Foods: Facts and Principles - PDF

FOODS FACTS AND PRINCIPLES-  FOODS FACTS AND PRINCIPLES (THIRD REVISED EDITION) N. Shakuntala Manay BA,M.S., Ph.D. Formerly, Head of the Department Food and Nutrition...

FOODS FACTS AND PRINCIPLES-  FOODS FACTS AND PRINCIPLES (THIRD REVISED EDITION) N. Shakuntala Manay BA,M.S., Ph.D. Formerly, Head of the Department Food and Nutrition Central Institute of Home Science Bangalore (Karnataka) M. Shadaksharaswamy Formerly, Professor of Biochemistry Head of the Department of Chemistry Dean Faculty of Science Bangalore University Bangalore (Karnataka) PUBLISHING FOR ONE WORLD NEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS New Delhi Bangalore Chennai Cochin Guwahati Hyderabad Jalandhar Kolkata Lucknow Mumbai Ranchi Visit us at www.newagepublishers.com Copyright © 2008,2001, New Age International (P) Ltd., Publishers Published by New Age International (P) Ltd., Publishers Second Edition: 2001 Third Edition: 2008 Reprint: 2013 All rights reserved. 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PUBLISHING FOR ONE WORLD NEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS 7/30 A, Darya Ganj, New Delhi-110002 Visit us at www.newagepublishers.com  Foreword I am happy that a revised and updated version of the excellent book by Drs' N. Shakuntala Manay and M. Shadaksharaswamy is being published. The earlier two editions of this book have proved to be important sources of information and guidance to all food scientists and scholars. We owe a deep debt of gratitude to the authors for revising the second edition and bringing the facts up to date. I also congratulate the Publishers, New Age International (P) Ltd. Among the UN Millennium Development Goals (2000-2015) the highest priority has been given to the elimination of hunger and poverty. Hunger is the extreme manifestation of poverty or inadequate purchasing power. Maternal, foetal and adult malnutrition is unacceptably high in our country. We are having problems in making proportionate advance in achieving the goal of reducing hunger by half by 2015. Hunger has multiple dimensions. Chronic Hunger arising from inadequate economic access to balanced diet. We have to fight the famine of work, to leading to endemic poverty, to win the battle against chronic protein - energy undernutrition. Hidden hunger caused by the deficiency of micronutrients in the diet like iron, iodine, zinc and vitamin A. Transient Hunger caused by disruption of communication, drought, floods and other natural calamities. The following aspects of food security will also have to be dealt with in an integrated manner in order to identify feasible and affordable solutions. Availability of Food: this is related to food production and where necessary food imports so that there is adequate stock of food in the market. Access to Food: this is an function of economics and purchasing power. This is where steps like the Public Distribution System (PDS) and the National Rural Employment Guarantee Programme will help. There are age specific nutrition intervention programmes like Integrated Child Development Scheme (ICSS) and support for nursing mothers. ICDS should be restructured so as to react to infants in the 0-2 age group. Absorption of Food in the body which is a function of access to safe drinking water, environmental hygiene, sanitation and primary healthcare and nutrition education. Thus, sustainable food and nutrition security involves the physical, economic, social and environmental access to balanced diet and clean drinking water. Inspite of many steps taken by the Government of India and the State Governments, we have not been able to achieve the goal set by Mahatma Gandhi in 1948 that "the God of Bread should be available in every home and hut of our country". Child malnutrition is even higher in India than in sub-saharan Africa. It is in this context that the present book assumes great importance. The authors have given detailed information on all aspects of food viz., production, post-harvest technology, culinary aspects, and fortification and enrichment. A perusal of the book will provide  V1 Foreword affordable guidelines for solving the problem of endemic and hidden hunger. For example, there are simple horticultural remedies for major nutritional maladies. Using the knowledge contained in this book, nutrition gardens can be set up in all schools in order to spread nutrition literacy. Similarly, steps can be taken for biofortification and for preparing diets based on locally available grains for ensuring that all the essential macro and micro nutrients available. There is ample data to show that malnutrition not only reduces work output but enhances vuhierability to diseases. For example, a drug based approach alone will not be adequate in the cjjse of HIV/AIDS, Tuberculosis, Leprosy and other diseases requiring prolong treatment. I therefore hope that this book will become a catalyst for building a sustainable nutrition security system for our country. The book should be read by policy makers, teachers, students, nutritionists and all concerned with achieving the goal of balanced diets for all. We owe the authors a deep debt of gratitude for their commitment to assisting malnourished children, women and men of our country. M.S. Swaminathan Chairman Swaminathan Research Foundation, 3rd Cross Taramani Institutional Area, Chennai-600113  Preface to the Third Edition This edition of "Foods: Facts and Principles" is a revision of the second edition with emphasis on Food Products, Food Processing and. Food Consciousness and updating of information on production, yield and area of cultivation of individual foods. In the first edition of this book we focused on the philosophy "Food is more than Nutrients," in the second edition we discussed the relationship between the dynamic forces of food and the dynamic force of one's total being. In this edition we have expanded our discussion to cover the psycho-physiological effects of food intake and its regulations. This edition introduces the concept of Genetically Modified Foods and Organic Foods. We have also introduced new foods and food products (viz., Beverages, Foods, Vegetables, Cereals, Pulses, Nuts, Oil & Fats, Milk & Milk Products, Eggs, Sea Foods) that have entered the Indian markets as a consequence of globalization of the economy. Special emphasis has been laid on the various nutritional aspects of the newly introduced foods, especially flaxseed and the most talked about co-3-fatty acids. In the food processing sections, we have laid stress on controlling the food quality conforming to ISO 9000 Standards. In the last edition a new chapter on Nutrition, Health and Food Consciousness was included; in addition to it we have dealt with body, mind, emotion and its relation to Spiritual Health. Health as a constant growth of spirit is a progressive unfoldment, a growth which is teleotic (Teleosis), and the limitation of the present concept, backed up with scientific study in connection with the same, has been cited, thus bridging the gap between pure science and spiritual life. This edition also contains an exhaustive index and references of useful websites. Our special thanks are acknowledged to Dr. A. M. Natrajan (Retd; HOD, Microbiology, NDRI, and now Head, K. C. Das, R & D Centre, Church Street, Bangalore) for his valuable inputs and support in the revision of the Milk and Milk Products chapter. N. Shakuntala Manay M. Shadaksharaswamy  Preface to the Second Edition This edition of Foods: Facts and Principles is essentially a version of the original, except for updating information regarding the production, yield and area of cultivation of individual foods. In the first edition of this book we wrote, "Food is more than Nutrients." In addition to nursing our body and promoting good health foods have an affect on our mind, emotion and spiritual life. There is a relationship between the dynamic forces of the food and the dynamic force of one's total being. This has been recognized for thousands of years by great Indian Seers and referred to by spiritual leaders of all faith. There is of late, a great awareness in the relationship of food ad spiritual life. Hence, a new chapter on Nutrition, Health and Food Consciousness has been included in this editon. N. Shakuntala Manay M. Shadaksharaswamy  Preface to the First Edition This book is written to meet the needs of students of Indian universities pursuing courses in foods, nutrition and allied courses, at the honours and postgraduate levels. The books currently used as text and reference books, mostly written by western authors, do not satisfy the needs of Indian students. Such books deal at great length of foods like beef, cheese, etc, which are important in their diet, but hardly deal with foods like pulses, spices, etc., which are important in ours. Our book eliminates this imbalance.. The production, processing and utilization of each food is discussed from the Indian context. Indigenous food products from cereals like idli, dosa and chapatti, milk products like dahi and khoa, and Indian confectionery (sweetmeats) have received special attention. Nutritional foods mixes from oilseed cakes and pulses, which are important to overcome malnutrition of infants and young children of developing countries like India, are considered. Indian food laws and the role of Indian Standards Institute in regulation of food standards are discussed. The book gives a comprehensive account of foods. It consists of three parts. Part 1, constituting Chapters 1-11, deals with the constituents of foods and the basic science required for understanding foods. Chapters 12-26, constituting Part 2, deal with different kinds of foods, some individual members of each kind of food, their composition, and nutritive value and uses. The final four chapters (Part 3) deal with method of food preparation, food quality, and processing and preservation of foods. We trust that the book would be of interest to all students and scientists working in the field of food, and nutrition. Suggestions for improvement of the book are welcome. The authors wish to convey their grateful thanks to Dr. M.S. Swaminathan, Director General, International Rice Research Institute, Manila, Philippines, and President of the International Union of Conservation of Nature and Natural Resources, for writing the Foreword. Our thanks are also due to Prof. P.S. Veerabhadrappa and Sri L. Goverdhan of the Biochemistry Department, Bangalore