Functional Foods: Principles and Technology (Woodhead Publishing Series in Food Science, Technology and Nutrition)

Functional foods: principles and technology Dr Mingruo Guo Professor Nutrition & Food Sciences Department University of Vermont Burlington, Vermont

CRC Press Boca Raton Boston New York Washington, DC

England

New Delhi

Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, England www.woodheadpublishing.com Woodhead Publishing India Pvt Ltd, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA Published 2009, Woodhead Publishing Limited and CRC Press LLC © 2009, Woodhead Publishing Limited The author has asserted his moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the author and the publishers cannot assume responsibility for the validity of all materials. Neither the author nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Woodhead Publishing ISBN 978-1-84569-592-7 (book) Woodhead Publishing ISBN 978-1-84569-607-8 (e-book) CRC Press ISBN 978-1-4398-0897-9 CRC Press order number: N10083 Printed in the United States of America

PREFACE The subject of functional foods is one of the hottest topics in food science and nutrition. This trend will continue for a long time. I have been teaching Functional Foods-Principles and Technology at University of Vermont since 2000. The course is getting more and more popular on the campus. Students in my classroom keep asking to have a textbook for study and for future reference. Although there are a number of books on functional foods available on the market, none of them are written for classrooms. In 2005, I decided to take a one-half year sabbatical leave to write a textbook for my class (I now realize that six months was not sufficient to complete this task). The structure of the book is based on my lecture notes. This textbook consists of nine chapters and laboratory manuals as an appendix. Chapter 1 describes the definition, history, and global aspects of functional foods. Chapters 2, 3, 4, 5 and 6 deal with some of the foundations of functional foodsantioxidants, dietary fiber, pre- and probiotics, functional fatty acids, and vitamins and minerals, respectively. Chapter 7 discusses the chemistry and health benefits of soybeans and soy products. Chapter 8 deals with aspects of biochemistry and formulation of sports drinks. The last chapter (9) discusses human milk chemistry and infant formula formulation. I sincerely thank my research associates Dr. Sumagala Gokavi (Chapters 2, 3, 4, and 7), Dr. Mohamed Alam (Chapter 5 and 6), Dr. Frank Lee (Chapter 8), Ms. Beth Rice (Chapter 8), and my friend Dr. Gregory Hendricks of the Medical School of University of Masssachusetts (Chapter 9) for their help and their expertise to get my lecture notes together. I would also like to thank my graduate students and the undergraduate students who attended my functional foods class during the years for their valuable comments and feedback about my lectures on functional foods. Finally, I am grateful to Randy Gerstmyer, the President of CTI Publications, for his interest in this book and his patience while working with me on this exciting project. Mingruo Guo Burlington, Vermont

While the recommendations in this publication are based on scientific study and industry experience, references to basic principles, operating procedures and methods, types of instruments and equipment, and food formulas, are not to be construed as a guarantee that they are sufficient to prevent damage, spoilage, loss, accidents or injuries, resulting from use of this information. Furthermore, the study and use of this publication by any person or company is not to be considered as assurance that that person or company is proficient in the operations and procedures discussed in this publication. The use of the statements, recommendations, or suggestions contained, herein, is not to be considered as creating any responsibility for damage, spoilage, loss accident or injury, resulting from such use.

DEDICATION I dedicate this work to Ying, Fei, and Mike for their love, support and encouragement, and to my late mother who played a critical role in my education.

This Book Belongs To:

CONTENTS Chapter One – Introduction __________________________ 1 Chapter Two – Antioxidants __________________________ 9 Chapter Three – Dietary Fiber ______________________ 63 Chapter Four – Prebiotics & Probiotics ______________ 113 Chapter Five – Lipids ______________________________ 161 Chapter Six – Vitamins ____________________________ 197 Chapter Seven – Soy _______________________________ 237 Chapter Eight – Sports Drinks _____________________ 279 Chapter Nine – Human Milk _______________________ 299 Appendix – Laboratory Manual _____________________ 339 Laboratory 1 - Iced Tea ___________________________ 339 Laboratory 2 - Symbiotic Yogurt __________________ 342 Laboratory 3 - Yogurt Beverage ___________________ 342 Laboratory 4 - Sports Drink ______________________ 347 Laboratory 5 - Soy Milk and Tofu _________________ 350 Index _____________________________________________ 353

Chapter 1 INTRODUCTION Definition, History and Market A food may have three functions: (1) providing energy in the form of carbohydrates, proteins and/or lipids, and basic nutrition; (2) giving us pleasure, i.e., enjoyable aroma, color, and taste; (3) having health benefits. A functional food may be similar in appearance to, or is a conventional food, is consumed as a part of normal diet, and has physiological benefits and/or reduces the risk of chronic disease beyond basic nutrition. Functional foods are also called “nutraceuticals”, “medical foods”, or “designer foods” in the literature. The terminology, functional foods, for these beneficial foods is preferred due to the self descriptive nature of the term. Some examples are iodized salt, vitamin A and D fortified milk, yogurt, folic acid enriched bread, tomatoes, broccoli, soy products, blueberries, cranberries, garlic, wheat bran, and oats. Functional foods can be the foods which are natural, fortified, enriched, or contain functional ingredients. The term functional food was coined by Japanese scientists in the 1970’s and was introduced to the European scientific community in the 1980’s. Functional foods did not receive much notice in the U.S. until the 1990’s, where they first gained popularity in the west coast. However, the roots could be traced back to the Chinese who used foods as medicine for thousands of years. The market sale value for functional foods was over $10 billion in 2005 in the U.S. according to a strict definition. In fact, the functional foods market will reach about $36 billion in 2006, and it will jump up to $60 billion in 2009 (NMI, 2005). Based on my personal calculations, current functional foods market value will exceed $100 billion if a general definition for functional foods is applied. It is increasing with a growth rate of 10% annually. The global functional foods market will continue to be a dynamic and growing segment of the food industry. Functional foods are considered to be the foods for the next century.

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Awareness of Functional Foods The good news is that the concept of functional foods is becoming more widespread. More than 90% of Americans could name a functional food and its associated benefits in 2005 up from 77% in 1998, and 84% in 2002 (IFIC, 2005). The vast majority of Americans believe foods have health benefits beyond basic nutrition. Through education and media exposure, the benefits of functional foods are more widely understood by the population. A survey to identify which functional foods’ benefits were recognized by the majority of the population revealed that while some foods were clearly identified with their benefits, others were not. An example of these results is presented in Figure 1.1. FIGURE 1.1 — Awareness of Functional Foods and Disease Association Calcium for the promotion of bone health Fiber for maintaining a healthy digestive system Vitamin D for the promotion of bone health Whole grains for reducing risk of heart disease Probiotics for maintaining a healthy digestive system Soy for reducing risk of heart disease Plant sterols for reducing risk of heart disease

93% 92% 88% 83% 49% 41% 30%

(Adapted from IFIC, 2005)

Figure 1.1 indicates that while more than 90% of respondents were aware of the association of calcium and bone health, less than 50% were aware of the benefits of probiotics (the living organisms that can be found in yogurt) supporting a healthy balance of microflora in the human digestive tract. These will be addressed in greater detail later in the course. Only about 40% of respondents were familiar with or associated soy protein with reducing the risk of heart disease. Despite the low level of awareness of certain functional food benefits, the overall awareness is growing, which explains the increase in consumption of functional foods. Consumers want to learn more about the health benefits offered by foods that have health benefits beyond nutrition. Figure 1.2 shows that awareness for health benefits of some functional foods are gaining ground. The awareness comes from several sources such as the government, health care providers, personal health concerns, and friends and family. The source of information about health and nutrition is primarily from the media accounting for 72%, medical sources ranking second with 44%, and 20% obtained from friends and family or self. Diet and health books account for only 13%. With the growing awareness of these benefits, the food industry has shown an interest in meeting the growing demand for functional foods. What foods will people want to be fortified with these functional

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ingredients? The foods we consume everyday such as juices and milk are the answers. Health officials in the government and in hospitals also are interested in finding ways to deliver more of these benefits to the population. Some examples of government intervention in delivering functional food were a move to iodize salt and to add fluoride to public drinking water. Research has been done on what food sources would be most acceptable to the population for the delivery of antioxidants (often found in less popular foods such as fruits and green vegetables). A large majority of the people would find fruit juice fortified with antioxidants appealing while only about 1/3 would like it in candy, indicating more Americans are interested in natural and functional foods. FIGURE 1.2 — Top Five Sources of Information About Health and Functional Foods Media (Internet, magazines, TV, newspapers, newsletters) Medical sources (Physicians, nutritionists, dietitians, nurse/PA) Friends/family/self Diet/health books Researchers/scientists

72% 44% 20% 13% 4%

(Adapted from IFIC, 2005).

Evolution of Health Care and Functional Foods This increasing interest in functional foods represents a paradigm shift from eliminating “bad” to increasing the “good” components that one consumes. It is a widely held belief that most people have control over their health and a large part of that is controlling their diet. In a way our method of ensuring health and long life has come full circle (Figure 1.3). One explanation of this is that we have not had many large infectious disease outbreaks. Therefore, most of the population is more concerned with non-infectious diseases; obesity, diabetes, heart disease, cancer, etc. The diseases that are commonly associated with what we eat are heart disease, diabetes, high blood pressure (hypertension), dental diseases, gastrointestinal disease, anemia, and obesity (65% of U.S. residents are overweight, and the instance of obesity is 25% of the population). The life expectancy in the U.S. is increasing, and the older population is increasing with it. Currently 12% of the population is over 65, by 2030 it is expected that 20% of the population will be over 65. The key to maintaining good health is a healthy balanced nutritious diet, especially when health care comes at such a great financial burden for the U.S. population.

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FIGURE 1.3 — Evolution of Mankind's Health Care

Health Claims Approved by FDA The Nutrition Label Education Act (NLEA) allows certain claims to be made by food manufacturers. This is another advantage for functional foods development and manufacturing. The list of approved claims (claims adequately backed by scientific research) includes: Calcium and osteoporosis; Dietary lipids and cancer; Dietary saturated fat and cholesterol and risk of coronary heart disease (CHD); Sodium and hypertension; Fiber containing grains, fruits and vegetables and cancer; Fruits and vegetables, and cancer; Fruits, vegetables, and grain products and risk of CHD; Noncarcinogenic carbohydrate sweeteners and dental caries; Folic acid and neural tube defects; Soluble fiber from certain foods and risk of CHD; Soy protein and cardiovascular disease; Plant sterol/stanol ester and CHD.

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Human Body System and Functional Foods The human body is an open system. It is influenced by what one encounters, and what one consumes. The human body is exposed to toxins, viruses and bacteria, as well as hostile environments (heat, cold, air, UV rays, radiation, etc.). We are protected from the environment by our defense systems: 1) skin and hairs, 2) immune systems, 3) microfloral systems, and 4) antioxidative mechanisms. We are what we eat. Food and diet may affect all of the defense mechanisms (Figure 1.4). We consume tons of food in our lifetime, with nutrients and functional components, but they also contain pathogens, toxins, and antigens. As seen in Figure 1.4, the foods we eat not only provide energy and nutrients, but have an impact on our health. There are around 200-400 different types of microbes in the human GI (gastrointestinal) tract (there are more than 1000 species of microbes in the colon reported by a study published in June 2, 2006 issue of Science).

FIGURE 1.4 — The Relationship of Human Health & Diet

They number 10,000,000,000,000 (1013) per gram of content in the colon, 106 in the stomach, and 107 in the upper GI tract. Maintaining a healthy balance is important to maintaining good health. Diet can either positively or negatively affect this balance. Therefore, people should eat functional foods and a balanced diet. Consuming 25-30 grams of fiber a day and probiotics containing foods will help to maintain the

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healthy level of 70% healthy microflora in the colon. If the number drops below 50% problems will develop, as minor as diarrhea, or may weaken the defense system leading to serious health problems. Therefore, it is recommended that you put functional foods to work immediately. Here are the selected foods you should consume weekly. Tomatoes (lycopene); Spinach (folic acid); Broccoli (fiber, antioxidants, vitamins, sulfur compounds); Nuts (Vitamin E); Oats (soluble fiber/prebiotics); Yogurt (probiotics); Pink color fish like salmon (omega-3 fatty acids); Berries such as blueberries (antioxidants); Garlic (antioxidants); Green Tea (antioxidants); Soy foods (isoflavones). Syllabus This book is designed for the students majoring in nutrition and food sciences. It may also be used for students in nursing, medical, and other health related fields. Students will be presented with definitions and concepts pertaining to different categories of functional foods. They will learn the importance of chemical structures and properties of nutrients and functional components as well as the non-nutritive functions of several different foods in these categories. Students will also learn the laboratory techniques needed to create their own functional foods. This textbook consists of nine chapters and five laboratory exercises. Introduction: Students will learn the definition of Functional Foods. They will explore both the industry and the consumer roles involved in this growing field. Antioxidants: Students will learn the chemical makeup of free radicals, antioxidants and biochemical functions of antioxidants. Foods explored in this unit will be cranberries, tomatoes, garlic, and different iced teas. The students will learn the chemical composition of these foods, and have the opportunity to sample them. The first lab of the semester will be part of this unit. The students will have the opportunity to make their own functional iced teas.

INTRODUCTION

7

Dietary Fiber: Students will learn about soluble and insoluble fiber, resistant starch, and how important these food components are to human health. The biochemical functions of dietary fiber will be explored, and oats and oat products will be the main example used in the classroom. Pre- and Probiotics: Students will learn the definition of both preand probiotics, and their physiological functions. They will learn how to develop prebiotics and probiotics, pre- and probiotics will be used together as symbiotics. The second and the third labs of the semester will be part of this unit. The students will create their own symbiotic yogurt and beverage. Lipids and Their Health Benefits: Students will learn the structure and function of essential fatty acids. The chemistry and health benefits of w-3 fatty acids, phytosterols, and CLA will be discussed. Olive oil and fish oil will be used as an example of a functional food product bearing essential fatty acids. Vitamins and Minerals: In this chapter, the chemistry, functions, and sources of functional vitamins and minerals will be discussed. Proposed functional claims are also discussed. Soy Products and Their Health Benefits: Students will learn the history of soy products around the world as well as the health benefits that soy foods have contributed to the American diet. The chemistry and biological functions of isoflavones will be discussed. Tofu, tempeh, soy milk, and other soy products will be discussed in this unit. The fourth lab of the semester will be part of this unit. The students will make their own soymilk and tofu. Sports Drinks: In this unit students will learn principles of sports drinks formulation. Electrolytes and carbohydrates and their functions will be a large part of the discussion. The last lab of the semester will be conducted during this unit, at which time the students will have an opportunity to formulate and make their own sports drink like Gatorade. Human Milk and Infant Formula: Students will learn the chemistry and biological properties of human milk and principles and the ingredients and formulation techniques of infant formula, and all aspects of the product that make it a functional food. Students will learn recent progress in infant formula formulation.

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Laboratory Experience: This course will include 5 short laboratory exercises. Laboratory sessions include iced tea formulation, symbiotic yogurt and symbiotic beverage making, soy milk and tofu preparation, and sports drinks formulation. References IFIC (International Food Information Council). 2005. Quantitative Research on Functional Foods. NMI (Natural Marketing Institute). 2005. Health and Wellness Trends Database.

Chapter 2 ANTIOXIDANTS AND ANTIOXIDANT RICH FOODS Oxidation is one of the metabolic reactions in the body and in foodstuffs essential for the survival of cells. Normal metabolism is dependent on oxygen, a free radical. Through evolution, oxygen is thought of as the terminal electron acceptor for respiration. The dependence on oxygen for normal metabolism results in the production of other oxygen-derived free radical species, such as superoxide or hydroxyl radicals, formed during metabolism, energy production in the body or by ionizing radiation. These oxygen-derived free radical species are stronger oxidants and are, therefore, dangerous which cause oxidative damage leading to cell and tissue injury. These free radicals are involved in both human health and disease. Free radicals are atoms or molecules having unpaired electrons. The unpaired, or odd, electron is highly reactive as it seeks to pair with another free electron. Free radicals are involved in enzyme-catalysed reactions, electron transport in mitochondria, signal transduction and gene expression, activation of nuclear transcription factors, oxidative damage to molecules, cells and tissues, antimicrobial action of neutrophils and macrophages, aging and disease. When an excess of free radicals is formed, they can overwhelm protective enzymes such as superoxide dismutase, catalase and peroxidase and cause destructive and lethal cellular effects (e.g., apoptosis) by oxidizing membrane lipids, cellular proteins, DNA and enzymes, thus shutting down cellular respiration. Oxidation in foods is one of the major causes of chemical spoilage resulting in rancidity and/or deterioration of the nutritional quality, color, flavor, texture and safety of foods. It is estimated that half of the world’s fruit and vegetable crops are lost due to post harvest deteriorative reactions. This chapter deals with autoxidation and mechanisms leading to autoxidation in food and biological systems, lipid

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oxidation, sources of natural and synthetic antioxidants, their chemistry and mechanism of action to prevent autoxidation, and health benefits of some antioxidative foods. AUTOXIDATION Autoxidation is a chain reaction that degrades hydrocarbons in products such as polymers, lubricants and lipids, proteins, and DNA in living organisms. Mechanisms Leading To Autoxidation In Food Systems Autoxidation is propagated by peroxyl radicals formed by reaction of atmospheric molecular oxygen and organic molecules. In food systems, naturally occurring antioxidants impart a certain amount of protection against oxidation. However, natural antioxidants are often lost during processing or storage, necessitating the addition of exogenous antioxidants. Antioxidants effectively retard the onset of lipid oxidation in food products. Lipids deteriorate in food products during processing, handling, and storage. Oxidation of unsaturated lipids in the food system is catalyzed by heat, light, ionizing radiation, trace metals, and metallo-proteins and also enzymatically by lipoxygenase. Lipid oxidation is the major cause of the development of off-flavor compounds and rancidity as well as a number of other reactions that reduce the shelf life and nutritive value of food products. In recent years, the possible pathological significance of dietary lipid oxidation products has attracted the attention of biochemists, food scientists, and health professionals. Studies indicate that lipid oxidation products have cytotoxic, mutagenic, carcinogenic, atherogenic, and angiotoxic effects. Mechanisms Leading To Autoxidation In Biological Systems In biological systems, various biochemical defense mechanisms involving enzymes, trace minerals, and antioxidant vitamins or compounds protect the cellular components from oxidative damage. The formation of reactive free radicals is mediated by a number of agents and mechanisms such as high oxygen tension, radiation, and xenobiotic metabolism. The free radicals formed are highly reactive with molecular oxygen, forming peroxy radicals and hydroperoxides thus initiating a chain reaction. Prooxidant states cause cellular lesions in all major organs by damaging cellular components, including polyunsaturated fatty acids, phospholipids, free cholesterol, DNA, and proteins. The health implications of tissue lipid oxidation are numerous and well documented.

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11

Lipid Oxidation Lipids form one of the major bulk constituents in some foods and other biological systems. Lipids in biological systems can undergo oxidation, leading to deterioration. In foods, these reactions can lead to rancidity, loss of nutritional value from the destruction of vitamins (e.g., A, C, and E) and essential fatty acids, and the possible formation of toxic compounds and colored products. Unsaturation in fatty acids makes lipids susceptible to oxygen attack leading to complex chemical changes that eventually manifest themselves in the development of off-flavors in food. In addition to the role of autoxidation in food deterioration, there is growing interest in the problem of lipid oxidation as related to health status. Lipid oxidation is believed to play an important role in coronary heart disease (CHD), atherosclerosis, cancer, and the aging process. A complex antioxidative defense system normally protects cellular systems from the injurious effects of free radicals. Mechanism Of Lipid Oxidation In A Food System The major lipid components involved in oxidation are the unsaturated fatty acid moieties, oleic, linoleic, and linolenic. The rate of oxidation of these fatty acids increases with the degree of unsaturation. The overall basic mechanism of lipid oxidation consists of three phases: (1) initiation, the formation of free radicals; (2) propagation, the free-radical chain reactions; and (3) termination, the formation of nonradical products. Initiation The autoxidation of a lipid is thought to be initiated with the formation of free radicals (reactive oxygen species) (Figure 2.1). When in contact with oxygen, an unsaturated lipid gives rise to free radicals (Eq. a). Initiation reactions take place either by the removal of a hydrogen radical from an allylic methylene group of an unsaturated fatty acid or by the addition of a radical to a double bond. RH ➞ R• + H• (a) The formation of lipid radical R• is usually mediated by trace metals, irradiation, light, or heat. Also, lipid hydroperoxide, which exists in trace quantities prior to the oxidation reaction, breaks down to yield radicals as shown by Eqs. (b and c) RH + O2 ➞ R• + HO• (b); 2ROOH ➞ RO• + HO• (c) where RH is any unsaturated fatty acid; R• is a free radical formed by

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removing a labile hydrogen from a carbon atom adjacent to a double bond; and ROOH is a hydroperoxide, one of the major initial oxidation products that decompose to form compounds responsible for off-flavors and odors. Such secondary products include hexanal, pentanal, and malonaldehyde. FIGURE 2.1 — Reactive Oxygen Species Species •

HO • HO2 • O2 • 1 O2 • RO • ROO • NO H 2O 2 HOCl

Common Name

Half-life (37oC)

Hydroxyl radical Hydroperoxyl radical Superoxide anion radical Singlet oxygen radical Alkoxy radical Peroxyl radical Nitric oxide radical Hydrogen peroxide Hypochlorous acid

1 nanosecond unstable enzymatic 1 microsecond 1 microsecond 7 seconds 1-10 seconds Stable Stable

R = lipid, for example linoleate

The hydroperoxides undergo homolytic cleavage to form alkoxy radicals (RO • ) or undergo bimolecular decomposition. Lipid hydroperoxides are formed by various pathways including the reaction of singlet oxygen with unsaturated lipids or the lipoxygenase-catalyzed oxidation of polyunsaturated fatty acids. Propagation Free radicals are converted into other radicals. Thus, a general feature of the reactions of free radicals is that they tend to proceed as chain reactions, that is, one radical begets another and so on. Thus, the initial formation of one radical becomes responsible for the subsequent chemical transformations of innumerable molecules because of a chain of events. In fact, propagation of free-radical oxidation processes occurs in the case of lipids by chain reactions that consume oxygen and yield new free-radical species (peroxy radicals, ROO•) or by the formation of peroxides (ROOH) as in (d) and (e). R• + 3O2 ➞ ROO• (d) ROO• + RH ➞ ROOH + R• (e) The products R • and ROO • can further propagate free-radical reactions.

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Termination Lipid oxidation terminates when two radicals interact. R• + R ➞ R-R ROO• + ROO• ➞ ROOR + O2 RO• + R• ➞ ROR ROO• + R• ➞ ROOR 2RO• + 2ROO• ➞ 2ROOR + O2 Free radicals are considered to be bonding-deficient and hence structurally unstable. They, therefore, tend to react whenever possible to restore normal bonding. That is why a free radical is highly reactive. When there is a reduction in the amount of unsaturated lipids (or fatty acids) present, radicals bond to one another, forming a stable nonradical compound. Thus the termination reactions lead to interruption of the repeating sequence of propagating steps of the chain reaction. Mechanism Of Lipid Oxidation In The Biological System Lipid oxidation is a normal biological process by which we obtain energy from fat. Deleterious lipid oxidation occurring in the body generally is called peroxidation. Uncontrolled oxidation of lipids in biological membranes is a major contributor in several disease states such as atherosclerosis, cancer, and neurodegeneration. Fatty acid hydroperoxides (LOOHs) are the primary products of the oxidation of polyunsaturated fatty acids (PUFAs). The elevated levels of LOOHs observed during instances of cellular injury have been correlated to the disruption of biological membranes, inactivation of enzymes, and damage to protein and DNA molecules. To understand the mechanism of lipid peroxidation in the biological system, isolated microsomes from liver are used. Initiation and propagation of lipid peroxidation are catalyzed by iron and microsomal NADPH-cytochrome P-450 reductase. This enzyme is responsible for the formation of a superoxide anion, formed by the addition of an extra electron onto the diatomic oxygen molecule, which catalyzes the reduction of iron ions. Aust and Svingen (1982) suggested a mechanism for lipid peroxidation in microsomes. NADPH-dependent microsomal lipid peroxidation is considered to take place in two stages: initiation

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and propagation. Initiation reactions proceed by a NADPH-cytochromeP-450-reductase catalyzed reduction of ADP-Fe+3, subsequently reacting with oxygen to form a ADP-perferyl radical. The perferyl radical is then responsible for initiating lipid peroxidation and forming lipid hydroperoxides. In this manner, the evanescent hydroxyl radical need not be invoked, nor is a significant hydrogen peroxide flux required. Propagation reactions proceed by the interaction of lipid hydroperoxides with cytochrome P-450, which catalyzes their decomposition to peroxy or alkoxy radicals. In this regard, EDTA or DTPA chelates of iron are also capable of catalyzing the propagation reaction. The cyclical reduction by P-450 reductase, and reoxidation of the iron chelates serves to maintain the propagation reaction. Sources Of Free Radicals Sources of free radicals can be classified into two categories – endogenous and exogenous sources. Endogenous sources (Figure 2.2) which account for most of the free radicals produced by cells are: 1. Normal aerobic respiration – As a result of normal aerobic respiration, mitochondria consume molecular oxygen, reducing it by sequential steps to produce water. The formation of O2•-, H2O2, and •OH occurs by successive additions of electrons to O2• Cytochrome oxidase adds four electrons fairly efficiently during energy generation in mitochondria, but some of the toxic intermediates are inevitable by-products. In a study conducted on rats, about 1012 oxygen molecules are processed by each rat cell daily, and the leakage of partially reduced oxygen molecules is about 2%, yielding about 2x1010 superoxide and hydrogen peroxide molecules per cell per day (Ames et al., 1993). 2. Peroxisomes, which are organelles responsible for degrading fatty acids and other molecules, produce H2O2 as a by-product, which is then degraded by catalase. Under certain conditions, some of the peroxide escapes degradation, resulting in its release into other compartments of the cell and in increased oxidative DNA damage. 3. Cytochrome P-450 enzymes in animals constitute one of the primary defense systems against natural toxic chemicals from plants, the major source of dietary toxins. The induction of these enzymes, prevent acute toxic effects from foreign chemicals, but also results in oxidant by-products that damage DNA. 4. Phagocytic cells destroy bacteria or virus-infected cells with an

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oxidative burst of NO, O2•-, H2O2, and OCl-. Chronic infection by viruses, bacteria, or parasites, results in a chronic phagocytic activity and consequent chronic inflammation, which is a major risk factor for cancer. Chronic infections are particularly prevalent in third world countries. FIGURE 2.2 — Cellular sources of free radicals. Free radicals are produced by cells through the action of various soluble and membrane bound enzymes. The capacity of specific pathways to produce free radicals varies with the cell type, but all aerobic cells appear capable of producing some level of free radicals.

The large endogenous oxidant load may significantly be influenced by exogenous sources which are: 1. Cigarette smoking: The oxides of nitrogen (NOx) in cigarette smoke (about 1000 ppm) cause oxidation of macromolecules, and deplete

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antioxidant levels. This is likely to contribute significantly to the pathology of smoking. Smoking is a risk factor for heart disease as well as a wide variety of cancers in addition to lung cancer. 2. Dietary factors: Iron and copper salts promote the generation of oxidizing radicals from peroxides. Men who absorb significantly more than normal amounts of dietary iron due to a genetic defect (hemochromatosis disease) are at an increased risk for both cancer and heart disease. It has, therefore, been argued that too much dietary copper or iron, particularly heme iron (which is high in meat), is a risk factor for cardiovascular disease and cancer in normal men. 3. Normal diets contain plant food with large amounts of natural phenolic compounds, such as chlorogenic and caffeic acid, that may generate oxidants by redox cycling. 4. Radiation/UV light: UVA rays constitute 90-95% of the ultraviolet light reaching the earth. They have a relatively long wavelength (320-400 nm) and are not absorbed by the ozone layer. UVA light penetrates the furthest into the skin and is involved in the initial stages of sun tanning. UVA tends to suppress the immune function and is implicated in premature aging of the skin. UVB rays are partially absorbed by the ozone layer and have a medium wavelength (290-320 nm). They do not penetrate the skin as far as the UVA rays do and are the primary cause of sunburn. They are also responsible for most of the tissue damage which results in wrinkles and aging of the skin and are implicated in cataract formation. UVC rays have the shortest wavelength (below 290 nm) and are almost totally absorbed by the ozone layer. As the ozone layer thins UVC rays may begin to contribute to sunburning and premature aging of the skin. All forms of ultraviolet radiation are believed to contribute to the development of skin cancer. 5. Strenuous work or exercise: During exercise the increase in whole body oxygen consumption of 10-20 fold causes a severe disturbance of various biochemical pathways. The oxygen flux in individual muscle fibers is believed to increase by as much as 100-200 fold. This tremendous increase in oxygen consumption results in an increased leakage of electrons from the mitochondrial respiratory chain, forming various one-electron oxygen intermediates, such as superoxide anion, hydrogen peroxide and hydroxyl radicals. These reactive oxygen species

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(ROS) are capable of triggering a chain of damaging reactions in the cell, such as lipid peroxidation, inactivation of certain enzymes, alteration of cellular oxidoreductive status, and oxidative damage to proteins and DNA. A role for free radicals has been proposed in the pathogenesis of many diseases (Figure 2.3). The free radical reactions which involve biological molecules (DNA, protein, and lipids) appear to occur constantly as a consequence of the aerobic environment in which we live. Cells have developed a battery of defenses to prevent and repair the injury associated with oxidative changes to DNA, protein, and lipids. These include superoxide dismutases, catalase, the glutathione system, vitamin E, ascorbic acid, urate, and perhaps several others such as lipases to remove oxidized fatty acids, DNA repair of enzymes, and proteases to degrade damaged proteins (Figure 2.3). It is only when the homeostatic mechanisms fail to keep pace with these reactions that detrimental effects become evident. FIGURE 2.3 — Possible Free Radical Related Diseases/Tissue Injury Lung Detrimental Effect

Chemical Agent

Normobaric hyperoxic injury Bronchopulmonary dysplasia Asbestosis Adult respiratory distress syndrome Ideopathic pulmonary fibrosis

Inhaled oxidants – SO2, NOX, O3 Inhaled oxidants – SO2, NOX, O3 Paraquat, Bleomycin Emphysema Cigarette smoke

Heart and Cardiovascular System Detrimental Effect

Chemical Agent

Reperfusion - after infarction or transplant Atherosclerosis

Ethanol, Doxorubicin Selenium deficiency

GI Tract Detrimental Effect

Chemical Agent

Reperfusion

Nonsteroidal anti-inflammatory agents Blood

Detrimental Effect Protoporphyrin photoxidation Malaria Various anemias (Sickle cell, favism)

Chemical Agent (Phenylhydrazine, primaquine and related drugs, sulfonamides, lead)

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FIGURS 2.3 — Possible Free Radical Related Diseases/Tissue Injury Continued Miscellaneous Aging Detrimental Effect

Chemical Agent

Radiation injury

Alloxan (diabetes), iron overload, radiosensitizers

Rheumatoid arthritis and other autoimmune diseases such as Lupus

Inflammation in general Brain

Detrimental Effect

Chemical Agent

Hyperboric hyperoxic injury Parkinson’s disease

Neurotoxins (eg., 6-hydroxydopamine, MPTP) Vitamin E deficiency Neuronal ceroid lipofuscinoses Traumatic injury/inflammation/ reperfusion Kidney

Detrimental Effect

Chemical Agent

Autoimmune nephrosis (Inflammation)

Aminoglycosides, Heavy metals

Liver Detrimental Effect

Chemical Agent

Reperfusion

Halogenated hydrocarbons, quinines, iron, acetaminophen, ethanol Endotoxin Eye

Detrimental Effect

Chemical Agent

Retinopathy of prematurity (oxygen) Photic retinopathy, Cataracts Skin Detrimental Effect

Chemical Agent

Radiation (solar or ionizing) Thermal injury Contact dermatitis

Photosensitizers (tetracyclines) Porphyria Muscle

Detrimental Effect

Chemical Agent

Muscular dystrophy, Multiple sclerosis Exercise

Antioxidants The onset of lipid oxidation can be delayed by adding antioxidants to food or by including them in our diet. The major role of antioxidants in

ANTIOXIDANTS

19

the food industry is to prevent off-flavors, rancidity and to maintain their nutritional value. These undesirable characteristics are related to lipid peroxidation or peroxidation initiated by the action of lipoxygenase enzymes in the plant. Food antioxidants are inhibitors of lipid peroxidation and consequent food deterioration. On the other hand, in the human gastrointestinal tract and within the body tissues, oxidative damage to proteins and DNA is as important as damage to lipids. Oxidative DNA damage could be a major risk factor for the development of tumors, so that dietary antioxidants able to decrease such damage in vivo would be expected to have cancer prevention effects. Hence, antioxidants are defined as substances when present in foods at low concentrations compared with those of an oxidizable substrate markedly delay or prevent the oxidation of the substrate (Halliwell, 1999). FIGURE 2.4 — Biological antioxidant defense systems. All aerobic cells contain a spectrum of chemical and enzymatic antioxidants that work in concert to minimize undesirable oxidative reactions within cells.

20

FUNCTIONAL FOODS

The term oxidizable substrate encompasses almost everything (except water) found in foods and in living tissues and includes proteins, lipids, carbohydrates and DNA molecules. An antioxidant may be able to protect one biological or food system but it may fail to do so in others. For example, antioxidant inhibitors of lipid peroxidation may not protect other molecular targets such as DNA and protein against oxidative damage and may sometimes aggravate such damage. This may not matter much in foods, because damage to DNA and proteins, unless extensive, will not normally alter the taste or texture of food or affect nutritional quality. However, essential amino acids, such as tryptophan and methionine are destroyed by certain reactive species and oxidative damage to sulfur containing amino acids can sometimes create offflavors. But in biological system, oxidative DNA and protein damage are of great importance in the cells of the human gastrointestinal tract and within the body. Oxidative DNA damage is a risk factor for cancer development, and protein damage by reactive species is involved in cancer, cardiovascular, and neurodegenerative diseases. Antioxidants have become an indispensable group of food additives. The use of antioxidants dates back to the 1940s. Gum guaiac was the first antioxidant approved for the stabilization of animal fats, especially lard. Natural Antioxidants Antioxidants in food are important for four reasons. First, endogenous antioxidants may protect components of the food itself against oxidative damage. For example, spices rich in antioxidants have been used for centuries to delay oxidative deterioration of foods during storage or cooking. Second, dietary antioxidants may be absorbed into the human body and exert beneficial effects. For example, quercetin and catechins can be absorbed to some extent in humans and they and their metabolites can reach plasma concentrations in the range of 0.1 – 1
M. Such concentrations can, in vitro, delay the process of lipid peroxidation in liposomes, microsomes and low-density lipoproteins (LDL). Third, food derived antioxidants could exert beneficial effects, without being absorbed, in the gastrointestinal tract itself. Fourth, there is great interest in plant extracts for therapeutic use as antiinflammatory, anti-ischemic, and antithrombotic agents. An extract of the ornamental tree Ginkgo biloba has been used in herbal medicine for thousands of years: the extract has antioxidant properties in vitro, apparently largely from the flavonoids present, which include rutin, kaempferol, quercetin, and myricetin.

