used in the body to contribute to vitamin A activity. The well-known orange pigment of carrots (betacarotene), which shows some vitamin activities itself, is an important source of vitamin A. (2) General properties. Usually, vitamin A is a light yellow viscous liquid. Pure vitamin A1 is a light-yellowish (or primrose yellow) crystal and melts at 62°C-64°C. Vitamin A is fat soluble and is stable under alkali conditions or inert atmosphere. Vitamin A is thermal stable and is not destroyed in temperatures up to 120°C-130°C, but it is liable to decomposition in exposure to air, oxidants and ultraviolet radiation. Both vitamin A and carotene can react with antimony trioxide in ethanol to produce dark blue yellow. This property has been used for the spectrometric determination of vitamin A and carotene. (3) Physiological functions and deficiency symptoms. Vitamin A promotes the growth of human body. Besides, it maintains the integrity and health of epithelial tissue and ensures the normal vision. Deficiency of vitamin A for a long time can cause night blindness and loss of dark adaptation. In subjects suffering severe vitamin deficiency, the keratinized epithelial cells at gastrointestinal tract and eye cornea can even not be produced. The obvious characteristic of vitamin A deficiency is xerophthalmia, such as cornea hyperemia, inflammation and sclerosis. This is why vitamin A is also known as the antixerophthalmic vitamin. Excessive intake of vitamin A (more than 65,000~500,000 international units) can result in poisoning, but the symptoms can disappear several days later if vitamin A is excluded from the diet. (4) Stability and degradation. All naturally occurring carotenoids are present in the trans conformation. The compounds can transform to the cis conformation and lose its activity when heating. The isomerization of carotenoids occurs readily during storage, as shown in Table 6-3. In addition, exposure to light, acids and watery iodine also causes the transform from cis- to trans-conformation of carotenoids and retinols. The destruction mechanisms of vitamin A precursors vary with processing conditions (Figure 6-1). The cis-trans transformation of beta-carotenes upon heating occurs only in the absence of oxygen. Beta-carotene is decomposed to a series of aromatic hydrocarbons at higher temperatures and the major decomposition product is lonene, which has important implications on food flavor. Carotene is often intentionally added to foods to produce aroma during food processing.

Vitamins Table 6-3. Distribution of β-carotene isomers in fresh fruits and processed vegetables [1] Products

Status

Sweet potato

Fresh Canned Fresh Canned Fresh Canned Fresh Canned Fresh Canned Fresh Pasteurized Fresh Canned Fresh Canned Dehydrated Canned Fresh Fresh

Carrot Pumpkin Spinage Collard Cucumber Pickld cucumber Tomato Peach Apricot Nectarine Plum

Figure 6-1. β-Carotene degradation [2].

Percentage of total β-carotene (%) 13-cis All trans 9-cis 0 100.0 0 15.7 75.4 8.9 0 100.0 0 19.1 72.8 8.1 15.3 75.0 9.7 22.0 66.6 11.4 8.8 80.4 10.8 15.3 58.4 26.3 16.3 71.8 11.7 26.6 46.0 27.4 10.5 74.9 14.5 7.3 72.9 19.8 0 100.0 0 38.8 53.0 8.2 9.4 83.7 6.9 6.8 79.9 13.3 9.9 75.9 14.2 17.7 65.1 17.2 13.5 76.6 10.0 15.4 76.7 8.0

195

196

Yibin Zhou, Dongfeng Wang and Ping Dong Table 6-4. Contents of beta-carotene in fresh and dehydrated carrot [3] Carrot Fresh Vacuum freeze-drying Conventional air-drying

Content (mg/kg) 980-1860 870-1125 636-987

Oxygen, light, enzymes, and lipid hydroperoxide enhance the oxidative degradation of carotene. Oxidation of beta-carotene first involves the formation of the 5, 6-epoxide, which will isomerize to beta-carotene oxide, namely 5, 8-epoxide. Vitamin A and provitamin A are usually inactivated in dehydrated foods, but the loss varies with the drying technology (Table 6-4). Maintenance of Aw and oxygen partial pressure in low levels can reduce the loss of carotenoid and vitamin A.

2.2. Vitamin D Vitamin D is also known as anti-rickets or anti-rachitis vitamin. Six members have been ascertained, including Vitamin D2, Vitamin D3, Vitamin D4, Vitamin D5, Vitamin D6, and Vitamin D7, among which Vitamin D2 and Vitamin D3 are the most important. Vitamin D in food always coexists with vitamin A. Fat in fishes or liver in animals are rich in vitamin D. The content of Vitamin D in fish oil from seafood is much high, followed by egg yolks, milk, and butter. Sunlight can promot the synthesis of vitamin D in animals. Thus, the levels of vitamin D in milk and cream produced in summer are more than those in winter due to the stronger sunlight in summer. (1) Structure. The vitamin D members are steroids that contain the cyclopentanoperhy drophenanthrene ring structure and they are distinguished by their structures of the branch chain R, as shown in Figure 6-2. Vitamin D is found only in animal tissues, but most plants contain sterols, which can be converted to vitamin D by ultraviolet light. (2) General properties. Vitamin D members are colorless crystal and fat soluble. Vitamin D is quite stable against acids, alkali, and oxygen, but is readily destroyed by light and temperatures higher than 160°C. (3) Physiological functions and deficiency. Vitamin D regulates the metabolism of calcium and phosphorus and maintains the level of calcium and phosphorus in blood. Hence, the deficiency of calcium and phosphorus is often related to the level of vitamin in human body. The increased requirements of pregnant and lactating women on calcium and phosphorus can be realized by vitamin D-reinforced diets. Vitamin D deficiency leads to osteomalacia and rickets. However, excessive intake of vitamin D can cause posioning. The symptoms of acute vitamin D poisoning include loss of appetite, nausea, vomiting, diarrhea, headache and polyuria and those of chronic poisoning include weight loss, albinism, and even soft-tissue calcification or serious kidney failure.

197

Vitamins CH 3 19

11 12 13 14 9 8

CH 2 2 3

1 4

10 5

6

R 17 16 15

7

HO

Vitamin D2

R=

CH

CH

CH

CH

CH3 Vitamin D3

R=

CH

CH

CH3

CH3

CH2

CH2

CH2

CH

R=

CH

CH2

CH2

CH

CH

CH3 Vitamin D5

R=

CH

CH2

CH3

Vitamin D6

R=

CH

R=

CH CH3

CH

CH 2C H 3 CH

CH

CH3 Vitamin D7

CH

CH

CH

C H 2C H 3 CH2

CH CH3

CH2

CH

CH3 CH3

CH3 CH2

CH2 CH3

CH3 Vitamin D4

CH2

CH3 CH3

CH3 CH3 CH3 CH3

Figure 6-2. Chemical structures of vitamin D members [4].

2.3. Vitamin E Vitamin E is also called as anti-sterility factor or tocopherol and is a family of eight separate compounds, in which, α-, β-, γ-, δ-tocopherols are the most important and αtocopherol is the most active. Hence, tocopherol sometimes refers solely to α-tocopherol. The contents of tocopherol in some foods are listed in Table 6-5. The content of vitamin E is often expressed as international unit (IU). One IU of vitamin E is equal to 1 mg of D, Lα-tocopherol acetate or 0.667 mg of D-α-tocopherol. (1) Structure. Vitamin E is derivatives of benzo-3 H-pyran and its general structure is shown in Figure 6-3. The members of the vitamin E family differ in the structures of the three side chains, as listed in Table 6-6. (2) General properties. Pure vitamin E is a transparent and light yellow oily liquid. It is fat soluble and is stable against acid, alkaline and heating (up to 200°C in the absence of oxygen), but it is susceptible to oxidation by oxygen. Hence, vitamin E is an important natural antioxidant in foods. Vitamin E is quiet stable under white light, but is easily destroyed by ultraviolet light. (3) Physiological function and deficiency.

198

Yibin Zhou, Dongfeng Wang and Ping Dong Table 6-5. Contents of tocopherol in plant oil and some foods [4,5] Foods

α-T

α-T3

β-T

β-T3

γ-T

γ-T3

δ- T3

56.4 0.013 17.9 40.3 27.2 9.0 9.1

0.013 0.007 0.021 0.002 5.37 0.008 5.19

2.45 0.039 2.80 0.196 0.214 0.16 0.153

0.207 0.394 0.437 0.87 1.1 0.417 0.4

0.43 13.1 60.4 38.3 56.6 0.471 0.84

0.023 0.03 0.078 0.089 6.17 0.026 13.2

0.087 0.922 37.1 0.457 2.52 0.043 0.002

4.0

16.4 6.9

Plant oil (mg/100g) Sunflower seed Peanut oil Bean oil Cottonseed oil Corn germ oil Olive oil Palm oil Other foods (μg/ml or g)

Spinage 26.05 Beef 2.24 Flour 8.2 Barley 0.02 Notes: T-tocopherol; T3- tocotrienol.

9.14 1.7 7.0

2.8

Figure 6-3. Structure of vitamin E [6].

Table 6-6. Members of the vitamin E family and their Physiological values [4,7] Side chain

[

[

]3

]3

Name

R1

R2

R3

Occurrence

Relative biological value

α- tocopherol

-CH3

-CH3

-CH3

Wheat embryo

1

β- tocopherol γ- tocopherol δ- tocopherol Σ- tocopherol η- tocopherol

-CH3 -H -H -CH3 -H

-H -CH3 -H -CH3 -CH3

-CH3 -CH3 -CH3 -H -H

Wheat embryo Corn Bean Rice Rice

0.5 0.2 0.1 0.5 0

ε- tocopherol

-CH3

-H

-CH3

Corn

0.5

Σ1- tocopherol

-CH3

-CH3

-CH3

Rice

0.5

Vitamin E is involved in the conversion of arachidonic acid to prostaglandins and slows down the aggregation of blood platelets. Vitamin E deficiency can cause damage of the genitals of human and infertility.

Vitamins

199

Figure 6-4. Oxidation pathway of α-tocopherol initiated by peroxide free radicals [6].

(4) Stability and degradation. The majority of vitamin E in foods is lost during processing and storage. For example, about 80% vitamin E is lost in the processing of wheat, corn, oat and rice. Degreasing, dehydration, lipid refinement and oxidation significantly affect vitamin E contents in foods, but the specific losses depend on the processing technology and starting material. For example, 36~45% of α-tocopherol is lost in the dehydration of chicken and beef, but there is almost no loss in the process of dehydration of pork. Rinsing with water does not reduce tocopherol contents in foods due to it fat solubility. The stability of vitamin E in foods is related to aw, temperature and duration during storage. Vitamin E is a strong antioxidant and can retard or prevent lipid oxidation. In the presence of peroxide free radicals, vitamin E is oxidized to α-tocopherol oxides, α-tocopherol quinone and α-tocopherol hydroquinone, as shown in Figure 6-4. In addition, singlet oxygen can also attack the epoxy system in tocopherol molecule, resulting in the formation of a hydrogen peroxide derivative. The derivative can generate tocopherol quinone and α-tocopherol quinone-2-3-epoxide after rearrangement (Figure 6-5).

2.4. Vitamin K Vitamin K is found due to its blood coagulation functions. Two kinds of vitamin K have been identified, including vitamin K1, which is extracted from alfalfa, and vitamin K1, which is obtained from degraded fishes.

200


Figure 6-5. Oxidation pathway of α-tocopherol initiated by singlet oxygen [7].

Figure 6-6. Structures of vitamin K1 and vitamin K2.

Vitamin K occurs mainly in plant tissues. Green leafy vegetables, such as alfalfa, cabbage, cauliflower, spinach and cabbage, contain more vitamin K1 than vitamin K2, with the latter derived mainly from the metabolism of intestinal bacteria. (1) Structure. Vitamin K1 and K2 are the derivatives of 2-methyl-1, 4-naphthoquinone and the two compounds differ in their side chains, as shown in Figure 6-6. (2) General properties. Vitamin K1 is a viscous yellow oily liquid can be crystallized from alcohol solution under low temperature, and its melting point is -20 °C. Vitamin K2 is a yellow crystal and melts in the temperature range from 53.5°C to 54.5°C. Both vitamin K1 and K2 are stable

201

Vitamins

against heating, but are readily destroyed by alkali and sunlight. K2 is more susceptible to oxidation than K1. (3) Physiological function and deficiency. Vitamin K promotes blood coagulation and is essential for the synthesis of prothrombin in liver. Vitamin K deficiency leads to reduced content of prothrombin in plasma and prolonged duration of blood coagulation. Vitamin K will lose its promotion of prothrombin synthesis in liver if liver dysfunction occurs. In addition, vitamin K also enhances intestines peristalsis and secretion.

2.5. Vitamin B1 Vitamin B1 is also known as thiamin. Yeast is reported to have the highest content of vitamin B1, followed by muscle, drupe, and eggs. Vitamin B1 in cereals occurs mainly in cortex and embryo. Table 6-7 lists the contents of vitamin B1 in some cereal products. (1) Structure. Vitamin B1 contains a pyrimidine ring that contains an amino group and a thiazole ring thatn contains a sulfur residue, as shown in Figure 6-7. (2) Properties. The hydrochloride salt of vitamin B1 is a white crystal. The crystal is stable at 100°C for 24 hours, but is decomposed in temperatures higher than 249°C. Vitamin B1 is water soluble and one gram of vitamin B1 can dissolve in 1mL water, 18 mL glycerin, 100 mL 95% alcohol or 315 mL anhydrous alcohol, but is insoluble in ethyl ether, acetone, chloroform or benzene. Vitamin B1 has a smell similar to that of yeast and tastes bitter. Table 6-7. Contents of vitamin B1 in some cereal products (mg/100g Dry)[8] Name Wheat

Vitamin B1 content 0.37-0.61

Names Brown rice

Vitamin B1 content 0.3-0.45

Bran Embryo Flour (85% yield) (73% yield) (60% yield) Potato

0.7-2.8 1.56-3.0 0.3-0.4 0.07-0.1 0.07-0.08 0.08-0.1

Cortex Rice embryo Endosperm Corn Soybean Pea

1.5-3.0 3.0-8.0 0.03 0.3-0.45 0.1-0.6 0.36

Figure. 6-7. Structure of thiamin.

202


Figure 6-8. Structure of thiochrome.

Figure 6-9. Structure of thiamin pyrophosphate (TPP).

Vitamin B1 is stable in solutions with pH less than 3.5. Under these conditions, the vitamin can maintain its activity even when heated at 120°C; however, it is easily destroyed when the pH exceeds 3.7. For example, 25% of thiamin is lost when heated in 97°C at pH 4.3 for 1 hour and the value reaches 80% when the pH is further increased to 7.0. Though vitamin B1 is readily damaged by high temperatures in alkaline conditions, cooking is insufficient to completely destroy the vitamin in rice. During bread bakery, about 15~30% of the total amount of vitamin B1 in flour is lost. Both vitamin B1 and its pyrophosphate salt can be oxidized by mild oxidant (such as the alkaline solution of potassium ferricyanide) to produce the dark blue fluorescence-emitting thiochrome. This reaction has been used to quantitative determination of vitamin B1. The structure of thiochrome is shown in Figure 6-8: Thiamine can be converted to thiamine pyrophosphate (TPP, Figure 6-9) by thiamine kinase in the presence of ATP. TPP plays an important role in sugar metabolism. (3) Physiological function and deficiency. Vitamin B1 participates in the oxidative decarboxylation of α-keto acid, such as pyruvic acid and α- ketoglutaric acid, as cocarboxylase. Vitamin B1 deficiency results in the accumulation of pyruvic acid in blood. Meanwhile, the accumulated pyruvic acid inhibits the activity of dehydrogenase and leads to the accumulation of lactic acid. Hence, vitamin B1 is essential for sugar metabolism. Vitamin B1 is of special importance for consumers with starch as the main energy source. It is reported that the amount of vitamin B1 required is propontional to the amount of carbohydrates consumed and it is recommended that 1 microgram of vitamin B1 is needed for the metabolism of 1 gram of carbohydrates. The early symptom of vitamin B1 deficiency includes nervous system disorder, mental and physical fatigue, dyspepsia and loss of appetite. If vitamin B1 cannot be supplemented in time, the patients develop polyneuritis or beriberi. In this stage, the patients become poor in heath status and other symptoms include lower limb edema, nerve paralysis, loss of muscle contraction capacity, and even death. (4) Stability and degradation. Vitamin B1 is the most unstable among all the vitamins identified and is susceptible to the influence of aw, pH, temperature, ionic strength, buffer solution and the presence of other compounds. The degradation of vitamin B1 is initiated by the nucleophilic substitution of the

Vitamins

203

methylene carbon between the two rings. Therefore, vitamin B1 can be readily destroyed by strong nucleophiles such as HSO3-. Vitamin B1 is stable at low aw and room temperature. For example, nearly no loss of vitamin B1 is observed when cereal foods with aw ranging from 0.1 to 0.65 are stored at 37°C. In storage temperature 45°C and aw 0.2~0.5, the decomposition of vitamin B1 increases. The maximum vitamin B1 degradation is observed in aw 0.5. As the aw further increases, the loss of vitamin B1 decreases, as shown in Figure 6-10. Temperature significantly affects the retention of vitamin B1 during storage (Table 6-8). In 1.5°C, no loss of vitamin B1 is detected in tomato juice, pea and orange juice after storage for 12 months. When the storage temperature is elevated to 35°C, up to 50% of vitamin B1 is lost. The relationship between vitamin B1 degradation rate and pH is shown in Figure 6-11. The same relationship is observed in either phosphate buffer or cereal. That is, the degradation of vitamin B1 increases with the rise of pH. Similar to other other-water soluble vitamins, rinsing and boiling cause great loss of vitamin B1. Table 6-9 lists the effect of various processing methods on the retention of vitamin B1 in various foods. The thermal degradation of Vitamin B1 can generate special flavor compounds, such as hydrogen sulfide, furan, and dihydro-thiophenol. These compounds impart foods with special flavor.

2.6. Vitamin B2 Vitamin B2 is also known as riboflavin and is found mainly in yeast, liver, milk, muscle and yolk. In addition, Greenery vegetables, grain and germinated seed also contain vitamin B2.

Figure 6-10. Relationship between vitamin B1 degradation rate and aw of cereal foods when stored at 45°C [9].

204

Yibin Zhou, Dongfeng Wang and Ping Dong Table 6-8. Retention of vitamin B1 in some foods during storage [10]

Species

Retention after storage for 12 months (%) 1.5°C 35°C

Species

Retention after storage for 12 months (%) 1.5°C 35°C

Apricot

35

72

Tomato juice

60

100

Mungbean Lima bean

8 48

76 92

Pea Orange juice

68 78

100 100

Table 6-9. Effect of treatment methods on the retention of vitamin B1 [11] Products

Treatment

Retention (%)

Cereal Potato

Extrusion, Cook Fried after immerse in water for 16 hr Fried after immerse in sulfurous acid solution for 16 hr Boiled in water or carbonate after immerse in water Various heat treatments Various heat treatments Various heat treatments

48-90 55-60 19-24 23-52 82-97 80-95 77-100

Soybean Smashed potato Vegetable Freeze or fried fish

Figure 6-11. Relationship between vitamin B1 degradation and pH.

Vitamins

205

(1) Structure. Riboflavin is the condensation product of D-ribitol and 7, 8-dimethyl-isoalloxazine. The structure of vitamin B2 is showed in Figure 6-12. (2) General properties. Riboflavin is an orange needle crystal and tastes bitter taste. This vitamin melts in 282 °C and sparingly soluble in water, but readily soluble in alkaline solutions. The aqueous solution of riboflavin generates yellow fluorescence in wavelength range 440~500nm. This property can be used to spectrometrically determine the content of the compound. Riboflavin is more thermal stable than vitamin B1. After dried yeast is heated in 120 °C for several hours, nearly all vitamin B1 is lost, but vitamin B2 is completely retained. Riboflavin is unstable in the presence of light and ultraviolet, especially in alkaline and high temperature conditions. In the presence of light, riboflavin in alkaline solutions is converted to lumiflavin. This product has the same color and fluorescence emission capability as riboflavin. In acidic or neutral solutions, riboflavin is transformed to lumichrome, which can emit blue fluorescence. The structures of the two compounds are illustrated in Figure 613. (3) Physiological function and deficiency. Vitamin B2 is an essential cofactor for enzymes involved in cellular respiration and dehydrogenation. Vitamin B2 deficiency leads to reduced respiration capacity and retarded metabolism. The symptoms of vitamin B2 deficiency include stomatitis, angular cheilitis, eye keratitis, and dermatitis. (4) Stability and degradation. Riboflavin is stable against thermal treatment and oxygen. Riboflavin is resistant to light in acidic solutions, but is readily decomposed by light in alkaline solutions. In light-induced decomposition, photochemical degradation occurs in ribitol to generate inactive lumiflavin and a series of free radicals (Figure 6-14). Lumiflavin has stronger oxidizability than riboflavin and its generation accelerates the destruction of other vitamins, especially Vc. The special off-odor of sunlight-exposed milk has been attributed to the light-induced decomposition of riboflavin.

Figure 6-12. Structure of vitamin B2.

206


Figure 6-13. Chemical structures of lumiflavin and lumichrome [12].

Figure 6-14. Photodegradation of riboflavin [12].

2.7. Pantothenic Acid Pantothenic acid (Vitamin B5) is a water soluble vitamin and occurs widely in foods, including yeast, liver, kidney, egg, muscle, skimmed milk, pea, peanut and sweet potato. The contents of pantothenic acid in unpolished rice, wheat, and corn are about 1.7 mg/100g, 1.01.5 mg/100g, and 0.46 mg/100g respectively. Intestinal bacteria and some plants can synthesize pantothenic acid. (1) Structure. The chemical name of pantothenic acid is N-(α, γ-dihydroxy-β, β-dimethyl-butyryl)- βalanine. Its structure is shown in Figure 6-15. (2) Properties. CH3 HOCH2

C

CH CO NH CH2

CH3 OH Figure 6-15. Structure of vitamin B5.

CH2

COOH

207

Vitamins 4 5 3 61 2

CONH2

COOH

N Nicotinic acid

N Nicotinamide

Figure 6-16. Structures of nicotinic acid and nicotinamide.

Pantothenic acid is light yellow and viscous. It is soluble in acetic acid and water, but insoluble in chloroform and benzene. In neutral solutions, pantothenic acid is resistant to heat, oxidation and reduction, but can be decomposed β-alanine and other compounds by acids, bases, and dry heating. The calcium salt of pantothenic acid is a colorless crystalline powder. Calcium pantothenate tastes slightly bitter and is water soluble. It is stable against light and air, but its aqueous solutions of pH 5~7 can be destroyed by heat. In organisms, pantothenic acid takes effect mainly in the form coenzyme A (CoA) form. (3) Physiological function. Pantothenic acid is the coenzyme of many enzymes involved in carbohydrate, lipid and protein metabolism and plays an important role in the transfer of the acetyl group. Pantothenic is also essential for the growth of some microorganisms. (4) Stability and degradation. Pantothenic acid is stable in neutral solutions, but is easily decomposed in acid solutions. In the pH range 6~4, its decomposition rate increases with the increase of acidity. Due to the heat liability and strong hygroscopicity of pantothenic acid, the calcium salt of pantothenic acid has been commercially used as an alternative. The changes of pantothenic acid contents in foods are affected by processing methods in addition to the materials. For example, the loss of pantothenic acid in pasteurized milk is usually less than 10%. The loss of pantothenic acid in vegetables is about 10-30 % and most of vitamin is lost during rinsing. The bioavailability of dietary pantothenic acid is about 51%.

2.8. Vitamin B3 Vitamin B3 is known as vitamin PP and is famous for its anti-pellagra capability. Two members of the family have been identified, including nicotinic acid and nicotinamide. Both te two compounds occur widely in yeasts, liver, muscle, milk, peanut, and soybean. The cortex and embryo of cereal grains also contain high levels of the two compounds. (1) Structure. Both nicotinic acid and nicotinamide are the derivates of pyridine and their structures are shown in Figure 6-16. (2) Properties. Both nicotinic acid and nicotinamide are colorless needle crystals. Nicotinic acid melts in 235.5-236°C. It is sparingly soluble in water but readily soluble in ethanol. The melting point of nicotinamide is 129-131°C and the compound is readily water soluble. Nicotinic acid is the most stable among all the vitamins identified and is not destroyed by light, air, heating, acids, or bases. Nicotinamide is converted to nicotinic acid in acidic solutions upon heating.

208


Figure 6-17. Oxidized and reduced states of nicotinamide.

Nicotinic acid can react with cyanogen bromide to yield a yellow-green compound. This reaction has been used for the quantitative analysis of nicotinic acid and nicotinamide. The double bond between C4 and C5 can be reduced. Hence, the two compounds can be present either in oxidized or reduced form. Nicotinamide implements its in vivo function as nicotinamide dinucleotide (NAD, also known as coenzyme I) or nicotinamide dinuclotide phosphate (NADP, CoII). NADP can be converted from NAD by phosphorylation with ATP. (3) Physiological functions and deficiency. Nicotinamide is a main component of CoI and CoII, which are the coenzymes of many dehydrogenases. Nicotinamide in the two enzymes occur either in the reduced and oxidized states and the transformation between the two states realize the transfer of hydrogen (Figure 17). Nicotinamide deficiency results in pellagra. The symptoms in the early phase include symmetrical dermatitis in hands, cheeks, foreheads and other naked areas, gastrointestinal disorders, mouth and tongue inflammation, indigestion and diarrhea. If nicotinamide is not supplemented in time, the patients might suffer neurological disorder and even death. (4) Stability and degradation. As mentioned previously, nicotinic acid is the most stable vitamin. However, nonchemical processing of vegetables, such as dressing and rinsing, can cause loss of this vitamin as other water-soluble ones. The loss of nicotinic acid of pork and beef during storage is attributed to biochemical reactions, roasting seems to have no effect the content of nicotinic acid. No loss of nicotinic acid is detected in processed diary products.

2.9. Vitamin B6 Vitamin B6 is also called adermine and includes pyridoxine, pyridoxal, and pyridoxamine. Vitamin B6 widely distributes in animals and plants. Wheat germ, rice bran, soybean, peanut, yeast, liver, fish and meat are the major storage pool of this vitamin. (1) Structure Pyridoxine, pyridoxal and pyridoxamine are the derivatives of pyridine and their structures are shown in Figure 6-18. (2) Properties. Pyridoxine, pyridoxal and pyridoxamine are colorless crystals and are readily soluble in water and alcohol. The three compounds are stable in acidic conditions, but are easily destroyed in alkaline conditions. Pyridoxine is heat tolerant, but the other two compounds are not.

Vitamins

209

Figure 6-18. Structures of pyridoxine, pyridoxal and pyridoxamine.

Figure 6-19. Structures of pyridoxal phosphate and pyridoxamine phosphate.

Figure 6-20. Structures of Schiff bases of pyridoxal and pyridoxamine [13].

Pyridoxine can be converted to pyridoxal or pyridoxamine and all the three compounds can be phosphorylated, of which, pyridoxal phosphate and pyridoxamine phosphate (Figure 6-19) are the major active forms of vitamin B6 and they are the coenzymes of amino acid decarboxylases and amino transferases. (3) Physiological functions and deficiency. Vitamin B6 is the coenzyme of amino acid decarboxylases and amino transferases and participates in many metabolism reactions. Long-term deficiency of vitamin B6 may cause skin irritation and damage of the central nervous system and hematopoietic organ. (4) Stability and degradation. The three members of vitamin B6 exhibit excellent thermal stability, of which, pridoxamine is the most stable and is selected in food fortification. In the presence of oxygen, vitamin B6 can be transformed to inactive 4-pyridoxine acid by UV irradiation. Vitamin B6 is easily degraded in alkaline solutions, but is stable in acidic conditions. Vitamin B6 can react with amino acids to produce Schiff base when heated (Figure 6-20). In acidic solutions, the formed Schiff base undergoes further dissociation. In addition, the Schiff base can rearrange to yield many cyclic compounds. Vitamin B6 suffers more or less loss during processing. For example, about a half of the vitamin is lost in sterilized liquid milk and the loss further increases during storage for 7~10 days. High temperature-short time pasteurization (92°C, 2~3s) causes only a loss of 30% in milk, but the values reaches up to 84% when sterilized in 119~120°C for 13 to 15 min.

210


2.10. Vitamin H Vitamin H is also called biotin and includes α- and β-biotin, of which, the former exists in yolk and the later in liver. Biotin widely distributes in plant and animal tissues. Most of vitamin H is bound with proteins and only a little portion occurs in the unbound state. Many organisms can synthesize biotin on their own. In human body, part of this vitamin is synthesized by intestinal bacteria. (1) Structure. Both the two members of biotin are thionic compounds and they are differentiated by their side chains, as shown in Figure 6-21. (2) Properties. Biotin is a colorless needle crystal and its melting point is 232~233°C. Biotin is soluble in hot water and ethanol, but is insoluble in ether or chloroform. It is stable against light, heating, acids, but can be destroyed by high temperatures and oxidants. (3) Physiological functions and deficiency. Biotin is the cofactor of many carboxylases. It is the carrier of CO2 in many CO2 fixation reactions. Because biotin can be synthesized intestinal microorganisms in addition to intake from foods, biotin deficiency occurs rather occasionally compared with other vitamins. The symptoms of biotin deficiency include dermatitis, muscle pain, hypersensitivity, fatigue, anorexia, and slight anemia.

2.11. Vitamin B11 Vitamin B11 is also called as folic acid or folate. Folic acid occurs widely in nature. Green leaves, liver, kidney, cauliflower, yeast, beef and kernels are found to have high contents. (1) Structure. Vitamin B11 is composed of pteridine, p-aminobenzoic acid and L-glutamate, as shown in Figure 6-22. (2) Properties.

Figure 6-21. Structure of biotin [14].

211

Vitamins


Folic acid is a bright yellow crystal and is sparingly soluble in water. It is easily destroyed by light in aqueous solutions. Caron atoms in positions 5, 6, 7, 8 can be reduced by NADP·H2, positions to yield tetrahydrogen folic acid (THFA). THFA is the carrier of many one-carbon groups, such as formyl, formaldehyde, and methyl and plays important role in the synthesis of proteins and nucleic acids. (3) Physiological function and deficiency. Folic acid is involved in the synthesis of proteins and nucleic acids. Its deficiency can cause blood diseases and the symptoms include pernicious anemia, glossitis and gastrointestinal problems. Because intestinal bacteria can synthesize folic acid, the incidence of folic acid deficiency is very low. (4) Stability and degradation. Dihydrofolate (FH2) and tetrahydrofolate (FH4) are easily oxidized in air and are sensitive to pH. The two compounds are the most stable in pH ranges 8-12 and 1-2. In neutral solution, FH4 and FH2, and folic acid are quickly oxidized to p-aminobenzoyl-L-glutamic acid (PABG), pteridine, xanthine, 6-methyl pterin and many other pterin derivatives. FH4 is oxidized more quickly in acidic solutions than in alkaline solutions and its oxidation products include PABG and 7,8-dihydro-pterin-6-carboxyl aldehyde. Reducing agents, such as thiol and ascorbic acid, can slow down the oxidation of FH2 and FH4. The stability of several derivatives of THFA follows the decreasing order of 5-formyltetrahydrofolate > 5-methyl-tetrahydrofolate > 10-methyl-tetrahydrofolate > tetrahydrofolate. Table 6-11. Loss of folic acid in some foods during processing [11] Foods Eggs Liver Atlantic plaice Cauliflower Carrot Meat Grapefruit juice Tomato juice

Corn Flour Meat or vegetables

Processing methods Oil fried or cooking Cooking Cooking Boiling Boiling γ-radiation Canned or storage Canned Storage in dark for 1 year Exposure to light for 1 year Refining Milling Canned storage for 3 years Canned storage for 5 years

Loss of folic acid activity (%) 18~24 None 46 69 79 None Negligible 50 7 30 66 20~80 Negligible Negligible

212


Figure 6-23. Structure of vitamin B12..

The loss of folic acid in foods varies with food species and processing technologies, as listed in Table 6-10.

2.12. Vitamin B12 Vitamin B12 contains cobalt and is therefore also called cobalamine. To present, at least five members of this family have been identified, but vitamins B12 sometimes refer solely to cyanoccbalamin. Liver has the highest vitamin B12 content, followed by milk, meat, egg, and fish. Vitamin B12 is not found in plants. Because only bacteria can synthesize vitamin B12, vitamin B12 in animal tissues derives either from foods or intestinal microbes. Vitamin B12 are bound to proteins in foods and are absorbed only after it is dissociated from the complex by heating or protease hydrolysis. (1) Structure. Vitamins of the B12 family are polycyclic compounds and contain one trivalent cobalt ion (Figure 6-23). According to the structure the R group, cobalamide is divided into cyanocobalamin (R = CN), hydroxocobalamin (R = OH), adenosylcobalamin (R = Ado), nitrosocobalamin (R = nitroso), methylcobalamin (R = CH3), etc. (2) Properties. The compounds of the B12 family are pink needle crystals and melt in temperatures higer than 320°C. The compounds are soluble in water, ethanol and propanol, but insoluble in chloroform. Vitamin B12 in either crystals or aqueous solutions (pH 4.5~5) are quite stable, but are readily destroyed strong acids/bases, sunlight, oxidants and reducing agents. (3) Physiological functions and deficiency.

Vitamins

213

Vitamin B12 is the coenzyme of many enzymes and participates in a variety of reactions. For example, it is the carrier of methyl and is involved in the biosynthesis of methionine and thymine. Besides, vitamin B12 increases the bioavailability of folic acid. Vitamin B12 synthesized by intestinal microbes must combine with the special mucoprotein (also known as endogenous factor) secreted by gastricism for its adsorption. Lack of the endogenous factor retard the absorption of vitamin B12 and causes pernicious anemia and lesions in nervous system, tongue and gastricism. (4) Stability and degradation. Vitamin B12 in aqueous solutions is stable in pH range 4~6 in dark environments and room temperature, but it is quantitatively destroyed in alkaline solutions upon heating. Reducing agents in low levels protect vitamin B12 from destruction, but those in high concentrations have opposite effects. Trivalent iron salts can stabilize vitamin B12, but bivalent iron salts rapidly destroy the vitamin.

2.13. Lipoic Acid Lipoic acid occurs either in the reduced and oxidized forms and their structures are shown in Figure 6-24). Lipoic acid, which is necessary for the growth of microorganisms and protozoa and dissolves in water, is generally classified as a vitamin B. Lipoic acid is rich in liver and yeast, and the human body can synthesize it. Lipoic acid is a coenzyme of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, plays the role as acyl transfer. Lipoic acid is water soluble. It is essential for the growth of microorganisms and protozoa and is generally considered a member of the B family. Human can synthesize lipoic acid on their own. Lipoic acid is the coenzyme of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and participates in acyl transfer.

2.14. Vitamin C (1) Structure. Vitamin C, or ascorbic acid, is an acid derivative of hexose. Vc occurs widely in fresh fruits and vegetables. Only L-ascorbic acid is bioactive. The structure of vitamin C is shown in Figure 6-25. (2) Properties. Vc is a colorless sliced crystal and melts in 190-192°C. It is soluble in water and ethanol and its aqueous solutions taste sour. Vc is unstable against heating, sunlight exposure, and metal ions such as such as Cu2+, Fe3+. However, it is quite stable in oxalic acid or metaphosphoric acid solutions. Vc is a strong reducing agent and can be reversibly oxidized to the dehydrogenated form (Figure 6-25). Vitamin C has been widely used as an antioxidant in the food industry. L-ascorbic acid can reduce the blue dye 2, 6-dichlorophenol-indophenol to the colorless leuco and react with 2,4-dinitrophenylhydrazine to yield colored hydrazone. These reactions can be used for the qualitative and quantitative analysis of Vc.

214


Figure 6-24. Structure of lipoic acid [15].

Figure 6-25. Structure of Vitamin C.

(3) Physiological functions and deficiency. Vc enhances the formation of supportive tissues and intercellular adhesion molecules. Vc functions as both hydrogen donor and receptor and plays important role in the redox reactions in human body. In addition, Vc is also involved in disease resistance and detoxification. Because human body cannot synthesis Vc, Vc deficiency causes a variety of symptoms, of which, the most notably symptom is scurvy. The symptoms in the early stage of Vc deficiency include local inflammation of skin, loss of appetite, dyspnea and general fatigue. In the later phase, the patients might suffer microvessel fracture in visceral, subcutaneous tissue, metaphyseal and gingiva and even death. (4) Stability and degradation. The stability of Vc depends largely on temperature, pH, oxygen, enzymes, metal ions such as Cu2+ and Fe3+, aw, initial concentration, and Vc to dehydrogenated Vc ratio. Although the nonenzymatic browning reaction of Vc occurs slowly in anaerobic conditions, Vc is degraded in the pathway shown in Figure 6-26 in weak acid and weak alkaline conditions. Diketo-gulonic acid can be further decomposed to produce a variety of compounds, including reducing ketones, furfural, and furan-2-carboxylic acids. In the presence of amino acids, Vc, dehydroascorbic acid, and their degradation products undergo the Maillard reaction, as shown in Figure 6-27.

R-CO-CO-COOH

Figure 6-26. Formation of diketo-gulonic acid through the degradation of Vc.

Vitamins

215

Figure 6-27. Vc browning pathway [16].

Figure 6-28. Relation of Vc content in cabbage and blanching time [17].

Vc has been used as natural antioxidant in foods. For example, because Vc can reduce oquinone compounds and thus inhibit enzymatic browning, it has been used as a bread improver. In addition, Vc can protect folic acid and other oxidizable compounds from oxidation. Vc is also an effective free radical scavenger. It can remove singlet oxygen, reducing oxygen and carbon-centered radicals, and regenerate other anti-oxidants such as copherol. The changes of Vc content during food storage in an indicator of food quality change. Because Vc is water soluble and is sensitive to heat, pH, and oxygen, processing and storage cause serious Vc loss in foods. Figure 6-28 shows the effects of heating time on Vc

216


content, Figure 6-29 indicates the retention of Vc in various processing methods, and Figure 6-30 illustrates the relationship between Vc destruction rate and aw.

Figure 6-29. Remained Vc content after various processes.

Figure 6-30. Relation between water activity and the rate of Vc destruction. O: Orange juice crystal; ●: △ Sucrose solution; : Corn and soybean mixture; □: Wheat flour.

Treatment of food materials with sulfur dioxide reduces the loss of Vc. Besides, sugar and sugar alcohols protect Vc from oxidative degradation, possibly due to their combination with ions.

217

Vitamins

3. CHANGES OF VITAMIN CONTENTS IN FOODS 3.1. Stability of Vitamins The contents of various vitamins in foods are affected by many factors, such as maturity of raw materials, growth environment, soil conditions, fertilizer and water management, illumination time and intensity, as well as post-harvest or post-slaughter handling. The properties of the vitamins have been reported by many publications; however, only few efforts have been made on the changes of the vitamins in complex food systems during storage and processing. The stability of various vitamins is summarized in Table 6-12.

3.2. Effect of Maturity on Vitamin Contents According to Table 6-13, the maturity of tomato significantly affects the content of Vc.

3.3. Change of Vitamins During Postharvest Processing and Storage The nutrient compositions in foodstuff change significantly after harvest or slaughter. Many enzymes, especially endoenzymes such as oxidase and hydrolase, are released from cells.

Table 6-12 Stability of vitamins [11] Items

Light

Oxidant

Reductant

Heat

Moisture

Acid

Base

VA

+++

+++

+

++

+

++

+

VD

+++

+++

+

++

+

++

++

VE

++

++

+

++

+

+

++

VK

+++

++

+

+

+

+

+++

VC

+

+++

+

++

++

++

+++

B1

++

+

+

++

++

+

+++

B2

+++

+

++

+

+

+

+++

B5

+

+

++

+

+

+

+

B6

++

+

+

+

+

++

++

B12

++

+

+++

++

++

+++

+++

Folic acid Biotin

++

+++

+++

+

+

++

++

+

+

+

+

+

++

++

Note: +; good stability; ++, moderate stability; +++, poor stability.

218

Yibin Zhou, Dongfeng Wang and Ping Dong Table 6-13. Contents of Vc in tomato as a function of maturity tomato [18]

Weeks after bloom

Color

Vitamin C content (mg%)

2

Average weight of a single fruit/g 33.4

green

10.7

3 4 5 6 7

57.2 102.5 145.7 159.9 167.6

green green-yellow red-yellow red red

7.6 10.9 20.7 14.6 10.1

These enzymes might change the chemical structure and activity of vitamins. For example, dephosphorylation of VB6, thiamine or riboflavin significantly affects the activities of the vitamins The most important factors that affect vitamin stability during storage and processing are temperature and time, because the activities of enzymes are directly relevant to temperature and reaction time. For example vitamin C oxidase specifically catalyzes the oxidation of vitamin C; the loss of beta carotenes in dehydrated food is 15% after storage for 10 days and the value reaches up to 98% after storage for 60 days. Foods undergo multiple reactions during storage and processing and these reactions significantly affect the sensory properties and vitamin contents of the foods. For example, lipid oxidation produces hydrogen peroxide and epoxide, which initiate the oxidation of carotenoid, tocopherol, and vitamin C; furthermore, hydrogen peroxide is decomposed to carbonyl compounds, which can react with thiamine, vitamin B, and pantothenic acid and lead to loss. In addition, carbonyl compounds generated in non-enzymatic browning of carbohydrates also partially contribute to the loss of some vitamins. The stability of vitamins is storage method dependent (Table 6-14).

3.4. Losses of Vitamins during Cereal Grinding Cereals are often grinded before further processing. Grinding generates heat and hence is a potential threat of vitamin stability. Figure 6-31 shows that effect of grinding time (indicated by flour yield) on the retention of various vitamins.

3.5. Losses of Vitamins During Rinsing and Blanching Rinsing and blanching causes the loss of water-soluble and heat-liable vitamins. For example, blanching at 100°C for 2 min leads to the loss of 65% of vitamin C in cabbage and vitamin C is completely lost after the 10 min of blanching. Steaming and boiling lead to different vitamin losses and water-soluble vitamins are better retained in steaming than in boiling (Table 6-15).

219

Vitamins Table 6-14. Losses of vitamins in different storage technologies [18] Methods

Frozen storage Storage after sterilization

Number of vegetable speciess 10(2) 7(3)

Loss rate (%) (1) Vitamin B2 Vitamin B5

Vitamin A

Vitamin B1

Vitamin C

12(4)

20

24

24

26

0-50 10 0-32

0-61 67 56-83

0-45 42 14-50

0-56 49 31-65

0-78 51 28-67

Note: (1) All vegetables were heated and dehydrated before storage. (2) Vegetables including asparagus, lima bean, kidney bean, brocoli, cauliflower, green pea, potato, spinage, cabbage and tender corn. (3) Vegetables treated with hot water including asparagus, lima bean, kidney bean, green pea, potato, spinage, and tender corn; (4) Average value.

Figure 6-31. Relationship between vitamins retention ratio and wheat flour yield [20].

Table 6-15. Retention of various vitamins in potato slices during steaming and boiling [21] Vitamins Vitamin C Vitamin B1 Vitamin B5 Vitamin B6 Folic acid

Boiling 60% 88% 78% 77% 66%

Steam 89% 90% 93% 97% 93%

220


3.6. Losses of Vitamins During Chemical Treatment Various chemicals are often added during the storage and processing of foods and these compounds affect the stability of some vitamins. For example, decolourants in wheat flour causes oxidation and consequently loss of some sensitive vitamins; sulfur dioxide, sulphites, bisulfite, and pyrosulfites are inhibitors of both enzymatic and non-enzymatic browning reactions and protect vitamin C from oxidation. Nitrite is added as a color fixative and preservative in bacons. This compound can react with vitamin C and destroy thiamine and folic acid.

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[6] [7] [8] [9] [10] [11]

[12]

[13]

[14]

Chandler, LA; Schwartz, SJ. HPLC separation of cis-trans carotene isomers in fresh and processed fruits and vegetables. Food Science, 1987, 52, 669-672. Pesck, CA; Warthesen, JJ. Kinetic model for photoisomerization and concomitant photo-degradation of ß-carotenes. Journal of Agricultural and Food Chemistry, 1990, 38, 1313-1315. Dellamonica, ES; McDowell, PE. Comparison of beta-carotene content of dried carrots prepared by three dehydrated process. Food Technology, 1965, 19, 1597-1599. Van Niekerk, PJ. Determination of vitamins, in HPLC in Food Analysis. 2nd ed. San Diego: Academic Press, 1988. Thompson, JN; Hatina, G. Determination of tocopherols and tocotrienols in foods and tissues by high performance liquid chromatography. Journal of Liquid Chromatography, 1979, 2, 327-344. Herbert, V. Present Knowledge in Nutrition. 6th edition. Washington DC: International Life Science Institute – Nutrition Foundation, 1988. Machlin, LJ. Vitamin E in commercial processing on nutrients. In Machlin LM. Handbook of Vitamins. 2nd ed. New York: Marcel Dekker, 1991 Cort, WM; Borenstein, JH. Nutrient stability of fortified cereal products. Food Technololgy, 1976, 30, 52-62. Dennison, DJ; Kirk, J. Storage stability of thiamin and riboflavin in a dehydrated food system. Journal of Food Processing and Preservation, 1977, 1, 43-54. Freed, MS; Brenner, S; Wodicka, VO. Prediction of thiamin and ascorbic acid stability in canned stored foods. Food Technology, 1949, 3, 148-151. Emilia, L; Jana, K. Vitamin losses: Retention during heat treatment and continual changes expressed by mathematical models. Journal of Food Composition and Analysis, 2006, 19, 252–276. Woodcock, EA; Warthesen, JJ. Riboflavin photochemical degradation in pasta measured by high performance liquid chromatography. Food Science, 1982, 47, 545555. Weisinger, H; Hinz, HJ. Kinetic and thermodynamic parameters for Schiff base formation of vitamin B6 derivatives with amino acids. Archives of Biochemistry and Biophysics, 1984, 235, 34-40. Bonjour, JP. Biotin. In: Machlin LJ, ed. Handbook of Vitamins. 2nd edition: New York: Marcel Dekker, 1991.

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[15] Flavia. NI. Lipoic acid: a unique antioxidant in the detoxification of activated oxygen species. Plant Physiology and Biochemistry, 2002, 40, 463–470. [16] Tannenbaum, S. Ascorbic acid. In: Fennema, O. (Ed.), Principles of Food Science Part I Food Chemistry. Marcel Dekker, New York, 1976, 477–544. [17] Plank, R. Handbuch der Kältetechnik. Bd. XIV. Berlin: Springer-Verlag, 1966. [18] Maleski, W; Markakis, P. A research note. Ascorbic acid content of developing tomato fruit. Journal of Food Science, 1971, 36, 537-539. [19] Lund, D. Nutritional Evaluation of Food Processing. 3rd editon. New York: Van Nostrand Reihhold, 1988. [20] Moran, R. Biotin. Present Knowledge in Nutrition. International chemical form. Nutrition, 1959, 122, 533-545. [21] Zhao, H. The loss of vitamin in the process of cooking and food processing. Foreign medical and hygiene anthology, 2003, 30, 221.



Chapter 7

MINERALS Dongfeng Wang1, Lina Yu2, Haiyan Li1, Bin Zhang1, Shuhui Wang3 and Xingguo Liang1 1

College of Food Science and Engineering, Qingdao, China 2 Shandong Peanut Research Institute, Qingdao, China 3 Biosystems Engineering Department, College of Agriculture - Ginn College of Engineering, Aubum University, Auburn, AL, US

ABSTRACT Among the 92 natural chemical elements identified to present, 81 of them have been found in human body and 25 elements are found to be essential to life, which are termed essential minerals. Essential minerals are widely involved in metabolisms in human body as cofactors or activators of enzymes and some play important role in maintaining osmotic pressure and cell membrane integrity. Besides, some minerals are important constituents of human tissues, such as calcium and phosphorus in bone and teeth. This chapter concerns the classification, distribution, property, and function of minerals and their occurrence in foods. Besides, the changes of minerals in foods during harvesting, processing, and storage are also highlighted in this chapter.