University, for help at various stages of the preparation of the manuscript. One of us (N.S.M) thanks the U.G.C for financial assistance for writing the book. N. Shakuntala Manay M. Shadaksharaswamy  Contents Foreword v Preface to The Third Edition vii Preface to The First Edition ix 1. Introduction 1-10 1.1 Food as a Source of Nutrients 1 1.1.1 Nutrients 1 1.2 Foods is More Than Nutrients 3 1.3 Food Intake and Its Regulations 4 1.3.1 Hunger, Appetite and Satiety 4 1.3.2 Regulation of Hunger 4 1.4 Food Patterns 6 1.5 Population and Food Production 7 1.5.1 Population Growth 7 1.5.2 Food Production 7 1.6 Food and the Future 8 1.6.1 Genetically Modified foods 9 1.6.2 Organic foods 10 PARTI: FOOD CHEMISTRY 2. Carbohydrates 11 - 26 2.1 Introduction 11 2.2 Monosaccharides 12 2.2.1 Structure 12 2.2.2 Properties of Monosaccharides 14 2.2.3 Monosaccharide Derivatives 16 2.3 Oligosaccharides 17 2.4 Functions of Sugars in Foods 17 2.4.1 Browning Reaction (Nonenzymic) 19 2.5 Polysaccharides 20 2.5.1 Properties of Polysaccharides 20 2.5.2 Starch 21 2.5.3 Dextrin 24 2.5.4 Glycogen 24 2.5.5 Cellulose 24 2.5.6 Hemicelluloses 24 xii Contents 2.5.7 Pectic Substances 25 2.5.8 Gums 25 3. Lipids 27-34 3.1 Classification 27 3.2 Role of Lipids 27 3.3 Fatty Acids 28 3.3.1 Saturated Fatty Acids 28 3.3.2 Unsaturated Fatty Acids 28 3.3.3 Essential Fatty Acids 29 3.4 Triglycerides 29 3.5 Physical Characteristics 30 3.5.1 Polymorphism 30 3.6 Reactions of Fats 31 3.6.1 Rancidity 31 3.6.2 Polymerization 33 3.7 Phospholipids 33 3.8 Lipids in Foods 33 4. Proteins 35-51 4.1 Amino Acids 35 4.1.1 Classification of Amino Acids 3 6 4.1.2 Properties 3 6) - O -a-D-glucopyranosyl-(l —> 2) - p - D -ffuctofuranoside Fig. 2.2 Conformational formula of common oligosaccharides 2.4.1 Browning Reactions (Non-enzymic) The non-enzymic browning reactions are responsible for the colour and flavour of foods, such as dates, honey and chocolate. The distinctive flavours that coffee beans, groundnuts, cashewnuts and breakfast cereals develop after roasting is due to browning reactions. The presence of reactive reducing sugars is responsible for browning in foods. On heating they undergo ring opening, enolization, dehydration and fragmentation. The unsaturated carbonyl compounds that are formed react to produce brown polymers and flavour compounds. The heat-induced browning reactions occur in two ways; caramelization and the Maillard reaction. Caramelization: Sugars in dry condition or their syrups, when heated, undergo a number of reactions, depending upon the temperature and presence of catalysts. Generally, there will be reactions leading to equilibration of anomeric and ring forms, inversion of sucrose, condensation reactions leading to the formation of oligosaccharides and polysaccharides, isomerization, dehydration and fragmentation, leading to the formation of unsaturated polymers with brown colour. With the use of proper catalysts it would be possible to conduct caramelization to provide either flavouring or colouring caraiftel for food use. For flavouring purposes, sucrose in concentrated syrups is caramelized. For the manufacture of caramel colours for use in beverages, glucose syrups treated with dilute sulphuric acid partially neutralized with ammonia are used. Maillard reaction: The Maillard reaction is actually a complex group of many reactions. The carbonyl group of acyclic sugars readily combines with the basic amino groups of proteins, peptides, 20 Foods: Facts and Principles and amino acids, resulting in sugar-amines. The set of various reactions sugar-amines undergo, resulting in browning, is known as the Maillard reaction. The sugar-amines form a brown colour at a lower temperature than that for the formation of the colour by caramelization. Hence, Maillard-reaction products predominate in browned foods. The condensation product of sugar and amines undergoes enolization and rearrangement and then undergoes condensation and polymerization forming red-brown and dark-brown compounds. The brown to black, amorphous unsaturated heterogeneous polymers are called "melanoids." Inhibition of the Maillard-browning reaction can be accomplished by keeping the pH below the isoelectric pH of the amino acids, peptides and proteins and by keeping the temperature as low as possible during processing and storage. Use of non-reducing sugars, such as sucrose, under conditions not favouring inversion, also helps bring down Maillard browning. Sulphur dioxide and sulphites used in extending the storage life of dehydrated foods, fruit juices and wines also inhibit the browning reaction. Sweetness of sugars: The most important function of sugars in food concerns their role as sweeteners. For an account of sweetness of sugar see Chap. 9 2.5 Polysaccharides Most of the carbohydrates in nature occur as polysaccharides. They are formed by a few types of hexoses, hexose derivatives (uronic acids and sulphate esters) and pentoses. They are high-molecular-weight substances composed of a large number of monosaccharide units combined to form one large molecule or polymer. They consist of a primary chain and, in some, side chains or branches may exist. The primary chain consists usually of one, but sometimes two, types of monosaccharide units. The side chains may consist of sugars different from those of the main chain, with different types of glycosidic linkages. Polysaccharides commonly found in foods are starch, dextrins, glycogen, cellulose, hemicellulose, pentosans and pectic substances. The generic name of polysaccharides is "glycans." If they are composed of a single type of monosaccharide unit they are homoglycans, and if of two or more monosaccharide units, they are heteroglycans. Glycans that release only glucose on hydrolysis are glucans (hexosan) and xylose, xylans (pentosan). Similarly, other homoglycans are called mannan, galactan, fructan and araban depending upon the sugar released on the hydrolysis of the polysaccharide. Heteroglycans composed of two sugars are called galactomannan, glucomannan, arabinogalactan, etc., depending on the sugars present. The names of some well-known polysaccharides end in "in," for example, chitin, dextrin, inulin (fructan) and pectin. Polysaccharides serve plants and animals in three ways: Cellulose, hemicellulose, pentosans and pectic substances provide structural material (cell walls, fibres, seed coats, peels and husks). Chitin and mucopolysaccharides (polysaccharides containing amino sugars) serve this purpose in animals. Structural polysaccharides are indigestible substances; nevertheless, they are important for human health. They provide the bulk in the diet and aid excretion. Starch, dextrin and fructan in plants, and glycogen in animals, provide food reserves. These polysaccharides are digested and utilized by the human body. They are nutritionally important. Polysaccharides attract and retain water so that life's enzymic processes are not impeded under dehydrating conditions. 2.5.1 Properties of Polysaccharides There are great differences in the solution properties of various polysaccharides. Some disperse rapidly in water, some are quite insoluble, and others disperse as swollen particles or globules. A few  Carbohydrates 91 form clear solutions at moderate concentrations. Some form gels at low concentrations, some gel only at high concentrations and others do not gel at all. Some form gels that are translucent and others opaque. The differences in the solution properties of polysaccharides are related to their monosaccharide composition, type of chemical linkages among sugar units, hydrogen bonding and ionic interaction between and among polymers and the gross conformation of the hydrated polymer in solution. The structure of polysaccharides influences their interaction with water. Hydrogen bonds are formed between the hydroxyl groups of the polysaccharide and water. Layers of water molecules adjacent to the hydroxyl groups of polysaccharides are, therefore, partially ordered and immobilized, and assist the dissolution or dispersion of the large molecules. As the hydrated macromolecules move in circles the water aggregates can be rearranged or displaced: By folding or coiling, the polysaccharide molecule can associate with itself to form loops. Hydrogen bonding between sections of different molecules can result in the formation of crystalloid regions (micelles). Linear polysaccharides can associate through intramolecular hydrogen bonding to form gels and, if the solution is dilute, it may result in precipitation (retrogradation). Branching side-chains or negative charges like those of carboxyl groups of uronic acids distributed along the linear polysaccharide molecules inhibit intramolecular association. At the same concentration, solutions of linear polysaccharides have greater viscosities than those of the branched polysaccharides of the same molecular weight. If the polysaccharide contains carboxyl groups the viscosity is more pH-dependent. At low pH values carboxyl groups are un- ionized. Therefore, solutions of polysaccharides containing acidic groups can form gels upon the addition of polyvalent cations since the ions readily form intermolecular salt bridges. 2.5.2 Starch This is the principal food-reserve polysaccharide of the plant kingdom, where it occurs in cereal grains, pulses, tubers, bulbs and fruits in varying amounts from a few per cent to over 75 per cent in the case of several grains. Starch provides the major source of energy in the diet of man. Much of the starch is consumed without being isolated from the plant material in which it occurs. Refined starch, either natural or modified, also plays an important role in food preparations. Starches contain only glucose residues. They are mixtures of the structurally distinct polysaccharides, amylose and amylopectin. Cereal starches usually contain 25 per cent amylose and 75 per cent amylopectin. Waxy or glutinous starches like waxy corn starch contain little or no amylose. Waxy varieties of corn and sorghum starches have found use in food-pie fillings and puddings. Amylose has great industrial possibilities and hence, by breeding, corn with starch of high amylose content (about 85 per cent), has been obtained. In addition to these components, starch granules commonly contain small amounts of proteins, fatty substances and inorganic material. The non-carbohydrate constituents are of relatively little importance in relation to food use, but they affect the physical properties of starch. Amylose consists of glucose units linked bya -(l->4) bonds, i.e., the anomeric hydroxyl of one glucose unit is in glycosidic linkage with carbon-atom 4 of the next glucose unit (Fig. 2.3). The chain length of amylose varies and it is considered to have a molecular weight of 1.1 to 1.9 million. Amylose is not truely soluble in water but forms hydrated micelles. In such micelles the long chain is twisted into a helical coil. It is this structure of amylose that is responsible for the blue colour produced by iodine with starch. The structure of amylose contributes to the gelling characteristics of cooked and cooled starches. 