ANTIOXIDANTS

21

Of the natural antioxidants, two important groups, the tocopherols and ascorbic acid, are highly effective in many food products. Due to concern over the safety of synthetic compounds, extensive work is being carried out to identify novel naturally occurring compounds as replacements for potentially toxic synthetic antioxidants. Natural antioxidants occurring in foods may be used as a component for food formulations in order to stabilize them or may be extracted and added to foods. As an example, oat and amaranth oils contain high levels of antioxidants such as tocopherols and squalene. These oils might be added to certain other oils in order to stabilize them. Furthermore, extracts of green tea, rosemary and sage might be used in a variety of foods in order to control oxidation. In addition, mixed tocopherols as well as combination of tocopherols with lecithin and ascorbic acid may be employed to retard oxidation of bulk oils, emulsions and other products. Chemical Classification Of Food Antioxidants 1. Phenols a. Tocopherol derivatives b. Flavonoid derivatives Flavanol – Epicatechin, catechin, epigallocatechin, epicatechin gallate Flavanone – Naringin, taxifolin Flavonol – Kaempferol, quercetin, myricetin Flavone – Chrysin, apigenin Anthocyanidins – Malvidin, cyaniding, apigenidin Phenyl propanoids – Ferulic acid, caffeic acid, β-coumaric acid, chlorogenic acid c. Gallic acid derivatives d.Cinnamic acid derivatives e. Coumarin derivatives f. Ellagic acid derivatives g.Tannin derivatives h.Phenoilc terpenoids i. Lignan derivatives j. Resins and polyphenols 2. β- Diketones 3. Nucleic acid bases 4. Amino acids, peptides and amines 5. Phospholipids 6. Ascorbic acid and reductones 7. Sulphur and selenium compounds

22

8. 9. 10. 11. 12. 13. 14. 15. 16.

FUNCTIONAL FOODS

Carotenoids Melanoidines Hydroquinones Organic acids Porphine compounds Protease inhibitors Terpenes Indoles Isothiocyanates

Classification Of Antioxidants Based On Their Function 1. Primary or chain breaking antioxidants (scavenger antioxidants): These antioxidants can neutralize free radicals by donating one of their own electrons, ending the electron “stealing” reaction. The resultant antioxidants which become free radicals, because of one electron left in their outer shell, are relatively safe, stable and in normal circumstances insufficiently reactive to initiate any toxic effect, e.g., -tocopherol. 2. Secondary or preventive antioxidants: These antioxidants act through numerous possible mechanisms like: a) sequestration of transition metal ions which are not allowed to participate in metal catalyzed reactions; b) removal of peroxides by catalases and glutathione peroxidase, that can react with transition metal ions to produce ROS; c) removal of ROS, etc. These antioxidants which are also called as synergistic antioxidants can be broadly classified as oxygen scavengers and chelators. Oxygen scavengers such as ascorbic acid, ascorbyl palmitate, sulfites and erythorbates react with free oxygen to remove it in a closed system. Chelators like ethylenediaminetetraacetic acid (EDTA), citric acid, and phosphates are not antioxidants, but they are highly effective as synergists with both primary antioxidants and oxygen scavengers. An unshared pair of electrons in their molecular structure promotes the chelating action. They form stable complexes with prooxidant metals such as iron and copper, which promote initiation reactions and raise the energy of activation of the initiation reactions considerably. 3. Tertiary antioxidants: These antioxidants remove damaged biomolecules before they can accumulate and before their presence results in altered cell metabolism and viability. For example, methionine sulphaoxide reductase repairs damaged DNA, proteolytic enzyme system remove oxidized proteins and lipases, peroxidases and acyl transferases act on oxidized lipids.

23

ANTIOXIDANTS

Classification Of Antioxidants Based On The Site Of Synthesis Some antioxidants are synthesised within the cells themselves which are called as endogenous antioxidants and others are found in food referred to as natural antioxidants (Figure 2.5). FIGURE 2.5 — Examples of Endogenous Antioxidants and Natural Antioxidants Endogenous Antioxidants

Natural Antioxidants

Polyamines Melatonin Oestrogen Superoxide dismutase (SOD) Glutathione peroxidase Catalase Lipoic Acid Caeruloplasmin Albumin Lactoferrin Transferrin

Vitamin E Vitamin C Carotenoids Polyphenols Selenium Flavonoids

FOODS RICH IN ANTIOXIDANTS Berries Small berries constitute an important source of potential healthpromoting phytochemicals. These include fruits of the Vaccinium, Ribes, Ribus and Fragaria genera. Examples of Vaccinium genus are lowbush and highbush blueberry, bilberry, cranberry and lingonberry. Examples of Rubus genus are blackberries, red and black raspberries. Gooseberries, jostaberries and currants belong to the Ribes genus and strawberry to the Fragaria genus. These fruits are rich sources of flavonoids and other phenolics that display potential health-promoting effects. For example, over 180 Vaccinum-based Pharmaceuticals have been introduced to the market. Grapes (Vitis vinifera L) are one of the world’s largest berry crops. Cranberries and grapes are discussed in detail in the following sections. Cranberries (Vaccinium macrocarpon) The fruits of American cranberries, Vaccinium macrocarpon and European cranberries, Vaccinium oxycoccus have been associated with a variety of health benefits. There are reports of its use by American Indians to dress wounds and prevent inflammation. In the early 20th century, cranberries were thought to help relieve the symptoms of

24

FUNCTIONAL FOODS

urinary tract infections, or perhaps even prevent their occurrence. Cranberries possess a distinctive flavor and a bright red color. They are sold fresh or processed into sauce, concentrates, and juice. Chemical composition of cranberries: • Proanthocyanidins and anthocyanins make up the pigment of the leaves, and produce the color of the berries. More importantly, proanthocyanidins are responsible for the cranberry’s best-known medicinal effect, preventing bladder and urinary tract infections by inhibiting bacterial colonization. They may also help relieve diarrheal symptoms. • Organic acids, including quinic, malic, and citric acids. Quinic acid is considered the most important among these organic acids. These compounds, which are responsible for the sour taste of cranberries, acidify the urine and prevent kidney stones. • Vitamins and minerals. Cranberries are rich sources of vitamins including vitamin A, carotene, thiamine, riboflavin, niacin, and vitamin C. They also contain many essential minerals such as sodium, potassium, calcium, magnesium, phosphorus, copper, sulfur, iron, and iodide. These vitamins and minerals are strong antioxidants that enable cranberries to help protect the body against such infections as colds or influenza. Because of their high vitamin C content, cranberries were used in the past to prevent a vitamin C deficiency known as scurvy. • Cranberries are also a good source of fiber. Antioxidants in Cranberries Cranberry fruits serve as an excellent source of anthocyanins, flavonol glycosides, proanthocyanindins and phenolic acids. Cranberries contain about 1g/kg of fresh weight of phenolic acids predominantly as glycosides and esters. Twelve phenolic acids have been identified in cranberries (Figure 2.6). Sinapic, caffeic and p- coumaric acids are the most abundant bound phenolic acids and coumaric, 2,4dihydroxybenzoic and vanillic acids the predominant free phenolic acids found in cranberry. Resveratrol (0.25 mg/kg) has also been detected in cranberry fruit. THERAPEUTIC EFFECTS OF CRANBERRY Urinary Tract Infections (UTIs) The term urinary tract infection (UTI) refers to the presence of microorganisms in the urinary tract, including the bladder, prostate, collecting system, or kidneys. Common symptoms include frequent and urgent need to urinate, painful urination, cloudy urine, and lower back

ANTIOXIDANTS

25

pain. Escherichia coli is the most common urinary pathogen, accounting for 85% of UTIs. Other pathogenic bacteria (Enterococcus, Staphylococcus, Proteus, or Klebsiella) can also be responsible. UTIs account for 9.6 million doctor visits annually. The cost of diagnostic work-up and treatment has been estimated at $100 per visit. The treatment of choice is an antibiotic, generally effective within three days. UTIs are one of the most common infections in females, more prevalent among women than men. Avorn et al (1994) conducted a 6-month randomized, double-blinded, placebo-controlled trial with 153 elderly, institutionalized women. Subjects consumed 10 ounces of either a low-calorie cranberry juice cocktail (CJC) or a specially-prepared placebo drink that contained no cranberry, on a daily basis. Biomarkers assayed for urinary tract infections included bacteria in the urine (bacteriuria) and white blood cells in the urine (pyuria). They found that bacteriuria with pyuria was reduced by nearly 50% with consumption of CJC. Additionally, women in the test group with a positive urine culture in a given month had only 27% likelihood in comparison to the control group for having their urine remain positive in the following month. This trial also investigated the effect of drinking CJC or the placebo drink on urinary acidification. They found that the mean pH was actually lower in the placebo group, indicating that urinary acidification was not the mechanism for cranberry’s beneficial effect. Walker et al (1997) conducted an intervention trial using solid cranberry dietary supplements prepared from spray-dried cranberry juice. The study was a randomized double-blinded placebo-controlled crossover study using a population of 19 sexually active women (mean age of 37) who consumed two 400 mg capsules of cranberry solids or placebo capsules daily for three months, with opposite treatment for the next three months. A statistically significant reduction in risk for urinary tract infections when taking the cranberry supplement was found with the 10 subjects who completed the study. Kontiokari et al (2001) conducted a randomized trial investigating the effect of either a cranberry-lingonberry juice beverage or a Lactobacillus GG drink (LGG) on the incidence of urinary tract infections. One hundred-and-fifty young, sexually active women (average age of 30) with a history of at least one symptomatic UTI participated. Subjects were randomly allocated into three groups of 50, and received either 50 ml of the cranberry beverage daily for six months, or 100 ml of the LGG drink five days per week for a year, or served as open controls.

26

FUNCTIONAL FOODS

FIGURE 2.6 — Phenolic Acids In Cranberries Phenolic acids

Structure

HOOC

H

OH o-Hydroxybenzoic acid

H H

COOH

OH

m-Hydroxybenzoic acid H H

COOH

OH

p-Hydroxybenzoic acid H

OH

O

p-Hydroxyphenyl acetic acid O

HO

OH

HO

2,3-Dihydroxybenzoic acid O

HO HO OH

2,4- Dihydroxybenzoic acid

O

27

ANTIOXIDANTS

FIGURE 2.6 — Phenolic Acids In Cranberries - Continued Phenolic acids

Structure OCH 3

COOH

OH

Vanillic acid H

OH

o-hydroxycinnamic acid OH

O

O OH

HO Caffeic acid

OH

HO

p-Coumaric acid

O

OH

HO

Ferulic acid

OH

O O

28

FUNCTIONAL FOODS

FIGURE 2.6 — Phenolic Acids In Cranberries - Continued Phenolic acids

Structure

O

HO

Sinapic acid

O

OH

O

HO OH

Resveratrol

OH

The outcomes measured were the first recurrence of symptomatic UTIs with positive confirmation by urine culture. At 6 and 12 months, the LGG drink showed no beneficial effect. At six months, eight (16%) of the women in the cranberry group and 18 (36%) in the control group had at least one recurrence. At 12 months, the cumulative occurrence of the first episode of recurrent UTI was still significantly different between the control and cranberry groups, even though the test group had stopped consuming the cranberry beverage group after six months. This outcome is significant in providing support for a hypothesis that consumption of the cranberry beverage in the first 6 months had changed the microbial flora in the gastrointestinal tract, and reduced the uropathogenic E. coli colonization in the gut. Potentially, the load of uropathogenic E. coli in the stool would be lowered, thereby reducing the external migration of these bacteria from the GI to the urinary tract, and reducing the chance of a UTI. Thus, cranberry may be acting

ANTIOXIDANTS

29

in both the gut (the source of most uropathogens) and in the bladder in preventing colonization of certain uropathogenic bacteria. Stothers (2002) presented a study investigating the effectiveness of either cranberry juice or cranberry tablets vs. placebo as a prophylaxis against UTIs. A prospectively randomized blinded one-year trial was conducted with 150 sexually active women, ages 21–72 with a history of at least two symptomatic UTIs. Both groups consuming cranberry juice and cranberry tablets showed significant decreases in the mean number of symptomatic UTIs compared with those consuming the placebo. Total antibiotic consumption was significantly decreased in the two cranberry groups as well. Mechanism Of Action Of Cranberries In Urinary Tract Infections Earlier it was thought that cranberry’s effect on acidification of the urine as the possible mechanism for cranberry’s antibacterial effect in the urinary tract, but this theory was not substantiated by other research (Avorn et al., 1994). For UTIs to occur, bacterial entry and proliferation must occur. Proliferation requires attachment to urinary tract mucosal surfaces. The latest research supports the hypothesis that cranberry juice acts to promote urinary tract health by inhibiting bacterial adherence to mucosal surfaces (Henig and Leahy, 2000; Leahy et al., 2001). These studies measured the ability of various bacteria to adhere to uroepithelial cell surfaces using in vitro techniques and evaluated this activity in both human and animal urine after subjects drank cranberry juice. Bacteria have different types of adhesions on the fimbriae and pili that attach to epithelial cells. Cranberry juice contains a relatively unique component that inhibits certain adhesions (P-fimbriae) of some uropathogenic strains of E. coli. Using bioassay-directed fractionation techniques, Howell et al (1998) identified proanthocyanidins (PACs, also known as condensed tannins) as the compounds in cranberries that are responsible for preventing P-fimbriated E. coli from adhering to the urinary tract. Vaccinium PACs are polymers of catechin and epicatechin. The higher molecular weight trimers and oligomers had the greatest anti-adhesion activity, while monomers and dimers had little. Structural characterization using NMR indicates that cranberry and blueberry PACs have a unique A-type linkage not found in other foods (e.g., tea, grapes, wine, and cocoa) which have the more common B-linkage (Foo et al., 2000a). Three A-linked cranberry PAC trimers have been shown to prevent adhesion of P-fimbriated E. coli to bladder cells in vitro (Foo et al., 2000b) (Figure 2.7). Questions remained as to

30

FUNCTIONAL FOODS

bioavailability and absorption of these compounds. Recently, a study was completed in which extracts of purified cranberry PACs were fed to mice. The urine was found to exhibit anti-adhesion activity against P-fimbriated E. coli, providing the first in vivo evidence that cranberry PACs and/or metabolites can be absorbed into the blood, and into urine, thereby eliciting this anti-adhesion effect (Howell et al., 2001). This is also significant in suggesting bioavailability for other potential health benefits. FIGURE 2.7 — Chemical Structures Of Proanthocyanidins

While orange juice, pineapple juice, and cranberry juice cocktail exhibited anti-adhesion activity against type 1 fimbriated E. coli, containing a mannose-sensitive (MS) adhesion, only those juices from

ANTIOXIDANTS

31

the Vaccinium genus tested (cranberry and blueberry) contained the mannose-resistant (MR) adhesion inhibitor (Ofek et al., 1991). Oral Cavity Health Various bacteria appear to be major causative factors in the etiology of both dental caries and periodontal gum disease. Of the hundreds of bacteria and bacterial pairs that could comprise the dental plaque, Weiss et al (1997) isolated a wide variety of bacteria from the human gingival crevice, and used a coaggregation assay to measure both aggregation and the reversal of aggregation in the presence and absence of a selected cranberry fraction in vitro. Using this assay with over 80 pairs of the recovered bacteria, they reported that the isolated cranberry fraction was able to inhibit the coaggregation of 70% of the bacterial pairs tested when at least one was Gram negative. Also highly noteworthy was their finding that the fraction was able to actually reverse the coaggregation of 50% of those pairs. As an example, they showed that the cranberry fraction, but not apple juice, caused complete reversal of aggregation of S. oralis HI and F. nucleatum PK1594. The authors concluded that the cranberry fraction would be an excellent candidate for further animal and clinical studies to assess its ability to influence plaque development and the resultant effects on periodontal gum disease. Other Benefits In vitro and in vivo animal studies have found anti-inflammatory, anticarcinogenic, antiplatelet aggregation, vasodilatory, and other effects of several of antioxidant compounds. Preliminary research on various proanthocyanidins suggests that they may act as antioxidants, and have cardioprotective and anticarcinogenic activities (Ho et al., 1999). Phenolic acids may contain antibacterial, antifungal, anticancer effects and activity. Ellagic acid has been shown to have a broad range of anticarcinogenic activity. Both in vitro and in vivo studies have shown inhibition against a broad range of carcinogens in several different tissues (Barch et al., 1996). The newest research suggests that cranberries may also play another potential role in maintaining gastrointestinal health. A recent in vitro study investigated cranberry’s potential in inhibiting the adhesion of some strains of H. pylori to human mucosal cells (Burger et al., 2000). A cranberry fraction was found to inhibit adhesion of three strains of H. pylori that is mediated by a sialic acid-specific adhesion. Research is going on to determine cranberry’s activity against many other strains. Helicobacter pylori infections have been implicated as a major cause of gastric, duodenal, and peptic ulcers.

32

FUNCTIONAL FOODS

GRAPES Grapes belonging to the species Vitis vinifera L. are predominantly cultivated in Europe, while those belonging to species Vitis labrusca and Vitis rotundifolia are grown in North America (Mazza, 1995). Approximately 80% of the total crop is utilized for wine making; 13% is consumed as table grapes and 7% processed into juice and raisins. Antioxidants In Grapes The main phenolics in grapes are listed in Figure 2.8. Anthocyanins are the predominant phenolics of red table grape varieties, while flavanols are the main phenolics in white table grape varieties (Cantos et al., 2002). The total content of phenolics in some table grape varieties is presented in Figure 2.9. These phenolics contribute to the sensory quality of grape products. Pomace (skin and seeds), a by-product from processing grape to juice and wine, comprises about 13% of the amount of processed berries (Torres and Bobet, 2001) and may also contain stems when wines are made from nondestemmed crop. Grape seeds, stems and skins are a rich source of health-promoting flavonoids such as proanthocyanidins, flavonols and flavan-3-ols. Proanthocyanidins are the major polyphenols in grape seeds, stems and skins. Procyanidins are the predominant proanthocyanidins in grape seeds, while procyanidins and prodelphinidins are dominant in grape skins and stems (Souquet et al., 2000). The total contents of dimers, dimer gallates and trimers in seeds from grape cultivars grown in Ontario are 0.16 to 3.75 g of B2 equivalents, traces of 1.08 g of B2-3'-(9-gallate equivalents, and traces of 0.84 g of B2 equivalents/kg of seeds (Fuleki and da Silva, 1997). Approximately 55% of grape seed procyanidins are of polymeric type (degree of polymerization, DP > 5), while the ratios of polymeric procyanidins (DP > 4) to monomeric (catechin + epicatechin) are 5.2 to 8.9 (Peng et al., 2001). Grape seeds contain polymeric procyanidins from 33.2 to 50.7 g/kg in seeds or from 1.68 to 3.19 g/kg in berries. The mean degree of polymerization for proanthocyanidins isolated from the seeds of grapes ranges from 4.7 to 17.4. For those isolated from grape skin, it is between 9.3 and 73.8 and for those extracted from grape stems between 4.9 and 27.6 (Souquet et al., 2000). Other phenolics detected in whole grape berries, grape skins and stems include phenolic acids: caftaric acid (trans-caffeoyltartaric acid), coutaric acid (p-coumaryltartaric acid), trans-Fertaric acid (Figure 2.10), flavonols: quercetin 3-glucuronide, quercetin 3-glucoside, myricetin 3glucuronide, myricetin 3-glucuronide (Figure 2.11), and flavanonols:

33

ANTIOXIDANTS

astilbin (dihydroquercetin 3-rhamnoside), engeletin (dihydrokaempferol 3-rhamnoside) (Figure 2.12) (Souquet et al., 2000). FIGURE 2.8 — Main Phenolics Identified In Grapes Group

Phenolics

Phenolic acids

p-hydroxybenzoic, o-hydroxybenzoic, salicylic, gallic, cinnamic, p-coumaroylartaric (= coutaric), caffeoyltartaric (= caftaric), feruloylartaric (= ertaric), p-coumaroyl glucose, feruloylglucose, glucose ester of coutaric acid

Anthocyanins

Cyanidin 3-glucoside, cy 3-acetylglucoside, cy 3-p-coumaryl-glucoside; peonidin 3-glucoside; pn 3-acetylglucoside, pn 3-p-coumarylglucoside, pn 3-caffeylglucoside, delphinidin 3-glucoside, dp 3-acetylglucoside, dp 3-p-coumarylglycoside, petunidin 3-glucoside, pt 3-p-coumarylglucoside, malvidin 3-glucoside, mv 3-acetylglucoside, mv 3-p-coumaryglucoside, mv 3-caffeylglucoside

Flavonols

Kaempferol 3-glucoside, k 3-glucuronide, k 3-glucosylarabinoside, k 3-galactoside, quercetin3-glucoside, q 3-glucoronide, q 3-rutinoside, q 3-glucosylgalactoside, q 3-glucosylxyloside, iso-rhamnetic 3-glucoside

Flavan-3-ols and tannins

(+)catechin, (-)epicatechin, (+)gallocatechin, (-)epigallocatechin, epicatechin-3-O-gallate, procyanidins Bl, B2, B3, B4, Cl, C2, polymeric forms of condensed tannins

Flavanonols

Dihydroquercetin 3-rhamnoside (= astilbin), dihydrokaempferol 3-rhamnoside (= engeletin)

FIGURE 2.9 — Total Phenolic Contents In Some White And Red Table Grape Varieties Phenolics

Red Globe (R)

Flame Crimson Napoleon Superior Dominga Moscatel (R) (R) (R) (W) (W) Italica (W)

225.4 8.4 61.3 40.4 115.3

361.2 47.6 53.8 109.1 150.7

Mg/Kg Fresh Weight Phenols1 Hydroxycinnamates2 Flavonols3 Flavan-3-ols4 Anthocyanins5

131.9 9.5 12.8 41.1 69.5

135.9 9.5 32.4 18.3 75.7

135.7 9.0 64.0 62.7 -

114.9 25.0 32.7 57.2 -

145.1 16.3 47.7 81.1 -

R = Red; W = white 1 Total phenols = total hydroxycinnamates + total flavonols + total flavan-3-ols + total anthocyanins 2 Total hydroxycinnamates expressed as chlorogenic acid equivalents 3 Total flavonols expressed as quercetin 3-rutinoside equivalents 4 Total flavan-3-ols expressed as catechin equivalents 5 Total anthocyanins expressed as cyanidins

34

FUNCTIONAL FOODS

FIGURE 2.10 — Chemical Structures Of Caftaric, Coutaric And trans-Fertaric Acids

ANTIOXIDANTS

FIGURE 2.11 — Chemical Structures Of Quercetin 3-Glucuronide, Quercetin 3-Glucoside and Myricetin 3-Glucuronide

35

36

FUNCTIONAL FOODS

FIGURE 2.12 — Chemical Structures Of Astilbin (dihydroquercetin 3rhamnoside) and Engeletin (dihydrokaempferol 3-rhamnoside)

ANTIOXIDANTS

37

Grape berries contain from 0.27 to 0.47 g/kg of total hydroxycinnamoyltartaric acids (HCAs). Caftaric acid (0.12 to 0.37 g/ kg) and trans-coutaric acid (55.3 to 93 mg/kg) are the predominant HCAs in berries, while cis-coutaric acid (11.8 to 21.0) and fertaric acids (1.7 to 16.8 mg/kg) are minor HCAs present (Vrhovsek 1998). Stilbenes such as trans- and cis-resveratrols (3,5,4'trihydroxystilbene), trans-and cis-piceids (3-O-β-D-glucosides of resveratrol), trans- and cis-astringins (3-O-β-D-glucosides of 3'hydroxyresveratrol), trans- and cis-resveratrolosides (4'-O-β-Dglucosides of resveratrol) pterostilbene (a dimethylated derivative of stilbene) are grapevine phytoalexins found in grape leaves and berries. Raisins are important processed grape products. Italy, France and the U.S. are the world’s largest producers of raisins. Karadeniz et al. (2000) evaluated the effect on the composition of phenolic in raisins of sun-drying grapes (sun-dried raisins), dipping grapes into hot water (87 to 93°C) for 15 to 20 s before drying at 71°C for 20 to 24 h (dipped raisins), and dipping grapes into hot water followed by 5- to 8-h treatment with sulfur dioxide and then drying at 63°C (golden raisins). Oxidized hydroxycinnamic acids, formed upon the action of polyphenoloxidases, were only found in sun-dried and dipped raisins. The loss of hydroxycinnamic acids and flavonols during processing of grapes to raisins is in the order of 90 and 62%, respectively; procyanidins are degraded completely (Karadeniz et al., 2000). Drying grape pomace may be an essential step in the utilization of this by-product for the production of pharmaceuticals. Therapeutic Effects of Grapes Antioxidants in grapes are believed to protect the body from certain cancers and heart disease. These exhibit antioxidant properties and wine is a major source of these nutrients. Resveratrol has anti-infective, antiinflammatory and antioxidant properties in humans. This compound helps battle cancer in various stages, from initiation to promotion to progression. Studies propose that eating resveratrol-rich foods may reduce the risk of cardiovascular disease, lower total cholesterol and lower LDL cholesterol. The compound’s antioxidant properties may also play a part in slowing the oxidation of LDL cholesterol. Resveratrol is water- and fat-soluble so it lends itself to a variety of applications. It’s believed to improve circulation, promote healing and help prevent wrinkles. Grapes’ antioxidant properties have been shown to strengthen blood vessels, boost immunity and inhibit allergies.

38

FUNCTIONAL FOODS

TOMATO The tomato (Lycopersicon esculentum) is one of the world’s major vegetables with 4.4 million hectares under production and 115 million tons produced worldwide in 2004 (FAOSTAT, 2004). Tomatoes are consumed both fresh and processed (in multiple forms) around the globe in many countries by many cultures and are available year round. Americans each eat more than 16 pounds of fresh tomatoes a year and consume the equivalent of 79 pounds in processed tomatoes annually. Tomatoes are a rich source of antioxidants, including vitamin C and lycopene. The chemical composition of tomato is given in Figure 2.13 and the range of lycopene content for several tomato products is shown in the Figure 2.14. FIGURE 2.13 — Chemical Composition Of The Tomato Constituent Moisture (%) Protein (%) Ash (%) Ascorbic acid (mg/100 g) Vitamin E (mg/100 g) b-carotene (mg/100 g)

-carotene

(mg/100 g) Phenolic (mg/100 g) Lycopene (mg/100 g) Lutein (mg/100 g) Phytoene (mg/100 g) Na (mg/kg) K (mg/kg) Ca (mg/kg) Mg (mg/kg) Fe (mg/kg) Cu (mg/kg) Zn (mg/kg) Mn (mg/kg) pH Brix degree Refractive index Acidity (%)

Range 93.1-94.2 0.7-1.0 0.40-0.52 16.0-24.2 0.80-1.22 0.30-0.52 0.04-1.61 8.4-17.0 0.90-9.30 0.04-0.10 0.49-2.80 102-186 2158-3192 38.4-58.0 63.3-96.1 0.44-2.58 0.19-0.71 0.67-1.01 0.45-0.67 4.06-4.22 4.50-6.62 1.3395-1.3427 0.48-0.56

39

ANTIOXIDANTS

FIGURE 2.14 — Lycopene Contents Of Common Tomato-based Foods (mg/g weight) Tomato products Fresh tomatoes Cooked tomatoes Tomato sauce Tomato paste Tomato soup (condensed) Tomato powder Tomato juice Pizza sauce Ketchup

Lycopene 8.8 – 42.0 37.0 62.0 54.0-1500.0 79.9 1126.0-1264.9 50.0-116.0 127.1 99.0-134.4

Antioxidants In Tomato Lycopene, the carotenoid pigment responsible for the red color, is the most distinctive compound present in tomatoes and has been recognized as the most effective antioxidant among the carotenoids (Figure 2.14). In addition to lycopene, tomatoes also contain other compounds which are recognized as antioxidants. The total flavonol content of tomatoes grown in different countries ranges from 1.3 to 36.4 mg of quercetin/kg of fresh weight (Dewanto et al., 2002). Quercetin conjugates are the predominant form of flavonols found in tomatoes, but smaller quantities of kaempferol conjugates and traces of free aglycons have also been detected. Flavonols of tomatoes are a mixture of quercetin 3-rhamnosylglucoside (rutin), quercetin 3-rhamnosyldiglucoside, kaempferol 3-rhamnosylglucoside and kaempferol 3rhamnosyldiglucoside. Presence of p-coumaric acid conjugate of rutin has also been reported. Of these, rutin is the major flavonol in tomatoes (Stewart et al., 2000). FIGURE 2.15 — Chemical Stucture Of Lycopene

Frying, boiling or microwaving removes 35 to 78% of quercetin conjugates originally present in tomatoes. These losses may be due to the degradation or extraction of flavonols from tomato by water. Tomato juice and puree are a rich source of flavonols. Processing tomatoes to juice and puree increases the content of free quercetin by up to 30%, an increase

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that may be brought about by enzymatic hydrolysis of quercetin conjugates. Tomato juice and puree contain 15.2 to 16.9 mg/L and 16.6 to 72.2 mg/kg of fresh weight of flavonols, respectively. On the other hand, canned tomatoes are a poor source of flavonols (Stewart et al., 2000). The unique quality about the composition of tomatoes and tomato products with respect to other fruits and vegetables is their high content of lycopene, the acyclic carotenoid containing 11 conjugated double bonds. There is a small amount of lycopene in just a few other foods such as watermelon, pink guava, pink grapefruit, strawberry, papaya but tomatoes and tomato products are the major sources in the diet. The lycopene content can vary greatly depending on the variety of the tomatoes considered and obviously on the type of processing method. Apart from lycopene, the tomato is also a good source of vitamin C, providing a significant contribution to dietary intake. Raw tomato contains more vitamin C than processed tomato, and there is a higher loss of the vitamin during the production of tomato concentrates than in tomato juice or whole canned tomatoes. Therapeutic Effects Of Tomatoes Lycopene and β-carotene have been shown to act as powerful antioxidants in humans. A diet containing moderate amounts of lycopene has been associated with the prevention of cardiovascular disease and cancers of the prostate and gastrointestinal tract (Rao and Agarwal, 2000). Increasing levels of dietary lycopene through the consumption of fresh tomatoes and tomato products has been recommended by many health experts. One 6-year, prospective, epidemiological study of approximately 47,000 men, the Health Professional Follow-up Study (HPFS), concluded that 2 to 4 servings per week of raw tomatoes significantly reduced the risk of prostate cancer by 26% compared to no servings per week. Additionally, eating tomato products such as pizza and tomato sauce 2–4 times per week significantly reduced the risk by 15% and 34%, respectively, compared to not eating these foods. The HPFS study period was extended an additional 6 years. The results supported the early findings, and concluded that tomato sauce consumption was associated with a 23% reduction in prostate cancer risk when two or more servings were compared with less than one serving per week. Subgroup analysis revealed an inverse association between serum lycopene concentration and prostate cancer risk, which was most evident in men older than 65 years and in those with no family history of prostate cancer. The authors concluded that tomato and lycopene intake may demonstrate stronger

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protection in cases of sporadic prostate cancer rather than in cases with a strong genetic component (Campbell et al., 2004). The antiproliferative effect of tomato polyphenol on LNCaP, a human prostate cancer cell line, and on Hepa1c1c7, a mouse hepatocyte cell line was studied by Campbell et al (2004). Polyphenols can be attached to a molecule of sugar, in which case they are in the “glycone” form. When the polyphenol is not attached to a molecule of sugar it is said to be in the “aglycone” form. Both cell lines were inhibited in a dosedependent manner (10–50 µmol/L) by the aglycone forms of quercetin, kaempferol, and naringenin, but not as glycones. Interestingly, treating the cell lines with a combination of the aglycone polyphenols (25, 40 and 50 µmol/L total) produced greater inhibition than treating them with the aglycone polyphenols individually, suggesting a synergism exists between the polyphenols. The hypothesis that a synergism might exist between the compounds was tested by the same authors by studying the effects of tomato powder versus lycopene alone on a prostate cancer rat model. They fed rats diets of 10% tomato powder, 0.025% lycopene, 20% dietary energy restriction, or control rats allowed to eat ad libitum. Rats fed the tomato powder had a significant 26% decrease in prostate cancer-specific mortality, while the lycopene-fed rats had a nonsignificant 9% decrease in mortality. Rats on the caloric restricted diet had a decrease in prostate cancer-specific mortality by 32% compared with the rats fed unrestricted amounts of food. When they segmented their data into 45-week intervals, energy restriction diets decreased the risk of prostate cancer by 48% during the first 45 weeks, but had no effect after 45 weeks. Tomato powder and lycopene had a nonsignificant effect during the first 45 days, but decreased the risk of prostate cancer by 56% and 44%, respectively, after 45 weeks. Although the role of all carotenoids in humans has yet to be fully determined, 25 carotenoids and 9 metabolites have been identified and characterized in human serum; breast milk; and several organs, including the breast, lung, liver, cervix, colon, skin, and prostate. Of all the organs studied, the prostate contains the highest concentration of lycopene. Experimentation in vitro demonstrated that cis lycopene is absorbed more readily than all-trans-lycopene. The role of cis versus trans lycopene in human physiology has not yet been determined. In a trial of 32 men with diagnosed prostate cancer, supplementation with 30 g/d tomato sauce resulted in a tripling of total lycopene in the prostate. Two other studies concluded that dietary intervention and supplementation with 15 mg lycopene and smaller quantities of other tomato carotenoids, including phtyoene, phytofluene, -carotene, and

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-carotene twice daily positively altered serum markers of prostate cancer progression. Serum prostate specific antigen (PSA) levels, a marker of tumor activity, decreased in both trials, and tomato oleoresin supplementation altered biomarkers of cell growth and differentiation in the one study in which it is was tested. Cancer cells show a decrease in cellular differentiation, and are sometimes said to “revert back” to a more undifferentiated, embryonic-type cell. If tomato carotenoids can increase cellular differentiation, they may be important in the treatment of prostate cancer. The Mechanisms Accounting For Health Benefits Of Lycopene There are five mechanisms that researchers propose may account for the beneficial effects of tomato phytochemicals and their metabolites. These mechanisms may complement each other and have overlapping functions. Lycopene is the strongest antioxidant compared with other commonly consumed carotenoids. Decreased DNA damage has been reported in white blood cells after 15 days of supplementation with tomato and tomato juice. Second, lycopene alters the biotransformation of xenobiotics, which are pharmacologically, endocrinologically, or toxicologically active substances not produced by the body that must be metabolized to a different compound before being eliminated in the stool or urine. Xenobiotics are metabolized by two families of enzymes, called cytochrome P-450 enzymes, via two pathways, called phase I and phase II detoxification pathways. The study showed lycopene significantly induced phase I enzymes in a dose-dependent manner and doubled hepatic quinone reductase (QR), a phase II enzyme. Tomato flavonoids also affect these enzyme systems. Kaempferol and naringenin inhibit the cytochrome P450-IA enzyme, while quercetin inhibits this same enzyme while also increasing QR activity. Cooked tomatoes and lycopene alone alter hormone and growth factor signaling in prostate cells. This includes alterations in insulin-like growth factor-1 (IFG-1) activity. IFG-1 stimulates cellular proliferation and decreases apoptosis, which is a mechanism by which normal cell death happens. Cancer cells are said to be immortal—they proliferate indefinitely. Eating cooked tomatoes was associated with a 31.5% decrease in serum IGF-1 levels in a case-controlled study of 112 men. Beneficial alterations of IGF-1 concentrations or its ability to stimulate cell division have also been found in rats and in healthy men. An in vitro study showed lycopene and tomato polyphenols, including

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quercetin, kaempferol, and rutin, to interfere with IGF-1 signaling thus preventing the growth factor from stimulating cell proliferation. In a number of cancer cell lines, including breast cancer cells, endometrial cancer cells, and in normal prostate cells, lycopene halted cellular replication in vitro. Lastly, lycopene and its metabolites may help fight some cancers by increasing connexin 43 levels. Connexin 43 is a molecule involved in cell-to-cell communication, which is important in the regulation of uncontrolled, rapid cell growth. In a metastatic prostate cancer cell line (PC-3MM2), lycopene did not increase connexin 43; however, it did in another prostate cancer cell line (PC-3), a breast cancer cell line (MCF-7), and oral cancer cells (KB-1). The inhibition of connexin 43 in these cell lines was associated with an inhibition of cell growth, suggesting that upregulation of connexin 43 may be important to the anticancer action of lycopene. Since a synergistic effect appears to exist between tomato phytochemicals, recommending the consumption of supplements made from whole tomatoes and/or the consumption of 2 to 4 or more servings per week of tomatoes and tomato products may reduce the incidence of prostate cancer and health care costs in our aging population. GARLIC Garlic has been called Russian penicillin. It belongs to the Lily family. Garlic is not just spice, herb or vegetable but a combination of all the three. Americans consume <0.6 g/week compared with 16 g/day in parts of China with no side effects. It is a chemically complex herb containing more than 200 different compounds, 100 of which are sulfur compounds, which confer — at least anecdotally — a number of medicinal benefits. Antioxidants Found in Garlic Garlic has many functions. Some of sulfur containing compounds in garlic are E-ajoene, Z-ajoene, allicin, allixin, allyl mercaptan, allyl methyl sulfide, diallyl disulfide, diallyl sulfide, diallyl trisulfide, s-allyl cysteine and s-allylmercaptocysteine. Among these, the chief active ingredient is allicin. The characteristic flavor of garlic is due to presence of allicin (thiol-2-propene-1-sulfinic acid s-allylester) and oil soluble compounds formed when bulb is crushed or damaged. An enzyme alliinase is released which converts alliin to allicin (Figure 2.16) which is decomposed to high volatile sulfur compounds. The names and chemical structures of the principal organosulphur compounds present in different garlic preparations are summarized in Figure 2.17.