1. INTRODUCTION 1.1. Definition and Classification To present, 115 chemical elements have been discovered, including 23 artificial and 92 natural ones. Of the 115 elements, 81 have been discovered in human bodies. According to their importance to human body, elements are divided into 3 classes: Essential elements Essential elements support the normal biochemical processes of human body. Deficiency of elements of this class leads to impaired functions of human body and the functions can be

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restored in the early stage of the deficiency if the elements are supplemented in time. Essential elements have special physiologic functions and cannot be replaced by other minerals. To present, 29 elements have been found essential for the human body, including oxygen (O), carbon (C), hydrogen (H), nitrogen (N), calcium (Ca), phosphorus (P), potassium (K), sodium (Na), chlorine (Cl), sulfur (S), magnesium (Mg), ferrum (Fe), fluorine (F), zinc (Zn), cuprum (Cu), vanadium (V), stannum (Sn), selenium (Se), manganese (Mn), iodine (I), nickel (Ni), molybdenum (Mo), chromium (Cr), cobalt (Co), bromine (Br), arsenic (As), silicon (Si), boron (B), and strontium (Sr). The former 11 elements account for up to 99.95% of the total amount of the 29 elements in human body and are termed main or macro elements. The remaining 18 elements are trace elements and account for only 0.05% of the total amount. Potential beneficial elements or supplementary elements Elements of this class are beneficial to the physiological activities of human body when their contents are in normal levels. However, the elements are toxic when they are present in high levels. Rubidium (Rb), aluminum (Al), niobium (Nb), zirconium (Zr), lithium (Li), and some rare earth elements (REEs) belong to this class. Toxic elements Toxic elements are also referred to as contamination elements or toxic trace elements. These elements exert their toxicity even in very low concentrations. Bismuth (Bi), stibium (Sb), beryllium (Be), cadmium (Cd), mercury (Hg), plumbum (Pb), and thallium (Tl) are important toxic elements. The elements except C, H, O, and N are termed minerals. Based on the contents in foods, minerals are divided into main minerals, trace minerals, and ultra-trace minerals. Main minerals include Na, K, Ca, P, Mg, S, and Cl; trace minerals include Fe, F, I, Zn, Se, Cu, Mn, Cr, Mo, Co, and Ni; and ultra-trace minerals include Al, As, Ba, Bi, B, Br, Cd, Cs, Ge, Hg, Li, Pb, Rb, Sb, Si, Sn, Sm, Ti, Sr, Tl, W, and V.

1.2. Distribution of Minerals in Human Body The toxicity of main elements arranged from left to right in the same period and from top to bottom in the same group in the element periodic table increases with their atom number (Figure 7-1). Therefore, the argument that the effect of an element on the biological eukaryotic cells closely relate to the periodic law of the elements is proposed. The study on the subgroup elements shows that the change of their optimal concentration stimulated cells growth with their abundance in the seawater is established (Figure7-2). Figure 7-1 shows that all the 11 main elements have atom number less than 20 and they are mainly distributed on the top of the S and P areas in the element periodic table. The essential trace elements have the tendency to be at the position of the fourth period, especially at the d and ds areas. Most toxic elements except Be and Ba lie in the bottom of the P area. The majority of essential trace elements are in the top of the element periodic table and their atom numbers are less than 35 except I and Mo. Almost all the toxic elements are located in the lower part of the element periodic table, especially in the fifth and sixth period.

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The distribution of minerals in the environment and human body is displayed in Figure 72. Living organisms have evolved regulatory measures to maintain essential mineral contents in normal ranges. That is to say, a living organism can maintain a mineral in a constant range when the element intake is insufficient or excessive.

Figure 7-1. Distribution of macro and trace elements in the element periodic table [8].

Figure7-2. Distribution of elements in the seawater (upper), human body (middle) and crust (lower). (Data in graph are log c; the unit of c is mg/kg) [5].

Table 7-1. Functions of main minerals [4] Mineral B F Fe Zn I Cu Se Mn Mo

Functions Growth promotion and essential for plant growth Closely correlated with bone growth Component of hemoglobin, myoglobin and cytochrome Involvement in enzyme activity, nucleic acid structure and protein synthesis Component of thyroxine Cofactor of many metalloenzymes Component of glutathione peroxidase and participation in liver function and muscle metabolism Activation of enzyme and participation in hematopoiesis Component of molybdoenzymes

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Dongfeng Wang, Lina Yu, Haiyan Li et al. Table 7-1. (Continued)

Mineral Cr Mg Si P Co Ca S K Na Cl

Functions Effect of insulin intensive and glucose utilization promotion Activation of enzyme and component of bone Participation in skeletogeny Component of ATP Component of VB12 Component of bone and involvement in neural transmission Component of proteins Electrochemical and messenger function and extracellular cations Electrochemical and messenger function and extracellular cations Electrochemical and messenger function and extracellular anions

1.3. Functions of Minerals in the Living Body Table 7-1 lists the functions of main minerals for living organisms. Minerals play important roles in the human body and their functions often involve complex mechanisms. The functions of the minerals are affected by their interactions (such as coordination and antagonism) with other food constituents and the compounds in human body. For example, P and Ca mutually affect their absorption and their absorption rates are the highest when the two minerals are present in molar ratio 1:1 in foods. Fe increasingly antagonizes the absorption of Zn in Fe/Zn ratios range from 1:1 to 22:1 in diets and Zn reduces the absorption of Cu. Many researches have revealed that both deficiency and imbalance of minerals cause a number of abnormalities in living organisms. In contrast to most organic constituents, minerals cannot be synthesized in the human body and must be ingested from foods and drinking water. Besides, minerals cannot be metabolized and are repelled from the body by excreting. Therefore, dietary structure significantly influences the contents of minerals and their proportions in the body.

2. THE EXISTENCE STATES OF MINERALS IN FOODS Minerals in foods can be present in the dissolved or non-dissolved, colloidal or noncolloidal, organic or inorganic state, ionic or non-ionic state, or complexed and noncomplexed state. The existence states of minerals significantly influence their bioavailability and the safety of foods. For instance, iron in heme is absorbed much easier than other iron forms and its bioavailability is much less affected by other factors in diets, including iron absorption inhibitors. The absorption of minerals in foods is also affected by the presence of other food components. For example, vitamin C enhances the absorption of iron, but phytic acid and polyphenols inhibit iron absorption. The toxicity of toxic elements, such as Cd, Pb, and Hg, is affected by the presence other constituents in foods. For example, vitamin C decreases the toxicity of Cr6+ by reducing it to Cr3+; phytic acid, polyphenols, and proteins reduce the toxicity of Pb and Cd by complexing with them. The toxicity of heavy metals can be antagonized by other minerals. Zinc is the

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metabolic antagonist of Cd, competing with Cd for the thiol of metallothionein. Pb is more poisonous in the absence of Fe and Cr in the diet; Se can complex with Hg and reduce Hg toxicity. The valence significantly influences the toxicity of metals. As3+ is much more toxic than As5+; methyl arsenic acid and dimethyl arsenic acid are moderately toxic, while arsenobetaine and arsenocholine are almost non-toxic. Cr3+ is an essential trace element, while Cr6+ is toxic and Cr2O72- is a strong carcinogen. Hence, in evaluating the bioavailability and safety of a mineral in foods, its existence state must be taken into consideration. Generally, only very few minerals are present as free ions in foods.

2.1. Combination with Monosaccharides and Amino Acids According to the Lewis acid-base theory, metals are Lewis acids which can provide empty orbitals. Small molecules that are rich in N, S, and O atoms, such as sugars, amino acids, nucleic acids, chlorophylls, and hemoglobins, contain lone pair electrons and can act as Lewis bases. Therefore, minerals can form complexes with above-mentioned biomolecules. α-Amino acids are bidentate ligands and bond to metal ions via an oxygen of the carboxylic acid group and the nitrogen of the amino group to form a five-membered ring, as shown in Figure 7-3. Under certain conditions, the side chains of amino acids also participate in the coordination. In addition to carboxyl terminal peptide and certain groups of amino acid side chains, the carboxyl in peptide and imino groups are also involved in coordination [10]. O

O C

H N

H2 C

CH2

Zn N H

O

C O

Note: Left: Zn(Gly)22H2O, Midmost Glycine tripeptide mineral complex, M = Cu (II) or Ni (II); Right: Glycine tetrapeptide cupric complex. Figure 7-3. Structures of mineral-peptide complexes.

Both plant- and animal-derived foods contain a large number of monosaccharides, carbohydrate derivatives and amino acids. As long as the adjacent hydroxyls in sugars are arranged in favorable spatial configurations, coordination with metals occur readily. For example, the three hydroxyl groups on pyranose in the axial-transverse-axial configuration and the three hydroxyl groups on furanose in the cis-cis-cis structure favor the coordination with divalent or trivalent minerals. The presence of carboxyl and imino groups in carbohydrates increases the stability of their complexes with minerals by several orders of magnitude. Yunlan Su, et al. determined the molecular structure of the lanthanum-galactose acid complex by infrared spectroscopy and found two complexes with different structures. Iron can generate multi-core complexes with glucose, fructose and other monosaccharides. The osamines generated during the Maillard reaction, such as glycosyl amines and glucosyl amines, can react with minerals to form stable complexes. Nagy L [6] et al found that the Amadori isomer formed during the Maillard reaction between glucose and amino acids at high temperatures is a more stable mineral ligand. Structural analysis of complexes formed between Cu(II) and aminosaccharide reveals that all the aminosaccharides are the binary ligands, in which -NH2 is the main coordination group

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for Cu(II) and other divalent minerals and the -OH group plays less role in coordination with Cu(II). The –OH group on 1-C of GlcNH2GalNH2 is the main coordination group for divalent minerals, but if the -OH on 1-C is not available due to steric hindrance, such as that in aminosaccharide derivatives, other –OH groups participate in the coordination. Nagy L et al. [6] studied the structural parameters of carbohydrate-mineral complexes by EXAFS (extended X-ray absorption fine structure spectroscopic) and the results are given in Table 72. It could be seen that minerals in foods can form various complexes with carbohydrates and their derivatives. Table 7-2. Structural parameters of carbohydrate-mineral complexes Ligands Fru

Rib

GlcNH2

Adenosine

Uridine

HyA

N-D-glugly

N-D-glugly N-D-glugly

PHTAc

PHTAc

Binding Interaction Fe-O Fe…C Fe…Fe Cu-O(eq) Cu-O(ax) Cu…C Cu-O(eq) Cu-O(eq) Cu-O(ax) Cu…C Cu-O(eq) Cu-O(ax) Cu…C Cu-O(eq) Cu-O(ax) Cu…C Cu-O(eq) Cu-O(ax) Cu…C Zn-O Zn…C Cu-O(eq) Cu-N(eq) Cu-O(ax) Cu…C Cu…Cu Ni-O,N Ni…C Co-O,N Co…C Co…Co Zn-O,N Zn…C Zn…O,C,S Mn-O,N Mn…C Mn…O,C,S

N 6 4 1 4 2 2 3 1 2 2 4 2 2 4 2 2 4 2 2 4 2 2 2 2 4 1 6 6 6 6 1 4 6 6 6 6 8

γ/pm 195 277 310 191 230 271 193 193 234 274 191 232 275 192 234 279 191 234 313 202 299 190 190 215 270 297 204 285 200 290 303 205 290 286 216 305 375

σ/pm 9.8 7.9 7.0 7.5 13.0 10.0 7.7 7.7 13.0 10.0 5.4 7.8 8.4 7.9 7.9 9.6 8.2 3.2 13.0 8.1 13.2 5.6 5.6 2.5 2.8 11.4 7.6 5.4 11.9 11.7 7.6 8.0 10.0 14.0 9.5 11.0 18.0

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Minerals Ligands PHTAc

PHTAc

LacA Mal GlcA Sacc Mal GlcA

Binding Interaction Ag-N Ag-S Ag…C Ni-O,N Ni…C Ni…O,C,S Mn(IV)-O Mn(IV)-O Mn(IV)-O Mn(III)-O Mn(III)-O Mn(III)-O

N 1 1 4 6 6 8 6 6 6 6 6 6

γ/pm 203 230 294 203 284 390 208 208 209 209 206 208

σ/pm 5.0 4.6 19.0 8.0 9.5 15.0 2.3 3.5 1.9 3.6 1.0 3.3

Note: N, coordination number; γ, atomic distance; σ, Debye-Waller factor; Fru, D- Fructose; Rib, DRhamnose; GlcNH2, 2-amino-2-deoxy-D-Glucose; HyA, Hyaluronic Acid; N-D-glugly(N-Dgluconylglycine, a pseudopeptide derivative glucono-delta-lactone and glycine); PHTAc, [2(polyhydroxyalkyl)thiazolidine-4-carboxylic acid]; LacA, D-lactobionic acid; Mal, maltitol 4-O-ζD-glucopyranosyl-D-glucitol; GlcA, D- Gluconate; Sacc, D- Sucrose; Ara, L- or D- Arabinose.

2.2. Combination with Oxalic Acid and Phytic Acid Oxalic acid in plant-derived foods is an important mineral chelator. When oxalic acid and phytic acid are present in high levels in foods, the bioavailability of some essential minerals and the toxicity of some toxic elements will be significantly reduced. Phytic acid can form insoluble complexes with Ca, Fe, Mg, Zn and many other minerals. Besides, phytic acid is also able to form ternary complexes with proteins and minerals. The reaction not only reduces the bioavailability of proteins, but also weakens the absorption of minerals. About 10% of P in vegetables cannot be absorbed by bodies because of its combination with phytic acid. In cereals, the percentage is as high as about 40% and even up to 90% in some species, as shown in Table 7-3.

2.3. Combination with Nucleotides The biological functions of certain nucleotides are closely related to minerals. For example, in the presence of Mg, ATP can be hydrolyzed to ADP and phosphate. All the three components in nucleotides have the ability to coordinate with minerals and their coordination capacities decrease in the following order: bases > phosphate groups > pentose. Bases coordinate with minerals through N-3 of pyrimidine and N-7 of purine base. Ca2+, Mg2+, Cu2+, Mn2+, Ni2+ and Zn2+ are common coordination minerals for nucleotides. In the case of coordination with ATP, Ca2+ and Mg2+ bond only to the phosphate groups, whereas Cu2+, Mn2+, Ni2+ and Zn2+ combine with both phosphate groups and N-7 of adenine. The stability constants of ATP-divalent minerals follow the following order: Cu2+ > Zn2+ > Co2+ > Mn2+ > Mg2+ > Ca2+ > Sr2+ > Ba2+ > Ni2+. NMR and Raman spectroscopy reveal that the molar ratio of the Mg2+-ATP complex is 1:1 (Figure 7-4). 1H-NMR and 31P-NMR indicate that Cu2+ binds to the N-7 of purine or the N-3 of pyrimidine on ATP. Cu2+, Co2+, Ni2+ and Cd2+ coordinate with the phosphate and N-7 of adenine in ATP in two ways, with the resultant structures termed macrochelated inner sphere (Figure 7-5A) and

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macrochelated outer sphere (Figure 7-5B) respectively. In the former structure, N-7 of adenine directly binds to the mineral, but the α-phosphate group coordinates through H2O. In the later structure, N-7 of adenine coordinates with mineral through H2O, whereas the α, β, γphosphate groups directly link with the mineral (Figure 7-5). Table 7-3. Content of phosphorus coordinated with phytic acid in plant food Food

Oat Wheat Barley Rye Rice Corn Peanut

Phosphorus coordinated with Phytic Acid mg/100g % 208~355 50~88 170~280 47~86 70~300 32~80 247 72 157~240 68 146~353 52~97 205 57

Figure 7-4. Complex of Mg with ATP [4].

a)

Figure 7-5 (continued)

Food

Potato Kidney Bean Carrot Orange Lemon Walnut Soybean

Phosphorus coordinated with Phytic Acid mg/100g % 14 35 12 10 0~4 0~1 295 91 120 81 120 24 231~575 52~68

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b)

Figure 7-5. Two simplified structural models of (ATP) 2-Chelate inner ring (A) and external ring (B) coordination sphere (M indicates a mineral ion) [7].

2.4. Combination with Ring Ligands Minerals can complex with planar ring ligands in addition to low molecular weight carbohydrates, amino acids and peptides mentioned above. Take porphyrins as example. Porphyrins are the derivatives of porphine consisting of four pyrrole rings connected by four carbon atoms (Figure 7-6a, b). When H atoms in numbered positions of the porphine ring are replaced by other groups, porphyrins are generated (Table 7-4). a)

b)

Figure 7-6. Structure of porphine. In (a) and (b), the structure is numbered in different ways.

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Dongfeng Wang, Lina Yu, Haiyan Li et al. Table 7-4. Important Porphyrins Porphyrins

Substituents 1 2 3 4 5 6 7 Protoporphyrin IX M V M V M P P Mesoporphyrin IX M E M E M P P Deuteroporphyrin IX M H M H M P M Hematoporphyrin IX M B M B M P P Spirograhic porphyrin IX M F M V M P P Corproporphyrin III M P M P M P P Aetioporphyrin III M E M E M E E Uroporphyrin III A P A P A P P Note: A=-CH2COOH, B=-CH (OH) CH3, E=-C2H5, F=-CHO, M=-CH3, H=-H, P=-CH2CH2COOH, V=CH=CH2.

Porphyrins can form complexes with Fe2+, Fe3+, Zn2+, Co2+, Cu2+, Mg2+ and other mineral ions, such as Fe in hemoglobin and Mn in chlorophyll. Heme (Figure 7-7) consists of an iron atom and a planar porphyrin ring. The iron in the center is connected with the N-atoms of four pyrrole rings through coordination bonds, and the fifth coordination point is provided by a histidine residue in globin, and the remaining sixth coordination point comes from negatively charged atoms in other ligands, such as H2O. Heme can connect with globin to form myoglobin (Figure 7-8). Three types of hemes, namely heme a, heme b and heme c, have been found. Heme a is the prosthetic group of cytochrome c oxidase; heme b is an iron-protoporphyrin IX complex (Table 7-4) and it is the prosthetic group of hemoglobin, myoglobin, cytochrome b, cytochrome p-450, catalase and peroxidase; heme c is the prosthetic group of cytochrome c.

Figure 7-7. Structure of heme.

Chlorophyll is a green pigment in the higher plants and photosynthetic organisms. It has a planar molecule and consists of four pyrrole rings connected through the methylene bridge. The Mg in center is connected with the N-atoms of four pyrrole rings through coordination bonds (Figure 7-9). Many chlorophyll species have been identified, such as chlorophyll a, b, c and d, as well as bacteria chlorophyll and chlorobium chlorophyll. Chlorophyll a and b occur widely in plant-derived foods in the ratio 3:1. Chlorophyll is transformed to pheophytin during food processing. In acidic conditions, the Mg atom in chlorophyll is replaced by

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hydrogen atoms and the resultant pheophytin is dark olive-brown. Heating accelerates the transformation. In addition to hydrogen, Mg can also be replaced by other divalent minerals, such as zinc and copper.

Figure 7-8. Structure of myoglobin.

Figure 7-9. Structure of chlorophyll.

Vitamin B12 consists of a corrin ring and has four pyrrole subunits. Because cobalt is essential for its bioactivity, Vitamin B12 is also known as cobalamin. Vitamin B12 is a red crystal and its systematic name is α-(5,6-dimethylbenzimidazolyl)cobamidcyanide. In the structure, the cobalt atom combines with four inner nitrogen atoms in the corrin ring. If the sixth coordination site is replaced by cyanide, VB12 is transformed to cyanocobalamin; if the cyanogroup connected with cobalt is replaced by the hydroxyl group, VB12 is converted to hydroxocobalamin, which is the ubiquitous form of VB12 in nature. If the cyano group is replaced by the nitroso group, nitriocobalamin is generated. Nitriocobalamin is found mainly in some bacteria. The structure of Vitamin B12 is given in Figure 7-10.

2.5. Combination with Proteins Protein consists of amino acids. In addition to peptide bonds, terminal amino groups and carboxyl groups, as well as some side groups of amino acid residues, such as the hydroxyl group in serine and threonine, the phenolic hydroxyl group in tyrosine, the carboxyl group in

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acidic amino acids, the amino group in basic amino acids, the imidazolyl group in histidine, the sulfhydryl group in cysteine, and the thioether group in methionine, can coordinate with minerals. However, only the groups in suitable sites can form complexes with minerals.


Figure 7-13. schematic diagram of Zn2+ in carboxypeptidase A [3].

Figure 7-11 is the schematic diagram of carboxypeptidase A. Zn2+ coordinates with the protein constituent through two imidazole groups and a carboxyl group in the protein. Metals are involved in the activity of a large number of enzymes and such enzymes are known as metalloenzymes. Zn, Fe, Cu, Mn, Mg, Mo, Co, K, and Ba are the most important cofactors for such enzymes. Living organisms synthesize a large variety of proteins for the storage and transport of minerals.

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Ferritin Ferritin is mainly distributed in the spleen, liver and bone marrow of animals, as well as in plants chloroplasts and certain fungi. Its main physiological function is to store unused or excessively absorbed iron. Transferrin Transferrin is mainly distributed in body fluids and cells of vertebrates. A large variety of transferrins have been identified, such as serotransferrin in serum and lactotransferrin in milk and lacrimal gland secretion. Serotransferrin is a class of mineral-binding glycoproteins with molecular weight about 7.7~7.4 × 104. After the transferrin releases all Fe3+ irons, the protein constituent can combine with other divalent or trivalent ions other than iron, such as Cu2+, Zn2+, Cr3+, Mn3+, Co3+, and Ga3+. Iron-sulphur protein Iron-sulphur proteins are characterized by the presence of the Fe-S chromophore and their molecular weight is about 10 kD. All the Fe atoms in the proteins have variable valence. The proteins participate in redox reactions in organism as the electron transfer group and are involved in biological oxidation, nitrogen fixation and photosynthesis. Cuprein Cupreins are copper-containing proteins and superoxide dismutase is a typical example of such proteins. To present, more than forty kinds of cupreins have been found. Cupreins with blue color are called blue copper proteins and those without the blue color are called nonceruloplasmins. Minerallothionein Metallothionein (MT) was firstly separated from horse kidney in 1957. Later, it was found that MT is widespread in nearly all organisms. MTs are a category of induced protein with a molecular weight about 6~10 kD. MTs are characterized by their high Cys contents, reaching up to 25~35%. The functions of MTs include anti-oxidation, free radicals scavenging, heavy metal complexing, and mineral storage. The structures of MTs are highly conserved. Many minerals, such as Cd, Pb and Zn, can induce the synthesis of MTs. One of the most important functions of MTs is to the alleviate toxicity of heavy metals. The affinity of MTs to some heavy metals follow the order Cd2+ > Pb2+ > Zn2+. Because the MT level is positively correlated with contaminant level, it is widely used as an indicator of environmental pollution. Capasso, C. et al [1] determined the three-dimensional structure of MT-nc obtained from the Antarctic fish (Notothenia coriiceps). NMR analysis showed that MT-nc is composed of the α- and β- domains. The α-domain in the C-terminal consists of eleven Cys residues and four mineral atoms, and the β-domain in the N-terminal has nine Cys residues and three mineral atoms (Figure 7-12). The ninth cystine of MT-nc in the α domain is different from that of mammals.

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The terminal amino acid sequence of mammal-derived MTs is CXCC (C denotes Cys, X denotes any amino acid), while the sequence of fish MT is CXXXCC. This difference attributes to the different structures of their α domains. Except few exceptions, the N-terminals of mammal-derived MTs are acetyl Met, and the carboxyl-terminals are Ala. Each MT molecule contains 20 Cys residues and the Cys residues are in the same positions. The Cys residues can form five Cys-X-Cys units, one Cys-Cys-XCys-Cys unit and one Cys-X-Cys-Cys unit. Although the amino acid compositions of MTs in different animal are quite similar, the MTs differ in the metal binding capacity. Generally, each MT molecule can bind seven mineral ions and the affinities of some metals to MTs follow the increasing order Zn2+ < Pb2+ < Cd2+ < Cu2+ < Ag+ < Hg+. a)

b)

a b

The main chain composite structure of 31-60 residues in α domain; The main chain composite structure of 2-28 residues in β domain.

Figure 7-12. NMR diagram of MT-nc.

Phytochelatins Phytochelatins are firstly found in plants and have similar metal binding capabilities as MTs. It is estimated that more than 90% of Cd2+ in plant cells are bound with phytochelatins. The general formula of phytochelatins is (γ-Glu-Cys)n-Gly. Phytochelatins are major heavy metal binding peptides in plants and some yeast species. The biosynthesis of phytochelatins is induced rapidly after exposure to heavy metals, especially Cd2+ and Hg2+. Heavy metals in living organism are present in both the soluble and insoluble forms, in which, soluble heavy metals bind mainly to phytochelatins. Phytochelatins from different crops have the following common characteristics: (1) the molecules have similar structure, as

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Minerals

shown in Figure 7-14; (2) the Glu residue locates in the N-terminal; (3) the amino acid connected with Glu-γ-oxo is Cys; (4) the γ-Glu-Cys unit occurs repeatedly in the structure.

Figure 7-14. Structure diagram of phytochelatins.

2.6. Complexation with Polysaccharides Polysaccharides can complex with metals through thiol, amino, and carboxyl groups in addition to hydroxyls. Complexation with metals enhances the biological functions of polysaccharides and provides an effective way for removing harmful minerals. Polysaccharide chains contain a large number of groups for coordination with metals, such as pectin and alginate of the plated ribbon-type conformation (Figure 7-15). The conformation of polysaccharides significantly affects the metal-binding capability. In the case of alginate, Ca2+ facilitates the maintenance of the egg-box conformation, as shown in Figure 7-16.

Figure 7-15. Plated ribbon-type conformation of pectin and alginate.

Ca

Ca

Figure 7-16. Sketch map of egg box conformation.

Ca

Ca

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3. PHYSICOCHEMICAL PROPERTIES, TROPHISM AND SAFETY OF MINERALS IN FOOD 3.1. Physicochemical Properties 3.1.1. Solubility The transfer and metabolism of most minerals in all biological organisms occur in aqueous solutions. Therefore, the bioavailability and activity of minerals in foods, to a great extent, depend on their solubility in water. Mg, Ca and Ba are in the same group (II A) and their halogenides are readily soluble in water. However, their hydroxids, carbonates, phosphates, sulphates, oxalates and phytates, are sparingly soluble. Besides, the presence of phytic acid significantly reduces the bioavailability of some minerals, such as Fe, Zn, Ca, Mg and Mn. The solubility of minerals is influenced by pH and other constituents in foods. Generally, minerals are present in the dissolved state in pH below 6 and their complexation with proteins, amino acids, organic acids, nucleic acids, nucleotides, peptides and carbohydrates markedly improve their solubility and bioavailability. Harmful minerals can be removed by the formation of insoluble complexes. For example, citric acid is sometimes used as a chelator in lead poisoning treatment. 3.1.2. Oxidability and Reducibility Minerals might occur in multiple valences in foods and their valences are subject to changes due to the presence of other food components. The valence of a metal largely affects its significance for human body. For example, Fe2+ is beneficial for the biological process of living organisms, but Fe3+ are toxic; Cr2+ and Cr3+ are not toxic or only moderately toxic, but Cr6+ is a well-known strong carcinogen (LD50 6~8 g). Cr6+ in blood stream can be oxidized by oxygen to chromium oxide and transform hemoglobin to methemoglobin, leading to decreased oxygen level in blood. Inhale of a small amount of chromic salt or chromic acid (+6) cause great pathological changes in the kidney, liver, nervous and blood. 3.1.3. Activity The reactivity of an ion in biochemical reactions depends on its activity instead of its concentration. The definition of the activity of minerals is as follows: ai= fi·Ci where: ai denotes the activity of the ion, fi denotes the activity coefficient, and Ci denotes the ion concentration. The activity coefficient (fi) increases as the ion concentration decreases. No method has been developed to determine the accurate activity of minerals in foods. Because the ion activity is positively correlated with ion concentration, the ion activity can be presented by ion concentration. Indeed, all mineral-involved biochemical reactions are related to the concentrations, states, valence state, dietary patterns, and so on (Figure 7-18).

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239

Figure 7-18. Schematic relationship between biological activity and relative concentration of minerals.

3.1.4. Chelating Effect Most metal ions in foods are present as complexes by coordinating with organic molecules. According to Werner‘s coordination theory, coordination compounds are divided into two parts. Take [Cu(NH3)4]SO4 as an example. Cu(NH3)42+ is defined as the internal limiting ion, Cu as the central ion, NH3 as the ligand, and SO42- as the external ion. In foods, most internal limiting ions are transition elements, such as Fe, Co, Mg, and Cu. The central ions are connected to certain atoms on ligands, such as O, N and S, which are called coordination atoms. C, H, VA group elements, and VIA group elements are important coordination atoms. A chelate complex is characterized by the presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central atom. Chelate complexes are contrasted with coordination complexes composed of monodentate ligands, which form only one bond with the central atom. Many minerals exert their functions by chelating or coordination with organic ligands. For example, Fe in heme is essential for oxygen transport and Mg in chlorophyll enables photosynthesis. In addition, many metals, such as Fe2+, Mg2+, Co2+, Mo2+, Mn2+, Cu2+ and Ca2+, can coordinate with specific groups on the side chains of amino acids in metalloenzymes.

3.2. Nutrition and Toxicity 3.2.1. Nutrition Atomic absorption spectrophotometry is a conventional method for determining the total content of a mineral in foods. However, the bioavailability and nutritional value of minerals are affected by multiple factors and the information on the total content is insufficient for bioavailability and nutrition evaluation. The utilization of minerals is discussed below by taking Fe as an example. Fe in diets is present in two forms: heme-iron and non-heme iron. Heme-iron occurs primarily in animal-derived foods. The heme-iron is bivalent and can be absorbed directly and stored in intestinal ferritin for human utilization. Non-heme iron is the major form of Fe in plant-derived foods, such as cereals food, vegetables, fruits and legumes. Non-heme iron is trivalent and cannot be easily absorbed unless it is reduced to its bivalent form. Some plant-

240


derived foods contain high contents of phosphates, oxalic acid and tannic acids and these compounds can precipitate Fe by complexing with it, which further reduces Fe utilization. The presence of reducing agents in plant-derived foods improves Fe utilization. Generally, the bioavailability of Fe in animal-derived foods is higher than that in plant-derived foods, as shown in Figure 7-19. The utilization of Fe is associated with its sources, existence state and diet structure. For example, phosphoric acid in milk can precipitate Fe and reduces its utilization; polyphenols in tea can complex with Fe and affect its utilization; diet with insufficient Cu decreases Fe absorption, because Cu is essential for hemoglobin synthesis. The absorption of Fe is also affected by individual or physiological factors. People suffering Fe deficiency or anemia absorb Fe easily and women generally have a better absorption than men. Besides, the possibility of Fe deficiency decreases with age increase.

3.2.2. Toxicity All minerals are toxic when they are present in high levels in foods. In addition to contents, the toxicity of minerals is also affected by the following factors.

Figure 7-19. Utilization of Fe element in different sources. 1 to 12 represents rice, spinach, beans, corn, lettuce, wheat, soybean, iron, protein, bovine, fish, hemoglobin, and beef, respectively.

Synergistic or antagonistic actions between minerals The presence of a metal might enhance or decrease the toxicity of another. For example, As increases the toxicity of Pb; Cu increases the toxicity of Hg; Cu decreases the toxicity of Hg; Mo significantly reduces Cu absorption; Co increases the toxicity of S; Se decreases the toxicity of Cd and Ni; Cd antagonizes the absorption of Zn and Cu. Valence The toxicity of an element is closely related its valence. The toxicity of elements depends on their coordination with biological macromolecules and the valence markedly influences the coordination. For example, Cr3+ is an essential trace element, while the Cr6+ is highly toxic. Chemical form The toxicity of an element is also related to its chemical forms. For example, the LD50s (mg/kg) of various arsenides are arsenite 14.0, arsenate 20.0, methyl arsenate 700~1800, 2-

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241

methyl arsenate 700~2600, arsenocholine 6500 and betaine arsenate complex >10,000. It could be seen that volatile inorganic arsenic is the most toxic. Arsenocholine and betaine arsenate are often considered non-toxic. Poisoning mechanism of heavy metals The poisoning mechanisms of heavy metals are rather complex and affected not only by their contents in the body, but also by the exposing pathway, metabolism, and health conditions of ingesters. In general, heavy metals exert their toxicity in one or more of the following mechanisms: Heavy metals disrupt the functional groups in biomolecules. For example, Hg(II) and Ag(II) can bind with –SH of Cys residues in enzymes and block the participation of –SH in enzymatic reactions. Heavy metals replace the essential metals in biomolecules. The activity of many enzymes is closely related to the metal cofactors. The activity of metalloenzymes is easily destroyed by the replacement of metal factors. For example, Be(II) can replace the Mg(II) in Mg(II)-containing enzymes. Heavy metals change the conformation of biomolecules. Heavy metals can bind to various biomolecules, such as proteins, nucleic acids and change their conformations. For example, Pb2+, Cd2+ and Ni2+ can bind to double stranded DNA (dsDNA) and this interaction leads to different modifications in the dsDNA structure.

3.2.3. Effect of Existence State The existence states of minerals tremendously influence their nutrition and toxicity. The existence state determines mineral solubility. Minerals must be in the dissolved state for their nutrition or toxicity. Minerals often occur as complexes with various organic ligands in foods and the ligands tremendously influence mineral solubility and bioavailability. For example, Ca in protein-Ca complexes is absorbed easily, but that in the Ca-oxalic acid complex is insoluble and possibly causes urinary calculus. The solubility of minerals also depends on the counter ion. For a same heavy metal, its oxide is less poisonous than its soluble chloride and nitrate. Generally, the counter ion influences the toxicity of a metal in the following order: Nitrate > Chloride > bromide > acetate > iodide > perchlorate > sulfate >> phosphate > carbonate > fluoride > hydroxide > oxides. The solubility of metal salts in aqueous solution decreases with the increase of atomic weight of the metals.

The existence states decide the physiological functions of minerals. The physiological functions of minerals are closely related with their valences. Cr3+ plays an important role in glucose, fat, and cholesterol metabolisms. In contrast, Cr6+ is highly toxicity and its in vivo transformation to Cr3+ is nearly negligible. When the body accumulates an excessive amount of Cr6+, poisoning symptoms appear.

242

Dongfeng Wang, Lina Yu, Haiyan Li et al. The coexistence of other minerals changes the nutrition and toxicity a metal.

Hg, Pb and Cd are well-known toxic metals, but the intake of certain components may strengthen or weaken their toxicity. For example, lipopolysaccharides increase the accumulation of Hg in mice and augment the Hg-induced nephrotoxicity. Table 7-5. Minerals contents in some foods [2, 9] Food Fried eggs Wheat bread Graham bread Salt-free macaroni Cooked rice Instant rice Mature black soybean Red cashew Whole milk Skim milk /nonfat milk Ameican cheese Sayda cheese Farmhouse cheese Low fat yogurt Vanilla ice-cream Overbark baked potato Underbark boiling otato Palm cabbage, crude stem Palm cabbage, cooked stem Crude shattering carrot Cooked freezing carrot Fresh entire tomato Canned tomato juice Attains (defrosting) Attains Apple (skin) Banana (peeling) Roasted beef (cans) Roasted veal (cans) Roasted chicken breast Roasted chicken drumstick Cooked salmon Canned salmon with bone

Ca 57 35 20 5 10 10 24 25 291 302 261 305 63 415 88 20 10 216 249 15 21 6 17 17 52 10 7 5 6 13 10 6 203

Mg 13 6 26 13 42 42 61 40 33 28 10 12 6 10 9 55 26 114 130 8 7 14 20 18 13 6 32 21 28 25 20 26 25

P 269 30 74 38 81 81 120 126 228 247 316 219 139 326 67 115 54 297 318 24 19 30 35 30 18 10 22 176 234 194 156 234 277

Content / mg/100g Na K Fe 290 138 2.1 144 31 0.8 180 50 1.5 1 22 1.0 5 42 0.4 5 42 0.4 1 305 2.0 2 356 3.0 120 370 0.1 126 406 0.1 608 69 0.2 264 42 0.3 425 89 0.1 150 531 0.2 58 128 0.1 16 844 2.8 7 443 0.4 123 1470 4.0 141 1575 4.5 19 178 0.3 43 115 0.4 11 273 0.6 661 403 1.0 2 356 0.2 0 237 0.1 1 159 0.3 1 451 0.4 50 305 1.6 68 389 0.9 62 218 0.9 77 206 1.1 56 319 0.5 458 231 0.9

Zn 2.0 0.2 1.0 0.4 0.6 0.6 1.0 0.9 0.9 0.9 1.3 1.3 0.4 2.0 0.7 0.7 0.4 2.0 2.1 0.1 0.2 0.1 0.3 0.1 0.1 0.1 0.2 3.7 3.0 0.8 2.4 0.4 0.9

Cu 0.06 0.04 0.10 0.07 0.01 0.01 0.18 0.21 0.05 0.05 0.01 0.01 0.03 0.10 0.01 0.62 0.23 0.40 0.23 0.03 0.05 0.09 0.18 0.08 0.06 0.06 0.12 0.08 0.13 0.04 0.07 0.06 0.07

Se 8 8 16 19.0 13.0 13.0 7.9 1.9 3.0 7.6 3.8 7.0 7.3 5.5 4.7 1.8 1.2 0.9 1.1 0.8 0.9 0.6 0.4 0.4 1.2 0.6 1.1 — — — — — —

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Minerals

4. CONTENTS OF MINERALS IN FOODS The contents of minerals in foods are governed by multiple factors, such as place of production, cultivation methods, and processing techniques. For example, the Cu content in rice is influenced by the Cu content in soil, climate, water source, use of fertilizer, insecticide, pesticide and fungicide, and processing equipment. The mineral contents of some foods are shown in Table 7-5. Besides, the utilization of minerals in foods is closely associated with the dietary structure due to the interactions with other food components. This section concerns only the impact of material, processing, and storage on mineral contents in foods.

4.1. Effect of Raw Material on Mineral Contents in Foods The contents of minerals in plant-derived foods are subject to the influences of soil, water and fertilizer management, antagonistic effect among minerals and climate. As shown in Table 7-6, the contents of some minerals in black glutinous rice of three different provinces in China differ greatly. The contents of minerals in animal-derived foods depend on feed, animal health status and environment, in which, the mineral content of feed is the most important determinant. As shown in Table 7-7, the supplementation of trace elements in feed markedly increases the contents of K, Ca, and P in milk.

4.2. Effect of Processing on Mineral Contents in Foods Processing method, water used, processing equipment, and food additives also change the contents of minerals in foods. Table 7-8 lists the effects of four processing methods on the mineral contents of instant fiddlehead. It could be seen that the four treatments slightly increase the contents of Ca, but more or less decrease the contents of other minerals, of which, salting-out dehydration plus blanching leads to the highest loss of Mg, Mn, and Zn, and salting-out dehydration without blanching causes most Fe and Cu losses. Table 7-9 illustrates the loss of some minerals in spinach after blanching. K and Na suffer the highest loss after blanching, but the Ca content is nearly not affected. Hence, minerals in free state, such as K and Na, are lost easily during bleaching, but minerals occuring in insoluble state (such as Ca) are not lost. Table 7-6. Contents of some minerals in black glutinous rice sampled from three provinces in China (mg/100g) producing area Hunan Zhejiang Guizhou

Zn 19.48 19.47 17.64

Cu 1.779 2.549 0.702

Fe 17.18 20.13 24.97

Mn 15.46 24.25 25.36

Ca 27.59 59.48 32.00

Mg 12.27 12.00 11.42

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Table 7-7. Effect of trace element supplementation in cattle feed on the minerals content of milk (mg/100g) Fe Cu Zn Mn K Na Ca Mg P 0.122 0.032 0.417 0.008 81.60 83.70 144.0 11.00 98.60 0.137 0.007 0.442 0.010 68.39 85.34 77.67 10.06 82.09

Experimental group Control group

Table 7-8. Effect of different processing methods on some trace elements content in instant fiddlehead (mg/100g dry weight) Processing methods Ca Mg Fe Mn Cu Zn Before processing 62.5 238.0 32.0 8.1 27.4 9.5 (1) 80.0 140.9 30.6 7.3 22.4 7.1 (2) 80.1 169.5 21.1 7.3 20.3 7.0 (3) 80.6 127.0 27.6 5.1 20.2 5.7 (4) 88.0 157.3 20.7 7.7 15.5 7.9 Note: (1) natural dehydration plus blanching; (2) natural dehydration without blanching; (3) salting-out dehydration plus blanching; (4) salting-out dehydration without blanching.

Table 7-9. Effect of steam blanching on the loss of minerals in spinach Mineral

g/100g

Loss (%)

Not steam blanching

Steam blanching

K

7.9

3.0

56

Na

0.5

0.3

43

Ca Mg P Nitrite

2.2 0.3 0.6 2.5

2.3 0.2 0.4 0.8

0 36 36 70

Table 7-10. Effect of processing method on the Cu content in potato (mg/100g fresh weight) Processing method

Cu

Material

0.21

Water boiling

0.10

Fluctuati on (%) 0.00 -52.38

Baking Potato chip

0.18 0.29

-14.29 +37.20

Processing method

Cu

Potato mesh

0.10

French frying potato crisp

0.27

Fluctuati on (%) -52.38 +28.57

Fast potato Peeling potato

0.17 0.34

-19.05 +61.90

The effect of processing method on Cu content in potato shown in Table 7-10. Cu content is slightly increased in the frying potato crisp and peeling potato.

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Minerals

4.3. Effect of Storage on Mineral Contents in Foods Minerals in foods can migrate from packaging materials. The contents of some minerals in canned liquid and solid foods are given in Table 7-11. The contents of Al, Sn and Fe in solid foods are evidently increased because solid foods repeatedly collide with the packing material. Table 7-11. Contents (g/Kg) of trace elements in some vegetable cans Vegetable Mung bean

Can La

State Al Sn L 0.10 5 S 0.7 10 Bean La L 0.07 5 S 0.15 10 Small green pea La L 0.04 10 S 0.55 20 Parsley heart La L 0.13 10 S 1.50 20 Sweetcorn La L 0.04 10 S 0.30 20 Mushroom P L 0.01 15 S 0.04 55 Note: a. La = Lacquered can; P = T in plate can; b. L = Liquid, S = Solid.

Fe 2.8 4.8 9.8 26 10 12 4.0 3.4 1.0 7.4 5.1 16

REFERENCES [1] Capasso, C; Carginale, V. Solution structure of MT-nc, a Novel Metallothionein from the Antarctic Fish Notothenia coriiceps. Structure, 2003, 11, 435-443. [2] ESHA Research. The Food Processor Plus, ESHA Research, Salem, OR, 1992. [3] Ji, L; Huang, J; Mo, T. Bioinorganic Chemistry an Introduction (Second), Guangdong: Zhongshan University Press, 2001 [4] Kan, JQ. Food Chemistry. Beijing: China Agricultural University Press; 2008. [5] Liu, Y. An idea on trace elements as nutrients of organism. Journal of Peking University (Natural Science), 1986, (3): 121. [6] Nagy, L; Szorcsik, A. Equilibrium and structural studies on metal complexes of carbohydrates and their derivatives. Journal of Inorganic Biochemistry, 2002, 89, 1-12. [7] Sigel, H; Sigel, A. Metal Ions in Biological Systems. 1st edition. New York: Marcel Dekker, 1995. [8] Tang Renhuan. On the Biological Element Spectrum in Organism. Acta Scientiarum Naturalium Universitatis Pekinensis, 1996, 32, 790-803. [9] U. S. Department of Agriculture (1976-1986). Composition of Foods. Agriculture Handbook Nos.8-1 to 8-16. Human Nutrition Information Service, USDA, Hyattsville, MD. [10] Yang, P. Introduction of Bioinorganic Chemistry, Xi‘an: Xi‘an Jiaotong University Press, 1991.



Chapter 8

FOOD FLAVORS Xiaoxiong Zeng1 and Guaoqing Huang2 1

College of Food Science and Technology, Nanjing Agriculture University, Nanjing, China 2 College of Food Science and Engineering, Qingdao Agriculture University, Qingdao, China

ABSTRACT The special tastes or aromas of foods are contributed by abundant small-molecular weight compounds and these compounds significantly affect the acceptability of foods by consumers. This chapter firstly concerns the major tastes and their perception by human beings. Then, the major flavor compounds in various foods, including fruits, vegetables, and meats are elucidated in detail. Besides, reactions that contribute to the formation of flavors during food processing and storage are also presented in detail in this chapter. Foods should not only meet the needs of consumers on nutrition, but also provide desirable flavors so that consumers can enjoy the foods. Flavor is an important aspect that determines food quality, and affects the intake and absorption of nutrients in foods. Flavor is an overall integrated perception of all contributing senses (smell, taste, sight, feeling, and sound) at the time of food consumption. The evaluation and preference to flavors differ significantly among individuals, areas, and ethnics. Flavor together with nutritional value and security determine consumers‘ acceptance of foods. The flavor of food is contributed by multiple compounds in addition to environmental factors. The development of modern analysis techniques, such as chromatography and mass spectrometry, provides great convenience for further study of food flavor. However, since food flavor is a physiological perception, neither qualitative nor quantitative methods can accurately measure or describe a food flavor yet.

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TASTE COMPOUNDS IN FOODS Taste and Taste Compounds Food Taste A taste is a feeling caused by the stimulation of food compounds on taste receptors in the mouth. Taste buds are the most important taste receptors in the mouth, followed by free nerve endings. Taste buds locate on the surface of the tongue and the back of the oral cavity. Each taste bud is the cluster of approximately 30-50 taste cells and taste receptors are distributed on the membranes of these cells. Each taste bud is connected to the oral cavity through a hole on the top. When foods are ingested, the taste compounds get contact with the corresponding receptors in the taste cells through the hole and consequently a taste is perceived. Free nerve ending acts as a micro-receiver and can identify different chemical substances. It is scattered in the entire oral cavity and surrounded by sacs. Different parts of the tongue are sensitive to different tastes. Each of the primary tastes is associated with a specific area in the tongue. The tip of the tongue is most sensitive to sweet and salty tastes, while sour seems to register more strongly on the sides of the tongue. Far to the rear of the tongue are most of the receptors for bitter tastes. The sensitivity to a taste is often described by threshold, which is the lowest concentration required for eliciting a sensation. Threshold values are usually determined using individuals representative of the general populations, and each panelist indicates whether or not the compound can be detected. The concentration range where at least half (sometimes greater) of the panelists can detect the compound is designated as the flavor threshold. Table 8-1 lists the threshold values of some taste compounds. Three kinds of threshold value are proposed based on measurement methods. Absolute threshold value, also called sensory threshold value, is the lowest level of a taste compound that can be perceived. It is determined by tasting a series of diluted taste compound solutions. Differential threshold value is the smallest change in taste compound concentration that consumers can detect. Terminal threshold value is the minimum concentration of a taste compound, whose further increase does not enhance the perception. In this chapter, all thresholds value mentioned refer to the absolute threshold value, except special instructions. Taste classification. Different taste classifications have been proposed due to the differences in culture and dietary habits. In Japan, the primary tastes include sweet, sour, bitter, salty and pungent. In European and American countries, six primary food tastes are identified, including sweet, sour, bitter, salty, pungent, and astringent. In India, up to eight tastes are recognized as primary tastes and they are sweet, sour, bitter, salty, pungent, insipid, astringent and abnormal. In China, the original standard specifies only five primary tastes, including sweet, sour, bitter, salty and pungent, but the astringent and delicious tastes are added later. From the physiological point of view, only the sweet, sour, bitter and salty tastes can be perceived by taste receptors. The pungent taste is the pain feeling when pungent substances stimulate oral mucosa, nasal mucosa, skin and trigeminal nerve. The astringent taste is the response of tactile nerves to protein aggregation. Both the tastes affect the flavor of foods independently. The presence of delicious compounds enhances the sensation of other tastes. In European and American countries, the delicious taste is excluded from the primary tastes.

249

Food Flavors Factors affecting taste perception.

The perception of tastes is affected by many factors. In addition to dietary habits, health status, age and other individual factors, taste perception is also affected by the following: Temperature: the optimum temperature for taste sensation is between 10-40 °C and taste receptors are the most sensitive at 30 °C. When the temperature is lower than 10 °C or higher than 50 °C, the sensation becomes dull. It can be seen from Table 8-1 that the salty taste is the most sensitive to temperature variation and the sour taste of citric acid is the least sensitive. Solubility: the intensity of a taste is related to the solubility of the taste compound, because taste receptors or free nerve endings are stimulated only by dissolved compounds. Table 8-1. Threshold values of several taste compounds Taste compounds

Taste

Quinine sulfate Sucrose Sodium chloride Citrate

Bitter Sweet Salty Sour

Threshold (%) 25 °C 0 °C 1.0×10-4 3.0×10-4 0.1 0.4 0.05 0.25 2.5×10-3 3.0×10-3

Generally, taste compounds that dissolve quickly are perceived in a very short time and the sensation disappears very fast. For example, sucrose is readily soluble. Its sweetness is perceived rapidly after ingestion, but the sensation lasts only a short time. In contrast, the sweetness of saccharin is perceived slowly, but the sensation can last for a relative long time. Presence of other taste compounds: Interactions might occur between different taste compounds. a.

Enhancing: the presence of a taste compound could enhance the intensity of another taste. For example, table salt in concentration 0.017% enhances the sweetness of sucrose solution (15 %). b. Modification: the presence of a taste compound might change the taste of another compound. For example, water tastes sweet if table salt or quinine has been perceived before. c. Elimination: the presence of a taste compound reduces or eliminates the intensity of another taste. For example, any one of sucrose, citric acid, table salt, and quinine can reduce the taste intensity of the other three compounds. In wines or beverages, the sweetness of sugars weakens the sour taste and vice versa. d. Multiplication: when two taste compounds are present at the same time, the intensity of the tastes increases significantly. For example, the mixture of sodium glutamate and 5‘-inosinic acid has markedly improved delicious taste than alone and maltol significantly enhances the sweet taste in beverages and candies. e. Adaptation: adaptation refers to the gradual decline of taste intensity with prolonged stimulation. The time required for the recovery of taste sensation varies with tastes. The time needed for recovery of sour taste sensation after adaptation is 1.5~3 min and that for sweetness, bitterness, and salty is 1~5 min, 1.5~2.5 min and 0.3-2 min respectively.