22 Foods: Facts and Principles CH2OH CH2OH O CH2OH OLOH Fig. 2.3 Partial structure of amylose Amylopectin also has a backbone of a -(1—^linkages but, in addition, the molecule is branched through a -(l->6) linkages to the extent of 4-5 per cent. The length of the linear unit in amylopectin is about 20-25 glucose units. Amylopectin has a brushlike structure and its molecular weight is over 10 million. Amylopectin is responsible for the thickened properties of starch preparations but it does not contribute to gel formation. The partial structure of amylopectin is given in Fig. 2.4. CH2OH CH2OH CH2OH HO CH2OH Q H oCHLOH 2 ^ L Fig. 2.4 Partial structure of amylopectin Granular structure: Starch occurs as granules in the cytoplasm of the cells. The granules remain essentially intact during most types of processing, such as milling, separation and purification of starch, and even during most types of chemical modification. The microscopic appearance of starch granules from different sources is different, and thus they can be identified by this method alone. The unique characteristics of granules are their sizes, shapes, uniformity, location, and the appearance of granules under polarized light (birefringence). Within the starch granules, a mixture of linear and branched molecules is arranged in concentric shells. When parallel association exists between adjacent linear molecules or between the outer branches of the branched molecules, they are held together by hydrogen bonds resulting in crystalloid regions or micelles, and this causes the granules to be birefringent. More loosely packed regions are amorphous and they exist between the micelles in each concentric shell. Water can easily enter the amorphous regions of the grain. Gelatinization: Cereal starches under normal conditions usually have a moisture content of 12-14 per cent. Cold water can penetrate amorphous regions of starch without disturbing the micelles and a maximum water content of about 30 per cent can be obtained. If the mixture is heated the intramolecular hydrogen bonding is broken, the grains absorb more water and swell. The swelling causes loss of birefringence. The temperature at which the granules begin to swell rapidly and lose birefringence is called the "gelatinization temperature." This change is completed with a given starch sample with a rise of a few degrees in temperature after gelatinization sets in, because the individual granules within any starch sample differ not only in size and shape but also in the energy required for bringing about swelling. Thus, it is more appropriate to call the temperature of gelation the gelatin- Carbohydrates 23 ization temperature range. The gelatinization temperature range is somewhat specific for starches from different sources and this property is useful for the purposes of identification of starch. As the temperature of the starch suspension is increased above the gelatinization range, the granules continue to swell, if sufficient water is present. Additional swelling increases the viscosity as the swollen granules begin to collide frequently. In some cases the fragile granules may be torn into fragments, causing a reduction in viscosity. The viscosity changes depend on the temperature, initial concentration of starch suspension, size of the granules, the internal forces holding the molecules together within the granule and the effect of the other ingredients in the system. When a starch-thickened mixture is allowed to cool without stirring, there is a tendency for intramolecular bonds to form resulting in the formation of a more or less rigid gel. This is because the hydrogen bonding between molecules becomes more extensive, thereby increasing the micellar regions. The growth of micelles results in the gel becoming firm. When only amylopectin molecules are present, as in waxy starches, the branches prevent the degree of association for gel formation, except when an extremely high starch concentration of about 30 per cent or more is present. Waxy starches form a soft thick mass rather than stiff gels. This characteristic, together with translucence and slow retrogradation, make waxy corn and sorghum starches useful in pie fillings and similar products. In a very dilute solution of starch, the individual amylose molecules are not sufficiently entangled to produce a gel so that the growing micelles eventually cause precipitation. This is known as "retrogradation." The rate and extent of retrogradation is also, influenced by temperature, size, shape of starch granules and other ingredients present. Retrogradation is more rapid at temperatures near 0° C. Even waxy starches, which do not form gels except at very high concentration, retrograde under frozen conditions. Amylose can form strong and flexible films which are water soluble and edible. Presence of amylopectin decreases the intermolecular binding and prevents the formation of films. This difficulty can be overcome by using high-amylose starch. Foods can be coated with a thin film of amylose to improve water retention and decrease surface thickness in dehydrated fruits. Modified starches: As has already been discussed, unmodified starches from different sources differ in size, shape, appearance and gelation temperature and gel-forming ability. Increased knowledge of the molecular and granular structure of starches has helped modify starches to meet the specific needs of food and other industries. Modified starches are not toxic and their digestibility appears to be little affected. Pregelatinized starches are those that are cooked and dried to give products that readily disperse in cold water to give moderately stable suspensions. Dispersions of pregelatinized starch have a less desirable texture than cooked dispersions prepared from untreated starch. Nevertheless, pregelatinized starch dispersions have some characteristics of the original starches. This has led to their wide use in the preparation of instant puddings, pie fillings, soup mixes, salad dressings, etc. Starches are modified by suspending granular starch in a very dilute acid at a temperature below its gelatinization temperature. This results in "thin boiling" starch. This method hydrolyzes sufficient molecules in the granules so that, on heating with water, a product with low viscosity but which can form a firm gel upon cooling and ageing is produced. Acid-modified starches are used in the manufacture of starch gum confections (gum drops). The extent of swelling and ultimate breakdown of starch granules during cooking can be controlled by introducing cross bonding between starch molecules, using suitable reagents. Chemical crosslinking can induce gel formation in the hydrated starch. By appropriate treatments, starch 24 Foods: Facts and Principles granules whiter in colour, improved gel strength and clarity, greater paste stability and paste viscosity, can be obtained. 2.5.3 Dextrin These are products of the partial breakdown of starches. Dextrins are intermediate in size between starches and sugars and exhibit properties that are intermediate between these classes of materials. These are formed, amongst other methods, when starch is subjected to dry heat. The toasting of bread converts a part of starch to dextrin. The sweet taste of toast is due to this change. 2.5.4 Glycogen This is the storage polysaccharide in animals, and hence is sometimes called animal starch. This is mainly present in the liver and skeletal muscle. Liver glycogen rapidly hydrolyzes to D-glucose after slaughter of animals. Glycogen is a branched-chain polysaccharide resembling amylopectin rather than amylose, but is more branched than amylopectin. 2.5.5 Cellulose Cellulose makes up more than 25 per cent of cell walls in higher plants and this one polysaccharide is said to account for nearly half the world's supply of carbon. In woody tissues, cellulose molecules are associated into partially crystalline microfibrils. However, in vegetable pulp cells it has little fibrous character. Cellulose is found embedded in an amorphous gel composed of hemicellulose and pectic substances. In the case of fruits and vegetables, texture is more important than flavour. Changes in cell wall constituents surrounding the cellulose, and in the interstitial matrix, are largely responsible for the changes in texture of fruits and vegetables during ripening, storage and processing. Cellulose, like amylose, is composed of long chains of glucose residues with (1—>4) linkages, but the linkages in cellulose are p-rather than a-configuration of amylose. As with amylose, cellulose has a very high molecular weight. The fibrous polysaccharide does not dissolve in water to any extent. Cellulose is not digested in the human system and acts as roughage or dietary fibre. It can be broken down to glucose by certain microbial enzymes. This is the way cellulose is hydrolyzed in the rumen of animals to provide them with sugar. Some modified celluloses find use in food industries. Avicel is partially hydrolyzed cellulose and is sometimes added to foods to contribute bulk without calories. Carboxymethyl cellulose (CMC) is used in the manufacture of ice cream. It contributes a good body and smooth texture, retards the enlargement of ice crystals during storage and improves the melting characteristics of ice cream. CMC also finds numerous other uses in foods. Other derivatives of cellulose-hydroxypropylmethyl cellulose, methyl-methyl cellulose, etc., also find use in food industries. 