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FIGURE 2.16 — Conversion Of Alliin To Allicin

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FIGURE 2.17 — Major Organosulphur Compounds Found In Different Garlic Preparations Garlic preparations

Compounds

Garlic homogenate

Allicin

CH 2

S

S

CH 2

O Methyl-allyl Thiosulfinates

CH 2

S

S

CH 3

O 1- propenyl allyl thiosulfinate

CH 2

S

S

CH 3

O

L-glutamyl-S-alkyl-L-cysteine O

HO R

H N S

NH 2 HO

Heat treated garlic and garlic powder

O O

Alliin

H 3C

NH 2

S O

HO

O

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FIGURE 2.17 — Major Organosulphur Compounds Found In Different Garlic Preparations - Continued Aged garlic extract

S-allyl cysteine

H 2C

NH 2

S HO

O

S-allyl mercaptocysteine

O

HO H 2C

Steam distilled garlic oil

S

S

Diallyl disulfide

CH 2

S

S

CH 2

Diallyl trisulfide

CH 2

S

S

CH 2

S

Allyl methyl trisufide

CH 2

S

S

S

CH 3

Allyl methyl disulfide

CH 2

S

S

CH 3

Diallyl tetrasulfide

CH 2

S

S

S

S

CH 2

Allyl methyl tetrasulfide

H 2C

S

S

S S

CH 3

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FIGURE 2.17 — Major Organosulphur Compounds Found In Different Garlic Preparations - Continued Dimethyl trisulfide

H 3C

S

S

CH 3

S

Diallyl sulfide

H 2C

CH 2

S

Oil-macerated/ether-extracted garlic oil

2-Vinyl-4-H-1,3-dithiin S CH 2 S

3-Vinyl-4-H-1,2-dithiin S

S CH 2

E-ajoene

H 2C

S

S

S

CH 3

O Z-ajoene

H 2C

CH 2

S S

S

O

Major metabolite of raw garlic in blood

Allyl meracaptan

H 2C

SH

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Therapeutic Effects Of Garlic Research on the health benefits of garlic has tended to focus on fresh garlic powders, aged garlic extracts, distilled oils (from steaming), and other forms. Research studies on garlic discussed here cover a wide topical area, from cardiovascular benefits to garlic’s anticarcinogenic properties. Garlic’s benefits include the reduction of harmful LDL cholesterol and triglycerides; the prevention of blood clots and blood platelet clumping; the ability to block chemical carcinogens; the stimulation of various immunological factors that may help combat cancer as well as fungal infections; and the protection of cells against various oxidizing agents. The therapeutic effects of garlic can be grouped into four main areas: 1. Antimicrobial properties 2. Cardiovascular effects 3. Anticarcinogenic components 4. Other benefits Antimicrobial Properties Of Garlic These include antibacterial, antifungal, antiprotozoal and antiviral properties. Garlic was used as a remedy for indigestion and diarrhea, to destroy microorganisms for bacterial, fungal, and viral infections, and to expel intestinal worms and parasites before World War II. Allicin is responsible for these beneficial properties of garlic. Allicin is a strong antibiotic agent and 1 mg allicin is equivalent to the antibiotic activity of 15 I.U. of penicillin. Garlic is shown effective against both Grampositive and negative bacteria, Staphylococcus aureus, Salmonella typhi, E. coli, Listeria monocytogenes. Garlic has been reported to inhibit Aerobacter, Aeromonas, Bacillus, Citrella, Citrobacter, Clostridium, enterobacter, Escherichia, Klebsiella, Lactobacillus, Leuconostoc, Micrococcus, Mycobacterium, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus and Vibrio. A garlic oil preparation showed good antituberculosis activity in guinea pigs with a intraperitoneal dose of 0.5 mg/kg (Jain, 1993). As a result of the bactericidal activity of garlic, toxin production by the bacteria is also prevented (Dewitt et al., 1979). Garlic has been used in folk medicine for years to treat fungal and yeast infections. Several studies indicate allicin is the active compound in fighting fungal infections and preventing further growth. American researchers tested 139 different types of fungi and yeasts for sensitivity. After 21 days, some microorganisms grew while some showed no growth

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at all. These results indicate that the use of garlic in treating infections is very specific. The use of aged garlic extract (containing no allicin or allicin-derived compounds) was ineffectual in treating fungal and yeast infections. Russian researchers in the late 1920s, observed that the volatile constituents of fresh garlic and onion strongly inhibit and even kill various microorganisms. Ensuing experiments with ingredients of these plants, and later of other plants, were mainly performed in protozoa, especially Paramecium caudatum, and with eggs and larvae of mollusks and frogs. The favorable results prompted some to coin the descriptive word “phytoncides” for the antibiotically active components of higher plants. Although there is not much research dealing with garlic as a treatment against viruses, it has been used in China and India as a cheaper alternative to expensive Western medication. In Russia, the use of garlic is so widespread that it is known as “Russian penicillin.” During one acute influenza epidemic in Russia, the Soviet Union imported 500 tons of garlic cloves to treat it. In a patent filed in 1992, it is claimed that an allicin-urotropin treatment can be used against viral infections, including AIDS. Cardiovascualr Benefits Of Garlic Garlic may protect blood vessels from the negative effects of free radicals, may have a positive influence on blood lipids, increase capillary flow, and decrease elevated blood pressure levels if consumed in appropriate amounts. The combined result is that garlic can prevent arteriosclerosis or ameliorate an existing condition. Garlic’s cardiovascular effects were rediscovered in the late 1960s by Western scientists, but it has been used in Asian folk medicine for heart problems for years. Garlic has also been used in Europe for some time. Studies have repeatedly shown that garlic strengthens the heart and stimulates well being. Advocates’ claims that garlic prevents arteriosclerosis are based on studies conducted on thrombocyte adhesiveness and aggregation, which is reportedly decreased significantly by the effective constituents of garlic. At the same time, the dissolution of coagulated blood, plaques, and clots is enhanced. Garlic decreases the lipoproteins circulating in the blood as LDL (lowdensity lipoprotein). This occurs through the increased production of HDL (high-density lipoprotein) at the expense of LDL. Russian researchers reported on the cholesterol-reducing benefits of garlic. They noted that garlic indirectly affects artherosclerosis by

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reducing hyperlipidemia, hypertension, and likely, diabetes mellitus, and prevents thrombosis formation. They deduced that its effects are the result of garlic’s capacity to reduce lipid content in arterial cells and prevent intracellular lipid accumulation. The results from a collection of 40 studies with 43 groups of patients and volunteers show a mean decrease in serum cholesterol levels of 10.6%. The forms of garlic used in the studies were fresh garlic, garlic powder tablets, oils (predominantly ether-extracted garlic), and steam-distilled oils. They all showed decreases in cholesterol ranging from 4 to 16%. The steam-distilled oils had the lowest decrease, although the amounts used varied considerably, and the effect increased as the dose increased. The lipid-lowering effect is attributed to the active compound allicin. Additionally, 33 human studies were conducted using garlic cloves and garlic powder tablets. Three of those studies showed no effect on lipid lowering, and the reason given is that the study used tablets that contained either no allicin potential (spray-dried) or low allicin potential (Koch and Lawson, 1996). Clinical trials with garlic oils which contain only allicin-derived garlic compounds plus added vegetable oils have lowered serum lipids. Anticarcinogenic Functions Of Garlic For decades, garlic has been touted as very effective against various forms of cancer. A study conducted 60 years ago concluded that the occurrence of cancer is lower in countries that have high garlic and onion consumption. The general belief is that garlic is effective as a cancer-inhibitor due to its ability to prevent putrification of the intestines and activate secretions, leading to an increase in the body’s resistance. Garlic’s ability to activate gastric secretions makes it a possible agent for preventing gastrointestinal cancer. Gastric cancer is the major cancer in the developing world and one of the top two worldwide. Helicobacter pylori is a bacterium implicated in the etiology of stomach cancer. The incidence of stomach cancer is lower in individuals with a high intake of allium vegetables in developed and developing (high risk) countries. Because allium vegetables, particularly garlic, have antibiotic activity, the antimicrobial activity of garlic against H. pylori was investigated by Sivam et al (1997). An aqueous extract of a known variety of garlic (Oswego white) was used. The extract was standardized for thiosulfinate concentration. The minimum inhibitory concentration was found to be 40 µg/ml. At this concentration, the control organism Staphylococcus aureus was not inhibited by the garlic extract. Thus H. pylori is more susceptible to garlic extract. Cellini et al (1996)

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reported a similar study. They tested 16 clinical isolates of H. pylori and showed 90% inhibition of the isolates with aqueous garlic extract at 5 mg/ ml. The concentration used in that study is the total weight of garlic per milliliter. However, calculations show that the minimum inhibitory concentrations reported in the two studies are comparable. It is plausible that the sensitivity of H. pylori to garlic extract at such a low concentration may be related to the reported lower risk of stomach cancer in those with a high allium vegetable intake. The inhibitory concentration of garlic reported in the two studies above is achievable in the stomach by consuming a medium size clove of garlic or equivalent amount of garlic supplements. Thus, this finding may identify a strategy for low-cost intervention for stomach cancer. A wide variety of components in garlic work synergistically to provide various health benefits. Garlic cloves contain a limited number of organosulfur compounds (OSCs), such as alliin and -glutamyl-Sallylcysteine. However, processing initiates a cascade of chemicals conversions of OSCs. Processed garlic preparations contain both oiland water-soluble OSCs and their exact chemical makeup is significantly affected by the processing method, i.e., different processing methods yield not only different preparations but also different constituents with differing effectiveness and safety. Aged garlic exhibits antioxidative activities whereas other preparations may stimulate oxidation. Other Benefits a. Immunomodulatory effects: In vitro and animal studies suggest that garlic may play an important role in enhancing the immune system. The human studies are encouraging, because they show a positive link between garlic and enhanced immunoreactions and phagocytosis. A study conducted on rats revealed garlic’s antiarthritic effect when used in the form of commercial steamdistilled garlic oil. The effect of garlic was synergistically increased when boron was added to the diet. Other studies showed garlic extract’s necrosis-inhibiting effect along with the compounds of allicin and diallyl disulfide. b. Hypoglycemic effects: Studies conducted from the 1920s to the 1970s show that the consumption of onions and garlic can lower elevated blood glucose concentrations. In 1924, a study by Mahler and Pasterny gave two diabetic patients 10-15 g of fresh garlic sliced and distributed to them throughout the day in three

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portions. Blood sugar decreased and the urine sugar excretion in one patient was reduced from 3.2 to 1%, and from 3.8% to zero in the second patient. Garlic oil was also found to increase insulin and urea levels and to change the activities of key liver enzymes in ethanol-fed rats. The results of another study showed that the glucose level in the blood was lowered by garlic, while the concentrations of glycogen level in the liver and insulin in the blood increased. In a study with diabetic rats, the compound allicin was shown to exert significant antidiabetic effects. c. Hormone-like effects: In older medical literature, garlic is mentioned to confer hormone-like effects. Aphrodisiac properties are linked with garlic because of the belief that it stimulates male and female sex hormones. Garlic is also assumed to stimulate the pituitary gland, which in turn influences other glands and has an effect on fat and carbohydrate metabolism. d. Enhancement of thiamin absorption: In 1924, the Medical Academy in Paris confirmed garlic’s effectiveness for scurvy. In the 1800s, the Japanese established garlic’s effectiveness for beriberi. e. Effects on organic and metabolic disturbances: Garlic is ideal for self-medication. In the 1980s, researchers noticed that people in their mid-40s started using garlic as a preventive tool in strengthening their defense mechanisms. Garlic contains many unknown factors that block enzymes and modify the functions of biological membranes. Most studies conducted on garlic to determine these compounds and their effectiveness were conducted in vitro and must be viewed with caution. Allicin is known to be metabolized very rapidly in blood to allyl mercaptan. f. Antihepatotoxic effects: Studies on rats showed that several known compounds in garlic were able to provide adequate liver protection. In livers with high amounts of lipid peroxides and accumulation of triglycerides, levels were significantly decreased in the animals treated with garlic extract. An aged garlic extract proved to be effective in controlling hepatopathy and liver damage induced by hepatoxins during acute hepatitis. g. Dyspepsia and indigestion: Throughout the ages, garlic has been used in the treatment of stomach and intestinal troubles. The resistance of the people in the Balkans to all kinds of intestinal diseases is credited to their high consumption of garlic. Garlic

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53

oil activates higher secretions of hydrochloric acid and digestive enzymes in the stomach because of its stimulating effect on the mucous membrane of the stomach. The effect of dried garlic on gastrointestinal motility by means of roentgenography was investigated and demonstrated the elimination of flatulence and meteorism, gaseous colic, and nausea, in clinical experiments by Koch and Lawson (1996). h. Respiratory diseases: Recent reports indicate that garlic may help people with respiratory dysfunction, including problems due to high altitude. Third-World countries have successfully used garlic for years to treat such respiratory ailments as tuberculosis, bronchiectasis, gangrene of the lungs, and whooping cough. The former Soviet Union and Bulgaria also use garlic to treat flu and inflammation of the throat. Mechanism Of Action Of Allicin On Microbes Thiosulfinates play an important role in the antibiotic activity of garlic. Hughes and Lawson (1991) showed that the antimicrobial activity of garlic is completely abolished when the thiosulfinates (e.g., allicin) are removed from the extract. Also, upon reduction of allicin to diallyl disulfide, the antibacterial activity is greatly reduced (Reuter et al., 1996). Feldberg et al (1988) showed that allicin exhibits its antimicrobial activity mainly by immediate and total inhibition of RNA synthesis, although DNA and protein syntheses are also partially inhibited, suggesting that RNA is the primary target of allicin action. The structural differences of the bacterial strains may also play a role in the bacterial susceptibility to garlic constituents (Tynecka and Gos, 1975). The cell membrane of Escherichia coli contains 20% lipid, whereas that of Staphylococcus aureus contains only 2% lipid. The lipid content of the membranes will have an effect on the permeability of allicin and other garlic constituents. On the basis of this hypothesis, it is interesting to recall the difference in susceptibility that was observed between gramnegative H. pylori (40 µg/ml) and gram-positive Staphylococcus aureus (>160 µg/ml) to garlic extract (Sivam et al., 1997). TEA The tea plant Camellia sinensis is native to Southeast Asia but is currently cultivated in more than 30 countries around the world. Tea has been used as a daily beverage and crude medicine in China for thousands of years. Tea is consumed worldwide, although in

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greatly different amounts; it is generally accepted that, next to water, tea is the most consumed beverage in the world, with per capita consumption of about 120 ml per day. Of the total amount of tea produced and consumed in the world, 78% is black, 20% is green, and less than 2% is oolong tea. Black tea is consumed primarily in Western countries and in some Asian countries, whereas green tea is consumed primarily in China, Japan, India, and a few countries in North Africa and the Middle East. Oolong tea production and consumption are confined to southeastern China and Taiwan. Green, black, and oolong teas undergo different manufacturing processes. To produce green tea, freshly harvested leaves are rapidly steamed or pan-fried to inactivate enzymes, thereby preventing fermentation and producing a dry, stable product. For the production of black and oolong teas, the fresh leaves are allowed to wither until their moisture content is reduced to about 55% of the original leaf weight, which results in the concentration of polyphenols in the leaves. The withered leaves are then rolled and crushed, initiating fermentation of the polyphenols. During these processes, the catechins are converted to theaflavins and thearubigins. Oolong tea is prepared by firing the leaves shortly after rolling to terminate the oxidation and dry the leaves. Antioxidants in Tea The tea plant contains many kinds of polyphenols, catechins being particularly prolific. Catechins belong to those groups of compounds generically known as flavonoids, which have a C 6-C 3-C 6 carbon structure and are composed of two aromatic rings. Currently, the tea plant is known to contain seven kinds of major catechins and traces of various other catechin derivatives. They are divided into two classes: the free catechins, (+)-catechin, (+)-gallocatechin, (-)epicatechin, (-)-epigallocatechin; and the esterified or galloyl catechins, (+)-catechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-gallocatechin gallate (Figure 2.18). While the galloyl catechins are astringent with a bitter aftertaste, free catechins have far less astringency, leaving a slightly sweet aftertaste even at 0.1% aqueous solutions. These catechins are present in all parts of the tea plant; 15-30% are present in the tea shoots, and there is also a high content in the second and third leaves. Epicatechins are the main compounds in green tea, accounting for its characteristic color and flavor.

ANTIOXIDANTS

FIGURE 2.18 — Antioxidants In Tea

55

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FUNCTIONAL FOODS

Therapeutic Effects of Consuming Tea a. Anticarcinogenic effect: The relatively low rates of cancer found in Asian populations that regularly consume green tea have recently instigated hundreds of scientific studies. The results of the research suggest, time and time again, that tea is linked to preventing cancer in humans, including bladder, breast, colon, esophagus, pancreas, rectum and stomach cancers. Much of the cancer-preventive effects of green tea are mediated by epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea. One cup (240 ml) of brewed green tea contains up to 200 mg (EGCG). Many consumer products, including shampoos, creams, drinks, cosmetics, lollipops, and ice creams, have been supplemented with green tea extracts and are available in grocery stores and pharmacies. b. Antibacterial effect: Studies show the positive effects tea can have on oral health; scientific and medical findings show that tea fights the cavity-causing bacteria on teeth. Also, tea naturally contains fluoride, which protects teeth from cavities. c. Antiatherosclerotic effect: A group of studies suggests that heavy tea drinkers (those who drink two to three cups of either green or black tea daily) are 44 percent less likely than non-drinkers to die after having a heart attack. Also, the antioxidants in tea help prevent LDLs (“bad” cholesterol) from building up in the blood, making tea drinkers less likely to get heart disease (Mukamal et al., 2002). d. Anti-inflammatory and arthritis preventing effect: Recent studies report possible anti-inflammatory and arthritis-preventing effects of green tea. Case Western University scientists suggest green tea antioxidants postpone the beginning of and decrease in the severity of one type of arthritis in mice (Haqqi et al., 1999). e. Preventing weight gain: The antioxidant ECCG (epigallocatechin gallate) found in green tea helps the body burn fat. A study in Switzerland found that drinking the equivalent of two to three cups of green tea daily caused the participants to burn 80 extra calories each day, without increasing their heart rate and factoring out the tea’s caffeine content (Dulloo, 1999). f. Protection of liver: The research into the health benefits derived from drinking tea continues to expand. Some preliminary studies

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57

suggest that green tea helps protect the liver by triggering the immune system and by defusing the effects of harmful toxins such as alcohol and cigarette smoke. Other fruits and vegetables such as strawberries, cherries, nectarines, peaches, plums, prunes, apples, pears, banana, citrus fruits, mango, passion fruits, pomegranate, star apple, carrot, onions, parsnip, potato, red beetroot, sweet potato, asparagus, celery, endive, lettuce, spinach, swiss chard, avocado and pepper, beverages such as beer, coffee and cereals contain similar types of antioxidants. SYNTHETIC ANTIOXIDANTS Synthetic antioxidants are mainly phenolic, for example, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butyl hydroquinone (TBHQ), and the gallates. Polymeric antioxidants such as Anoxomer, Ionox-330, and Ionox-100, a derivative of BHT, have also been introduced, but they are not being used commercially. In general the use of primary antioxidants is limited to 100-200 ppm of BHA, BHT, or TBHQ or 200-500 ppm of the gallates for the stabilization of fats and oils. Commercially a number of ready-to-use formulations containing a food grade solvent (propylene glycol or glycerol monooleate), a synergist like citric acid, and one or more phenolic antioxidants are available. MECHANISMS OF ACTION OF ANTIOXIDANTS There are many mechanisms by which antioxidants protect food and human body including: • Scavenging reactive oxygen and nitrogen free radical species; • Decreasing the localised oxygen concentration thereby reducing molecular oxygen’s oxidation potential; • Metabolising lipid peroxides to non-radical products; • Chelating metal ions to prevent the generation of free radicals. Antioxidants exhibit specific benefits by limiting the free radical damages from: • Oxidising Low Density Lipoprotein (LDL) cholesterol, which may increase the risk of athersclerosis; • Promoting platelet adhesion, which can lead to thrombosis thereby increasing the risk of heart disease or stroke;

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• Damaging the cell’s DNA, which may lead to cancer; • Blocking the normal endothelial cell function and vasodilatation in response to nitric oxide, a potential mechanism for heart disease and cancer; • Triggering inflammation; • Impairing immune function. SUMMARY Autoxidation in food and biological systems has varied implications not only for human health and nutritional status but also for the vast area of food science and technology. Autoxidation of lipids and the generation of free radicals are natural phenomena in biological and food systems. However, when an excess of free radicals is formed, they can be responsible for the occurrence of many chronic diseases. The damaging effect of excessive free radicals can be prevented by dietary antioxidants. Antioxidants are substances when present in foods at low concentrations compared with those of an oxidizable substrate markedly delay or prevent the oxidation of the substrate. Antioxidants are classified as natural and synthetic antioxidants. They are also classified based on their chemical nature, function and site of synthesis. Sources of natural food antioxidants include most of the fruits and vegetables among which cranberries, grapes, and tomato are researched to a great extent. Apart from these garlic and tea are also rich in antioxidants. Cranberries are known to relieve the symptoms of urinary tract infections. It is a good source of anthocyanins, flavonol glycosides, proanthocyanidins and phenolic acids. Grape antioxidants are thought to protect the body from some cancers and heart diseases. Lycopene, the pigment responsible for red color of tomato, has been recognized as the most effective antioxidant among the carotenoids. Garlic is a combination of spice, herb and vegetable with many functions. It contains many sulfur containing compounds among which allicin is the chief active ingredient. Garlic is known to have antimicrobial properties, cardiovascular effects and anticarcinogenic components. Tea is another herb that is known for health promoting properties due to its richness in antioxidants catechins. Synthetic antioxidants that are commonly used in food industries are mainly phenolic which include BHA, BHT and TBHQ.

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References Ames, B. N., Shigenaga, M. K. and Hagen, T. M. 1993. Oxidants, Antioxidants, and the Degenerative Diseases of the Aging. Proc Natl Acad Sci USA; 90:7915-22. Aust, S. D. and Svingen, B. A. 1982. In: Free Radicals in Biology (Pryor, W.A., ed.), vol. 5, pp. 1-28. Academic Press, New York, NY. Avorn, J., Monane, M., Gurwitz, J. H., Glynn, R. J., Choodnovskiy, I. and Lipsitz L. A. 1994. Reduction of Bacteriuria and Pyuria After Ingestion of Cranberry Juice The J. Am. Med. Assoc. 271(10): 751-754. Barch, D. H., Rundhaugen, L. M., Stoner, G. D., Pillay, N. S., and Rosche, W. A. 1996. Structure-Function Relationships of the Dietary Anticarcinogen Ellagic Acid. Carcinogenesis 17(2), 265. Burger, O., Ofek, I., Tabak, M., Weiss, E. I., Sharon, N. and Neeman, I. 2000. A High Molecular Mass Constituent of Cranberry Juice Inhibit Helicobacter pylori Adhesion to Human Gastric Mucus. FEMS Immunol. Med. Microbiol. 29:295-301. Campbell J. K., Canene-Adams, K, Lindshield, B. L., Boileau, T. W. M., Clinton, S. K. and Erdman, Jr J. W. 2004. Tomato Phytochemicals and Prostate Cancer Risk. J. Nutr. 134:3486S-3492S. Cantos, E., Espin, J. C., and Tomas-Barberan, F. 2002. Varietal Differences Among Polyphenols Profiles of Seven Table Grape Cultivars Studied by LC-DAD-MS-MS. J Agric. Food Chem., 50:5691-5696. Cellini L., Di Campli E., Masuli M., Di Bartolomeo S. and Allocati N. 1996. Inhibition of Helicobacter pylori by Garlic Extract (Allium sativum). FEMS Immunol. Med. Microbiol. 13:273-277. Dewanto, V., Wu, X., Adom, K. K., and Liu, R. H. 2002. Thermal Processing Enhances the Nutritional Value of Tomatoes by Increasing Total Antioxidant Activity. J. Agric. Food Chem. 50:3010-3014. Dewitt, J. C., Notermans, S., Gorin, N. and Kampelmacher, E. H. 1979. Effect of Garlic Oil or Onion Oil on Toxin Production by Clostridium botulinum in Meat Slurry. J. Food Prot. 42:222-224. Dulloo, A. G., Duret, C., Rohrer, D., Girardier, L., Mensi, N., Fathiu, M., Chantre, P., and Vandermander, J. 1999. Efficacy of a Green Tea Extract Rich in Catechin Polyphenols and Caffeine in Increasing 24-Hour Energy Expenditure and Fat Oxidation in Humans. Am. J. Clin. Nutr. 70:1040-1045. FAOSTAT, 2004. Accessed at http://apps.fao.org/faostat/collections?version=ext&hasbulk=0&subset=agriculture. Feldberg R. S., Chang S. C., Kotik A. N., Nadler M., Neuwirth Z., Sundstrom D. C. and Thompson N. H. 1988. In Vitro Mechanism of Inhibition of Bacterial Growth by Allicin. Antimicrob. Agents Chemother. 32:1763-1768. Foo, L. Y., Lu, Y., Howell, A. B. and Vorsa, N. 2000a. The Structure of Cranberry Proanthocyanidins Which Inhibit Adherence of Uropathogenic P-fimbriated Escherichia Coli in Vitro. Phytochem. 54(2):173-181. Foo, L. Y., Lu, Y., Howell, A. B. and Vorsa, N. 2000b. A-type Proanthocyanidin Trimers From Cranberry That Inhibit Adherence to Uropathogenic P-fimbriated Escherichia Coli. J. Nat. Prod. Chem. 63(9):1225-1228. Fuleki, T. and da Silva, R. J. M. 1997. Catechin and Procyanidin Composition of Seeds From Grape Cultivars Grown in Ontario. J. Agric. Food Chem., 45:1156-1160. Halliwell, B. 1999. Antioxidant Defense Mechanisms: From the Beginning To The End (Of The Beginning). Free Radical Res. 31:261-272. Haqqi, T., Anthony, D. D., Gupta, S., Ahmad N, Kumar GK and Mukhtar H. 1999. Prevention of Collagen-induced Arthritis in Mice by a Polyphenolic Fraction Found in Green Tea. Immunol. 96(8): 4524-44529.

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Henig, Y. S. and Leahy, M. M. 2000. Cranberry Juice and Urinary-tract Health: Science Supports Folklore. Nutr. 16(7–8): 684–687. Ho, K.Y., Huang, J. S. Tsai, C. C., Lin, T. C., Hsu, Y. F., and Lin, C. C. 1999. Antioxidant Activity of Tannin Components From Vaccinium Vitis-idaea. L. J. Plumn. Pharmacol. 51(9), 1075. Howell, A. B., Leahy, M., Kurowska, E. and Guthrie, N. 2001. In Vivo Evidence That Cranberry Proanthocyanidins Inhibit Adherence of p-fimbriated E. coli Bacteria to Uroepithelial Cells. FASEB. J. 15:A284. Howell, A. B., Vorsa, N., Der Marderosian, A. and Foo, L. Y. 1998. Inhibition of The Adherence of p-fimbriated Escherichia coli to Uroepithelial-cell Surfaces by Proanthocyanidin Extracts From Cranberries. N Engl J Med. 339(15): 1085-1086. Hughes B. G. and Lawson L. D. 1991. Antimicrobial Effects of Allium Sativum L. (garlic), Allium Ampeloprasum (elephant garlic), and Allium Cepa (onion), Garlic Compounds and Commercial Garlic Supplement Products. Phytother. Res. 5:154-158. Jain, R. C. 1993. Antitubercular Activity of Garlic Oil. Indian Drugs 30:73-75 Karadeniz, F., Durst, R.W., and Wrolstad, R. E. 2000. Polyphenolic Composition of Raisins. J. Agric. Food Chem. 48:5343-5350. Koch, H. P. and Lawson L. D. (eds.). 1996. Garlic: The Science and Therapeutic Application of Allium sativum L. and Related Species, 2nd ed. Baltimore: Williams & Wilkins Publishing Co. Kontiokari, T., Sundqvist, K., Nuutinen, M., et al. 2001. Randomized Trial of CranberryLingonberry Juice and Lactobacillus GG Drink for the Prevention of Urinary Tract Infections in Women. Brit. Med. J. 322(7302): 1571–1573. Leahy, M., Roderick, R. and Brilliant, K. 2001. The Cranberry — Promising Health Benefits, Old and New. Nutr. Today 36(5): 254–65. Mazza, G. 1995. Anthocyanins in Grapes and Grape Products. CRC Crit. Rev. Food Sci. Nutr. 35:341-371. Mukamal, K. J., Maclure, M., Muller, J. E., Sherwood, J. B. and Mittleman, M. A. 2002. Tea Consumption and Mortality After Acute Myocardial Infarction. Circulation. 105:2476-2481. Ofek, I., Goldhar, J., Zafiri, D., Lis, H., Adar, R. and Sharon, N. 1991. Anti-Escherichia Adhesin Activity of Cranberry and Blueberry Juices. N. Eng. J. Med. 324(22):1599. Peng, Z., Hayasaka, Y.( Hand, P. G., Sefton, M., Hoj, P., and Waters, E. J. 2001. Quantitative Analysis of Polymeric Procyanidins (Tannins) From Grape (Vitis vinifera) Seeds by Reverse Phase High-Performance Liquid Chromatography. J. Agric. Food Chem. 49:26-31. Rao, A. V. and Agarwal, S. 2000. Role of Antioxidant Lycopene in Cancer and Heart Disease. J. Am. College Nutr. 19(5): 563-569. Reuter, H. D., Koch, H. P. and Lawson D. L. 1996. Therapeutic Effects and Applications of Garlic and its Preparations. In: Garlic: The Science and Therapeutic Applications of Allium sativum L. and Related Species. 2nd ed. (Koch, H. P. & Lawson, D. L., eds.), pp. 135–212. William & Wilkins, Baltimore, MD. Sivam G. P., Lampe J. W., Ulness, B., Swanzy S. R. and Potter J. D. 1997. Helicobacter Pylori—in vitro Susceptibility to Garlic (Allium sativum) Extract. Nutr. Cancer 27:118-121. Souquet, J. M., Labarbe, B., Le Guerneve, C, Cheynier, V., and Moutounet, M. 2000. Phenolic Composition of Grape Stems. J. Agric. Food Chem. 48:1076-1080. Stewart, A. J., Bozonnet, S., Mullen, W., Jenkins, G. I., Lean, M. E. J., and Crozier, A. 2000. Occurrence of Flavonols in Tomatoes and Tomato-based Products. J. Agric. Food Chem. 48:2663-2669. Stothers, L. 2002. A Randomized Trial to Evaluate the Effectiveness and Cost

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Effectiveness of Naturopathic Cranberry Products as Prophylaxis Against Urinary Tract Infection in Women. Can J Urol. 9:1558-1562. Torres, J. L. and Bobet, R. 2001. New Flavanol Derivatives From Grape (Vitis vinifera) Byproducts. Antioxidant Aminoethylthio-flavan-3-oI Conjugates From a Polymeric Waste Fraction Used as a Source of Flavanols. J. Agric. Food Chem. 49:4627-4634. Tynecka Z. and Gos Z. 1975. The Fungistatic Activity of Garlic (Allium sativum) in Vitro. Ann. Univ. Mariae Curie-Sklodowska Sect. D Med. 30:5-13. Vrhovsek, U. 1998. Extraction of Hydroxycinnamoyltartaric Acids From Berries of Different Grape Varieties. J. Agric. Food Chem. 46:4203-208. Walker, E. B., Barney, D. P., Mickelsen, J. N., et al. 1997. Cranberry Concentrate: UTI Prophylaxis. J Family Pract. 45:167–8 [letter]. Weiss, E. I., Lev-Dor, R., Kashman, Y., Goldhar, J., Sharon, N. and Ofek, I. 1998. Inhibiting Interspecies Coaggregation of Plaque Bacteria With a Cranberry Juice Constituent. JADA. 129:1719-1723. (Guo and Gokavi)

Chapter 3 DIETARY FIBER AND DIETARY FIBER RICH FOODS Introduction Dietary fiber (DF) has been consumed for centuries and most food labels in the supermarket now list dietary fiber. Even though fiber is not considered a nutrient, health professionals and nutritionists agree that fiber is required in sufficient amounts for the proper functioning of the gastrointestinal tract. DF is 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. The reductions in LDL-cholesterol, attenuating glycemic and insulin response, increasing stool bulk, and improving laxation have been associated with DF intake through the consumption of foods rich in this dietary component, such as vegetables, fruits, whole grains, and nuts. DF consumption has established the basis for associating high-fiber diets in epidemiological studies with reduced risk of most of the major dietary problems in the U.S.A.; namely, obesity, coronary disease, diabetes, gastrointestinal disorders, including constipation, inflammatory bowel diseases like diverticulitis and ulcerative colitis, and colon cancer (Jones, 2000). Despite the understanding of health benefits of DF and its association with reduced risk of many diseases, the intake remains low in many parts of the world, in particular in the U.S.A. One of the reasons for this may be the difficult challenge to increase fiber consumption in the diet. The fiber sources usually used in foods have not made high-fiber foods with acceptable sensory properties. A product development technologist who makes foods, using high fiber ingredients needs to realize that a product not only supply fiber, but also provide enhanced functional properties to make high-fiber foods taste better, thus encouraging continued intake of this type of product.