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The interactions between tastes are very complicated and are affected by both psychological and physicochemical factors. The mechanisms involved are not well understood yet to present.

Sweet Taste Sweetness is the most popular taste for consumers. It improves the palatability and certain properties of foods. The intensity of sweetness is expressed in relative sweetness by using 10 % sucrose solution as reference. Table 8-2 lists the relative sweetness of several sweeteners. The mechanism of sweet taste can be explained by the AH/B through proposed by Robert Shallenberger and Terry Acree in 1963. It is supposed that the initial event in the perception of sweetness is the hydrogen bonding of a stimulant to receptors on the tongue. Each sweet compound possesses an electronegative atom A (usually N or O) covalently connected with a hydrogen atom (AH). Hence, the AH can be hydroxyl (-OH), imino (-NH) or amino (-NH2) group. AH functions as the proton donor. The sweet compound contains another electronegative atom B (usually N, O, S, or Cl), which is 0.25-0.4 nm away from the AH group and acts as the proton receptor. The sweetness receptor contains the AH/B unit. When the AH/B unit of a sweet compound binds to the AH/B unit in the receptor through hydrogen bonding, the taste nerve is stimulated and the sweet taste is perceived. Figure 8-1 shows the AH/B structure of chloroform, saccharin and glucose. This theory has been successfully used to explain the sweet sensation of many compounds. However, this theory has some limitations. It can not explain why the sweet intensities of sugars and D-amino acids that contain the AH/B structure differ significantly and why the optical isomers of amino acids have different tastes. Table 8-2. Relative sweetness of several sweeteners (with sucrose as 1.0)

β-D-fructose α-D-glucose α-D-galactose β-D-mannose

Relative sweetness 1.0-1.75 0.40-0.79 0.27 0.59

Xylitol

0.9-1.4

Sweetener

Chloroform

Sweetener

Relative sweetness

D-tryptophan glycyrrhizic acid saccharin Naringin dihydrochalcone

35 200-250 200-700 100

Neohesperidin dihydrochalcone

1500-2000

Saccharin

Figure 8-1. AH/B relationships of chloroform, saccharin, and glucose [1].

Glucose

Food Flavors

251

Sweet receptor

Figure 8-2. Schematic showing the relationship between AH/B and γ sites in the saporous sweet unit for β-D-fructopyranose [1].

To solve these problems, Kier extended the AH/B theory. He proposed that each sweet compound also contains a lipophilic region γ in addition to the AH/B structure. The lipophilic region might be methylene (-CH2-), methyl (-CH3) or phenyl (-C6H5) groups. All the three active units (AH, B and region γ) must be arranged correctly in space so that the units can contact with receptor molecules. The relationship between the three groups can be illustrated in Figure 8-2.

Bitter Taste The bitter taste is unpleasant on its own, but its combination with other tastes can provide foods with special flavor, such as in tea, coffee, beer, and balsam pear. Strychnine is the bitterest substance (threshold 0.0016 %) ever found and quinine is often used as reference in the assessment of bitter intensity of other substance. The sensation of the bitter taste is similar to that of the sweet taste. Bitter compounds posses the AH/B entity and can interact with receptors through hydrogen bonding. The difference is that the proton donor (AH) in bitter compounds are –OH, –C(OH)COCH3, – CHCOOCH3, or –NH and the proton donor (B) is –CHO, –COOH, or –COOCH3. In addition, the distance between AH and B is 0.15 nm, which is much less than that in sweet compounds. Naturally occurring bitter compounds are alkaloids, terpenoids, and glycosides in plants and bile in animals. Caffeine, theobromine, theophylline: caffeine, theobromine and theophylline are derivatives of purine and are important bitter substances in foods. Caffeine occurs in tea, coffee and cacao, and theophylline is found in cacao and tea (Figure 8-3). All the three alkaloids can stimulate the central nervous system. Naringin and neohesperidin: naringin and neohesperidin are main bitter taste compounds in citrus fruits and are mainly distributed in the peel of the fruits. Both the two compounds are flavanone glycosides and are water soluble. The bitterness of naringin is related to the l→2 glycosidic bond between rhamnose and glucose. Enzymatic hydrolysis of the linkage yields products without bitterness (Figure 8-4).

252

Xiaoxiong Zeng and Guaoqing Huang O

R1

N

O

N

N

N

R3

R1=R2=R3=CH3 caffeine R1=H R2=R3=CH3 theobromine R1=R2= CH3 R3= H theophylline

R2

Figure 8-3. Structure of caffeine, theobromine and theophylline.

Enzyme

Figure 8-4. Debittering site of naringin by naringinase.

humulone

cis-isohumulone

trans-isohumulone

Figure 8-5. Thermal isomerization of humulone to isohumulone [2]. R3 COOH

R1=R2=OH R3=H chenocholic acid R1=R3=OH R2=H deoxycholic acid R1=R2=R3= OH

R1

cholic acid

R2

Figure 8-6. Structures chenocholic acid, deoxycholic acid and cholic acid.

Bitter substance in beer: the bitter taste of beer is contributed by the bitter substances originally contained in hops and those generated during brewing. The major bitter substances in beer are α-acids, such as humulone, cohumulone, adhumulone. Hops are often added as the mashed malted barely (or malt extract) is boiled in water. During this boiling process the modestly bitter α-acids undergo thermal isomerization to form extremely bitter iso-α-acids. (Figure 8-5). Bile acid: bile acid is an extremely bitter fluid secreted by the liver of most vertebrates. It is involved in the digestion of lipids in the small intestine and the adsorption of fat-soluble vitamins. The major bitter compounds in bile acid are chenocholic acid, deoxycholic acid, and cholic acid (Figure 8-6). If bile acid leaks from gallbladder during the processing of livestock, poultry and aquatic products, the bitter taste cannot be removed even after repeated washing.

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Sour Taste The sour taste is produced by hydrogen ion of organic acid, inorganic acids and acidic salts. Moderate sour intensity yields pleasant sensation and enhances appetite. Generally, the intensity of the sour taste is positively related to the concentration of hydrogen ion in solutions. When the H+ concentration is too high (pH <3.0), the sour taste becomes intolerable and sour intensity changes can no longer be perceived. The sensation of the sour taste is affected by counter ion species and the buffering capacity of food medium. For example, the sour intensities of five common acids in the same pH are in the following decreasing order: acetate acid > formic acid > lactic acid > oxalate acid > hydrochloride yield. Anions decide the characteristics of the sour taste, which is why the sour taste of sour compounds differs. Acidulants are important additives in food processing. The compounds not only impart foods with desired sour taste, but also inhibit the growth of microorganisms. The most commonly used acidulant in food industry is acetic acid, followed by citric acid, lactic acid, tartaric acid, gluconic acid, malic acid, fumaric acid and phosphoric acid. Acetic acid is the major active component of vinegar. Citric acid is the most widely used acidulant in food industry. Malic acid is used to mask the lingering bitterness of synthetic sweeteners. Gluconic acid-δ-lactone can produce gluconic acid upon heating and is hence a slow-acting acidulant. It can be used as leavening agent in biscuit processing and curing agent in tofu preparation. Phosphoric acid is mainly used in the production of cola-type beverages. Salty Taste The salty taste is contributed by neutral salts and it is an indispensable and the most fundamental taste in foods. This taste is the compromise of the effects of dissociated anions and cations. Cations produce salty taste, while anions suppress the sensation of salty taste and elicit other undesirable tastes. Whether an inorganic salt is bitter or salty depends on the diameters of cations and anions. If the sum of the diameters of cation and anion of a compound is less than 0.65 nm, the salt tastes salty; otherwise, the salt tastes bitter. For example, the sum of the diameters of Mg2+ and Cl- is 0.85 nm and MgCl2 tastes quite bitter.

L-sodium glutamate L-sodium glutamate (MSG)

(MSG)

Inosine-5‘-monophate (5‘-IMP). Inosine-5'monophosphate (5′-IMP)

Sodium chloride is the only substance known to evoke a purely salty taste in any concentration that is suprathreshold. The optimum concentration of NaCl in liquid foods is 0.8-1.2 %. Because excessive intake of NaCl can cause negative impacts on health, the development of NaCl substitutes has attracted much attention. For example, a mixture of 20 % of KCl and 80 % NaCl has been developed as a low-sodium salt. Delicious Flavor The delicious flavor is a very complex sensation. The flavor enhances the appetite of consumers and makes foods tasted more delicious. When the concentration of a delicious

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compound in foods is higher than threshold value, it evidently increases the delicious taste of the food, and enhances the original flavor of the food even its concentration is lower than its threshold value. Hence, delicious compounds are also known as flavor enhancers. Delicious compounds can be divided into amino acid, nucleotide and organic acid types and their typical representatives are L-sodium glutamate (MSG), 5‘-inosine monophosphate (5‘-IMP), and sodium succinate respectively. MSG is the first discovered and commercially available flavor enhancer. It occurs widely in nature and is abundant in kelps. 5‘-IMP is widely found in chickens, fishes and broths and 5‘-IMP in animals is mainly generated by the degradation of ATP in muscles. 5‘-Guanosine monophosphate (5‘-GMP) is the major delicious component in mushrooms. Sodium succinate has been identified in poultry, livestock and mollusks and the highest content is detected in shellfishes. It is also present in low levels in fermented products, such as soy sauce, sauce, and yellow rice wine. Aspartic acid and its sodium salt are the main delicious substances in bamboo shoots, though their intensities are lower than that of MSG. The combination of IMP, GMP and MSG significantly enhance the delicious taste of MSG. For instance, the delicious intensity of the mixture containing 1 % IMP, 1 % GMP, and 98 % MSG is four times of that of MSG alone.

Pungency Certain compounds found in some spices and vegetables cause characteristic hot, sharp and stinging sensations that are known collectively as pungency. Pungent compounds enhance the appetite and increase the secretion of digestive juice of consumers and they are indispensable condiments in daily life. Based on sensation, natural pungent compounds are divided into hot, aromatic and irritative types. Hot compounds: pungent compounds of this type are odorless and can cause the burning feeling in mouth. The pungent compounds in chili, black pepper and zanthoxylum belong to this type. Chili. Capsaicinoids are the vanillyl amides of monocarboxylic acids with varying chain length (C8~C11) and capsaicin is the active component in chili pepper. The contents of capsaicin in chili peppers vary significantly with species. The contents of capsaicin in red chili, horn red chili, Sam chili, and Uganda chili are 0.06 %, 0.2 %, 0.3 %, and 0.85 %, respectively.

Capsaicin

Black pepper. Piperine is the major pungent compound in black pepper. Pepperine is an amide compound and has three isomers, of which the isomers with more cis double bonds possess

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Food Flavors

high pungency intensity. The liability of black pepper to light and storage is due to the isomerization of piperine isomers.

Pipirine

Chinese prickly ash. The main pungent compound in Chinese prickly ash is suberosin and it also an amide compound. Aromatic compounds: pungent compounds of this type are volatile and aromatic. Ginger. Zingiberols are the main active components responsible for the pungency of fresh ginger. The carbon chain length on the lateral side of hydroxyl in the side chain on the ring is different (n=5-9). When fresh ginger is dried, zingiberols are converted to more pungent gingerols through dehydration. When ginger is heated, the side chain of zingiberols breaks to produce zingerone, which is a moderate pungent compound.

Zingiberols

Zingerols

Zingerone

Cloves and nutmeg. The main pungent components in the two species are eugenol and isoeugenol respectively.

Eugenol

Isoeugenol

Irritative compounds: in addition to tongue and oral mucosa, compounds of this type are also irritative to nose and eyes.

256

Xiaoxiong Zeng and Guaoqing Huang Mustard, radish, horseradish.

The pungency and irritability of these materials are contributed by mustard oil that is generated through the hydrolysis of sinigrin. It is the collective of isothiocyanate. The following active substances have been identified in mustard, radish, and horseradish: CH2=CH CH2- NCS CH3CH=CH-NCS allyl isothiocyanate propylene isothiocyanate. CH3(CH2)3- NCS C6H5CH2-NCS butyl isothiocyanate benzyl isothiocyanate. Welsh onion, garlic, leek, and onion.

Disulfides are the major pungent and irritative substances in welsh onion, garlic, leek, and onion. The pungent components of garlic are generated by the decomposition of alliin and include diallyl disulfide and allyl propyl disulfide. The pungent components in leek and onion are also sulfur-containing compounds. These substances are converted to sweet thiol compounds when heated. This is why cooked onion and garlic taste sweet other than pungent.

Astringency Astringency is the sensation of the aggregation of proteins in oral mucosa. Tannins and polyphenols are major components in foods that cause astringency. Besides, some salts (such as alum), aldehydes, organic acids (such as oxalic acid), and quinine acid are sometimes also involved in astringency sensation. Mature fruits taste less astringent than immature fruits, because polyphenols are decomposed, oxidized, or polymerized during maturation. Tea also contains polyphenols, but their contents vary with processing methods. Red tea is less astringent than green tea, because polyphenols are oxidized during fermentation. Astringency is a characteristic taste of red wine. To obtain an acceptable astringency, measures must be taken to reduce the contents of polyphenols in wines.

FLAVOR COMPONENTS IN FOODS Aroma Compounds in Plant-Derived Foods Fruits The characteristic aromas of fruits are mainly contributed by esters, aldehydes and terpenes, followed by alcohols, ethers and volatile acids. The contents of these compounds increase gradually as fruits get mature. Table 8-3 lists the aromatic compounds in some fruits. Vegetables Generally speaking, the intensity of the aroma of vegetables is weaker than that of fruits. However, some vegetable, such as onion, garlic, leek, and onion, possess unique and strong aromas.

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Food Flavors Table 8-3. Main aroma components in some fruits Fruit Apple

Major aroma components Isoamyl acetate

Pear Banana Muskmelon Peach

Isoamyl formate Isoamyl acetate, isoamyl isovalerate Diethyl sebacate Ethyl acetate, agarolactone

Apricot Grape

Amyl butyrate Methyl o-aminobenzoate

Citrus

Butyraldehyde, octyl aldehyde, decanal, linalool Formic acid, acetaldehyde, alcohol, acetone Phenylethyl alcohol, formic acid, ethyl acetate

Peel Juice

Minor aroma components Volatile acids, ethanol, acetaldehyde, geranium alcohol Volatile acids Hexanol, hexenal Volatile acids, acetaldehyde, advanced aldehyde C4~C12 fatty acid esters, volatile acids

Fresh vegetables. Many fresh vegetables have the smell of soil. This aroma is generated by methoxy alkyl pyrazines, such as 2-methoxy-3-isopropyl pyrazine in fresh tomato and pee, 2-methoxy-3isobutyl pyrazine in green pepper, and 2-methoxy-3-sec-butyl pyrazine in red beet root. These compounds are synthesized from the precursor leucine. The biosynthesis pathway of pyrazine compounds in plants is shown in Figure 8-7. enzyme

leucine 2-methoxy-3-isobutyl pyrazine

Figure 8-7. Biosynthesis pathway of methoxy alkyl pyrazines in plant tissues [3].

Unsaturated fatty acids in vegetables can be oxidized by endogenous lipoxygenase to produce peroxides, which are further decomposed to produce aromatic aldehydes, ketones and alcohols. Vegetables of the Liliaceae family. The aroma of these vegetables is generated by sulfur compounds, such as dialkyl thioethers, dialkyl disulfides, dialkyl trisulfides, and dialkyl tetrasulfides. In addition, thio-

258


propanal, thiocyanate, thiocyanate esters, mercaptan, dimethylthiophene, and thiosulfinate are also involved in aroma formation. These compounds are transformed by enzymes from their precursors when plant tissues are damaged. The aroma precursor of onion is S-(1-propenyl)-L-cysteine sulfoxide, which is converted from cysteine. In aroma formation, the precursor undergoes rearrangement with the help of allinase and generates 1-propenyl sulfenic acid and pyruvate. 1-Propenyl sulfenic acid is unstable.

acetone alkyl garlic alcohol ammonia

1-allyl-1-subsurfuracid

S-propionaldehyde-S-oxidate

Figure 8-8. Reactions involved in the formation of onion aroma [4].

Figure 8-9. Reactions involved in the formation of garlic flavor compounds [5].

Part of the compound is rearranged to lacrimatory sulfoxide thio-propanal and part is converted to mercaptan, disulfide compounds, tri-sulfur compounds and thiophene (Figure 88). All these compounds are involved in the characteristic aroma of onion. 2-Amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) is the aroma precursor of garlic and it is degraded in a similar way to that of S-(1-propenyl)-L-cysteine sulfoxide in onion (Figure 8-9). The generated 2-propene-1-sulfinothioic acid S-2-propenyl ester (allicin) has a strong irritating smell. Allicin also undergoes rearrangement to produce mercaptan, disulfide compounds and other aromatic compounds. Allicin together with diallyl disulfide and methyl allyl disulfide produce the characteristic aroma of garlic. The characteristic smell of chives flavor is contributed by dimethyl disulfide, dipropyl disulfide and propyl propenyl disulfide and that of asparagus is generated by 1,2-disulfide-3cyclopentene and 3-hydroxy-butanone. 5-Methyl-2-hexyl-3-dihydro-furanone and propylmercaptanare contribute to the characteristic smell of leek. Vegetables of the Cruciferous family. Cruciferous vegetables, such as mustard, radish, and horseradish, have strong pungent smells. These smells are caused by isothiocyanate esters, such as 2-vinyl isothiocyanate, 3-

Food Flavors

259

propenyl isothiocyanate and 2-styryl isothiocyanate. Isothiocyanate esters are generated by the enzymatic hydrolysis of glucosinolates. In addition to isothiocyanate esters, allyl isothiocyanate (R-S-C=N) and nitrides are also formed during the hydrolysis (Figure 8-10). Mushroom.

Figure 8-10. Reactions involved in the formation of Cruciferae flavors [6].

Figure 8-11. Reactions involved in the formation of lenthionine [7].

The aroma precursor of mushrooms is lenthionine acid. The precursor can be hydrolyzed by S-alkyl-L-cysteine sulfoxide lyase to produce the aromatic lenthionine (Figure 8-11). In addition, benzyl isothiocyanate, phenethyl isothiocyanate, and benzaldehyde cyanohydrin are also involved in the aroma formation of mushroom. Other vegetables. The major aromatic compounds in cucumber are carbonyl substances and alcohols and its characteristic aroma is contributed by 2-trans-6-cis-nonadienal, trans-2-nonene aldehyde and 2-trans-6-cis-nonadienol. Besides, 3-cis-hexenal, 2-trans-hexenal, and 2-trans-nonnenal also affect the aroma of cucumber. These flavor compounds are synthesized from linoleic acid and linolenic acid. More than 80 kinds of volatile compounds have been identified in tomato, in which, 3cis-hexenal, 2-trans-hexenal, β-ionone, hexanal, β-damascenone, 1-penten-3-one, 3-methyl butyraldehyde, are major active compounds for tomato aroma. In heated products such as

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ketchup, the aroma changes due to the formation of dimethyl sulfide, the increase of βionone, β-damascenone and the decrease of 3-cis-hexenal and hexanal. Potato contains only trace amounts of aromatic compounds. Pyrazines, including 2isopropyl-3-methoxy-pyrazine, 3-ethyl-2-methoxy-pyrazine and 2, 5-dimethoxy-pyrazine, are the major active components in fresh potato. Volatile compounds in cooked potato are carbonyl compounds (including saturated and unsaturated aldehydes, ketones and aromatic aldehydes), alcohols (C3~C8 alcohols, linalool, neroli and geraniol), sulfur compounds (mercaptan, sulfide and thiazole) and furan compounds. A large variety of terpenes have been identified in the volatile oil of carrot, mainly including γ-bisabolene, caryophyllene, and terpinolene. cis-γ-Bisabolene, trans-γ-bisabolene and hypoxanthine contribute to the characteristic smell of carrot.

Aromatic Components in Tea Aroma is an important factor that determines the quality of tea. The aroma type and characteristic aroma compounds of teas are associated with tea varieties, growing conditions, harvesting time, maturity and processing methods. Only tens of aroma compounds have been identified in fresh tea leaves, but up to more than 500 aromatic compounds are found in processed teas. Aroma components in green tea. Green tea does not undergo fermentation during processing and exhibits the typical roasted and fresh smell. The first step of green tea processing is water removing and enzymes are deactivated in this step. Hence, most aromatic components of green tea are original from fresh leaves and only a few are formed during processing. The main volatile components in fresh tea leaves are leaf alcohols (3-cis-hexenol, 2-cishexenol) and leaf aldehydes (3-cis-hexenal and 2-cis-hexenal), which have a strong flavor of grass. During processing, part low boiling point substances, such as leaf alcohols and leaf aldehydes, are evaporated, while part leaf alcohols and leaf aldehydes are isomerized to transleaf alcohols and trans-leaf aldehydes. These products have the faint scent and are the main body of the aroma of green tea. The smells of high boiling point aromatic compounds, such as linalool, benzyl alcohol, phenylethanol, and acetophenone, get exposed with the volatilization of low boiling point substances. Linalool is an important active component and accounts for 10 % of total aromatic components in green tea. These high boiling point compounds have pleasant smells and are important aromatic substances of green tea. The characteristic aroma of teas harvested right before or after Ching Ming Festival is contributed by disulfide ether and leaf alcohols and the aroma disappear gradually during prolonged storage. Semi-fermented tea. Oolong tea is the representative of semi-fermented tea and its major aromatic components include leaf alcohol, cis-jasmone, jasmine lactone, methyl jasmonate, nerolidol, benzyl alcohol cyanohydrin, and ethyl acetate.

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Food Flavors HO

CH2COOCH 3

CH CN

CH 2CH=CHCH 2CH3 O

OH

O

O

O

Cis-jasmone

Jasmine lactone

Methyl jasmonate

Nerolidol

Benzyl alcohol cyanohydrin

Red tea Red tea undergoes fermentation during processing and has strong aroma. During processing, tremendous reactions occur to produce hundreds of aromatic components. Hence, red tea has obviously different aroma from green tea. Alcohols, aldehydes, acids and esters are major constituents of the aroma of red tea and violet ketone plays important role in the formation of the characteristic aroma. O

O

O

+

O2

Cis-theaspirane

β-carotene

β-ionone

β-damascenone

Figure 8-12. Oxidation and decomposition of β-carotene in red tea.

Carotenoids, amino acids, and unsaturated fatty acids are the aroma precursors for red tea. During processing, β-carotene is oxidized and decomposed to ionone (Figure 8-12), which is further oxidized dihydroaclinidiolide and theaspirone. Unsaturated fatty acids in tea, especially linolenic acid and linoleic acid, undergo enzymatic oxidation during processing to produce C6-C10 aldehydes and alcohols. Meanwhile, the fatty acids are also esterified by alcohols to yield esters with different aromas, such as benzyl acetate, ethyl phenylacetate, methyl benzoate, and methyl salicylate. These compounds have important effects on the aroma of tea. Amino acids are also decomposed by enzymes to produce aldehydes, alcohols, and acids. These compounds also contribute to the aroma of red tea.

Aromatic Compounds in Animal-Derived Foods Livestock and Poultry Meat Raw pork contains more than 300 kinds of volatile compounds, of which, most are hydrocarbons, aldehydes, ketones, alcohols, esters, furan compounds, nitrogenous compounds and sulfur compounds. The aroma of different raw meats varies and depends mainly on lipid composition. Raw beef or pork has no special odor, but raw mutton and dog meat possess special smells. The goaty flavor of mutton is contributed by methyl fatty acids, such as 4methyl-capryli acid, 4-methyl-pelargonic acid and 4-methyl-capric acid, while the fishy smell of dog meat is closely related to the presence of trimethylamine and lower fatty acids. The special smell of sexually maturated male livestock is due to the secretion of gonad. For example, two compounds, namely 5α-male-16-en-3-one and 5α-male-16-ene-3α-ol (Figure 813), are responsible for the strong odor of the meat of emasculated male pig.

262


enzyme

Pregnenolone

5α-male-16-en-3-one

Figure 8-13. Formation of characteristic aromatic components in boar [8].

Aromatic compounds in cooked meats are generated in three ways: lipid oxidation and hydrolysis; Maillard reaction between amino acids or proteins and reducing sugars; and further decomposition or recombination of flavor compounds. The aroma compositions of cooked meats vary with the cooking temperature and processing methods. Cooked pork contains reduced volatile compounds (mainly aldehydes, ketones, carboxylic acids and sulfur compounds). Some non-volatile compounds, including free amino acids, peptides, carbohydrates, vitamins and nucleotides, are important aroma precursors of livestock and poultry meats. When heated, the precursors undergo various chemical reactions to yield characteristic aromas. When fat-containing beef is cooked, abundant volatile compounds are produced, including fatty acids, aldehydes, ketones, alcohols, ethers, furan, pyrrole, lactones, aromatic hydrocarbons, sulfur compounds (thiazole, thiophene, alkyl sulfur, sulfide, disulfide compounds) and nitrogenous compounds (oxazole, pyrazine). To present, more than 600 volatile compounds have been identified in cooked beef, of which acidic compounds have only minor effect on the aroma. Instead, thiophenes, furan, pyrazine compounds and pyridine compounds contribute largely to the characteristic aroma of cooked beef. The composition of the characteristic aroma of cooked pork is similar to that of beef, except that the contents of γor δ-lactones converted from 4 (or 5)-hydroxyl fatty acid precursors are higher than in cooked beef. Besides, cooked pork contains more unsaturated carbonyl and furan compounds than cooked beef. Because mutton contains less free fatty acids and unsaturated fatty acids than beef or pork, cooked mutton with less carbonyl compounds. The characteristic aroma of cooked chicken meat is provided by sulfide and carbonyl compounds, in which, carbonyl compounds, such as 2-trans-4-cis-decadien-1-al, are the most important active components. The aroma of boiled meats is provided mainly by neutral compounds, such as sulfides, furan-type and benzene-type compounds, while that of roasted meats is contributed mainly by basic compounds, such as pyrazine, pyrrole, pyridine, and carbonyl compounds. However, regardless of the processing method, sulfur compounds are the most important active components for meat aroma. If sulfur compounds are removed, cooked meats will lose their aroma. The content of hydrogen sulfide significantly affects the aroma of cooked meat. When the content is too high, the odor of rotten egg is perceived; however, when the content is too low, the aroma intensity is reduced. Smoked meats have unique aroma and tastes. The smoke used contains phenols, formaldehyde, acetaldehyde, acetone, cresol, fatty acids, alcohols, pyromucic aldehydes, and guaiacol, in which, fatty acids, phenols, and alcohols play important role in the formation of the unique taste and aroma of smoked meats. Lipids play an important role in the formation of the aroma of livestock meats. When tallow is heated, it is decomposed and generates abundant compounds, including esters, hydrocarbons, alcohols, carbonyl compounds, lactones, pyrazine and furan compounds, which

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263

are important active components of beef aroma. The same decomposition products have also been detected in heated lard. When pork is cooked at temperatures lower than 100 °C, flavor compounds derived from fat accounts for more than 50 % of total aroma compounds.

Aquatic Products Volatile compounds in fresh aquatic products. Generally, fresh marine fishes and freshwater fishes have only light odor and the odor is mainly provided by volatile carbonyl compounds and alcohols, including aldehyde (C6, C8, and C9), ketones and alcohols (such as 1-octene-3-one, 2-trans-nonyl aldehyde, cis-1-5octadiene-3-one and 1-octene-3-ol). These compounds are generated from the oxidation of unsaturated fatty acids by lipoxygenase. As the freshness decreases, the compositions of the odor change gradually and a characteristic fishy smell is perceived. This smell is caused by δamino-valeraldehyde, δ-amino-valeric acid and hexahydropyridine compounds in fish skin mucus, which are synthesized from basic amino acids. δ-Amino-valeraldehyde and δ-aminovaleric acid have strong fishy smell. Because fish blood also contains δ-amino-valeraldehyde, fish blood also smells fishy.

Volatile compounds in rotting fish. Rotted aquatic products have disgusted odor. Ammonia, dimethylamine (DMA), trimethylamine (TMA), methyl mercaptan, indole, skatole and fatty acid oxidation products are major constituents of the odor. All these compounds are alkaline and can be neutralized by acetic acid to eliminate the unpleasant odor. In fresh fish, ammonia can also be generated in the formation of inosine monophosphate (IMP) through the hydrolysis of adenine nucleotide (AMP) by AMP deaminase (Figure 8-14). As the rot proceeds, free amino acids, urea and proteins are decomposed to produce large amount of ammonia, such as the muscle of cartilaginous fishes contains high content of urea, which can be decomposed by microbial urease to produce ammonia and carbon dioxide (Figure 8-15). Hence, the fishes have strong smell of ammonia. TMA is a main representative compound that causes the unpleasant odor of rotten fish and its threshold is as low as 300-600 μg/kg. Fresh fishes contain trimethylamine oxide (TMAO) instead of TMA. TMAO is odorless and is important in maintaining the osmotic equilibrium between blood and tissues. It is found only in marine fishes. TMAO can be reduced to TMA under the action of enzymes or microbes (Figure 8-16). TMA has been used as an indicator of the degradation of unfrozen fishes. DMA and formaldehyde are also the decomposition products of TMAO (Figure 8-16). The odor intensity of DMA is lower than that of TMA.

264


Figure 8-14. Generation of ammonia in AMP hydrolysis.

Figure 8-15. Formation of ammonia from urea.

Figure 8-16. Formation of volatile amines in marine fishes [9].

Volatile sulfur compounds are always detected in degraded marine fishes, such as hydrogen sulfide, methyl mercaptan, dimethyl sulfide and diethyl sulfide. These compounds are also involved in the unpleasant odor of marine fishes. Marine fishes might exhibit the smell of oxidized fish oil or cod liver oil during storage due to the oxidation of polyunsaturated fatty acids. Linolenic acid, arachidonic acid and docosahexaenoic acid are the main unsaturated fatty acids in fish oil, their auto-oxidation decomposition products can cause unpleasant odor. The smell of the oxidation products depends on the degree of oxidation. In the early phase of oxidation, the products have the smell of cucumber. As the oxidation proceeds, the smell of cod liver oil appears.

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Food Flavors

FLAVOR COMPOUNDS FORMATION PATHWAYS Lipoxygenase-Catalyzed Reactions Lipid Oxidation Lipoxygenase occurs widely in plants and catalyzes the oxidation of polyunsaturated fatty acids. The produced peroxides are then decomposed by lyase to yield aldehydes, ketones, alcohols and many other flavor compounds. Hexanal, synthesized by linoleic acid as precursor (Figure 8-18.), is the flavor compound of apple, strawberry, pineapple, and bananas. Lipid oxidation can also generate undesirable flavors. For example, the oxidation of linoleic acid leads to the formation of the characteristic beany flavor of soybean. 2-trans-Hexenal and 2-trans-6-cis-nonadienol are the characteristic aroma compounds of tomato and cucumber, and both the compounds are synthesized with linolenic acid as precursor (Figure 8-19). Among the flavor compounds generated in the lipoxygenase-catalyzed pathway, C6 compounds has the fragrance of grass, C9 compounds generate aroma similar to that of cucumber and watermelon, and C8 compounds possess the smell of mushroom or violet. C6 and C9 compounds are generally aldehydes and primary alcohols and C8 compounds are often ketones and secondary alcohols. The pleasant aroma of mature pear, peach, apricot and other fruits is generally contributed by the β-oxidation products of long-chain fatty acid (C8-C12).

linoleic acid

lipoxygenase

peroxidelyase

hexanal

Figure 8-18. Formation of hexanal by the oxidation of linoleic acid.

+lipoxygenase +aldehyde lyase ++--yde lyase

oxygen acid

2-trans-hexenal

2-trans-6-cis-nonadienal aldehyde

Figure 8-19. Lipoxygenase catalyzed formation of aldehydes from long-chain polyunsaturated fatty acids [10].

266


Figure 8-20 β-oxidation of linoleic acid [11].

Figure 8-21. Pathways of aromatic compounds formation from leucine [11].

For example, 2-trans-4-cis-decadienoic acid ethyl ester is generated by the β-oxidation of linoleic acid (Figure 8-20) and it is the characteristic aroma compound of pear. The βoxidation of lipids also generates C8-C12 hydroxyl acids. These compounds can be cyclized to γ-lactones or δ-ketones by enzymes, of which, C8-C12 lactones have aroma similar to that of coconut and peach.

Degradation of Branched-Chain Amino Acids Branched-chain amino acids are important flavor precursors of mature fruits. The characteristic branched-chain carboxylic acid esters formed in the post-ripening process of banana, pear, kiwi fruit, and apples, such as isoamyl acetate and 3-methyl ethyl butyrate, are derived from branched-chain amino acids (Figure 8-21). Shikimic Acid Pathway Shikimic acid is the precursor of the three essential aromatic acids. In addition to its involvement in aromatic amino acids synthesis, it can also produce various volatile compounds, as shown in Figure 8-22:

267

Food Flavors

enzyme

eugenol

enzyme enzyme

p-cresol

cinnamyl alcohol lignin polymer 3-methoxy-4-hydroxybenzaldehyd(vanillin)

Figure 8-22. Volatile compounds produced in the synthesis of shikimic acid [12].

Figure 8-23. Structures of several important aromatic terpenoids.

In the synthetic pathway of shikimic acid, aromatic compounds (phenylalanine and other aromatic amino acid) can be produced from intermediate product in the pathway. Besides aromatic amino acid, the pathway can also form other volatile compounds related to essential oil. Aromatic components which are formed by fumigation of food, some of them are also formed from compounds in the shikimic acid pathway as precursors, such as vanillin. Cinnamyl alcohol is an important aroma component in cinnamon perfume. Eugenol is the main flavor and pungent ingredients in clove. Some important flavor compounds in shikimic acid pathway are showed in Figure 8-22.

Terpenoids Synthesis Terpenoids are important aromatic compounds of citrus fruits. Terpenoids containing two or more isoprene units are nonvolatile and do not directly involve in aroma sensation. Sesquiterpenes neral and ngcuka ketone are the characteristic aroma components of orange and grapefruit respectively. Citral and limonene are monoterpenes and possesses the unique smell of lemon and sour orange respectively. The enantiomers of terpenes may exhibit quite different smells. For example, l-carvone [4(R)-(-) carvone] has a strong aroma of spearmint, while d-carvone has the characteristic aroma of sweet wormwood (Figure 8-23).

268


Heterolactic Fermentation Pathway Microbial fermentation can yield abundant favor compounds and these compounds significantly influence the taste and aroma of dietary products and alcoholic beverages. Figure 8-24 shows the production of various flavor compounds in the heterolactic fermentation of glucose and citric acid. The products of microbial fermentation constitute the main body of the flavor of alcoholic beverages. The flavor of beer is influenced by the presence of alcohols, esters, aldehydes, ketones and sulfides, in which, isoamyl alcohol, α-phenyl ethanol, ethyl acetate, isoamyl acetate, and ethyl benzene are the most important active components. Acetaldehyde, diacetyl and hydrogen sulfide impart beer with the flavor of green apple and their contents must be reduced to allowed ranges in post-fermentation. The flavor of Chinese white spirit is affected by alcohols, esters, carbonyl compounds, phenols, and ethers. Aldehydes (mainly aldehyde) are predominant aroma compounds in newly distilled wines and these components make the wines tasted pungent. Furfural is usually not good for the flavor of wine, but it is an important component which constitutes the sauce flavor of Maotai wine and its content reaches up to 29.4 mg/L. Esters, especially the ethyl esters and iso-amyl esters of C2-C12 fatty acid, ethyl phenylacetate, ethyl lactate, and phenethyl acetate, are determinants for the flavor of Chinese white spirits.

Non-Enzymatic Reactions Maillard Reaction The Maillard reaction can yield a large number of aromatic compounds and their contents and proportion vary with substrate type, heating duration, and temperature. When the reaction occurs in a low temperature for a short time, aromatic l actones, pyran compounds and furan compounds as well as Strecker aldehydes are produced. When the reaction proceeds in high temperatures for a long time, pyrazine, pyrrole and pyridine compounds with baking aroma are formed. Pyrazine compounds are important flavor compounds in all bakery foods and thermally processed foods. It is generally believed that pyrazine compounds are generated through the Strecker degradation between amino acids and α-dicarbonyl compounds, which are the intermediates of the Maillard reaction (Figure 8-25). Small sulfides formed in the Maillard reaction also influenced the flavor of foods. For example, methionic aldehyde is the characteristic aroma compound of boiled potatoes and cheese biscuits. Methionic aldehyde is unstable and can be easily decomposed into methane thiol and dimethyl disulfide, resulting in the increase of low molecular weight sulfides. The thermal degradation produces H2S and NH3 are also involve in the flavor formation through Maillard reaction. For example, H2S, NH3, and acetaldehyde are the thermal degradation products of cysteine. They can react with hydroxy ketones formed in the Maillard reaction to generate thiazoline, which has the flavor of cooked beef (Figure 8-26).

269

Food Flavors

Figure 8-24. Formation of volatile compounds in the heterolactic fermentation of citric acid and glucose.

Figure 8-25. Formation of an alkyl pyrazine and small sulfur compounds through the Maillard reaction [13].

cysteine

3-hydroxyl-2-butanone Fig. 9-24. Methionine reacts with carbonyl compounds to form thiazoline

2,4,5-trimethyl-3-thiazoline

Figure 8-26. Formation of a thiazoline through the reaction between the thermal degradation products of cysteine and carbonyl compounds [13].

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Thermal Degradation Reaction Thermal decomposition of carbohydrates, proteins and fats. Carbohydrates can be decomposed by heat in the absence of amines to produce a serious of flavor compounds. The thermal decomposition of monosaccharides and disaccharides produce mainly furan compounds, accompanied by a small proportion of lactones, cyclodiones and other substances. The compounds can be further decomposed to methylglyoxal, glyceraldehyde and other low molecular weight volatile compounds. Starch, cellulose and other polysaccharides are decomposed to furan compounds, furfural compounds, maltol, as well as organic acids at temperatures below 400 °C. The thermal degradation products of proteins and amino acids include hydrogen sulfide, ammonia, pyrrole compounds, pyridine compounds, thiazole, thiophene, and many other sulfur-containing compounds. Most of the products have strong smells. For the thermal degradation of lipids, please refer to related contents in Chapter 4. Degradation of vitamin. When vitamin Bl is heated, it is decomposed and produces a large number of sulfurcontaining compounds, furan and thiophene and some of the products have meat flavor. Ascorbic acid is unstable. When it is heated in the presence of oxygen, it is degraded to yield furfural, glyoxal, glycerol aldehyde and other low-molecular aldehydes, of which furfural compounds are important constituents of the aroma of cured tea, peanut and cooked beef. Fat oxidation. The non-enzymatic oxidation of lipids can cause rancidity. Nevertheless, moderate oxidation of lipids imparts foods with desired flavor, such as in breads. Please refer to Chapter 4 for the mechanism of lipid oxidation.

REFERENCES [1] Shallenberer, RS; Acree, TE. Molecular theory of sweet taste. Nature, 1967, 216, 480482. [2] DeTaeye, L; DeKeukeleire D; Siaeno E; Verzele M. Recent developments in hop chemistry. In European Brewery Convention Proceedings. Amsterdam: European Brewing Congress, 1977; 153-156. [3] Morgan, ME; Libbey, LM; Scanlan, RA. Identity of the musty-potato aroma compound in milk cultures of Pseudomonas taetrolens. Journal of Dairy Science, 1972, 55, 666 [4] Whitfield, FB; JH. Last Vegetables. In: Maarse, H. Volatile Compounds in Foods and Beverages. New York: Marcel Dekker, 1991; 203-269. [5] Shankaranarayana, ML; Raghaven, B; Abraham, KO; Natarajan, CP. Sulphur compounds in flavours. In: Morton, ID; Macleod, AJ. Food Flavours, Part A, Introduction. Amsterdam: Elsevier Scientific, 1982; 169-281.

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[6] Govindarajan, VS. Pungency: the stimuli and their evaluation. In: Boudreau, JC. Food Taste Chemistry. Washington DC: American Chemical Society, 1979; 52-97. [7] Hiraider, M; Miyazaki, Y; Shibata, Y. The smell and odorous components of dried shiitake mushroom, Lentinula edodes I: relationship between sensory evaluations and amounts of odorous components. Journal of Wood Science, 2004, 50, 358-364. [8] Gower, DB; Hancock, M Bannister, LH. Biochemical studies on the boar pheromones, 5a-androst-16-en-3-one and 5a-androst-16-en-3a-ol, and their metabolism by olfactory tissue. In: Cagan, RH; Kare, MR. Biochemistry of Taste and Olfaction. New York: Academic Press, 1981; 7-31 [9] Hebard, CE; Flick, GJ; Martin. Occurrenceand significance of trimethylamine oxide and its derivatives in fish and shellfish. In: Martin, RE; Flick, GJ; Ward, DR. Chemistry and Bilchemistry of Marine Products. Westport: AVI Publishing, 1982; 149-304. [10] Blank, I; Lin, J; Vera, FA; Weli, DH; Fay, LB. Identification of potent odorants formed by autoxidation of arachidonic acid: structure elucidation and synthesis of (EZZ)-2,4,7tridecatrienal. Journal of Agricultural and Food Chemistry, 2001, 49, 2959-2965. [11] Tressl, R; Holzer, D; Apetz, M. Biogenesis of volatiles in fruit and vegetables. In: Maarse, H; Groenen, PJ. Aroma Research: Proceedings of the International Symposium on Aroma Research, 1975 in Zeist, the Netherlands. Wageningen: Centre for Agricultural Publishing and Documentation, 1975; 41-62. [12] Wittkowski, R; Ruter, J; Drinda, H; Rafiei-Taghanaki, F. Formation of smoke flavor compounds by thermal lignin degradation. In: Teranishi, R; Takeoka, GR; Guntert, M. Flavor Precursors: Thermal and Enzymatic Conversions. Washington DC: American Chemical Society, 1992; 232-243. [13] Mussinan, CJ; Wilson, RA; Katz, I; Hruza, A; Vock, MH. Identification and some flavor properties of some 3-oxazolines and 3-oxazolines and 3-thiazolines isolated from cooked beef. In: Charalambous, G; Katz, I. Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors. Washington DC: American Chemical Society, 1976; 133-145.



Chapter 9

FOOD ADDITIVES 1

Linwei Liu1 and Shiyuan Dong2

Northwest AandF University, Yangling, Shaanxi, China College of Food Science and Engineering, Ocean University of China, Qingdao, China

2

ABSTRACT Food additives are substances intentionally added by manufacturers to foods to preserve flavor or enhance taste and appearance. A substantial amount of food additives with different functions have been widely used in the food industry and these compounds contribute largely to the development of the industry. Due to the increased concern on food safety and the rapid development of analysis techniques, the definition of food additive has evolved greatly. This chapter firstly describes the evolution of the definition of food additives. Then, various food additives are introduced briefly according to their classification and the functions, properties and related aspects of important additives are described one by one.

1. INTRODUCTION 1.1. Definition Food additives are regulated substances and therefore defined in law. The definition of food additives was firstly proposed by the FAO/WHO Joint Expert Committee for Food Additives (JECFA) in 1955 as ―non-nutritive substances added intentionally to food, generally in small quantities, to improve its appearance, flavor, texture, or storage properties‖. This definition covered a rather narrow range and did not include flavorings and nutrients. Since then, the definition evolves gradually and various definitions have been proposed by different countries or organizations. According to the Chinese Hygienic Standards for the Use of Food Additives (GB27602007), food additives refer to ―artificially chemosynthetic or natural substances to be added to foods in order to improve food quality and color, flavor and taste, and meet the need of

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preservation and processing technology. Nutritional fortification substances, flavoring agents and processing aids, are also included in this definition as well‖. The official definition of food additives in the European Economic Community (EEC) is ―any substance does not normally consumed as a food in itself and not normally used as a characteristic ingredient of food, whether or not it has nutritive value, the intentional addition of which to a food for a technological purpose in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food results, or may be reasonably expected to result, in it or its by-products becoming directly or indirectly a component of such foods‖. Federal Food Drug and Cosmetic Act, US (FFDCA) defines food additives as ―any substance the intended use of which results or may reasonably be expected to result, directly or indirectly, in its becoming a component or otherwise affecting the characteristics of any food, including any substance intended for use in producing, manufacturing, packing, processing, preparing, treating, packaging, transporting, or holding food, if such substance is Generally Recognized As Safe (GRAS)‖. Though the definitions vary, they specify three common functions for food additives. Firstly, food additives improve food quality and meet consumers‘ requirements on flavor, color, and taste. Secondly, food additives make food processing more reasonable, more hygiene, more convenient and enhance the mechanized, automated, and scaled production of foods. Thirdly, food additives contribute to saving resources, reducing cost, and providing significantly social and economic benefits.

1.2. Classification According to origin or source, food additives are divided into natural and artificial ones. Food additives are also classified by functions in most cases. In China, food additives are subdivided into 21 categories, including acidulant, anticaking agent, antifoaming agent, antioxidant, bleaching agent, bulking agent, chewing gum base, coloring agent, color fixative, emulsifier, enzyme preparation, flavor enhancer, flour treatment agent, coating agent, water retention agent, nutrition enhancer, preservative, stabilizing and coagulating agent, sweeter, thickener and others. Among the 1500 kinds of food additives approved for use in China, more than 700 kinds are spices and essence oils.

1.3. Regulation of Food Additives To guarantee food safety, use of food additives is strictly regulated by national and international laws. According to the Chinese Hygiene Standards for Use of Food Additives (GB2760-2007), the use of food additives must comply with the following principles: 1. Food additives must be nontoxic, do not generate toxic compounds upon decomposition and do not cause chronic poisoning symptoms after long-term intake of dose allowed. 2. Food additives do not destroy the nutritional components of foods, reduce food quality, or generate toxic compounds upon decomposition. 3. Food additives can not be applied for the purpose of adulteration or for shielding the facts of food spoilage and food deterioration.

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4. Food additives cannot be applied for the purpose of concealing the quality deficiency caused by the food itself or by processing. 5. The application ranges, dosages, and residues of food additives must comply with related national standards and regulations, and try to minimize the using amount to bring about the desired result. 6. Processing aids must be removed after processing, except those with allowed residues.

2. ACIDULANTS 2.1. Functions Acidulants or acidity regulators are some acids and their salts, which serve a variety of functions shown as the following: (1) Flavoring to provide a desired taste and serve to intensify, enhance, blend of modify the overall flavor of the product. (2) Reduction of the pH to prevent or retard the growth of microorganisms and the germination of spores, and to increase the lethality of the process. (3) Maintainer or establishment of pH by serving as buffering agents. Usually a combination of free acids and salts are used. (4) Chelation of metal ions (Cu, Fe) to assist in minimizing lipid oxidation, reducing color changes and controlling texture in some fruits and vegetables. (5) Alteration of the structure of foods including gels made from gums (pectin, carrageenan), and proteins. (6) Interaction with proteins and emulsifiers to modify the structure of foods such as doughs, alter the heat stability of proteins, and to serve as an emulsifier in processed cheese. (7) Modification of sugar crystallization in hard candy manufacturing.

2.2. Acidulants The usual use of acidulants in food are acidic, phosphoric, citric, malic, succnic, tartaric, lactic, gluconic, glycolic, fumaric and adipic acid. The pKa or pKa1 of them are 4.75, 2.1, 3.08, 3.4, 4.2, 3.2, 3.86, 3.60, 3.03 and 4.43, respectively. Their structures are shown in Figure 9-1: The differences between acidulates are flavor, acidity, metal chelating and antimicrobial activity, solubility, hydroscopicity and cost. Citric, malic, tartaric and gluconic acids have similar taste which compliant with making beverage and candy. But for cola drink, using phosphoric acid is more suitable. Fumaric and acitic acid have good antimicrobial activity, and acetic acid (vinegar) is an important condiment and functional agent for Chinese food, mayonnaise and pickles. Tartaric acid is often used to make leavening (bulking agent) owing to its lower solubility.

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Figure 9-1. Sketch of structures of some acidulants added in food.

Gluconolactone can be used to make Doufu and also used as a ingredient for making the leavening because it can change to gluconic acid gradually in hot water. Lactic acid has special taste, which compliant with many dairy products and fermented food, such as yoghourt and alcohol. Phosphoric, citric, malice, succinct and tartaric acids are metal chelating agents, they can be used as antioxidant synergist.

3. SWEETENERS 3.1. Type and Functions There are two types of sweeteners: caloric (nutritive) and noncaloric (non-nutritive). The caloric sweeteners provide about 4 calories per gram. The noncaloric varieties provide zero calories. Caloric sweeteners provide sweet flavor and bulk when added to food. They also maintain freshness and contribute to product quality. Caloric sweeteners act as a preservative in jams and jellies, and a flavor enhancer in processed meat. They provide fermentation for breads and pickles, bulk to ice cream, and body to carbonated beverage. Some caloric sweeteners are rich in natural resources, e.g., sucrose and fructose. Some are mainly made by processing sugar or other small molecule compounds, e.g., molasses and sugar alcohols. Sugar alcohols are low glycemic sweetener that is safe for diabetics, and helps manage healthy glucose levels, and does not promote tooth decay. Non-caloric sweeteners are used in place of caloric sweeteners in some foods. They do not provide calories, but they do provide the sweet taste. Most non-caloric sweeteners are chemically sythenic. Some of them, e.g., saccharin, acesulfame K and sucralose are more stable than sugar to high temperature and other conditions of food processing, e.g., they do not go in for non enzyme browning. Almost all of them can use in the diet for diabetics and obesity patients.