2.5.6 Hemicelluloses Hemicelluloses are a poorly defined group of substances. They cover the polysaccharides, other than cellulose, starch and pectic substances, that occur in land plants. They are insoluble in water but, unlike cellulose, are soluble in alkali. Some hemicelluloses are composed predominantly of pentoses (xylose); others contain hexuronic acids and occasionally L-rhamnose. Hemicellulose helps cement together closely packed cellulose microfilaments. Together with cellulose, hemicellulose forms a portion of undigested carbohydrate and is therefore a dietary fibre. Carbohydrates 25 2.5.7 Pectic Substances Pectic substances occur as constituents of plant cell walls and in the middle lamella. In the latter, they serve as cementing material to hold cells together. They are mixtures of polysaccharides formed from galactose, arabinose and galacturonic acid, the last being predominant. The carboxyl groups of galacturonic acids are partly esterified with methyl groups and partly or completely neutralized by one or more bases (Fig. 2.5). Fig. 2.5 Partial structure of pectic substances The water-insoluble parent pectic substance that occurs in plants is protopectin. This, in fruits, decreases during ripening and soluble pectin increases. Protopectin on restricted hydrolysis, gives pectinic acid and pectin. Pectinic acid forms a colloidal solution and contains more than a negligible proportion of methylester groups. It is capable of forming gels with sugar and acid. Pectin is water-soluble pectinic acid with a varying methyl ester content and degree of neutralization. Pectin forms gel with sugar and acid under suitable conditions. Pectic acid is mostly galacturonic acid and is essentially free from methyl ester groups. Commercial pectin is obtained by extraction of citrus peels (or from apple pomace) with dilute acid. The product in which at least 50 per cent of the carboxyl groups of galacturonic acids are methylated is used in the manufacture of jams and jellies. For rapid-set of jellies the degree of methylation should be high (about 60). For gel formation, sugar (65-70 per cent), acid (pH 2.3-3.2), water and pectin must be present. The acid increases the unionized carboxyl groups which decrease the attraction between pectin and water molecules, thus decreasing the ability of pectin to remain in a dispersed state. Sugar further decreases the hydration of pectin by competing for water. The unstable dispersion of the less hydrated pectin forms a gel when cooled. Pectin with a low methoxyl content forms a gel by the cross-linkages established between free carboxyl groups and divalent cations like calcium. Gel-formation does not require the presence of sugar in such cases. 2.5.8 Gums Gums are hydrophilic substances that give a viscous solution or dispersion when treated with hot or cold water. Most gums are polysaccharides and included in this definition are starches, pectic substances and some derivatives of starches and cellulose, which have been already considered. Other natural polysaccharide gums are seed gums, plant exudates and seaweed gums. Gums are incorporated to improve the texture, water retention and rehydration of many dehydrated, frozen and "instant" convenient foods. Ice cream, gelled desserts, salad dressings, baked goods, processed cheese and encased ground meats are some of the examples of the foods whose qualities have been improved by the addition of gums. Gums function as thickeners for gravies and sauces, moisture retention agents in baked goods, emulsion stabilizers in salad dressings, protective colloids in chocolate milk and syrup, foam stabilizers in whipped popping and beer, clarifying agents for wines and beer, flavour fixing agents and lubricants. 26 Foods: Facts and Principles The seed gums commonly used are guar (guaran) obtained from the common Indian pulse guar (Cyamopsis tetragonoloba) and locust bean gum (from Ceratonica siliqua). These are long-chain polysaccharides of p -D mannose and a -D-galactose (galactomannans), with branched units. These give colloidal dispersions of high viscosity which are stable over the pH range of foods and are not affected by heat processing, freezing, acids, salts or proteins in the food product. They form complexes with other gums which help modify the texture of their gels. The best known plant exudates are gum arabic (Acacia), karaya (Sterculia) and tragacanth. These are mixtures of heteroglycans containing the monosaccharides D-galactose, L-arabinose, D-galacturonic acid, L-fucose and L-rhamnose. They are generally contaminated with proteinaceous and phenolic substances, glycosides and foreign matter. Gum arabic is more soluble in water giving a solution of low viscosity compared with other gums. Because the quality, quantity and markets of exudate gums are variable, these natural gums are being replaced by derivatives of starch and cellulose to provide more uniform properties and more dependable supplies, Agar is an extract from red and brown algae. It is mainly P -D- and a -L-galactan where galactose residues are sulphated at various hydroxyl positions. Neutral fractions called "agarose" or "agaran" can be separated from agar. Agar forms the strongest and most stable gels at lowest concentration. Agar gels are transparent and reversible upon heating and cooling. Therefore, agar finds several uses in stabilizing food products. Carrageenans are also sulphated galactans from seaweed extracts. There are both gel-forming and non-gel-forming carrageenan fractions. Both form stable complexes with proteins and other gums. Carrageenans are especially useful in dairy products because they form stabilizing complexes with milk proteins and suspended cocoa powder in milk, and give a more acceptable texture to processed cheese and cream. Algin is a product of brown algae. It is composed of D-mannuronic and D-galacturonic acids. The carboxylic groups are neutralized by various natural cations. If they are replaced by a single cation (sodium, potassium, ammonium, etc.) the product is called an "alginate." When the cations are removed insoluble alginic acids are produced. Alginates give widely variable sols and viscosity properties in acid and salt solutions. They form gels and films and find a number of food uses. Some microbial gums also find use in foods. Two important ones are dextran and xanthan gums. Dextran is formed by the action of Leuconostoc mesenteroids on sucrose. It is a polysaccharide consisting of a -D-glucose units, 95 per cent of the units being linked by a -(1^6) linkages and 5 per cent a -(1^3) linkages. Dextran of molecular weight below 100,000 is readily soluble in water and is used in many food products. Xanthans are formed by the action of the bacterium Xanthomonas comestris on D-glucose. The gum contains D-glucose, D-mannose and D-galacturonic acid and has a high molecular weight. It dissolves in cold water and finds use in food as a stabilizer, emulsifier, thickener, suspending agent, bodying and foam enhancer.  CHAPTER Lipids The term "lipid" is used to denote diverse types of compounds which are insoluble in water but soluble in nonpolar solvents, such as chloroform, carbon disulphide, benzene and ether. The characteristic insolubility of lipids in water is, in several cases, due to the presence in them of one or more fatty acids which contain long aliphatic hydrocarbon chains. Lipids are widely distributed in nature. They rarely occur in an organism in the free state, but are more usually combined with proteins (lipoproteins) or carbohydrates (lipopolysaccharides or glycolipids). 3.1 Classification Lipids are broadly classified into two groups, simple lipids and compound lipids. Simple lipids include fatty acids, fats which are esters of fatty acids with glycerol (triglycerides), and waxes which are esters of fatty acids with long chain monohydroxy alcohols. All other lipids are included under compound lipids. These include phosphoglycerides (phospholipids or phosphatides), steroids, carotenoids, and lipids functioning as vitamins or hormones. Foods may contain any or all these substances but those of greatest interest are fats (acylglycerols or triglycerides) and phospholipids. The term "fat" is generally applied to all triglycerides regardless of whether they are normally non-liquid or liquid at room temperature. Triglycerides from animal sources contain a higher percentage of saturated fatty acids and are normally solids at room temperature and known as fats. The plant triglycerides are rich in unsaturated fatty acids, are generally liquids at room temperature and called "oils". 3.2 Role of Lipids One of the major roles of lipids in foods is to serve as fuel molecules. Triglycerides constitute a pool of stored energy. They are a more highly concentrated form of metabolic energy than carbohydrates. When oxidized, a gram of fat gives more than twice (9 Kcal) as much energy as proteins or carbohydrates. This is because fats are more highly reduced than carbohydrates and proteins. It is desirable that lipids supply 20 to 30 per cent of the total calorie requirement. When energy supply from foods is in excess of energy demand, the excess is preferentially stored as fats in the adipose tissue. As a storage fuel, fats have an advantage as they can be stored in an anhydrous form while glycogen, the stored polysaccharide, is heavily hydrated. Taking into account these two properties of fats, a gram of fat stores more than six times as much energy as a gram of glycogen and this energy is mobilized for use by the body when needed. Carbohydrates and proteins can be converted into fat when energy and growth requirements have been satisfied. Fatty acids are not converted into proteins or carbohydrates. These complex processes are co-ordinated by hormones. 28 Foods: Facts and Principles Lipids in the form of triglycerides, phospholipids, cholesterol and cholesterol esters, are important to the structure, composition and permeability of membranes and cell walls. They perform the function of energy storage in seeds and fruits. Lipids are a major component of adipose tissue. Skin waxes act as insulation barriers to avoid thermal and physical shock. Some fats play a solvent-like role—the dietary fats help carry fat-soluble vitamins so that they are efficiently absorbed. Fats serve as vitamins, emulsifiers, and flavour and aroma components. 33 Fatty Acids A fatty acid is a long-chain carboxylic acid, CH3 (CH2)n COOH. A considerable variety of fatty acids occur in nature. Most natural fatty acids contain an even number of carbon atoms due to their mode of biosynthesis, but odd-numbered fatty acids do occur naturally. Most fatty acids are linear though some branched-chain and cyclic fatty acids are also found in nature. 