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Why is fiber important? What does fiber do? This chapter will answer these questions in detail. It is the purpose of this chapter to provide an overview of important oligosaccharides and polysaccharides that function as DF, to explain in detail their occurrence and structures and their various physiological effects and health implications, and also to describe the role high fiber ingredients play in food development. Definition Establishing a definition for dietary fiber has a long history. The term ‘dietary fiber’ was coined by Hipsley in 1953 and since then its definition has undergone several revisions. The history of the definition of DF is presented in Figure 3.1. While defining dietary fiber, it was intended to balance between nutritional knowledge and analytical method capabilities. While the physiologically based definitions most widely accepted have generally been accurate in defining the dietary fiber in foods, scientists and regulators have tended, in fact, to rely on analytical procedures as the definitional basis. As a result, incompatibility between theory and practice has resulted in confusion regarding the components that make up dietary fiber. In November 1998, the president of American Association of Cereal Chemists (AACC) International appointed a scientific review committee and assigned the task of reviewing, and if necessary, updating the definition of dietary fiber. The updated definition includes the same food components as the historical working definition used for almost 30 years. But the updated definition more clearly describes the makeup of DF and its physiological functionality. This definition typically includes the fiber components; nonstarch polysaccharides (NSP) and resistant oligosaccharides (RO), lignin, substances associated with the NSP and lignin complex in plants, and other analogous carbohydrates, such as resistant starch (RS) and dextrins, and synthesized carbohydrate compounds, like polydextrose (Tungland and Meyer, 2002). Finally, dietary fiber is defined as the edible parts of the plant and analogous carbohydrate that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. It includes polysaccharides, lignin and associated plant substances. Dietary fiber exhibits one or more of the following: laxation (fecal bulking and softening; increased frequency; and/or regularity), blood cholesterol attenuation, and/or blood sugar regulation.

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FIGURE 3.1 — History Of Definition Of Dietary Fiber Over The Years Organization

Year

Definition

Hipsley

1953

Coined term “dietary fiber” as a shorthand term for nondigestable constituents making up the plant cell wall.

Trowell and others

1972-1976

Used Hipsley term in conjunction with a dietary fiber hypothesis related to health observations. The term was defined as: “consisting of the plant polysaccharides and lignin which are resistant to hydrolysis by digestive enzymes of man.”

Asp, Schweizer, Furda, Theander, Bakker, Soutgate and others

1976-1981

Developed methods directed at quantifying food components meeting definition

Prosky

1979

Began process of developing worldwide consensus on fiber definition and methodology for dietary fiber

Canadian Association of Official Analytical Chemists Workshop

1981

Consensus on fiber definition and analytical approach

Prosky, Asp, Furda, Schweizer, DeVries and Harland

1981 -1985

Validate consensus methodology in multinational collaborative studies

AOAC

1985

Official Method of Analysis 985.29, Total dietary Fiber in Foods-Enzymatic-Gravimetric Method, adopted, becoming de facto working definition for dietary fiber

Health and Welfare Canada

1985

Defined dietary fiber as: “the endogenous components of plant material in the diet which are resistant to digestion by enzymes produced by humans. They are predominately nonstarch polysaccharides and lignin and may include, in addition, associated substances.

Scientific community

1985-1988

Developed methodology and collaboratively studied these for various types of fiber.

US-FDA

1987

Defined dietary fiber as the material isolated by AOAC method 985.29

Life Sciences Research Office (LSRO)

1987

Defined dietary fiber as: the endogenous components of plant materials in the diet that are resistant to digestion by enzymes produced by humans

Health Canada

1988

Defined (dietary fiber) as: being the endogenous components of plant material in the diet which are resistant to digestion by enzymes produced by man: they are predominately nonstarch polysaccharides and lignin. The composition varies with the origin of the fiber, and includes soluble and insoluble substances. Defined (novel fiber or novel source) as: (1) a food that has been manufactured to be a source of dietary fiber, and has not traditionally been used for human consumption to any significant extent, or (2) had been chemically processed (oxidized), or (3) had been highly concentrated from its plant source.

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FIGURE 3.1 — History Of Definition Of Dietary Fiber Over The Years - Continued Germany

1989

Defined fiber as: substances of plant origin, that cannot be broken down to resorbable components by the body’s own enzymes in the small intestine. Included are essentially soluble and insoluble nonstarch polysaccharides (cellulose, pectin, hydrocolloids) and lignin and resistant starch. Substances like some sugar substitutes, organic acids, chitin and so on, which either are not or are incompletely absorbed in the small intestine, are not included.

Lee, Mongeau, Li, Theander and others

1988-1994

Various fiber methodologies fitting definition of dietary fiber developed, validated and brought to an Official Method status

Japan

1990

Dietary fiber defined as: material isolated by a modified method of AOAC 985.29

AOAC

1991

Official Method of Analysis 991.42, Insoluble Dietary Fiber in Foods and Food Products, Enzymatic-Gravimetric Method-Phosphate Buffer, adopted.

International Fiber Survey

1992

Reaffirms consensus on physiological dietary fiber definition.

Belgium

1992

Defined dietary fiber as: the components of food that are not normally broken down by the body’s own enzymes of humans

International Fiber fiber Survey

1993

Reaffirmed consensus on physiological dietary

Italy

1993

Defined dietary fiber as: the edible substance of vegetable origin which normally is not hydrolyzed by enzymes secreted by the human digestive system

AOAC International

1995

Workshop on definition of complex carbohydrates and dietary fiber reaffirms consensus on physiological dietary fiber definition and inclusion components

FAO/WHO

1995

(Codex Alimertarius Commission) Defined dietary fiber as: the edible plant or animal material not hydrolyzed by the endogenous enzymes of the human digestive tract as determined by the agreed upon method. Approved AOAC methods 985.29 & 991.43.

China

1995

Defined dietary fiber as: the sum of food components that are not digested by intestinal enzymes and absorbed into the body

Denmark

1995

Defined dietary fiber as: the material isolated by AOAC methods 985.29 and 997.08 (fructan method)

Committee on Medical Aspects (UK)

1998

Defined dietary fiber as: nonstarch polysaccharide as measured by the Englyst method of Foods [Committee on Medical Aspects of Food and Nutrition Policy (COMA)]

Finland

1998

Defined dietary fiber as: part of the carbohydrate obtained using AOAC Methods 985.29 and AOAC 997.08.

definition and reaffirms inclusive components

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FIGURE 3.1 — History Of Definition Of Dietary Fiber Over The Years - Continued Norway

1998

Defined dietary fiber as: material isolated by AOAC Method 985.29 and inulin and oligofructose

AACC

1998

Assigns Scientific Committee to review and develop definition of Dietary Fiber

Sweden

1999

Defined dietary fiber as: edible material that cannot be broken down by human endogenous enzymes and determined with AOAC Methods 985.29 and/or 997.08 (fructan method)

Food Standards Agency (U.K.)

1999

Defined dietary fiber as: material isolated by AOAC methods 985.29 and 997.08 (fructan method)

AACC

2000

Defined dietary fiber 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 plant substances. Dietary fibers promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood glucose attenuation.

Australia New Zealand Food Authority (ANZFA)

2001

Following the lines of the AACC definition, defined dietary fiber as: that fraction of the edible part of plants or their extracts, or analogous carbohydrates, that are resistant to digestion and absorption in the human small intestine, usually with complete or partial fermentation in the large intestine. The term includes polysaccharides, oligosaccharides (DP > 2), and lignins. Dietary fiber promotes one or more of these beneficial physiological effects: laxation, reduction in blood cholesterol, and/or modulation of blood glucose. They accepted by use of AOAC methods 985.29 and 997.08 (fructan method) for labeling.

National Academy of Science (NAS)

2002

2002 Panel on the Definition of Dietary Fiber defined the dietary fiber complex to include dietary fiber consisting of nondigestible carbohydrates and lignin that are intrinsic and intact in plants, functional fiber consisting of isolated, nondigestible carbohydrates which have beneficial physiological effects in humans, and total fiber as the sum of dietary fiber and functional fiber.

Chemistry Of Dietary Fiber The physical properties of dietary fiber are predominated by the shape (conformation) of the individual chains, and the way in which they interact with one another. Each dietary fiber molecule typically contains several thousand monosaccharide units which are often arranged in a linear sequence, like a very long string of beads, although more complex branched arrangements also occur. In contrast to globular

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proteins, polysaccharides normally have structures based on regular repeating sequences. The simplest arrangement is where all the monosaccharides are the same, and are linked together in the same way along the chain. Disaccharide repeating consequences (-A-B-A-B-) are also common, and larger repeating units (up to octasaccharide) can occur, particularly in polysaccharides produced by bacteria. The constituent monosaccharides have a ring structure, which can be either five-membered or six-membered, and are linked together by ‘glycosidic bonds’ with a shared oxygen atom between adjacent sugars. The polysaccharides of greatest practical importance, both as commercial hydrocolloids and as constituents of dietary fiber, are built up from six membered (pyranose) rings consisting of one oxygen atom and five carbon atoms, which are numbered sequentially from the ring oxygen as C-1 to C-5, and with a sixth carbon atom, numbered as C-6, lying outside the ring. As a consequence of the tetrahedral bonding arrangement of carbon, and the requirement to avoid steric clashes between adjacent groups, the pyranose ring is locked in a fixed, chairlike geometry, and the overall shape of the polysaccharide molecule is dictated by the torsional angles characterizing the relative orientation of neighboring sugars. These angles may be either fixed at the same values for equivalent linkages along the polymer chain, giving regular, ordered chain geometry, or constantly fluctuating, to give the disordered ‘random coil’ geometry typical of polysaccharide solutions. The chemical structures of different dietary fibers are given in Figures 3.2 and 3.3. Physical Properties Of Dietary Fiber When considering the action of cooking on cell wall structure and comparing cooked and raw plant foods, the different solubility characteristics of cell wall polysaccharides should be considered. Cell wall structures are degradable to varying degrees, depending on the structure and the conditions used. An important function of insoluble fibers is to increase lumenal viscosity in the intestine. It is not yet clear whether the soluble fibers in food have the same effect. Other polymeric components of the diet (proteins, gelatinized starch) and mucus glycoproteins liberated from the epithelia contribute to viscosity. Particulate materials present in chyme, such as insoluble fiber or hydrated plant tissues, also contribute to a lesser extent to overall viscosity. Digesta viscosity is highly sensitive to changes in ionic concentration that are due to intestinal secretion or absorption of aqueous fluids. Raw apples undergo little damage of cells upon ingestion

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FIGURE 3.2 — Chemical Structures Of Starch And Other Polysaccharides

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FIGURE 3.3 — Chemical Structures Of Polyfructans

and mastication. Gastric hydrochloric acid only solubilizes a small proportion of the pectin. Cooking the apples results in cell damage, and hence significant proportions of the middle lamellae pectic polysaccharides are solubilized. These make the digesta more viscous. Vegetables undergo structural change during cooking and mastication, e.g., cellular disintegration. The cells in the intact carrot are each bounded by an intact cell wall; after cooking most, if not all, the cell walls have been ruptured and the cell contents lost. The grinding of

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foods before cooking and ingestion may also have pronounced effects on fiber action. Cell walls may be disrupted, and the reduced particle size of some fiber preparations such as wheat bran may be less biologically effective. The effects of other cooking processes, e.g., Maillard reactions, are not known. Controlled drying of a heated starch gel can produce any of the different X-ray diffraction patterns, depending on the temperature. On cooling, gelatinized starchy foods will retrograde. During retrogradation, solubility of the starch molecule decreases and so does its susceptibility to hydrolysis by acid and enzymes. Chain length and linearity are important factors affecting retrogradation. The longer the starch chains, the greater the number of interchain hydrogen bonds formed (Dobbing, 1989). Classification Of Dietary Fiber Several different classification systems have been used to classify the components of dietary fiber: based on their role in the plant, based on the type of polysaccharide, based on their simulated gastrointestinal solubility, based on the site of digestion, and based on products of digestion and physiological classification. However, none is entirely satisfactory, as the limits can not be absolutely defined. The most widely used classification for dietary fiber has been to differentiate dietary components on their solubility in a buffer at a defined pH, and/or their fermentability in an in vitro system using an aqueous enzyme solution representative of human alimentary enzymes. However, there is still debate regarding the most appropriate means to classify dietary fiber. Since most fiber types are at least partially fermented, it is suggested that it may be most appropriate to refer to them as partially or poorly fermented and well fermented. Classification Based On Solubility Based on solubility, dietary fiber is classified into two types – soluble and insoluble. Soluble fiber dissolves in water. This includes gums, mucilages, pectin and some hemicelluloses. These fibers are found in all types of peas and beans like lentils, split peas, pinto beans, black beans, kidney beans, garbanzo beans, and lima beans, as well as oats, barley, and some fruits and vegetables like apples, oranges, and carrots. Fiber from psyllium seed is also in this group. For people with diabetes, eating foods that contain soluble fiber can help control or lower the level of sugar in their blood and decrease insulin needs; and, studies have shown that including one or two servings of beans, oats, psyllium, or other sources of soluble fiber help

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lower fasting blood sugar levels. It may also help lower blood cholesterol levels, especially LDL-cholesterol or the “bad” cholesterol. Fiber decreases blood cholesterol by binding to bile acids, which are made of cholesterol, in the gastrointestinal tract and carrying them out of the body as waste. Researchers have found that soluble fibers in beans, psyllium fiber, oats, and oat bran help lower blood cholesterol levels in many groups of people. Insoluble fiber does not dissolve in water. Cellulose, lignin, and the rest of the hemicelluloses, are all insoluble fibers. These fibers provide structure to plants. Whole grains, wheat and corn fiber, and many vegetables like cauliflower, green beans, and whole potatoes are good sources of insoluble fiber. The skins of fruits and vegetables are also good sources of insoluble fiber. And, wheat bran is a good source of insoluble fiber, which is why it is added to many dry breakfast cereals. Insoluble fiber, also known as “roughage”, aids digestion by trapping water in the colon. The water that is trapped by insoluble fiber keeps the stool soft and bulky. This promotes regularity and prevents constipation. Wheat bran, for example is high in insoluble fiber, and also helps prevent two kinds of intestinal diseases, diverticulosis and hemorrhoids. Classification Based On Fermentability Fibers that are well fermented include pectin, guar gum, acacia (gum arabic), inulin, polydextrose, and oligosaccharides. The less wellfermented types include cellulose, wheat bran, corn bran, oat hull fiber, and some resistant starches. The fiber types based on fermentability are listed in Figure 3.4. Generally, well fermented fibers are soluble in water, while partially or poorly fermented fibers are insoluble. Classification Based On The Way The Monomeric Units Present Indigestible polysaccharides (fiber components) consist of all nonstarchy polysaccharides (NSP) resistant to digestion in the small intestine and fermentable in the large intestine. These polysaccharides are typically long polymeric carbohydrate chains containing up to several hundred thousand monomeric units. The polysaccharides differ by the number and type of monomeric units linked together, the order in the chain, the types of linkages between the various monomers, the presence of branch points in the backbone of the molecule, and those having acidic groups present (for example, uronic acids in pectins). Examples of these NSP compounds are cellulose with beta-glycosidic bonds, nonglucose sugars (hemicelluloses such as arabinoxylans and arabinogalactans), sugar acids (pectins), gums, and mucilages. Resistant

DIETARY FIBER

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FIGURE 3.4 — Classification Of Fiber Components Based On Fermentability Characteristic

Fiber component

Main food source

Partial or low fermentation

Cellulose

Plants (vegetables, sugar beet, various brans) Cereal grains Woody plants Plant Fibers Fungi, yeasts, invertebrates Plants (corn, potatoes, grains, legumes, bananas) Bacterial fermentation

Hemicellulose Lignin Cutin/suberin/other plant waxes Chitin and chitosan, collagen Resistant starches Curdlan Well fermented

β-glucans Pectins Gums

Inulin Oligosaccharides/analogues

Animal origin

Grains (oat, barley, rye) Fruits, vegetables, legumes, sugar beet, potato Leguminous seed plants (guar, locust bean), seaweed extracts (carrageenan, alginates), plant extracts (gum acacia, gum karaya, gum tragacanth), microbial gums (xanthan, gellan) Chicory, Jerusalem artichoke, onions, wheat Various plants and synthetically produced (polydextrose, resistant maltodextrin, fructooligosaccharides, galactooligosaccharides, lactulose) Chondroitin

oligosaccharides, such as the fructans [inulin and fructooligosaccharides (FOS)] (Figure 3.4) are characterized as carbohydrates with a relatively low degree of polymerization (DP), as compared to the NSP. FOS differ from fructopolysaccharides (inulin) only in chain length. The strict definition of an oligosaccharide is a chain of monomeric units with a DP of 3-10. Lignin is a phenylpropane polymer, and not a carbohydrate that is covalently bound to the fibrous polysaccharides (cellulose) of plant cell walls. Lignin has a heterogeneous composition ranging from 1 or 2 units to many phenyl propanes that are cyclically linked. It is likely these two characteristics have established the basis for it being included as a dietary fiber. Another group of compounds, found in several physiological definitions, the analogous carbohydrate(s), refer to compounds that are

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analogous to those of naturally-occurring dietary fibers. These compounds demonstrate the physiological properties of the respective materials for which they are analogous to, but are not obtained by eating the whole or part of the native originating plant, such as fruits, vegetables, grains, legumes, and nuts. They can be produced during food processing by chemical and/or physical processes, or by purposeful synthesis or isolation as a concentrated form from the native plant. These “analogous” carbohydrates can include, but are not limited to, those isolated from Crustacea and single-cell organisms, polydextrose, resistant maltodextrins and starch, and the modified celluloses. Resistant starch (RS) is defined as the sum of starch and starch products of starch degradation that is not broken down by human enzymes in the small intestine of healthy individuals. A classification of these starches based on the origin of their resistance to digestion has been proposed by Englyst et al (1992). Resistant starch is not a homogenous entity, but rather the resistance is dependent on a number of natural or processing phenomena which make up the subcategories RS1, 2, 3, and 4. RS1 relates to resistance conferred due to physical entrapment of starch, as found in partly milled grains or chewed cereals, seeds, or legumes. RS2 includes starch granules that are highly resistant to digestion by alpha-amylase until gelatinized. This form is typically found in raw or uncooked potato, banana (particularly when green), and high amylose maize starch. RS3 relates to the retrograded starch polymers from food processing of the above mentioned sources. RS4 includes chemically modified, commercially produced resistant starches that are likely degraded by amylases to alcohol soluble fractions and are used in many baby food applications. RS may have the similar health benefits as dietary fiber. Also included in the fiber component list are the associated plant substances, such as waxes and cutin. These components are found as waxy layers at the surface of the cell walls, made up of highly hydrophobic, long chain hydroxy aliphatic fatty acids. Suberin, another one of these associated substances, even though not fully characterized, is speculated to be a highly branched, crosslinked molecule containing polyfunctional phenolics, polyfunctional hydroxyacids, and dicarboxylic acids, having ester linkages to the plant cell walls. Analysis Of Dietary Fiber Adoption of the proposed definition for regulatory, research, and nutrition purposes will result in little change of analytical methodology, food labels, or food databases from the current situation. While several

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methods have been developed for analyzing dietary fiber, two primary methods are now used for content labeling: enzymatic gravimetric methods (for example, the AOAC procedure), and enzymatic chemical methods (for example, the Englyst and Southgate procedures). The AOAC procedures primarily measure NSP, lignin, and a portion of RS, as does the Southgate method, while RS and lignin are not measured by the Englyst method. Due to method limitations of these primary methods, other, more specific, methods must be used to measure other components of dietary fiber, such as inulin, FOS, RS, and lignin. Current methodologies will continue to accurately quantitate the amount of fiber in the majority of foods, the exception being those foods containing a significant amount of dietary fiber which is soluble in a solvent mixture of 4 parts alcohol and 1 part water. This exceptionally soluble dietary fiber has heretofore been excluded from the quantity of dietary fiber reported on food labels and entered into database(s) for analytical, as opposed to definitional, reasons. Additional methods, or adjustments to current methods, which assure inclusion of the exceptionally soluble dietary fiber, will increase the reported dietary fiber level of a few foods, particularly foods high in fructans such as onions and leeks. Methods accurately fitting the definition will minimize regulatory confusion and result in accurate nutrition labeling of food products. Method Requirements Adoption of the definition for dietary fiber, i.e. “Dietary fiber is the remnants of the edible part of plants and analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the human large intestine. It includes polysaccharides, oligosaccharides, lignin and associated plant substances. Dietary fibers exhibit one or more of either laxation (fecal bulking and softening; increased frequency; and/or regularity), blood cholesterol attenuation, and/or blood glucose attenuation,” will result in relatively few method changes or changes in food labels or food databases. Analytically inclusive components fitting this definition include cellulose, hemicellulose, lignin, gums, mucilages, oligosaccharides, pectins, waxes, cutin, and suberin. Analytical methodology useful for food labeling needs to effectively quantitate all of these components, while excluding all other food components. The analytical method also must quantitate the dietary fiber using a set of standardized conditions which will convert the food to the state of the food as it is most likely to be consumed. That is, the method should not

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quantitate “resistant starch” as dietary fiber merely because the starch is resistant to digestion because it is ungelatinized as it is found in the food product as labeled and sold, when there is a chance it will be cooked prior to consumption. Thus, a starch gelatinization step is necessary in any method developed for dietary fiber analysis as is a sample digestion step with enzymes that simulate the human digestion system to the closest extent possible in the laboratory. Applicable Methods In the 1981 definition, “Dietary Fiber consists of the remnants of edible plant cells, polysaccharides, lignin and associated substances resistant to (hydrolysis) digestion by the alimentary enzymes of humans” as in the proposed definition, dietary fiber is the remnants of the edible parts of plants resistant to digestion in the human small intestine. This resistance to digestion was, and remains, the key focus of the analytical method requirements. The first Official Method of Analysis developed based on the 1981 consensus definition was AOAC 985.29. This method is based on the premise of resistance to digestion. Human digestive enzymes are known to digest fats, proteins, and starch. Using 985.29, the food samples are defatted, then heated to gelatinize the starch (the primary form of starch in foods as consumed), then subjected to enzymatic digestion by protease, amylase, and amyloglucosidase (glucoamylase) to remove the digestible components of the food. The residues are quantitated, and adjusted for protein and ash to assure against a protein contribution from the enzymes, and assure that inorganic materials present in the sample are not quantitated as dietary fiber. The enzymes utilized for starch and protein digestion are required to completely digest representative starch and proteins. The method and the enzymes must also pass a purity of activity test to assure against extraneous enzymatic activity, i.e. to assure that the method does not destroy, and the enzymes do not digest any of the dietary fiber components listed above. Substrates to use to assure against extraneous enzymatic activity are listed in the referenced table and section. Other AOAC Official Methods of Analysis and AACC Approved Methods of Analysis adopted since that time have the same or similar method performance requirements, and are listed in Figure 3.5. Additional Methods Requirements Since the time of the adoption of the consensus definition in 1981, and the adoption of Official Method of Analysis 985.29 in 1985, dietary fiber research has expanded dramatically. This expanded knowledge

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FIGURE 3.5 — Official And Approved Methods For Dietary Fiber Analysis AOAC Official Method of Analysis

AACC Approved Method of Analysis

Designation

Title

Designation

Title

AOAC 985.29

Total Dietary Fiber in Foods Enzymatic-Gravimetric Method

AACC 32-05

Total Dietary Fiber

AOAC 991.42

Insoluble Dietary Fiber in Foods and Food Products Enzymatic-Gravimetric Method, Phosphate Buffer

AACC 32-20

Insoluble Dietary Fiber

AOAC 991.43

Total, Soluble, and Insoluble Dietary Fiber in Foods Enzymatic-Gravimetric Method, MES-Tris Buffer

AACC 32-07

Determination of Soluble, Insoluble and Total Dietary Fiber in Foods and Food Products

AOAC 992.16

Total Dietary Fiber, Enzymatic-Gravimetric Method

AACC 32-06

Total Dietary Fiber Rapid Gravimetric Method

AOAC 993.19

Soluble Dietary Fiber in Food and Food Products, Enzymatic-Gravimetric Method (Phosphate Buffer)

AOAC 993.21

Total Dietary Fiber in Foods and Food Products with <2% Starch, Nonenzymatic Gravimetric Method

AOAC 994.13

Total Dietary Fiber (Determined as Neutral Sugar Residues, Uronic Acid Residues, and Klason Lignin) Gas Chromatographic Colorimetric-Gravimetric Method (Uppsala Method)

AACC 32-25

Total Dietary Fiber Determined as Neutral Sugar Residues, Uronic Acid Residues, and Klason Lignin (Uppsala Method)

AACC 32-21 Insoluble and Soluble Dietary Fiber in Oat Products Enzymatic Gravimetric Method

includes the discovery of “resistant starch,” expanded knowledge of the physiological and chemical properties of fructans, including inulin, and the technical capabilities to produce edible carbohydrate-based polymers that are analogous to dietary fiber in their digestive and fermentative behaviors. The analytical methodology adopted in 1985 depends upon the fiber fraction isolated being insoluble in a mixture of 4 parts alcohol and 1 part water. This 4:1 solvent mixture is a traditional chemical means of separating simple sugars and other compounds from the more complex starches and proteins in the samples prior to the analysis of

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the simpler compounds. In the early 1980s, the 4-part alcohol, 1-part water solvent mixture was believed adequate for precipitating and isolating the dietary fiber from the enzyme digestion media. It is now evident that this mixture is not sufficient for the isolation of all dietary fibers, and additional methods need to be used in conjunction with AACC 32-05 (AOAC 985.29) or their equivalents to address those fibers not precipitated. Fructan(s), because of the conformation of the molecule(s), are nearly 100% soluble in the 4-part alcohol, 1-part water mixture. As a result, they will not be isolated as part of the precipitate using 985.29 or equivalent methods. Fructans are part of the “remnants of the edible part of plants that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the human large intestine.” Because fructans are not isolated as part of contemporary methodology, the recently adopted AOAC Official Method of Analysis 997.08, Fructans in Food Products, Ion Exchange Chromatographic Method (AACC Proposed Method of Analysis 32-21), or AOAC 999.03, Measurement of Total Fructan in Foods, Enzymatic/Spectrophotometric Method (AACC Proposed Method 32-32), must be used. In addition, a small amount of inulinase enzyme must be added during the enzymatic digestion steps of the contemporary methods to digest the small amount of fructan that co-precipitates with the rest of the fiber to avoid duplicate quantization. Fructans are nonexistent, or occur in small quantities in most foods such as whole grains, fruits, and vegetables which are consumed in significant quantity. It is likely that there will be little impact on the food labels of these foods on a per-serving basis. A few foods, such as onions and leeks, contain high levels of fructans, so the food label of these products may need to be adjusted slightly on a per-serving basis when the inulin content is added to the fiber quantized by contemporary methodology. Polydextrose, like fructans, is also nearly 100% soluble in the 4-part alcohol, 1-part water mixture, due to the highly branched nature and relatively low molecular weight of the molecule. No significant amount of polydextrose is measured as dietary fiber by AOAC Official Method 985.29 or equivalent, therefore AOAC Official Method of Analysis 2000.11, Polydextrose in Foods by Ion Chromatography, has recently been approved. For foods that contain polydextrose, this method can be used as an adjunct to AOAC 985.29 or equivalent methods in order to determine polydextrose as dietary fiber. Advancing technical capabilities now allow the production of edible

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carbohydrate-based polymers that are analogous to dietary fiber in their digestive and fermentative behaviors. Since it is impossible to completely predict the analytical behavior of these analogous carbohydrates relative to the behavior of naturally occurring dietary fibers, methods for the analysis of other, currently available, analogous carbohydrate materials, or for those that may be developed in the future cannot currently be prescribed. It will be good if those involved with the research and production of such materials who are best equipped with the knowledge and resources develop appropriate analytical methods for their respective materials when used as an ingredient, and quantized when used as part of a food product. Subsequent to the adoption of Official Method of Analysis 985.29, researchers discovered that, in some foods, primarily processed grain products, a small percentage of the starch becomes resistant to the enzymatic digestion procedure of the method. This starch is truly resistant to digestion, resisting digestion in the human intestine (actually, additional quantities of starch typically pass into the large intestine with the resistant starch) and during the analytical processes for quantizing dietary fiber. In addition, starch in other foods also resists digestion, either because it is in a granular form, or because it has retrograded into a digestion-resistant crystalline domain. For labeling purposes, it is not clear what portion of this starch, if any, should be considered as dietary fiber. In some cases, the resistant starch is a component of a not fully ripened plant material. In other cases it is the result of incomplete cooking, or of heating and cooling the food product. In any of these cases, there is no consistent means of producing data for labeling purposes. Less than ripe plant materials can be ripened, or can be at various stages of ripeness when consumed. Less than fully cooked, or heated and cooled products can be cooked, or reheated or at various stages of cooling and crystallization, and the quantity of resistant starch changed before consumption. Therefore, for labeling purposes, utilizing the standardized methodology of AOAC 985.29 or equivalent provides the most reliable and accurate assessment of the quantity of digestively resistant starch that can consistently be delivered to the consumer at the time of consumption. For research purposes, other definitions for resistant starch and other methods for the quantization of the resistant starch thus defined may be suitable. But for labeling purposes, the starch that is truly resistant to digestion in a method that standardizes the treatment of the sample to simulate the likely state of the food at the time of consumption and digests the sample with enzymes that simulate the human small intestine is in order.

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DIETARY FIBER METABOLISM IN GASTROINTESTINAL TRACT Physicochemical Characteristics And Physiological Effects The gastrointestinal tract is the primary area of action of dietary fiber, most notably in the large intestine. The physiological effects of dietary fiber depend on a myriad of variables, but generally they depend on the type (partially fermentable or highly fermentable), the dose of a specific fiber consumed, the composition of the entire fiber-containing meal, and the individual physiological profile of the subject consuming the fiber-containing meal. However, the major physiological effects of dietary fiber originate from the interactions with colonic content throughout its fermentation. Through its varying physicochemical properties, dietary fiber intake influences several metabolic processes, including the absorption of nutrients, carbohydrate and fat metabolism, and cholesterol metabolism. It further has influence on colonic fermentation and affects the production of stools. In the large intestine, dietary fiber influences the colonic structure and barrier function, and as the large intestine encompasses a significant body of the human immune system, it is also likely to have influence on elements of immune function. Dietary fibers, as mentioned previously, differ in their water solubility and can have rheological effects; many well fermented fibers form viscous solutions in the gut (for example, guar gum), but notable exceptions are gum arabic and inulin. Some form gels (pectins), while others have a high water holding capacity (WHC) (for example, cellulose). A changed rheology of the intestinal contents can also have physiological effects. A high viscosity is generally connected with a delayed gastric emptying and increased small intestinal transit time. Fibers with a high WHC (that is, partially fermented fibers) can directly influence the volume and bulk of the intestinal content. The solubility of a fiber is related to its fermentability. Almost all of the fiber sources are fermented to some degree by the resident microorganisms present in the colon. The most notable exceptions are cellulose derivatives, such as carboxymethyl cellulose (CMC), which is soluble but almost nonfermentable by the human colon flora. The physical properties of fiber having primary influence on physiology are its dispersibility in water or WHC; its intestinal bulk due to nondigestibility; its ability to increase viscosity (rheology change) as associated with the more fermentable fiber component; its ability to adsorb or bind bile acids; and its fermentability by microorganisms in the gut.