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3.2. High Fructose Corn Syrup (Hfcs) This sweet enhancer is derived from cornstarch and is a mixture of 55% fructose and 45% sucrose. Starches are treated with enzymes that convert glucose to fructose, which results in a sweeter product. The sweeters are used in many mass-produced foods, including soft drinks, baked goods, jelly, syrups, condiments (like ketchup), fruits and desserts.

3.3. Sugar Alcohols Sugar alcohols belong to polyols family which has many functions in food, such as increasing viscosity, modifying sugar crystallization, being water binder, plasticizer, cryoprotectant, and non-cariogenic sweetener. In China and many other countries, xylitol, maltitol, sorbitol and mannitol are allowed to add into food. Xylitol is odorless and a white crystalline powder with a pleasant and sweet taste, and its energy value is 16.72 kJ/g. It has the same sweetness and bulk as sucrose with one-third fewer calories and no unpleasant aftertaste. It quickly dissolves, produces a cooling sensation in the mouth and reduces the development of cavities. Xylitol occurs naturally in many fruits and vegetables and is even produced by the human body during normal metabolism. It is produced commercially from plants such as birch and other hard wood trees and fibrous vegetation, or from xylose hydrogenation. It can be used into any food with the amount as the required of food making because its safety level is high. Maltitol (4-O-α-glucopyranosyl-D-sorbitol) has 75-90% of the sweetness of sucrose and its food energy value is 8.8 kJ/g. It is used to replace table sugar because it has fewer calories, and does not promote tooth decay and has a somewhat lesser effect on blood glucose. Maltitol is made by hydrogenation of maltose obtained from starch. It is especially used in production of sweets: sugarless hard candies, chewing gum, chocolates, baked goods, and ice cream. Its similarity to sucrose allows it to be used in syrups with the advantage that crystallization is less likely. Maltitol may also be used as a plasticiser in gelatine capsulesand as a humectant.http://en.wikipedia.org/wiki/Maltitol - cite_note-1#cite_note-1 Maltitol does not brown and caramelize after liquifying by exposing to intense heat. It is somewhat more slowly absorbed than sucrose which makes it somewhat more suitable for people with diabetes than sucrose. It can use with quantity stipulated by use standard for different food categories. Sorbitol, also known as glucitol, is a sugar alcohol that the human body metabolises slowly. It is obtained by the reduction of glucose changing the aldehyde group to an additional hydroxyl group. It also occurs naturally in many stone fruits and berries from trees of the genus sorbus. It provides dietary energy 2.6 kJ per gram. and is often used in diet foods including diet drinks and ice cream, mints, cough syrup, and sugar-free chewing gum. Sorbital sometimes is used as a sweetener and humectant in cookies. Ingesting large amounts of sorbitol can lead to abdominal pain, gas, and mild to severe diarrhea. In China, the maximum level of use to each food is 5.0 g/kg. Mannitol is also used as a sweetener for diabete patient. Since mannitol has a positive heat of solution, it is used as a sweetener in "breath-freshening" candies, the cooling effect contributing to the fresh feel. The pleasant taste and mouth feel of mannitol also make it as a popular excipient for chewable tablets. It can be used a little in food coating for flavor.

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3.4. ISO-Maltulose Iso-maltulose, also known by the trade name Palatinose, is a disaccharide that is commercially manufactured from sucrose hydrolysed via bacterial fermentation. It is a natural constituent of honey and sugar cane and has a very natural sweet taste. It is particularly suitable as a non-cariogenic sucrose replacement. Iso-maltulose is fully absorbed in the small intestine as glucose and fructose. Like sucrose, it is fully digested and provides the same caloric value of approximately 4 kcal/g. However, it is low-glycemic and low-insulinemia. Because isomaltulose is released to the blood slowly, this sweetener avoids the sudden increase of drop of blood glucose level. This leads to a more balanced and prolonged energy supply in the form of glucose. In China, iso-maltulose is allowed to use in many foods and the quantity of use is decided by the needing of food manufacture.

3.5. Noncaloric Sweeteners There are several noncaloric sweeteners, such as saccharin, aspartame, acesulfame K, and sucralose and their chemical structures are shown in Figure 9-2: (1) Saccharinhttp://en.wikipedia.org/wiki/Saccharin - cite_note-1#cite_note-1 is a colorless, crystal and artificial sweetener. The basic substance, benzoic sulfimide, has effectively no calories and is many times sweeter than sucrose (about 300-700 times as sucrose) [1], but has an unpleasant bitter or metallic aftertaste, especially at high concentrations. In countries where saccharin is allowed as a FD, it is used to sweeten products such as drinks, candies, medicines, and toothpaste. In China, the maxium usage quantity of saccharin for food is 0.15 g/kg. Many studies have been performed on saccharin, some showing a correlation between saccharin consumption and increased frequency of cancer in rats (especially bladder cancer) and others finding no such correlation. No study has ever shown a clear causal relationship between saccharin consumption and health risks in humans at normal doses, though some studies have shown a correlation between consumption and cancer incidence. (2) Aspartame is 200 times sweeter than sugar in typical concentrations. Aspartame has a caloric value of 17 kJ per gram, so the minimum quantity of aspartame which needs to produce a sweet tast is so small that its caloric contribution is negligible. Therefore, it is a popular sweetener for those trying to avoid calories from sugar. The taste of aspartame is not identical to that of sugar. Blends of aspartame with acesulfame K taste more like sugar, and to be sweeter than either substitute used alone. Like many other peptides, aspartame could be hydrolyzed into its constituent amino acids under conditions of high temperature or pH. This makes aspartame undesirable as a baking sweetener.. At room temperature, it is the most stable at pH 4.3, so that it is reasonably stable in soft-drinks. In the powdered beverages, the amine in aspartame can undergo Maillard reaction with the aldehyde groups present in certain aroma compound. The safety of aspartame has been studied thoroughly, and the scientific evidence indicates it is safe at current level of consumption as a non-nutritive sweetener. (3) Acesulfame K, also known as cesulfame potassium, is a white crystalline powder. It is 180-200 times sweeter than sucrose with a slightly bitter aftertaste, especially at high concentrations.

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Figure 9-2. Structures of some noncaloric sweeteners.

Acesulfame K is often blended with other sweeteners (usually sucralose or aspartame) which give a more sugar-like taste, and each of them masks the other aftertaste. It is stable under heat even under moderately acid or basic conditions, allowing it to be used in baking, and it has been also used in beverages, tabletop sweeteners, desserts, puddings, baked goods, soft drinks, dairy products, candies and canned foods to replace part sucrose. Research shows that acesulfame K is safe to consume if the concentration in any edible form is less than 3%. (4) Sucralose is approximately 600 times as sweet as sucrose (table sugar), twice as sweet as saccharin, and 3.3 times as sweet as aspartame. Unlike aspartame, it is stable under heat and over a broad range of pH conditions. Therefore, it can be used in baking or products that require a longer shelf life. The commercial success of sucralose-based products stem from its favorable comparison to other low-calorie sweeteners in terms of taste, stability, and safety. In China, the maxium usage quantity of sucralose for many foods is 0.25 g/kg or less. (5)There are other noncaloric sweeteners being used in different countries. Sodium cyclamate is a water soluble chemical powder. It is 30 times as sweet as sucrose and stable in most conditions of food processing. It is allowed to use in cold drink and pickle with the ML equal to 0.65 g/kg. Alitame, made from amino acids, is an artificial sweetener about 2000 times sweeter than sucrose and has no aftertaste. It is relatively stable. Alitame was approved for use in Mexico, Australia, New Zealand and China. Leaves of Stevia rebaudiana are a source of several sweet glycosides of steviol. The major glycoside, stevioside, diterpenoid glycoside are used in oriental countries as food sweetener. Its medical use is also reported as a heart tonic. Besides, it is used against obesity, hypertension, and stomach burn and to lower uric acid levels.

4. PRESERVATIVES 4.1. Definition Preservatives or antimicrobial agents play an important role in today's supply of safe and stable foods. Preservative means any substance which is capable of inhibiting, retarding or arresting the process of fermentation, retarding acidification or other deterioration of food or masking any of the evidence of putrefaction. Preservatives work by preventing the growth of

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microbes (fungi, bacteria) or by killing the microbes. Food preservatives, low toxicity, are commonly used in food processing. They should not pose significant health effects on consumers during normal consumption.

4.2. Use and Limitation To prolong the shelf-life of food against deterioration caused by microorganisms, the preservatives are indispensable in the food industry. In several decades, increasing demand for convenient foods and reasonably long shelf life of processed foods makes the use of chemical food preservatives imperative because they are more effective and cheaper than biopreservative. Traditional preservatives, such as wood smoke or vinegar, have been used for centuries. However, they are not always safe. Smoking may introduce carcinogenic materials into the food. Some of the commonly used preservatives, belong to inorganic chemicals, such as sulfites, nitrate, and salt have also been used for centuries in processed meats and wine. The use quantity of them are limited strictly to avoid their harmful affects on human health. Many bio-preservatives are produced by microorganism; a few others are made from plant. They also have some unhealthy effects on people. Since there are not any preservatives being using in food without any danger, some countries have set up their mandatory use standard of food preservatives. The using levels must be obeyed the items of the standards to minimize risk.

4.3. The Choice of Preservative The choice of antimicrobial agent has to be based on the antimicrobial spectrum of the preservative, the chemical and physical properties of both food and preservative, the conditions of storage and handling, and the assurance of a high initial quality of the food to be preserved. Many preservatives have broad bacteriostasis spectrum, but some others can only affect on some species of microorganism. Salts of acids, such as sodium acetate, calcium citrate, sodium benzoate, calcium propionate, and sodium nitrite all have antimicrobial properties. Sulfur dioxide can be derived from a various sulfur additives such as bisulfite, and produce a multitude of effects in food. Most preservatives only have antimicrobial effects at certain range of pH because their states can be changed with the change of pH. Some preservatives are unstable in high temperature, and some are dissoluble in water or oil. Therefore, the user must know those properties before using them. Table 9-1 shows properties of some common preservatives.

4.4. Preservatives in Common Use 4.4.1. Benzoic Acid Benzoic acid naturally occurs in many types of berries, plums, prunes, and some spices. As an additive, it is used as benzoic acid or as benzoate.

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Food Additives Table 9-1. Properties of some preservatives [2,3] Compound Benzoic acid (benzoate) Sorbic acid (sorbate) Parabens Dehydroacetic acid Sodium propionate Sulfites Nitrites

Effective against Yeast, mold, bacteria Yeast, mold, bacteria, Bacteria,mold, yeast Mold,yeast, bacteria Mold

Effective pH range 2.5-4.0

Bacteria,mold, yeast Bacteria,mold, yeast

2.5-5.0

Water easily, Oil slightly Water easily, Oil slightly Ethnol easily, Water slightly Ethnol easily, Water slightly Water easily, Ethnol easily, Water easily

4.0-5.5

Water easily

3.0-6.5 3.0-9.0 3-10 6-8

Solubility

Some food applications Jam,juice,sauce Meat, juice, bread Sauce, beverage, baking product Pickles, orange pulp Baking product, meat Wine, dried fruits Meat

The latter is used more often because benzoic acid is sparsely soluble in water, and sodium benzoate is more soluble. The undissociated form on benzoic acid is the most effective antimicrobial agent. The optimum pH range ranges from 2.5 to 4.0, which makes it an effective antimicrobial in highly acidic foods. Benzoic acid and sodium benzoate are widely used in fruit drinks, cider, carbonated beverages, sauce, jams and pickles with 1.0 g/kg or less as the ML for different foods.

4.4.2. Sorbic Acid and Potassium Sorbate Sorbic acid is a straight-chain, trans-trans unsaturated fatty acid, 2, 4-hexadienoic acid, and it has a low solubility in water at room temperature. The salts of sorbic acid, such as sodium, or potassium are more soluble in water. Sorbates are stable in the dry form; they are unstable in aqueous solutions because they decompose through oxidation. The rate of oxidation increases at low pH, high temp, light exposure. Sorbic acid and other sorbates are so effective against yeasts and molds that they can be used in many foods, such as wine, fruit juice, dried fruit, cottage cheese, meat, and fish products. Sorbates are most effective in products with low pH including salad dressings, tomato products, carbonated beverages. In China, its ML in most foods is 1.0 g/kg. 4.4.3. Propionic Acid, Sodium Propionate and Calcium Propionate Propionic acid and its salt are effective against molds, but no effect on yeast. These additives are mainly used in baked foods. The calcium salt is the most popular form used. In China, its ML in many foods is 2.5 g/kg [4]. 4.4.4. Sulfites Sulfur dioxide and sulfites have been used as preservatives serving both as antimicrobial substance and antioxidant. Instead of sulfur dioxide solutions, a number of sulfites, such as sodium bisulfite, sodium metabisulfite, sodium sulfite, potassium bisulfite, sodium hydrogen sulfite, sodium hyposulfite and potassium metabisulfite can be used. When dissolved in water, they all yield active SO2 (see the following equations)[3]. The most widely used of these sulfites is potassium metabisulfite.

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Linwei Liu and Shiyuan Dong SO2(gas) ↔ SO2(aq) SO2(aq) ↔ H2O + H2SO3 H2SO3 ↔ H+ + HSO3HSO3- ↔ H+ + SO322HSO3- ↔ S2O52- + H2O

All of these forms of sulfur are known as free sulfur dioxide. Meanwhile, the bisulfite ion (HSO3-) can react with aldehydes, dextrins, pectic substances, proteins, ketones, and certain sugars to form addition compounds, which are known as bound sulfur dioxide. Sulfurous acid inhibits molds and bacteria to a lesser extent yeasts. For this reason, SO2 can be used to control undesirable bacteria and wild yeasts in fermentations without affecting the SO2- tolerant cultured yeasts. The antiseptic activity of SO2 highly depend on the pH. The lower the pH, the greater the antiseptic action of SO2. The amount of SO2 added to foods is self-limiting because the product may develop an unpleasant off-flavor at concentration from 200 to 500 ppm. The use of SO2 is not permitted in foods that contain significant quantities of thiamine, because this vitamin is destroyed by SO2. Because SO2 is volatile and easily lost to the atmosphere, the residual levels may be much lower than the amounts originally applied. In China, the ML of sulfites are calculated by means of the residual levels in the finished food. Different food has different maxium residual quantity. For example, the maximum residual quantities for fresh and dried fruits processed are 0.05 g/kg and 0.2 g/kg, respectively.

4.4.5. Natamycin Natamycin (Figure 9-3) is a naturally occurring antifungal agent produced by Streptomyces natalensis. Natamycin has a very low solubility in water; however, it is effective at very low levels. Natamycin has been used for decades in the food industry as a hurdle to fungal outgrowth in dairy products, meats, and other foods. It has a neutral flavor impact, and less dependence on pH for efficacy. It may be applied by spraying a liquid suspension, by dipping the product in an aqueous suspension, or by mixing it into the product in a powdered form along with cellulose on whole, shredded, or soft cheeses. 4.4.6. Nisin Nisin is an antimicrobial polypeptide, with a molecular weight of 3500, produced by some strains of Lactococcus lactis. Nisin-like substances are wide products from lactic acid bacteria.

http://upload.wikimedia.org/wikipedia/commons/a/a3/Natamycin.svg Figure 9-3. Structure of natamycin [5].

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These inhibitory substances are known as becteriocins. Nisin can be used as a processing aid against gram-positive organisms. It has been effectively used in preservation of processed cheese. It is also used in the heat treatment of nonacid foods and in extending the shelf life of sterilized milk [6].

5. ANTIOXIDANTS 5.1. Definition and Classification Food antioxidants in the broadest sense are all of the substances that have some effects on preventing or retarding oxidative deterioration that leads to rancidity, loss of flavor, color and nutritive value of foodstuffs in foods. Antioxidants can be classified into a number of groups: (1) Primary antioxidants which can terminate free radical chains reactions and function as electron donors including the phnolic antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butyl hydroquinone (TBHQ), propylgallate (PG), natural and synthetic tocopherols and so on. (2) Oxygen scavengers which can remove oxygen in a closed food system. Most widely used compounds are Vitamin C, and related substances, ascorbyl palmitate, and erythorbic acid (the D-isomer of ascorbic acid). (3) Chelating agents or sequestrants. They remove metallic ions, especially copper and iron, that are powerful pro-oxidants. Citric acid is widely used for this purpose. Amino acids and ethylene diamine tetraacetic acid (EDTA) are examples of chelating agents. (4) Enzymatic antioxidants which can remove head space oxygen dissolved, such as glucose oxidase. Superoxide dismutase can be used to highly remove oxidative compounds from food systems. (5) Natural antioxidants which are present in many spices and herbs. Rosemary and sage are the most potent antioxidant spices.

5.2. Mechanism of Antioxidation Primary antioxidants are mostly phenolic compounds. They can react with free radicals, which scavenge the active radicals and change themselves into lower active radicals. The lower active radicals can react each other or with other radicals until becoming stable molecules. That is why they can terminate free radical chains reaction, which is the main oxidation process of lipids. Phenolic compounds are a mixture and in combination with a chelating agent because different antioxidants have the synergistic effect. This kind antioxidant should be added into food before serious oxidation has occurred and used with small quantity. Otherwise, it may act as a pro-oxidant. For example, phenolic radicals can promote ROOH decomposition to produce ROOO. ROO

AH 2

ROO AH AH AH

ROOH ROOH A AH 2

AH A

Chelating agents which have ability to bind metal ions have contributed significantly to stabilization of food color, aroma and texture because traces of heavy metal ions can act as

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catalysts for lipid oxidation. Many natural constituents of food can act as chelating agents, e. g., carboxylic acids (oxalic, succinic), hydroxy acids (lactic, malic, tartaric, citric), polyphosphoric acids (ATP, pyrophosphates), amino acids, peptides, proteins and porphyrins. Both ascorbic acid and glucose plus glucose oxidase and catalase can deplete O2 in food system. When they react with oxygen, ascorbic acid becomes to dehydroascorbic acid, and glucose becomes to gluconic acid, and H2O2, another product of the oxidation of glucose, can be broken down to water by catalase. When the oxygen has been depleted completely, the food oxidation will stop. However, owing to VitC, oxidation is based on a complex mechanism with some active substance giving birth to the midway of the reaction, adding less quantity of VitC will prooxidant.

5.3. Common Antioxidants The common used antioxidants in food are tocopherols, ascorbic acid, ascorbyl palmitate, sodium isoascorbate, tert-butylhydroxyanisole (BHA), di-tert-butylhydroxytoluene (BHT), tertiary butyl hydroquinone (TBHQ), propylgallate (PG), tea polyphenols, antioxidant of glycyrrhiza, and so on. The structures of some of the antioxidants are shown in Figure 9-4. Tocopherols and ascorbic acid are two vitamins, and their use in food have no danger but expensive. Tocopherols and ascorbic acid, which are supplyed either natural or synthetic equivalents, are less restricted in food using. In China, they can be used in food with any quantities as the needs for food process. Ascorbyl palmitate, a derivative of ascorbic acid, has good solubility in oil, so as to be a wide use as antioxidant which contains abundant oil or fat. Sodium isoascorbate has almost the same function as ascorbic acid except it is not a vitamin and cheaper than ascorbic acid. The use standard of FDs of China permit those antioxidant to be added into many food with ML being 0.2-0.5 g/kg. BHA, BHT and PG have been used, with powerful antioxidant function, for many years in most countries. The first two are typical lipophilic artificial antioxidants and the last one has some polarity. The first two are very stable during food process, but PG is unstable at high temperature and discolour in presence of iron and metal complexes. The use of these synthetic antioxidants is very restricted. The ML of them in most food categories is 0.1 g/kg according to hygienic standards for uses of FDs in China. TBHQ is more stable and less volatile than BHA at high temperature. TBHQ gives excellent antioxidant potency to oils and fats.

Figure 9-4. Structure of some commercial antioxidants.

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It can maintain freshness and quality of crude oils during long distance transportation, and can offer protection to fried foods, thus enhancing their storage life and freshness. The highest level of TBHQ permitted in the GSFA (USA) is 1 g/kg for frozen fish, fish fillet, and fish products, and in the FDs use standard (China) is 0.2 g/kg for edible oils and fats, fried food, curing meat class, and dried fishes. It is often used in conjunction with BHA, BHT and PG to provide a synergistic antioxidant effect. Tea polyphenols, antioxidant of glycyrrhiza and antioxidant of bamboo leaves are natural antioxidants being extracted from tea, liquorice and bamboo. Tea polyphenols are water soluble. Antioxidant of glycyrrhiza is unsoluble in water but in ethanol and ethyl acetate. The antioxidant of bamboo leaves is soluble in ethanol. They are phenols substance, such as catechins, flavonoids and phenolic acids. The ML of TP, AG and ABL in the FDs use standard (China) are 0.3, 0.2 and 0.5 g/kg, respectively. Rosemary extract is another natural antioxidant. The active ingredients are ursolic acid, rosmarinic acid, conorsic acid, and conorsol. The ML of it in the FDs use standard (China) is 0.3 g/kg. EDTA is abbreviation of disodium ethylene-diamine-tetraacetate, a famous chelating agent in analysis chemistry. Phytic acid (inositol hexaphosphoric acid), orthophosphoric acid, pyrophosphoric acid, triphosphoric acid, hexameta-phosphoric acid, tartaric acid, citric acid, gluconic acid, and their salts are also good chelating agents. All of those chelating agent have certain ability of antioxidant. In the use standard of FDs of China, the ML of EDTA and phytic acid are 0.07 and 0.2 g/kg repectivesly. The orthophosphoric acid, pyrophosphoric acid, triphosphoric acid, hexameta-phosphoric acid are classified also as water retention reagents which can be use with high levels as 3-5 g/kg. The other chelating agents meationed above can be used in food without level limits.

6. EMULSIFIERS AND STABILIZERS 6.1. General Description The purpose of emulsifiers and stabilizers is mix together of different ingredients that normally would not mix, namely fat and water. This mixture of the aqueous and lipid phases is then maintained by stabilizers. For example, these additives are essential in the production of mayonnaise, chocolate products and fat spreads. The manufacture of fat spreads (reducedfat substitutes for butter and margarine), has made a significant contribution to consumer choice and dietary change, which would be impossible without the use of emulsifiers and stabilizers. Other reduced and low-fat versions of a number of products are similarly dependent on this technology. In addition to this function, the term stabilizers is also used for substances that can stabilise, retain or intensify an existing colour of a foodstuff and substances that increase the binding capacity of the food to allow the binding of food pieces into reconstituted food. Some emulsifier and a few stabilizers used in food are synthetic compounds, while many of them are natural substances. Hygeian use of these additives need to be known and controlled their negative effects on people health. Although almost each of them is generally recognized as safe (GRAS), some of them still strict the level of use. Moreover, the increasing awareness of problems with food allergy and intolerance has led to the requirement to state the source of certain emulsifiers on food labelling. For example, lecithin derived from soya is

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not suitable for an individual with an allergy to soya, so labelling of the source of the ingredient is vital to aid in consumer choice in products safe for individuals with specific dietary requirements.

6.2. Principle of Emulsifying and Stabilization When two immiscible liquids, e.g., oil and water are mixed by stirrer throughly together, there are great interface appearing between them. Because the surface tension of the interface send up the free energy of the system greatly, the system, named as disperse system, is high unstable and will separate water and oil quickly. Emulsifier, an amphiphilic substance, has usually a hydrophilic head and a hydrophobic tail. When emulsifier moleculars are adsorbed to the interface, the head and the tail will point into the water and oil respectively, which will decrease the surface tension and increase stability of the disperse system. If water, oil and emulsifier are mixed together, the disperse system will formed much easier than before. That is why emulsifier has the funtion to make and stablize emulsion and other disperse system. Stabilisers may have a little emulsifier's function or not, but they can enhance the stability of disperse system by other effects. It may increase the viscosity of the system, which decrease the thermal motion speed of the droplets so as to retard their aggregation. It may be adsorptive as the second layer on the first adsorptive layer on the surface of the droplets, which increase the steric restriction to aggregation. Addition from the fundamental function, emulsifier has other useful functions for food. Formation or deformation of foaming also need emulsifier to be an accelerant for forming or deforming the airtight film on the bubble. Another function is wetting effect, which means moisten the solid surfaces. Solid material is mixed with an emulsifier or its surface is spread with emulsifier, the surface then becomes hydrophilic. For example, chewing gum is apt to stick to teeth. We can prevent adhesion by wetting the surface of chewing gum by adding emulsifier. Emulsifier can act on starch and protein, which is important also. When emulsifier combine with the amylose helix structure, the α-starch can not change to β-starch, which prevent degradation of starch in bread and other millet cake, so that extending shelf life of bread products. When making sponge dough, emulsifier can modify gluten molecules and enhance its film-forming power, resulting in good spreadability and improvement of working efficiency. Thus, easy-rising bread can be obtained. Emulsifiers stabilise the emulsion in lowfat spreads providing the right stability and mouthfeel and reduce spattering in frying margarine also.

6.3. Kinds of Emulsifiers 6.3.1. Kinds of Emulsifiers Only food emulsifiers defined as food additives are usable to food by law. Those emulsifiers are shown in Table 9-2. Some of the emulsifiers have been used ordinarily.

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Table 9-2. Food emulsifiers Name Glycerin Fatty Acid Esters Acetic Acid Esters of Monoglycerides Lactic Acid Esters of Monoglycerides Citric Acid Esters of Monoglycerides Succinic Acid Esters of Monoglycerides Polyglycerol Esters of Fatty Acids Sorbitan Esters of Fatty Acids Polyoxyethylene sorbitan esters of fatty acids Propylene Glycol Esters of Fatty Acids Sucrose Esters of Fatty Acids Lecithin

Common Name Monoglyceride (MG) Acetylated Monoglyceride (AMG) Lactylated Monoglyceride (LMG) CMG SMG PolyGlycerol Ester (PGE) Sorbitan Ester (SOE) Tween PG Ester (PGME) Sugar Ester (SE) Lecithin (LC)

When an emulsifier will be used for a purpose, knowing its classification is important because different classes of emulsifier have different properties, function and safety. For the student to learn the basical knowledge, the concept of HLB (hydrophile-lipophile balance) should be understood. The Hydrophilic-lipophilic balance of a surfactant is a measure of the degree to decide whether it is hydrophilic or lipophilic, determining by calculating values for the different regions of the molecule, as described by Griffin in 1949 and 1954. Griffin's method for non-ionic surfactants as described in 1954 works as follows: HLB = 20Mh / M Where, Mh is the molecular mass of the hydrophilic portion of the Molecule, and M is the molecular mass of the whole molecule, giving a result on an arbitrary scale of 0 to 20. An HLB value of 0 corresponds to a completely hydrophobic molecule, and a value of 20 would correspond to a molecule made up completely of hydrophilic components. The HLB value can be used to predict the surfactant properties of a molecule: Value from 0 to 3 indicates an anti-foaming agent. Value from 4 to 6 indicates a W/O emulsifier. Value from 7 to 9 indicates a wetting agent. Value from 8 to 18 indicates an O/W emulsifier. Value from 13 to 15 is typical of detergents. Value of 10 to 18 indicates a solubiliser or hydrotrope. Some of emulsifies' HLB values are listed in Table 9-3.

6.3.2. Common Used Emulsifiers (1) Glycerine fatty acid esters. Glycerin fatty acid esters (mono[di,tri] glycerides of fatty acids) are made from glycerin and animal and plant oils/fats or their fatty acids. Those are generally produced by interesterification method. Monoglycerides have various characteristics depending on the kind and the content of its fatty acid. They are applied to many different fields being as an emulsifier, foaming agent, anti-foaming agent, anti-tack agent, starch-modifying agent and anti-bacterial agent, so you need to select the most appropriate type of monoglycerides for respective

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purposes. In China, those emulsifiers are extensively used in dairy, pasta, butter, and coffee drinks, with ML from 5 g/kg to production need. (2) Acetic acid esters of monoglycerides. Acetic acid esters of monoglyceride, also called as acetylated monoglyceride, are an emulsifier in which acetic acid is bound with monoglyceride. It has little emulsifying activity but there are many characteristics and application fields as follows: Soft acetylated monoglyceride is able to expand by more than 8 times with tension. It is an extremely stable oil of which peroxide value does not increase even when heated at 97.7 °C for 1000 hours. The combination of liquid acetylated monoglyceride and hydrogenated fats can improve the quality of fats, for example, margarine characterized with small temperature changes and wide plasticizing range, can be produced with them. It is a liquid characterized by being less oily even at low temperatures and available as a solvent, lubricant, etc. Although it has no function as a good emulsifier, it is usable for foaming fats and oils by itself or in combination with other emulsifiers because of its stable alpha-crystal structure. Practically, it is used as powdered foaming agents, solvents, plasticizers for gums and coating agents for food. Table 9-3. HLB values of some emulsifiers [7] Emulsifier Acetylated monoglycerides Sorbitan trioleate Glycerol dioleate Sorbitan tristearate Propylene glycol monostearate Glycerol Monoleate Glycerol monostearate Acetylated monoglycerides (stearate) Sorbitan monooleate Propylene glycol monolaurate Sorbitan monostearate Calcium stearoxyl-2-lactylate Glycerol monolaurate Sorbitan monopalmitate Soy lecithin Diacetylated tartaric acid esters of monoglycerides Sodium Stearoyl lactylate Sorbitan monolaurate Polyoxyethylene (20) sorbitan tristearate Polyoxyethylene (20) sorbitan trioleate Polyoxyethylene (20) sorbitan monostearate Sucrose monolaurate Polyoxyethylene (20) sorbitan monooleate Polyoxyethylene (20) sorbitan monopalmitate

HLB value 1.5 1.8 1.8 2.1 3.4 3.4 3.8 3.8 4.3 4.5 4.7 5.1 5.2 6.7 8.0 8.0 8.3 8.6 10.5 11.0 14.9 15.0 15.0 15.6

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(3) Lactic acid esters of monoglycerides. Lactic acid esters of monoglyceride are called lactylated monoglyceride in which lactic acid is bound with monoglyceride (Figure 9-4). Its foaming ability is stronger than emulsifying ability. It is used in shortening for cakes, desserts and in foaming for cream by itself or in combination with monoglyceride. Lactic acid esters of monoglyceride are a mixture of isomers even only one kind of fatty acid being its fatty acid constituents (see the Figure below this paragraph). That is why these kind emulsifiers have wide HLB from 2.0 to 15. Lactic acid esters of monoglyceride are a very safe emulsifier which can be used in appropriate level based on the need of food production according to the Chinese GB2760. (4) Citric acid esters of monoglycerides. Citric acid esters of monoglyceride are called citrated monoglyceride, in which citric acid is bound with monoglyceride and the obtainable products are mixtures containing a few monoglycerides. It is a highly hydrophilic emulsifier and a stable alpha-crystal structure used for margarine, dairy products, such as coffee whitener and cream. It also used as an emulsion stabilizer for mayonnaise and dressing by utilizing its strong acid-resistance. (5) Succinic acid esters of monoglycerides. Succinic acid ester of monoglyceride is called succinylated monoglyceride, in which succinic acid is bound with monoglyceride. It is insoluble in cold water, dispersible in hot water, and soluble in hot alcohol, fats and oils. Succinylated monoglyceride forms a complex with starch which is able to react with protein. It is used as a dough modifying agent and an emulsifier for shortening. (6) Polyglycerol esters of fatty acids. Polyglycerol esters of fatty acids are called polyglyceryl ester, an emulsifier in which fatty acid is bound by esterification with polyglycerine, and generally it is dispersible in water and soluble in oil. Its hydrophilicity and lipophilicity greatly change with the degree of its polymerization and the type of fatty acid. Its HLB ranges from 3 to 13. It has a variety of functions depending on these conditions, and is usable for various purposes. It is used in many types of food as an O/W and W/O emulsifier for milk products containing acid and salt and a modifier to control the crystallization of fats. In China, tripolyglyceryl monostearate is used in ice-cream, bread and pastry with ML being 3.0, 0.1 and 0.1 g/kg. (7) Sorbitan esters of fatty acids. Sorbitan esters of fatty acid are called sorbitan ester, which is produced by esterification of sorbitol and fatty acid. It is a mixture of sorbitol ester and sorbide ester, which are simultaneously produced as well as sorbitan ester. There are many types of sorbitan esters with the kinds of fatty acids and various degrees of esterification. Those are generally used as emulsifier for cream, W/O emulsion, etc. It is applied in limited fields by itself because its special characteristics other than emulsifying capability are few; however, it is widely used as a major emulsifier in combination with other emulsifiers with different functions.

Figure 9-4. Sketch of two isomers of lactic acid esters of monoglyceride.

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In China, sorbitan monolaurate, monopalmitate, monostearate, tristearate, and monooleate (brand name are Sorbitan 20,40,60,65 and 80) are widely used in many foods such as hydrogenated vegetable oil, dairy, bean products, wheaten food, and drinks with ML from 0.5-10.0 g/kg according to the Chinese GB 2760. (8) Polyoxyethylene sorbitan esters of fatty acids. Polyoxyethylene sorbitan esters of fatty acids are called polyoxyethylene sorbitan ester, which is produced by the combination of polyoxyethylene and sorbitan ester. Polyoxyethylene sorbitan monolaurate, monopalmitate, monostearate, monooleate are called Tween 20, 40, 60, and 80 (brand name) respectively. They are generally used as emulsifier and stabilizer for O/W emulsion system, such as flavored milk and other liquid milks that use non-diary ingredients. According to the Chinese FDs standard, they can be used in dairy, drinks, bean products and bread and its ML is about 0.5-2.5 g/kg. (9) Propylene Glycol Esters of Fatty Acids (PG ester). Propylene glycol esters of fatty acids is called PG ester, in which propylene glycol and fatty acid are linked by ester bonding and the product obtained from inter-esterification, is a mixture of monoester and diester. To isolate the monoester with surface effects, high purity product of monoester is produced by molecular distillation and distilled monoglyceride. PG ester has little emulsifying action, but has a tendency to keep its alpha-crystal structure. Since it is usable as a foaming agent when combined with monoglyceride, it is used as a powder-foaming agent for cakes and desserts, liquid shortening, etc, with the exception of whole or partially skimmed milk. The ML is 5.0 g/kg according to the Chinese GB2760. (10) Sucrose Esters of Fatty Acids. Sucrose esters of fatty acids are called sucrose ester, prepared from sucrose and methyl and ethyl esters of food fatty acids or extracted from sucroglycerides. They occur as white to yellow-brown powdery or massive substances, or as colorless to red-brown, viscous resinous or liquid substances. They are odorless or have a slight, characteristic odor, sparingly soluble in water, soluble in ethanol. The solubility in oil increases with the increase of the degree of esterification. Because sucrose esters have many sub-categories and most of them are complex sucrose and different fatty acids, the products of sucrose esters, generally being mixtures of different components, have HLB ranging from 1 to 16. Owing to the wide ranging HLB, sucrose esters have many different functions. It is used as an emulsifying and dispersing agent for foods, such as cream, and bactericidal agents for canned coffee. The ML is from 1.5 g/kg for fats and oils essentially free from water to 10.0 g/kg for vegetable oils and fats according to the Chinese GB2760. (11) Calcium Stearoyl-Lactate and sodium stearoyl lactylate. Calcium (or sodium) stearoyl-2 lactate is called CSL. It is a product obtained by bonding two lactic acids and stearic acid and partially neutralized with calcium (or sodium). It is a mixture including the original stearic acid and salt. It is an anionic emulsifier having a strong ability to bind protein and is used as a dough modifier for flour foods like bread. In China, it also used in wheaten foods, meat enema and dairy drinks with ML being 2.0 g/kg. Sodium stearoyl lactylate can be used as emulsifier or stabilizer for setting up water-inoil food emulsions containing at least 80% fat. The maximum level of use is 1.5 g/kg according to the Chinese GB2760. Sodium stearoyl lactylate can be used as emulsifier or stabilizer for making breads, rolls and pastries. The ML is 2.0 g/kg according to the Chinese GB2760.

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(12) Lecithin (LC). Lecithin is a mixture containing phospholipids as the major component and widely found in animals and plants such as soybeans, corn, rapeseed and egg yolk. It has long been used as a natural emulsifier. The products in the market are paste lecithin or powdered lecithin of high purity. In the food industry, lecithin has multiple uses: in confectionery it reduces viscosity, to replace more expensive ingredients, control sugar crystallization and the flow properties of chocolate, improve the homogeneous mixing of ingredients and shelf life for some products, and be used as a coating. In emulsions and fat spreads, it stabilizes emulsions, reduces spattering during frying, and improves texture of spreads and flavor release. In doughs and bakery, it reduces fat and egg requirements, helps even distribution of ingredients in dough, stabilizes fermentation, increases volume, protects yeast cells in dough when frozen, and acts as a releasing agent to prevent sticking and simplify cleaning. It improves wetting properties of hydrophilic powders (e.g. low-fat proteins) and lipophilic powders (e.g. cocoa powder), controls dust, and helps complete dispersion in water. It can be used as a component of cooking sprays to prevent sticking and as a releasing agent. For example, lecithin is the emulsifier that keeps cocoa and cocoa butter in a candy bar from separating. In margarines, especially those containing high levels of fat (>75%), lecithin is added as an 'anti-spattering' agent for shallow frying. Most major religions have no dietary restrictions on the use of lecithin. In China, this emulsifier can be used in appropriate level based on the need of food production according to the Chinese GB2760.

7. STABILIZERS, THICKENERS AND GELLING AGENTS 7.1. Definition All the stabilizers, thickeners and gelling agents are known and described as food hydrocolloids implying that functional properties are obtained by mixing them with water. They are macromolecules, such as carbohydrate polymers, proteins and their modified forms, which are water soluble. These additives include traditional materials such as starch, cellulose, gums, obtained from many land plants, and gelatin, an animal by-product, and microbial polysaccharides of xanthan, gellan and pullulan, and their modified forms. Even at low concentrations, they are able to increase a system's viscosity, to form gels and to stabilize emulsions, suspensions or foams. These compounds are also active as crystallization inhibitors and are suitable for aroma encapsulation, which is often needed for dehydrated food.

7.2. Classification Raw material origin has been used to classify hydrocolloids, for example as seaweed extracts, seed gums, fermentation products, plant exudates, animal gelatin or microorganic gums. Their general functional properties may also be used to classify them as thickeners, stabilizers or gelling agents. The resourceful plant polysaccharoses, like starch and cellulose, are derived by some chemical methods are classified modify polysaccharoses. Natural

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stabilizers, thickeners and gelling agents coming from plants can be classified based on their sources shown in Table 9-4.

7.3. Functional Properties The following is a brief overview of the key functional properties for gelling agents, stabilizers or thickeners used. These ingredients need to be added to commercially-produced food to maintain their structure and physical properties during processing. For example, fruits such as strawberries, raspberries and cherries need the addition of a small amount of pectin to form a jam. Nutritional properties are relatively new and nutraceutical or health-enhancing properties are even more recent. Further work is sure to advance the use of hydrocolloids beyond modification of the rheology of foods.

7.3.1. Viscosity Adjustment Viscosity is probably one of the most widely used properties. In this respect, hydrocolloids are widely used to thicken sauces, soups, stews and other dishes with a high liquid content, and the thickened water simply adds body, texture and mouth feel to a food such as table syrups, particularly low-calorie syrups. In other cases, hydrocolloids are often used also in systems where the oil or fat content has been reduced or eliminated through substitution with water. The hydrocolloid thickens water, which, in turn, replaces the fat or oil to give a product with similar properties to the full-fat food. A typical application for this function is reduced-fat salad dressings. 7.3.2. Emulsion Stabilization If oil or fat is partially removed from a formulation or replaced with thickened water, an emulsion is usually formed. Often the function of the hydrocolloid is to stabilize the emulsion, to prevent separation and, in the case of frozen foods, to control ice crystal formation. New technology and new ingredients have been developed specifically to address the problem of ice crystals in frozen foods, but hydrocolloids will continue to play a role. Virtually every ice cream product sold in retail outlets is stabilized with carrageenan, locust bean gum and/or guar gum. Low-fat salad dressings also benefit from emulsion-stabilizing properties. Table 9-4. Groups of plant-derived hydrocolloids [8] Group Seeds gum Plant exudates Fruits extract Plant materials Seaweed extract

Compounds examples Guar gum, Locust bean gum, Tara gum Acacia gum, Tragacanth gum Pectin Cellulose, Starches Agar, Alginates, Carrageenan

7.3.3. Suspension Stabilization If insoluble particles are included in the thickened product, then separation and settling should be eliminated or at least minimized. Some hydrocolloids create solutions with a ''yield point'' that will keep particles immobilized in suspension. Salad dressing is a good example of

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this and xanthan gum is the typical hydrocolloid to supply this functionality. For beverage with pulp suspended, like orange juice with pulp particle, precipitation can be prevented by adding suitable amount agar to crate a small yield point which is big enough to stop the pulp settling during the shelf-life and small enough to keep the beverage's flow property like normal drink.

7.3.4. Gelation One of the key texturising aspects of hydrocolloids is the ability to gel and solidify fluid products. For example, in gelled milk desserts, even low levels of carrageenan will form a solid milk gel. Other classic gelling agents are pectin, gelatin and agar. However, others will form a gel under specific conditions. Certain grades of alginates form gels with calcium ions. Xanthan and locust bean gum alone do not gel, but their combination display synergy and form a strong cohesive gel. Methyl cellulose and hydroxypropylmethyl cellulose are unusual in forming solutions that reversibly thicken or gel when heated. The food industry has a myriad of gelling applications ranging from soft, elastic gels to hard and brittle gels. 7.3.5. Nutritional and Nutraceutical Function There is already a wide use of some hydrocolloids. For example, Arabic and guar gum are sources of soluble dietary fiber. Much research has been conducted in the nutraceutical benefits of hydrocolloids. Potential benefits range from cholesterol reduction to cancer risk prevention.

7.4. Common Used Stabilisers, Thickeners and Gelling Agents The following agents are widely used in food industry. Almost every one is permitted to be used in any food categories, except those special mentioned in appropriate level, based on the need of production according to the Chinese GB 2760.

7.4.1. Acacia Gum Acacia gum, also known as gum arabic, is a natural gum exudate obtained from acacia trees in the 'African sub-Sahelian zone'. The gum is a complex mixture of polysaccharides and glycoproteins that have a highly branched compact arabinogalactan structure which gives a low-viscosity solution together with a central protein fraction that provides good emulsification properties. The powder hydrates readily in water and concentrations up to 4050% can be handled easily. Its applications include a range of confectionery products, flavored oil emulsions and capsules and health foods as a stabilizer and a source of soluble fiber with prebiotic properties. 7.4.2. Agar Agar is extracted from red seaweed (Rhodophyceae) and has been used in foods for more than 350 years. Agar consists of a mixture of agarose and agaropectin. Agarose is a linear polymer, made up of the repeating monomeric unit of agarobiose. Agarobiose is a disaccharide including D-galactose and 3, 6-anhydro-L-galactopyranose. Agaropectin is a heterogeneous mixture of smaller molecules that occur in lesser amounts. Agar is insoluble in cold water and hydrates when boiled. Cooling solutions below about 40 °C produce very firm brittle gels which can be melted by heating above 85 °C. Food applications include water

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dessert gels, aspics, confectionery jellies, canned meats gels, a vegetarian gelatin substitute, a thickener for soups, and as a clarifying agent in brewing.

7.4.3. Alginates Alginates are derived from various species of brown seaweed found off the coasts of the North Atlantic, South America and Asia. They are produced as a range of salts, but sodium and potassium alginate are predominantly used in foods. It is a polysaccharide, consisting primarily of D- and L-galactose units. About every tenth D-galactopyranose unit contains a sulphate ester group. Calcium, magnesium, potassium or sodium cations are also associated with the polysaccharide. It is insoluble in cold water and soluble in boiling water. Sodium or potassium alginate hydrates in cold or hot water to give viscous solutions. The controlled interaction between sodium or potassium alginate and calcium salts gives cold-setting gels that are shear irreversible and heat stable. Control is affected using citrate or phosphate sequestrants, or by processing at temperatures above about 70 °C and cooling. Typical food applications include reformed foods such as onion rings and olive fillings, cold-setting bakery cream fillings and heat-stable bakery and fruit fillings. 7.4.4. Carrageenan Carrageenan is obtained from red seaweeds. Different species provide a number of carrageenan extracts. Carrageenan is a hydrocolloid consisting mainly of the ammonium, calcium, magnesium, potassium and sodium sulphate esters of galactose and 3, 6anhydrogalactose polysaccharides. These hexoses are alternatively linked -1, 3 and -1, 4 in the copolymer. The relative proportions of cations existing in carrageenan may be changed during processing to the extent that may become predominant. Depending on the source of seaweed, the functional components are kappa-carrageenan, iota-carrageenan or lambdacarrageenan, each of which is characterized by the content of sulphate groups and gelling properties. Carrageenan is soluble in water of about 80 °C, forming a viscous, clear or slightly opalescent solution that flows readily. It is insoluble in ethanol. Kappa carrageenan and furcellaran form thermally reversible firm, brittle gels. Iota carrageenan gives soft, elastic gels. Kappa carrageenan interacts synergistically with polymannans, such as locust bean gum and konjac, to give strong cohesive gels. Blends of kappa with iota carrageenan or polymannans are used to give a range of gel textures used in injected meats, canned meats and water dessert gels.. Hybrid (kappa/iota) carrageenan is particularly suitable for firm, creamy textures in dairy desserts. Lambda carrageenan is non-gelling but thickens in drinks and dairy desserts. A specific interaction between kappa carrageenan and kappa casein is widely used to stabilise dairy products including milk beverages, ice cream mixes and processed cheese products. 7.4.5. Microcrystalline Cellulose Microcrystalline cellulose is produced by converting fibrous cellulose to a fine-particle form crystalline cellulose using acid hydrolysis. This material can be readily dispersed in water using high shear. This dispersion will reconstitute to deliver a colloidal form which is unique when compared to other soluble food hydrocolloids. It exhibits a variety of desirable characteristics including suspension of solids, heat stability, ice crystal control, emulsion stabilization, foam stability, texture modification and fat replacement. Food applications include frozen desserts and ice cream, whipped toppings, low-fat mayonnaise and salad

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dressings, ambient-stable dairy and non-dairy beverages, nutritional drinks, ready-to-use bake-stable bakery fillings and fruit fillings and dairy and non-dairy creams.

7.4.6. Cellulose Derivatives Cellulose derivatives are substances commonly made from cellulose by chemical modification. The common used cellulose gum in food is carboxymethyl cellulose and hydroxypropylmethyl cellulose. In food applications, cellulose gum is an effective thickener and moisture binder used for clear beverages, dairy products, such as ice cream, bakery and other prepared foods. Hydroxypropylmethyl cellulose exhibits thermogelation, utilized for bake-stable sauces, fillings and formed foods. Hydroxypropylmethyl cellulose is also a surface-active agent to be used to stabilize foams and emulsions. 7.4.7. Starch Derivatives Starch derivatives are wide substances commonly made from starch by chemical or phsical modification. The common used starch derivatives in food are starch acetate, distarch phosphate, hydroxypropyl distarch phosphate, acid treated starch, oxidized starch, oxidized hydroxypropyl starch, acetylated distarch phosphate and acetylated distarch adipate. Starch derivatives are easier gelatination and more anti-aging than common strarch. The viscosity of their solution is also more stable than the common starch. Starch derivatives can be used in any food to substitute starch to make the food more stable. 7.4.8. Gelatine Gelatine is a protein material obtained from animal connective tissue using hydrolysis in acidic (type A) or basic (type B) solution followed by hot water extraction. Gelatine hydrates readily in warm or hot water to give low-viscosity solutions that have good whipping and foaming properties. Concentrated solutions containing up to 40 % gelation are made for use in confectionery. After cooling, the network of polypeptide chains associates slowly to form clear, elastic gels that are syneresis free. The thermoreversibility of gelatine gels gives them unique properties: the melting point is below 37 °C so they melt in the mouth to give smooth textures with excellent flavour releasing. Gelatine is used in a wide range of food, pharmaceutical and photographic applications. Food products include jelly candies and aerated confectionery, yogurt and other cultured desserts, dairy desserts and creams, low-fat spreads, canned meat products, water dessert gels and decorative aspics. 7.4.9. Seed Gums The origin of seed gums, such as guar gum and carob or locust bean gum, are produced by removing the outer coating of the seed and grinding the endosperm. The composition and structure of the galactomannans are described and linked to their functional properties. Seed gums are very effective thickening agents in water. In addition, guar gum interacts synergistically with xanthan to increase viscosity. Locust bean gum consists mainly of high molecular weight (approximately 50,000-3,000,000) polysaccharide composed of galactomannans. It is soluble in hot water and insoluble in ethanol. Carob gum forms cohesive, elastic gels with xanthan gum and it increases the strength and elasticity of kappa carrageenan and agar gels. The main applications are for thickening convenience foods, dairy products, including frozen products such as ice cream, soft drinks and fruit juices, bread and pastry, fruit preserves, baby food, instant products including puddings, flans and pudding powder, and for dietary fibre in baked goods and pet foods.