3.3.1 Saturated Fatty Acids Although straight-chain saturated fatty acids containing from 2 to 80 carbon atoms per molecule are known, generally those containing C2 to C2o and particularly those containing Ci6 and Cig are found in most animal and vegetable fats. Table 3.1 gives the trivial name, systematic name and structure of some saturated fatty acids found in food lipids. Table 3.1 Some saturated fatty acids found in food lipids Trivial name Systematic name Structure Butyric Tetranoic acid CH3 (CH2)2 COOH Valeric Pentanoic CH3 (CH2)3 COOH Caproic Hexanoic CH3 (CH2)4 COOH Enanthic Heptanoic CH3 (CH2)5 COOH Caprylic Octanoic CH3 (CH2)6 COOH Capric Decanoic CH3 (CH2)8COOH Laurie Dodecanoic CH3 (CH2)10 COOH Myristic Tetradecanoic CH3 (CH2)i2 COOH Palmitic Hexadecanoic CH3(CH2)14COOH Stearic Octadecanoic CH3(CH2)16COOH Arachidic Eicosanoic CH3 (CH2)i8 COOH Behenic Docosanoic CH3 (CH2)20 COOH Lignoceric Tetracosanoic CH3(CH2)22COOH Saturated fatty acids with more than 24 carbon atoms seldom occur in food triglycerides, but do so in waxes. The lower members C4—C10 occur mainly in milk fat. Those of chain-length Cn—C24 are found in most animal and vegetable fats. Fatty acids with odd-numbered carbon atoms are found in animal and vegetable fats but they seldom exceed 1-2 per cent of the total fat. The most commonly encountered saturated acids are lauric, myristic, palmitic and stearic. 3.3.2 Unsaturated Fatty Acids Unsaturated fatty acids differing with respect to number of carbon atoms and double -bond characteristics are found in lipids. Unsaturated acids may have one to six double bonds. Polyunsatu- Lipids 29 rated acids usually have a methylene (CH2) group between double bonds. Double bonds occur in many locations but a regularity of position is seen in most of the acids commonly found in food fats. The unsaturation in different acids is at a definite location with respect to the carboxyl carbon number one or CH3 ("GO" carbon atom) and they have cis configuration. The unsaturated fatty acids found in lipids are given in Table 3.2. Table 3.2 Some unsaturated fatty acids found in food lipids Trivial name Systematic name Structure Myristoleic 9-Tetradecenoic CH3(CH2)3CH=CH(CH 2)7COOH v Palmitoleic 9-Hc ^Hecenoic CH3(CH2)5CH=CH(CH 2)7COOH Oleic 9-Octadecenoic CH3(CH2)7CH=CH(CH 2)7COOH Linoleic 9,12-Octadecadienoic CH3(CH2)4CH=CH CH2CH=CH (CH2)7COOH Linolenic 9,12.15-Octadecatrienoic CH3(CH2CH=CH)3(CH2)7COOH Arachidonic 5,8,11,14-Eicosatetraenoic CH3(CH2)3(CH2CH=CH)4(CH2)3COOH Oleic acid is found in almost all fats. It is a dominant compound of olive oil in which it is present up to a concentration of 75 per cent. Linoleic acid is present in high concentration in many fats and oils. It is present to the extent of 60-80 per cent in safflowerseed oil. This acid enters into a number of reactions involving oxidation, polymerization and lipoprotein interaction. Linolenic acid comprises 50-60 per cent of the fatty acid in linseed oil. Arachidonic acid which is C2o tetraenoic acid is found primarily in animal sources. It is a component of many phospholipids and a precursor of prostaglandins. 3.3.3 Essential Fatty Acids Fatty acids containing more than one double bond (polyunsaturated) are derived from those containing one double bond. The way in which the new double bond is introduced between the existing double bond and the carboxyl group differs in plants and animals. Linoleic acid, which contains two double bonds, and certain acids derived from it, are necessary to maintain animals in a healthy condition. Animals are unable to desaturate oleic acid to linoleic acid. Therefore, the latter must be supplied in the diet from plant sources. Linoleic acid and other unsaturated acids formed from it are, therefore, known as essential fatty acids. 3.4 Triglycerides Triglycerides are the acyl derivatives of glycerol. The position of an acyl group in glycerides is indicated by the term "Sn" (stereospecifically numbered) in which the carbon atoms of glycerol are projected in a vertical line, and the secondary hydroxyl is shown to the left and the top carbon atom is C-l. Thus, a triglyceride containing palmitic, oleic and stearic acid residues in positions 1, 2 and 3 respectively of glycerol is named Sn-glycerol-1 palmi-to-2-oleo-3-stearate or simply palmito-oleo-stearate. In triglycerides, all the glycerol hydroxyl groups are esterified. Partial glycerides with only one or two hydroxyl groups esterified are called mono- and diglycerides respectively. The structures of these are given in Fig. 3.1. Rb R2 and R3, represent fatty acid residues. If Ri R2, and R3, are the same, i.e., only one fatty acid species is present, the triglyceride is a simple glyceride. If they are different, the glyceride is referred to as "mixed triglyceride." Natural triglycerides are mixtures of mixed triglycerides. 30 Foods: Facts and Principles H2 H2OOH O H2 O O- H2 OO* OR2 HOC2H 1 R^OO-CH R OO-CH O H2 C3 OH H2C OH H 2 O OH H2O O C R3 Glycerol- Sn-Glycerol-2- monoglyceride Sn-Glycerol Sn-Glycerol-1, 1,2-diglyceride 2,3-triglyceride Fig. 3.1 Structure of glycerides 3.5 Physical Characteristics A knowledge of the physical properties of fats is useful for evaluating the utility of a fat for a specific purpose and for determining the stage of processing. As fats are mixtures of triglycerides they do not have a sharp melting point; they have a melting range. Fats with a narrow range or sharp melting point are required for confectionery purposes, while for shortenings, fats with a broad range or gradual melting range is required. The specific heat of fats is useful in processing operations. It increases with the increasing unsaturation of fatty acids. Viscosity increases with an increase in the average chain-length of fatty acids in triglycerides and decreases with increasing unsaturation. Viscosity is a factor that must be considered when designing systems for handling fats. A knowledge of the density of fats helps determine the solid-liquid ratio in commercial fats. This could be determined more rapidly by nuclear magnetic resonande (NMR) spectroscopy. The cold test, which is a measure of the time required for an oil to develop a cloudy appearance when held in an ice bath, is useful to determine the use of the oil for salad dressing. The refractive index of fat increases with the increasing chain-length of fatty acids in the triglycerides or with increasing unsaturation. The refractive index can, therefore, be used to control the process during hydrogenation. 3.5.1 Polymorphism Polymorphism is the phenomenon in which a substance occurs in different crystalline forms, e.g., the existence of carbon in common black form or as diamond. Certain triglycerides are found to exist in polymorphic forms. The forms have different melting points. Tristearin can exist in three forms with melting points 53°, 64.2° and 71.7°C. Polymorphs also differ in X-ray diffraction patterns and infrared spectra. This is because of different modes of molecular packing in the crystal. The X-ray analysis of polymorphic crystals provides information about the packing density of the molecules and, therefore, a measure of the degree of randomness of molecular orientation in a given polymorphic form. Infrared spectroscopy gives the characteristics of the subcell packing in the crystals. Based on this information a monoacid trisaturated glyceride shows three forms—a, (5 'and p. Triglycerides containing different fatty acids with varying chain-lengths and degrees of unsaturation exhibit even greater complex polymorphic behaviour. Tempering: Newly processed fats may not be in the polymorphic form and physical state in which they are most useful. A process which permits the formation of the proper polymorphic form is known as tempering. This is done by the controlled removal of the heat of transformation during Lipids 31 crystallization. Tempering at different temperatures allows the formation of different sets of mixed crystals, melting over a wide range of temperatures. Shortening: The quality of shortening of fat depends on the incorporation of air, plasticity and consistency, and solid-liquid ratio. Polymorphs differ in their abilities to incorporate air during plasticizing (the whipping of air into fat during the crystallization stage, to give whiter, creamier, smoother and more uniform creaming). P * crystals assist in the incorporation of an abundant quantity of very small air bubbles, while P crystals result in the incorporation of a small amount of large bubbles. Air incorporation in batters is important, since the volume, texture and tenderness of baked products depend upon the size aH number of air bubbles in the batter, p' crystals are best for use in cakes, while p crystals are very unsatisfactory. Vegetable fats, partially hydrogenated in a manner that favours P' crystal formation, have good air incorporation properties. 3.6 Reactions of Fats The stability of a fat or fatty food is important to maintain a fresh taste or odour during storage and use. The stability is related to the composition of the lipid moiety and the changes it is subjected to. Fats with substantial unsaturation in the fatty acids are usually unstable. The presence of agents causing oxidation (pro-oxidants) or preventing oxidation (anti-oxidants) and the methods of storage determine their stability. Vegetable fats are usually more stable than some of the animal fats, such as lard even though the total unsaturation of the vegetable oils may be greater, because of the natural anti-oxidants usually present in them. 3,6.1 Rancidity The change that a lipid undergoes leading to an undesirable flavour and odour is known as rancidity. This is brought about in two ways: hydrolysis and oxidation. The ester linkages of lipids are hydrolyzed to yield fatty acids. Hydrolysis is catalyzed by acids, bases, enzymes or thermal effects. When a base is the hydrolyzing agent, the liberated fatty acids are converted into their salts, or soaps, and the hydrolysis is termed "saponification." The other agents release free fatty acids and the reactions brought about by them are collectively known as lipolysis, "lipolytic rancidity"or "hydrolytic rancidity." Hydrolytic rancidity occurs in raw milk and is important for the flavours of various milk products. The flavour of rancid milk is due to even numbered fatty acids from C4 to C12, and u not just due to butyric acid, as is commonly believed. Lipolysis seriously degrades the quality of cooking and frying of oils. The smoke point (temperature at which smoke is seen over the surface of heated fat) of a fat is seriously depressed due to lipolysis. For example, the smoke point of cottonseed oil decreases from 232.2°C to 160°C in the presence of 1 per cent free fatty acids. Foods fried in such oils exhibit cracked surfaces, have increased tendency for browning, and increased fat absorption. Fats, when in contact with air, react with oxygen producing products with undesirable characteristics. This is known as "oxidative rancidity." This type of rancidity is more of a problem with foods than hydrolytic rancidity. In the case of the latter, the undesirable flavour is only owing to short-chain fatty acids. In oxidative rancidity, a large number of intermediates are formed and their condensation products lead to rancidity. This type of rancidity is promoted by heat, light, ionizing radiations, catalysts and enzymes (lipoxygenases). Usually, fats with substantial unsaturation in the fatty acids are more prone to oxidative rancidity. 