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Colonic Fermentation And Its Consequences The large intestine plays a role in managing and conserving water and electrolytes, to further the digestion of residual material passing from the small intestine, and provides a route for residual, nondigestible material and toxins to pass. The large intestine is the most heavily colonized region of the digestive tract, with up to 1011 to 1012 anaerobic bacteria for every gram of intestinal content (Gibson and Roberfroid 1995). These bacteria produce enzymes that further the digestion of proteins and carbohydrates (fiber) passing undigested from the small intestine. Many variables can influence the extent of the fiber fermentation and, consequently, the nature and amount of the various end products produced from the fermentation, including gases (methane, hydrogen, carbon dioxide), short chain fatty acids (SCFA) (C2-C4 organic acids), and an increased bacterial mass. The extent of fermentation typically ranges from completely fermented (many watersoluble fibers) to little fermentation, for example, cellulose particles. However, of the many factors influencing the extent of fermentation, the primary influence is the physicochemical nature of the fiber. Green et al (1998), while working with human fecal slurries and different fiber sources, observed significantly different levels of SCFA and gas produced from the fiber sources. Botham et al (1998) further noted that the degree of fermentation and concentration of the various end products, particularly the SCFA, change due to the chemical structure and nature of the fiber source. Cellulose composed of β-1,4-linked Dglucose is hardly fermented, whereas starch, which has
-1,4 and
-1,6 linked D-glucosyl residues, is much more susceptible. It is also clear that amylose film shows limited fermentation, due to the limited accessibility to fermentative enzymes, whereas an amylose gel having better access for enzymes is fermented to a greater extent. Increases in microbial mass from fiber fermentation contribute directly to stool bulk, which is a large part of the stool weight. Bacteria are about 80% water and have the ability to resist dehydration, and thus contribute to water-holding in fecal material. Gas production from colonic fermentation can also have some influence on stool bulk. Trapping of gas can contribute to increased volume and a decrease in fecal transit time. WHC of dietary fiber was originally thought to be an important element for maintaining water content of stool. However, water content of stool is relatively constant at about 25%, and the WHC of dietary fiber likely does not have a direct influence on stool bulk. Bourquin et al (1996) reported that the dispersibility and WHC of fiber determine the ability of microorganisms to penetrate undigested food

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and degrade the fiber for growth. As such, WHC has an indirect relationship to stool bulk through its influence on the fermentation of fiber. Hence, fiber sources having high WHC, such as gums or pectin, generally tend to be more readily fermented than those with lower WHC, such as wheat bran, that contain higher levels of insoluble cellulose material. Poorly fermented fibers, such as cellulose, exert an indirect stool bulking effect that causes shorter fecal transit times, a greater fecal mass, and laxation effects. Several researchers working with various fiber sources containing both poorly fermented and well fermented fiber, such as wheat and barley bran, cellulose, soy fiber, and inulin, have shown stool bulking/laxation related effects (Causey et al., 2000). In addition, different fiber types can yield different fermentation products. Poorly fermented cellulose produces very little acid during its fermentation, most of which is only acetic acid; by contrast, in the case of more fermentable fibers, large quantities of SCFA are formed, including propionic, butyric, and acetic acids, in varying proportions. Thus, within a given gut microbiota environment, it may be possible to manipulate several of the key variables of the fermentation, notably the feeding of different fiber sources and combinations of fiber, to manipulate the specific types and amount of the SCFA (Silk et al., 2001). The potential to modify the amounts and distribution of the SCFA and the site of their production in the colon may be important, as each has different physiological influence, with varying implications on human health. In addition, as various gut microorganisms have specificity for fibers of different chemical structure, utilizing fiber sources, such as inulin, that promote bifidobacteria more selectively can provide a means to modify colonic microbiota to a more healthy balance, thereby potentially increasing host health. The metabolic end products of fermentation including the gases, SCFA, and increased microbiota, play a pivotal role in the physiological effects of fiber and implications for local effects in the colon and systemic effects. The gases produced from fiber fermentation by strict anaerobic species, such as bacteriodes, some nonpathogenic species of clostridia and yeasts, anaerobic cocci, and some species of lactobacilli, are mostly released as flatulence or are absorbed and subsequently lost from the body through the lungs. However, some of the hydrogen and carbon dioxide produced from these microorganisms may be further metabolized to methane (CH4) by methanogenic bacteria, thus reducing intestinal gas pressure. Of these anaerobic microorganisms, the clostridia, eubacteria, and anaerobic cocci are the most gas producing, while the bifidobacteria are the only group of the

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common gut microbiota that do not produce any gases. The SCFA resulting from the fermentation process provide a certain amount of energy from their metabolism in the liver. The energy content of a fiber is, from a scientific standpoint, dependent on the degree of fermentation. Fibers that are not fermented to any extent have a caloric content approaching 0 kcal/gram, while data from caloric studies indicate that the average energy yield from dietary fiber fermentation in monogastric species is in the range of 1.5 to 2.5 kcal/g (Smith et al., 1998). It should be noted that legal values for nutritional labeling can be different. The primary SCFA generated by fermentation are acetate, propionate and butyrate, accounting for 83 to 95% of the total SCFA concentration in the large intestine, which ranges from about 60 to 150 mmol/L. The concentrations of these acids are highest where concentrations of microbiota are also highest, namely in the cecum and transverse colon. Corresponding to these higher acid levels, the pH is also typically lowest in the transverse colon (5.4 to 5.9) and gradually increases through the distal colon to 6.6 to 6.9. At the colonic level, the fermentation of fiber increases the concentrations of these health-promoting SCFA and endogenous microbiota, exerting potential health effects, such as inhibiting the growth of pathogens, increasing mineral absorption, or producing vitamins. The SCFA absorbed into the portal blood system and reaching the liver and kidneys can further influence metabolism. This can lead to systemic effects, such as changes in glycemia, lipidemia, uremia, and overall nitrogen balance. Influence on lipids is an example of a potential health effect; a high serum lipid level is connected with a increased risk of cardiovascular disease. This risk may be lowered by the consumption of fermentable fibers. PHYSIOLOGICAL FUNCTIONS OF DIETARY FIBER Dietary Fiber And Cancer Colon cancer is one of the leading causes of cancer morbidity and mortality among both men and women in the Western countries, including the U.S.A. Historical observational and epidemiological studies from around the world have long supported that increased consumption of fruits and vegetables and high fiber intake provide a protective relationship between dietary fiber intake and colon cancer incidence (Byers, 2000). Of particular interest is the utilization of fermentable fiber by the colonic microbiota that can result in changes to the numbers and types

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of bacteria and, more importantly, changes to their metabolic activities in terms of the formation of genotoxins, carcinogens, and tumor promoters. Reddy (1999) emphasized synergistic effect when both probiotics and prebiotics are used together for possible mechanistic effects for cancer inhibition. Selective prebiotic fiber sources, such as inulin, resistant starches, and some oligosaccharides, act as selective substrate for bacteria that produce specific SCFA and can lower the intestinal pH. The SCFA butyrate has been shown to increase apoptosis in human colonic tumor cell lines (Scheppach, 1998). Possible mechanisms for the anticarcinogenic and antitumorigenic effect of highly fermentable fibers are not completely understood and require further research. However, it is likely that some or all are involved in a metabolic chain reaction for the inhibitory effect to occur. The primary mechanisms involved with these effects are proposed to be: a reduction in the production of carcinogenic substances by decreasing the amount of pathogenic bacteria in the colon; and/or lowering the colonic pH to affect pH-dependent enzymatic reactions; for example, secondary bile acid formation; and/or reducing the amount of carcinogenic substances available to colonic mucosa by adsorption of the substances to the cell wall of the microorganisms, by speeding up the intestinal transit time and by increasing colonic contents and thus diluting all components; and/or exerting inhibiting effects on initiation and promotion stages in colon cancer formation in which SCFA, particularly butyric acid, may play a key role. Dietary Fiber And Carbohydrate Metabolism An association between insufficient dietary fiber intake and increased risk of diabetes has been postulated since 1970s. While a direct linkage between insufficient dietary fiber intake and diabetes has not been established, evidence that indicates decreased risk of the disease with increased dietary fiber consumption continues to grow (Chandalia et al., 2000). Well fermented viscous fibers, either as part of a food or as a supplement that is well mixed with food, appear to offer the greatest potential benefit to reduce glycemic response and to increase insulin sensitivity. Glycemic Index And Glycemic Load The glycemic index (GI) is a numerical system of measuring how much of a rise in circulating blood sugar a carbohydrate triggers—the higher the number, the greater the blood sugar response. So a low GI food will cause a small rise, while a high GI food will trigger a dramatic spike. A GI of 70 or more is high, a GI of 56 to 69 inclusive is medium,

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and a GI of 55 or less is low. The glycemic load (GL) is a relatively new way to assess the impact of carbohydrate consumption that takes the glycemic index into account, but gives a fuller picture than does glycemic index alone. A GI value indicates only how rapidly a particular carbohydrate turns into sugar. It doesn’t show how much of that carbohydrate is in a serving of a particular food. It is necessary to know both to understand a food’s effect on blood sugar. That is where glycemic load comes in. The carbohydrate in watermelon, for example, has a high GI. But there isn’t a lot of it, so watermelon’s glycemic load is relatively low. A GL of 20 or more is high, a GL of 11 to 19 inclusive is medium, and a GL of 10 or less is low. Epidemiologic evidence suggests that a diet with a high glycemic load or glycemic index may increase the risk of type 2 diabetes. The beneficial physiological effects of viscous fiber sources on blood glucose concentrations have been consistently demonstrated over the last 2 decades. The mechanisms explaining the influence fibers have on reducing postprandial glycemia and fiber’s potential for enhancing carbohydrate metabolism over a longer term remain unclear. However, most likely causes for these influences are those related to small intestinal viscosity and nutrient absorption, and systemic effects from colonic-derived SCFA. Relative to postprandial glycemia, fibers that provide high viscosity in the small intestine (for example, guar gum, pectin) generally offer greater effect. By contrast, the SCFA, produced in the colon from well fermented fiber (for example, inulin), likely influence hepatic cholesterol synthesis, the production of glucose and its utilization later in the day (Luo et al., 1996). As the small intestinal transit times for mixed meals is relatively long (about 6 h), the colonicderived SCFA likely do not explain the acute effects of slowing small intestinal carbohydrate absorption typical of the postprandial effects following the intake of viscous well fermented fibers. The SCFA, namely, acetate, produced from fiber fermentation and absorbed into the peripheral blood may influence systemic metabolic functions. Acetate may influence serum fatty acid levels, which may directly inhibit adipose tissue lipolysis. Studies involving acetate, whether derived from fiber fermentation during chronic feeding studies, or given orally, or given by rectal infusion have all shown acetate to reduce serum fatty acid levels. But none has shown acetate to improve carbohydrate tolerance. However, propionate is gluconeogenic in the liver, potentially increasing blood glucose levels. Even though a clear mechanism for fiber effects on carbohydrate metabolism is yet undefined, the beneficial effects in support of high fiber intake for type 2 diabetes mellitus continue to

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increase. While the reduced small intestinal absorption may play a greater role in this effect than the colonic effects, further research is needed in the area of specific fiber sources and carbohydrate tolerance to more fully elucidate potential synergistic effects from SCFA. Dietary Fiber, Lipid Metabolism, And Cardiovascular Disease Total serum cholesterol and low-density-lipoprotein (LDL) cholesterol levels are generally accepted as biomarkers, indicative of potential risk for developing the disease. As such, research has primarily focused on their reduction as a means to reduce the risk of developing cardiovascular disease (CVD). Substantial experimental data support that blood cholesterol can be lowered using well fermented fiber types that produce relatively high viscosity, and epidemiological evidence supports the relationship between higher dietary fiber intake and reducing the risk of cardiovascular disease (Anderson et al., 2000). They concluded that high-fiber diets may protect against obesity and CVD by lowering insulin levels. A meta-analysis of 67 controlled studies using viscous well fermented fibers (oat-25, psyllium-17, pectin-7, and guar gum-18) showed reduction in serum cholesterol with higher rather than lower fiber levels (Brown et al., 1999). Less viscous fiber sources, like inulin, have generally shown consistent lipid lowering in animal studies, but human studies have shown variable results (Boeckner et al., 2000). When considering the results of human lipid studies, a number of factors need to be addressed, including: individual variation, duration of administration of the fiber source, fermentation rates of the various fiber types and chain fractions, intakes of dietary fat and carbohydrate in the background diet; and prior serum lipid levels. Mechanisms for cholesterol-lowering ability of water-soluble fiber have been suggested, but no consensus has been reached. Studies indicate effects on cholesterol formation due to certain well fermented fibers, such as psyllium, citrus pectin, oat bran, and rolled oats (Anderson et al., 1984), bind bile acids increasing their excretion and decrease cholesterol in the liver. Certain fibers, such as pectins and galactomannans that have high WHC and generate higher viscosity, also have influence on small intestine absorption of nutrients. It has also been suggested that the hypocholesterolemic effect of dietary fibers might also be mediated by the SCFA from fiber fermentation. SCFA are absorbed from the colon; butyrate and propionate are extracted by the colonic mucosa and liver, respectively, whereas acetate reaches peripheral circulation. Propionate primarily is reported to inhibit fatty acid metabolism, which plays a key role in the synthesis of cholesterol (Demigne et al., 1995). Kok et al

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(1998) postulated that lower glucose and insulin levels found after feeding inulin at a dose of 10% to rats contributed to reduced hepatic fatty acid and triglyceride synthesis. This would positively influence lipid metabolism by increasing the secretion of intestinal hormones, namely, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). These gut hormones are known to regulate postprandial insulin release and also to have direct insulinlike actions on lipid metabolism. Dietary Fiber, Mineral Bioavailability And Bone Health Certain fiber sources from fruits and vegetables that have cation exchange capacity from unmethylated galacturonic acid residues and phytic acid from cereal fibers, have been found to depress the absorption and retention of several minerals. However, certain highly fermentable fibers have resulted in improved metabolic absorption of certain minerals, such as calcium, magnesium, and iron, even when phytic acid is present at lower concentrations (Lopez et al., 1998). These compounds include pectin, various gums, resistant starches, cellulose, certain oligosaccharides like soy and fructooligosaccharides, inulin, lactulose, and related sugars. Mineral absorption has generally been accepted as stemming from diffusion in the small intestine. However, studies now indicate that highly fermentable fibers, such as inulin and fructooligosaccharides, also promote mineral absorption in the colon. Through their fermentation by colonic microbiota and subsequent SCFA production, these fiber components stimulate the proliferation of epithelial cells in the ceco-colon and reduce the luminal pH (Younes et al., 1996). The SCFA and lower pH may, in turn, dissolve insoluble mineral salts, especially calcium, magnesium, and iron, in the luminal content and increase their diffusive absorption via the paracellular route. In particular, the accumulation of calcium phosphate in the large intestine and the solubilization of minerals by SCFA are likely to play an essential role in the enhanced mineral absorption in the colon. Also, a recent study has demonstrated that fructooligosaccharides (inulin) stimulates the transcellular route of calcium absorption in the large intestine, as indicated by increased concentrations of calbindin-D9k, a calcium binding protein that plays an important role in intestinal calcium transport (Ohta et al., 1998). Dietary Fiber, Nitrogen Utilization And Bile Acid Metabolism Fiber also has influence on nitrogen balance. Fiber acts as substrate for increased microbial mass, which utilizes high fecal nitrogen and

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creates a marked enlargement of the cecum. The SCFA produced from fermentation of fiber and their associated lowering of colonic pH provide an added effect by protonating potentially toxic ammonia (NH3) to produce ammonium ion (NH4+), a form that is nondiffusible into the portal blood system (Younes et al., 1995). The consequence of this process is higher nitrogen retention in the cecum, increased fecal nitrogen excretion, lower blood ammonia levels, and decreased uremia. Studies in both animals and humans have shown that fecal nitrogen excretion is increased during consumption of a high soluble fiber diet (Vanhoof and De Schrijver 1996). Nitrogen balance, however, is not compromised due to a concomitant decrease in renal nitrogen excretion, likely due to a strong transfer of urea nitrogen to the intestine, to depress the plasma uremia. This shift does not appear to alter protein bioavailability, and seems more evident when the dietary protein level is moderate. The acidification of the luminal content by the SCFA may also potentially modify the metabolism of bile acids. Of particular interest is reducing the conversion of primary to secondary bile acids, as these are believed to be associated with increased risk of colon cancer. Dietary Fiber, Role In Gut Barrier Function And Gastrointestinal Disorders The health of the large intestinal wall and its microbial ecosystem play a key role in gastrointestinal health. Through its fermentation, dietary fiber and SCFA are important elements for both protecting the health of the large intestinal wall and stimulating repair in a damaged colon. The health of this organ is very important as it, in addition to serving as a primary site for water reabsorption, plays an important role as a major immune organ and functions as a barrier to prevent foreign materials from dietary or microbial origin from crossing into the internal body cavity. The integrity of the intestinal barrier changes with stress, starvation, and in several clinical situations where the gut is damaged; for example, Crohn’s disease, celiac sprue, extensive burn injury, antibiotic therapy, parasites, rheumatoid arthritis and indomethacin-associated enteritis, and intestinal obstruction (Lipman, 1995). Many of these disorders result indirectly from the loss of barrier function and are directly related to bacterial translocation. Various researchers showed that specific well fermented fibers, such as inulin, encourage the growth of healthpromoting bacteria and minimize the growth of pathogens and the

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production of subsequent harmful byproducts of protein degradation (that is, ammonia, phenolic products, amines, and N-nitroso compounds), which have been linked to various types of cancer and ulcerative colitis (Birkett et al., 1996). Further, researchers working with animals and in vitro testing have suggested a role of various fibers on intestinal immune function (Meyer et al., 2000). The SCFA, from fiber fermentation, particularly butyrate, play a key role in the health of the colon. They are suggested to be of influence in both stimulation of cell division and regulation of apoptosis (Wasan and Goodlad, 1996). Unlike the small intestine, which can derive energy from various endogenic sources, such as glutamine from muscle breakdown or ketone bodies from hepatic ketogenesis, during periods of starvation, the colon epithelial cells derive their energy from SCFA, particularly butyrate. Butyrate, is a preferred nutrient for colonocytes, prevents colonic mucosal atrophy, which develops within a day of oral starvation. Intestinal permeability or “leaky gut syndrome” is viewed as an indicator for sub-clinical disease states. As gut barrier function is lost, the formation of chronic bowel inflammation may appear, since exposure of the mucosa to luminal antigens can be responsible for inflammation. In order to keep the colonocytes healthy and minimize potential for chronic bowel inflammation, it is important to maintain a positive balance between crypt cell growth and luminal cell loss by apoptosis or sloughing (Lynn et al., 1994). While different fiber types have different effects on epithelial cell growth, it is generally accepted that more fermentable fibers have greater influence on cell growth, as being promoted via SCFA production. Poorly fermented fibers, such as cellulose, appear to maintain muscle layer, independently from the mucosal layer, while a fermentable substrate is required for mucosal layer growth. Poorly fermented fibers may also influence colonocyte proliferation by direct abrasive action (Folino et al., 1995). The mucus layer is important to the gut lining, provides lubrication and protects the gut from enzymatic acid and toxin attack. It is also food for several microbial species, helps remove microorganisms, and further serves as an antioxidant (Satchithanandan et al., 1996). Highly fermentable fibers appear more likely than poorly fermentable fibers to alter intestinal mucus composition, either through their direct mechanical effects or indirectly by regulating mucosal metabolism via SCFA derived from fiber fermentation.

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FUNCTIONAL FOODS

PROPERTIES AND PHYSIOLOGICAL EFFECTS OF SELECTED NON DIGESTIBLE POLYSACCHARIDES (NDP) AND NON DIGESTIBLE OLIGOSACCHARIDES (NDO) An overview of many different NDPs, their chemical makeup and occurrence, and a summary of the effects on the human intestine is given in Figure 3.6. A description of the physicochemical structures of selected NDOs and NDPs will provide a further understanding of possible physiological effects of these isolated dietary fiber ingredients based on human studies. Resistant Starch Resistant starch is part of our daily diet or can be added as an ingredient to various food products. The colonic fermentation of resistant starch in humans is well documented (Heijnen et al., 1998). Stool bulking effects for resistant starch were found as well, probably as a consequence of the increased bacterial mass in the feces. Further, consumption of resistant starch (and/or its subset resistant maltodextrin) may also stimulate the growth of specific bacteria purported to provide beneficial health effects, that is, the bifidobacteria and lactobacilli. Potential health benefits of resistant starch, such as blood sugar and cholesterol control, weight control and energy management, gut disorders, and colon cancer were reported by Baghurst et al (1996). Fermentation of resistant starch gives rise to relatively high levels of butyric acid, which, as mentioned earlier, may have implications in tumorigenesis and gut barrier health. Animal model studies show that resistant starch inhibits chemically induced carcinogenesis in the colon of rats (Sakamoto et al., 1996). Pectins Pectins are present in plant cell walls, and are polysaccharides with
-1, 4-linked D-galacturonic acid backbone with a variable amount of neutral sugars (arabinose, galactose, xylose) present as side-chains. Rhamnose may also be present in the backbone. The galacturonic acid residues are substituted with acetyl and methyl ester groups. The main applications for pectins as food additives are as gelling and thickening agents in many food products. Most pectins for food use are extracted from citrus or apple. Due to their gelling behavior, these soluble polysaccharides may decrease the rate of gastric emptying and influence small intestinal transit time. This explains their hypoglycemic properties. Various human studies show that pectins are fermented to a large extent in the colon.

By alkaline deacetylation of crustacean chitins. Found in fungi cell walls of Zygomycetes sp. Extracted from plants (wood pulp, bamboo, wheat, cottonseed hulls)

2-amino-2-deoxy-βD-glucose

β-1-4-D-glucose

Chitosan

Microcrystalline Cellulose

Produced by fermentation using Pseudomonas elodea

Produced by fermentation using Xanthomonas campestris

β-1-4-D-glucose (backbone), β-D-glucuronic acid, D-rhamnose

β-1-4-D-glucose (backbone), β-D-glucuronic acid, β-D-rhamnose

Gellan gum

Xanthan gum

Chemical reaction of cellulose with caustic followed by reaction with substituting reagent

Produced by fermentation using Alcaligenes faecalis var. myxogenes

β-1-3-D-glucose

Curdlan (insoluble β-glucan)

Modified cellulose Various functional groups (MC, CMC, MHPC) substituted for OH- of cellulose

Cereal (barley, oats)

Occurrence & Production

β-1-4-D-glucose and β-1-3-D-glucose

Composition

β-glucan

Name

Increases water holding capacity, increased fecal bulk, partially fermented in human gut.

Increases fecal excretion of neutral steroids. Interferes with intestinal absorption of cholesterol.

Increased water holding capacity

Fermented in large intestine, strong butyrate production, blood lipid effects

Human Physiological Effects

Adds viscosity, fermented to short-chain fatty acids in human gut Pastry fillings, sauces Adds viscosity, fermented to and gravies, salad short chain fatty acids in dressings, dairy products, human gut beverages, puddings

Icings, fillings, dessert gels, low-sugar jams and jellies, puddings and confections

Toppings, fillings, icings, Partial fermentation in the breadings, soups, sauces, human gut gravies, baked goods, biofilms

Dressings and sauces, beverages, baked goods, whipped toppings

High viscosity limits food uses. Decreased hepatic cholesterol and triglycerides

Processed meat, poultry, seafood products, noodles and pasta, sauces and dressings desserts, biofilms, jellies

Breakfast cereals, functional food products

Food Applications

FIGURE 3.6 — Commercial Nondigestible Polysaccharides

DIETARY FIBER 91

Not in nature, in Japan by transglucosylation of glucose.

Cyclic molecules of
-1-4 linked D-glucose, (
-cyclodextrin-hexamer, β-cyclodextrin-hetamer & g-cyclodextrin-octamer)

Mixture of β-1-6 linked D-glucose oligomers

Mixture of
-D-glucose

β-Cyclodextrins

Gentiooligosaccharides (GeOS)

Glucooligosaccharides (
-GOS)

By transglucosidation using an
-glucosidase from Leuconostoc mesenteroides

Not in nature

4-O-β-D-glucopyranosyl-D -glucose [(4-O-β-D-glucopyranosyl)n -D-glucose]

Not in nature, degradation of cellulose

Produced by pyrolysis of corn starch with HCl, further enzymatic hydrolyzed

Cellobiose (CEL) Cellodextrins

Cellobiose and cellodextrins

Mixed and random linkages,
-1-4 and 1-6 glucosidic bonds from starch and 1-2 and 1-3 bonds, from transglucosidation

Resistant maltodextrin

By vacuum thermal polymerization of glucose, sorbitol and citric acid

Occurrence & Production

Mixed and random glycosidic linkages, (1,6-bonds)-D-glucose

Composition

Polydextrose

Name

Sauces, salad dressings, ice cream, frozen desserts, partially hydrolyzed used as highly soluble fiber

No information

No information

No information

Nutritional beverages, functional foods, low viscosity fiber source

Low calorie, bulking agent for sugar replacement in foods (confections, desserts, dairy, baked goods, fillings)

Food Applications

Animal studies show influence of intestinal microbiota. No human studies

Scarce data, no conclusion on effects on intestinal microbiota

May have effect based on fermentation, no published studies on effects

No studies on prebiotic effects

Partially digestible, laxation and colonic fermentation, effects on lipid levels

Fermented to produce microbiota, SCFA stool bulking and stool softening in intestine

Human Physiological Effects

FIGURE 3.6 — Commercial Nondigestible Polysaccharides - Continued

92 FUNCTIONAL FOODS

Composition

Dried exudates from stems and branches of African bush Acacia senegal

β-D-galactose (backbone), L-arabinose, L-rhamnose, D-glucuronic acid (highly branched)


-1-4-D-galacturonic acid backbone/neutral sugar side chains, some ester groups

Acacia (gum Arabic)

Pectin

Fruits and vegetables (apples, citrus, sunflowers, sugar beet)

Extracted from red algae, Dairy products, Rhodophyceae (Gelidium sp. confectionery, baked and Gracilaria sp.) products, meat analogues, desserts

β-1-3-D-galactose and 3,6-anhydro-β-L-galactose

Agar

Jams and jellies, low-sugar or sugar free jams and jellies, beverages, milk products, biofilms

Spray dried flavors, confectionery products, jellies, (dry mixed puddings, desserts cake mixes)

Decrease gastric emptying and small intestine transit time (hypoglycemic properties) Fermented in large intestine No effects on stool weight, decrease in serum cholesterol

Fermented in the human gut. Prebiotic

Adds viscosity, fermented in the human gut to short chain fatty acids

Adds viscosity, decreases gastric emptying and small intestine transit time (hypoglycemic properties) Fermented in large intestine to short chain fatty acids

Both animal and human studies indicate influence of intestinal microbiota

Human Physiological Effects

Dairy products, bakery products, dessert gels, processed meat, low-sugar jams and jellies

Extracted from red algae (Rhodophyceae)

Mixture of sulfated polysaccharides made up of
-D-galactose & 3, 6-anhydro-D-galactose

Carrageenan

Dairy products, bakery products, dessert gels, processed meat, low-sugar jams and jellies

Food Applications

Linear
-1-6 linked glucose residues, some 1-4 linkages, 6-O-
-D-glucopyranosyl-Dglucose, 6-O-
-Dglucopyranosyl-(1-6)-
-Dglucopyranosyl-D-glucose, 6-O-
-D-glucopyranosyl(1-6)-
-D-glucopyranosyl(1-4)-D-glucose

Occur in miso, soy sauce, sake and honey. Prepared commercially by transglucosylation of glucose residue. By transglucosidase (
-glucosidases)

Occurrence & Production

Isomaltose - IMA Isomaltose - IMT Panose - PAN

Isomalto-oligosaccharids

Name

FIGURE 3.6 — Commercial Nondigestible Polysaccharides - Continued

DIETARY FIBER 93

Human Physiological Effects

O-D-galactopyranosyl(1-4)-O-β-Dgalactopyranosyl-(1-4)D-glucopyranose

Not in nature, produced in Japan by action of Crytococcus laurentii on lactose

Functional foods in Japan

Unaffected by human enzymes. Likely to have effect on composition and metabolic activity in human intestinal microbiota

A study in rats indicates effects on composition and metabolic activity of intestinal flora, no human data are available

4’-galactosyllactose (GLL)

Functional food development in Japan

4-O-β-D[galactopyranosyl]nD-sorbitol

Lactitol oligosaccharides (LTOS)

Not in nature, Transgalactosylation of lactitol using Aspergillus oryzae β-galactosidase

Not in nature, alkaline Drug status in EU, Gas production is relatively isomerization of glucose not approved for food use large, due to fermentation by moiety of lactose to fructose, clostridia, Kl. Pneumoniae. marketed as laxative and Studies suggest effects on gut health aid bacterial composition and activity

Studies indicate intestinal flora effects on composition and activity

4-O-β-D-galactopyranosylD-fructose

Sugar replacement, bulking agent, functional foods, prebiotic

Sugar replacement, May have significant effect on bulking agent, functional composition and activity of foods, prebiotic intestinal flora

Food Applications

Lactulose (LAT)

Extraction from soybeans, legumes

Occurrence & Production

Likely some in nature, enzymatic transgalactosylation of lactose

RAF, O-
-Dgalactopyranosyl-(1-6)-
D-glucopyranosyl-β-Dfructofuranoside

Composition

β-galactoβ-D-galactopyranosyl-(1-6) oligosaccharides or -[β-D-galactopyranosyl]ntransgalactoolig(1-4)
-D-glucose osaccharides (TOS)


-galactooligosaccharides (raffinose, stachyose/other soy oligosaccharides)

Name

FIGURE 3.6 — Commercial Nondigestible Polysaccharides - Continued

94 FUNCTIONAL FOODS

Composition

Levan-type

Fructans

Neogalactobiose (NGB) Isogalactobiose (IGB) Galsucrose (GAS) Isolactose I (IL1) Isolactose II (IL2) Isolactose III (IL3)
lactose trimer

β-D-(2,6)-fructofura-nosyl)n
-D-glycopyranoside

β-D-galactopyranosyl-βD-glucopyranoside β-D-galactopyranosyl-
D-glucopyranoside
-D-galactopyranosylβ-D-glucopyranoside
-D-galactopyranosyl-βD-fructofuranosyl-(2-6)-βD-fructofuranoside

Synthetic galactooligosaccharides

Name

Produced by Bacillus polymyxa on sucrose

Chemically by the Koenigs-Knorr reaction

Occurrence & Production

No primary commercial application

Proposed for functional foods

Food Applications

Data indicate that addition of inulin-type fructans affect the bacterial composition and the metabolic pattern of the intestinal microbiota. Studies have shown production of short chain fatty acids and a relatively high production of propionate and butyrate, necessary for colonic health and systemic influences on blood glucose and lipids

No animal or human data exists. Unknown if these products are digested in the upper GI tract

Human Physiological Effects

FIGURE 3.6 — Commercial Nondigestible Polysaccharides - Continued

DIETARY FIBER 95

Composition

β-1-4-D-mannose (backbone), Guar gum (Cyamopsis Sauces, salad dressings,
-1-6-D-galactose tetragonolobus), locust ice cream, frozen desserts, bean gum (Ceratonia siliqua) partially hydrolyzed used as highly soluble fiber

Galactomannan residues

Galactomannan (guar gum, locust bean gum)

Guar gum oligosaccharides

Produced by partial hydrolysis of guar gum

Functional foods

Fermented by colon microbiota. Lipid lowering, plasma glucose lowering

Readily fermented in human gut with bifidogenic effects, improves bowel function, shows hypolipidemic effects reduces postprandial glycemia

Fat/sugar replacement, Data indicate that addition of texture modification. inulin-type fructans affect the Dressings and sauces, bacterial composition and the beverages, baked products, metabolic pattern of the fillings, icings, frozen intestinal microbiota. Studies desserts, dairy products, have shown production of processed meats, low fat short chain fatty acids and a spreads, toppings, relatively high production of extruded products, propionate and butyrate, dietary supplements necessary for colonic health (prebiotic functional foods) and systemic influences on blood glucose and lipids

Mixtures of 1- kestose, nystose & 1f-β-fructofuranosylnystose β-D(2,1)-fructofuranosyl)n β-D-fructofuranoside

Fructooligosaccharides

Produced by transfructosylation of a β-fructosidase of Aspergillus niger on sucrose. By partial enzymatic degradation of native inulin

Human Physiological Effects

Fat/sugar replacement, Data indicate that addition of texture modification. inulin-type fructans affect the Dressings and sauces, bacterial composition and the beverages, baked products, metabolic pattern of the fillings, icings, frozen intestinal microbiota. Studies desserts, dairy products, have shown production of processed meats, low fat short chain fatty acids and a spreads, toppings, relatively high production of extruded products, propionate and butyrate, dietary supplements necessary for colonic health (prebiotic functional foods) and systemic influences on blood glucose and lipids

Food Applications

β-D-(2,1)-fructofuranosyl)n
-Dglucopyranoside Naturally occurring in Jerusalem artichokes, chicory, onion, and so on. Produced by extraction from chicory root.

Occurrence & Production

Inulin-type

Fructans - Continued

Name

FIGURE 3.6 — Commercial Nondigestible Polysaccharides - Continued

96 FUNCTIONAL FOODS

Naturally occuring as the husk of the psyllium seed

Extracted from seeds of tamarind tree (Tamarindus indica)

Partial enzymatic hydrolysis Functional foods of polyxylan by xylanase from Trichoderma sp.

Dehydration and purification of konjac tubers (Amorphophallus konjac)

Polymer of arabinoxylans with 1,4 and 1,3 linkages

β-(1-4) linked D-glucose, partially substituted with
-D-xylopyranose.