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7.4.10. Pectin Pectin is a polysaccharide that is naturally present in most land plants, although commercial pectin is primarily extracted from citrus peel and apple pomace. Two forms of commercial pectin are available: high methyl- and low methyl-esterified pectin; and two versions of the latter exist: a conventional and an amidated form. High methyl-esterified pectin forms gels in high soluble solids and acidic systems, whereas low methyl-esterified pectin forms gels in a much broader pH and soluble-solids range, but requires the presence of divalent cations for gelling. As a consequence, each type has its own particular function. Nevertheless, general attractive features include excellent flavor release, good processing characteristics and stability at low pH. Its traditional and major function is to act as a gelling agent in foods, but, nowadays, it also serves as a thickening and stabilizing agent. The application of pectin is diverse and covers fruit-based products, dairy products, acidified milk drinks and other beverages, confectionery, bakery products, various fine foods and spreads. Additionally, pectin is used in the pharmaceutical industry. Finally, increasing consumer awareness of healthy life-style habits and the emerging trend to produce functional foods increases the significance of the status of pectin as a water-soluble dietary fiber. 7.4.11. Xanthan Gum Xanthan gum is a high-molecular-weight polysaccharide of which D-glucose and Dmannose are the dominant hexose units, along with D-glocuronic acid and pyruvic acid, secreted by the microorganism Xanthomonas campestris and produced commercially in a batch fermentation process. It exists as the sodium, potassium or calcium salts. Its solutions are neutral. It is soluble in water, and insoluble in cold water. The solution is a neutral and viscous one with pseudoplastic flow behaviour. This gives excellent suspension and cling at low shear and excellent mouthfeel and pouring properties at high shear. The xanthan gum molecule has a cellulosic backbone with side chains that wrap around the backbone protecting it and conferring excellent stability across a wide pH range and tolerance of high salt concentrations and ingredients, such as glycerol and alcohol. The rigid backbone helps to maintain viscosity during heating. Xanthan gum shows synergistic thickening with guar gum and forms very elastic cohesive gels with locust bean gum and konjac mannan. Typical applications include sauces and dressings, baked goods, beverages, desserts and ice creams. 7.4.12. Gellan Gun Gellan gum is a fermentation polysaccharide produced by the microorganism Sphingomonas elodea. It has a straight chain structure based on repeating glucose, rhamnose and glucuronic acid units with side groups of acryl groups. Gellan gum hydrates in hot water and the low-acyl form also hydrates in cold water with sequestrants. On cooling, native highacyl gellan gum gives gels that are soft and elastic. Low-acyl gellan gum gels at very low concentrations using both monovalent and divalent cations gives firm, brittle textures with excellent thermal stability. Combinations of the two kinds of gellan gum can be used to control syneresis and form a range of textures from soft and elastic to firm and brittle. A major food application is water dessert gels, particularly for Asian desserts. Other significant applications include confectionery, dairy desserts and bakery fillings. At levels too low to form a demoldable gel, gellan gum can form fluid gels that can suspend particulates in sauces and dressings and fruit pulp in beverages.

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7.4.13. Konjac Glucomannan Konjac flour yields a high molecular weight, viscous polysaccharide: konjac glucomannan. Glucomannan is a highly soluble, neutral plant polysaccharide that has been used as a gelling agent for 2000 years in China. It gels alone or in association with other polysaccharides where it shows strong synergistic effects in viscosity, gel strength and elasticity. Konjac has a greasy mouthfeel and chewy texture that resemble fat. It is resistant to digestion and has a very low calorific value. As well as being a healthy food, it is a preferred texturing ingredient in Asia. Glucomannan has generally recognised as safe (GRAS) status in the USA and it is well established as an additive in the EU food industry. Cost-effective as a thickener, the effects on post-prandial sugar and lipid levels, low digestibility and prebiotic fermentation have placed glucomannan amongst the most exciting hydrocolloids on the 'emerging' world nutraceutical market. 7.4.14 Tragacanth Gum Tragacanth is a dried exudation obtained from the stems and branches of Astragalus gummifer Labilliardiere and other Asiatic species of Astragalus (family Leguminiosae).Tragacanth consists mainly of high molecular weight polysaccharides (approximately 800.000) i.e. galactoarabans and acidic polysaccharides, which are hydrolyzed to galacturonic acid, galactose, arabinose, xylose, and fucose. Small amounts of rhamnose and glucose (derived from traces of starch and/or cellulose) may be present. Gum tragacanth is a viscous, odorless, tasteless, water-soluble mixture of polysaccharides. It is used in pharmaceuticals and foods as an emulsifier, thickener, stabilizer, and texturant additive. 7.4.15. Chitin and Chitosan Chitin and chitosan have the similar chemical structure. Chitin is made up of a linear chain of acethylglucosamine groups. Chitosan is obtained by removing enough acethyl groups (CH3-CO-) for the molecule to be soluble in most diluted acids. This process, called deacetylation, releases amine groups (-NH) and gives the chitosan a cationic characteristic. This is especially interesting in an acid environment where the majority of polysaccharides are usually neutral or negatively charged. Chitin can use in non-dairy creamer, ice creams, jams, marmalades, peanut butter, sesame paste, mayonnaise, lactobacillus drink, and beer with ML 0.4-5 g/kg based on GB2760. Chitosan can be an effective complement to help lose weight during diet period or to stabilise ones weight, and used as clarifier in wine industry. 7.4.16. Others Polydextrose, propylene glycol alginate, fenugreek gum, sesbania gum, tamarind seed polysaccharide gum, sodium carboxymethyl starch, ablmoschus manihot gum, artemisia gum, gleditsia sinensis lam gum, linseed gum, polyglycerol esters of fatty acid, hydroxypropyl starch phosphated distarch phosphate, methyl cellulose and ethyl cellulose are other thickeners, stabilisers and gelling agents used in food in China. They are used in some appointed food categories with limited amount as ML in GB2760.

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8. PHOSPHATE AND POLYPHOSPHATES Phosphates' buffering capacity, chelating metal ions, water-holding capacity, and interacting with long chain polyelectrolytes such as protein, make them widely used as FDs. Phosphates are one of the most widely used functional food ingredients. Applications of the various salt forms of phosphates, such as leavening agents in baked goods, moisture loss inhibitors in frozen and processed meats, emulsifiers in dairy products, and buffering agents for many different food formulations, are included. For example, monocalcium (MCP) and dicalcium phosphate dihydrate act as leavening agents in baked goods. MCP is also useful as a dough conditioner, and can be used to strengthen the gel formation in instant pudding products. Tricalcium phosphate is very useful in dry powder mixtures, to prevent the adsorption of moisture and allow the powders to flow properly. When these phosphates are used as functional food ingredients, they also provide the fortification of calcium and phosphorus. Phosphates are used in pasteurized cheese products, ice cream, frozen custard, breads, rolls, buns, flour, macaroni products, fruit jellies, preserves and jams, frozen eggs, vanilla powder, as well as many other foods. Polyphophates are added to flesh foods, such as meat, fish and poultty, in order to increase the retention of water and the solubility of proteins to improve texture. A range of phosphoric acids and their sodium potassium, and calcium salts, are used as FDs, from the simple acid orthophosphoric acid, H3PO4, to complex polyphosphates are permitted for use in food. Because too much of them in diet leads to loss of calcium in bones and onset of osteoporosis, almost every kind of those FDs are used with special limited ML for different food catogries according to the GB2760, and is banned in organic foods and drinks in some countries.

8.1. Phosphoric Acid Phosphoric acid (other names: orthophosphoric acid.) can only be obtained pure in the crystalline state and slowly undergoes dehydration to diphosphoric acid. Phosphoric acid is added to food to enhance the antioxidant effects of other compounds present, and also as an acidity regulator. Typical products include carbonated beverages, processed meat, chocolate, fats and oils, beer, jam and sweets. Phosphoric acid is a highly acidic ingredient in cola drinks.

8.2. Sodium Phosphate (1) Monosodium phosphate is a sodium salt of phosphoric acid. It is added to food to act as an antioxidant synergist, a stabiliser and a buffer. Typical products include processed meat products, processed cheese products. It is also used in bread, rolls, buns, artificial sweetened fruit jelly, canned potatoes, canned sweet peppers, and canned tomatoes and as a jelling agent. No food safety problems have been shown to occur with this chemical at the levels commonly used in foods, but high intakes may upset the calcium/phosphorus equilibrium. (2) Disodium phosphate is a sodium salt of orthophosphoric acid and is used as an antioxidant synergist, stabiliser and buffering agent in food. It is also used as an emulsifier in

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the manufacture of pasteurised processed cheese. Disodium phosphate is added to powdered milk to prevent gelation. Typical products include processed meat products, processed cheese products, powdered milk. (3) Trisodium phosphate is the sodium salt of orthophosphoric acid and used as an antioxidant synergist, stabiliser and buffering agent in food. Typical products include processed meat products, processed cheese products.

8.3. Potassium Phosphates (1) Monopotassium phosphate is a potassium salt of phosphoric acid used as an antioxidant synergist, buffer and emulsifier in food. Typical products include sauce and dessert mixes, jelly products. (2) Dipotassium phosphate is a potassium salt of phosphoric acid used as an antioxidant synergist, buffer and emulsifier in food. Typical products include cooked and other cured meats, milk and cream powders, drinking chocolate. (3) Tripotassium phosphate is a potassium salt of phosphoric acid used as an antioxidant synergist, buffer and emulsifier in food. Typical products include cooked and other cured meats, milk and cream powders, drinking chocolate.

8.4. Calcium Phosphates (1) Monocalcium phosphate is commercially available in the anhydrous or monohydrate form. Both are used as a leavening acid to replace cream of tartar in foods. Mono-calcium phosphate is used extensively in the fertiliser industry. Typical products include self-raising flour, baking powder, cake and pastry mixes, cakes and other pastry products as a baking agent. (2) Dicalcium phosphate - Manufactured from phophoric acid, dicalcium phosphate is used as an antioxidant in food, as well as being a firming agent. It is available in the anhydrous or dihydrate forms. Typical products include tinned and packaged fruit deserts, granular food products. In the canned products it provides calcium which has been shown to maintain the firmness of fruits and vegetables during the canning process. (3) Tricalcium phosphate is added to table salt, sugar, baking powder and fertilisers to give a 'free-flowing' quality. Typical products include salt, sugar and other granular foods, packet sauce mixes, cake mixes etc.

8.5. Sodium Triphosphate Sodium triphosphate is beside potassium triphosphate, one of the polyphosphates that are allowed as FDs. It is used in food for soften water, as emulsifying salt for process cheese or for the preparation of cooked sausages, surimi or fish fingers. It has antimicrobial function also.

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9. COLORANTS Dyes and pigments used in the food are known collectively as food colours, or more tersely, simply as colours. They are added to foods to make it more attractive to the purchaser or consumers or to replace natural colours lost during the food processing. Canned strawberries, for example, would be grayish-brown if colours are not added and canned peas would be brownish green without colouring with green colorant. In China, only 56 colorants are permitted for use as FDs. Most of them are natural, and include saffron, cochineal, carotenes (and the closely related xanthophylls) and anthocyanins. Other permitted colorants include synthetic or semi-synthytic coumpound, such as carotene structure identical, sodium copper chlorophyllin and erythrosin, and inorganic substances, such as the white pigment titanium dioxide and finely divided aluminum, silver and gold which are used in cake decoration. The list of permitted colours includes 9 synthesis dyes and 47 natural pigments or extracts. Most of them are used in foods with strictly limits. For example, colouring matter must not be added to raw meat, fish, poultry, fruit, cream, milk, honey, vegetables, wine, coffee, tea and condensed or dried milk in most countries. Also, by agreement with the manufacturers, colours are not used in foods made for babies and infants. The 9 synthesis colours use in China are amaranth, ponceau 4R, erythrosin, new red, lemon yellow, indigo blue, sunset yellow, brilliant blue and crimson. Sodium copper chlorophyllin is a semisynthesis colour which is also permitted in China. The 47 natural colours are β-carotene (zymotechnics), bect-root red, turmeric yellow, curcumin, safflower yellow, http://www.chinaadditives.com/Carthamus-Red.htm lac dye, cowberry red, papika extract include capsorubin and capsanthin, chilli orange, caramel, carthamus yellow, gardenia yellow, coreopsis yellow extract, wild groundnut red, broomcorn red, maize yellow include zeaxanthin and crtotixabthin, radish red, cacao husk pigment, ang-kak, monascorubin, vinespinach red, black currant red, neutral mustard red, gardenia blue, hippophae rhamnoides(sea backthern) yellow, hibiscus sabaariffa(roselle) red, acorn husk brown, NP red (NP), tanoak brown, mulberry red, natural amaranth, cherokee rose brown, wide jujube skin extract, peanut underwear red, grape skin red, uguisukagnra color, spirulina blue, vegetable carbon black, pale butterflybush flower yellow, gromwell red, theaflavin, tea green pigment, citrus yellow, bixin, cochineal red and iron oxide red or black. All of the 9 synthesis colours and most of natural colours used in China are water soluble and purchased in a powder format that exhibits coloring solutions when they are dissolved. Therefore, their using are very easy. Aluminum lake pigments, made of aluminum and colorants, are water insoluble material but oil dispersible (but generally not oil soluble) and thus can be mixed with oils and fats. They can also be dispersed in other carriers such as propylene glycol, glycerin and sucrose (water and sugar). Aluminum lake pigments are used in a variety of applications: (1) To color a fat based product, such as chocolate or compound coatings. For these, we produce a concentrated dispersion in a high quality and very stable vegetable oil. The dispersion is added directly to the chocolate to dye it accordingly. (2) For "hard panning" (to dye the outside of a product such as a gum ball or a pill). In this case, we produce a dispersion usually using sucrose (sugar and water) that is applied to the candy or food as it is being tumbled and dried. Multiple layers are applied to produce the desired shade.(3) Lakes tend to resist bleeding. Dyes have a tendency to "bleed", or migrate from one part of the product to another. This can be a problem in candy canes or any product where there are defined borders such as

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the food with desired stripes. While Dyes are normally used in hard candy, lakes are sometimes substituted if bleeding is a problem.

10. FOOD FLAVORANTS AND FLAVOUR MODIFIERS 10.1. Food Flavorants Flavourings, or flavors, spices and essence, are substances or food flavor additives used to give taste and/or smell to food. There are different types of flavourings, such as natural, natural-identical or artificial flavouring substances, flavouring preparations of plant or animal origin, processing flavourings which evolve flavour after heating and smoke flavourings. The expression "natural flavouring" may only be used for flavouring substances or flavouring preparations which are extracted from vegetable or animal materials. Generally, different flavors are specially formulated for various food items to make them more delightful. Flavorants contains many flavour compounds. Only the Generally Recognized As Safe flavour compounds can be used as food flavor additives. Flavour compounds often classify into 18 groups, shown in Table 9-5. Each of the 18 groups contains substances that have similar chemical structure at least partly, but have not similar odors mostly. Table 9-5. Groups of flavouring substances Group 1

Chemical substances Isothiocyantaes (except for those generally recognized as toxic).

2

Indole and its derivatives.

3 4 5

Ethers. Esters. Ketones.

6 7 8 9

Fatty Acids. Aliphatic Higher Alcohols. Aliphatic Higher Aldehydes (except for those generally recognized as toxic) Aliphatic Higher Hydrocarbons (except for those generally recognized as toxic)

10 11 12 13

Thioethers (except for those generally recognized as toxic) Thiols(Thioalcohols) (except for those generally recognized as toxic) Terpens Phenol Ethers (except for those generally recognized as toxic)

14 15 16 17

Phenols (except for those generally recognized as toxic) Furfural and its derivatives (except for those generally recognized as toxic) Aromatic Alcohols Aromatic Aldehydes (except for those generally recognized as toxic)

18

Lactones (except for those generally recognized as toxic)

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10.2. Flavour Modifiers A flavour modifier is a substance which is capable of enhancing or modifying the taste or odour, or both, of a food. Flour modifiers or enhancers are themselves practically tasteless but they intensify the flavour of soups, meat and other savoury foods. The most widely used flavour enhancers are monosodium Glutamate (MSG) and sodium 5-ribonucleotide. MSG is present in most dehydrated soups, stock bouillon cubes and other meaty prepared foods. Some people have an alergy to MSG, which has come to be known as the "Chinese restaurant syndrome", if they eat food containing it. This manifests itself in a variety of ways including palpitations, chest or neck pain and dizziness. The cause is not known and the ill effects soon disappear. It has also been reported in potter, N.N. (1986) of brain damage resulting when MSG was injected under the skin of young mice. Though MSG is not thought to be harmful according to Fox B.A. and Cameron, A.G (1989), precautionary measures are still been taken for not adding it to babies and infants food. Sodium 5'-ribonucleotide is another powerful permitted flavour enhancer. Ribonucleotides are compounds formed from the sugar ribose, phosphoric acid and an organic base such as guanine. They occur in all animal tissues and are present in yeast extracts and contribute substantially to their characteristic meaty taste. Ribonucleotide flour enhancers are used in soups, meat and fish pastes, all types of canned meat products, sausages, meat pies and other processed food products of which the main ingredients is meat or fish. Their flavour enhancing power is 10 times greater than that of MSG. Hydrolyzed proteins, broken apart into amino acids, are used by the food industry to enhance flavor. The chemical breakdown of proteins may result in the formation of free glutamate that joins with free sodium to form MSG. In this case, the presence of MSG does not need to be disclosed on labeling. However, labeling is required when MSG is added as a direct ingredient.

REFERENCES [1] [2] [3] [4] [5]

[6] [7]

Salant, A. Nonnutritive sweeteners. In: Furia, TE. Handbook of Food Additives. Cleveland: CRC Press, 1972. Luo, AS; Chun, Z; Luo, AX; Fan, YJ; Ge, SR. The survey and progress of the food preservative. China Food Additives, 2005, 55-57. Wedzicha, RL. Chemistry of Sulphur Dioxide in Foods. 1st edition. London: Elsevier, 1984. Chichester, DF, et al. Antimicrobial food additives. In: Furia, TE. Handbook of Food Additives. Cleveland: CRC Press, 1972. Zsolt, F, et al. A synthetic and in silico study on the highly regioselective Diels-Alder reaction of the polyenic antifungal antibiotics natamycin and flavofungin. Tetrahedron Letters, 2010, 51, 4968-4971. Ma, LR; Zhang, ZL; Li, H. New type food preservative-NISIN. Journal of Hebei Academy of Sciences, 2001, 18, 180-182. Griffin, WC. Classification of Surface-Active Agents by HLB. Journal of the American Oil Chemists' Society, 1949, 311.

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Ren, P; R, YW; Wang, LD. Properties of Plant Gum and Its Application in Food Industry. Food Research and Development, 2004, 39-44.



Chapter 10

TOXICANTS IN FOODS Wang Dongfeng1, Guoqing Huang2 and Shuhui Wang3 1

College of Food Science and Engineering, Ocean University of China, Qingdao, China 2 College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, China 3 College of Agriculture - Ginn College of Engineering, Aubum University, Auburn, AL, US

ABSTRACT Microorganisms, plants and animals have evolved multiple defense systems against predators. Among the systems, the chemical defense system is of special interest for the food industry. Some of these compounds are toxic to specific microorganisms, insects, birds, and mammals, but most of those are toxic to humans and can give rise to chronic or acute symptoms of poisoning. Though some of the compounds occur in very low concentrations, the compounds have a cumulative harmful effect if they constitute a part of the normal diet over a prolonged period. Besides, food materials might be contaminated by exogenous toxins. Intrinsic toxins and contaminants have been recognized as the major components that affect food safety. This chapter introduces the occurrence and properties of the most important toxicants in foods, including allergens, toxic glycosides, toxic amino acids, lectins, saponins, toxins in aquatic organisms, various antinutrients, heavy metals, pesticide residues, dioxins, veterinary and fish drug residues, benzopyrenes as well as heterocyclic amines. Besides, measures for preventing the generation of some toxicants are proposed.

1. INTRODUCTION According to structure and biological effects on human body, toxicants in foods are divided into toxic substances, harmful substances, and antinutrients. A toxic compound shows its toxicity even in a very low dose, and a harmful substance is toxic only when its content exceeds a certain threshold. An antinutrient may interfere in or inhibit the absorption of other nutrients in foods. The definitions of these terms, however, are subject to changes due to the

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improvement of analysis techniques and the availability of more data on their biological effects. It has been found that some toxicants are beneficial in certain contents and some substances are toxic or hazardous only in certain cases. For example, phenols in foods are often recognized as antinutrients since they inhibit the adsorption of proteins. However, phenols have the anti-oxidation and free radical scavenging capabilities and can be used as natural antioxidants in foods. In addition, some well-known nutrients may be risky to certain individuals, such as lactose, which may be deleterious to lactose-intolerant individuals. According to origin, hazards in foods can be divided into inherent toxicants (or endogenous toxicants) and contaminants (or exogenous toxicants). Endogenous toxicants are metabolites produced via biosynthesis by food organisms under normal growth or metabolites produced via biosynthesis by food organisms that are stressed. Contaminants refer to toxicants that directly contaminate foods, toxicants that are absorbed from the environment by food-producing organisms, toxic metabolites produced by food organisms from substances that are absorbed from the environment, and toxicants that are formed during food preparation [1]. Table 10-1. Endogenous toxicants in foods Toxicants Glucosinolates (goitrogens)

Cyanogenic glycosides Lectins

Source Cruciferae seeds, oilseeds, mustard seed, kale, radish, cabbage, peanut, soybean, cassava, onion, et al. Cassava, sweet potato, nuts, kidney bean, limabean, millet, broomcorn millet, et al Papilionaceae, cereals, soybean, and other beans

Glycoalkaloids

Potato, tomato, and other immature fruits

Gossypol

Cottonseed

Tetrodotoxin

Globefish

Shellfish toxins Histamine

Mussels, Mytilus edulis, scallops, Meretrix meretrix, et al. Scomber japonicus, tunny, sardine, et al. Toxic mushrooms

Mycotoxins

Effects on human upon ingestion Thyroid enlargement, thyroxine synthesis decrease, metabolism impairment, reduced iodine adsorption and protein digestion Cellular inspiration block, stomach upset, sugar and calcium transfer block, and iodine uptake interference Intestinal epithelial cells damage, nutrient adsorption retardation, enzyme inhibition, B12 and lipid adsorption impairment, et al. Cholinesterase inhibition, stomach upset, hematolysis, kidney function impairment Metal binding, iron adsorption decrease, enzyme inhibition Neuron paralysis, dyspnoea and even death Nerve paralysis and liver poisoning Allergy, including blush, dizziness, and tachypnea Gastroenteritis, nerve system injury, hematolysis, visceral parenchymal injury

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Toxicants in Foods Table 10-2. Endogenous antinutrients in foods Antinutrient

Source

Effect on human upon ingestion

Oxalic acid

Beet root, spinach, celeriac, Rhubarb, amaranth, tomato

Interference in the adsorption of Ca, Fe, Zn and other metals and Ca metabolism

Phenols

Vegetables, fruits, grape wines, cereals, soybeans, potato, tea, et al.

Block of thiamine adsorption and decrease of metal bioavailability

Phytates

Papilionaceous plants, cereals, and the seeds of all plants

Decrease of the bioavailability of Ca, Mg, Fe, Zn, Cu, and other metals and that of proteins and starch

Protease inhibitors

Papilionaceous seeds, peanut, cereals, rice, maize, potato, apple, sweet potato, et al.

Inhibition of trypsinase, chymotrypsin, glycopeptidase, and amylase

Saponins

Papilionaceous plants, spinach, lettuce, sugarbeet, soybean, tea, peanut

Complexation with proteins and lipoids, hematolysis, and gastroenteritis Note that most saponins are nontoxic.

Tannins

Plant-derived foods, such as most fruits, tea, and coffee

Inhibition of pancreatic enzymes, bioavailability decrease of thiamine, proteins, cobalamin, and Fe

Non-protein amino acids

Legume, seaweed

Interference with protein metabolism, bone and nerve poisoning

Table 10-3. Exogenous toxicants in foods Hazards

Source

Effect on human upon ingestion

Heavy metals

Plants and animals cultivated in polluted rivers, estuaries, and soils Foods polluted by organophosphorus pesticides

Inappetency, gastroenteritis, insomnia, dizziness, muscle soreness, anemia, and many other symptoms

Veterinary drugs

Diary and meat products

Microbial toxins

Microorganism-polluted foods and materials

Allergy, drug-resistance development, microecological environment change, early maturity, physiological disorder, chronic disease symptom exacerbation Multiple adverse effects

Organophosphoru s pesticides

Enzyme inhibition, muscle vibration, spasm, myosis, blood pressure elevation, heartbeat acceleration, dyspnea, pneumonedema, and coma

It should be noted that all chemicals are potentially toxic, and that the dose alone determines whether or not a toxic effect occurs. Indeed, toxicant (or toxin) is recently defined as a ―substance that has been shown to present some significant degree of possible risk when consumed in sufficient quantity by humans or animals‖ [2]. For example, pure water drunk to excessive can induce an electrolyte imbalance and lead to death, which is called the ―water toxicity‖ that is due in part to decreased renal capacity to excrete water [3]. The innumerable

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naturally occurring chemicals in food are potentially capable of inducing toxic effects, but few are ordinarily present in sufficient concentrations to actually show toxic effect [2, 4]. Hence, we should distinguish between ―toxic substance‖ and ―toxic effect‖. The most frequently occurring toxicants in foods are listed in Table 10-1~10-3. A substantial number of toxic substances have been isolated and identified This chapter concerns only the most important toxicants that have been identified in foods.

2. ENDOGENEOUS TOXICANTS Endogeneous toxicants include any substances produced by food materials (either plants or animals) that are harmful to human beings. Lectins, saponins, and toxic peptides mentioned above are endogenous toxicants.

2.1. Allergen An allergen refers to any substance that can cause an allergy. Most immune responses of caused by food components are IgE-mediated immediate allergic reactions. Upon exposure to allergens, the lymphatic system of human body acts to avoid or alleviate the adverse effects. Most antigens can be metabolized to monosaccharides, amino acids and lower fatty acids after antigen-antibody reactions. However, complete-antigens, which are characteristic of both immunogenicity and immunoreactivity, can cause immune reactions. The intake of allergen-containing foods can cause allergic symptoms, such as pruritus, functional gastrointestinal disorder and fever. Many foods have been found to contain allergens, but the sensitivity to these foods varies with local diet, environmental factors and possibly genetics. Epidemiological investigations reveal that the incidence of food-induced allergy in infants and children is higher than that in adults and the incidence decreases as age increases. Food allergens share the following characteristics: 1. Allergens are present in most foods, such as milk, eggs, and soybean, of which, milk and egg are the most important source of allergens. Though allergens occur widely in foods, 90% of food allergies are induced by only a small number of foods. 2. Only few food components are allergenic. For example, up to 23 types of glycoproteins have been identified in white egg, but only ovalbumin, conalbumin, and ovomucin are allergic. 3. The allergenicity of foods is subject to change after processing. For example, heat treatment inactivates most allergens and the increase of acidity or the presence of digestive enzymes reduces the allergenicity of foods. 4. Sensitive consumers could suffer allergic cross reactions. Some proteins contain the same antigenic determinant and these proteins can cause allergic cross reactions. For instance, at least 50% of patients allergic to cow‘s milk also react to goat‘s milk and patients allergic to chicken eggs might be allergic to the eggs of other birds.

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2.2. Toxic Glycosides 2.2.1. Properties of Toxic Glycosides Toxic glycosides are also known as cyanogenic glycosides. These compounds often contain glucose or rhamnose as their sugar components and are widely present in cassava, sweet potato, nuts, kidney bean, limabean, millet, and many other cereals. Excessive intake of toxic glycosides may lead to gastrointestinal malaise and affect the transfer of sugars and calcium. The contents of toxic glycosides in plants vary with species and cultivation technologies. Tables 10-4 and 10-5 list the distribution of toxic glycosides and their contents in various cereals. Glycosides can be hydrolyzed by enzymes to produce thiocyanates, isothiocyanates, and perthiocyanates. All these products are toxic and can cause goiter. Table 10-4. Distribution and hydrolytic products of some cyanogenic glycosides in food plants [5] Glycoside Amygdalin Dhurrin Linamarin Lotaustralin Prunasin Cicianin

Source Almond, cherry, peach, plums, apples Sorghums Linseed, clovers, cassava, lima beans Linseed, cloves, cassava, lima beans Cherry, almond Vetches

Hydrolytic products HCN, gentobiose, and benzaldehyde HCN, glucose, and hydroxybenzaldehyde HCN, glucose, and acetone HCN, glucose, and 2-butanone HCN, glucose, and benzaldehyde HCN, vicianose, and benzaldehyde

Table 10-5. Thiocyanate contents in typical vegetables (mg/100g edible part of fresh leaf) Plant Cabbage Savoy Brussels sprout Cauliflower Kidney bean, turnip Kohlrabi Colza Swedish turnip Lettuce, Spanish, onion

Content 3~6 18~31 10 4~10 <1 2~3 2.5 9 <1

Figure 10-1. Structure of glucosinolates; side group R varies.

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Figure 10-2. Hydrolysis of glucosinolates by myrosinase.

2.2.2. Glucosinolates Structure and main species Glcosinolates, also known as β-thioglucoside N-hydroxysulfates, contain one branch and one glucopyranose residue bound via a sulfur atom, as shown in Figure 10-1. The side group R may be sulfur-containing groups, straight-chain alkanes, side-chain alkanes, olefins, saturated alcohols, ketones, aromatic groups, benzoates, indole, polyglucose groups, or others. Among the glucosinolates that have been identified, one third of the compounds possess sulfur-containing R groups and the sulfur atoms are present in multiple forms, such as methyl sulfanes, methyl thionyl alkanes, and methyl sulfuryl alkanes. Plants of the Cruciferae family have high levels of glucosinolates and the side chain R may be alkanes, ω-methyl sulfanes, aromatic groups, or heterocyclic groups. Glucosinolates that act as progoitrins are the source of organic nitriles, isothiocyanates, and SCN ions. Some of these compounds have been shown to be quite harmful if consumed in sufficient amounts by humans and animals. Enzymatic hydrolysis and changes in processing Glucosinolates are quite stable. They are the precursors of isothiocyanates and are present in much higher levels than their hydrolyzates in fresh plants. Glucosinolates can produce a large variety of products when exposed to myrosinase as shown in Figure 10-2. In the case of fresh plants chewing or tissue injury during cultivation, harvest, transport, and processing due to scratching or freezing-thawing, glucosinolates get contact with myrosinase (EC 3.2.3.1) and are hydrolyzed to toxic isothiocyanates. Glucosinolates and their enzymatic hydrolyzates are water soluble. During cooking, more than a half of the compounds are released to water, but the specific loss caused by processing varies with vegetable species. Myrosinase can be activated by Vc. Many facts have proved that myrosinase is inactive in the absence of Vc. The activation is not due to the reducing property of Vc, instead due to a nucleophilic group provided by Vc. Vc improves the Vmax and Km of myrosinase on its substrates [6].

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2.3. Toxic Amino Acids Toxic amino acids are rare amino acids that do not participate in protein synthesis, such as homoserine, jenkolic acid, and 5-hydroxytryptophan. Toxic amino acids are usually the structural analogs of protein amino acids and exert their toxicity as antimetabolites. Nonproteinic amino acids are present in plants in quite low levels and certain non-proteinic amino acids are found only in specific plants. For example, theanine is found only in the Camellia family and the highest content is recorded in tea seeds, reaching 1% of dry weight; 5hydroxytryptophan is present only in the pea family and its content in Griffonic seeds amounts to 14% by dry weight. It should be noted that not all non-proteinic amino acids are toxic. Some rare amino acids, such as theanine and alliin, can impart foods with special characteristics and healthy functions. A summary of some unusual amino acids in food plants is given in Table 10-6.

2.3.1. Classification Up to hundreds of non-proteinic amino acids have been identified in animals and plants and most of them are the intermediate or final products of certain metabolisms in animals and plants. Toxic amino acids can be divided into two categories according to their toxicity: Table 10-6. Some toxic amino acids commonly occurring in plant foods and forages [7,8] Amino acid N-(3-hydroxypyridone-4)-2-aminopropionic acid (mimosine) 2-Amino-4-(guanidinooxy)butyric acid (canavanine) L-2-Amino-6-amidinohexanoic acid (indospicine) 5-Hydroxy-L-tryptophan (5-HTP) 2-Amino-3-methylaminopropionic acid L-3,4-dihydroxyphenylalanine (L-DOPA) 3-Methylenecyclopropylpropionic acid (hypoglycin A) Seleno-amino acids (methylselenocysteine, selenocystathionine, selenocystine) 3-Cyanoalanine and 4-Glutamylcyanoalanine δ-Acetyl ornithine Homoserine and o-oxalylhomoserine Lathyrine, γ-methyl glutamic acid, γhydroxynorvaline, and γ-hydroxyhomoarginine Homoarginine and pipecolic acid

Source Many species of Mimosoidae (a subfamily of Leguminosae) Many species of Papilionoidae (a subfamily of Leguminosae Tropical legume Indigofera spicata Griffonia simplicifolia Cycas spp. Vicia and some other legumes Plants of Sapindaeceae family Plants grown on selenium-rich soils Vicia spp. Lathyrus spp. Lathyrus spp. Lathyrus spp. Lathyrus and Vicia spp.

Bone poisoning-causing amino acids β-aminopropionitrile and β-(N–γ-glutamyl)- aminopropionitrile are the representatives of this category. Both the two amino acids exert their toxicity by inhibiting the formation of cross linkages between the polypeptide chains during the synthesis of collagen and elastin [9].

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Wang Dongfeng, Guoqing Huang and Shuhui Wang Neurotoxic amino acids

The representatives are α, γ-diaminobutyric acid, β-cyano-L-alanine, β-N-oxalyl-α, βdiamino-propionic acid and L-homoarginine.

2.3.2. Toxicity Toxic amino acids are not essential for human body and can interfere with the normal amino acid metabolism. Djenko sickness is a disorder of the urinary tract and most cases are caused by the intake of djenko bean, which contains the toxic djenkolic acid. In some legume species, the contents of djenkolic acid can reach 1~2% and its content in black varieties is as high as 3~4%. Djenkolic acid shares a similar structure with cysteine and thus can interfere in the metabolism of this essential amino acid.

2.4. Lectins 2.4.1. Classification Lectins occur widely in plants, animals, and microorganisms. Exogenous lectins are also known as vegetable haemagglutinin. They are sugar-binding proteins or glycoproteins of nonimmune origin and can lead to selective red blood cell agglutination in human blood. Lectins can reversibly bind to specific monosaccharides or oligosaccharides. Most lectins contain more than one sugar-binding site for cross connection with oligosaccharides. The molecular weight of lectins ranges from 91 kDal to 130 kDal. Lectins are natural antigen of red blood cells and are heat stable. Some lectins are highly toxic to experimental animals. For example, when mouse are orally fed with 80 mg/kg garlic lectin for consecutively 7 days, both the appetite and body weight are significantly reduced. Vegetable hemagglutinin is firstly discovered in castor seeds. To present, a large number of lectins with different properties have been identified. According to structure, lectins are divided into merolectin, hololectin, chemerolectin, and superlectin. Based on the specificity to sugars, lectins can be of the fucose type, the galactose/N-acetyl galactosamine type, the Naceytl glucosamine type, the mannose type, the sialic acid type, or the glycoconjugate type. According to the origin, lectins can be divided into legume-derived lectins, mannose-binding lectins, chitin-binding lectins, type-2 ribosome-inactivating proteins (RIP), and lectins derived from other crops. According to the agglutination to red blood cells, lectins can be categorized as specific and non-specific lectins. Based on evolution and structure correlation, lections can be divided into 7 families. Table 10-7 lists the structure, sugar specificity, and distribution of the 7 families in crops. Based on reported researches, Peumans W J divided the lectins that have been identified in crops into 5 categories: (1) legume lectins. Legume lectins are a family of structurally related lectins that occur exclusively within the legume family. In Spite of their structural and evolutionary relationships, legume lectins exhibit a wide range of carbohydrate-binding specificities. (2) Monocot mannose-binding lectins. Monocot mannose-binding lectins occur in at least five different families (Amaryllidaceae, Alliaceae, Araceae, Liliaceae and Orchidaceae). They all have a similar molecular structure and sugar-binding specificity. (3) chitin-binding lectins.

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Toxicants in Foods Table 10-7. Properties of the 7 families and their distribution in crops Family

Structure

Leguminous lectin

Β-sandwich

Monocot mannose-binding lectin

Specificity Man/Glc Gal/Gal NAc (GlcNAc)n Fuc Siaα2,3 Gal/GalNAc complex

Β-barrel

Man

Family Hevein domaincontaining chitinbinding lectin Type-2 ribosomeinactivating proteins

Structure Hevein domain

Specificity (GlcNac)n

β-trefoil

Gal/GalNac Siaα2,6 Gal/GalNAc

Cucurbitaceae Phloem Lectins Breadfruit lectin

Unknown

(GlcNac)n

Β-prism

Gal/T-antigen Man

Amaranthaceae lectin

Β-trefoil

GalNAc/T-antigen

Distribution Leguminosae Orchidaceae, Liliaceae, Amaryllidaceae, Araceae, Alliaceae, Bromeliaceae, Iridaceae Distribution Gramineae, Solanaceae, Urticaceae, Loranthaceae Euphorbiaceae, Caprifoliaceae, Loranthaceae, Iridaceae, Ranunculaceae, Lauraceae, Passifloraceae, Liliaceae, Leguminosae Cucurbitaceae Moraceae, Convolvulaceae, Compositae, Musaceae, Gramineae, Cruciferae Amaranthaceae

Table 10-8. Legume lectins [10] Species (common name)

Tissue

Oral toxicity

Heat stability a

Harmful lectin in food Raw Processed

Seed

Content in raw material (g/kg) 0.2-2

Arachis hypogaea (peanut) Glycine max (soybean) Lens culinaris (lentil) Phaseolus coccineus (scarlet runner bean) Phaseolus lunatus (lima bean) Phaseolus acutifolius (tepary bean)

Slight

Unstable

Yes

Yes

Seed

0.2-2

Slight

Low

Yes

No

Seed

0.1-1

Slight

Unstable

Yes

No

Seed

1-10

High

Moderate

Yes

Possibly

Seed

1-10

High

Moderate

Yes

Possibly

Seed

1-10

High

Moderate

Yes

Possibly

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Wang Dongfeng, Guoqing Huang and Shuhui Wang Table 10-8. (Continued)

Species (common name)

Tissue

Content in raw material (g/kg) 1-10

Oral toxicity

Heat stability a

Harmful lectin in food

Phaseolus Seed High Moderate Yes Possibly vulgaris (kidney bean) Pisum sativum Seed 0.2-2 Minor Unstable Possible No (garden pea) Vicia faba (fava Seed 0.1-1 Minor Unstable Possible No bean) Note: a Heat stability (of purified lectin in aqueous solution); very high: withstands 90°C; high: withstands 80°C; moderate: withstands 70°C; low: withstands 6OT; unstable: denatured at 60°C. The definitions also apply to Table 10-6.

Chitin-binding lectins are found in five taxonomically unrelated families (Gramineae, Solanaceae, Urticaceae, Papaveraceae and Amaranthaceae). Although there are some differences in their overall molecular structure, all chitin-binding lectins are composed of similar domains and exhibit comparable specificity. (4) A dozen type-2 ribosome-inactivating proteins (RIP) have been isolated from plants belonging to taxonomically unrelated families. All type-2 RIP are chimeric proteins that are composed of a catalytically active A chain that is covalently linked through a disulphide bridge to a carbohydrate-binding chain. With the exception of a lectin from elderberry, all type-2 RIPs exhibit specificity towards galactose or N-acetylgalactosamine. (5) Other plant lectins [10]. Table 10-9. Mannose-binding lectins [10] Species (common name) Allium ascalonicum (shallot) Allium cepa (onion) Allium porrum (leek) Allium sativum (garlic) Allium ursinum (ramsons) Colocasia esculenta (taro) Xanthosoma sagittifolium (taro)

Tissue

Content in raw material (g/kg)

Oral toxicity

Heat stability


Bulb

0.01-0.1

Nontoxic

Moderate

No

No

Bulb

<0.01

Nontoxic

Moderate

No

No

Leaf

<0.01

Nontoxic

Moderate

No

No

Bulb

0.5-2

Nontoxic

Moderate

No

No

Bulb

1-5

Nontoxic

Moderate

No

No

Tuber

1-5

ND

Moderate

No

No

Tuber

1-5

ND

Moderate

No

No

2.4.2. Contents and Properties Based on reported researches, Peumans W J et al. summarize the contents, heat stability, and harmfulness to human body of the 5 categories of lectins, as shown in Tables 10-8 to 1012. It could be seen that among the legume lectins, runner bean, lima bean, tepary bean, and kidney bean have higher lectin contents and the corresponding processed foods are possibly toxic. Soybean foods that are improperly processed, such as by insufficient heating, may lead

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to lectin poisoning. Experiments have proved that when mouse are fed lectin-containing crude soybean powder, the mouse will suffer either enterocyte breakdown, leading to intestine malfunction, or reduced activity of hydrolytic enzymes in stomach and intestine, leading to decreased nutrient adsorption and inhibited growth. Table 10-10. Chitin-binding lectin [10] Species (common name)

Tissue

Oral toxicity

Heat stability


Seed

Content in raw material (g/kg) <0.01

Hordeum vulgare (barley) Oryza sativa (rice) Secale cereale (rye) Triticum vulgare (wheat) Triticum vulgare (wheat) Amaranthus caudatus (amaranth seeds) antifungal protein Cyphomandra betacea (tamarillo) Lycopersicon esculentum (tomato) Solanum tuberosum (potato)

ND

High

Yes

Possible

Seed Seed

<0.01 <0.01

ND ND

High High

Yes Yes

Possible Possible

Seed

<0.01

Moderate

High

Yes

Yes

Germ

0.1-0.5

Moderate

High

Yes

Yes

Seed

0.1

Nontoxic

Very high

Unkown

Unkown

Seed

<0.01

ND

Unkown

Unkown

Unkown

Fruit

<0.01

Nontoxic

High

Possibly

Possibly

Tuber

0.01-0.05

No

High

Not for direct consumption

Possibly

Table 10-11. Type-2 ribosome-inactivating proteins [10] Species (common name)

Tissue


Oral toxicity

Heat stability

Harmful lectin in food Raw

Processed

None

Possibly

Ricinus communis (castor seeds)

Seed

1-5

Lethal

Unstable

Not for direct consumpti on

Sambucus nigra (elderberry)

Fruit

0.01

ND

Moderate

Yes

2.5. Saponins Saponins are a complex group of compounds and consist of saponin, sugar, uronic acid or other organic acids. Most saponins are white and amorphous powders and tastes bitter and spicy. Saponins are insoluble in nonpolar solvents but are soluble in water-containing polar solvents. Saponins can irritate the mucous coat of digestive tract and lead to local hyperemia,

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swelling, and hemorrhagic inflammation. The symptoms of saponin poisoning include nausea, vomiting, diarrhea, abdominal pain. Table 10-12. Other plant lectins [10] Species (common name) Amaranthus caudatus (grain amaranth) Artocarpus integrifolia (jact fruit) Cucurbita pepo (pumpkin) Musa paradisiac (banana)

Tissue


Oral toxicity

Heat stability

Harmful lectin in food Raw

Processed

Seed

0.1-0.5

Nontoxic

Unstable

No

No

Seed

0.5-2

ND

ND

Unknown

Unknown

Fruit

<0.01

ND

ND

Possibly

Unknown

Fruit

<0.01

ND

ND

Unknown

Unknown

Saponins are widely distributed in plants as well as in some marine animals. The reports on saponins-induced food poisoning are emerging in recent years. For example, poisoning caused by the ingestion of improperly cooked kidney bean is quite common in China and saponins have been confirmed as a cause of the poisoning. One of the most intensively studied saponins is tea saponins and their structure, composition, and physiologicalbiochemical properties have been well elucidated. The following describes the structure, properties, and toxicity of saponins by taking tea saponins as an example.

2.5.1. Structure of Tea Saponins A saponin consists of ligand, glycoside, and organic acid. Chemically, a saponin has a triterpene or steroid backbone linked to one or more sugar groups. There are thus two broad subdivisions of saponins: those with triterpenoid aglycones and those with steroid aglycones. Tea saponins contain one of the following four ligands: R1-barrigenol, theasapogenol B, theasapogenol D, and A1-barrigenol. Of the four ligands, R1-barrigenol and A1-barrigenol are present only in theafolisaponin and cannot be found in tea seed saponins. The structure and properties of the saponins are illustrated in Figure 10-7 and Table 10-13. Both theafolisaponin and tea seed saponins have triterpenoid aglycones as their ligands. Table 10-13. Chemical properties of theafolisaponin Ligand

Melting point (°C)

R1-barrigenol Theasapogenol B Theasapogenol D A1-barrigenol

303~308 284~288 285~286 285~287

Molecular weight 506 490 474 490

Empirical formula C30H50O6 C30H50O5 C30H50O4 C30H50O5

Toxicants in Foods

R1-barrigenol

Theasapogenol D

317

Theasapogenol B

A1-barrigenol

Figure 10-7. Ligands of theafolisaponin.

Figure 10-8. Glycoside structure of tea saponins.

Theafolisaponins consist of sapogenin, glycoside, and organic acid. In addition to the four ligands mentioned above, three more ligands have been found, but their structures remain unclear. The organic acid in tea seed saponins might be angelic acid, tiglic acid (E-2-methylbut-2enoic acid), or acetic acid. In theafolisaponins, the organic acid is one of angelic acid, tiglic acid, or cinnamic acid. Arabinose, xylose, galactose, and glucuronic acid are major glycosides contained in theafolisaponins and tea seed saponins (see Figure 10-8).

2.5.2. Properties and Toxicity of Tea Saponins Tea saponins are colorless crystals with fine cylindrical structure. The saponins taste bitter and spicy with strong foamability. Crystal tea saponins are insoluble in ether, chloroform, benzene or other nonpolar solvents, less soluble in cold water, anhydrous methanol or anhydrous ethanol, soluble in warm water, carbon disulfide, and ethyl acetate, and readily soluble in water-containing ethanol, water-containing methanol, n-butanol, glacial acetic acid, acetic anhydride, pyridine, and many other polar solvents. Tea saponins produce green color after reacting with 5-methyl resorcinol hydrochlorate and their aqueous solutions are acidic in the methyl red test.

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The toxicity of saponins is often attributed to their hemolysis-inducing capability. Tea saponins can damage the red blood cells of animals and result in hemolysis. The toxicity of saponins is thus measured by the maximum valid dilution that still yields hemolysis. The hemolytic activity of tea saponins is lower than Camellia sasanqua saponins, but comparable to those derived from Camellia sinensis and Camellia japonica. Tea saponins exhibit toxicity only to red blood cells (including the nucleated red cells of fish blood and chicken blood and non-nucleated human blood) and are non-toxic to leucocytes. The hemolytic mechanism of tea saponins is reported related to the permeability change of cell membrane. Tea saponins can injury the cholesterol-containing cell membrane and lead to cytoplasm leakage and cell disintegration. The contact between saponins and blood is the prerequisite of hemolysis. Hence, oral ingestion of saponins does not cause damage in animals or human body. Tea saponins are highly toxic to cold-blood animals, such as fish, frog, and blood sucker, even in low concentrations, but are non-toxic to higher animals when orally administered. Camellia sasanqua saponins are the most toxic to fishes, followed by those derived from Camellia japonica and Camellia sinensis in sequence. Their LD50s are 0.25 mg/L, 3.8 mg/L, and 4.5 mg/L respectively. High water salinity improves the toxicity, as evidenced by the LD50 of 5 mg/L for freshwater fishes and that of less than 1 mg/L for seawater fishes. Osmotic pressure also affects tea saponin toxicity. In the same tea saponins concentration, fishes display low mortality in the salinity range 4‰~10‰, but a further increase or decrease of the salinity accelerates fish death. Besides, the toxicity to fish increases with elevated temperature. Tea saponins can be hydrolyzed in alkaline conditions and thus inactivated. Since seawater is slightly alkaline, tea saponins are degraded within 48 h and do not cause the environmental pollution concern. Tea saponins injury gills and induce hemolysis in fishes. Tea saponins have no adverse effect on shrimps, which also use gill for respiration. There are two reasons for the selective toxicity. The shrimp gill is a cuticle layer zone that develops from the cuticle layer. The epidermis of shrimp gill consists mainly of chitin and protein, which differs significantly from that of fish gill in structure and composition. Besides, the oxygen carrier in fish blood is heme with Fe2+ as its core. In contrast, the oxygen carrier in shrimp blood is hemocyanin and its core is Cu2+. The specific toxicity of tea saponins to fishes has been utilized in the aquaculture industry. Tea saponins have been used as pond-clearing agent of shrimp rearing ponds for removing harmful fishes. Tea saponins have been successfully in hundreds of hectares of sea prawn rearing ponds along the coastlines of the East China Sea, the Yellow Sea, and the Bohai Sea.