32 Foods: Facts and Principles Mechanism ofoxidative rancidity: The mechanism of oxidative rancidity has been extensively studied. Rancidity takes place through auto-oxidation because the rate of oxidation increases as the reaction proceeds. Oxidation takes place through a free-radical chain mechanism involving three stages: (1) initiation, formation of free radicals, (2) propagation, free-radical chain reaction, and (3) termination, formation of nonradical products. In the initiation stage, an unsaturated hydrocarbon loses a hydrogen atom to form a radical and oxygen adds on at the double bond to form a diradical: RH->R+H- O-O- 0-0" R-C-C-R -» R-C-C-R HH HH In some cases, initiation takes place by the direct incorporation of oxygen between a labile hydrogen atom to form a hydroperoxide: RH + 0 2 ->R00H The free radicals formed during the initiation reaction react with oxygen and the hydrocarbon, forming peroxy radicals, hydroperoxides and new hydrocarbon radicals R+0 2 ->R00 -ROO +RH->R00H + R-Termination occurs when two radicals interact: R.+R.->RR ROO +R00- -> ROOR + 02 R 0 + R - > R 0 R ROO +R-> ROOR 2R0 +2R00 -> 2R00R + 02 Hydroperoxides formed by the above method are readily decomposed by heat, metal catalysts or enzyme activity. 2R00H -> RO ROO +H20 The radicals formed from hydroperoxides undergo a series of reactions leading to several products, such as hydroxy acids, keto acids and aldehydes. The short-chain aldehydes and the short-chain acids derived by their oxidation are largely responsible for the undesirable flavours and odours characteristic of rancid foods. Anti-oxidants: An anti-oxidant is a substance that is added to fats or fat-containing foods to retard the oxidative breakdown of fat and thus prevent spoilage of foods. Naturally, an anti-oxidant should be fat soluble and should not contribute any objectionable flavour, odour or colour to the fat or foods in which it is used. Anti-oxidants function by combining with free radicals and thus interrupting the free-radical chain mechanism. Free-radical and anti-oxidant complexes have been isolated. Anti-oxidants, such as ascorbic acid, function by being preferentially oxidized and they afford poor protection. Tocopherols are naturally occurring anti-oxidants. For antioxidants used in foods, see Chap. 29. Lipids 33 3.6.2 Polymerization Unsaturated fatty acids in lipids undergo polymerization owing to heat, oxidation, and the presence of free radicals or polar catalysts. Heating of fats and oils, as in frying operations, can produce changes in molecular weight, colour, viscosity or refractive index due to polymerization. Polymers decrease the heat transfer efficiency of an oil and also affect the quality of products fried in oil. Under extreme conditions it can also adversely affect the nutritional quality of oils and their wholesomeness. 3.7 Phospholipids Phospholipids or phosphoglycerides differ from triglycerides in that one of the terminal hydroxyls of glycerol is esterified with phosphoric acid and the other two hydroxyls by fatty acids. The fatty acids occupying Sni, and Sn2 positions vary in chain length and in unsaturation. Acids in position 2 are usually highly unsaturated and those in position 1 can be saturated or monoenoic.The phosphoric acid in position Sn3 is,in turn, esterified to another moiety x, 2 O i R/C-O-CH O Hf -OP-OX Phospholipids In the above structure, x may be choline HO-CH2-CH2-N+(CH3)3,ethanolamine (HO-CH2-CH2-NH2), serine or inositol. Phosphoglycerides containing these components are all found in foods. The phospholipid containing choline (phosphatidylcholine) is known as "lecithin." Lecithin is a very good emulsifier. Egg yolk is rich in lecithin and is thus used in the preparation of emulsions. H 2 O O O It , f? > II + / CH 3 H2C-0 P- OCH2*N CH-C-COO CH, 1 + NH3 Proline (Pro) H2TT r/C\ /COO" 2 NH Phenylalanine (Phe) H 1 +NH3 Tryptophan (Trp) H ^ \ - ----- pCH2-C-COO" Nn +NH3 (Contd...) Proteins 37 Methionine (Met) H CH3-S- CH2- CH2- C- COO" +NH3 Polar uncharged R Group (name) Structure (pH-7) Glycine (Gly) H +l Serine (Ser) H1 HO-CH-C-COO" +NH3 H Threonine (Thr) CH-CH-C-COCT OH + NH3 Cysteine (Cys) H HS-CH-C-COO" +NH3 Tyrosine (Thr) H 1 HQX~^~ C% C-COO" +NH3 Asparagine (Asn) \2 1 C-CH2-C-COO" 0 +NH3 Glutamine (Gin) C- CHr CHrC-COO" // | (Contd...) 38 Foods: Facts and Principles Negatively charged R groups (name) Structure (pH-7) Aspartic Acid (Asp) -O H _ C-CH,-C-COO # x1 + 0 NH3 Glutamic Acid (Glu) "K\ of the carboxyl group, cation and zwitterion will exist in equal concentration. Similarly, at the pH equal to pK2, of the amino group, zwitterion and anion will be present in equal concentration. The pH at which the amino acid exists as the zwitterion is called the isoelectric pH (pi) and is the mean of p^and pK2 i.e., P The aromatic amino acids—tryptophan, tyrosine and phenylalanine—absorb light significantly in the ultraviolet range. Since most proteins contain tyrosine, the spectroscopic measurement of light absorption at 280nm is a convenient and rapid method for estimating the protein concentration of a solution. Amino acids undergo the usual reactions of carboxyl and amino groups. Thus, the a-carboxyl group participates in reactions leading to the formation of amides, esters and acylhalides. Similarly, the amino group reacts with ninhydrin, dinitrofluorobenzene, phenylisothiocyanate, dansyl chloride, nitrous acid, etc. In addition, amino acids show reactions characteristic of the side-chain hydroxyl, sulphydryl, amino, carboxyl, indoyl, imidazoyl and guanidino groups. 4.2 Peptides When the a -amino group of one amino acid reacts with the a -carboxyl group of another, a peptide bond is formed with the elimination of a molecule of water. C0 NH3 NHL 3 N °" RrC-COO \ +R2-C-COO H I I H H When a few (2-10) amino acids are joined together by peptide bonds the compound is called an oligopeptide. With more than ten amino acid residues the compound is termed a polypeptide. When 40 Foods: Facts and Principles the polypeptide contains about 100 amino acid units, it is called a protein. The general structure of a polypeptide can be represented thus: Rv H O H H H HR The linear peptide chain has two terminal residues, one terminal residue possessing a free amino group, called the N-terminal residue, and the other a free carboxyl group, called the C-terminal residue. The terminal residues are usually free to ionize. Peptides undergo the same k ind of chemical reactions as those given by constituent amino acids. Peptides give a colour reaction that is not given by free amino acids, the "biuret" reaction. Treatment of a peptide or protein with Cu 2+ in alkaline solution yields a purple colour, which has an absorption at 540nm. This property is used in the estimation of peptides and proteins. Most peptides are the partial hydrolytic products of proteins. However, a few peptides are of metabolic importance and are found free in nature. Carnosine (P -alanyl-L-histidine) and anserine (|3-alanyl-L-methylhistidine), are peptides found in muscle. Glutathione (y -glutamyl-cysteinyl-glycine) is found in mammalian erythrocytes and functions in oxidative metabolism. The peptide bonds due to p- and y -carboxyls in the above peptides are not found in proteins. Other natural peptides function as antibiotics and hormones. Oxytocin and vasopressin are examples of peptide hormones. 4,3 Proteins Classification: An infinite number of proteins could be synthesized from the twenty odd natural amino acids. As proteins perform a wide variety of biological functions, they are classified in a number of ways. They are classified on the basis of their function as catalytic proteins, contractile proteins, structural proteins, etc. On a compositional basis, proteins can be classified into two general classes — simple proteins and conjugated proteins. Simple proteins yield only amino acids on hydrolytic degradation, while conjugated proteins yield, in addition to amino acids, an organic or inorganic moiety called the prosthetic group. The simple proteins are further subclassified on the basis of their solubility as shown in Table 4.2. Most enzyme and hormone proteins belong to either the albumin or globulin class. Plantseed proteins are mostly glutelins. The prosthetic groups of conjugated proteins are carbohydrates, lipids, nucleic acids, metal ions or phosphates. They are bound to the protein by linkages other than salt linkag es. The protein component of a conjugated protein is stabilized by combination with the prosthetic group. Lipoproteins are proteins complexed with lipids and are found in cells and the blood stream. The lipids are very firmly held to the proteins and canno t be easily removed. The lipids in lipoproteins are triglycerides, phospholipids, cholesterol or derivatives of cholesterol. Lipoproteins serve as transporters of lipids in blood. Based on density, lipoproteins are grouped into three groups—high-density, low-density and very low-density lipoproteins. Glycoproteins are proteins whose prosthetic groups are heterosaccharides containing hexosamine, galactose, mannose, fucose and sialic acid. A covalent bond joins the protein to the Proteins 41 Table 4.2 Classification of simple proteins Solubility in JName Water Salt Solution Acid/Alkali Alcohol 80% Albumins Soluble Soluble — — Milk, egg, plant and animal cells Globulins — Soluble — — Milk, eggs, meat, plant cells, particularly in seed proteins Glutelins — — Soluble — Cereal grains and related plant materials Prolamines — — — Soluble Cereal grains and related plant materials. Histones Soluble — Soluble — Glandular tissues, pancreas, thymus, fish Protamines Soluble — Soluble — Fish sperm Sclero-proteins — — — — (albuminoids) Meat heteropolysaccharide by either O-glycosidic (with serine or threonine) or N-glyosidic bonds (with asparagine). Glycoproteins are found in mucous secretions of mammals and they function as lubricating agents. Egg-white contains ovomucoid which cannot be coagulated by heat and is a glycoprotein. Certain fractions of pulse proteins are glycoproteins. Metalloproteins are complexes of proteins and heavy metals. In most metalloproteins the metal is loosely bound and can be easily removed. However, in some proteins such as haemoglobin and myoglobin, the metal iron is firmly bound to the prosthetic group. Liver and spleen contain the metalloproteins ferritin and haemosiderin with about 20 per cent iron content. These are storage forms of iron in animals which release iron from the proteins when required. Conalbumin from egg can form a complex with iron and it also combines with copper and zinc. Nucleoproteins are complexes of proteins and nucleic acids. Nucleic acids readily combine with proteins to form the complexes. Phosphoproteins are proteins conjugated with inorganic phosphate. The most widely known phosphoproteins are the milk protein casein and the enzyme pepsin. 