β-D-((1,4)-xylose)n

β-1,4-linked D-glucose and D-mannose (glucomannan)

Psyllium seed husk

Xyloglucan

Xylooligosaccharides

Konjac (flour) mannan

Binder in meat, often used with k-carrageenan and xanthan gum for gelling

Food additives in Japan, sauces, dressing, ice cream, mayonnaise

Functional food development as a fiber source

Functional food development, dietary supplements

Highly branched (β-1-3 & (β-1-6) D-arabinose and D-galactose sub-units) 6:1 ratio

Arabinogalactan

Extracted from the pulp of Western Larch trees

Complex mixture of polymers Dried exudates of Asiatic sp. Salad dressings, pickle of D-galacturonic acid, of Astragalus, Leguminosae relish, pulpy beverages, galactose, arabinose, xylose, (Astragalus gummifer) milkshakes, ice cream traces starch and cellulose

Human Physiological Effects

Ability to reduce serum cholesterol and serum triglyceride levels and influence glucose and insulin responses

Not hydrolyzed by human enzymes, Changes in metabolic pattern of intestinal flora observed in rats. Blood lipid effects

Fermented in the human colon. Adds viscosity in the small intestine

Reduced risk of coronary heart disease (health claim, at least 1.7 g per RA. Reduced cholesterol

Data indicate gut fermentation, increasing Lactobacilli populations. Studies show immunological properties

Adds viscosity, fermented in the human gut

Powdered doughnuts, Adds viscosity, fermented French dressings, ice in the human gut pops, cheese spreads, ground meats, meringues

Food Applications

Gum tragacanth

Dried exudate of the Indian tree Sterculia urens

Occurrence & Production

Acetylated galacturonic acid+rhamnose+galactose

Composition

Gum karaya

Name

FIGURE 3.6 — Commercial Nondigestible Polysaccharides - Continued

DIETARY FIBER 97

High in mannuronic and glucuronic residues

Mannanoligosaccharide mixture containing less than 50% oligosaccharides

Alginate oligosaccharides

Mannanoligosaccharides

Adapted from Tungland and Meyer, 2002

β-1-4-D-mannuronic acids and
-1-4-L-guluronic acid

Composition

Alginate

Name

By Saccharomyces cerevisiae on sucrose

Produced by enzymatic degradation of alginate

Extracted from brown algae (Phaeophyceae)

Occurrence & Production

Human Physiological Effects

Limited data, algal oligosaccharides have an effect on either the metabolism or composition of the intestinal microbiota

Used as growth promoter No data available on in animal feed industry fermentation

Functional foods

Dairy products, bakery Adds viscosity, fermented in products, salad dressings, human gut to short chain dessert puddings, foam fatty acids. stabilization, fabricated foods, dietetic products

Food Applications

FIGURE 3.6 — Commercial Nondigestible Polysaccharides - Continued

98 FUNCTIONAL FOODS

DIETARY FIBER

99

Guar Gum Guar gum is a galactomannan isolated from the seed of Cyamopsis tetragonolobus (guar). In its unmodified form, this food additive is used as a thickener in a large variety of food products. Partial enzymatic hydrolysis results in a product that can be used as a soluble dietary fiber. The physiological effects of this fiber source comply with what might be expected from a soluble fiber. Guar gum is readily fermented by the human fecal microbiota, and it has bifidogenic effects, at least with enteral feeding (Okubo et al., 1994). It improves bowel functioning, reducing diarrhea in enterally fed patients and relieves constipation in patients. It shows a hypolipidemic effect in humans, lowering both serum cholesterol and triglycerides, and it reduces postprandial glycemia. Gum Arabic This is exudate from the acacia tree and is a complex arabinogalactan polysaccharide in admixture with a glycoprotein. It has a high molecular weight and it is used as an additive in many food applications as a stabilizer and emulsifier. The physiological effects from human studies include its complete fermentation in the human colon with indications for a bifidogenic effect and its ability to lower serum triglyceride and cholesterol levels (Michel et al., 1998). Fructans Fructans can be divided in 2 classes. Levans are β-2,6 -linked fructans with variable degrees of β-2,1 -linked side chains that are produced by a large variety of bacteria. Inulins, on the other hand, are composed of β-2,1 -linked fructosyl units, and are produced by many dicotyledonous plants as a reserve carbohydrate. Fructans are discussed in detail in the chapter “Prebiotics, probiotics and symbiotics”. Galactooligosaccharides These oligosaccharides are produced from lactose by the transglycosylating activity of β-galactosidase. They consist of a number of β-1,6-linked galactosyl residues linked to a terminal glucose unit via an
-1,4-bond. Galactooligosaccharides are not digested in the human alimentary tract, acting as soluble dietary fiber. Reports show a change in colon flora composition and activity following consumption of these compounds (Alles et al., 1999). Various human studies show that these oligosaccharides may relieve constipation, improve calcium absorption, and retard the development of colon cancer in rat model systems.

100

FUNCTIONAL FOODS

Another type of galactooligosaccharides is natural isolates from soybeans. These
-galactooligosaccharides (galactosylsucroseoligosaccharides) include raffinose, stachyose, and verbascose and consist of galactose residues linked
-1,6 to the glucose moiety of sucrose. Their physiological effects appear similar to the β-linked galactose oligomers, such as gut pathogen reduction with corresponding effects, as expected from this change in colon microbiota. However, no data are available at present. Lactulose Lactulose is a disaccharide of D-galactose linked β-1,4 to fructose. It is manufactured from lactose, where alkali isomerization is used to convert the glucose moiety in the lactose into a fructose residue. The disaccharide is not digested by humans, and promotes growth of bifidobacteria in the colon (Strohmaier, 1996). Lactulose is mostly used as a pharmaceutical in prevention of constipation and in portosystemic encephalopathy. It is not allowed for food use in Europe, due to its drug status, but seems to find its way into the food supplement market (mainly in The Netherlands). Although its use is clearly for physiological reasons and it has well-established health effects, its legal status limits use in food. The physiological effects of lactulose are exploited in Japan. Other Oligosaccharides As indicated in Figure 3.6, there is a wide variety of oligosaccharides, all of which have properties characteristic of dietary fiber, that have effects on the activity and/or composition of the human colon flora. PROPERTIES OF ISOLATED FIBER IN FOOD APPLICATIONS Dietary fiber components, isolated from their native plants, provide many functional properties that affect the technological function of foods. These functional properties also influence the food product’s properties during its processing and its final product quality and characteristics. The primary properties provided by isolated fiber ingredients for food development are related to their solubility, viscosity and gelation-forming ability, water-binding capacity, oil-binding capacity, and mineral and organic molecule-binding capacity. The solubility of fiber as a technological property refers to its solubility in water. Four primary structural features of the polymeric backbone primarily increase solubility. More branching generally results in greater solubility (for example, gum acacia); the presence of ionizing

DIETARY FIBER

101

groups also tend to increase solubility (for example, pectin methoxylation); the potential for inter unit positional bonding (for example, β-glucans with mixed β-1-3 and β-1-4 linkages) also increase solubility; and alterations of the monosaccharide units or their molecular form (
- or β-form) further increase solubility (for example, gum acacia, arabinogalactan, and xanthan gum). Viscosity is another technological property of fiber that provides rheological change in food systems. Generally, as the molecular weight or chain length of the fiber increases, the viscosity of the fiber in solution increases. However, the concentration of the fiber in solution, the temperature, pH, shear conditions of processing, and ionic strength all substantially depend on the fiber used. Primarily, long chain polymers, such as the gums (for example, guar gum, locust bean gum, tragacanth gum, etc.), bind significant water and exhibit high solution viscosity. These are used as thickening agents in foods at low concentrations. While these fiber sources are typically necessary to make food-based delivery systems functional, they are limited, due to their high water binding capacity, in their ability to be used at high levels that may provide significant benefit as a fermentable food source for colon microorganisms. However, in general, highly soluble fibers, those that are highly branched or are relatively short chain polymers, such as gum arabic, isolated arabinogalactans, inulins, and oligosaccharides have low viscosities. These low viscosity fibers are generally used to modify texture or rheology, manage water migration, influence the colligative properties of the food system, and improve the marketability of the food product as a health-promoting or functional food product. These fiber sources can be used in food products at relatively high levels, as they typically enhance the food product’s taste, mouthfeel, and shelf life without significantly altering the specific application characteristics. For example, sugar-free and fat-free products also have potential for high fiber claims and marketed as supplements. Gelation is an important attribute of some fiber ingredients as a means to add form or structure to various food products. Gelation represents the association of polymer units to form a network of junction zones. The gel formed by this process encapsulates water and other components in solution to form a firm 3-dimensional structure. Gel formation depends on the type of gum, its concentration, temperature, presence of ions (for example, calcium), pH, and the presence of other rheology modifiers in the food system. Gums, including guar gum, gum arabic, karaya,  carrageenan, and tragacanth gum, as molecular gel forming polymers, are typically used as rheology modifiers or stabilizers

102

FUNCTIONAL FOODS

in food systems. Particle gel forming polymers, such as starch and inulin, are typically used at much higher concentrations than the molecular gel forming gums, and are used with the gums in systems to influence system rheology and overall texture. Fiber ingredients interact with water differently, dictating how the fiber is used and how it functions in a food system. This interaction is generally described as water uptake, hydration, adsorption, absorption, binding, or holding, with the 2 most common being water-binding capacity (WBC) and water-holding capacity (WHC). While being used interchangeably, the terms are differentiated based on the ability of a fiber to retain water under stress. WHC refers to the amount of water the gel system retains within its structure without pressure or stress, while WBC refers to the amount of water the gel system retains after it is stressed, as following centrifugation. The WBC likely has greater practicality, because food manufacturing/processing typically uses some form of physical stress (for example, extrusion, mixing or kneading, homogenization, and so on). The fiber source does not directly influence the WBC, but rather the source determines the physicochemical properties of the fiber ingredient, such as fiber length, particle size, and porosity. These properties in turn influence the WBC and its use and the conditions of its use in food development. However, other factors in the food system can also influence WBC, such as pH, ionic strength, concentration of the fiber component, and interaction with other waterbinding ingredients (that is, sugar, starches, and so on). By contrast, water interactions with soluble fibers are more greatly influenced by pH and ionic strength. Many dietary fibers are fat and/or oil dispersible, and some also bind oil. Oil binding is in part related to its chemical composition, but is more largely a function of the porosity of the fiber structure rather than the affinity of the fiber molecule for oil. By hydrating a fiber with water, the water occupies the fiber pores, significantly reducing oilbinding. This technique is used successfully with batters and film coatings to reduce the oil uptake during frying operations, and reduces the total fat content of the final food product, enhancing crispiness. Some dietary fibers, fruit and vegetable fiber that have cation exchange capacity (CEC) from unmethylated galacturonic acid residues and phytic acid from cereal fiber, are also able to bind cations such as calcium, cadmium, zinc, and copper. The CEC is influenced by the type of fiber (its makeup), the system pH and ionic strength, and the chemical nature of the cation. Cho et al (1997) reported that some dietary fibers have also been shown to absorb organic molecules (for example, lignin

DIETARY FIBER

103

binds bile acids, and wheat bran binds certain carcinogens like benzopyrazine). However the effect is pH dependent. These primary functional properties of several isolated fiber sources provide the means to make high-fiber foods with high eating quality, The main technological functions in food of isolated fiber components, such as pectin and guar gum, are as gelling and thickening agents. However, other food (fiber) ingredients are also available to modify or stabilize the texture of food product (for example alginates, carrageenans, cellulose and its derivatives, modified starch). Due to their relatively low level of use in prepared food systems, these polysaccharides are used mainly for their technological properties rather than their physiological significance. Yet they fit the general definition of dietary fiber and are considered as dietary fibers according to their physiology. In contrary to the fiber polysaccharides, NDO, and, more specifically, inulins, are used in food products because of their physiological functionality as prebiotic fiber as well as their technological characteristics. These dietary components function particularly well when used in sugar and fat replacer systems, as having synergy with high-water binding thickening agents to add texturizer, provide bulk, and enhance rheology in food product development. These combined properties provide a means to enhance health promoting, high-fiber food product development, without compromising taste. Worldwide Fiber Recommendations And Intake Most data in literature regarding intake of total dietary fiber (TDF) are dependent on the method(s) used to define their dietary content and are estimates using standard food tables. These data are derived from analysis of foods using official methods for fiber labeling, such as AOAC method 985.29. Typically, there has been little indication of the individual components making up this TDF value. More recent studies have used various analytical methods to determine intakes of various individual segments of the dietary fiber, such as the insoluble and soluble nonstarch polysaccharides. While specific official analytical methods are now available for meeting the requirements for measuring these dietary fiber components, only limited data are available. The National Cancer Institute (NCI) recommended the adult fiber consumption should be increased to 20 to 30 g daily, not to exceed 35 g due to research data suggesting fiber-containing foods provide some protection against colon and rectal cancer (Butrum et al., 1988). It was suggested that if these guidelines were followed, they may help reduce risk of these

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cancers. Reviewing recommendations for healthy populations from other agencies and countries suggest that fiber intakes should be increased, but the recommendations are somewhat unclear as to the amounts and types of fiber being recommended. Beneficial Claims For DF (1) A grain product, fruit, or vegetable that contains dietary fiber; low fat and good source of dietary fiber (without fortification) may be beneficial in preventing some types of cancer. (2) A grain product, fruit, or vegetable that contains dietary fiber: low saturated fat, low cholesterol, and low fat, particularly soluble fiber (0.6 g per Reference Amount [RA] without fortification), may reduce the risk of coronary heart disease. (3) A fruit or vegetable, low in fat with good source of vitamin A, vitamin C or dietary fiber (without fortification) may reduce the risks of some types of cancer. Soluble fiber must be labeled. (4) Soluble fiber from: (1) β-glucan soluble fiber from oat bran, rolled oats (oatmeal) and whole oat flour; and (2) psyllium husks may reduce the risk of heart disease if they are low in fat, saturated fat, cholesterol and include 0.75 g of whole oat soluble fiber or 1.7 g of psyllium husk soluble fiber per RA. Soluble fiber must be labeled. (5) Diets rich in whole grain foods and other plant foods and low in total fat, saturated fat, and cholesterol may reduce the risk of heart disease and some cancers. The food must contain 51% or more whole grain ingredients by weight per serving, and a dietary fiber content of at least 3.0 per RA of 55 g, 2.8 g per RA of 50 g, 2.5 g per RA of 45 g, 1.7 g per RA of 35 g, and be low in fat. OATS Oats (Avena sativa) is a whole kernel cereal consumed as groats milled to yield various products – rolled oats, oatmeal, oat flour. FDA has allowed a health claim for an association between consumption of diets high in oat meal, oat bran, or oat flour and reduced risk of coronary heart disease. This is the first health claim for a specific food under Nutrition Labeling and Education Act (NLEA). Oats based functional foods, oats yogurt, and a symbiotic oats based beverage have been developed in the author’s laboratory. Oat Bran Oat bran is the food which is produced by grinding clean oat groats or rolled oats and separating the resulting oat flour by sieving,

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bolting and/or other suitable means into fractions so that the oat bran fraction is not more than 50% of the starting material, and has total βglucan content of at least 5.5% (dry weight basis) and a total dietary fiber content of at least 16% (dry weight basis), and so that one third of the total dietary fiber is soluble fiber (AACC). Oat bran concentrate Natureal GI from GTC Nutrition, Golden, Colo., is high in β-glucan and said to slow the uptake and release of energy from a meal and exert positive control over healthy blood glucose levels and subsequent inulin response. Tappy et al (1996) found that 5 g of βglucan reduced glycemic response by 50% in a 35 g carbohydrate meal. The soluble fiber in Natureal oat bran concentrate has been associated with benefits for blood sugar control and satiety, and with weight management. The effectiveness of ingredient for control of glycemic response is attributed to the viscous nature of β-glucan. Viscosity is critical to slowing stomach emptying and regulating the uptake of energy from a meal. This effect is important to controlling glycemic response and blunting the after-meal insulin surge, which is thought to have positive effects for healthy weight maintenance. β -glucan β-glucan is distributed throughout the endosperm and located in the endosperm cell walls and constitutes 75%. It is a linear, unbranched polysaccharide composed of 4-O-linked β-D-glucopyranosyl units (70%) and 3-O-linked β-D-glucopyranosyl units (30%), MW=1.5-3.0 X 106. There is 3-11% in barley, 3-7% in oats and <1% in wheat. β -glucan Isolate There is no pure form of oat β-glucan. A new product high in β-glucan called Oatrim by Quaker Oats is used as soluble fiber or a fat replacer. Oatrim is prepared by extraction of oats or oat bran with hot water containing heat soluble
-amylase. Oats Based Functional Foods Dietary oats have been shown to confer a number of significant physiological effects in the prevention or alleviation of disease and thus may be considered as a multifunctional food. Soluble fiber favors the growth of probiotic bacteria. Oats contain a high percentage of desirable complex carbohydrates that may reduce the risk of certain cancers and constipation and also promotes a good balance of fatty acids. The key cholesterol lowering ingredient in oats is soluble fiber. It works by binding cholesterol-containing bile acids produced in the liver and speeding their exit from the body. It also helps control diabetes by

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preventing erratic swings in blood sugar levels. Soluble fiber slows down the absorption of sugar from the intestines into the blood. It also increases the cell’s insulin sensitivity and assists the cells in drawing sugar from the blood. Due to these benefits, there is a great interest in increasing the consumption of products based on oats that contain both soluble and insoluble fibers. However, consumption of oat-based products is low mainly due to the lack of acceptable food products based on oats. So oats based symbiotic beverage and yogurt were developed in the author’s laboratory with the idea of preparing products having two healthy components, dietary fiber of oats and probiotic lactic acid bacteria wherein the oats is fermented by probiotic bacteria itself. Symbiotic Oats Beverage Oats flour (3-7%, w/v), sugar (4-8%, w/v), inulin (0.2%, w/v) and whey protein concentrate (0.5%, w/v) were blended in water to make a homogenous slurry. The slurry was slowly heated at the rate of 1°C per min. to boiling and boiled for 3 min. to break down the starch content of oats. The cooked slurry was sterilized at 121°C for 15 min. The slurry was then cooled to 37°C stirring at intervals to avoid formation of a layer on the surface. This was inoculated with probiotic culture and mixed well so that the culture was distributed evenly in the slurry. Fermentation was carried out at 37°C in aerobic environment. Oats flour was the main ingredient in the product. We chose to use 5% because less than that would not provide the required amount of dietary fiber, and it was required to add more of inulin to meet the dietary fiber requirement. But if more was used the product would be more viscous, and it would not be a beverage. 0.2% inulin was used to increase the soluble dietary fiber content to meet the requirement (0.75 g/serving). Inulin acts as a water binder, stabilizer and texturizer in addition to being a prebiotic. Whey protein concentrate was used at the level of 0.5%. This was just enough to make a homogenous product, and more than this would coagulate the slurry when it was autoclaved. Whey protein concentrate acts as a stabilizer that makes the product homogenous. Sugar (4%) was added as energy source for the cultures and to give a balanced taste of sweet and sour to the product. All the ingredients and water were blended in a blender to make a homogenous slurry. The slurry was cooked and autoclaved. The beverage was homogenous, free flowing and had smooth texture compared to the unfermented slurry. The pH, titratable acidity and viscosity of the control sample (without fermentation) were 6.3, 0.032% and 420 mPas, respectively. After fermentation for 12 h, there was a

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significant change in pH which reduced to 3.63, titratable acidity which increased to 0.21% and viscosity that decreased to 222 mPas. The product had a good balance of sweet and sour tastes. The beverage provides 0.80 g of soluble dietary fiber per serving (150 mL) which meets the FDA requirements (0.75 g per serving). Since this product contains only plant origin raw materials and whey protein concentrate, it can be considered as low in saturated fat and cholesterol free. It is also suitable for enrichment with traditional flavors. This oats based beverage is typically a non-dairy vegetarian product containing no milk. It serves as an alternative to both dairy and soy beverages and suits a healthy life-style whether vegetarian-oriented or not. Symbiotic Oats Yogurt Symbiotic oats yogurt was formulated as a multifunctional product that delivered dietary fiber and viable probiotic lactic acid bacteria, in which the bacteria fermented the oats, and prepolymerized whey proteins were utilized to form a gel. Preparation of symbiotic oats yogurt is presented in Figure 3.7. The oats yogurt provides 1.68 g of soluble dietary fiber per serving (240ml) which meets the FDA requirement (0.75 g per serving) for labeling. Since this product contains only raw materials from plant origin and whey protein isolate, it can be considered low in saturated fat and cholesterol free. Other oats-based functional foods are being developed in the author’s laboratory. SUMMARY Dietary fiber, an essential part of a healthy diet, provides many health benefits which have been researched to a large extent. This chapter deals in detail about its role in human health and also its functionality in foods as a functional ingredient and/or food. Dietary fiber also known as roughage or bulk includes all parts of plant foods that our body can’t digest or absorb. The term ‘Dietary fiber’ was coined by Hipsley in 1953 and is defined as the edible parts of the plant and analogous carbohydrate that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. It includes polysaccharides, lignin and associated plant substances. Dietary fiber exhibits one or more of laxation (fecal bulking and softening; increased frequency; and/or regularity), blood cholesterol attenuation, and/or blood sugar regulation.

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FIGURE 3.7 — Preparation Of Symbiotic Oats Yogurt

Dietary fiber is classified into different types based on solubility, fermentability and the arrangement of monomeric units. There are also different methods of analysis of dietary fiber which can be modified based on which fiber type we want to analyse. The physiological benefit of various dietary fiber types depends on how they are metabolized in the body which in turn depends on their physicochemical characteristics. Dietary fiber has been found to have

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an important role in preventing chronic diseases such as cancer, cardiovascular disorders, diabetes, gastrointestinal disorders and also in improving mineral bioavailability. The primary properties provided by isolated fibers for food product development are solubility, viscosity, and gelation-forming ability, waterbinding capacity, oil-binding capacity, and mineral and organic moleculebinding capacity. Oats have been popularly known as a functional food. The first health claim that FDA has allowed for a specific food under The Nutrition Labeling and Education Act (NLEA) is a health claim for an association between consumption of diets high in oat meal, oat bran, or oat flour and reduced risk of coronary heart disease. The main component that makes oats a functional food is β-glucan, a soluble dietary fiber. Focusing on the physiological health aspect of dietary fiber and the types of fibers meeting a physiological definition will help to make the consumer understand the importance of fiber in their diets. References Alles, M. S., Hartemink, R., Meyboom, S., Harry, J. L., van Laere, K. M. J., van Nagengast, F. M. and Hautvast, J. C. A. J. 1999. Effect of transgalactooligosaccharides on the composition of the human intestinal microflora and on putative risk markers for colon cancer. Am. J. Clin. Nutr. 69:980-991. Anderson, J. W., Story, L., Sieling, B., Chen, W. J. L., Petro, M. S. and Story, I. 1984. Hypocholesterolemic effects of oat-bran or bean intake for hypercholesterolemic men. Am. J. Clin. Nutr. 40:1146-1155. Anderson, J. W., Allgood, L. D., Lawrence, A., Altringer, L. A., Lerdack, G. R., Hengehold, D. A. and Morel, J. G. 2000. Cholesterol-lowering effects of psyllium intake adjunctive to diet therapy in men and women with hypercholesterolemia: meta-analysis of 8 controlled trials. Am. J. Clin. Nutr. 71:472-479. Baghurst, P. A., Baghurst, K. I. and Record, S. J. 1996. Dietary fiber, nonstarch polysaccharides and resistant starch: a review. Food Australia 48(3):1-36S. Birkett, A., Muir, J., Phillips, J., Jones, G. and O’Dea, K. 1996. Resistant starch lowers fecal concentrations of ammonia and phenols in humans. Am. J. Clin. Nutr. 63:766772. Boeckner, L. S., Schnepf, M. I. and Tungland, B. C. 2000. Inulin: A review of nutritional and health implications. Adv. Food Nutr. Res. 43:1-63. Bourquin, L. D., Titgemeyer, E. C., Garleb, K. A. and Fahey, G. C. 1996. Fermentation of various dietary fiber sources by human fecal bacteria. Nutr. Res. 16:1119-1131. Brown, L., Rosner, B., Willett, W. W. and Sacks, F. M. 1999. Cholesterol-lowering effects of dietary fiber: a meta-analysis. Am. J. Clin. Nutr. 69:30-42. Butrum, R. R., Clifford, C. K. and Lanza, E. 1988. Dietary guidelines: Rationale. Am. J. Clin. Nutr. 48:888-895. Byers, T. 2000. Diet, colorectal adenomas, and colorectal cancer. N. Engl. J. Med. 342(16):1206-1207. Causey, J. L., Feirtag, J. M., Gallaher, D. D., Tungland, B. C. and Slavin, J. L. 2000. Effects of dietary inulin on serum lipids, blood glucose and the gastrointestinal environment in hypercholesterolemic men. Nutr. Res. 20(21):191-201.

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Chandalia, M., Garg, A., Lutjohann, D., von Bergmann, K., Grundy, S. M., Brinkley, L. J. 2000. Beneficial effects of high dietary fiber intake in patients with type 2 diabetes mellitus. N. Engl. J. Med. 342:1392-1398. Cho, S., DeVries, J. W., Prosky, L. 1997. Dietary fiber analysis and applications. Maryland: AOAC International. Demigne, C., Morand, C., Levrat, A. M., Besson, C., Moundras, C. and Remesy, C. 1995. Effects of propionate on fatty acid and cholesterol synthesis and on acetate metabolism in isolated rat hepatocytes. Br. J. Nutr. 74:209-219. Dobbing, J. 1989. Dietary Starches and Sugars in Man: A Comparison. London: SpringerVedag. 256 pp. Englyst, H. N., Kingman, S. M. and Cummings, J. H. 1992. Classification and measurement of nutritionally important starch fractions. Eur. J. Clin. Nutr. 46(Sup. 2):S33-S50. Folino, M., McIntyre, A. and Young, C. P. 1995. Dietary fibers differ in their effects on large bowel epithelial proliferation and fecal fermentation-dependent events in rats. J. Nutr. 125:1521-1528. Gibson, G. R. and Roberfroid, M. R. 1995. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. Nutr. 125(61):1401-1412. Green, C. J., Van Hoeij, K. A. and Bindels, J. G. 1998. Short chain fatty acid (SCFA) and gas production of individual fiber sources and a mix typical to a normal diet using an in vitro technique. J. Pediatr. Gastroenterol. Nutr. 26:591. Heijnen, M. L. A., Amelsvoort, J. M. M. van, Deurenberg, P. and Beynen, A. C. 1998. Limited effects of consumption of uncooked (RS2) and retrograded (RS3) resistant starch on putative risk factors for colon cancer in healthy man. Am. J. Clin. Nutr. 67:322-331. Jones, J. M. 2000. Dietary advice in North America: the good, the bad and the unheeded. In: McCleary BV, Prosky L, editors. Proceedings of the 1st International Conference On Dietary Fiber; Dublin, Ireland; May. Oxford, U.K.: Blackwell Science. P 30. Kok, N. N., Morgan, L. M., Williams, C. M., Roberfroid, M. B., Thissen, J. P. and Delzenne, N. M. 1998. Insulin, glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide and insulin-like growth factor I as putative mediators of the hypolipidemic effect of oligofructose in rats. J. Nutr. 128:1099-1103. Lipman, T. O. 1995. Bacterial translocation and enteral nutrition in humans: an outsider looks in. J. Parenter. Enteral Nutr. 19:156-165. Lopez, H. W., Coudray, C., Ballanger, J., Younes, H., Demigne, C. and Remesy, C. 1998. Intestinal fermentation lessens the inhibitory effects of phytic acid on mineral utilization in rats. J. Nutr. 128:1192-1198. Luo, J., Rizkalla, S. W., Alamowitch, C., Boussairi, A., Blayo, A., Barry, J. L. Laffitte, A., Cuyon, F., Bornet, F. R. J. and Slama, G. 1996. Chronic consumption of shortchain fructooligosaccharides by healthy subjects decreased basal hepatic glucose production but had no effect on insulin-stimulated glucose metabolism. Am. J. Clin. Nutr. 63:939-945. Lynn, M. E., Mathers, J. C. and Parker, D. S. 1994. Increasing luminal viscosity stimulates crypt cell proliferation throughout the gut. Proc. Nutr. Soc. 53:227A. Meyer, P. D., Tungland, B. C., Causey, J. L. and Slavin, J. L. 2000. The immune effects of inulin in vitro and in vivo. Agro Food Ind Hi-Tech Nov./Dec.:18-20. Michel, C., Kravtchenko, T. P., David, A., Gueneau, S., Kozlowski, F. and Cherbut, C. 1998. In vitro prebiotic effects of Acacia gums onto the human intestinal microbiota depends on both botanical origin and environmental pH. Anaerobe 4:257-266. Nordgaard, I. and Mortensen, P. B. 1995. Digestive processes in the human colon. Nutr. 11:37-45.

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Ohta, A., Motohashi, Y. and Sakuma K. 1998. Dietary fructooligosaccharides change the concentration of calbindin-D9k differently in the mucosa of the small and large intestine of rats. J. Nutr. 128:934-939. Okubo, T., Ishihara, N., Takahashi, H., Fujisawa, T., Kim, M., Yamamoto, T. and Misuoka T. 1994. Effects of partially hydrolyzed guar gum intake in human intestinal microflora and its metabolism. Biosci. Biotechnol. Biochem. 58:1364-1369. Sakamoto, J., Nakaji, S., Sugawara, K., Iwane, S. and Munakata, A. 1996. Comparison of resistant starch with cellulose on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats. Gastroenterol. 110:116-120. Satchithanandam, S., Vargofcak-Apker, M., Calvert, R. J., Leeds, A. R. and Cassidy, M. M. 1990. Alteration of gastrointestinal mucin by fiber feeding in rats. J. Nutr. 120:1179-1184. Scheppach, W. 1998. Butyrate and the epithelium of the large intestine. In: Guillon, F., Amado, R., Amaral-Collaco, M. T., Andersson, H., Asp, N. G., Bach Knudsen, K. E., Champ, M., Mathers, J., Robertson, J. A., Rowland, I., Van Loo, J. editors. Proceedings of the Profiber Conference: Functional Properties of Nondigestible Carbohydrates. Lisbon, Portugal; Feb. P 215. Silk, D. B. A., Walters, E. R., Duncan, H. D., Green, C. J. 2001. The effect of a polymeric enteral formula supplemented with a mixture of six fibres on normal human bowel function and colonic motility. Clin. Nutr. 20:49-58. Smith, T., Brown, J. C. and Livesey, G. 1998. Energy balance and thermogenesis in rats consuming nonstarch polysaccharides of various fermentabilities. Am. J. Clin. Nutr. 68:802-819. Strohmaier, W. 1996. Lactulose, an innovative food ingredient - physiological aspects. In: Proceedings of FIE Conference; Paris; 12-14 Nov. 1996. Maarssen, Netherlands: Miller Freeman. P 69-72. Tappy, L., Gugolz, E. and Wursch, P. 1994. Effects of breakfast cereals containing various amounts of beta-glucan fibers on plasma glucose and insulin responses in NIDDM subjects. Diabetes Care. 19:831-834. Tungland, B. C. and Meyer, D. 2002. Nondigestible Oligo- and Polysaccharides (Dietary Fiber): Their Physiology and Role in Human Health and Food. Comprehensive Reviews in Food Science and Food Safety. 3:73-92. Vanhoof, K. and De Schrijver, R. 1996. Nitrogen metabolism in rats and pigs fed inulin. Nutr. Res. 16:1035-1039. Wasan, H. S. and Goodlad, R. A. 1996. Fiber-supplemented foods may damage your health. Lancet 348:319-320. Younes, H., Garleb, K., Behr, S., Remsey, C. and Demigne, C. 1995. Fermentable fibers or oligosaccharides reduce urinary nitrogen excretion by increasing urea disposal in the rat caecum. J. Nutr. 125:1010-1016. Younes, H., Demigne, C. and Remesy, C. 1996. Acidic fermentation in the caecum increases absorption of calcium and magnesium in the large intestine of the rat. Br. J. Nutr. 75:301-314. (Guo, M.R., Gokavi, S.)