2.6. Toxins in Aquatic Organisms 2.6.1. Tetrodotoxin (TTX) TTX is a neurotoxin found in a variety of fish species. Pure TTX is odorless and colorless crystals and its formula is C11H17O8N3, corresponding to the molecular weight of 319.27. TTX is insoluble in water or organic acids, but soluble in weakly acidic aqueous solutions. TTX is unstable in strong acid and alkaline solutions, but is resistant to UV and sunlight. Exposure to UV for 48 h or to sunlight for one year does not change the toxicity of TTX. TTX is not degraded by pepsin, trpsin or amylase and is stable against salts. TTX is heat stable in neutral and faintly acidic conditions. This is why cooked globefishes still cause TTX poisoning.

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319

TTX is an extremely toxic natural toxin and is roughly 100 times more poisonous than potassium cyanide. The LD50 (i.v.) to mice is 8.7μg/kg.

2.6.2. Paralyfric Shellfish Poisons Paralyfric shellfish poisons (PSPs) are the derivatives of tetrahydropurine. These compounds are produced by Gonyaulax catenella and other dinoflagellate species, and the algal toxins (phycotoxins) contaminate shellfish during the filter-feeding of bivalve molluscs. To present, more than 20 PSPs have been identified. Based on structure, PSPs can be divided into carbamoyl type, such as saxiltoxins (STX), neosaxitoxins (neoSTX) and gonyautoxins (GTX 1-4); N-sulfocarbamoyl type, such as C1-4, GTX5, and GTX6; and decarbomyl type, such as dcSTX and doGTX2-3. PSPs are soluble in water, methanol, and ethanol and are stable against acid and heat treatment. PSPs are nerve and muscle paralyzants and take effect by disrupting the Na/K channel. Symptoms of PSP poisoning include nausea, vomiting, diarrhea, abdominal pain, and tingling or burning of lips, gums, tongue, face, neck, arms, legs, and toes. 2.6.3. Ciguatera Toxins Ciguatera is a foodborne illness caused by eating reef fishes whose flesh is contaminated with toxins originally produced by dinoflagellates such as Gambierdiscus toxicus. The poisons causing ciguatera are termed ciguatera toxins and are composed of cigatoxins (CTXs), maitotxin (MTX), and scaritoxin (STX), of which, CTX and MTX are the major constituents. CTXs consist of 13 fused ether rings and are colorless, heat resistant, and amorphous. CTXs are soluble in polar organic solvents, such as methanol, ethanol, and acetone, but insoluble in benzene or water. CTXs are susceptible to oxidation. Its LD50 (i.v.) to mice is 0.45 μg/kg and is 20 times poisonous than TTX. MTX is a polyether and contains up to 31 ether rings. It is ahighly polar molecule and is soluble in water, methanol, ethanol, and dimethylsulfoxide but insoluble in chloroform, acetone or acetonitrile. Pure MTX is white crystal and is extremely susceptible to oxidation. Incubation with 1mol/L hydrochloric acid or ammonium hydroxide solution with heating does not change MTX toxicity. Its LD50 (i.v.) to mice is 0.15μg/kg, which is 2 times more poisonous than CTXs. STX is fat soluble and shares similar chemical and chromatographic properties with CTXs, but differ in polarity as evidenced by TLC and DEAE chromatograph analysis. STX has no absorbance in wavelengths above 220nm and its structure is still not elucidated to the present. 2.6.4. Diarrhetic Shellfish Poisons Diarrhetic shellfish poisons (DSPs) are fat soluble polyether compounds. Based on their backbone structures, DSPs are divided into two groups: OA-group toxins: This group includes okadaic acid (OA) and its derivatives dinophysistoxin-1 (DTX-1) and dinophysistoxin-2. OA is a derivative of C38 fatty acid and its formula is C44H68O13. The structures of OA, DTX-1, and DTX-2 are given in Figure 10-9. Toxins of this group are lipophilic and heat stable. They are produced by dinoflagellates and can be found in various species of shellfish, mainly in filter-feeding bivalve molluscs such as oysters, mussels, scallops, and clams.

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PTX-group toxins: Pectenotoxins (PTXs) are a group of polyether-lactone toxins. To date, more than 15 different analogues have been isolated and characterized. PTX-group toxins are exclusively produced by Dinophysis species. They can be found in filter-feeding bivalve molluscs such as oysters and mussels. PTX-group toxins are heat stable, but they are easily destroyed in strong alkaline and acidic solutions. The characteristic symptoms of diarrhetic shellfish poisoning include diarrhoea, nausea, vomiting and abdominal pain. The LD50 of OA to rat is 160μg/kg. In additoin to causing diarrhea, OA is strongly cancerigenic. PTXs are much more toxic (with LD50 in the range 1677μg/kg) and can cause liver damage.

2.6.5. Actinotoxins Actinotoxins are toxin derived from extracts of the tentacles of sea anemones. To present, more than 60 kinds of cytolysin toxins have been isolated from sea anemones. Cytolysins are substances or antibodies raised by microorganisms, plants or animals that are specifically toxic to individual cells. The molecular weights of sea anemone-derived cytolysins range from 15000 to 20000 and can selectively bind to the lipids on cell membranes, causing pain, inflammation, and muscular paralysis. The most intensively studied actinotoxin is the cytolysin isolated from anthoplerin. The molecular weight of the cytolysin is 17000 and is characterized by a long hydrophobic β-sheet section and 5 short hydrophobic β-hydrophobic sections in the N terminal, in which, 60%-70% of amino acids are hydrogen bonded and form a transmembrane structure. The C terminal is highly hydrophilic and locates on the outside surface of membrane, which forms a transmembrane channel.

Figure 10-9. Structures of OA and DTX-1, and DTX-2. OA: R1=CH3, R2=H, R3=H, DTX-1: R1=CH3, R2=CH3, R3=H, DTX-2: R1=H, R2=CH3, R3=H.

2.6.6. Conotoxins Conotoxins (CTXs) are a group of neurotoxic peptides isolated from the venom of the marine cone snail, genus Conus. CTXs are highly poisonous to human body and the selectivity varies with the living habits of cone snails. Among the CTXs identified, that isolated from Conus gegraphus Linnaeus is the most poisonous. The structures of more than 100 CTXs have been elucidated. A majority of CTXs characterized from venoms so far are 12–30 amino acids (AA) in length and contain one or more disulfide bonds. CTXs are initially translated as propeptide precursors, approximately 80–100 AA and the biologically active toxins are produced by proteolytic cleavage from the precursors. Conotoxins can act on a wide range of targets and are highly tissue specific. CTXs have been used as probes in the classification and identification of ion channels and receptors. CTXs are potential drugs or leading compounds for new drug development.

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2.6.7. Cyanotoxins Cyanotoxins are produced by cyanobacteria (also known as blue-green algae). The mechanisms of cyanobacterial toxicity currently described and understood are very diverse and range from hepatotoxic, neurotoxic and dermatotoxic effects to general inhibition of protein synthesis. Cyanotoxins fall into three broad groups of chemical structure: cyclic peptides (including microcystins and nodularins), alkaloids (including neurotoxic anatoxins and saxitoxins, cytotoxic cylindrospermopsin and demethoxy-cylindrospermopsin, and dermatotoxic aplysiatoxins and lyngbyatoxin) and lipopolysaccharides.

3. EXOGENOUS TOXICANTS IN FOODS During the processing, package, storage, and transport of foods, non-intrinsic components are inevitably added or generated. Besides, plants and animals might accumulate hazardous compounds for the environment and some of which might be absorbed by consumers and pose potential risks. The intrinsic components and various additives that are supplemented in compliance with regulations impart foods with nutrition and flavor. However, the contaminants produced during food processing, package, storage, and transport or due to environmental pollution are unnecessary for foods and most of the contaminants might cause potential risk on food safety.

3.1. Heavy Metals The nutritional status of plants and animals are closely associated with the environment and fertilizer or feed. When minerals in the environment and fertilizer or feed are deficient or excessive, the minerals in the corresponding foods are also in shortage or excess. Plants and animals accumulate both essential and harmful minerals and finally affect the mineral contents of top predators in the food chain. For example, the Keshan disease and kashin-beck disease suffered by residents in Se-deficient areas are possibly due to Se deficiency in foods. If the human body intakes excessive minerals, especially heavy metals, poisoning occurs. The world-famous Minamata disease is a result of Hg pollution. In addition to water and air, the contents of heavy metals in human body are mainly affected by their contents in foods. When human beings consume foods derived from plants or animals with strong heavy metal accumulation capabilities, the metals will exceed thresholds and cause potential harm on human body.

3.1.1. Content and Toxicity of Heavy Metals All metals are toxic when their contents exceed certain thresholds. The deficiency of essential metals can lead to growth retardation, declined propagation, and even death. In the early stage of essential metal deficiency, the growth and propagation can be recovered by supplementing the corresponding elements. The toxicity of metals depend on their contents and valence and the presence of other metals as well. For example, Cu increases the toxicity of Hg but reduces the toxicity of Mo. In turn, Mo significantly reduces Cu adsorption and causes Cu deficiency. Cd interferes in the adsorption of Cu and Cu in low valence reduces the tolerance to Cd toxicity.

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Living bodies do not show deficiency for non-essential metals, such as Be, Hg, Pb, and Sb. However, once the contents of these metals exceed certain thresholds, toxicity symptoms appear.

3.1.2. Poisoning Mechanism The poisoning mechanisms of metals are very complex. In addition to their contents in human body, the toxicity of metals is also affected by their ingestion pathway, solubility, presence form, physic-chemical properties, and the status of human body. Generally, metals exert their toxicity through one or more of the following mechanisms: 1. Heavy metals disrupt the functional groups of bioactive molecules. For example, Hg(II) and Ag (II) can bind to the –SH of cysteine residues in enzymes and interrupt the enzymatic reactions mediated by the –SH group. 2. Heavy metals replace the metal cofactors in biomolecules. The activity of metalloenzymes is closely associated with metals. Due to the different macromoleculebinding stability of metals, a metal with a higher stability constant replaces another metal with lower stability constant and consequently inactivate the metalloenzyme. 3. Heavy metals change the conformation or higher-order structures of biomolecules. For example, polynucleotides are units for genetic information storage and transfer. Heavy metals can bind to the units and change their structures. This is why heavy metals readily cause cancer and teratogenesis. Proteins, phosphatides, some sugars and nucleonic acids can provide atoms for coordinating with metal ions, such as the nitrogen atom in imidazole (histidine), NH2 (lysine), purine and pyrimidine bases in DNA and RNA, oxygen atom in the hydroxyl group (serine, tyrosine), COO- (glutamic acid and aspartic acid), PO43- (phospholipids, nucleic acids), and sulfur atom in –SH (cystine) and SR (methionine, CoA). The three atoms are good ligands for metals. In metal activated enzymes, the prosthetic metals often bind loosely to the enzymes. The metals can be easily replaced by other metals and the functions of the enzymes are destroyed as a result. In metalloenzymes, metals bind tightly to the enzymes and their replacement by other metals is difficult. However, during enzyme synthesis, metal replacement may occur readily. Cell wall, cell membrane, and organelle membrane are the main barrier of metal intake. Generally, lipophilic metal ions with less charge can permeate the barriers easier. For example, CH3Hg+ has a higher permeability than Hg2+ and the permeability of (CH3)2Hg is greater than CH3Hg+. Based on these facts, it can be deduced that M2+ coordinated with a organic ligand can enter cells easier than its hydrate form [M2+(H2O)n]. Metals can change the permeability of cellular membranes by initiating lipid peroxidation and the changes vary with the affinity of metals to the ligands on membrane surface. The peroxidative degradation of lipids in cell and organelle membranes is lethal to life systems. For example, the increase of permeability and hemolysis of red cell has been related to the peroxidative degradation of lipids. Lipid peroxidation is a special form of cell damage and occurs mainly to polyunsaturated fatty acids in membranes. Under normal conditions, the damage caused by low metal levels can be repaired by intracellular molecules such as vitamin E and Se-containing glutathione peroxidase.

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3.2. Pesticide Residues [11] Pesticides are the collectives of pure or mixture compounds derived from organisms or other natural substances for the prevention, diminish, or control of diseases, insects, grasses, and other harmful organisms that adversely affect agriculture and forestry or for regulating the growth of plants and insects. Based on purposes, pesticides are subdivided into insecticides, fungicides, herbicides, nematicides, acaricides, rodenticides, defoliants, and plant growth regulators, of which, insecticides, fungicides, and herbicides are the most widely used. According to chemical composition and structure, pesticides can be organophosphorus, carbamate, organochlorine, pyrethroid, organoarsenic, or organomerurial.

3.2.1. Organochlorine Pesticide According to structure, organochlorine pesticides are divided into dichlorodiphenyl trichloroethane (DDT) and its homologens, hexachlorocyclohexane (HCH), cyclopentadiene and related compounds, and toxaphene and related compounds. Pesticides of the same type share similar chemical structure and pharmacological actions, but differ significantly in toxicity. DDT and its homologens and HCHs are highly toxic and are degraded very slowly in the environment. Most species of the two classes have been prohibited for use in agriculture. 3.2.2. Organophosphorous Pesticides Due to advantages of economy, efficiency, and convenience for agricultural insect control, organophosphorous pesticides are currently widely used for insect control.

Organophosphorous pesticides have the similar backbone , in which, groups R1 and R2 are simple alkyl or aryl groups. The two groups can bind directly to the P atom or through –O– and –S–. In phosphoramidate, the C atom is connected to the P atom through – NH. The X group might be fatty, aromatic, or heterocylic groups connected to P through –O– or –S–. According to structure, commercial organophosphorous pesticides are divided into phosphates (not containing the sulfur atom, such as dichlorvos, dipterex), monothiophosphate (containing one sulfur atom, such as fenitrothion and prothiofos), and dithiophosphate (containing two sulfur atoms, such as rogor). Most organophosphorous pesticides are provided as esters.. Hydrolysis Due to the presence of polar P=O or P=S bond in phosphoryl compounds, the P atom posses valid positive charge and is highly electrophilic. Hence, substitution reactions occur easily to electrophilic reagents. The hydrolysis susceptibility of organophosphorous pesticides to alkaline, neutral and acidic conditions is related with their structures. Hydrolysis leads to the breakdown of the P-O-C or P-S-C bond and activity loss. All phosphate pesticides contain the phosphate bond P-O-C. Two electrophilic centers (P and C) exert effects in the hydrolysis of the bond. According to the Lewis‘s acid-base theory, P serves as a hard acid and C serves as a soft acid. The hard base –OH attacks the P atom with priority and breaks the P-O linkage. The soft base H2O attacks the C atom with priority and lead to the breakdown of the O-C linkage.

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Phosphate pesticides are hydrolyzed slowly in neutral and acidic medium. Thiophosphates are more resistant to hydrolysis than the corresponding phosphates, because the S atom is less electronegative than the O atom. Phosphorylation The substitution reaction between phosphoryl compounds and nucleophilic agents may yield two types of products. If the P atom is attacked, a phosphoryl compound is produced; if the α-C atom is attacked, an alkyl product is yielded, as shown in Figure 10-5. The phosphorylation reaction is essentially the nucleophilic substitution reaction occurred in the P atom. The H atom connected to O, S, and N of nucleophilic reagents can be substituted by phosphoryl group to yield new phosphoryl products. The phosphorylation reaction is important for the preparation of natural phosphoryl compounds and their biochemical aspects. The inhibition of esterase and the toxicity against animals by organophosphorous pesticides are attributed to the phosphorylation reaction.

Figure 10-5. Substitution reactions between phosphoryl compounds and nucleophilic agents.

Alkylation As mentioned above, when a nucleophilic agent attacks the α-C of the phosphate group, the alkyl group is detached from the phosphate and the phosphate anion and alkylated compound are generated. Many nucleophilic reagents can react with phosphoryl compounds for alkylation. For example, amine compounds and sodium iodide have been used in the production of demethylated derivatives of organophosphorous pesticides. Many sulfide compounds, such as dithiophosphates, dithiocar-bamates, thiol, thioether, thiocyanates, and thiourea are dealkylation agent of phosphates. Oxidation and reduction Oxidation of P=S to P=O is an important reaction for organophosphorous pesticides. Oxidation increases the activity of pesticides, because the resultant products are strong cholesterase inhibitors. Hydrogen nitrate, hydroxides, bromine water, peroxides, and many peroxides catalyze the oxidation of organophosphorous pesticides. It should be noted other groups on ester groups and side chains are also easily oxidized during P=S oxidation. Reduction often leads to activity loss. For example, the S atom of the P=S bond can be reduced to hydrogen sulfide when boiled in 48% bromic acid. The resultant hydrogen sulfide can react with dimethyl-p-phenylenediamine and ferric chloride to produce methylene blue. This reaction has been used to detect the presence of diazinon, rogor, and azinphos methyl.

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The nitro group in the benzene ring of parathion, fenitrothion, and ethyl-p-nitrophenyl can be easily reduced to amine and the reducing products are no longer effective. Photo and thermal degradation

Figure 10-6. Photo degradation of chlorphrifos.

The energy carried by light is sufficient to change organophosphorous pesticides. The changes include the oxidation of thiophosphoryl and thioether groups, breakdown of ester linkage, rearrangement of P=S to P-S-, cis- and trans- transformation, and polymerization. Photo degradation is affected by light intensity, wavelength, exposure time, pesticide state, medium or solvent properties, pH, coexistence with air or water, and presence of photo sensitizer. Methyl parathion, rogor, and ethyl-p-nitrophenyl are susceptible to photo degradation and the P=S bond can be oxidized to P=O or rearranged to P-S-. The thioether group on side chains are also oxidized to sulfoxide and sulfone after exposure to light. The susceptibility to photo degradation has been identified in dialkyl thioethers (thimet, disulfoton and phosphorodithioate), alkylaryl thioethers (fenthion and trithion), and diaryl thioether (abate). UV radiation induces the conversion between trans and cis structures. For example, mevinphos is susceptible to photo catalysis and either forms of Z- and E- mevinphos are converted to the mixture of 30% Z and 70% E configurations. If UV light and water are present at the same time, hydrolysis occurs in the acidic ether bond. When chlorphrifos is hydrolyzed, trichlorohydroxy pyridine is produced at first. The product is then further hydrolyzed to multihydroxyl pyridine. The latter is quite unstable and is finally degraded to carbon dioxide, as shown in Figure 10-6. It could be seen that organophosphorous pesticides are very unstable and are subject to degradation in alkaline condition, UV exposure, oxidation, and thermal treatment. Besides, organophosphorous pesticides can be hydrolyzed by esterases including acid phosphatase and microorganism-derived organophosphor hydrolases. The instability to heat and enzyme can be used to reduce organophosphorous pesticide residues during food processing.

3.2.3. Pyrethroids Pyrethroids are high-efficay and low-toxicity insecticides and acaricides. Pesticides of this category include deltamethrin, prallethrin, cyphenothrin, and cyhalothrin. The effective doses of pyrethroids are less than 10 gram per hectare. In environment, pyrethroids are destroyed mainly by photo degradation (leading to isomerization, ester linkage breakdown, and dehalogenation), followed by hydrolysis and oxidation. However, insects can evolve resistance to pyrethroids in a short time. Hence, pyrethroids are often used together with other pesticides to reduce the resistance issue. Pyrethroids are divided into two groups based on their chemical structure and action mechanism. Group I pyrethroids do not contain the cyano group. Commonly used group I pyrethroids include allethrin, bifenthrin, tetramethrin, ethofenprox, and permethrin. Group I

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pyrethroids tend to produce repetitive action potential as a result of the increase in depolarizing after-potential [12]. When the depolarization potential exceeds a threshold, a series of action potentials are initiated. Group II pyrethroids contain the cyano group and include fenvalerate, cypermethrin, deltamethrin, cyfluthrin, and cyhalothrin. These pesticides can cause conduction block and elongate the depolarization phase. In this case, membrane is gradually depolarized without the occurrence of action potential and the nerve conduction is blocked as a result. The increase of depolarization potential or the elongation of the depolarization phase is possibly due to the change of the three-dimensional structure and permeability of biomembranes. After the pesticides attach to the membranes, the functions of the Na/K pump are affected and the Na+ channel in membranes are persistently opened or closed. As a result, Na+ continuously enters the cell and lead to abnormalities. Pyrethroids change the mobility of membranes (group I pyrethroids increase the membrane mobility, whereas group II pyrethroids reduce the index), stimulate the release of neurotransmitters (glutamic acid and aspartic acid) and cGMP, and interfere in the normal functions of cytochrome C and electron conduction systems. Commercial pyrethroids are the mixture of their optical isomers and the isomers differ markedly in toxicity.

3.2.4. Carbamates Pesticides of this category include tricarnam, aldicarb, bufencarb, carbofuran, methomyl, and propoxur. Carbamates are characterized by quick action, high selectivity, low toxicity to homothermal animals, fishes, and human, easy degradation by soil microorganisms, and less accumulation in organisms. Carbamates exert their toxicity as cholinesterase inhibitors, which is similar to that of organophosphorous pesticides. After carbamates are hydrolyzed, the activity of cholinesterase can be partially restored.

3.3. Dioxins and Dioxin-Like Compounds The term dioxin is commonly used to refer to a family of toxic chemicals that all share a similar chemical structure and a common mechanism of toxic action. This family includes 7 of the polychlorinated dibenzo dioxins (PCDDs), 10 of the polychlorinated dibenzo furans (PCDFs) and 12 of the polychlorinated biphenyls (PCBs). Dioxin is defined by World Health Organization (WHO) and United Nations Environment Program (UNEP) as persistent organic pollutants (POPs). POPs are chemical substances that persist in the environment, bioaccumulate through the food web, and pose a risk of causing adverse effects to human health and the environment. Due to the similarities in biochemistry and toxicological properties, some halogenated aromatic hydrocarbons, such as polychlorinated diphenyl ethers (PBDEs), polychlorinated naphthalene (PCNs), polybrominated dibenzodioxin/furan (PBDD/Fs), polybrominated biphenyls (PBBs), and other halogenated aromatic hydrocarbon mixtures are also classified as dioxin-like compounds.

3.3.1. Dioxins Dioxins are a group of planar aromatic hydrocarbons that are substituted by multiple chlorides and share similar structure and physic-chemical properties. Dioxins are oxygen containing chlorinated tricyclic aromatics. Seventy five PCDDs and 135 PCDFs have been defined as dioxins. The most intensively studied toxic dioxins are 17 congeners, whose –H atoms in positions 2, 3, 7, and 8 are substituted by chloride. 2,3,7,8-Tetrachlorodibenzo-p-

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dioxin (TCDD) is the most toxic dioxin among all known congeners (oral LD50 1µg/kg bw). TCCD is highly carcinogenic and can interfere in endocrine in extremely low concentrations. Dioxins are the by-product of combustion and various industrial processes. The sources of environmental dioxins include chlorphenols used in wood preservation and blood fluke prevention, combustion, defoliant, insecticides preparation, paper bleaching, and automobile exhaust.

3.3.2. Polychlorinated Biphenyls Polychlorinated biphenyls (PCBs) are a group of organic compounds with a theoretical number of 209 congeners. Most PCBs are nonplanar compounds. However, some PCBs have the planar dioxin-like structures and are quite similar to 2,3,7,8-TCDD in biochemical and toxicological properties. Pure PCBs are crystals and their mixtures are oily liquids. Most industrial PCBs are mixtures and contain both coplanar and nonplanar congeners. PCBs are highly stable and are resistant to acid, alkaline, corrosion, heat, and oxidation. PCBs do not corrode metals and are fire retardant and insulating. PCBs have been widely used in industries and business for more than 40 years in open (such as in paint, printing ink, copy paper, adhesive, sealant, and lubricant) and closed conditions (as special heat transfer medium in the insulating fluids of transformers and capacitances and the medium in heat transfer systems and hydraulic systems). The production and use of PCBs have been banned in many countries since 1970s. However, the PCBs that have been used can still enter the environment; besides, PCBs generated by waste burning and dioxin-like PCBs are still potential environment pollutants. 3.3.3. Chemical Properties PCDD/Fs have similar physical and chemical properties. They are colorless, odorless, lipophilic and insoluble in water with high boiling and melting points. Thermal stability. PCDD/Fs are extremely heat stable. The lowest temperature for degradation is 800 °C and substantial degradation occurs in 1000 °C and higher temperatures. Low vapour pressure (from 4.0×10–8 mm Hg to 8.2×10–13 mm Hg) PCDD/Fs can exist in both the gaseous phase and bind to particles. The two key parameters, the congener‘s vapor pressure and the ambient air temperature, govern the partitioning between gaseous phase and particle. Fat solubility PCDD/Fs are extremely lipophilic. The log of the distribution coefficient between octane and water reaches up to 6. Hence, PCDD/Fs transfer and accumulate through the food chain mainly via lipids. Environmental stability

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PCDD/Fs are resistant to physical, chemical, and biochemical degradations and can persist in environment. UV light can rapidly destroy PCDD/Fs. However, PCDD/Fs in air are absorbed on gasoloid particles and hence quite resistant to UV degradation. In soil, PCDD/Fs are stable against physical, chemical, and biological degradation and the average half life reaches 9 years.

3.3.4. Toxicity PCDD/Fs are extremely toxic. In contrast to common acute toxicants, death occurs several weeks post PCDD/Fs intake. After ingestion, the body weight of animals decrease sharply in the early phase, accompanied by emaciation syndromes, including significant muscle and fat tissue reduction. The uptake of sublethal dose leads to body weight loss and the effect is dose dependent. In experimental animals, sublethal dioxin induces thymic atrophy by reducing the number of lymphocytes in thymic cortex. According to published researches, human thymus is also sensitive to dioxins. A characteristic symptom of dioxin poisoning is chloracne [12]. Chloracne can cause skin accretion or hyperkeratosis [13] and pigmentation [14]. Dioxins are hepatoxic. Dioxins in high levels cause liver enlargement and subsequent degeneration and necrosis in experimental animals. Besides, dioxins have reproductive toxicity and carcinogenicity. It is reported that dioxins stimulate the hydroxylation of dihydrotheelin and decrease the dihydrotheelin concentration in blood. Dioxins can change the morphology of testis and affect the formation of sperm. The strong carcinogenicity of 2,3,7,8-TCDD has been evidenced in rodents and the minimum dosage for liver cancer in mice is as low as 10 pg/kg body weight.

3.4. Veterinary Drugs The veterinary drug residues in foods include both original drugs and their in vivo metabolites. Original drugs and their metabolites might covalently bind to intrinsic molecules as bound residues. The main veterinary drug residues include antibiotics, sulphonamides, furanes, coccidiostats, hormones and anthelmintics, of which, antibiotics and hormones are the most important healthy risks for human body.

3.4.1. Chemical Properties of Common Antibiotics Penicillins Penicillins refers to group of compounds containing the penicillin core and includes penicillin F, penicillin G, penicillin X, penicillin K and dihydropenicillin F. The penicillin core structure is shown in 10-7. Table 10-14 lists the structures of the R side chain of common penicillins and some properties. Penicillins are monoacid and can form salts with potassium, sodium, calcium, magnesium and many other ions. Penicillins are readily soluble in organic solvent such as alcohols, ethers, and esters. Penicillin salts are readily soluble in water. Free penicillins and their salts are quite unstable in aqueous solutions and are liable to heat treatment, but crystallized penicillins and their salts are quite stable and can be stored for several years in room temperature.

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The antibacterial potency of penicillins is related to the stability of the lactan ring, in which, the peptide bond can be broken by acids, bases, heavy metals or penicillin enzymes, causing activity loss. Figure 10-8 and Figure 10-9 illustrate the hydrolysis of penicillins under acidic and alkali conditions.

Figure 10-7. Structure of penicillin core.

Table 10-16. Side chains and physical constants of penicillins Trivial name

Chemical name

Side chain R

Molecular weight of sodium salt

Melting point (°C)

Penicillin F

2-pentene penicillin

CH3-CH2CH=CH-CH2-

334.2

204~205

Penicillin G

benzylpenicillin

356.4

215

Penicillin X

p-hydroxybenzylpenicillin

372.4

228~235

Penicillin K

n-heptylpenicillin

CH3-(CH2)5CH2-

364.3

Dihydropenicillin F

n-Amyl penicillin

CH3-(CH2)3CH2-

336.3

Penicillin V

phenoxymethylpenicillin

Figure 10-8. Hydrolysis of penicillin under acidic condition.

Figure 10-9. Hydrolysis of penicillin under alkali condition.

372.4

120~128

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Streptomycin: R=-CHO, Dihydrostreptomycin: R=-CH2OH. Figure 10-10. Structures of streptomycin and dihydrostreptomycin.

Penicillins can be modified in chemical or biological methods and the resultant derivatives might have improved properties or activity, such as α-ampicillin, cephalothin, cephaloridine. Streptomycin Streptomycin is an aminoglycoside antibiotic. Both streptomycin and its dihydrogenated derivative (Figure 10-10) are used as animal medicines. In acidic conditions, streptomycin is hydrolyzed to streptidine and streptobiosamine. Streptidine is the hexatomic cyclic alcohol inositol substituted by two guanidine groups. Streptobiosamine is a disaccharide consisting of streptose and 2-methylamino glucose. The aldehyde group in streptose determines the antibacterial efficacy of streptomycin and it can be destoryed by cysteine, Vitamin C, hydroxylamines. Dihydrostreptomycin does not contain the free aldehyde group and is therefore more stable than streptomycin. Streptomycin is an alkaline compound and is unstable, but its hydrochloride or sulphate is very stable. Commercial streptomycin is often provided as hydrochlorateor sulfate. Streptomycin and its salts are readily soluble in water, but are insoluble in organic solvents, such as alcohol, ether, and chloroform. Chloramphenicol Chloramphenicol is a broad-spectrum antibiotic and its structure is shown in Figure 1011. It contains two chiral carbon atoms and hence has 4 optical isomers, in which only the levoisomer posses the antibacterial activity. Chloramphenicol is white or colorless acicular or tabular crystal and melts in temperatures ranging from 149.7 °C to 150.7 °C. It is readily soluble in methanol, ethanol, tpropanol and ethyl acetate, slightly soluble in ether and chloroform, and insoluble in petroleum ether or benzene. Chloramphenicol is extremely thermal stable and remains its activity even after being boiled for 5h. Tetracyclines Tetracyclines are a class of broad-spectrum polyketide antibiotics and include aureomycin, terramycin and tetracyclin. Antibiotics of this class share the same core (Figure 10-12). Terramycin and aureomycin are the chlorinated and hydroxylated derivates of tetracycline (Figure 10-12).

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Tetracyclines are amphoteric. Tetracyclines and their salts are yellow or light yellow crystals and are extremely stable in dried state. They are soluble in diluted acid and alkaline solutions, slightly soluble in water and lower alcohols, and insoluble in ethers or petroleum ether.

3.4.2. Chemical Properties of Common Hormones Thyroxine Thyroxine stimulates the metabolism of sugars, proteins, lipids and salts, promotes growth and tissue differentiation, and significantly affects the central nervous system, circulatory system, hematopoiesis and muscular work of organisms. Female hormones Female hormones include hormones secreted by the ovarian follicle before ovum maturation such as estradiol and progesterone secreted by corpus luteum. The structures of common female hormones are shown in Figure 10-13. Male hormones Testosterone is the most important male hormone in human body. It is secreted by the interstitial cells of testicle. Testosterone can be metabolized to androsterone, which can be further converted to dehydroepiandrosterone (DHEA). The structures of these compounds are given in Figure 10-14. Among the male hormones mentioned above, testosterone is the most active and its activity is 5 times higher than that of androsterone. DHEA is the least active and its activity is only one-third of that of androsterone.

Figure 10-11. Molecular structure of chloramphenicol.

Note: R1=R2=H, tetracycline; R1=H, R2=OH, terramycin; R1=Cl, R2=H, aureomycin. Figure 10-12. Chemical structure of the core of tetracyclines.

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Figure 10-13. Structure of some female hormones.

Figure 10-14. Structures of testosterone and its metabolites.

Although female and male hormones have different functions in human body, they share quite similar structure. Both female and male hormones are derived from cholesterol and can be converted to each other.

3.4.3. Veterinary Drug Residues in Animal-Derived Foods The distribution and residue of veterinary drugs in animals depend on the drug application time (before or after meal), administration method (in feed or water, gavage or injection), and drug species. The organs or tissues of animals have different drug accumulation capabilities. Generally, high veterinary drug residues are found in tissues or organs that the drugs are metabolized in, such as liver and kidney. Theoretically, the percentages of veterinary drugs metabolized and excreted increases over time. In another word, veterinary drug residues in animals decrease gradually over time, but the half lives of the drugs vary with drug type and individuals. For instance, the half lives of most chicken drugs are less than 12h and the withdrawal time of most chicken drugs are 7 days. Generally speaking, animals receiving reasonable drug application do not cause healthy risks. However, long-term consumption of animal-derived foods with overstandard veterinary drug residues can cause poisoning. The incidence of precocious puberty in children has been partially attributed to the excessive intake of meats derived from animals overdosed with growth factor and sex hormones. 3.4.4. Fishery Drugs Fishery drugs are often classified based on purposes. Generally, fishery drugs include the following types: environmental modifiers, disinfectants, antimicrobial agents, insecticides,

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metabolic enhancers (hormones), Chinese herbal medicines, biological products, and others. According to preparation methods, fishery drugs are divided into chemical drugs, biological drugs, biochemical drugs, and feed additives.

4. ANTI-NUTRIENTS IN FOODS The nutritional values of animal- and plant-derived foods are determined by the nutritional composition of their edible parts. However, to meet the requirements for growth, propagation, and protection against invasions of microbes and predators, animals and plants have evolved effective protection systems, such as morphology change and chemical secretion. Besides, the accumulation of proteins or protein complexes in seeds or other edible parts is another measure against external attacks, such as enzyme inhibitor, amylase inhibitor, and sugar-binding proteins. These compounds are collectively termed anti-nutrients. If antinutrients are present in high levels, the adsorption and utilization of nutrients in foods will be affected.

4.1. Phytic Acid and Oxalic Acid Researchers have proved that the bioavailability of trace elements in animal-derived foods is higher than that of plant-derived plants. This is because that most trace elements in animal-derived foods are bound to proteins and anti-nutrients such as phytic acid and oxalic acid are absent in these foods.

4.1.1. Phytic Acid Phytic acid (1,2,3,4,5,6-hexakis (phosphonooxo) cyclohexane) is also known as inositol or myo-inosi tolhexakis (dihydrogen phosphate) and its structure is shown in Figure 10-20.

Figure 10-20. Structure of phytic acid.

Phytic acid is contained mainly in the seed, stem, and root trunk of plants and the highest content is found in the seed of legumes and the bran and embryo bud of cereals. Phytic acid can react with Ca, Fe, Mg, Zn, and other elements to form insoluble products and reduce their bioavailability. In addition, phytates also form complexes with proteins. Phytic acid occurs widely in plant-derived foods and is a main anti-nutrient that affects mineral absorption. Each phytic acid molecule has 12 dissociable protons, of which, 6 protons are highly acidic (pKa=1.84) and are completely dissociated in water, 2 protons are weak acidic groups

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(pKa=6.3) and the rest 4 protons are extremely weak acidic groups (pKa=9.7). Phytic acid can complex with most metals and the stability of the complexes is closely associated with the acidity of foods and the properties of metals. For example, the stability of some essential metal-phytic acid complexes in pH 7.4 is Cu2+ > Zn2+ > Co2+ > Mn2+ > Fe3+ > Ca2+. The complexes of phytic acid with proteins, Ca2+, and Mg2+ are water-soluble, whereas those with most heavy metals are only slightly water soluble. Due to the high contents in plant-derived foods, phytic acid can bind to endogenous Zn, Cu, and other metals in pancreatic juice and bile that are secreted by organs to small intestine. Hence, phytic acid not only affects the bioavailability of food-derived trace elements, but also hinders the re-adsorption of endogenous trace elements. Phytic acid has strong chelating capability. Each phytic acid molecule contains six negatively-charged phosphate groups and can complex with proteins as well. When the medium pH is lower than the pI of a protein, the protein carries positive charges. Due to strong electrostatic interaction, the protein binds to negatively-charged phytic acid and insoluble complex is formed. When the medium pH is higher than the pI of a protein, the free carboxyl groups and the unprotonated imidazolyl in histines are negatively charged. In this case, the protein forms a ternary complex with phytic acid by using Ca2+, Mg2+, or Zn2+ as the bridge. The formation of the ternary complex reduces the solubility of proteins and consequently decreases protein digestibility. For example, casein completely dissolves in pH 2 medium. If phytic acid is added, casein is nearly completely precipitated. The same effect also applies to proteins extracted from corn, sunflower seed, soybean meal, and rice bran. However, after phytic acid is degraded by phytase, the solubility of the proteins increase tremendously and some even exceed the original values, such as the proteins derived from fine rice bran and rapeseed.

4.1.2. Oxalic Acid Chemical property Oxalic acid is also termed ethane diacid and is widely present in plant-derived foods. Pure oxalic acid is colorless crystal and is readily soluble in water but not soluble in ether or other organic solvents. Oxalic acid is another strong metal chelator found in plant-derived foods. When oxalic acid binds to alkaline earth elements, the solubility of the metals is significantly reduced. For example, calcium oxalate is nearly insoluble in water (Ksp=2.6×10-9). Hence, oxalic acid significantly reduces the bioavailability of some minerals. It should be noted that formation of complex with some transition metals can significantly improve their solubility. For example: Fe3

3C2O4

20 [Fe(C2O4 )3 ]3 ( Kf=1.06×10 )

Toxicity Intake of excessive oxalic acid leads to urethral calculus and reduces the bioavailability of essential minerals.

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4.2. Polyphenols Polyphenols are widely distributed in plant-derived foods, but their species and contents vary significantly. Due to the presence of multiple hydroxyl groups, polyphenols are susceptible to oxidization and are natural antioxidants in foods. Besides, polyphenols have free radical scavenging, bacterostasis, and cancer prevention functions. Hence, polyphenols are known as functional components in foods. However, polyphenols can complex with some essential trace elements, precipitate proteins, and inhibit the activities of certain enzymes. In this sense, polyphenols are natural anti-nutrients.

4.2.1. Composition, Structure, and Properties Based on structure and biosynthesis pathway, polyphenols can be divided into flavonols, anthocyanins, flavones, phenolic acids, and other polyphenols. Flavones are the most abundant polyphenols in plant-derived foods and their core structure is shown in Figure 1016. The carbon atom in position 3 of flavones can be easily hydroxylated to form a nonphenolic hydroxyl group and the resultant products are called flavonols, which constitute a major class of flavonoids. As shown in Figure 10-16, when groups R1, R2, and R3 are –OH, the compound is kaempferol; if R1 is H, and R2 and R3 are –OH, the compound is quercetin; when R1, R2, and R3 are –OH, the compound is myricetin. Aurone, chalcone, flavanone, isoflavone, isoflavaone, and biflavonyl are important derivatives of flavones. Apigenin (5,7,4'-trihydroxyflavone), luteolin (3',4',5,7-tetrahydroxyflavon), and tricetin (3',4',5,5',7-pentahydroxyflavone) are also flavones. These compounds have similar structure as pelargonidin and anthocyania. In addition to these compounds, more than 60 flavones with different ligands have been identified and they are the hydroxylated and methoxylated derivatives of flavonols and flavones. Most flavones in plant-derived foods are present as glycosides and their ligands include glucose, rhamnose, galactose, arabinose, xylose, apiose, and glucuronic acid. 4.2.2. Physical-Chemical Properties Polyphenols are soluble in hot water and their glycosides are readily water soluble. Polyphenols are highly soluble in such organic solvents as acetic ester, ethanol, and methanol, but are insoluble in benzene and chloroform. Polyphenols have strong antioxidant capability and the specific antioxidant capability varies with structure. The spatial arrangement of substituents is a greater determinatant of antioxidant activity of flavones than the flavan backbone and the configuration and number of hydroxyl groups, O-methylation, 2-3 double bond and 4-oxo function, carbohydrate moieties, and degree of polymerization [15]. The chromone in polyphenols is originally colorless. After the benzene ring is introduced in C2, a cross conjugate system is formed and polyphenols display colors as a result of electron transfer, rearrangement, and conjugated chain elongation. Most flavones and flavonoid glycosides are yellow or slightly yellow. If the double bond between C2 and C3 is saturated by hydrogen, the cross conjugate system is disrupted or conjugates much less. As a result, the chromatic performance of those compounds, such as flavanol, is reduced. The stability of polyphenols is closely related to their structures. The increase of hydroxyl group number decreases the stability, but methylation and glycosylation improve the stability. In anthocyanins, the oxygen atom in the pyrannoid ring is tetravalent and anthocyanins are

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the most chemically active among polyphenols. Polyphenols are unstable and are easily destroyed by high pH, high temperature, and high oxygen concentrations. Besides, oxidases, oxidants, and metal ions also affect polyphenol stability.

Figure 10-16. Structure of flavones.

4.2.3. Antinutritional Effect and Toxicity Antinutritional effect The antinutritional effect of polyphenols is due to their metal-complexing and protein precipitation capabilities. The affinity of polyphenols to transition metals follows the decreasing order of A13+, Zn2+, Fe3+, Mg2+, and Ca2+. The complexation with these metals is one of the antioxidant mechanisms of polyphenols, though the interaction reduces the bioavailability of these metals. Polyphenols can bind proteins through hydrophobic interaction and hydrogen bonding. The formation of precipitates between polyphenols and proteins, amylases, or digestive enzymes reduces the adsorption of polysaccharides, proteins, and lipids. Besides, biomolecules in digestive tract can form complexes with polyphenols, leading to reduced digestion and absorption. Osman M A has attempted to remove catechin from sorghum by fermentation. The results showed that the content of catechin decreased during prolonged fermentation, but the adsorption of proteins increased gradually, as shown in Figure 10-17. Toxicity Polyphenols are sometimes intentionally added to foods by manufacturers as functional additives. Epidemiological survey reveals that excessive intake of polyphenols can cause potential health risks. When reduction, deoxidation, or free radical scavenging reaction occurs, polyphenols are converted to highly oxidized compounds such as quinones or free radicals. Quinones or free radicals are extremely unstable and can initiate the oxidation of other compounds or the generation of new free radicals. As shown in Figure 10-18, enzymatic and/or auto-oxidation of the flavonoid generates the flavonoid semiquinone radical, which may be scavenged by GSH, thereby regenerating the flavonoids and generating the thiyl radical of glutathione. This thiyl radical may react with GSH to yield a disulfide radical anion which rapidly reduces molecular oxygen to superoxide anion radicals [17].

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Figure 10-17. Relationship between catechin content and protein adsorption in fermented sorghum products [16].

Figure 10-18. Oxidation of phenolic flavone peroxides [17].

When positions 3‘ and 4‘ in ring B of flavones are connected with –OH, the flavones can easily form quinoid oxidates by enzymatic and automatic oxidation. The quinoid oxidates can complex with GSH, as shown in Figure 10-19.

4.3. Digestive Enzyme Inhibitors Trypsin inhibitor (LT1), chrymotrypsin inhibitor, and α-amylase inhibitor are major digestive enzyme inhibitors. These inhibitors are evolved by plants and animals as defensive measures against predators. The presence of these inhibitors affects the digestion and adsorption of nutrients and even threatens the health of consumers. For example, the intake of uncooked or under-cooked soybean products can cause nausea, vomiting, and other symptoms.

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Figure 10-19. Oxidation initiated by catechu flavonoid peroxide [17].

4.3.1. Properties Protease inhibitors are widely present in microbes and the tissues of plants and animals. Legume seeds have higher protease inhibitor contents than other sources. Protease inhibitors derived from legume seeds are divided into the Kunitz type and the Bowman-Birk type. Kunitz protease inhibitors have relatively higher molecule weights of about 20000 Dal and specifically bind to trypins. Bowman-Birk protein inhibitors have lower molecule weight of about 9000 Dal and two binding sites. Hence, Bowman-Birk protease inhibitors can bind two serine proteases, trypins, or chymotrypsins at the same time. Kunitz protein inhibitors are sensitive to heat treatment, but Bowman-Birk protein inhibitors are quite heat resistant. Protein inhibitors of difference sources are quite similar in amino acid composition. That is, cystine has the highest content, followed by aspartic acid, arginine, lysine, and glutamic acid in sequence. The high content of cystine facilitates the formation of intra-molecular disulfide bonds, which make the inhibitors heat and acid resistant. 4.3.2. Action Mechanism Digestive enzyme inhibitors inactivate proteases by forming complexes with enzymes. The action mechanism of digestive enzyme inhibitors is elucidated by taking trpsin inhibitor (LTI) as an example. LTI is mainly found in legume Leucaena leucocephala and belongs to the Kunitz family.

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Structure of LTI-trypsin complex Unlike other inhibitors, LTI has an exposed ring and this ring is not subject to the constraint of disulfide bonds and other secondary structures inside the molecule (Figure 1020). The linkage between Arg62-Ile63 in the ring locates in the bottom between the A4 chain and the β-pleated sheet in B1 chain and is susceptible to breakage. Although many similarities in structures, LTI and trypsin-LTI complex differ in the following: a) binding direction of P1; b) amino acids modification in the orthorhombic and tetragonal crystal. In STI, a total of 12 amino acids participate in the formation of the orthorhombic crystal of pig trypsin and the amino acids are Asp1, Phe2, Asn13, Pro61 (P3), Tyr62 (P2), Arg63 (P1), Ile64 (P1‘), Arg65 (P2‘), His71, Pro72, Trp117, and Arg119. In the tetragonal crystal, only 9 amino acids (except His71, Trp117, and Arg119) participate in complex formation and 8 amino acids in STI is involved in the formation of the tetragonal crystal of the complex, including Asp139, Asn136, Pro60, Tyr61, Arg62, Ile63, and Leu64 [18]. LTI provides two trypsin-binding sites. One site is in the reaction center (P3, P2, P1, P1‘, and P3‘) and another is the side chains of Asp139 and Asn136. As shown in Figure 10-20, LTI is presented in solid lines and trypsin is presented in pink lines. The reaction sites in the binding ring of LTI, including P1, P2, P3, P1‘, and P3‘, are presented in balls. Action mechanism of protease inhibitors The action mechanism of protease inhibitors against target enzymes is well elucidated in recent years. The interactions can be divided into three types: Complementary type: Inhibitors bind to the recognition and binding sites of target enzymes and block the activity center by hydrogen bonding with active groups. For example, trypsin inhibitor takes effect in this way.

Figure 10-20. Model structure of the LTI-trypsin complex [18].

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Wang Dongfeng, Guoqing Huang and Shuhui Wang a) "SH

"TO

"TO

b)

Figure 10-21. (a) Stereoview of the Ca-atom backbone of STI (a) and LTI (b). Only the positions of Arg63 and Asn11 are indicated in the structures. (b)

Interaction between Arg62 (P1) of LTI and S1 residues of trypsin (shown in pink color) [18].

Associated type: Inhibitors do not occupy the recognition sites of target enzymes. Instead, the inhibitors associate with target enzymes for hydrogen bonding with the active groups of target enzymes and block the substrate binding site. The interactions between thrombin inhibitors, such as hirudin, and their substrates are of this type. Covered type: Inhibitors cover the areas close to the active center of target enzymes as linear molecules to prevent the contact between active center and substrate. For example, papain inhibitor is of this type.

5. TOXICANTS GENERATED DURING PROCESSING AND STORAGE 5.1. Toxic Substances Generated During Roasting, Frying, and Smoking The high temperature used in roasting and frying can cause oxidization or polymerization of some compounds in foods, especially lipids. Lipid oxidation and thermal denaturation

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significantly affect the flavor, color, shelf life, and safety of lipid-containing foods. Many diseases have been associated with lipid oxidation and their thermal products.