4.3.1 Protein Structure The diverse biological functions of proteins are a result of their structure. The structure of a protein is normally considered under four levels—primary structure, secondary structure, tertiary structure and quaternary structure. Primary structure: The nature of amino acids and their linear sequence in the polypeptide chain is referred to as the primary structure. The determination of the primary structure, which was once a formidable job, has now become easy due to the modern analytical tools available for amino-acid analysis. The unique sequence of amino acids is responsible for many of the fundamental properties of the proteins, e.g., the replacement of one amino acid residue in the p -chains of haemoglobin, which 42 Foods: Facts and Principles contains 574 amino acid residues, can bring about profound changes in its biological properties. The sequence of amino acids also determines, to a large extent, the secondary and tertiary structures of proteins. Secondary structure: An extended peptide chain is not stable and it folds into itself. The three-dimensional manner in which relatively close members of the protein chain are arranged is referred to as the secondary structure. The native structure of a protein is that structure which possesses the lowest feasible free energy. Therefore, the structure of a protein is not random but somewhat ordered. The secondary structure of the protein depends upon the structural characteristics of the peptide bond which repeats itself along the chain. X-ray studies indicate that the peptide bond is slightly shorter than other single C-N bonds. This indicates that the peptide bond has some characteristics of a double bond. Thus, groups adjacent to the peptide bond cannot rotate freely. The rigidity of the peptide bond holds the six atoms shown in the diagram in a single plane. The amino (-NH-) group does not ionize between pH 0 and 14 due to the double bond properties of the peptide bond. In addition, the R-groups of amino acid residues, because of stearic hindrance, force oxygen and hydrogen of the peptide bond to exist in a trans configuration. Therefore, the backbone of peptides and proteins has free rotation in two of the three bonds between amino acids as indicated below. When the restrictions of the peptide bond are superimposed on a poly-amino acid chain of a globular protein, a right-handed coil, the a -helix, appears to be one of the most ordered and stable structures feasible (Fig. 4.1). The a-helix contains 3.6 amino acid residues per turn of the protein backbone, with the R-groups of the aminoacids extending outward from the axis of the helical structure. Hydrogen bonding occurs between the nitrogen of one peptide bond and the oxygen of another peptide bond in the four residues along the protein backbone structure. This arrangement enhances the stability of the structure. Another secondary structure found in many fibrous proteins is the P -pleated sheet configuration. In this configuration, the peptide backbone forms a zigzag pattern, with the R-groups of the amino acids extending above and below the peptide chain. Since all peptide bonds are available for hydrogen bonding, this configuration allows maximum cross-linking between adjacent polypeptide chains and thus ensures good stability. When the polypeptide chains run in the same direction, parallel-pleated sheets, and when they run in the opposite direction, antiparallel pleated sheets, are possible (Fig. 4.2). Silk and insect fibres are the best examples of the p -sheet structure. Most proteins do not have the regular secondary structure due to some factors which prevent the formation of long uninterrupted regions of a -helix or P-pleated sheets. For example, if a proline residue is present in the polypeptide chain, the a -helix is interrupted because the amide nitrogen of proline, when involved in peptide bond formation, does not possess an attached hydrogen bond. In addition, hydrogen bonding in both the pleated sheet and a-helical structures may be disturbed by Proteins 43 Fig. 4.1 Right-handed a-helix N N N =N (a) Antiparallel Fig. 4.2 Antiparallel and parallel p -pleated sheets 44 Foods: Facts and Principles unfavourable side-chain interactions, such as the presence of two bulky side-chains next to each other or the electrostatic repulsion produced by two similar groups. Another type of secondary structure of fibrous proteins is the collagen helix. Collagen, found in skin, tendons and numerous other parts of the body, accounts for one-third of the total body protein. Collagen contains one-third glycine and one-fourth proline or hydroxyproline residues. The rigid R-groups, and the lack of hydrogen bonds by peptide linkages involving proline and hydroxyproline, force the collagen polypeptide chain into an odd kink-type helix. Peptide bonds composed of glycine form interchain hydrogen bonds with two other collagen polypeptide chains and this results in a stable triple helix. This triple helical structure is called "Tropocollagen"(Fig.4.3) Fig. 4.3 Triple helix of Collagen Tertiary structure: The tertiary structure of proteins defines a specific three-dimensional configuration. This involves the folding of regular units of the secondary structure as well as structuring of areas of the peptide chain devoid of secondary structure. The folded portions are held together by hydrogen bonds formed between R-groups, by electrostatic interaction between chains possessing oppositely charged groups such as those of lysine, arginine, glutamic and aspartic acids, hydrophobic interaction between nonpolar legions and covalent disulphide linkages. In the formation of the tertiary structure, all the polar groups are on the surface of the molecule and the interior consists almost entirely of nonpolar hydrophobic residues, such as those of leucine, valine, methionine and phenylalanine. The presence of polar R-groups on the surface of proteins usually accounts for their solubility in aqueous solutions Quarternary structure: The three levels of structural organization discussed so far apply to all proteins with a single polypeptide chain. When a protein contains two or more polypeptide chains (subunits), the structure formed when individual polypeptide chains interact to form the native protein molecule, is referred to as the quaternary structure. The combination of different protein subunits has a number of advantages, such as in metabolic regulation. The bonding mechanisms holding protein chains together are generally the same as those involved in the tertiary structure, apart from the fact that disulphide bonds do not assist in maintaining the quaternary structure of proteins. 4.3.2 Properties of Proteins As already stated, proteins are molecules whose hydrophilic R-groups are on the exterior of the molecule, while their hydrophobic nonpolar R-groups are generally located in the interior. The presence of hydrophilic R-groups on the surface of proteins causes them to behave somewhat like amino acids. However, the behaviour of proteins is much more complex due to the multiplicity of hydrophilic R-groups. Proteins 45 Proteins behave as electrolytes and they conform to the same physical and chemical principles of electrolytes. The amino acid compositions of individual proteins greatly vary and thus their net charge varies. If the protein contains a high content of acidic groups (aspartic and glutamic) its isoelectric pH is low; if basic amino acids (arginine and lysine) are more, the protein has a high isoelectric point. The variation in the ionic properties of proteins leads to several methods for the fractionation of proteins from a biological system. Two methods have been successfully employed in the isolation and purification of proteins based on their ionic properties. They are electrophoresis, particularly zone electrophoresis on polyacrylamide gel, and ion-exchange chromatography. Electrical charges also influence protein solubility. Proteins in solution show changes in solubility as a function of pH, ionic strength, temperature and dielectric properties of the solvent. Using these variables protein mixtures can be resolved. Proteins can be hydrolyzed with acid, alkali or enzymes. Complete hydrolysis of proteins can be brought about by heating the proteins with 6 M HC1 for 8-10 hours at 120°C. Acid hydrolysis avoids recemization of amino acids. However, it results in the destruction of tryptophan. During acid hydrolysis, asparagine and glutamine are hydrolyzed to aspartic and glutamine acids, and sulphur-containing hydroxy amino acids undergo varying degrees of oxidation. Alkaline hydrolysis with 2 M sodium or barium hydroxide results in substantial recemisation of amino acids. The amino acids arginine and cysteine, and a portion of lysine, are destroyed, although tryptophan is retained. A complete hydrolysis of proteins can be brought about by enzymes without any destruction of amino acids. This type of hydrolysis takes place in the body during digestion of proteins. Micro-organisms cause deterioration of protein foods. Growth of spoilage organisms occurs under unsatisfactory conditions when the food is not properly processed. Microbial proteases bring about decarboxylation and deamination resulting in undesirable changes in flavour and texture, and production of offensive odours and toxins. These changes are referred to as putrefaction. 