Chapter 4 PREBIOTICS AND PROBIOTICS Among the most promising targets for functional foods are the gastrointestinal functions, including those that control transit time, bowel habits, and mucosal motility as well as those that modulate epithelial cell proliferation. Promising targets are also gastrointestinal functions that are associated with a balanced colonic microflora, that are associated with control of nutrient bioavailability, that modify gastrointestinal immune activity, or that are mediated by the endocrine activity of the gastrointestinal system. Also, some systemic functions such as lipid homeostasis that are indirectly influenced by nutrient digestion or fermentation represent promising targets. Bacteriotherapy is an alternative and promising way to combat infections by using harmless bacteria to displace pathogenic microorganism. Saliva and gastrointestinal secretions, as well as beneficial microbes (probiotics) and supplied fibers (prebiotics) are important for optimal function. It is common knowledge that the intestinal flora encompasses at least 500 different types of bacteria living in symbiosis with their host: both the host and the bacteria benefit from this symbiosis. The composition of the intestinal flora is fairly constant within an individual in spite of considerable intra-individual variation in the composition of the diet. The stable composition of the flora is indicative of a balanced ecosystem, at least in healthy individuals, which is not easily disturbed. This chapter discusses the significance of the intestinal flora to our health, how the composition of our diet can affect the intestinal flora and what impact a modified intestinal flora may have on our health. Also it reviews the basics of prebiotics and probiotics and scientific data showing that prebiotics and probiotics positively affect various physiological functions in ways that will permit them to be classified as functional foods. Bacterial counts per milliliter in intestinal contents increase in a distal direction in the gastrointestinal tract from 103–104 in the stomach

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to 106–107 in the distal ileum and 1011–1012 in the colon (Figure 4.1 and 4.2). Gram-positive bacteria are the dominant microflora in the stomach, whereas the bacteria in the distal small intestine and the colon are predominantly of the Gram-negative type. The numbers of anaerobic bacteria increase distally. The most important intestinal bacteria are bacteroides, bifidobacteria, Enterobacteraceae, lactobacilli, Grampositive cocci, Clostridium species and eubacteria. In addition, streptococci and various types of molds and yeasts are also found in the intestines. FIGURE 4.1 — Composition Of Human Gastrointestinal Microflora Bacterial flora Total bacterial count (per ml)

Stomach

Jejunum 5

Ileum 3

10 –10

Faeces 7

1010 - 1012

0–10

0–10

0–102

0–103

102–106

1010–1012

0–103 0–102 0–103 0–102

0–104 0–103 0–104 0–102

102–106 102–105 102–105 102–103

103 –1010 104 –107 106 –1010 102 –106

rare rare rare rare rare

0–102 0–103 0–103 rare rare

103–107 103–105 102–103 102–104 rare

1010–1012 108 –1012 108 –1011 106 –1011 109 –1012

Aerobic or facultative bacteria Enterobacteria Streptococci (including Peptostreptococcus) Staphylococci Lactobacilli Fungi Anaerobic bacteria Bacteroides Bifidobacteria Gram-positive cocci Clostridium spp. Eubacteria

FIGURE 4.2 — Density And Nature Of Bacteria In The Human Gastrointestinal Tract. Site Stomach and proximal ileum Terminal ileum Colon

Density 103 –104 /ml 106 –107 /ml 1011–1012/ml

Type predominantly Gram-positive predominantly Gram-negative predominantly Gram-negative

Studies with aseptically grown animals have provided insight into the physiological significance of the intestinal flora. The findings suggest that the intestinal flora affects both the immune system and the intestinal function. The intestinal flora has also been found to improve resistance to the colonization by enteropathogenic micro-organisms such

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as Salmonella bacteria. Further, the intestinal flora aids the digestion of food components, in particular of poorly digestible carbohydrates. The butyric acid formed in that process has a favorable effect on the intestinal epithelium. The intestinal flora also plays a role in fat metabolism (bile acid and cholesterol metabolism) and the synthesis of vitamin K. A variety of conditions may disturb the intestinal flora and induce colonization of the intestines by undesirable bacteria. Such conditions include treatment with antibiotics, food contamination (e.g., by Salmonella spp., Campylobacter spp. or Escherichia coli), viral infections, stress, shortage of gastric juice and diminished intestinal motility. The latter two factors play a major role in bacterial overgrowth in the small intestine leading to impaired food digestion (malabsorption of fats, carbohydrates, amino acids, and vitamin B12). A balanced intestinal flora is a precondition for a fairly stable ecosystem in which both host-related factors and antagonistic interactions among intestinal bacteria play a role (Figure 4.3). Food has limited influence on the composition of the intestinal flora, but a strong influence on their metabolic activity. FIGURE 4.3 — Schematic Presentation Of Interactions Between Food, Intestinal Flora And Host

With regard to host-related factors it should be noted that the intestines have a role in addition to their digestive function, namely that of a barrier against invading bacteria. The following factors are relevant in this respect: • gastric acid secretion; • bile and pancreatic juice; • intestinal motility and peristalsis;

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• rejection of epithelial cells; • secretion of immunoglobulins; • lysosomal and macrophagic activities. The antagonistic interactions among bacteria can be classified into indirect interactions and direct interactions. Indirect interactions (host-related factors) include: • deconjugation of bile salts; • formation of secondary bile salts; • induction of immune response (immunoglobulin A secretion); • stimulation of intestinal peristalsis. Direct interactions include: • competition for substrates; • competition for sites of attachment; • formation of growth-inhibiting metabolites (volatile fatty acids organic acids, sulfuric acid, bacteriocins); • decrease in pH of the environment. Recent studies have shown that metabolic activities of the intestinal flora could form potential carcinogens under specific conditions. It is essential, therefore, to have a thorough knowledge of dietary factors with a favourable effect on the composition and activity of intestinal flora. These dietary factors can be classified into three groups, namely prebiotics, probiotics and symbiotics (synergetic combinations of probiotics and prebiotics) (Figure 4.4). FIGURE 4.4 — Schematic Presentation Of Interactions In The Gastrointestinal Tract Between Probiotics/Prebiotics And Intestinal Flora

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PREBIOTICS Prebiotics are non-digestible food ingredients which beneficially affect the host by selectively stimulating the growth of and/or activating the metabolism of one or a limited number of health promoting bacteria in the intestinal tract, thus improving the host’s intestinal balance (Gibson and Roberfroid, 1995). All prebiotics to date have been carbohydrates, ranging in size from small sugar alcohols and disaccharides, to oligosaccharides and large polysaccharides. The defining criteria of prebiotics is: (1) A prebiotic should neither be hydrolyzed nor absorbed in the upper part of the gastrointestinal tract. (2) It should be a selective substrate for one or more potentially beneficial commensal bacteria in the large intestine. Colonization by an exogenous probiotic could be enhanced and extended by simultaneous administration of a prebiotic that the probiotic could utilize in the intestinal tract. As such it should stimulate that bacteria to divide, become metabolically active, or both. (3) Alter the colonic microenvironment toward a healthier composition. (4) Induce luminal or systemic effects that are advantageous to the host. The most studied non-digestible oligomers are galactooligomers, such as soya-derived raffinose and stachyose, and the fructooligomers or fructans. Chemistry Of Fructans A fructan is any compound where one or more fructosyl-fructose linkages constitute a majority of linkages (Englyst et al., 1992). Fructan is used to name molecules that have a majority of fructose residues whatever the number is. It even includes the disaccharide composed exclusively of two fructose residues, specifically the fructosyl-fructose or inulobiose but not sucrose, isomaltulose, and galactosucrose, etc. In addition, fructan is also sometimes either a cyclic or a branched molecule. Fructan is also known as polyfructosylfructose. All natural (plant and microbial) fructans are a mixture of oligomers or polymers or both, which is best described by the mean (or average) and the maximum number of fructose units, residues, or moieties, known as the average and the maximum degree of polymerization (DPav and DPmax), respectively. More than 50 generic names of fructans have appeared in old literature including, inulin, levan, and phlein but also

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fructoholoside, fructosan, graminin, inu-lenin, lavulan, levulosan, levosin, and pseudo-inulin, etc. (Suzuki, 1993). A fructan is defined as a carbohydrate that consists mostly of fructose plus a molecule of glucose or a molecule that has a majority of fructose residues and a glucose. A fructan can be linear [Inulin (2, 1 fructosyl-fructose), levan (2, 6 fructosyl-fructose)] or branched (2, 1 or 2, 6 and 2, 6) or cyclic. From a chemical point of view, the linear chain of fructans is either a (β-D-glucopyranosyl-[-β-D-fructofuranosyl]nl-(β-D-fructofuranoside (G py F n ) or a β-D-fructopyranosyl-[-β-D-fructofuranosyl] nl -β-Dfructofuranoside (FpyFn). The fructosyl-glucose linkage is always β-(2↔l) as in sucrose (the numbers indicate the linkage’s position on the C atoms of the fructose or glucose rings and the arrow points away from the reducing C atom (C2, in fructose or C1, in glucose) but the fructosylfructose linkages are either β-(l➞2) or β-(6➞2). In branched fructans the branching linkages are usually β-(2➞6). Fructans are mainly of plant origin, but they are also found in fungi and bacteria. In plant fructans the number of fructose monomers does not exceed 200, whereas in bacterial fructans it can be as high as 100,000, and it is highly branched. Inulin, levan, graminan, phlein, and kestoses are the general terms to describe fructans. Inulin: is a material that has mostly, or exclusively, the β-(l➞2) fructosyl-fructose linkage, and glucose may be present at the terminal position in the chain but is not necessary. Until recently, inulin was considered to be a linear molecule with β-(l➞2) linkages exclusively. However, using optimized permethylation analysis, it has been possible to demonstrate that even native inulin has a very small degree (1-2%) of branching (De Leenheer and Hoebregs, 1994). All fructans in dicotyledons, are inulin-type fructans, but only part of the fructans in monocotyledons are inulin-type fructans (Suzuki, 1993). Inulin exists also in a cyclic form that contains 6, 7, or 8 fructofuranose rings. Levan: is a material that has mostly, or exclusively, the β-(6➞2) fructosyl-fructose linkage. Like in inulin, glucose may be present at the terminal position in the chain but is not necessary. Levans are found mostly in bacteria (high molecular weight) but to some extent in higher plants also (short polymers). The levans of higher plants are heavily branched molecules through the formation of β-(2➞l) linkages. Phlein: has substantially the same meaning as levan, but the name has commonly been used to describe plant- (and not bacteria-) based material which contains, most exclusively, the β-(6➞2) fructosyl-fructose linkage; a glucose is allowed at position 1 in the chain but is not necessary. In general, plant-based fructans are of lower molecular

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weight (DP < 100) than those derived from bacteria, and thus this distinction has been useful. Phlein-type fructans occur mainly in monocotyledons, which represent the most frequently identified fructans. Graminan: is a material that has both β-(l➞2) and β-(6➞2) fructosylfructose linkages in significant proportions; glucose is allowed at position 1 in the chain but is not necessary. Kestoses or kesto-n-oses: are trimeric or oligomeric fructans containing one glucose and two or more fructose units linked by β-(l➞2) and/or β-(6➞2) fructosyl-fructose linkages. Bifurcose:
-D-glucopyranosyl-(l↔2)-β-D-fructofuranosyl-(6➞2)-βD-fructo-furanosyl-(1➞2)-p-D-fructofuranoside. Inulo-n-ose: Oligomeric fructofuranosyl-only fructans that have all(1➞2) linkages like inulobiose and inulotriose. Fructooligosaccharides, oligofructan, and oligofructose: These are oligomeric linear fructans with β-(l➞2) linkages. They can be of both (GpyFn) and (FpyFn) types. Among others, these terms include 1kestose, neokestose, and nystose. • 1-Kestose:
-D-glucopyranosyl-(l↔2)-β-D-fructofuranosyl-(l➞2)β-D-fructo-furanoside. • 6-Kestose:
-D-glucopyranosyl-(1↔2)-β-D-fructofuranosyl-(6➞2)β-D-fructo-furanoside. • Levan-n-ose: oligomeric fructofuranosyl-only fructans that have all β-(6➞2) linkages like levanbiose, levantriose, etc. • Neokestose: β-D-fructofuranosyl-(2➞6)-β-D-glucopyranosyl-(l↔2)β-D-fructofuranoside. • Nystose:
-D-glucopyranosyl-(1↔2)-β-D-fructofuranosyl-(1➞2)-βD-fructofuranosyl-(1➞2)-β-D-fructofuranoside. Natural Occurrence Of Fructans Fructans are reserve carbohydrates in at least 10 families of higher plants that store them in a soluble form in vacuoles in crowns, leaves, roots, stems, tubers, or kernels. The fructans and their linkage type and length differ greatly, depending on the plant and the plant organ. Moreover, the chain length of plant fructans can be modulated through changes in DP as a means to modulate osmotic pressure. Occurrence Of Fructans In Plants Fructan-containing plants are mainly angiosperms. The fructancontaining species belong to both mono- and dicotyledonous families. Some of these plants are eaten as vegetables such as artichoke, asparagus, chicory, garlic, Jerusalem artichoke, leek, onion, salsify, etc.

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In monocotyledons, fructans are widely present in the aerial parts of young seedings of Gramineae but significant concentration is found only in northern grasses (Pooideae), oat (Avena sativa), barley (Hordeum vulgare), rye (Secale sativa), and wheat (Triticum aestivum and Triticum durum). It is also present in the order Liliaceaes. Indeed, the bulbs, tuber, and tuberous roots of Amaryllidaceae, Agavaceae, Haemodoraceae, Iridaceae, Liliaceae, and Xanthorrhoeaceae produce and store fructans. Especially, fructans have been found in the family of Liliaceae, in the leaf and bulb of leek (Allium ampeloprasum), the bulb of onion, shallot (Allium cepa) and garlic (Allium sativum), and the tuber of asparagus (Asparagus officinalis and Asparagus racemosus) and in the family of Agavaceae in the tuber of palm lily (Cordyline terminalis) and Dracaena australis. In dicotyledons, the fructans-containing orders are the Asterales, the Campanulales, the Dipsacales, the Polemoniaceae and the Ericales. As far as is known, all members of the major family Compositae (Asterales order) store significant amounts of fructans in their underground storage organs such as tap roots and tubers but not in their leaves. This is the case for chicory (Cichorium intybus), elecampane (Inula hellenum), dandelion (Taraxacum officinale), Jerusalem artichoke (Helianthus tuberosus), murnong (Microseris lanceolata), salsify (Tragopogon porrifolius), and yacon (Polymnia sonchifolia). Occurrence Of Fructans In Fungi Fructans accumulate in various species of aspergillus, but some species also synthesize it extracellularly from sucrose. Specifically, it has been reported that Aspergillus sydowi synthesizes an inulin that has a molecular weight greater than that of plant inulin. However, fructan has not been demonstrated in penicillium, pestalotiopsis, myrothecium, or trichoderma. This observation correlates well with the fact that sucrose has not been confirmed as a fungal carbohydrate. Indeed, the most characteristic endogenous disaccharide of all fungal groups is trehalose (1-1-di-glucose). Occurrence Of Fructans In Bacteria With the exception of certain strains of Streptococcus mutans (a major component of dental plaque) that produce inulin-type fructans, the bacterial fructans are essentially of the levan type. Fructans or the genes for their synthesis appear essentially in five orders or families of bacteria, namely the Gram-negative aerobic (Pseudomonadaceae) and facultative, anaerobic (Enterobacteraceae) rods and cocci, the Gram-

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positive cocci (Streptococcaceae), endospore-forming rods and cocci (Bacillaceae), and Actinomycetaceae (Hendry and Wallace, 1993). INULIN The use of miscellaneous fructan- (but mostly inulin-) containing plants as food seems to be quite old, dating back to at least 5000 years, and one of the most commonly consumed vegetables in ancient times was onion (Allium cepa). It was in the 19th century that a German scientist discovered inulin after he had isolated a "peculiar substance of plant origin" from the boiling-water extract of Inula helenium.” That substance was called inulin. The first scientific report on the health benefits of inulin for humans also dates back to the last quarter of 19th century. Indeed, referring specifically to inulin, Kulz reported as early as 1874 that no sugar appears in the urine of diabetics who eat 50 to 120 g of inulin per day (Roberfroid, 2004). Inulin, structurally can be considered as a polyoxyethylene backbone to which fructose moieties are attached, as are steps to a winding stair. The degree of polymerization (DP) of inulin and the presence of branches are important properties that influence its functionality strikingly. Therefore, a strict distinction must be made between inulin of plant and bacterial origin. The DPmax of plant inulin is rather low (maximal DP < 200), but DPmax and DPav vary according to plant species, weather conditions, and the physiological age of the plant. Inulin is present in significant amounts in several fruits and vegetables (Figure 4.5). Chicory inulin Chicory (Cichorium intybus) is used today as an industrial crop and its fructan is known as chicory inulin. Native chicory inulin is a nonfractionated inulin, extracted from fresh roots, taking precautions to inhibit the plant’s own inulinase activity as well as acid hydrolysis. It always contains glucose, fructose, sucrose, and small oligosaccharides. Because of the beta configuration of the anomeric C2 in its fructose monomers, inulin is resistant to hydrolysis by human small intestinal digestive enzymes, which are specific for
-glycosidic bonds. It has thus been classified as “nondigestible” oligosaccharide (NDO). Production Of Inulin And Oligofructose And Related Products The production process involves extracting naturally occurring inulin from chicory roots by diffusion in hot water. The raw extract is then

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FIGURE 4.5 — Inulin Content And Chain Length Of Miscellaneous Plants Plant

Inulin (g/100g)

Asparagus Raw Boiled

2.0-3.0 1.4-2.0

Globe Artichoke (Cynara scolymus)

2.0-6.8

Banana (Musa cavendishii) Raw Raw-dried Canned

0.3-0.7 0.9-2.0 0.1-0.3

Barley (Hordeum vulgare) Raw Cooked

0.5-1.0 0.1-0.2

DP > 5 = 95% / DP > 40 = 87% DP < 5 = 100%

Chicory (Cichorium intybus) root

35.7-47.6

Dandelion greens (Taraxacum officinale) Raw Cooked

12.0-15.0 8.1-10.1

Garlic (Aliium sativum) Raw Dried

9.0-16.0 20.3-36.1

DP < 40 = 83% (DP 2-65) DP > 40 = 17%

DP > 5 = 75%

Jerusalem Artichoke (Helianthus tuberosus) 16.0-20.0

Leek (Allium ampeloprasum) Raw

Chain Length

DP < 40 = 94% (DP 2-50) DP > 40 = 6% DP 12 is most frequent

3-10

Onion (Allium cepa) Raw Raw-dried Cooked

1.1-7.5 4.7-31.9 0.8-5.3

DP 2-12

Wheat (Triticum aestivum) Bran – raw Flour – baked Flour – boiled

1.0-4.0 1.0-3.8 0.2-0.6

Rye - Baked

0.5-0.9

DP < 5 = 50%

DP ➞ Degree of polymerization. Adapted from Van Loo et al. (1995).

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refined by using technologies from the sugar and starch industries (e.g., ion exchangers), and then evaporated and spray dried (Figure 4.6). Chicory oligofructose is obtained by partial enzymatic hydrolysis of inulin, eventually followed by spray drying. Hydrolysis is catalyzed either by exo-inulinase (EC 3.2.1.80), by the combined action of exoand endo-inulinases, or solely by endoinulinase (EC 3.2.1.7). Although the best source of these enzymes is Kluyveromyces fragilis that produces only an exo-inulinase, most inulin-hydrolyzing enzymes of yeast origin have both exo- and endoinulinase activity (Uchiyama, 1993). The enzymes used for the commercial production of fructose and oligofructose come from Aspergillus niger or Aspergillus ficuum. The long-chain inulin or inulin HP is produced by using physical separation techniques to eliminate all oligomers with a DP < 10. The product known as Synergy 1 is obtained by mixing 30:70 (w/w) oligofructose and inulin HP. Other products are also made from inulin by intermolecular (depolymerizing) fructosyl-transferases (from Arthobacter globiformis, Arthobacter urefaciens, and pseudomonas) like DFA’s (difructose dianhydrides) and cyclic forms of difructose. Cyclofructans are also produced using an extracellular enzyme of Bacillus circulans. This enzyme forms mainly cycloinulohexaose (CFR-6), but also small amounts of cycloinuloheptaose and -octaose by an intramolecular transfructosylation reaction. Physicochemical and technological properties of chicory inulin, oligofructose, and their derivatives in powder form are presented in Figure 4.7 and their food applications are presented in Figure 4.8. Fructooligosaccharides are classified as prebiotics since they have the ability to selectively promote the growth of healthy intestinal bacteria (such as Bifidobacteria and Lactobacilli) at the expense of the putrefactive bacteria (such as bacteroides, clostridia, and other coliforms). Bifidobacteria produce acetic and lactic acids, which inhibit the growth of pathogenic bacteria and stimulate intestinal peristalsis. FOS facilitates the absorption of calcium, and possibly magnesium also, and may lower the risk of osteoporosis. They also suppress the activity of cancer causing enzymes in the large bowel. Because of these health benefits, these carbohydrates are being added to many processed foods. Sources Of Prebiotics Common food sources of prebiotics include whole grains, oatmeal, flaxseed, barley, dandelion greens, spinach, collard greens, chard, kale, mustard greens, berries, fruits and legumes (lentils, kidney beans, chickpeas, navy beans, white beans, black beans, etc), chicory, onion, leek, garlic, artichoke and asparagus. Yacon, which looks like a potato,

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FIGURE 4.6 — Inulin Production Process

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FIGURE 4.7 — Physicochemical And Technological Properties Of Chicory Inulin, Oligofructose, And Their Derivatives In Powder Form Inulin

Inulin HP Oligofructose

Chemistry

GpyFn DP 2-60

GpyFn DP 10-60

GpyFn and FpyFn DP 2-7

DP av Content (% dry matter) Dry matter (%) Sugars (% dry matter) pH (10% in H2O) Ash (% dry matter) Heavy metals (% dry matter) Color Taste

12 92 95 8 5-7 <0.2 <0.2 White Neutral

25 99.5 95 <0.5 5-7 <0.2 <0.2 White Neutral

Sweetness vs sucrose Water solubility (% at 25°C) Water viscosity (5% at 10oC) Food application (specific)

10% 12 1.6 mPa Fat replacers +Gelling agent

None 2.5 2.4 mPa Fat replacers +Gelling agent

4 95 95 5 5-7 <0.2 <0.2 White Moderately sweet 35% >75 <1 mPa Fat replacers +Intense sweetener

Food application (synergism)

Synergy 1 GpyFn and FpyFn DP 2-7 DP 10-60 95 95 5-7 <0.2 <0.2 White Moderately sweet

Adapted from Roberfroid (2004).

FIGURE 4.8 — Typical Examples Of Food Applications Of Chicory Inulin, Oligofructose, And Their Derivatives Food Products

Applications

Dairy products

Body and mouth feel, Foam stability, Sugar and fat replacement, Synergy with sweeteners

Frozen desserts

Sugar and fat replacement, Synergy with sweeteners Texture and melting

Table spreads

Fat replacement, Texture and spreadability, Emulsion stability

Baked goods and breads

Sugar replacement, Moisture retention

Breakfast cereals

Crispness and expansion

Fruit preparations

Sugar replacement, Synergy with sweeteners, Body and mouth feel

Meat products

Fat replacement, Texture and stability

Chocolate

Sugar replacement, Heat resistance

Adapted from Roberfroid (2004).

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is a root vegetable from Peru. It has a sweet, juicy taste. It is low in calories and rich in FOS. Onion and wheat are the major sources of FOS in American diet. TYPES OF PREBIOTICS OTHER THAN INULIN AND FOS Fiber gums are often used in such foods as yogurt to give the product a thicker consistency. They can be used as a prebiotic or as thickening material. Obviously, processing varies according to the desired outcome. Fiber gums are water-soluble and derived from such plants as acacia, carrageenan, guar, locust bean, and xanthan. Usually containing about 85% fiber, these gums help promote the production of large quantities of short-chain fatty acids, which are known to play several beneficial roles, including the development of such intestinal bacteria as Lactobacillus and Bifidobacteria. Isomalto-oligosaccharides are a mixture of glucose and other saccharide molecules. Produced by various enzyme processes, isomaltooligosacharides ultimately form several sugar molecules including isomaltose, panose, isomaltotetraose, isomaltopentaose, nigerose, kojibiose, isopanose and other higher branched oligosaccharides. They act to stimulate the growth of Bifidobacterium and Lactobacillus species in the large intestine. They are marketed in Japan as dietary supplements and used in functional foods. They are being developed in the United States for similar commercial uses. Lactilol is a disaccharide alcohol analogue of lactulose. Lactilol is used in many countries for treating constipation and hepatic encephalopathy, but not in the United States. In Japan, lactilol is also used as a prebiotic because it is resistant to digestion in the upper gastrointestinal tract and is fermented by a limited number of colonic bacteria. However, it is not approved as a prebiotic in the United States. In Europe, it is used as a food sweetener. Lactosucrose is a trisaccharide comprised of galactose, glucose, and fructose molecules. It is produced through enzyme action that results in sucrose. Resistant to digestion in the stomach and small intestine, lactosucrose acts on the intestinal microflora to increase significantly the growth of the Bifidobacterium species. Lactosucrose is widely used in Japan as a dietary supplement and in functional foods, including yogurt and is being developed in the United States for similar uses. Lactulose is a semisynthetic disaccharide comprised of galactose and fructose. Lactulose is resistant to human digestive enzymes and can be fermented by a limited number of bacteria in the colon, especially Lactobacilli and Bifidobacteria. Currently, lactulose is a prescribed drug

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in the United States for the treatment of constipation and hepatic encephalopathy, but it has not been proven to be a prebiotic substance. In Japan, it is marketed as a dietary supplement and used in functional foods. Lactulose has exhibited some ability to reduce infectious inflammatory bowel disorders, as well as some colonic tumors. Since it has some ability to improve glucose tolerance and is showing other improvements in carbohydrate metabolism, it is speculated that lactulose may be helpful in treating Diabetes Mellitus. In addition, it has significantly stimulated calcium absorption in postmenopausal women in preliminary clinical work. Oligofructose is a sweet product derived from native inulin and is approximately 30-60% as sweet as sugar. It is found on the market as an oligosaccharide because it consists mainly of fructose units with some glucose-terminated chains. It is also available as a mixture with inulin to reduce the amount of non-glucose terminated chains. The unbound fructose chains have prebiotic properties, but with a different fermentation profile than either inulin or FOS. However, it is fermented by a wider variety of probiotic bacteria than inulin. Unlike inulin, FOS has the ability to brown, making it a valuable addition to baked products. Pyrodextrins are a mixture of glucose-containing oligosaccharides derived from starch. Pyrodextrins are resistant to digestion in the upper gastrointestinal tract and have been found to promote the growth of Bifidobacteria in the large intestine and are being developed for the nutritional supplement market place. Soy oligosaccharides are those found mainly in soybeans, but can also be found in other beans and peas. There are two principal soy oligosaccharides: the trisaccharide raffinose and the tetrasaccharide stachyose. Raffinose is comprised of one molecule each of galactose, glucose and fructose. Stachyose is comprised of two molecules of galactose, one molecule of glucose and one molecule of fructose. Soy oligosaccharides act to stimulate the growth of Bifidobacterium species in the large intestine. They are marketed in Japan as dietary supplements and in functional foods and are being developed in the US for similar uses. Transgalacto-oligosaccharides (TOS) are a mixture of glucose and galactose oligosaccharides. They are produced from lactose via enzyme action obtained from Aspergillus oryzae, which can also be a pathogen. TOS are resistant to digestion in the upper gastrointestinal tract, and therefore able to stimulate the growth of bifidobacteria in the large intestine. TOS are marketed in Japan and Europe as dietary supplements and used in functional foods. They are being developed for similar use in the United States. TOS have demonstrated positive

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effects on calcium absorption and have prevented bone loss in some animal models. In preliminary studies, TOS have shown some ability to lower triglycerides. Xylo-oligosaccharides are comprised of oligosaccharides containing beta-linked xylose residues and obtained from enzymatic action. They are marketed in Japan as prebiotics and are being developed for similar use in the United States. Since xylo-oligosaccharides resist digestion in the upper gastrointestinal tract, they are able to function in the large intestine to increase the growth of Bifidobacterium species, thus improving gastric function. According to preliminary research, xylooligosaccharides have the potential to improve blood sugar levels and fat metabolism, restore normal intestinal flora following antibiotic, chemo, or radiation therapies, increase mineral absorption and vitamin B production, and reduce intestinal putrification. Beneficial Effects Of Prebiotics On Health Inulin, oligofructose, lactulose, galactooligosaccharides and synthetic FOS are probably the only prebiotics for which available scientific evidence would indicate limited and defined health benefits. The chemical structure of these prebiotics prevents their digestion in the small gut. Consequently, they reach the large bowel undigested and are fermented by bacteria. This fermentation stimulates the growth of Bifidobacteria, a species used as probiotics. The ability of these oligosaccharides to alter the gut microbial population towards a more beneficial composition has been consistently shown in human studies. The greatest benefit appears to be in those individuals with low levels of bifidobacteria. It must be pointed out that the daily intake of prebiotics can be increased by dietary means, which includes the regular consumption of leeks, artichokes, garlic, onions, wheat and wheat products, asparagus and bananas. The average daily intake of these prebiotics from food ranges from 1-4 g in the U.S.A. to 3-11 g in Europe. Although there is no daily recommendation for prebiotics, doses of 4-20 g per day have shown efficacy. Many other potential prebiotics are currently under investigation, including xylooligosaccharides, lactitol, soyoligosaccharides, pecticoligosaccharides, glucooligosaccharides, isomaltooligosaccharides and gentiooligosaccharides. Prebiotics have also been associated with a reduction in the risk for diarrhea, constipation, colon cancer, osteoporosis and heart disease. Their effect in improving constipation is largely attributed to increasing fecal bulk and improving gut motility.

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Mechanism Of Action Of Prebiotics In Reducing The Risk Of Colon Cancer The β 2-1 osidic bond of FOS, including the first glucose-fructose bond, is not readily hydrolyzed by mammalian digestive systems. However, they are food for Bifidobacteria, which possess β-fructosidases that can digest these compounds. As a result of this, short chain fatty acids acetic, butyric and lactic acids are produced which inhibit the growth of pathogenic bacteria and help to prevent colon carcinogenesis. Prebiotics stimulate the growth of endogenous Bifidobacteria, which after a short feeding period become predominant in human feces (Gibson et al., 1995). It has been suggested that the mechanisms by which fructooligosaccharides modulate human colon cancer incidence may involve multiple actions in the lumen and target tissue (Klurfeld, 1997). Reddy et al (1997) fed 10% oligofructose or inulin to rats given azoxymethane (AOM), a substance known to produce preneoplastic aberrant crypt foci (ACF) in the rat colon. At week 7 after the last dose of AOM, there were fewer ACF in the study groups (inulin, 78 ACF; oligofructose, 92 ACF) compared with the placebo group (120 ACF). The result may be due to the fact that Bifidobacteria contain a relatively small amount of enzymes βglucuronidase, azoreductase and nitroreductase that can convert precancerous substances into carcinogens (Hughes and Rowland, 2001). Indeed, trans-galactosylated oligosaccharides and oligofructose were found to suppress fecal activities of carcinogen metabolizing enzymes in humans and rats. Buddington et al (1996) noted significantly reduced nitroreductase activity while using 4 g/day of FOS. In addition, the study showed that reductive enzymes β-glucuronidase and glycoholic acid hydroxylase were decreased to 75% and 90%, respectively. β-Glucoronidase has implications in carcinogenesis through the release of aglycones from glycosides, while glycoholic acid hydroxylase is involved with the production of secondary bile acids, potentially linking it to the increased risk of cancer associated with high fat diets. A high fat, low fiber Western diet is responsible for reduced number of colonic apoptotic cells and is associated with tumorgenesis (Risio et al., 1996). In a study conducted by Hughes and Rowland (2001), rats were fed either a high fat diet alone, with oligofructans, or with inulin for 3 weeks. They were exposed to 1,2-dimethyl-hydrazine, and it was found that the mean number of apoptotic bodies was higher in the oligofructans and inulin groups than control. It is speculated that the bulking effects of these prebiotics contribute to their antineoplastic effect by decreasing exposure to carcinogenesis.

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Inulin and oligofructose are fermented by colonic microflora and behave as soluble fibers and selectively stimulate the growth of Bifidobacteria at the expense of Bacteroides, clostridia and coliforms. Bacterial fermentation of these prebiotics in the colon produces short chain fatty acids (SCFA) such as butyric acid which have been shown to increase apoptosis in human colonic cell lines (Campbell et al., 1997). Inulin is also found to inhibit preneoplastic lesions of the colon but the mechanisms involved are not fully known. Gibson et al (1995) have suggested that the effect of inulin proceeds through the modulation of microflora and production of SCFA in the colon. High butyrate levels following fermentation of soluble fibers may inhibit events in colon tumorigenesis by controlling the transcription expression and activity of key proteins involved in the apoptotic cascade. Verghese et al (2002) tested inulin, a known suppressor of azoxymethane (AOM)-induced aberrant crypt foci (ACF), for its ability to suppress preneoplastic ACF formation in mature rats. The authors found out that long-chain inulin dose dependently reduced ACF incidence in the colon (P<0.01). Compared with rats fed the control diet, the percentage of reductions of ACF in rats fed 2.5, 5.0 and 10 g inulin /100 g diets were 25, 51 and 65%, respectively. Because the long-chain oligosaccharides are fermented at a slower rate than short-chain oligosaccharides, they indeed may reach the more distal part of the colon where they can stimulate microbial metabolism. Altering the metabolic activity of the colonic microflora by inulin, which is bifidogenic reduction in cecal pH and stimulation of immune activity, may be the mechanisms by which the anticarcinogenic effect is exerted. Hsu et al (2004) evaluated the effects of xylooligosaccharides (XOS) and FOS on the precancerous colon lesions in male Sprague-Dawley rats. Both XOS and FOS markedly increased the total cecal weight and Bifidobacteria population. XOS had a greater effect on the bacterial population than did FOS. Moreover, both XOS and FOS markedly reduced the number of aberrant crypt foci in the colon of 1, 2dimethylhydrazine (DMH) treated rats. These results suggest that XOS and FOS dietary supplementation may be beneficial to gastrointestinal health. Taper and Roberfroid (1999) studied the influence of inulin and oligofructose on breast cancer and tumor growth. In a preliminary study on methylnitrosourea-induced mammary carcinogenesis in SpragueDawley female rats, 15% oligofructose added to the basal diet modulated this carcinogenesis in a negative manner. There was a lower number of

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tumor bearing rats and a lower total number of mammary tumors in oligofructose-fed rats than in the group fed the basal diet alone. The effect of dietary nondigestible carbohydrates (15% oligofructose, inulin or pectin incorporated into the basal diet) on the growth of intramuscularly transplanted mouse tumors, belonging to two tumor lines (TLT and EMT6), was also investigated. The results were evaluated by regular tumor measurements with a vernier caliper. The mean tumor surface in the experimental groups was compared with that in animals of the control group. Such nontoxic dietary treatment appears to be easy and risk free for patients, applicable as an adjuvant factor in the classical protocols of human cancer therapy. Prebiotics induce changes in the population and metabolic characteristics of the gastrointestinal bacteria, modulate enteric and systemic immune functions, and provide laboratory rodents with resistance to carcinogens that promote colorectal cancer. There is less known about protection from other challenges. Therefore, Buddington et al (2002) conducted a study in which mice of the B6CF1 strain were fed for 6 weeks a control diet with 100 g/kg cellulose or one of two experimental diets with the cellulose replaced entirely by the nondigestible oligosaccharides, oligofructose and inulin. From each diet, 25 mice were challenged by a promoter of colorectal cancer (1,2-dimethylhydrazine), B16F10 tumor cells, the enteric pathogen Candida albicans (enterically), or were infected systemically with L. monocytogenes or S. typhimurium. The incidences of ACF in the distal colon after exposure to dimethylhydrazine for mice fed inulin (53%) and oligofructose (54%) were much lower than in control mice (76%, P<0.05), but the fructans did not reduce the incidence of lung tumors after injection of the B16F10 tumor cells. Mice fed the diets with fructans had 50% lower densities of C. albicans in the small intestine (P<0.05). A systemic infection with L. monocytogenes caused nearly 30% mortality among control mice, but none of the mice fed inulin died, with survival intermediate for mice fed oligofructose. Mortality was higher for the systemic infection of S. typhimurium (>80% for control mice), but fewer of the mice fed inulin died (60%; P<0.05), with mice fed oligofructose again intermediate. The mechanistic basis for the increased resistance provided by dietary nondigestible oligosaccharide was not elucidated, but the findings are consistent with enhanced immune functions in response to changes in the composition and metabolic characteristics of the bacteria resident in the gastrointestinal tract.

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The Effect Of Fructan-Type Oligosaccharide Prebiotics On Lipid Metabolism In Humans In recent years, there has been increasing interest in the important nutritional roles of prebiotics as functional food ingredients. This interest has been derived from animal studies which have shown marked reductions in triacylglycerols (TAG) and, to a lesser extent, cholesterol levels when diets containing significant amounts of the prebiotic (FOS) were fed. However, studies conducted in humans examining the effects of prebiotics on plasma lipid levels have generated inconsistent findings. Prebiotics have been shown to be an ideal substrate for health promoting bacteria in the colon, notably bifidobacteria and lactobacilli (Gibson and McCartney, 1998). During the fermentation process, a number of byproducts are produced, including gases (hydrogen sulphide, carbon dioxide, hydrogen and methane), lactate and short chain fatty acids (acetate, butyrate and propionate). The short chain fatty acids acetate and propionate enter the portal blood stream where they are utilized by the liver. Acetate is converted to acetyl CoA in the liver and acts as a lipogenic substrate for de novo lipogenesis, whereas propionate has been reported to inhibit lipid synthesis (Demigne et al., 1995). Butyrate, on the other hand, is taken up by the large intestinal cells (colonocytes) and has been shown to protect against tumour formation in the gut. The type of short chain fatty acids which are produced during fermentation is dependent on the gut microflora that is stimulated by the prebiotic. Inulin, for example, has been shown to increase both acetate and butyrate levels (Van Loo et al., 1999). Inulin and FOS have been extensively studied to determine the mechanism of action of prebiotics in animals. Early in vitro studies using isolated rat hepatocytes suggested that the hypolipaemic action of FOS was associated with an inhibition of de novo cholesterol synthesis by propionate, following impairment of acetate utilization by the liver for de novo lipogenesis (Demigne et al., 1995). Fiordaliso et al (1995) demonstrated significant reductions in plasma TAGs, phospholipids and cholesterol in normolipidaemic rats fed a chow diet containing 10% (w/ v) FOS. The TAG-lowering effect was demonstrated after only 1 week of FOS and was associated with a reduction in very low density lipoprotein (VLDL) secretion. TAGs and phospholipids are synthesised in the liver by esterification of fatty acids and glycerol-3-phosphate before being made available for assembly into VLDL, suggesting that the hypolipidaemic effect of FOS may be occurring in the liver. The

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reduction observed in cholesterol levels in the rats was only demonstrated after long term feeding (16 wks) of FOS. Recent evidence has suggested that the TAG-lowering effect of FOS occurs via a reduction in VLDL TAG secretion from the liver due to a reduction in the activity of all lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, ATP citrate lyase and glucose-6- phosphate dehydrogenase), and, in the case of fatty acid synthase, via modification of lipogenic gene expression (Delzenne and Kok, 1998). However, further work is required to determine the mechanisms whereby short chain fatty acids lower cholesterol levels in humans. The Effect Of Prebiotics On Glucose And Insulin Levels It has been suggested that the mechanism of action of prebiotics on the lowering of glucose and insulin levels is associated with short chain fatty acids, especially propionate. A significant reduction in postprandial glucose concentration was observed following both acute and chronic intakes of propionate-enriched bread (Todesco et al., 1991). The effect of propionate intake on postprandial insulin levels was not investigated. A recent animal study has shown an attenuation of both postprandial insulin and glucose levels following 4 wks of feeding with FOS. These effects were attributed to the actions of FOS on the secretion of the gut hormones glucose dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1). These hormones are secreted from the small intestine (GIP) and the terminal ileum and colon (GLP-1), and contribute towards the secretion of insulin following a meal in the presence of raised glucose levels (Kok et al., 1998). Prebiotics In Infant Health And Nutrition Oligosaccharides are the third most abundant solid constituent of human milk in which these are believed to play two major roles, i.e., defense agents by acting as receptor analogues to inhibit the binding of enteropathogens to the host cell receptors and bifidogenic factors. At least 21 different kinds of these oligosaccharides have already been identified that are either linear or branched, composed of simple sugars like galactose, or sugar derivatives like uronic acids or uronic esters, some being acidic and others being neutral. The oligosaccharide secretion in mother’s milk is a complex, variable and dynamic process. The amount of oligosaccharides in human milk change during lactation and also the composition of their mixture vary among different samples, being influenced by many factors one of which is the mother’s diet. The highest amount of oligosaccharides is reached on day four after birth.