5.1.1. Lipid Auto-Oxidation Products and Toxicity The auto-oxidation of lipids is defined as the oxidation in normal temperature and pressure. Moderate lipid oxidation imparts foods with pleasant flavor, but excessive oxidation reduces the nutritional value of foods and imposes potential risk on human health. The autooxidation products of lipids can precipitate proteins or inactivate enzymes. It has been confirmed that lipid auto-oxidation products inhibit the activities of succinic acid dehydrogenase, ptyalin, and potato amylases. When mice are fed diet that contains low levels of superoxides, the development of mice is retarded. When excessive such diet is fed, mice death occurs. The toxicity of oxidized lipids is closely correlative to their peroxide values. 5.1.2. Thermal Products of Oils and Their Toxicity Oils are often heated during food processing. In high temperatures, the superoxides are degraded rapidly and hence only few superoxides are left in foods. However, the polymerization reaction in high temperatures cannot be ignored. Lipid polymerization changes the physical properties of lipids, such as viscosity, refractive index, and off-color and produces toxicants as well, such as dipropenyl cyclocompounds. When rats are fed basal diets containing 20% such cyclocompounds, death occurs with 3~4 days. 5.1.3. Polychlorinated Biphenyls Polychlorinated biphenyls (PCBs) can accumulate in foods due to their high stability and high solubility in lipids. Fishes are the main source of human PCB intake. PCBs are concentrated mainly in the fatty tissues and various organs of human beings. PCBs show only low acute toxicity and the clinic symptoms of PCB poisoning include erythra, pigmentation, edema, asthenia, and vomiting. The PCB contents in PCB poisoning patients range from 13.1 to 75.5 ppm. Although the PCB residues detected in human bodies cannot cause disease or death, whether the long-term accumulation of trace PCBs in human bodies have any adverse effects on human health or cause cancer, distortion, and mutation deserve intensive attentions. An international toxicity evaluation standard TEQ (toxic equivalent) has been proposed for the residue assessment of PCDDs and PCDFs in fired and roasted foods. Kim Y. measured the contents of PCDDs and PCDFs in two instant foods and found that the PCDDs contents in hamburg and fried chicken are 14 and 7 folds of the corresponding TEQ standards and those of PCDFs are 7 and 10 folds of the corresponding TEQ standards [19]. 5.1.4. Benzo(α)Pyrene Benzo(α)pyrene is a polycyclic aromatic compound that is often found in smoked and roasted foods. This chemical is present in very low level in common conditions. However, after foods are roasted or smoked, its content increases significantly. Benzo(α)pyrene is now recognized as one of the most carcinogenic, teratogenic, and mutagenic substances in the world. Physicochemical properties The systematic name of benzo(α)pyrene is 3, 4-benzo(α)pyrene (abbreviated as B-(α)P). B-(α)P is a polycyclic aromatic hydrocarbon (PAH) consisting of multiple benzene rings. B-

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(α)P has the most significant effect on food safety among PAHs. To date, a total of more than two hundred kinds of PAHs and PAH derivates have been identified and many of them are extremely carcinogenic and mutagenic. Figure 10-24 shows the structures of some common PAHs. Of the PAHs, 3,4-benzo(α)pyrene is the most widely distributed and is highly carcinogenic. 3,4-Benzo(α)pyrene is often regarded as the representative of PAHs.

Figure 10-22. Structure of dipropenyl cyclocompounds.

Figure 10-23. Structure of PCBs. (left) PCB; (right) chlorinated dibenzofuran (X can be substituted by chlorine).

Tetrabenz[a,c,h,j]anthracene Diabenzo(a,e) pyrene

Phenanthrene

Bezo(α)anthracene

Pyrene

Triphenylene

Diabenzo(a,i) pyrene

Bezo(α)pyrene

Figure 10-24. Structures of common PAHs.

The molecular formula of benzo(α)pyrene is C20H12 with molecular weight 250. Benzo(α)pyrene is solid in room temperature as yellow homocline or diamond tabular crystal. Both the two crystal forms are quite stable and insoluble in water, but soluble in benzene, methylbenzene, acetone, and other organic solvents. Benzo(α)pyrene is stable in alkaline medium, but is unstable in acidic medium and can react with nitric acid. Benzo(α)pyrene has strong affinity to chlorine, bromine and other halogen family elements. Benzo(α)pyrene displays a special yellow-green fluorescence, but the fluorescence is not visible after adsorption to such positively charged adsorbents as active carbon, charcoal, and ferric hydroxide. Bezo(α)pyrene cannot be absorbed by negativelycharged adsorbents. Toxicity Benzo(α)pyrenes are recognized as the most carcinogenic, teratogenic, and mutagenic compounds in the world. Experiments have confirmed that oral intake of 3,4-benzo(α)pyrene can cause cancer in experimental animals. High benzo(α)pyrene intake leads to higher

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incidence of cancer and shortened incubation period. In the case of mice, injection of 4-12 μg benzo(α)pyrene induces cancer and the half carcinogenic dosage is 80 μg. The carcinogenicity of PAHs is associated their chemical structures. Carcinogenicity is only observed in PAHs with four to seven benzene rings. PAHs with less than four or more than seven rings are not carcinogenic. Population and epidemiological investigations indicate that benzo(α)pyrenes and other PAHs can be absorbed through respiratory tract, digestive tract, and skin. B-(α)P can cause gastric cancer, lung cancer, or skin cancer in human. Iceland has the third highest incidence of gastric cancer and the reason has been associated with smoked foods in the diet of Iceland residents. Besides, the incidences of gastrointestinal tract and respiratory tract cancers are three times higher than those of inland populations. The carcinogenic incubation period of benzo(α)pyrene in human beings ranges from 20 to 25 years.

5.1.5. Heterocyclic Amines Heterocyclic amines are the thermal degradation products of proteins and amino acids generated during food processing and cooking and are carcinogenic and mutagenic. Based on chemical structures, heterocyclic amines are divided into aminoimidazo azaarens (AIAs) and amino-carboline congeners. AIAs can be further divided into quinoline congeners (IQ), quinoxaline congeners (IQx), pyridine congeners, and benzoxazine congeners. All the AIA congeners contain the imidazole ring with an amino group attached to the α position. ALA congeners can be converted to mutagenic and carcinogenic N-hydroxylated compounds. Because the amino groups in AIA congeners are resistant to 2 mol/L sodium nitrite, which is quite similar to the IQ congeners of firstly identified AIA congeners, AIAs are also known as IQ-type heterocyclic amines. Amino-carbolines are sub-grouped into α-, β, γ, and δ-carboline congeners. The amino groups in amino-carboline congeners are susceptible to diazotization reaction in 2 mmol/L sodium nitrite and can be converted to C-hydrolyzated compounds, which are termed non-IQ heterocyclic amines. Non-IQ heterocyclic amines are not mutagenic or carcinogenic [20]. The chemical structures of some heterocyclic amines are shown in Figure 10-25 and their important physic-chemical properties are listed in Table 10-15. 5.1.6. Acrylamide Acrylamide is carcinogenic and can cause nerve damage. A food can be contaminated by acrylamide if the food is processed by frying or roasting. Acrylamide has been identified in carbohydrate-rich foods that are processed in temperatures higher than 120 °C for a long time, such as fried potato chips. The contents of acrylamide in fried foods often exceed 1000μg/kg. In overcooked fried potato chips, the acrylamide level reaches up to 12800 μg/kg. Acrylamide is a simple molecule with molecular weight (CH2CHCONH2) of 71.09. Acrylamide melts in 87.5 °C and boils in 125 °C. Acrylamide is the block for polyacrylamid synthesis. Acrylamide is a white crystal. It is soluble in water, methanol, ethanol, acetone, dimethyl ether, and trichloromethane, and is insoluble in heptane or benzene. Acrylamide is stable in acidic medium, but is degraded in alkaline solutions. Self-polymerization occurs easily in the melting temperature or when exposed to UV light.

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Wang Dongfeng, Guoqing Huang and Shuhui Wang Table 10-15. Physicochemical properties of some heterocyclic amines Compound IQ 4-MeIQ 8-MeIQ 4-MeIQ 4,8-DiMeIQ PhIP AaC MeAaC Trp-P-1 Trp-P-2 Glu-P-1 Glu-P-2 Phe-P-1

Molecular weight 198.2 212.3 213.2 213.2 227.3 224.3 183.2 197.2 211.3 197.2 198.2 184.2 170.2

Formula C11H10N4 C12H12N4 C11H11N5 C11H11N5 C12H13N5 C13H12N4 C11H9N3 C12H11N3 C13H13N3 C12H11N3 C11H10N4 C10H8N4 C11H10N2

UVmax (nm) 264 257 264 264 266 315 339 345 263 265 364 367 264

pKa 3.8, 6.6 3.9, 6.4 <2, 6.3 <2, 6.3 <2, 6.3 5.7 4.6 4.9 8.6 8.5 6.0 5.9 6.5

Content in foods According to published reports, nearly all foods contain acrylamide. The acrylamide limit of WHO in tape water is 0.5 μg/kg (1993). However, investigations reveal that the acrylamide contents in foods that are rich in carbohydrates and processed by frying or roasting are much higher than the allowed value in tap water. For example, the average content of acrylamide in fried potato chip (slice) is 1000 μg/kg and that in infant biscuit is 600-800 μg/kg. Acrylamide is mainly found in foods that are processed in temperatures higher than 120 °C. High contents of acrylamide have been detected in most fried potato chips, part bread, cocoa powder, apricot kernel, coffee, and biscuits. Generation pathway The mechanism of acrylamide formation in heat-processed foods is still not well understood. It is recognized that acrylamide is generated through the Maillard reaction and the compounds involved include carbohydrates, proteins, amino acids, lipids, and other minor components in foods [21]. The possible mechanism is as follows: amino acids react with reducing sugars to produce dicarbonyl compounds and the products then react with amino acids to yield acrylaldehyde. Acrylaldehyde is oxidized to acrylic acid and acrylic acid is finally converted to acrylamide in the presence of amine or amino acids. Figure 10-27 illustrates the general pathway of acrylamide formation. It is reported that acrylamide can be directly converted from amino acids. In addition, many organic acids, such as malic acid, lactic acid, and citric acid, also participate in the formation of acrylamide. According to current data, the following conclusions can be drawn: (1) Amino acids are degraded in high temperatures and the degradation products react with reducing sugars to produce acrylamide; (2) The primary reaction product N-glucoside of the Maillard reaction plays important role in acrylamide formation; (3) α-Dicarbonyl compounds react with amino acids to release acrylamide;

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Figure 10-25. Structures of some common heterocyclic amines. AaC, 2-amino-9H-pyrido[2,3-b]indole; MeAae, 2-amino-9H-3-methyl-pyrido[2,3-b]indole; Trp-P-1, 2-amino-1,4-dimethyl -9H- pyrido[4,3b]indole; Glu-P-2, 2-amino-1-methyl -9H- pyrido[4,3-b]indole; Glu-P-1, 2-amino-6-methyl -9Hpyrido[1,2-a: 3‘, 2‘-d]imidazole; Glu-P-2, 2-dipyrido[1,2-a: 3‘, 2‘-d]imidazole; IQ, 2-amino-3-methylimidazole[4,5-f]quinoline; MeIQ, 2-amino-3,4-dimethyl-imidazole[4,5-f]quinoline; IQx, 2-amino-3methyl-imidazole[4,5-f]quinoxaline; PhIP, 2-amino-3,4-dimethyl-imidazole[4,5-b]pyridine; 4,8diMeIQx, 2-amino-3,4,8-trimethyl-imidazole[4,5-f]quinoxaline; MeIQx, 2-amino-3,8-dimethylimidazole[4,5-f]quinoxaline; 1,5,6-TMIP, 2-amino-1,5,6-trimethyl-6-phenyl-imidazole[4,5-b]pyridine; 1,6-DMIP, 2-amino-1,6-dimethyl-6-phenyl-imidazole[4,5-b]pyridine.

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Figure 10-26. Structure of acrylamide.

Figure 10-27. Pathway of acrylamide formation in foods [21].

(4) Strecker degradation facilitates the formation of acrylamide, because aldehydes are generated during the degradation; (5) Free radicals might also involve in acrylamide formation; (6) More than one pathway might contribute to acrylamide formation. In addition to intrinsic formation during food processing, acrylamide might come from other sources. Acrylamide can immigrate from polyacrylamide-based packaging materials to foods or from flocculating agent.

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Factors affecting acrylamide formation Temperature: Acrylamide is produced only in temperatures higher than 120 °C. When the equimolar (0.1 mol) mixture of asparagine and glucose is incubated, acrylamide is detected in temperatures higher than 120 °C and its content increases during temperature elevation until 170°C. When the temperature is further increased, acrylamide content tends to decrease and no acrylamide is detected when the temperature exceeds 185°C. Heating duration: When glucose is incubated with asparagines, glutamine, or methionine for 5~60 min, differences in acrylamide formation are found. The highest acrylamide content is detected in the asparagines-glucose mixture, but the content decreases after 5 min. In the glutamine-glucose system, the acrylamide content peaks within 10 min and then remains constant in subsequent incubation. In the case of the methionine-glucose system, the acrylamide content increases during prolonged incubation 30 min after the onset of the reaction, but reaches a constant level in subsequent heating. Carbohydrate species: When glucose, fructose, or lactose is incubated with asparagines, high acrylamide contents are detected in all the three systems, indicating that carbohydrate species plays less role in acrylamide formation. The carbonyl groups generated by lipid oxidation stimulate acrylamide formation. Amino acid species: When carbohydrates and amino acids are heated separately, no acrylamide is produced. However, when the two compounds are heated together, acrylamide formation is detected. Asparagine is the most susceptible to acrylamide formation with carbohydrates among all the amino acids. When asparagine is heated with glucose, the acrylamide content produced is hundreds to more than 1000 folds higher than that of the glutamine-glucose or methionie-glucose mixture. This is why fried potato chips have high acrylamide contents. Water content: The formation of acrylamide seems to be a surface reaction and the water contents of foods significantly influence acrylamide formation. High water contents facilitate the flow of reactants and products and consequently lead to more acrylamide formation. However, too high a water content causes diluted reactants and reduces the reaction rate. This phenomenon is quite similar to the Maillard reaction.

5.2. Nitrates, Nitrites, and Nitrosamines 5.2.1. Properties Nitrates can be converted to nitrites in mammals and nitrites can further react with amines, amino compounds, and amino acids to yield N-nitroso compounds. Nitrates are not poisonous, but nitrites and N-nitroso compounds are toxic to mammals. Hence, the conversion to nitrites and N-nitroso compounds must be considered in the safety evaluation of nitrates. Nitrites are oxidized to nitrates in the presence of reductants and reducants can be used to decrease nitrites accumulation and conversion. It has been proposed to add nitrites and Vc or Ve simultaneously in cured meats to reduce nitrite residue. R1 N

N

O

N-nitroso compounds share the R2 structure. Chemically, N-nitroso compounds can be divided into N-nitrosamines and N-nitrosamides. According to volatility, N-nitroso compounds can be classified as volatile and non-volatile N-nitroso compounds.

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In N-nitroso compounds, if groups R1 and R2 are the same, the compounds are symmetric; otherwise, the compounds are asymmetric. R1 and R2 can be alkyl groups, such as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), aromatic hydrocarbons, such as N-nitrosodiphenylamine (NDPhA), naphthenic bases, such as Nnitrosopyrolidine (NPRY), N-nitrosomorpholine (NMOR), N-nitrosopiperidine, and Nnitrosopiperazine, and amino acids, such as N-nitrosoproline (NPRO) and N-nitrososarcosine (NSAR). Low-molecular weight nitrosamines are yellow liquid in room temperature, and most high-molecular weight nitrosamines are solid. Most nitrosamines are insoluble in water, except some N-nitrosamines, such as NDMA, NDEA, and some N-nitroso amino acids. All N-nitrosamines are soluble in organic solvents and are stable against spontaneous hydrolysis. N-nitrosamines can be converted to carcinogenic compounds after being metabolized in human body. R1 N

N

O

The general structure of N-nitrosylamines is XCY , such as N-nitroso formylamine, N-nitroso methylurea, N-nitroso ethylurea, N-nitroso urethane, N-nitroso-N‘nitro-methylguanidine. N-nitrosylamines are unstable and can be directly decomposed to diazonium compounds, which are carcinogenic and mutagenic by binding DNA.

5.2.2. Sources of Nitrates and Nitrites Nitrites and nitrates in foods might either originate from intentional supplementation or migrate from excessively fertilized soils. Because nitrites inhibit the growth of Clostridium spp., the chemicals are previously used as preservatives. Nitrites can react with myoglobins in meats to yield nitrosomyoglobin and keep meat products in red color after heated. Besides, nitrites delay the formation of rancid flavor in meats during storage. Hence, nitrites as food additives can improve the commercial value of meat products. Nitrites are still used as colorants at present. In addition to intentional additions, the conversion from nitrates by nitrate reductase is another source of nitrite in cured and smoked foods. Some microbes, such as E. coli, Bacterium diphtheriae, and Staphylococcus aureus, contain highly active nitrate reductases. These exogenous enzymes together with intrinsic nitrate reductase calalyze the conversion. Curing conditions significantly affect the activities of nitrate reductases derived from these sources.

5.3. Chloropropanols With the increase of demand on flavorings, the soy sauce processing technique undergoes large evolutions during recent years. Hydrolyzed vegetable proteins have been used in soy sauce production to improve quality and reduce cost. If the hydrolysis condition is not well controlled, toxic chloropropanols may be generated. Chloroproanols can cause cancers in liver, kidney, and thyroid and affect fertility. Chloropropanols have turned to be another hotspot in the food pollutant area in addition to dioxins.

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5.3.1. Physiochemical Properties Chloropropanols are a collective of glycerol derivates whose hydroxyl groups are substituted by one or two chlorine. Chloropropanols have higher densities than water and boil at temperatures above 100 °C. Chloropropanols are liquid in room temperature and are soluble or miscible with water, acetone, benzene, glycerol, ethanol, ether, and carbon tetrachloride. There are 4 chloropopanols due to different substitution number and position: 3chloro-1,2-propanediol or monochloropropane-1,2-diol (3-MCPD), 2-chloro-1,3propanediolor monochloropropane-1,3-diol (2-MCPD), 1,3-dichloro-2-propanol (1,3-DCP or DC2P), and 2,3-dichloro-1-propanol (2,3-DCP or DC1P). Their structures are shown in Figure 10-28. Chloropropanols do not occur naturally in food materials and are mainly present in HClhydrolyzed proteins. Most protein materials contain lipids. During hydrolysis by HCl, side reactions occur and chloropropanols are formed. Generally, the contents of monochloropropanols are 100~10000 folds higher than that of dichloropropanols and 3MCPD is often several folds or tens of folds higher than 2-MCPD. The content of 3-MCPD is used as the main quality control index in protein hydrolyzate production. 5.3.2. Toxicity Because chloropropanols reduce the number and activity of sperms and inhibit the formation of male hormone, male contraceptives have been developed based on 3-MCPD. Due to the potential carcinogenicity, sperm inhibition, and kidney toxicity, many countries have set up limits for chloropropanol residue in foods.

5.4. Toxicants in Containers and Packaging Materials During processing, storage, and transportation, foods must contact various containers, utensils, and packaging materials. Some components in containers and packaging materials might migrate or dissolve in foods and cause chemical pollution in foods. Hence, containers and packaging materials are potential risks for food safety. Many types of containers and packaging materials have been used in the food industry in the history of China, such as bamboo wood, metals, glass, enamel, and ceramics. Years of practice have proved that most of them are safe for human body. However, with the rapid development of the food industry and chemical synthesis technologies, many new synthetic materials, such as plastics, coatings, and rubbers, have emerged and gained wide applications in food packaging. These synthetic materials contain potential toxicants and some can migrate into foods and cause poisoning.

Figure 10-28. Structures of the four chloropropanols.

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5.4.1. Plastics Plastics are made from synthetic resins and supplementary materials. Plastics and synthetic resins are polymerized from small blocks and the higher polymerization degree, the more stability and the less possibility of block migration into foods. If a polymer is synthesized from only one block, this polymer is a homopolyer; if two or more blocks are used, the polymer is a copolymer. Plastics that are currently allowed for food packaging include polyethylene (PE), polypropylene, polystyrene, urea formaldehyde, and melamino-formaldehyde. These materials are potential pollutants for foods. For example, polyvinyl chloride (PVC) is nontoxic, but the monomer vinyl chloride and its degradation products are toxic. PVC can be degraded by high temperature and UV light and the decomposition product can cause hepatic hemangioma. Free formaldehyde may exist in urea formaldehyde and melaminoformaldehyde plastics in case of incomplete reaction during production. Besides, urea formaldehyde and melamino-formaldehyde can be degraded by high temperature and acidic solutions to release formaldehyde and phenols. Formaldehyde is a protoplasmic poison and can cause focal necrosis of liver cells and lymphocytic infiltration if orally ingested by animals. 5.4.2. Other Packaging Materials Ceramic glazes or porcelain glazes that are coated on chinas and ceramics contain high levels of metallic salts, such as lead salts and cadmium salts. If the salts get in contact with foods for long periods, the salts dissolve in foods and poisoning occurs. The salts are readily soluble in acidic foods such as vinegar, fruit juices, and wines. Packaging paper used in food packaging cannot contain fluorescent brightener. Regenerated paper can cause potential microbial pollution in foods and recycled paper can lead to toxic substance contamination in foods. Paraffin used in paraffin paper must be pure and the contents of polycyclic aromatic hydrocarbons cannot exceed allowed values. Tinplate cans are widely used for packaging foods and beverages. Metal pollutions caused by food packaging materials and food containers are attracting more and more attentions in recent years. Ai et al [22] measured the contents of toxic metals in tinplate alloys by using ICP-AES and found that the alloys have high toxic metal contents, especially Pb, Cd, Cr, and Sn. Hence, how to control the contents of toxic elements in foods and reduce their potential damages deserves more attention.

REFERENCES [1] Pariza, MW. Toxic Substances. In: Owen R. Fennema. Food Chemistry. New York: Marcel Dekker, 1996; 825~841. [2] International Food Biotechnology Council (1990). Biotechnologies and food: Assuring the safety of foods produced by genetic modification. Regulatory Toxicology and Pharmacology, 1990, 12, S1-S10. [3] Chernoff, R. Thirst and fluid requirements. Nutrition Reviews, 1994, 52, S3–S5. [4] Strong, FM. Toxicants Occurring Naturally in Foods. 2nd edition. Washington DC: National Academy of Sciences, 1980. [5] Tewe, OO; Lyayi, EA. 1989. Cyanogenic glycosides. In: Cheeke PR. Toxicants of Plant Origin, Volume II: Glycosides. Boca Raton: CRC Press, 1989; 43–69.

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[6] Fenwick, G.R, et al., Glucosinolates and their breakdown products in food and food plants. Critical Reviews in Food Science and Nutrition, 1982, 18, 123–201. [7] Roy, DN; Spencer, PS. Lathyrogens. In: Cheeke PR. Toxicants of Plant Origin, Vol. III, Proteins and amino acids. Boca Raton: CRC Press; 1989; 169–194. [8] Evans, C. Naturally occurring food toxicants: Toxic amino acids. In: Rechcigl, M. Handbook of Naturally Occurring Food Toxicants. Boca Raton: CRC Press, 1983; 3–14. [9] Bell, EA. Nonprotein Amino Acids of Plants: Significance in Medicine, Nutrition, and Agriculture. Journal of Agricultural and Food Chemistry, 2003, 51, 2854-2865. [10] Peumans, WJ; Van Damme EJM. Prevalence, biological activity and genetic manipulation of lectins in foods. Trends in Food Science and Technology, 1996, 7, 132138. [11] Krieger, R. Handbook of Pesticide Toxicology: Principles and Agents. 2nd edition. Amsterdam: Elsevier Science, 2001. [12] Abraham, K; Geissler, K; Sator, MO; Stingl, G; Tschachler, E. Severe 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) Intoxication: Clinical and Laboratory Effects. Environmental Health Perspectives, 2001, 109, 865-869. [13] Geusau, A; Khorchide, M; Mildner, M; Pammer, J; Eckhart, L; Tschachler, E. 2,3,7,8Tetrachlorodibenzo-p-Dioxin Impairs Differentiation of Normal Human Epidermal Keratinocytes in a Skin Equivalent Model. Journal of Investigative Dermatology, 2005, 124, 275-277. [14] Heidebach, T; Först, P; Kulozik, U. Influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. Journal of Food Engineering, 2010, 98, 309-316. [15] Heim, KE; Tagliaferro, AR; Bobilya, DJ. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. The Journal of Nutritional Biochemistry, 2002, 13, 572-584. [16] Osman, MA. Changes in sorghum enzyme inhibitors, phytic acid, tannins and in vitro protein digestibility occurring during Khamir (local bread) fermentation. Food Chemistry, 2004, 88, 129-134. [17] Han, X; Shen, T; Lou, H. Dietary Polyphenols and Their Biological Significance. International Journal of Molecular Sciences, 2007, 8, 950-988. [18] Sattar, R; Ali, SA; Kamal, M; Khan, AA; Abbasi, A. Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling. Biochemical and Biophysical Research Communications, 2004, 314, 755-765. [19] Kim, Y; Yang, SH; Lee, SY; Kim, M. Levels of PCDDs and PCDFs in two kinds of fast foods in Korea. Chemosphere, 2001, 43, 851-855. [20] Jägerstad, M; Skog, K; Arvidsson, P; Solyakov, A. Chemistry, formation and occurrence of genotoxic heterocyclic amines identified in model systems and cooked foods. Zeitschrift für Lebensmitteluntersuchung und -Forschung A, 1998, 207, 419-427. [21] Mottram, DS; Wedzicha, BL; Dodson, AT. Food chemistry: Acrylamide is formed in the Maillard reaction. Nature, 2002, 419, 448-449. [22] Jun, A; Degang,T. Determination of harmful elements in aluminium alloys for the tin of beverage by inductively coupled plasma atomic emission spectrometry. Journal of Wuhan Institute of Chemical Technology, 2001, 23, 10-12.

INDEX A Abraham, 268, 349 accounting, 76 acetaldehyde, 255, 261, 266 acetic acid, 62, 66, 83, 206, 251, 261, 273, 286, 315 acetone, 47, 62, 67, 91, 201, 255, 261, 307, 317, 340, 341, 347 acetonitrile, 317 acetophenone, 258 acidic, 46, 48, 56, 59, 61, 66, 69, 70, 72, 74, 81, 82, 83, 95, 98, 138, 140, 145, 152, 165, 178, 184, 186, 205, 207, 208, 209, 211, 230, 232, 251, 260, 273, 279, 293, 294, 295, 296, 315, 316, 318, 321, 322, 323, 327, 328, 332, 340, 341, 348 acidity, 28, 47, 66, 98, 207, 273, 296, 306, 332 acrylic acid, 342 action potential, 324 activation energy, 31, 121, 179 active compound, 128, 258 active oxygen, 67 active radicals, 281 active site, 46 adaptability, 188 adaptation, 194, 247 additives, 158, 172, 184, 251, 271, 272, 273, 278, 279, 283, 289, 299, 319, 334 adenine, 227, 228, 261 adhesion, 159, 165, 284 adiposity, 100, 102 ADP, 227 adsorption, 50, 101, 102, 153, 159, 160, 162, 164, 165, 212, 250, 296, 304, 305, 313, 319, 331, 332, 334, 335, 340 adults, 103, 174, 176, 178, 306 advancement, 3, 40 adverse effects, 167, 305, 306, 324, 339 aflatoxin, 130, 131 agar, 88, 90, 91, 96, 100, 291, 293

age, 178, 238, 247, 306 agglutination, 169, 310 aggregation, 45, 151, 154, 157, 163, 164, 167, 168, 170, 183, 188, 198, 246, 254, 284 agricultural chemistry, 3 agriculture, xi, 321 alanine, 64, 66, 67, 70, 140, 170, 177, 180, 206, 310 Alaska, 185, 189 albinism, 196 albumin, 32, 146, 149, 153, 164, 165, 167, 171, 183 alcohols, 38, 39, 45, 49, 50, 55, 111, 124, 186, 254, 256, 257, 258, 259, 260, 261, 263, 266, 308, 326, 329 aldehydes, 48, 56, 58, 60, 66, 123, 124, 129, 181, 182, 184, 186, 187, 254, 256, 258, 259, 260, 261, 263, 266, 268, 280, 344 alfalfa, 199, 200 algae, 35, 38, 73, 101 alkaline media, 56, 60, 61 alkaloids, 249, 319 alkylation, 322 allergens, 303, 306 allergic reaction, 306 allergy, 283, 306 alters, 184 aluminium, 349 ambient air temperature, 325 amine, 138, 139, 140, 142, 181, 276, 295, 322, 323, 342 amines, 16, 68, 70, 138, 225, 262, 268, 303, 341, 342, 343, 345, 349 mino groups, 71, 95, 138, 181, 182, 231, 341 ammonia, 60, 132, 261, 262, 268 ammonium, 16, 59, 60, 138, 149, 292, 317 ammonium salts, 59 amphiphilic compounds, 17 amylase, 42, 48, 81, 82, 177, 305, 316, 331, 335 anemia, 210, 238, 305 anorexia, 210

354

Index

antagonism, 224 antibiotic, 328 antibody, 306 antigen, 306, 310, 311 antimony, 194 antioxidant, 61, 66, 67, 71, 126, 128, 131, 185, 189, 197, 199, 213, 215, 220, 272, 274, 279, 281, 282, 283, 296, 297, 333, 334 antitumor, 74 appetite, 251, 252, 310 apples, 71, 264, 307 aquaculture, 316 aqueous solutions, 34, 36, 51, 59, 85, 207, 210, 212, 213, 236, 279, 315, 316, 326 aqueous suspension, 84, 280 arabinogalactan, 291 arginine, 67, 68, 140, 149, 177, 336 aromatic compounds, 254, 256, 257, 258, 264, 265, 266 aromatic hydrocarbons, 194, 260, 324, 346 aromatic rings, 184 aromatics, 324 ARS, 135 arsenic, 222, 225, 239 ascorbic acid, 38, 61, 62, 63, 70, 211, 213, 219, 281, 282 aseptic, 179 Asia, 292, 295 asparagines, 345 asparagus, 74, 218, 257 Aspartame, 187, 276 aspartate, 147 aspartic acid, 140, 187, 320, 324, 336 assessment, 34, 249, 339 asthenia, 339 astringent, 46, 246, 254 atmosphere, 4, 194, 280 atmospheric pressure, 71 atomic emission spectrometry, 349 atoms, 11, 13, 41, 45, 108, 109, 126, 210, 225, 229, 230, 231, 233, 237, 308, 320, 321, 324 ATP, 202, 208, 224, 227, 228, 229, 252, 282 atrophy, 326 autolysis, 185 autooxidation, 55 awareness, 283, 294 B bacteria, 24, 74, 93, 94, 102, 173, 181, 185, 189, 200, 206, 209, 210, 212, 230, 231, 278, 279, 280 bacterial fermentation, 276 barriers, 320

base, 31, 55, 72, 151, 209, 220, 225, 227, 272, 300, 321 beef, 108, 156, 157, 174, 179, 188, 199, 208, 210, 238, 240, 260, 261, 266, 268, 269 beer, 5, 92, 158, 162, 249, 250, 266, 295, 296 Beijing, 7, 188, 189, 243 beneficial effect, 102 benefits, 7, 28, 272, 291 benzene, 62, 201, 206, 260, 266, 315, 317, 323, 328, 333, 339, 340, 341, 347 benzoyl peroxide, 184 beriberi, 192, 202 beryllium, 222 beta-carotene, 193, 194, 196, 219 beverages, 51, 74, 75, 91, 100, 169, 247, 251, 266, 276, 277, 279, 292, 293, 294, 296, 348 bile, 94, 101, 103, 249, 250, 332 bile acids, 103 bioavailability, 9, 68, 103, 178, 179, 180, 182, 207, 212, 224, 225, 227, 236, 237, 238, 239, 305, 331, 332, 333, 334 biochemical processes, 221 biochemistry, xi, xii, 2, 3, 324 biological activity, 191, 237, 349 biological stability, 5 biomass, 5, 173 biomolecules, 26, 32, 225, 239, 320, 334 biopolymer, 9 biopolymers, 34 biosynthesis, 44, 95, 212, 234, 255, 304, 333 biotechnology, xii biotic, 174 biotin, 209, 210 birds, 169, 303, 306 bladder cancer, 276 bleaching, 184, 241, 272, 325 bleeding, 298 blindness, 192, 194 blood, 40, 55, 94, 100, 101, 102, 149, 150, 192, 196, 198, 199, 201, 202, 210, 236, 261, 262, 275, 276, 305, 310, 316, 325, 326 blood plasma, 149 blood pressure, 102, 305 blood stream, 55, 236 body fluid, 192, 233 body weight, 310, 326 boils, 341 bonding, 10, 11, 12, 15, 16, 17, 18, 23, 27, 50, 52, 76, 79, 87, 91, 92, 101, 116, 145, 146, 153, 154, 156, 160, 165, 166, 168, 172, 248, 249, 288, 334, 337, 338 bonds, 10, 11, 13, 14, 15, 40, 42, 43, 46, 58, 76, 77, 85, 88, 98, 108, 109, 116, 124, 126, 127, 146, 147, 148, 151, 153, 155, 156, 159, 160, 166, 169, 172, 186, 230, 231, 237, 318, 336, 337

Index bone, 150, 170, 221, 223, 224, 233, 240, 305 bone growth, 223 bone marrow, 170, 233 bones, 296 botanists, 2 bounds, 17 brain, 300 brain damage, 300 branching, 77 breakdown, 58, 86, 124, 166, 184, 300, 313, 321, 323, 349 breast milk, 134, 180 bromine, 222, 322, 340 buns, 173, 296 burn, 116, 277 by-products, 272 C Ca2+, 14, 39, 41, 81, 87, 90, 92, 98, 102, 164, 167, 178, 227, 235, 237, 332, 334 cabbage, 10, 200, 215, 218, 240, 304 cacao, 249, 298 cadmium, 222, 348 caffeine, 68, 249, 250 calcification, 196 calcium, 39, 74, 91, 92, 95, 170, 188, 192, 196, 207, 221, 222, 278, 279, 288, 291, 292, 294, 296, 297, 304, 307, 326, 332 calculus, 239, 333 calorie, 85, 86, 100, 277, 290 cancer, 100, 276, 291, 320, 333, 339, 340, 341 cane sugar, 24 capillary, 51, 168 carbohydrate, xi, 40, 54, 55, 76, 84, 170, 181, 184, 207, 225, 226, 289, 310, 312, 333, 341, 345 carbohydrates, vii, xii, 1, 3, 5, 7, 32, 35, 36, 37, 46, 50, 51, 54, 58, 63, 66, 73, 100, 102, 103, 172, 184, 202, 217, 225, 226, 229, 236, 243, 260, 268, 342, 345 carbon, 28, 36, 37, 46, 49, 62, 63, 90, 92, 94, 102, 108, 109, 110, 111, 115, 116, 121, 122, 130, 131, 132, 143, 145, 163, 203, 210, 215, 222, 229, 253, 261, 298, 315, 323, 328, 333, 340, 347 carbon atoms, 36, 37, 90, 108, 109, 143, 229, 328 carbon dioxide, 28, 36, 102, 163, 261, 323 carbon tetrachloride, 92, 347 carbon-centered radicals, 215 carbonyl groups, 49, 72, 181, 345 carboxyl, 36, 37, 44, 48, 52, 53, 84, 102, 138, 141, 165, 211, 225, 231, 232, 234, 235, 332 carboxylic acid, 108, 138, 143, 214, 225, 227, 260, 264, 282 carboxylic acids, 138, 214, 260, 282

355

carboxylic groups, 87, 138, 139 carboxymethyl cellulose, 32, 293 carcinogen, 225, 236 carcinogenicity, 326, 341, 347 cardiovascular disease, 100 cardiovascular disease XE "cardiovascular disease" s, 100 carob, 96, 293 carotene, 131, 193, 194, 195, 196, 219, 259, 298 carotenoids, 191, 192, 194 cartilaginous, 261 casein, 32, 85, 91, 159, 162, 170, 171, 180, 184, 188, 292, 332, 349 catalysis, 7, 121, 323 catalyst, 126, 132, 133, 134 catalytic activity, 94, 126, 133, 152 category a, 112, 150 cation, 12, 87, 90, 164, 251 cattle, 242 causal relationship, 276 cauterize, 155 cell membranes, 318 cell surface, 74 cellulose, 32, 36, 40, 46, 49, 85, 86, 93, 94, 98, 100, 101, 102, 268, 280, 289, 291, 292, 293, 295 central nervous system, 209, 249, 329 ceramide, 111 cereal starches, 76 cerebrovascular disease, 6 chain molecules, 54 charge density, 154 cheese, 91, 96, 121, 141, 143, 158, 171, 177, 186, 240, 266, 273, 279, 281, 292, 296, 297 cheilitis, 205 chemical industry, 82 chemical properties, 2, 9, 39, 158, 320, 324, 325, 341 chemical reactions, 6, 9, 20, 30, 31, 107, 139, 177, 260 chemical structures, 276, 341 chemicals, 4, 11, 31, 121, 219, 278, 305, 324, 346 chicken, 143, 174, 199, 240, 260, 306, 316, 330, 339 children, 176, 178, 306, 330 China, vii, ix, xi, xii, 1, 7, 9, 35, 107, 128, 137, 167, 191, 221, 241, 243, 245, 246, 271, 272, 275, 276, 277, 279, 280, 282, 283, 286, 287, 288, 289, 295, 298, 300, 303, 314, 316, 347 Chinese restaurant syndrome, 300 chiral center, 37, 39 chitin, 74, 93, 94, 100, 310, 311, 312, 316 chitosan, 74, 93, 94, 95, 100, 101, 104, 295 Chitosan, 93, 94, 104, 295 chlorine, 222, 340, 347 chloroform, 62, 86, 201, 206, 209, 212, 248, 315, 317, 328, 333 chlorophyll, 115, 230, 231, 237

356

Index

cholesterol, 74, 94, 100, 101, 105, 107, 174, 239, 291, 316, 330 cholic acid, 250, 251 choline, 111 cholinesterase, 324 cholinesterase inhibitors, 324 chromatography, 245 chromium, 132, 222, 236 chromosome, 149 chymotrypsin, 177, 183, 184, 305 ciguatera, 317 civilization, 6 classes, 10, 13, 89, 90, 91, 221, 285, 321 classification, 35, 107, 191, 221, 246, 271, 285, 318 cleaning, 1, 289 cleavage, 318 climate, 173, 241 clusters, 170 CMC, 49, 85, 86 CO2, 66, 86, 93, 166, 210 coatings, 86, 95, 298, 347 cobalamin, 231, 305 cobalt, 212, 222, 231 cocoa, 91, 112, 117, 118, 134, 289, 342 cocoa butter, 112, 117, 118, 134, 289 coconut oil, 117 coenzyme, 207, 208, 209, 212, 213 coffee, 21, 22, 54, 68, 104, 249, 286, 287, 288, 298, 305, 342 coherence, 159 collagen, 32, 150, 156, 170, 183, 309 collisions, 31 colon, 73, 74, 101, 102, 108 color, 1, 2, 4, 5, 6, 7, 36, 37, 46, 55, 58, 59, 60, 63, 69, 71, 94, 95, 115, 130, 156, 158, 161, 170, 184, 191, 205, 219, 233, 271, 272, 273, 281, 298, 315, 338, 339, 346 coma, 305 combustion, 325 commercial, 33, 34, 74, 75, 86, 90, 95, 98, 134, 143, 172, 189, 219, 277, 282, 294, 321, 346 complement, 94, 295 complexity, 68, 103 compliance, 319 composition, 2, 4, 5, 6, 18, 20, 22, 29, 30, 36, 55, 58, 63, 65, 96, 101, 102, 104, 109, 111, 113, 114, 115, 117, 124, 129, 133, 148, 156, 159, 164, 166, 173, 174, 260, 293, 314, 316, 321, 331, 336 compression, 130 condensation, 39, 55, 58, 59, 62, 63, 65, 72, 124, 129, 139, 180, 181, 182, 204 conduction, 324 configuration, 10, 11, 37, 109, 110, 116, 124, 127, 143, 225, 333 conjugation, 116

connective tissue, 150, 170, 293 constant rate, 161 constituents, vii, 17, 18, 19, 28, 32, 171, 172, 221, 224, 236, 259, 261, 268, 282, 287, 317 consumer choice, 283, 284 consumers, 1, 3, 4, 5, 37, 55, 73, 130, 174, 176, 186, 202, 245, 246, 248, 252, 272, 278, 298, 306, 319, 335 consumption, 55, 60, 173, 174, 192, 245, 276, 278, 313, 330 containers, 29, 347, 348 contaminant, 233 contamination, 222, 348 conversion rate, 174 COOH, 138, 143, 187, 249 cooking, 83, 101, 130, 152, 173, 178, 183, 202, 211, 220, 260, 289, 308, 341 cooling, 29, 30, 45, 82, 91, 132, 169, 275, 292, 293, 294 coordination, 12, 224, 225, 226, 227, 229, 230, 231, 235, 237, 238 copolymer, 292, 348 copolymers, 58, 92 copper, 132, 150, 231, 233, 281, 298 cornea, 194 corpus luteum, 329 correlation, 33, 164, 276, 310 corrosion, 325 cortex, 201, 207, 326 cost, 48, 53, 272, 273, 346 covalent bond, 14, 17, 52, 146, 153, 183, 187 covalent bonding, 52 covering, xi crop, 173 crops, 76, 176, 234, 310, 311 crude oil, 283 crust, 223 crystal growth, 46, 83, 96 crystal structure, 85, 286, 287, 288 crystalline, 16, 22, 76, 77, 78, 117, 207, 275, 276, 292, 296 crystallization, 29, 30, 73, 79, 83, 97, 100, 117, 273, 275, 287, 289 crystals, 13, 18, 28, 38, 46, 117, 118, 134, 207, 208, 212, 290, 315, 316, 325, 329 cultivation, 241, 307, 308 culture, 246 curcumin, 298 cuticle, 316 cyanide, 47, 231, 317 cyanide poisoning, 47 cyanocobalamin, 212, 231 cyclodextrins, 39 cyclopentadiene, 321

Index cysteine, 71, 140, 144, 166, 179, 184, 232, 256, 257, 266, 267, 310, 320, 328 cystine, 165, 166, 179, 182, 184, 233, 320, 336 cytochrome, 223, 230, 324 cytoplasm, 316 D damages, 348 D-amino acids, 248 danger, 278, 282 DDT, 321 deacetylation, 93, 295 decay, 93, 274, 275 decomposition, 5, 6, 102, 116, 124, 125, 194, 203, 205, 207, 254, 259, 260, 261, 262, 268, 272, 281, 348 defecation, 101 defects, 13 deficiency, 173, 191, 192, 194, 196, 197, 198, 201, 202, 205, 208, 209, 210, 212, 213, 214, 222, 224, 238, 273, 319, 320 deformation, 29, 51, 118, 165, 284 degradation, 5, 6, 56, 57, 65, 66, 68, 71, 129, 181, 182, 184, 194, 195, 196, 199, 202, 203, 204, 205, 207, 208, 209, 210, 212, 214, 216, 219, 252, 262, 266, 268, 269, 284, 320, 323, 324, 325, 326, 342, 344, 348 degradation rate, 203 degumming, 131 dehydration, 9, 21, 22, 36, 58, 59, 60, 61, 66, 86, 143, 168, 179, 199, 241, 242, 253, 296 denaturation, 5, 6, 7, 137, 148, 151, 152, 153, 154, 156, 157, 159, 167, 168, 169, 172, 178, 180, 183, 184, 187, 338 dendrites, 13 dental caries, 73 Department of Agriculture, 243 dephosphorylation, 217 depolarization, 324 depression, 34, 83 derivatives, 36, 37, 49, 76, 82, 86, 107, 110, 111, 120, 138, 187, 197, 200, 208, 211, 220, 225, 226, 229, 243, 249, 269, 293, 299, 317, 322, 328, 333 dermatitis, 205, 208, 210 desorption, 22, 23, 29 destruction, 68, 151, 152, 183, 194, 205, 212, 215, 216 detectable, 152 detection, xii, 104 detergents, 285 detoxification, 71, 213, 220 developing countries, vii deviation, 113

357

diabetes, 6, 100, 275 dialysis, 68, 151 diarrhea, 73, 196, 208, 275, 314, 317, 318 dielectric constant, 156 diet, 6, 35, 107, 181, 183, 192, 194, 225, 238, 274, 275, 295, 296, 303, 306, 339, 341 dietary fiber, 37, 85, 86, 90, 100, 103, 105, 177, 291, 294 dietary habits, 1, 246, 247 dietary intake, 103 diffraction, 78 diffusion, 29, 30, 31, 33, 79, 81, 93, 125, 152, 153, 169, 185, 186 digestibility, 68, 157, 174, 176, 177, 178, 179, 184, 295, 332, 349 digestion, 100, 102, 177, 180, 183, 189, 250, 295, 304, 334, 335 digestive enzymes, 103, 182, 306, 334 dihydroxyphenylalanine, 177, 309 dilation, 119 dimethylsulfoxide, 317 dioxin, 324, 325, 326, 349 dioxin-like PCBs, 325 dipeptides, 140 dipole moment of, 11 discomfort, 102 diseases, 6, 100, 185, 191, 210, 321, 339 dislocation, 13 disorder, 202, 208, 305, 306, 310 dispersion, 17, 85, 154, 158, 169, 289, 292, 298 dissociation, 11, 71, 87, 95, 138, 169, 209 dissolved oxygen, 61 distillation, 7, 131, 288 distribution, 15, 40, 44, 96, 107, 110, 112, 113, 114, 115, 117, 124, 133, 134, 135, 146, 159, 161, 174, 176, 221, 223, 289, 307, 310, 311, 325, 330 dizziness, 300, 304, 305 DNA, 95, 239, 320, 346 docosahexaenoic acid, 262 donors, 11, 110, 128, 281 dosage, 133, 326, 341 double bonds, 58, 108, 109, 111, 116, 121, 122, 124, 126, 127, 131, 132, 253 double helix, 44, 45, 77 dough, 86, 91, 96, 165, 166, 167, 171, 172, 185, 284, 287, 288, 289, 296 drainage, 164 dressings, 83, 85, 86, 96, 97, 279, 290, 293, 294 drinking water, 94, 224 drugs, 305, 318, 326, 330, 331 dry matter, 27 drying, 2, 21, 29, 33, 83, 169, 179, 184, 186, 196, 349 DSC, 30, 156 dyes, 298

358

Index

dynamic mechanical analysis, 30 dyspepsia, 202 dyspnea, 214, 305 E edema, 202, 339 egg, 24, 41, 44, 45, 72, 87, 108, 152, 156, 158, 159, 164, 178, 183, 196, 206, 212, 235, 261, 289, 306 elastin, 32, 150, 170, 309 electrolyte, 305 electrolyte imbalance, 305 electron, 11, 79, 128, 179, 233, 281, 324, 333 electron pairs, 11 electrons, 11, 12, 225 electrophoresis, 138 elongation, 166, 324, 333 elucidation, 269 emergency, 3, 100 emission, 205 emulsifying agents, 82 emulsions, 91, 98, 119, 120, 125, 154, 158, 159, 160, 162, 169, 288, 289, 291, 293 enamel, 347 enantiomers, 37, 265 encapsulation, 73, 289 endocrine, 325 endosperm, 95, 171, 293 endothermic, 156 energy, 11, 12, 18, 31, 35, 37, 73, 86, 102, 107, 119, 120, 126, 128, 134, 153, 159, 162, 163, 170, 202, 275, 276, 323 energy consumption, 134 energy input, 159, 162, 163 energy supply, 276 engineering, xii, 1, 4 enlargement, 304, 326 entanglement network, 32 entropy, 188 environment, 1, 16, 17, 102, 138, 146, 168, 216, 223, 241, 295, 304, 305, 319, 321, 323, 324, 325, 326 environmental conditions, 159, 168, 183 environmental effects, 32 environmental factors, 245, 306 environmental stress, 151 environmental stresses, 151 environments, 50, 212 enzyme, 4, 5, 7, 31, 44, 46, 47, 71, 128, 151, 152, 163, 177, 180, 185, 223, 224, 272, 274, 304, 320, 323, 331, 335, 336, 349 enzyme inhibitors, 128, 177, 335, 336, 349 enzymes, 4, 28, 47, 48, 55, 68, 72, 77, 82, 85, 87, 88, 90, 120, 146, 150, 152, 153, 157, 165, 170, 176, 185, 191, 196, 205, 207, 208, 212, 214, 216, 217,

221, 232, 239, 256, 258, 259, 262, 264, 275, 305, 307, 313, 320, 327, 333, 336, 337, 338, 339, 346 EPA, 5, 109, 112 epidemiological investigations, 341 epidermis, 316 epinephrine, 107 epithelial cells, 194, 304 equilibrium, 19, 29, 30, 138, 168, 262, 296 equipment, 3, 162, 241 essential fatty acids, 107 ester, 49, 52, 54, 90, 111, 120, 139, 140, 150, 187, 194, 256, 264, 287, 288, 292, 322, 323, 333 ester bonds, 150 ethanol, 59, 62, 64, 91, 92, 93, 100, 142, 149, 165, 171, 172, 194, 207, 209, 212, 213, 255, 266, 283, 288, 292, 293, 315, 317, 328, 333, 341, 347 ethers, 49, 254, 260, 266, 324, 326, 329 ethyl acetate, 62, 255, 259, 266, 283, 315, 328 ethylene, 93, 281, 283 eukaryotic cell, 222 evacuation, 101 evaporation, 183 evidence, 16, 276, 277 evolution, 271, 310 EXAFS, 226 excretion, 95 experimental design, 6 exploitation, 4 exposure, 17, 118, 121, 126, 153, 183, 194, 213, 234, 279, 306, 323 extraction, 7, 88, 101, 130, 168, 169, 172, 293 extracts, 289, 292, 298, 300, 318 extrusion, 7, 83, 172 exudate, 96, 98, 291 F families, 310, 311, 312 fast food, 349 fat, 3, 5, 86, 91, 94, 100, 103, 107, 108, 111, 112, 113, 114, 118, 119, 131, 133, 134, 156, 158, 159, 161, 172, 191, 192, 193, 194, 196, 197, 199, 239, 240, 250, 260, 261, 282, 283, 284, 288, 289, 290, 292, 293, 295, 298, 317, 326 fat soluble, 191, 194, 196, 197, 317 fatty acids, xii, 16, 78, 83, 94, 102, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 120, 121, 122, 124, 126, 127, 129, 130, 131, 132, 133, 134, 182, 256, 259, 260, 261, 262, 285, 287, 288, 306 FDA, 100, 103, 187 feed additives, 331 fermentation, 5, 90, 100, 165, 173, 184, 185, 189, 254, 258, 259, 266, 267, 274, 277, 289, 294, 295, 334, 349