4.3.3 Denaturation The ordered three-dimensional structure of proteins is very delicate. It is subject to alterations by mild agents without any breaking of peptide bonds. The loss of native conformation brings about changes in specific properties characterizing the identity of proteins and this is known as "denaturation." Denaturation brings about many changes in a protein. It makes the peptide bonds of the protein more readily available for hydrolysis by proteolytic enzyme, solubility is decreased and biological properties such as catalytic, hormonal, etc., are lost. The crystallization of protein is no longer possible, and viscosity and optical rotation increase. The increase in viscosity suggests the unfolding of the molecule resulting in more asymmetry. This would expose more hydrophobic residues resulting in the decreased solubility of proteins. Denaturation is the result of the modification of the secondary, tertiary or quaternary structure of the protein molecule, excluding the breakage of covalent bonds. Denaturation is therefore a process by which hydrogen bonds, hydrophobic interactions and salt linkages are broken and the protein completely unfolds and assumes a random coil structure, in which state the protein molecules readily form aggregates (coagulation). Denaturation is usually reversible if drastic methods of denaturation are not used and if the molecular weight of the native protein is very large. However, reversible denaturation (renaturation) is also observed in some smaller proteins when milder methods of denaturation are used. 46 Foods: Facts and Principles Both physical and chemical agents bring about denaturation. Heat is the most important physical agent. For every 10°C rise in temperature the increase in denaturation rate is 600-fold. The effect of denaturation can be reduced by working at a reduced temperature. The rate of heat denaturation is affected by the water content of a protein, ionic strength, pH, and types of ions present in the solution. Layering of proteins at an interface can result in denaturation. Therefore, the creation of interfaces, such as those of foams, are to be avoided to preserve the native properties of proteins. Stirring, shaking, high pressure, and ultraviolet radiations bring about protein denaturation. Amongst the chemical agents, the pH of the medium has a profound effect on denaturation of proteins. Most proteins are stable within a fairly narrow pH range and exposure to pH values outside this range causes denaturation. A 6-8 M concentration of urea, and guanidine hydrochloride, that tends to break hydrogen bonds, also cause denaturation of proteins. Synthetic detergents, such as sodium dodecyl sulphate (SDS), are the most effective among the denaturing agents known. The phenomenon of denaturation is of great importance in food processing. When most foods are prepared for eating, they have been heat coagulated or denatured. In some cases, however, precautions have to be taken to prevent coagulation as in the processing of milk by pasteurization, evaporation and spray drying where it is desirable to retain the natural properties of proteins. 4.3.4 Protein Gels Dry proteins have the ability to absorb water. A few proteins can form gels capable of immobilizing (making nonflowable) water equal to nearly ten times the weight of the hydrated proteins. Proteins which form gels have structures with high degree of asymmetry. These long proteinaceous fibres form a three-dimensional matrix, primarily by the establishment of inter-protein hydrogen bonds and this cross-linked structure is sufficiently well developed to hold water in an immobilized state. Ionized functional groups on proteins also aid in immobilizing water. If the attractive forces on proteins are increased, e.g., by changing the pH to a value closer to the isoelectric point of the proteins, the gel tends to shrink. The shrinkage would expel some of the immobilized water (syneresis). Gelatin and casein coagulated by the action of enzyme rennin are good example of gel forming proteins. They are discussed in the chapters on meat and milk, respectively. 4.4 Food Proteins Both plants and animals require proteins for growth, survival and propagation of the species. Proteins are present in many of our foods in varying amounts. Most proteins have to be processed or modified prior to use by human beings. Some major food proteins are considered briefly in this section. For more details about proteins from different sources, the reader has to refer to the chapter dealing with the particular food. Animal proteins: Meat is an important source of protein from animal sources. Meat is the edible muscle of cattle, sheep and swine. These are designated as "red meat" because the colour of the beef, lamb mutton and pork is light or dark red due to the presence of the respiratory pigment myoglobin in them. Muscle from adult mammalian sources, stripped of all external fat, contains about 18-20 per cent protein on a wet basis. They are categorized on the basis of their origin and solubility as myofibrillar, sarcoplasmic and stroma (connective tissue) proteins which range from 49-55, 30-34 and 10-17 per cent, respectively, of the total amount of protein. Milk is an excellent source of protein in our diet. Cow milk contains about 3.5 per cent protein. Milk proteins are generally divided into two classes—casein and whey proteins. Casein which is Proteins 47 insoluble in water is a heterogenous group of phosphoproteins and accounts for 80 per cent of total milk protein. Whey proteins, making up the other 20 per cent, are the soluble proteins of milk. Egg contains 13-14 per cent protein, about 2/3 of which is present in egg-white and the rest in egg-yolk. Egg-white consists of a number of proteins which are readily denatured and coagulated. Yolk is a mixture of lipoproteins and phosphoproteins. Fish contains between 40 to 60 per cent edible flesh. The protein content of fish varies from 10 to 21 per cent. Fish muscle is similar to mammalian skeletal muscle with respect to structure and function but, unlike the latter, is easily damaged. The pigmented reddish brown muscle of fish contains enzymes which, following harvest, cause changes responsible for much of the instability of fish proteins. Vegetable proteins: Fresh vegetables are not good sources of proteins. Many contain less than 1 per cent protein. Potatoes and green beans contain about 2 per cent and fresh peas about 6.5 per cent protein. Proteins from potatoes are considered to be of good quality because they are relatively high in the levels of lysine and tryptophan. Cereal grains contain proteins ranging from 6 to 20 per cent. The protein content of rice is 7-9 per cent while that of wheat is 12-15 per cent. Proteins are found in various morphological tissues of the grains like embryo, germ, bran or seed coat and the endosperm. The germ proteins are mainly globulins or albumins and several enzymes are present in them. Bran is poorly digested by man and bran proteins are difficult to separate. Bran is mostly used as animal feed. The endosperm proteins act as structural components and also as a food reserve for the growing seedling. The endosperm proteins of wheat are mostly prolamines, gliadins and glutelins. Rice contains high levels of glutelins and low prolamine. Cereal proteins are generally of relatively poor nutritional quality. Seeds contain protein in excess of 15 per cent. Pulses (legumes) contain, on an average, more than 20 per cent proteins. Proteins of seeds are mostly concentrated in aleurone grains which are subcellular granules of the cotyledon cells. Seed proteins function as structural elements of cell walls and various membranes and also as food reserves. The proteins of most seeds are globular. Owing to a decrease in the availability of meat proteins, seed proteins are gaining importance. Newer techniques have been developed to produce textured or "shaped" proteins from seeds. Nutritionally, seed proteins contain less of lysine and are also deficient in methionine and threonine. Seed proteins contain some anti-nutritional factors. They should be subjected to proper heat treatment to destroy or inactivate anti-nutritional factors and maximize nutritional quality.( see sec 17.3) 4.5 Non-traditional Proteins People in many parts of the world are experiencing varying degrees of protein malnutrition, largely because of rapid increase in population. Therefore, there is a need to increase the production of proteins from traditional sources and to develop proteins from non-traditional sources. Some success has been achieved in the production of proteins from some unconventional sources. Micro-organisms grow rapidly, their yields are high, and their growing conditions can be controlled. Therefore, microbes have been used to obtain food proteins (single cell proteins). Two species of yeasts, Candida utilis (torula yeast) and Saccharomyces carlsbergensis (brewer's yeast), have been used for human food. Torula yeast grows well on sulphite waste liquor (waste product from paper industry) and wood hydrolysates, by utilizing pentoses as carbon source. Brewer's yeast can be collected after beer fermentation. These yeasts contain approximately 50 per cent of proteins on a dry-weight basis. They are, however, deficient in methionine but by adding 0.3 48 Foods: Facts and Principles per cent methionine their biological value can be increased to over 90. One disadvantage of food yeasts is their nucleic acid content. Nucleic acid metabolic products are relatively insoluble and may lead to the formation of kidney stones, or aggravate arthritic or gouty conditions. Strains of the yeast Candida lipolytica can grow on petroleum. The carbon and energy for the growth of the organism is provided by alkanes (straight- chain hydrocarbon molecules of petroleum). A commercial product named toprina has been prepared by this method. Toprina is toxicologically safe. However, the product has not been a success due to the rising cost of petroleum. The bacterium Methylophilus methylotrophus can oxidize and use methane (or methanol) as a source of carbon and energy. A product named pruteen is being produced using this method. Two genera of algae—Chlorella (green algae) and Spirulina (blue-gree algae, now classified as blue-green bacteria) - when grown un