359

Index ferritin, 237 fertility, 346 fever, 306 fiber, 35, 91, 100, 150, 170, 173, 291 fibers, 40, 100 films, 159, 163, 164, 165, 188 filtration, 4, 117, 131 fish, 24, 68, 92, 108, 148, 157, 169, 173, 174, 177, 178, 179, 180, 184, 185, 196, 204, 208, 212, 233, 234, 238, 261, 262, 269, 279, 283, 296, 297, 298, 300, 303, 316 fish oil, 196, 262 fission, 58 fixation, 210 flame, 179 flatulence, 73, 172 flavonoids, 45, 46, 283, 333, 334 flavour, 54, 103, 187, 293, 299, 300 flaws, 58 flexibility, 42, 52, 159, 160 flocculation, 120, 164 flora, 37, 75 flour, 10, 24, 86, 96, 165, 166, 167, 171, 172, 173, 177, 180, 184, 202, 216, 218, 219, 272, 288, 295, 296, 297, 300 fluid, 51, 85, 113, 157, 164, 169, 250, 291, 294, 348 fluorescence, 67, 104, 202, 205, 340 fluorine, 222 foals, 105 foams, 154, 158, 159, 162, 163, 164, 188, 289, 293 folate, 210 folic acid, 210, 211, 212, 215, 219 follicle, 329 food additive, vii, 5, 91, 241, 271, 272, 273, 284, 300, 346 food additives, vii, 5, 91, 241, 271, 272, 273, 284, 300, 346 Food and Drug Administration, 73 food chain, 319, 325 food industry, 1, 3, 4, 5, 6, 7, 39, 46, 48, 49, 73, 82, 85, 86, 87, 90, 91, 92, 93, 94, 97, 120, 132, 137, 168, 171, 178, 180, 184, 187, 213, 251, 271, 278, 280, 289, 291, 295, 300, 303, 347 food poisoning, 314 food production, 287, 289 food products, 297, 300 food safety, xi, xii, 2, 46, 271, 272, 296, 303, 319, 340, 347 food security, 3 food spoilage, 272 food web, 324 foodborne illness, 317 force, 17, 95, 101, 146, 147, 163, 168, 169 formaldehyde, 182, 210, 261, 262, 348

formula, 19, 37, 40, 41, 59, 75, 93, 141, 234, 314, 316, 317, 340 free energy, 119, 120, 141, 159, 284 free radicals, 26, 103, 121, 126, 128, 130, 182, 199, 205, 233, 281, 334 free rotation, 42 free volume, 31, 32 freezing, 2, 6, 13, 20, 28, 34, 45, 83, 84, 117, 240, 308 freshwater, 261, 316 friction, 152, 169 fructose, 4, 36, 47, 48, 49, 50, 55, 57, 59, 60, 74, 75, 82, 181, 225, 248, 274, 275, 276, 345 fruits, 4, 7, 18, 21, 24, 36, 55, 74, 87, 93, 95, 108, 195, 213, 219, 237, 245, 249, 254, 255, 263, 264, 265, 273, 275, 279, 280, 290, 297, 304, 305 functional food, 73, 86, 94, 294, 296 fungi, 74, 193, 233, 278 furan, 124, 203, 214, 258, 259, 260, 261, 266, 268, 324 G gallbladder, 251 gastroenteritis, 305 gastrointestinal tract, 194, 341 gel, 44, 51, 52, 53, 81, 82, 83, 84, 86, 87, 90, 91, 92, 98, 100, 167, 168, 171, 291, 292, 294, 295, 296 gel formation, 296 gelatinization temperature, 76, 80, 81, 82, 83, 84 gelation, 81, 87, 88, 98, 151, 158, 159, 160, 167, 168, 293, 297 genes, 145 genetic code, 145 genetic information, 320 genetics, 306 genitals, 198 genomics, 7 genus, 96, 98, 275, 318 geometry, 40, 41, 42, 43, 44 germination, 273 gill, 316 ginger, 253 gland, 233 glass transition, 23, 30, 32 glossitis, 210 glucoamylase, 48, 82 glucose, 36, 39, 40, 47, 48, 49, 50, 55, 59, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 82, 84, 86, 97, 98, 100, 101, 181, 224, 225, 239, 248, 249, 266, 267, 274, 275, 276, 281, 282, 294, 295, 307, 328, 333, 345 glucose oxidase, 48, 72, 281, 282 glucoside, 45, 342

360

Index

glucosinolates, 257, 307, 308 glutamate, 210, 247, 251, 252, 300 glutamic acid, 68, 140, 142, 211, 309, 320, 324, 336 glutamine, 69, 149, 171, 345 glutathione, 71, 223, 320, 334 glycans, 42, 150 glycerin, 201, 285, 298 glycerol, 92, 110, 111, 113, 120, 134, 268, 294, 347 glycine, 66, 67, 68, 70, 138, 140, 146, 170, 227 glycogen, 40, 52, 84 glycol, 286, 288, 295, 298 glycopeptides, 40 glycoproteins, 36, 40, 150, 165, 178, 233, 291, 306, 310 glycoside, 46, 48, 277, 314, 315 glycosylation, 334 goiter, 307 gonorrhea, 155 governor, 9 grades, 291 grain size, 79 granules, 76, 77, 78, 80, 164 graph, 223 GRAS, 272, 283, 295 grass, 177, 258, 263 grasses, 321 gravity, 119 green alga, 319 growth, 1, 9, 21, 23, 24, 25, 26, 31, 33, 74, 75, 94, 95, 102, 149, 176, 180, 181, 192, 193, 194, 207, 212, 213, 216, 222, 251, 273, 277, 304, 313, 319, 321, 329, 330, 331, 346 growth factor, 193, 330 growth rate, 176 Guangdong, ix, 243 guanidine groups, 328 guanine, 300 guidance, 4 H halogen, 340 haploid, 149 hardness, 53, 118, 159 harmful effects, 183 harvesting, 87, 221, 258 hazards, 304 headache, 196 health, xii, 101, 130, 149, 176, 194, 239, 241, 247, 251, 276, 278, 283, 290, 291, 334, 335 health condition, 176, 239 health effects, 278 health risks, 276, 334 health status, 241, 247

heart disease, 101 heat loss, 107 heat transfer, 179, 325 heavy metals, 101, 224, 233, 234, 239, 303, 319, 320, 327, 332 height, 39 helical conformation, 41, 42 hemangioma, 348 heme, 224, 230, 237, 316 hemicellulose, 32, 85, 86, 100 hemoglobin, 31, 147, 148, 150, 152, 161, 188, 223, 230, 236, 238 hemolytic anemia, 177 heptane, 341 herbal medicine, 331 heterogeneity, 40 high fat, 100 histidine, 67, 184, 230, 232, 320 histone, 140 histones, 149 history, vii, 3, 189, 347 HM, 87 homocysteine, 140, 142 Hops, 250 hormone, 329 hormones, 326, 329, 330, 331 human body, 1, 37, 73, 86, 94, 101, 102, 107, 133, 137, 143, 147, 176, 177, 182, 187, 192, 194, 196, 209, 212, 213, 214, 221, 222, 223, 224, 236, 275, 303, 306, 310, 312, 316, 318, 319, 320, 326, 329, 330, 346, 347 human health, 192, 278, 324, 339 humidity, 19, 50 hybrid, 73 hydrazine, 139 hydrocarbons, 16, 78, 111, 124, 129, 259, 261 hydrogen atoms, 10, 11, 122, 231 hydrogen bonds, 11, 13, 14, 15, 26, 31, 45, 46, 87, 156, 168, 169 hydrogen bridges, 11, 77 hydrogen peroxide, 182, 184, 199, 217 hydrogen sulfide, 203, 260, 262, 266, 268, 322 hydrogenation, 107, 132, 133, 275 hydrolysis, 5, 6, 25, 32, 33, 34, 46, 47, 48, 49, 74, 82, 83, 87, 90, 94, 101, 107, 108, 114, 120, 121, 151, 152, 161, 176, 177, 180, 182, 184, 185, 186, 212, 249, 254, 257, 260, 261, 262, 292, 293, 308, 321, 322, 323, 327, 346, 347 hydroperoxides, 26, 71, 121, 122, 124, 126, 127, 128, 129, 135, 182 hydrophilicity, 98, 287 hydrophobicity, 140, 141, 157, 167, 186 hydroquinone, 199, 281, 282 hydroxide, 239, 317, 340

361

Index hydroxyl, 36, 37, 39, 45, 46, 48, 49, 61, 62, 63, 67, 79, 83, 88, 90, 95, 102, 110, 111, 115, 116, 129, 139, 165, 225, 231, 248, 253, 260, 264, 275, 320, 333, 334, 347 hydroxyl groups, 36, 39, 45, 46, 49, 62, 63, 79, 83, 88, 95, 102, 225, 333, 347 hygiene, 220, 272 hyperemia, 194, 313 hypersensitivity, 210 hypertension, 100, 101, 277 hypothesis, 58 hysteresis, 23 hysteresis loop, 23 I Iceland, 341 ID, 268 ideal, 19, 94, 159, 176 identification, 5, 6, 115, 139, 318 illumination, 216 images, 3 immune reaction, 306 immune response, 306 immunity, 181 immunogenicity, 306 immunoglobulin, 180 immunoglobulins, 149 immunomodulatory, 185 immunoreactivity, 306 immunostimulatory, 74 impurities, 115, 116, 130, 131, 132, 133 in vitro, 349 in vivo, 72, 113, 148, 208, 240, 326 incidence, 73, 102, 210, 276, 306, 330, 341 incubation period, 341 India, ix, 107, 246 individual differences, 103 individuals, 102, 245, 246, 284, 304, 330 induction, 67, 121 induction period, 67, 121 industrial processing, 179 industries, 74, 87, 95, 133, 325 industry, 4, 5, 7, 90, 173, 179, 188, 251, 271, 294, 295, 297, 316, 347 infants, 155, 178, 180, 298, 300, 306 infertility, 198 inflammation, 192, 194, 208, 214, 314, 318 infrared spectroscopy, 225 ingestion, 75, 101, 102, 186, 247, 304, 305, 314, 316, 320, 326 ingredients, 5, 7, 37, 53, 73, 82, 94, 158, 265, 283, 288, 289, 290, 294, 296, 300

inhibition, 5, 67, 68, 91, 93, 304, 305, 319, 322, 347, 349 inhibitor, 67, 177, 183, 331, 335, 336, 337, 338, 349 initiation, 46, 116, 121, 124, 126 injury, 304, 308, 316 inositol, 38, 39, 111, 283, 328, 331 insecticide, 241 insects, 73, 174, 303, 321, 323 insertion, 44 insomnia, 305 instant noodles, 173 insulin, 147, 224 integrity, 194, 221 intercellular adhesion molecule, 213 interface, 119, 120, 125, 153, 154, 159, 160, 162, 163, 164, 169, 185, 284 interference, 44, 304 international law, 272 interphase, 160 intervention, 58 intestinal tract, 192 intestine, 37, 55, 94, 102, 103, 185, 313 invasions, 331 inversion, 161 investment, 173 iodine, 5, 129, 194, 222, 304 ion channels, 318 ionization, 153, 154, 168 ionizing radiation, 153 ions, 11, 14, 26, 70, 81, 87, 88, 91, 94, 98, 99, 102, 138, 158, 164, 178, 188, 216, 225, 230, 233, 234, 237, 281, 291, 308, 326 Iran, 96 iron, 60, 93, 150, 180, 212, 224, 230, 233, 237, 238, 281, 282, 298, 304 irradiation, 184 irreversible aggregation, 155 irritability, 178, 254 isoflavone, 333 isoleucine, 140 isomerization, 7, 48, 58, 66, 75, 122, 194, 250, 253, 323 isomers, 37, 39, 49, 113, 122, 124, 133, 135, 141, 195, 219, 248, 253, 287, 324, 328 isoprene, 265 isotherms, 22 isotope, 13 K K+, 14, 81, 90, 91, 98, 102 kaempferol, 333 keratin, 32

362

Index

ketones, 37, 58, 60, 66, 69, 124, 129, 181, 184, 187, 214, 256, 258, 259, 260, 261, 263, 264, 266, 280, 308 kidney, 177, 196, 206, 210, 218, 233, 236, 304, 307, 312, 314, 330, 346, 347 kidney failure, 196 kinetic model, 104 kinetics, 34 L labeling, 139, 300 lactase, 73 lactase deficiency, 73 lactic acid, 3, 66, 143, 173, 181, 185, 189, 202, 251, 280, 287, 288, 342 lactobacillus, 74, 295 Lactobacillus, 24, 74, 104 lactoferrin, 180 lactose, 50, 66, 67, 70, 73, 75, 304, 345 lactose intolerance, 73 lakes, 299 lamella, 164 lanthanum, 225 large intestine, 100, 102 lead, 70, 121, 129, 164, 167, 168, 177, 178, 179, 183, 217, 218, 236, 275, 305, 307, 310, 313, 316, 319, 321, 324, 345, 348 leakage, 93, 316 leaks, 251 lecithin, 110, 111, 283, 286, 289 legs, 317 legume, 76, 309, 310, 312, 336 lesions, 212 leucine, 67, 68, 140, 255, 264 Lewis acids, 225 ligand, 39, 46, 225, 237, 314, 320 light, 63, 88, 91, 92, 94, 121, 126, 141, 161, 192, 193, 194, 196, 197, 205, 206, 207, 209, 210, 211, 253, 261, 279, 323, 329 light scattering, 161 lignin, 85, 100, 269 linear molecules, 53, 338 linear polymers, 166 linoleic acid, 67, 108, 109, 110, 111, 112, 113, 114, 124, 126, 127, 133, 258, 259, 263, 264 lipases, 121 lipid metabolism, 192 lipid oxidation, 6, 26, 67, 103, 115, 121, 126, 128, 131, 184, 186, 199, 217, 260, 268, 273, 282, 339, 345 lipid peroxidation, 320 lipoid, 182 lipoproteins, 17, 150

liquid chromatography, 135, 219, 220 liquid phase, 29, 117, 118, 119 liquids, 53, 119, 179, 284, 325 lithium, 93, 222 liver, 36, 39, 40, 84, 94, 101, 170, 174, 196, 201, 203, 206, 207, 208, 209, 210, 212, 223, 233, 236, 250, 262, 304, 318, 326, 330, 346, 348 liver cancer, 326 liver cells, 348 liver damage, 318 livestock, 251, 252, 260, 261 loss of appetite, 196, 202, 214 low temperatures, 18, 45, 72, 118, 129, 153, 157, 286 lung cancer, 341 Luo, 300 lutein, 131, 174, 194 lymphatic system, 170, 306 lymphocytes, 326 lysine, 55, 66, 67, 68, 70, 71, 72, 140, 171, 179, 180, 181, 182, 320, 336 lysosome, 159 lysozyme, 158, 161, 181 M macromolecules, 9, 28, 37, 44, 52, 53, 68, 238, 289 magnesium, 39, 74, 150, 188, 222, 292, 326 magnitude, 23, 93, 225 Maillard reaction, 27, 29, 39, 46, 55, 56, 57, 58, 61, 64, 65, 66, 67, 68, 71, 72, 104, 181, 182, 184, 214, 225, 260, 266, 267, 276, 342, 345, 349 majority, 3, 36, 46, 58, 107, 199, 222, 295, 318 malaise, 307 malt extract, 250 maltose, 36, 39, 50, 73, 275 mammal, 234 mammals, 107, 111, 169, 233, 303, 345 management, 216, 241 manganese, 222 manipulation, 349 mannitol, 45, 50, 275 manufacturing, 187, 272, 273 marine fish, 185, 261, 262 masking, 277 mass, 21, 78, 161, 169, 245, 275, 285 mass spectrometry, 245 material surface, 180 materials, vii, 1, 3, 4, 5, 6, 21, 29, 32, 37, 65, 74, 76, 126, 131, 153, 158, 163, 165, 173, 174, 180, 182, 207, 216, 243, 254, 278, 289, 290, 299, 303, 305, 306, 344, 347, 348 matrix, 29, 53, 54, 61, 169, 170, 172 matter, 16, 177, 298

363

Index MCP, 296 measurement, 67, 119, 246 meat, 5, 10, 24, 49, 53, 66, 68, 72, 91, 92, 93, 98, 142, 143, 148, 158, 162, 169, 178, 179, 182, 184, 186, 208, 212, 260, 268, 274, 279, 283, 288, 293, 296, 297, 298, 300, 305, 346 mechanical stress, 163 media, 60, 71 medical, 220, 277 medication, 180 melt, 20, 117, 119, 212, 293 melting, 12, 30, 59, 111, 112, 116, 117, 118, 119, 132, 134, 162, 167, 201, 207, 209, 293, 325, 341 melting temperature, 341 melts, 59, 118, 119, 194, 201, 205, 207, 213, 328, 341 membranes, 93, 120, 246, 320, 324 mercury, 222 metabisulfite, 279 metabolism, 101, 177, 192, 196, 200, 202, 205, 207, 209, 223, 236, 239, 269, 275, 304, 305, 310, 329, 349 metabolites, 102, 107, 304, 326, 330 metabolized, 101, 187, 224, 306, 329, 330, 346 metal complexes, 243, 282 metal ion, 68, 92, 103, 128, 129, 150, 213, 214, 225, 237, 273, 281, 296, 320, 334 metal ions, 68, 92, 103, 128, 129, 213, 214, 225, 237, 273, 281, 296, 320, 334 metal salts, 155, 239 metalloenzymes, 223, 232, 237, 239, 320 metals, 70, 126, 133, 155, 184, 225, 233, 234, 235, 237, 239, 305, 319, 320, 325, 332, 334, 347 methanol, 12, 62, 91, 315, 317, 328, 333, 341 methodology, 22 methyl cellulose, 295 methylation, 333, 334 methylcellulose, 86 methylene blue, 322 Mexico, 277 Mg2+, 14, 39, 98, 164, 227, 230, 237, 251, 332, 334 mice, 240, 300, 317, 326, 339, 341 microbial cells, 95 microcrystalline, 80, 85 microcrystalline cellulose, 85 micrograms, 192 microorganism, 23, 101, 278, 294, 323 microorganisms, 1, 9, 18, 20, 24, 26, 31, 35, 72, 101, 102, 173, 181, 184, 185, 207, 210, 212, 213, 251, 273, 278, 303, 310, 318, 324 microscope, 79 microscopy, 78 microstructures, 18 migration, 6, 21, 29, 153, 348 milligrams, 192

Ministry of Education, xi mixing, 16, 165, 280, 289 model system, 63, 64, 66, 70, 71, 104, 184, 349 modelling, 349 models, 6, 78, 220, 229 modifications, 76, 82, 182, 187, 239 modified Clausius-Clapeyron equation, 19 moisture, 9, 18, 19, 21, 22, 23, 24, 25, 26, 29, 30, 31, 32, 50, 61, 172, 178, 180, 181, 184, 293, 296 moisture content, 21, 23, 31, 32, 180, 184 moisture sorption, 9, 21, 22, 25 molasses, 24, 274 mold, 173, 279 molds, 24, 26, 95, 279, 280 mole, 15 molecular biology, 2 molecular mass, 285 molecular mobility, 9, 23, 28, 29 molecular oxygen, 334 molecular structure, 169, 225, 310, 312 molecular weight, 32, 33, 39, 40, 46, 51, 52, 54, 58, 63, 68, 77, 85, 90, 92, 93, 94, 95, 96, 98, 129, 141, 159, 161, 166, 170, 171, 172, 178, 184, 185, 186, 187, 229, 233, 245, 266, 268, 280, 293, 295, 310, 316, 318, 340, 341, 346 mollusks, 252 molybdenum, 222 monolayer, 17, 18, 23, 27 monomers, 30, 40, 42, 43, 86, 95, 97, 147, 183 monosaccharide, 36, 37, 38, 39, 40, 43, 45, 69 monosodium glutamate, 142 morphology, 326, 331 mortality, 316 mosaic, 150 MR, 269 mRNA, 95 mucosa, 246, 254 mucus, 261 multiple factors, 153, 237, 241 muscle contraction, 202 muscles, 156, 252 mussels, 317, 318 mustard oil, 254 mutation, 339 myoglobin, 31, 146, 147, 170, 223, 230, 231 myosin, 156 N Na+, 14, 81, 90, 102, 324 NaCl, 14, 86, 162, 164, 165, 171, 172, 251 NAD, 208 naphthalene, 324 National Academy of Sciences, 348

364

Index

native starches, 76 natural compound, 187 natural food, 73 natural polymers, 156 natural resources, 274 nausea, 73, 177, 196, 314, 317, 318, 335 NCS, 254 necrosis, 326, 348 negative consequences, 68 negative effects, 283 nerve, 202, 246, 247, 248, 304, 305, 317, 324, 341 nervous system, 60, 202, 212 Netherlands, 269 neurotransmitters, 324 neutral, 46, 69, 90, 95, 98, 138, 150, 172, 186, 187, 205, 206, 207, 211, 251, 260, 280, 294, 295, 298, 316, 321, 322 New Zealand, 277 NH2, 72, 138, 144, 187, 226, 248, 320 nickel, 132, 222 nicotinamide, 206, 207, 208 nicotinic acid, 206, 207, 208 niobium, 222 nitrates, 345, 346 nitrides, 257 nitrite, 278, 341, 345, 346 nitrogen, 3, 17, 58, 138, 139, 168, 172, 176, 222, 225, 231, 233, 320 nitrogen fixation, 233 nitrogen gas, 139 nitrosamines, 345, 346 nitroso compounds, 345, 346 NMR, 64, 227, 233, 234 non-diffusion-limited properties, 33 nonequilibrium, 29 non-polar, 165 NPR, 346 nuclear magnetic resonance, 119 nucleic acid, 17, 150, 174, 210, 223, 225, 236, 239, 320 nucleophiles, 71, 203 nucleoprotein, 150 nucleotides, 227, 236, 260 nucleus, 46 nuisance, 163 nutraceutical, 290, 291, 295 nutrient, 75, 100, 179, 216, 304, 313, 332 nutrients, vii, 1, 5, 6, 9, 37, 177, 179, 189, 191, 219, 243, 245, 271, 303, 331, 333, 335 nutrition, vii, xi, xii, 1, 2, 3, 4, 5, 6, 7, 37, 63, 73, 75, 76, 100, 102, 130, 137, 149, 167, 176, 180, 237, 239, 240, 245, 272, 319 nutritional status, 319

O obesity, 101, 274, 277 octane, 325 OH, 11, 13, 14, 17, 31, 46, 63, 67, 152, 212, 226, 230, 248, 249, 321, 329, 333, 335 oil, 4, 5, 10, 67, 86, 100, 112, 117, 118, 119, 120, 125, 128, 131, 132, 159, 161, 162, 169, 172, 173, 176, 177, 182, 184, 185, 198, 258, 262, 265, 278, 282, 284, 286, 287, 288, 290, 291, 298 oilseed, 180 olefins, 308 oleic acid, 108, 110, 111, 112, 113, 115, 124, 127 oligomers, 74, 186 oligosaccharide, 4, 6, 36, 73, 74, 88 olive oil, 112, 117 optical activity, 152 oral cavity, 186, 246 organ, 209 organelle, 320 organic compounds, 36, 192, 325 organic food, 296 organic solvents, 92, 107, 156, 317, 328, 332, 333, 340, 346 organism, xii, 191, 223, 233, 234, 243 organs, 170, 330, 332, 339 ornithine, 140, 309 oscillation, 180 osmotic pressure, 102, 221 osteomalacia, 196 osteoporosis, 296 ovum, 329 oxalate, 251, 332 oxidation products, 61, 84, 103, 131, 211, 261, 262, 263, 339 oxidation rate, 26, 124, 125 oxidative reaction, 130 oxidizability, 205 oxygen, 6, 10, 11, 13, 26, 28, 31, 40, 41, 43, 45, 46, 61, 67, 93, 121, 122, 124, 125, 126, 127, 128, 129, 152, 182, 184, 194, 196, 197, 199, 200, 205, 209, 214, 215, 220, 222, 225, 236, 237, 268, 281, 282, 316, 320, 324, 334 oysters, 317, 318 P pain, 73, 210, 246, 275, 300, 314, 317, 318 palm oil, 112, 117 palpitations, 300 pancreas, 22 pantothenic acid, 206, 207, 217 parallel, 46, 77, 78, 146 paralysis, 202, 304, 318

Index pasta, 30, 220, 286 pasteurization, 178, 179, 209 patents, xii pathogens, 153 pathways, 56, 107 PCBs, 325, 339, 340 PCDD/Fs, 325, 326 pellagra, 192, 207, 208 penetrability, 45 penicillin, 326, 327 pepsin, 150, 180, 184, 185, 316 peptide, 15, 137, 141, 145, 146, 148, 159, 168, 182, 184, 185, 186, 189, 225, 231, 327 peptide chain, 146, 148, 160, 168 peptides, 4, 36, 69, 141, 142, 177, 181, 182, 185, 186, 229, 234, 236, 260, 276, 282, 306, 318, 319 perchlorate, 239 peristalsis, 201 permeability, 316, 320, 324 permit, 282 permittivity, 14 pernicious anemia, 192, 210, 212 peroxidation, 320 peroxide, 124, 128, 184, 199, 217, 286, 336, 339 pesticide, 241, 303, 323 petroleum, 328, 329 PGE, 285 pH, 28, 31, 39, 49, 52, 53, 61, 66, 70, 71, 72, 81, 82, 84, 85, 87, 88, 92, 95, 99, 102, 104, 138, 143, 146, 153, 154, 159, 162, 163, 168, 171, 172, 178, 188, 202, 203, 204, 207, 211, 212, 214, 215, 236, 251, 273, 276, 277, 278, 279, 280, 294, 323, 332, 334 pharmaceutical, 74, 293, 294 pharmaceuticals, 82, 87, 295 phase diagram, 29 phase inversion, 161 phase transformation, 119 phenolic compounds, 61, 63, 281 phenylalanine, 140, 153, 187, 265 phosphate, 39, 49, 52, 84, 144, 170, 192, 203, 208, 209, 227, 228, 239, 292, 293, 295, 296, 297, 321, 322, 331, 332 phosphates, 38, 83, 84, 92, 236, 238, 296, 321, 322 phosphatidylcholine, 111 phosphoinositides, 39 phospholipids, 74, 107, 110, 111, 120, 128, 130, 131, 150, 167, 289, 320 phosphorous, 93 phosphorus, 49, 77, 81, 83, 196, 221, 222, 228, 296 phosphorylation, 208, 322 photochemical degradation, 205, 220 photosynthesis, 36, 233, 237 physical chemistry, 2 physical interaction, 9, 17

365

physical properties, 39, 101, 172, 278, 290, 339 physical structure, 22 physicochemical properties, vii, 6, 53, 54, 76, 145 Physiological, 192, 194, 196, 197, 198, 201, 202, 205, 207, 208, 209, 210, 212, 213 physiological factors, 238 physiology, 5 pigmentation, 326, 339 pitch, 40, 42 plant growth, 223, 321 plants, 3, 35, 36, 37, 39, 87, 96, 100, 101, 108, 111, 112, 127, 130, 174, 176, 177, 196, 206, 208, 212, 230, 233, 234, 249, 255, 263, 275, 289, 290, 294, 303, 305, 306, 307, 308, 309, 310, 312, 314, 318, 319, 321, 331, 335, 336, 349 plasticity, 107, 118, 119 plasticizer, 9, 31, 275 plastics, 347, 348 platelets, 198 platinum, 132 poison, 348 polar, 12, 16, 17, 23, 26, 31, 54, 80, 93, 141, 156, 165, 168, 186, 313, 315, 317, 321 polar groups, 12, 16, 17, 31, 80, 141 polarity, 120, 282, 317 polarizability, 102 polarization, 11 pollutants, 6, 324, 325, 348 pollution, 233, 316, 319, 347, 348 polyacrylamide, 344 polybrominated biphenyls, 324 polychlorinated biphenyl, 324 polychlorinated biphenyls (PCBs), 324 polycyclic aromatic hydrocarbon, 339, 348 polyether, 317, 318 polymer, 2, 29, 30, 31, 34, 53, 63, 76, 77, 91, 93, 98, 166, 291, 348 polymer chain, 31 polymer matrix, 31 polymer systems, 30 polymeric materials, 21 polymerization, 5, 6, 36, 40, 51, 58, 59, 62, 76, 82, 85, 87, 92, 123, 124, 129, 130, 153, 165, 166, 182, 287, 323, 333, 338, 339, 341, 348 polymers, 23, 30, 31, 52, 58, 74, 82, 87, 141, 147, 166, 181, 289 polymorphism, 107, 117 polynucleotides, 320 polypeptide, 145, 146, 148, 154, 159, 160, 280, 293, 309 polypeptides, 145, 166 polyphenols, xii, 61, 224, 238, 254, 282, 283, 333, 334 polyphosphates, 296, 297 polypropylene, 348

366

Index

polysaccharide, 5, 6, 36, 40, 41, 43, 44, 45, 50, 51, 52, 53, 54, 55, 86, 87, 88, 91, 93, 97, 100, 103, 183, 292, 293, 294, 295 polysaccharide chains, 53 polysaccharides, 4, 9, 17, 31, 35, 36, 37, 40, 45, 46, 47, 49, 51, 52, 53, 73, 95, 96, 97, 99, 100, 167, 177, 183, 235, 268, 289, 291, 292, 295, 334 Polysaccharides, 36, 40, 42, 45, 48, 50, 51, 53, 97, 235 polystyrene, 348 polyunsaturated fat, 115, 127, 262, 263, 320 polyunsaturated fatty acids, 115, 127, 262, 263, 320 polyuria, 196 polyvinyl chloride, 348 ponds, 316 POPs, 324 population, 173, 174 porosity, 184 porphyrins, 229, 282 positive correlation, 166 potassium, 91, 202, 222, 276, 279, 292, 294, 296, 297, 317, 326 potato, 27, 28, 76, 77, 78, 79, 80, 81, 83, 163, 195, 204, 206, 218, 219, 240, 242, 258, 268, 304, 305, 307, 313, 339, 341, 342, 345 poultry, 108, 251, 252, 260, 298 precipitation, 85, 138, 291, 334 precocious puberty, 330 predators, 303, 319, 331, 335 predicate, 21, 28 preparation, 5, 48, 54, 85, 101, 172, 251, 272, 297, 304, 322, 325, 331 preservation, vii, xi, 4, 5, 28, 272, 281, 325 preservative, 93, 219, 272, 274, 278, 300 prevention, 5, 83, 91, 121, 128, 291, 321, 325, 333 principles, 272 probability, 160 probiotic, 73, 349 probiotics, 94 professionals, 1 progesterone, 329 programming, 80 proliferation, 24, 73, 74, 94 proline, 68, 139, 140, 149, 170 propagation, 102, 121, 174, 319, 331 propylene, 254, 288, 295, 298 prostaglandins, 198 protease inhibitors, 178, 336, 337 protection, 4, 5, 139, 150, 283, 331 protein components, 158, 171, 186 protein constituent, 172, 232, 233 protein folding, 17, 31 protein structure, 182 protein synthesis, 139, 149, 223, 309, 319 proteolytic enzyme, 185

prothrombin, 128, 201 protons, 332 pruritus, 306 pulp, 279, 291, 294 pure water, 13, 14, 15, 18, 19, 92, 305 purification, 168, 169 purity, 50, 82, 153, 288, 289 PVC, 348 pyridoxine, 208, 209 pyrimidine, 201, 227, 320 pyrophosphate, 90, 202 Q quality control, xii, 347 quantification, 139 quantum yields, 121 quercetin, 333 quinone, 199, 215 quinones, 334 R race, 110 radiation, 126, 153, 194, 211 Radiation, 153 radical formation, 153 radical reactions, 121 radicals, 26, 67, 121, 182, 281, 334, 344 radius, 14 Raman spectroscopy, 227 rancid, 121, 346 rape, 178 rape seed, 178 rare earth elements, 222 raw materials, 216 RE, 269 reactant, 9, 26 reactants, 26, 27, 31, 345 reaction center, 337 reaction mechanism, 58, 68 reaction medium, 9 reaction rate, 25, 26, 28, 48, 179, 345 reaction temperature, 69, 134 reaction time, 69, 181, 217 reactions, 2, 4, 5, 6, 7, 9, 20, 23, 26, 27, 31, 39, 46, 55, 58, 59, 60, 61, 63, 65, 66, 68, 69, 70, 71, 107, 121, 124, 127, 128, 129, 130, 133, 138, 165, 166, 177, 181, 182, 184, 208, 209, 210, 212, 213, 217, 219, 233, 236, 239, 245, 259, 281, 306, 320, 322, 347 reactive oxygen, 71 reactivity, 13, 16, 69, 167, 236 reagents, 139, 152, 283, 321, 322

367

Index receptor sites, 11 receptors, 53, 140, 141, 186, 246, 247, 248, 249, 318 recognition, 337, 338 recombination, 31, 260 recommendations, 173, 174 recovery, 247 recrystallization, 29, 81 red blood cells, 177, 310, 316 red wine, 254 reducing sugars, 55, 60, 69, 73, 177, 181, 260, 342 refractive index, 115, 132, 339 regenerate, 215 regulations, 273, 319 rehydration, 168 reproduction, 74 repulsion, 16, 52, 53, 77, 82, 120, 145, 154, 160, 168 requirements, 1, 3, 37, 196, 272, 284, 289, 331, 348 researchers, 58, 66, 181, 188 residues, 5, 17, 39, 40, 41, 42, 43, 44, 46, 55, 58, 74, 76, 84, 87, 95, 96, 98, 145, 146, 148, 153, 165, 166, 177, 181, 183, 231, 233, 234, 239, 273, 303, 320, 323, 326, 330, 338, 339 resins, 348 resistance, 51, 84, 85, 92, 96, 166, 169, 182, 213, 287, 305, 323 resorcinol, 315 resources, 2, 4, 173, 174, 185, 272 respiration, 93, 205, 316 response, 75, 246 restrictions, 289 retail, 10, 290 retardation, 71, 304, 319 reticulum, 170 retina, 193, 194 retinol, 191, 192, 193, 194 RH, 50, 121, 269 rheology, 53, 85, 290 riboflavin, 150, 203, 205, 206, 217, 219 ribose, 69, 144, 300 ribosome, 150, 310, 311, 312, 313 rickets, 192, 196 rings, 53, 58, 62, 203, 229, 230, 292, 317, 339, 341 risk, 278, 291, 305, 319, 324, 339 risks, 319, 326, 330, 347 RNA, 320 rodents, 326 ROOH, 121, 281 room temperature, 49, 115, 118, 203, 212, 276, 279, 326, 340, 346, 347 root, 37, 74, 255, 298, 305, 331 roots, 36 rubber, 23 rubbers, 347 rubbery state, 33 rules, 109, 140, 141

S saccharin, 247, 248, 274, 276, 277 safety, vii, xii, 2, 4, 5, 6, 19, 34, 35, 63, 68, 85, 107, 124, 130, 143, 224, 225, 275, 276, 277, 285, 339, 345, 348 salinity, 316 salmon, 240 Salmonella, 24 salt concentration, 149, 170, 294 salts, 9, 12, 16, 17, 39, 59, 60, 72, 81, 90, 91, 92, 98, 141, 142, 143, 163, 167, 168, 173, 184, 212, 251, 254, 273, 279, 283, 292, 294, 296, 316, 326, 328, 329, 348 saponin, 313, 314, 316 saturated fat, 108, 111, 112, 113, 114, 116, 129 saturated fatty acids, 108, 111, 112, 113, 114, 116, 129 saturation, 19 scanning calorimetry, 30, 156 scavengers, 71, 128, 281 scent, 258 school, 178 science, xi, xii, 1, 3, 4 sclerosis, 194 seafood, xii, 185, 196 secretion, 94, 201, 233, 252, 260, 331 security, 245 sedimentation, 91, 172 seed, 114, 176, 177, 184, 188, 198, 203, 289, 293, 295, 304, 314, 315, 331, 332 selectivity, 132, 133, 318, 324 selenium, 222, 309 sensation, 45, 54, 246, 247, 248, 249, 251, 252, 254, 265, 275 sensations, 252 senses, 245 sensitivity, 151, 153, 178, 246, 306 sequencing, 139 serine, 99, 111, 140, 170, 231, 320, 336 serum, 101, 153, 162, 163, 164, 171, 233 serum albumin, 153, 162, 163, 164, 171 sex, 107, 330 sex hormones, 330 SFI, 119 shade, 298 shape, 1, 2, 21, 22, 23, 46, 51, 52, 53, 79, 80, 91, 118, 145, 152, 153, 158, 159 shear, 51, 52, 98, 162, 165, 166, 169, 292, 294 shelf life, 30, 33, 82, 86, 95, 96, 120, 157, 277, 278, 281, 284, 289, 339 shellfish, 269, 317, 318 shortage, 319 showing, 249, 276 shrimp, 185, 316

368

Index

sialic acid, 310 side chain, 36, 44, 96, 98, 140, 141, 142, 145, 146, 148, 152, 156, 168, 182, 184, 197, 200, 209, 225, 237, 253, 294, 308, 322, 323, 326, 337 side effects, 55 silicon, 222 silver, 298 single chain, 156 skeletal muscle, 169, 170 skeleton, 65 skin, 10, 149, 156, 163, 170, 192, 209, 214, 240, 246, 261, 298, 300, 326, 341 skin cancer, 341 small intestine, 55, 73, 100, 102, 194, 250, 276, 332 smoking, 117 SO42-, 237 social development, 6 society, 3 sodium, 14, 24, 71, 82, 83, 84, 91, 92, 95, 99, 133, 141, 142, 144, 152, 161, 162, 167, 222, 247, 251, 252, 278, 279, 282, 288, 292, 294, 295, 296, 297, 298, 300, 322, 326, 327, 341 solid matrix, 23 solid phase, 118, 119 solid surfaces, 284 solubility, 30, 39, 45, 46, 68, 75, 83, 85, 92, 93, 94, 99, 100, 101, 102, 124, 126, 138, 149, 151, 152, 162, 163, 164, 168, 169, 170, 171, 172, 180, 182, 184, 185, 191, 199, 236, 239, 247, 273, 279, 280, 282, 288, 296, 320, 325, 332, 339 solution, 14, 19, 28, 30, 45, 46, 51, 52, 54, 59, 64, 85, 86, 91, 92, 95, 96, 98, 100, 152, 153, 156, 161, 163, 167, 172, 183, 201, 202, 204, 205, 211, 216, 239, 247, 248, 275, 291, 292, 293, 294, 312, 317 solvation, 52, 87 solvents, 90, 93, 156, 172, 286, 313, 315 sorption, 19, 22, 25 sorption isotherms, 22, 25 South America, 292 soybeans, 108, 177, 289, 305 species, 45, 67, 71, 77, 78, 88, 97, 98, 112, 113, 114, 117, 126, 133, 149, 152, 153, 159, 174, 187, 212, 220, 227, 230, 234, 251, 252, 253, 278, 292, 295, 307, 308, 309, 310, 316, 317, 318, 321, 330, 333, 345 spectrophotometry, 237 sperm, 149, 326, 347 spermatogenesis, 149 spin, 145 spleen, 233 sponge, 24, 52, 284 SSS, 109, 113 stabilization, 5, 40, 83, 91, 147, 154, 159, 160, 162, 164, 281, 292

stabilizers, 45, 84, 283, 289, 290 stable complexes, 225 stable states, 117 starch, 4, 5, 22, 27, 28, 29, 30, 32, 33, 34, 36, 45, 48, 49, 76, 77, 78, 79, 80, 81, 82, 83, 84, 92, 96, 98, 163, 165, 166, 167, 172, 173, 178, 202, 275, 284, 285, 287, 289, 293, 295, 305 starch granules, 77, 78, 79, 80, 81, 84, 165, 167, 178 state, 10, 11, 14, 29, 30, 31, 33, 45, 90, 119, 128, 132, 138, 153, 178, 188, 209, 224, 225, 236, 238, 239, 241, 283, 296, 323, 329 states, 9, 18, 29, 151, 167, 207, 208, 224, 236, 239, 278 sterile, 178 steroids, 196 sterols, 150, 196 stimulant, 248 stimulation, 53, 96, 246, 247 stock, 300 stomach, 102, 180, 277, 304, 313 stomatitis, 205 stress, 51, 163, 169 strontium, 222 structural changes, 156 structural characteristics, 187 style, 24, 84, 294 subcutaneous tissue, 214 substitutes, 86, 134, 143, 251, 283 substitution, 47, 83, 202, 290, 321, 322, 347 substitution reaction, 321, 322 substrate, 127, 173, 266, 338 substrates, 102, 121, 122, 126, 308, 338 sucrose, 22, 24, 32, 36, 37, 39, 45, 48, 49, 54, 55, 58, 59, 72, 73, 74, 75, 86, 87, 94, 95, 96, 120, 180, 181, 187, 189, 247, 248, 274, 275, 276, 277, 288, 298 sugar alcohols, 38, 45, 49, 50, 51, 54, 55, 216, 274 Sugar alcohols, 38, 39, 45, 50, 55, 274, 275 sulfate, 36, 43, 44, 52, 60, 90, 239, 247, 328 sulfites, 72, 278, 279, 280 sulfur, 132, 171, 176, 178, 182, 184, 191, 201, 216, 219, 222, 254, 256, 258, 259, 260, 262, 267, 268, 278, 279, 280, 308, 320, 321 sulfur dioxide, 216, 219, 279, 280 sulfuric acid, 88, 93 sulphur, 104, 233 Sun, v, ix supplementation, 167, 176, 241, 242, 346 surface area, 125, 168 surface properties, 171 surface tension, 163, 284 surfactant, 159, 167, 285 surfactants, 90, 120, 153, 159, 162, 167, 285 susceptibility, 9, 189, 321, 323 suspensions, 98, 289

369

Index sustainable development, 4 sweeteners, 55, 74, 94, 187, 248, 251, 274, 276, 277, 300 swelling, 23, 80, 81, 83, 84, 85, 90, 102, 159, 168, 169, 314 symptoms, 177, 191, 194, 196, 202, 205, 208, 210, 214, 240, 272, 303, 305, 306, 314, 318, 320, 335, 339 synergistic effect, 281, 295 synthesis, 37, 101, 196, 201, 210, 214, 233, 238, 264, 265, 269, 298, 304, 309, 320, 341, 347 Syria, 96 T tachypnea, 304 tannins, 349 target, 337, 338 techniques, 3, 107, 137, 241, 245, 271, 304 technological advancement, 4 technologies, 3, 4, 6, 7, 173, 218 technology, 3, 71, 179, 196, 199, 272, 283, 290 teeth, 221, 284 tension, 28, 31, 120, 286 terminals, 141, 234 terpenes, 254, 258, 265 testicle, 329 testis, 326 testosterone, 329, 330 tetracyclines, 329 textbook, vii, 4 textbooks, vii, 4 texture, 1, 4, 5, 6, 7, 9, 18, 37, 46, 53, 85, 87, 88, 91, 97, 156, 157, 158, 162, 163, 165, 169, 188, 271, 273, 281, 289, 290, 292, 295, 296 thallium, 222 thermal analysis, 30 thermal decomposition, 107, 129, 268 thermal degradation, 129, 203, 266, 267, 268, 323, 341 thermal expansion, 119 thermal stability, 74, 159, 209, 294 thermal treatment, 157, 188, 205, 323 thermodynamic equilibrium, 29 thermodynamic parameters, 220 thermodynamic properties, 156 thermograms, 156 thermolysis, 58 thermostability, 94 thiamin, 201, 202, 219 thickening agents, 97, 293 thinning, 82, 98, 169 thiophenol, 203 threonine, 140, 231

thrombin, 338 thymine, 212 thymus, 326 thyroid, 346 tin, 317, 349 tissue, 84, 86, 108, 170, 194, 196, 269, 308, 318, 326, 329 titanium, 298 tobacco, 147, 150 tocopherols, 197, 219, 281, 282 tofu, 152, 251 tones, 93 tonic, 277 tooth, 274, 275 toxic effect, 183, 305 toxic metals, 102, 240, 348 toxic substances, vii, 178, 303, 306 toxicity, 7, 132, 183, 188, 222, 224, 227, 233, 238, 239, 240, 278, 303, 305, 309, 311, 312, 313, 314, 316, 317, 319, 320, 321, 322, 323, 324, 326, 339, 347 toxicology, 2 toxin, 305, 317, 318 trace elements, 222, 223, 241, 242, 243, 331, 332, 333 trade, 276 transesterification, 5 transferrin, 233 transformation, 3, 12, 44, 117, 194, 208, 231, 240, 323 transformations, 165, 166 transition elements, 237 transition metal, 36, 41, 332, 334 transition temperature, 30 transmission, 224 transparency, 82, 83, 84 transport, 4, 6, 11, 31, 36, 150, 152, 232, 237, 272, 308, 319 transportation, 283, 347 treatment, 5, 71, 101, 153, 155, 156, 157, 164, 168, 172, 174, 177, 178, 179, 180, 182, 183, 187, 204, 220, 236, 272, 281, 306, 317, 326, 336 treatment methods, 204 trigeminal nerve, 246 triglycerides, 74, 94, 107, 133, 134 trypsin, 152, 177, 180, 183, 185, 337, 338, 349 tryptophan, 67, 68, 145, 153, 248, 309 Turkey, 96 tyrosine, 140, 153, 184, 231, 320 U ultraviolet irradiation, 151 United Nations, 324

370

Index

United States, vii, 4 urea, 15, 151, 156, 261, 262, 348 urethane, 346 uric acid, 277 uric acid levels, 277 urinary tract, 310 urine, 55 USDA, 243 UV, 126, 209, 316, 323, 326, 341, 348 UV irradiation, 209 UV light, 126, 323, 326, 341, 348 UV radiation, 323 V valence, 99, 225, 233, 236, 238, 319 valine, 140, 170 vanadium, 222 vapor, 10, 11, 18, 19, 20, 23, 116, 131, 325 variations, 39 varieties, 166, 258, 274, 310 vegetable oil, 288, 298 vegetables, 4, 18, 24, 70, 73, 74, 87, 95, 104, 108, 195, 200, 203, 207, 208, 211, 213, 218, 219, 227, 237, 245, 252, 255, 256, 257, 269, 273, 275, 297, 298, 307 vegetation, 275 vertebrates, 170, 233, 250 vibration, 51, 79, 84, 305 vinyl chloride, 348 viscera, 5 viscoelastic properties, 37, 164, 165, 167 viscosity, 23, 28, 30, 31, 32, 33, 45, 46, 48, 51, 52, 53, 54, 80, 81, 82, 83, 84, 85, 86, 91, 92, 95, 96, 97, 98, 99, 101, 102, 104, 120, 129, 137, 159, 161, 163, 164, 166, 169, 172, 185, 275, 284, 289, 291, 293, 294, 295, 339 vision, 193, 194 vitamin A, 192, 193, 194, 196 vitamin B1, 105, 201, 202, 203, 204, 205, 210, 211, 212, 232 vitamin B12, 105, 211, 212, 232 vitamin B2, 203, 204, 205 vitamin B6, 209, 220 vitamin C, 128, 191, 213, 217, 218, 219, 224 Vitamin C, 68, 213, 216, 217, 218, 219, 281, 328 vitamin D, 196, 197 vitamin E, 105, 128, 197, 198, 199, 320 vitamin K, 199, 200, 201 vitamins, vii, 1, 7, 103, 107, 111, 112, 131, 157, 173, 178, 191, 192, 202, 203, 205, 207, 210, 212, 216, 217, 218, 219, 250, 260, 282

volatility, 53, 345 volatilization, 258 vomiting, 156, 177, 196, 314, 317, 318, 335, 339 W Washington, 219, 269, 348 waste, 9, 325 water absorption, 96 water evaporation, 95 water structure, 12, 14, 28 water vapor, 25 wavelengths, 317 weight loss, 196, 326 wettability, 184 wetting, 284, 285, 289 wheat germ, 112 WHO, 100, 174, 175, 189, 271, 342 Wisconsin, 193 withdrawal, 330 wood, 104, 173, 275, 278, 325, 347 workers, 58 World Health Organization (WHO), 189, 324 worldwide, 6, 173 X xanthan gum, 32, 52, 98, 291, 293, 294 xerophthalmia, 192, 194 X-ray diffraction, 39, 43, 76, 78, 117 Y yeast, 26, 173, 201, 203, 205, 206, 208, 210, 212, 234, 279, 289, 300 yield, 4, 27, 35, 37, 46, 47, 48, 55, 60, 61, 66, 83, 87, 88, 98, 117, 121, 130, 132, 139, 169, 172, 173, 178, 180, 182, 184, 186, 201, 207, 209, 210, 213, 218, 251, 259, 260, 263, 266, 268, 279, 290, 322, 334, 342, 345, 346 yolk, 72, 108, 164, 178, 203, 209, 289 Z zinc, 222, 231 zirconium, 222 zoology, 2 zwitterions, 138

Food Chemistry

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