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At days 30 and 120 of lactation, the content decreases by 20 and 40%, respectively, and is compensated by an increase in lactose content. Cow’s milk is very poor in oligosaccharides and infant formulas made with cow’s milk are deficient in oligosaccharides. It has been hypothesized that supplementing infant formulas with oligosaccharides could improve the nutritional value of formula and help mimic some of the effect of mother’s milk, especially the bifidogenic effect. Studies have shown that the gut microflora of breast-fed infants is dominated by bifidobacteria, whereas the gut microflora of infants fed infant formula have a diverse composition (higher numbers of Bacteroides spp., Clostridium spp. and Enterobacteriaceae). The high proportion of bifidobacteria present in the gut of breast-fed infants is associated with lower risk of intestinal infection. There is evidence that human milk oligosaccharides may promote the proliferation of intestinal bifidobacteria and lactobacilli, thus contributing to the natural defense against infection. Since the composition and structure of human milk oligosaccharides cannot be entirely reproduced by the food industry, prebiotics are being considered for fortification of infant formulas. In South Africa, there are formulas fortified with prebiotics for the infants older than 6 months. Preliminary studies have reported that infants fed a cow’s milk formula supplemented with fructo-oligosaccharides and galacto-oligosaccharides had a significantly increased number of faecal bifidobacteria after 28 days of feeding. In addition to this, stool characteristics of the babies fed the supplemented formula were similar to those of the breast fed babies (Roberfroid, 2004). Breast feeding must remain the gold-standard and the common recommendation. But to help in improving the intestinal health and well-being of babies who are not breast fed at all, breast-fed only for a short period, or are mixed-fed, supplementing infant formulas with inulin-type fructans and other prebiotics is a promising approach. At present, some infant formulas are being fortified with prebiotics in China. Probiotics – Friendly Creatures The health benefits of bacteria in food were known as early as the Persian version of the Old Testament (Genesis 18:8), which states “Abraham owed his longevity to the consumption of sour milk.” The Russian Nobel prizewinner Elie Metchnikoff, in the beginning of the 20th century observed high life expectancy in Bulgarians who consumed large amounts of fermented-milk products. It was these observations that led to the concept of “probiotic,” derived from the Greek, meaning

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“for life” which evolved to apply to those bacteria that “contribute to intestinal balance.” The term probiotic, as an antonym to the term antibiotic, was originally proposed in 1965 by Lilley and Stilwell. The first probiotic species introduced into research were Lactobacillus acidophilus by Hull et al in 1984 and Bifidobacterium bifidum by Holcombh et al in 1991 (Tanboga et al., 2003). Probiotic food is defined as a preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora by implantation or colonization in a compartment of the host and by doing that exert beneficial health effects on the host (Schrezenmeir and deVrese, 2001). The probiotic microorganisms should be : • of human origin, • nonpathogenic in nature, • resistant to destruction during processing, • resistant to destruction by gastric acid and bile, • able to adhere to intestinal epithelial tissue, • able to colonize in the gastrointestinal tract, • able to produce antimicrobial substances, • able to modulate immune responses and • able to influence human metabolic activities (cholesterol assimilation, vitamin production, etc). The complex gut microflora, consists of >1 X 1011-13 living bacteria/g colon content and the bacteria with such beneficial effects are lactic acid bacteria (LAB). Probiotics And General Health The human body is a natural habitat for microorganisms and symbiosis with these microorganisms seems to be a condition for survival. A human individual has more prokaryotic organisms associated with skin, lung, and gut surfaces than human eukaryotic cells. A logical management approach to situations that alter our microbial ecology (e.g., diet, environment, antibiotics) would be to deliberately increase our association with specific non-pathogenic organisms to counter that alteration. Probiotics exert a wide spectrum of different effects ranging from direct antagonism against pathogens to influence upon intestinal epithelium and immune system of the host. Thus the use of probiotics constitutes a purposeful attempt to modify the relationship with our immediate microbial environment in ways that may benefit general health. Probiotic bacteria have been shown to influence the immune system through several molecular mechanisms (Gibson, 1998). A

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number of potential benefits arising from the use of probiotics have been proposed, including: • increased resistance to infectious diseases, • alleviating lactose intolerance, • prevention from gut, vaginal and urogenital infections, diarrhea and gastritis, • reduction in blood pressure and regulation of hypertension and serum cholesterol concentration, • reduction in allergy, respiratory infections and • resistance to cancer chemotherapy and decreasing risk of colon cancer. Today, research regarding probiotics concentrates essentially on L. acidophilus, L. casei, L. reuteri and B. bifidum. The growth in the production of probiotics by the dairy industry in some countries means that it is now increasingly difficult to purchase yogurts that do not contain probiotic bacteria such as L. acidophilus. Culture manufacturers recommend formulation of these products at 106 probiotic bacteria per gram or milliliter of dairy products, but viable counts may fall below these levels, especially at the end of shelf life. While defined as ‘medical probiotics’ (microbial preparation) and ‘other probiotics’ (functional food), probiotics are provided in products in one of four basic ways: • as a culture concentrate added to a beverage or a food • inoculated into prebiotic fibers • inoculated into a milk-based food (dairy products such as milk, milk drink, yogurt, yogurt drink, cheese, kefir, biodrink) and • as concentrated and dried cells packaged as dietary supplements (non-dairy products such as powder, capsule, gelatin tablets). Probiotics And Gastrointestinal Health Gastrointestinal infections and their consequences remain a major clinical problem despite numerous therapeutic improvements, especially in the field of antibiotics. In addition, there has been a dramatic increase in the incidence of antibiotic-resistant microbial pathogens. There is a concern that industry will no longer be able to develop effective antibiotics at a rate sufficient to compete with the development of microbial resistance to existing antibiotics. These factors have renewed interest in the possibility of deliberately feeding beneficial microorganisms to humans as an alternative to antibiotic therapy in gastrointestinal disorders. Probiotics are also an attractive treatment alternative because antibiotics further delay recolonization by normal colonic flora which can be avoided.

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Probiotics are usually targeted for use in intestinal disorders in which specific factors (such as antibiotics, medication, diet or surgery) disrupt the normal flora of the gastrointestinal tract, making the host animal susceptible to disease. Examples of such diseases include antibioticinduced diarrhea, pseudomembranous colitis and small bowel bacterial overgrowth. The goal of probiotic therapy is to increase the numbers and activities of those microorganisms suggested to possess healthpromoting properties until such time that the normal flora can be established. These diseases include traveler’s diarrhea, Helicobacter pylori gastroenteritis and rotavirus diarrhea. Intestinal Disorders Treated With Probiotics Antibiotic-induced diarrheal disease: Diarrhea is the most common side effect of antibiotic therapy. The pathogenesis of antibioticinduced diarrhea is not understood but is undoubtedly related to quantitative and qualitative changes in the intestinal flora. Several probiotics have been used in an attempt to prevent antibiotic associated diarrhea. These agents include Saccharomyces, Lactobacillus, Bifidobacterium, and Streptococcus. However, only S. boulardii, E. faecium and Lactobacillus have been shown to be clinically effective in preventing antibiotic-associated diarrhea. In a prospective, double blind, placebo-controlled study, treated 180 hospitalized patients were receiving antibiotic therapy concurrently with either placebo or S. boulardii. The overall incidence of diarrhea in these patients was 26%. There was significant difference between the placebo group and the S. boulardii group. Twenty two percent of the placebo group developed diarrhea whereas only 9% of the patients receiving S. boulardii treatment (Surawicz et al., 1989). In another study of 193 patients receiving at least one broad-spectrum β-lactam antibiotic, 97 patients received S. boulardii and 96 patients received placebo. Only 7.2% of the S. boulardii group developed antibiotic-associated diarrhea compared with 14.6% of the placebo group (McFarland et al., 1995). Clostridium difficile-associated intestinal disease. C. difficile is a classic example of opportunistic proliferation of an intestinal pathogen after breakdown of colonization resistance due to antibiotic administration. After antibiotic intake by animals and humans, C. difficile colonizes the intestine and releases two protein exotoxins, toxins A and B, which mediate the diarrhea and colitis caused by this microbe. Toxigenic C. difficile is the cause of ~20-40% of cases of antibioticassociated diarrhea (Fekety and Shah, 1993). In fact, this microorganism is the major identifiable cause of nosocomial diarrhea in the US,

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infecting 15-25% of adult hospitalized patients. This bacterium (C. difficile) can cause serious consequences, particularly in the elderly and debilitated; these include pseudomembranous colitis, toxic megacolon, intestinal perforation and even death. Standard treatment of C. difficile–associated intestinal disease, which involves either vancomycin or metronidazole, can be expensive and difficult. In addition, 25% of patients relapse with disease once treatment is discontinued (Fekety et al., 1989). Multiple relapses can occur and the relapses can be more severe than the original disease. The mechanism of relapse is unknown but is probably due to the survival of C. difficile spores in the intestinal tract until the antibiotic is discontinued. The spores then germinate and produce toxin. The antibiotic therapy prevents the normal flora from reestablishing itself. There is no uniform effective therapy to prevent further C. difficile recurrences in intractable patients. An attractive alternative to antibiotic therapy is to use probiotics to restore intestinal homeostasis. S. boulardii has demonstrated the most promise for use in C. difficile– associated intestinal disease. In a placebo-controlled study, McFarland et al (1994) examined standard antibiotic therapy (metronidazole or vancomycin) with concurrent S. boulardii or placebo in 124 adult patients, 64 patients with an initial episode of C. difficile disease and 60 patients with a history of at least one prior episode of C. difficile disease. The investigators found that in patients with an initial episode of C. difficile, there was no significant difference in the recurrence of C. difficile disease in the placebo or S. boulardii groups. However, in patients with prior C. difficile disease, S. boulardii significantly inhibited further recurrences of disease. The investigators concluded that in combination with standard antibiotics, S. boulardii is an effective and safe therapy for patients with recurrent C. difficile. Probiotic Treatment Of Infectious Diarrhea The two more common types of infectious diarrheal diseases are traveler’s diarrhea and rotavirus diarrhea. Traveler’s diarrhea: The incidence of diarrhea in travelers to foreign countries varies from 20 to 50% depending on the origin and the destination of the traveler, as well as the mode of travel. Although various infectious agents can cause traveler’s diarrhea, enterotoxigenic E. coli is the most common. Even small attacks can interrupt a holiday, and the traveling public has a great interest in medications that could be used to prevent traveler’s diarrhea. Thus, a safe, inexpensive and effective drug against traveler’s diarrhea would have important public

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health implications. Several probiotics have been examined for their ability to prevent traveler ’s diarrhea, including Lactobacillus, Bifidobacterium, Streptococcus and Saccharomyces (Hilton et al., 1997). These studies have involved several different groups of travelers such as Finnish travelers to Turkey, American travelers to Mexico, British soldiers to Belize and European travelers to Egypt. Rotavirus diarrhea: Rotaviruses are a significant cause of infant morbidity and mortality, particularly in developing countries. The principal means of treatment is oral rehydration, although an effective vaccine that should decrease dramatically the health impact of rotavirus infections has recently become available. Lactobacillus has demonstrated some promise as a treatment for rotavirus infection. Isolauri et al (1991) treated 74 children (ages 4–45 mo) with diarrhea with either Lactobacillus GG or the placebo. Approximately 80% of the children with diarrhea were positive for rotavirus. The investigators demonstrated that the duration of diarrhea was significantly shortened (from 2.4 to 1.4 d) in patients receiving Lactobacillus GG. The effect was even more significant when only the rotavirus-positive patients were analyzed. Helicobacter pylori gastroenteritis: H. pylori has recently been shown to be an important etiologic agent of chronic gastritis as well as gastric and duodenal ulcers. It has also been postulated that chronic H. pylori infection leads to stomach carcinoma. Lactobacillus has been shown to be antagonistic to H. pylori both in vitro and in a gnotobiotic murine model (Aiba et al., 1998). Hepatic encephalopathy: Hepatic encephalopathy is a neurologic disorder caused by increased blood levels of ammonia. The ammonia is produced in the intestine by the action of bacterial ureases. The ammonia is absorbed and, in healthy individuals, is detoxified by the liver. However, in patients with liver failure, the blood concentration of ammonia can reach toxic levels. Investigators have postulated that it may be possible to use probiotics to decrease intestinal urease activity. For example, patients treated with L. acidophilus and neomycin show a greater decrease in fecal urease activity than patients treated with neomycin alone (Scevola et al., 1989). The decreased fecal urease activities corresponded to lower serum ammonia levels and improvements in the clinical status of patients. HIV/AIDS diarrhea: Diarrhea is a very serious consequence of human immunodeficiency virus (HIV) infection. The etiology of this diarrhea is frequently unknown and there are no effective treatment modalities. However, S. boulardii was recently used to treat 33 HIV

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patients with chronic diarrhea (Born et al., 1993). In these double-blind studies, 56% of patients receiving S. boulardii had resolution of diarrhea compared with only 9% of patients receiving placebo. Sucrase-isomaltase deficiency: Sucrase-isomaltase deficiency is the most frequent primary disaccharidase deficiency seen in humans. It is an inherited condition that leads to malabsorption of sucrose. The resulting bacterial fermentation of the sucrose leads to an accumulation of hydrogen in the colon, producing diarrhea, abdominal cramps and bloating. A sucrose-free diet will lead to a disappearance of symptoms. However, not all patients will follow such a diet. Harms et al (1987) used Saccharomyces cerevisiae to treat eight children with sucraseisomaltase deficiency. These investigators demonstrated that in children given sucrose followed by S. cerevisiae, there was an improvement in both their hydrogen breath test and gastrointestinal symptoms. The investigators postulated that S. cerevisiae was supplying the missing enzymes. Lactose intolerance: People throughout the world suffer from a congenital deficiency of the enzyme β-galactosidase. This deficiency results in an inability to digest and absorb lactose. Bacteria in the gastrointestinal tract metabolize the lactose and the resulting by-products cause abdominal cramping, bloating, diarrhea and nausea. Lactasepositive strains of bacteria (e.g., Lactobacillus, Bifidobacterium and Streptococcus) are commonly added to pasteurized dairy products to increase digestibility of the lactose present in the dairy product. There are two probable mechanisms by which the addition of these bacteria is beneficial, i.e., the reduction of lactose in the dairy product through fermentation and the replication of the probiotic in the gastrointestinal tract, which releases lactase. Pouchitis: Pouchitis is a complication of ileal reservoir surgery occurring in 10–20% of the patients who undergo surgical treatment for chronic ulcerative colitis. Bacteria overgrow in the pouch, resulting in degradation of the mucus overlaying the epithelial cells. This results in inflammation and symptoms that include bloody diarrhea, lower abdominal pain and fever. Investigators have postulated that Lactobacillus GG may be an effective therapeutic agent for pouchitis because it does not demonstrate mucus-degrading properties (RuselerVan Embden et al., 1995). Irritable bowel syndrome: Irritable bowel syndrome is characterized by chronic, recurrent pain that occurs primarily during childhood. There is no specific treatment of this condition. However, a small, double blind, placebo-controlled, crossover study in Poland demonstrated a slight but

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significant reduction in the severity of abdominal pain in individuals receiving L. plantarum (Niedzielin and Kordecki, 1996). Small bowel bacterial overgrowth: Overgrowth of bacteria in the small intestine can have many causes, including blind loops, stenosis of the intestine, diverticula and motility disorders. Symptoms of small bowel overgrowth are frequently chronic and relapsing. Response to antibiotic treatment is often inadequate or incomplete. Surgical treatment is occasionally possible, but in many cases the underlying cause is not accessible for permanent treatment. Limited studies have suggested that L. plantarum and Lactobacillus GG may be helpful in eliminating the symptoms of small bowel bacterial overgrowth. Enteral feeding–associated diarrhea: Patients receiving nasogastric tube feeding frequently develop diarrhea. The mechanism of the diarrhea is not known, but investigators postulate that enteral feeding causes changes in normal flora that result in altered carbohydrate metabolism and subsequent diarrhea. Two separate studies (both placebo-controlled and double blind) demonstrated a significant reduction in diarrhea in these patients when they were given S. boulardii (Bleichner et al., 1997). Probiotics For Cancer Patients LAB play an important role in retarding colon carcinogenesis possibly by influencing metabolic, immunologic, and protective functions in the colon. Probiotics have anticarcinogenic-antimutagenic effects in vivo (Wollowski et al., 2001). In fact, Bifidobacterium longum supplementation reduces colon and liver carcinogenesis by 2-amino-3methylimidazo [4,5-f]quinoline as well as azoxymethane (AOM)-induced colon cancer in rats (Singh et al., 1997). Dietary supplements of Lactobacilli also increase the latency of induction of experimental colon cancer in rats, suggesting that Lactobacilli and Bifidobacteria may inhibit precancerous lesions and tumour development in animal models (Brady et al., 2000). Mechanism Of Action The mechanism by which probiotics exert the anticancer effect is unclear. However, potential mechanisms of anti-carcinogenicity of probiotics may include: • Alteration of the metabolic activities of intestinal microflora, • Alteration of physicochemical conditions in the colon, • Binding and degrading potential carcinogens, • Quantitative and qualitative alterations in the intestinal microflora,

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• Production of antitumorigenic or antimutagenic compounds, • Enhancing the host’s immune response, • Effects on the physiology of the host, • Fermentation of undigested food and the formation of metabolites Alteration Of The Metabolic Activities Of Intestinal Microflora Many foreign compounds which may be carcinogenic are detoxified in the liver which converts them to inactive glucuronides of procarcinogens. Subsequently those in bile enter the bowel and are excreted out of the body. But some intestinal flora possess enzymes such as β-glucuronidase, nitroreductase, azoreductase and choloylglycine hydrolase which hydrolyse glucuronides and liberate carcinogenic aglycones in the intestinal lumen. So when the diet is supplemented with probiotics, which have relatively low paucity of these enzymes, they may overtake the growth of the intestinal flora possessing higher activity of these enzymes. This alters the metabolic activities of intestinal flora resulting in inhibition of colon cancer (Rafter, 2002). Oral supplementation of the diet with viable L. acidophilus of human origin, which is bile resistant, caused a significant decline in three different fecal bacterial enzymes. The decline in fecal enzyme activity was noted in humans and rats. The bacterial enzymes that were affected included β-glucuronidase, azoreductase and nitroreductase. The effect of feeding of L. acidophilus strains NCFM and N-2 on the activity of three bacterial enzymes, i.e., β-glucuronidase, nitroreductase, azoreductase was studied in 21 healthy volunteers. Both strains had similar effects and caused a significant decline in the specific activity of the three enzymes in all subjects after 10 days of feeding. A reversal of the effect was observed within 10-30 days of ceasing L. acidophilus feeding, suggesting that continuous consumption of these bacteria was necessary to maintain the effect (Goldin and Gorbach, 1984). Consumption of milk with L. casei Shirota for 4 weeks temporarily decreased β-glucuronidase in 10 healthy subjects compared with 10 healthy controls (Rolfe, 2000). Another study using L. acidophilus, B. bifidum, S. lactis, and S. cremoris for 3 weeks demonstrated reduction of nitroreductase (Marteau et al., 1990). Thus, animal and human studies indicate that feeding certain lactic cultures can result in a decrease of fecal enzymes that may be involved in formation of carcinogens. Alteration Of Physico-chemical Conditions In The Colon Moddler et al (1990) have suggested that large bowel cancer could be influenced directly by reducing intestinal pH, thereby preventing the growth of putrefactive bacteria. In rats given inulin-containing diets

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with Bifidobacterium longum, an increase in fecal weight and a decrease in fecal pH were observed (Rowland et al., 1998). In a study by Biasco et al (1991), patients with colonic adenomas participated in a 3-month study, where L. acidophilus was administered together with B. bifidum. During this period, fecal pH and proliferative activity were reduced significantly after therapy with lactic acid bacteria. Binding And Degrading Potential Carcinogens One potential risk factor of colon cancer that is related to high meat consumption is the formation of heterocyclic amines formed during the cooking of meat. There are several reports of binding of carcinogens, such as heterocyclic amines, Aflatoxin B1 and benzopyrene, in vitro by LAB and other intestinal bacteria. Depending on the pH of the culture medium, LAB can bind to heterocyclic amines (Orrhage et al., 1994). When the dose of trypsin and bile acids was increased in a medium to simulate an in vivo situation in the intestine, the binding capacity of LAB decreased linearly and the negative influence of bile acids was more pronounced (Tanabe et al., 1994). Administration of L. acidophilus to healthy volunteers consuming a fried meat diet, known to increase fecal mutagenicity, resulted in a greater decrease in fecal mutagenic activity after 3 days than administration of ordinary fermented milk (Lidbeck et al., 1992). During L. acidophilus administration, the urinary mutagenic activity on days 2 and 3 was significantly lower compared to the ordinary fermented milk period. In most cases, an increase in the number of fecal Lactobacilli corresponded to a lower mutagen excretion, particularly in urine. Hayatsu and Hayatsu (1993) also demonstrated a marked suppressing effect of orally administered L. casei Shirota (LcS) on the urinary mutagenicity arising from ingestion of fried ground beef in man. It was estimated that the binding of mutagens could be attributed to the cell wall of bacteria and in view of the results of in vitro studies, it is possible that the lactic acid bacteria supplements are influencing excretion of mutagens by simply binding them in the intestine. Quantitative And Qualitative Alterations In The Intestinal Microflora The diet supplemented with products containing L. acidophilus has a beneficial effect on the intestinal microecology by suppressing the putrefactive organisms that are possibly involved in the production of tumor promoters and putative procarcinogens. Consumption of fermented milk containing L. acidophilus has been shown to significantly reduce the counts of fecal putrefactive bacteria such as

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coliforms and increase the levels of Lactobacilli in the intestine (Ayebo et al., 1980). Production Of Antitumorigenic Or Antimutagenic Compounds The antitumor activities of LAB may be due to the presence of an ex novo soluble compound produced by them during fermentation of milk or the microbial transformation of some milk components in a biologically active form. Although the protective activity of Lactobacilli observed in the colon may be explained by a local and direct effect on intestinal mucosa, some epidemiologic studies have also indicated a reduced risk of breast cancer in women who consume fermented milk products and/or the fermentative bacteria themselves may have chemoprotective effects. This was evident from studies using preimplanted cancer cells in animal models. Bogdanov et al (1978) observed that L. bulgaricus possessed a potent antitumour activity. They isolated three glycopeptides which had biological activity against sarcoma-180 and solid Ehrlich ascites tumour. Sekine et al (1994) reported that a single subcutaneous injection of whole peptidoglycan isolated from Bifidobacterium infantis strain ATCC 15697 significantly suppressed tumor growth. LAB significantly reduced the growth and viability of the human colon cancer cell line HT-29 in culture and dipeptidyl peptidase IV and brush border enzymes were significantly increased, suggesting that these cells may have entered a differentiation process (Baricault et al., 1995). L. bulgaricus prevented 1,2-dimethylhydrazine (DMH) induced DNA breaks in rats in vivo whereas S. thermophilus did not. However, both strains prevented DNA damage in vitro when rats were exposed to N-methyl-N-nitro-N-nitrosoguanidine (MNNG). Indeed extracts from the S. thermophilus were also effective in deactivating MNNG (Wollowski et al., 1999). The authors hypothesized that it is thiol-containing breakdown products of proteins created by bacterial proteases that deactivate various colonic mutagens. Milk fermented by B. infantis, B. bifidum, B. animalis, L. acidophilus and L. paracasei inhibited the growth of the MCF7 breast cancer cell line and the antiproliferative effect was not related to the presence of bacteria but due to the presence of an ex novo soluble compound produced by LAB (Biffi et al., 1997). It is expected that LAB or metabolites may prevent the carcinogens from inducing genotoxic effects. These preventive properties may be due to a scavenging of reactive carcinogen intermediates (by LAB or by their metabolites). Alternatively, LAB or LAB metabolites may affect carcinogen-activating and carcinogen-deactivating enzymes. Acetone

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extracts prepared from nonfermented milk, fermented milk, or L. acidophilus grown in De Man Rogosa Sharpe broth were investigated for their antigenotoxic activity in freshly isolated colon cells of rats treated with MNNG for 30 min. It was shown that fermentation resulted in short-lived metabolites that prevent DNA damage in these cells. The identity of these metabolites has not yet been characterized; however, protection by these metabolites was more pronounced than was protection observed by cellular components of LAB, eg., peptidoglycan or cytoplasma fractions (Wollowski et al., 2001). Enhancing The Host’s Immune Response The lactic acid bacteria are thought to suppress tumor formation by enhancing an immune response of the host. Sekine et al (1985) suggested that B. infantis stimulates the host-mediated response, leading to tumor suppression or regression. In addition there are studies to suggest that LAB play an important role in the host’s immunoprotective system by increasing specific and non specific mechanisms to have an antitumor effect (Schiffrin et al., 1995). Lactobacillus casei Shirota has been shown to have potent antitumor and antimetastatic effects on transplantable tumor cells and to suppress chemically induced carcinogenesis in rodents. Also, intrapleural administration of L. casei Shirota into tumorbearing mice has been shown to induce the production of several cytokines, such as interferon-, interleukin-1β and tumor-necrosis factor-
, in the thoracic cavity of mice resulting in the inhibition of tumor growth and increased survival. These findings suggest that treatment with L. casei Shirota has the potential to ameliorate or prevent tumorigenesis through modulation of the host’s immune system, specifically cellular immune responses. It has also been demonstrated that B. longum and B. animalis promote the induction of inflammatory cytokines in mouse peritoneal cells (Matsuzaki, 1998). Effects On The Physiology Of The Host Lactobacilli are one of the dominant species in the small intestine, and they presumably affect metabolic reactions occurring in this part of the gastrointestinal tract. They have been shown to increase colonic NADPH-cytochrome P-450 reductase activity (Pool-Zobel et al., 1996) and glutathione S-transferase levels (Challa et al., 1997) and to reduce hepatic uridine disphosphoglucuronyl transferase activity (Abdelali et al., 1995), enzymes which are involved in the metabolism of carcinogens in rats. It has been demonstrated that dietary administration of lyophilized cultures of B. longum strongly suppressed azoxymethane-

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induced colonic tumor development and that this effect was associated with a decrease in colonic mucosal and tumor ornithine decarboxylase and ras-p21 activities (Reddy, 1998). Fermentation Of Undigested Food And The Formation Of Metabolites A common characteristic of the microflora is fermentation. The anaerobic breakdown of substrates, such as undigested polysaccharides, resistant starch, and fiber, enhances the formation of short-chain fatty acids as fermentation products. Increased production of short-chain fatty acids leads to a decrease in the pH of colon content. A low pH in feces was associated with a reduced incidence of colon cancer in various populations (Segal et al., 1995). Depending on the nature, quantity, and fermentability of undigestible polysaccharides reaching the colon, the relation of the short-chain fatty acids acetate, propionate, and butyrate can vary. Resistant starch and wheat bran favor the production of butyrate, whereas pectin leads to a higher formation of acetate. Butyrate is associated with many biological properties in the colon (PoolZobel et al., 1996). One of the first observed effects of butyrate on the degree of methylation is probably associated with modified gene expression. Butyrate may also directly enhance cell proliferation in normal cells and suppress proliferation in transformed cells. In addition, apoptosis may be increased in transformed cells but inhibited in normal cells when butyrate is present (Marchetti et al., 1997). Butyrate is an important fuel for colon cells, which may explain the higher resistance of cells pretreated with butyrate to oxidative damage induced by hydrogen peroxide in comparison with cells not pretreated with butyrate. Butyrate has also been shown to increase glutathione transferase  in colon cells and may be a responsible factor for enhanced glutathione transferase  expression in colon tissue (Treptow-van Lishaut et al., 1999). Glutathione transferase  is the most abundant glutathione transferase species in colon cells and is an important enzyme involved in the detoxification of both electrophilic products and compounds associated with oxidative stress. Thus, enzyme induction by butyrate, or by the microflora and increased activity by prebiotics may be an important mechanism of protection against carcinogeninduced cancer. Probiotics For Oral Health Oral infections constitute some of the most common and costly forms of infections in humans. Dental caries and periodontal diseases occur in nearly 95% of the general public. Although fluoride and other

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preventive efforts have led to a dramatic decline in dental caries, the ability to control the actual infection has been limited (Caglar et al., 2005). The concept of microbial ecological change as a mechanism for preventing dental problems is an important one. The oral cavity is a complex ecosystem in which a rich and diverse microbiota has evolved. The wide range in pH, nutrient availability, shedding and non-shedding surfaces, salivary and crevicular fluids select localized, discrete microbial climax communities to fluctuate in composition and metabolic activity but reach a kind of homeostasis in balance with the host. Changes in the environment whether imposed by illness, debility, behavior, diet, or medications disturb the homeostasis and lead to endogenous infections or susceptibility to exogenous infections. The resident oral microflora is diverse, being comprised of species with differing nutritional (saccharolytic, proteolytic, secondary feeders), atmospheric (aerobic, anaerobic, facultative, micro-aerophilic, capnophilic) and physico-chemical (pH, co-factors) requirements. Dental disease may be a consequence of changes in the ecology stated above. If the local environment is perturbed, then potential pathogens may gain a competitive advantage and, under appropriate conditions, reach numbers that predispose a site to disease. Regarding elimination of pathogenic members of the oral cavity a new method such as probiotic approach (i.e., whole bacteria replacement therapy) is reported (Caglar et al., 2005). Possible Mechanisms Of Action Of Probiotics In Maintaining Oral Health As a result of cariogenic properties, lactobacilli have been of great interest to dental researchers for several decades. They are associated more with carious dentine and the advancing front of caries lesions rather than with the initiation of the dental caries process. Lactobacilli are the most common probiotic bacteria associated with the human gastrointestinal tract; therefore it may also play an important role in the eco physiology of oral microbiota. Various lactobacilli species (L. paracasei, L. gasseri, L. fermentum L. salivarius, L. plantarum, L. crispatus, and L. rhamnosus isolated) inhabit healthy mouths, although no species is specific to the mouths of healthy subjects. Development of new ways to block the pathogenesis of oral infections can reduce tissue destruction associated with oral infection and chronic inflammation. It is thought that probiotics particularly lactobacilli that hydrolyse proteins to amino acids and dipeptides, stimulate growth of streptococci which produce low pH conditions in the oral environment.

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Conversely, in recent studies, it was stated that probiotics might reduce the risk of the high level of Streptococcus mutans (Ahola et al., 2002) which are responsible for dental caries. Recently it was evaluated whether the oral administration of lactobacilli could change the salivary counts of these bacteria compared with placebo. Lactobacilli were administered in liquid and in capsule form to volunteer subjects to determine the role of direct contact with the oral cavity. It was found that the oral administration of probiotics, both in capsules and in liquid form, significantly increases salivary counts of lactobacilli while S. mutans levels were not modified (Montalto et al., 2004). There is a concept where these beneficial microorganisms can inhabit a bio-film and actually protect oral tissue from disease. It is possible that one of these biofilm’s mechanisms to keep pathogens out is to occupy a space that might otherwise be occupied by a pathogen. An in vitro study suggests that L. rhamnosus GG (LGG) can inhibit the colonization of streptococci caries pathogens, thus reducing the incidence of caries in children (Meurman et al., 1995). In a Swiss study, bacterial strains with potential properties as oral probiotics, were studied for the prevention of dental caries. From 23 dairy microorganisms studied, two were identified; which were able to adhere to saliva-coated hydroxyapatite beads to the same extent as Streptococcus sobrins OMZ176. Streptococcus thermophilus NCC1561 and Lactobacillus lactis NCC2211, were successfully incorporated into a bio-film mimicking the dental plaque. Furthermore, they could grow in such a biofilm together with five strains of oral bacterial species, representative of supragingival plaque. In this system, Lactococcus lactis NCC2211 was able to modulate the growth of the oral bacteria, and in particular to diminish the colonization of Streptococcus oralis OMZ607, Veillonella dispar OMZ493, Actinomyces naeslundii OMZ745 and of the cariogenic Streptococcus sobrinus OMZ176 (Comelli et al., 2002). From a periodontal view, a Russian study examined probiotic tablets in complex treatment of gingivitis and different degrees of periodontitis. The treatment of the patients of the control group was provided by drug ‘Tantum Verde’. The effect of probiotics to the normalization of microflora was found to be higher in comparison with Tantum Verde, particularly in the cases of gingivitis and periodontitis (Grudianov et al., 2002). There is no research regarding relationship between dental restorative materials and probiotics. However, in the larynx, the second barrier after the oropharynx, probiotics strongly reduce the occurrence of pathogenic bacteria in voice prosthetic bio-films (Free et al., 2001).

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Installation Of Probiotics In The Oral Cavity Probiotics should adhere to dental tissue for them to establish a cariostatic effect and thus should be a part of the bio-film to fight with cariogenic bacteria. For this action, installation of probiotics in the oral environment seems important. However, the contact time between probiotics and plaque would be short, that the activity is not sufficient to stop the growth of cariogenic bacteria. This activity increases if probiotics could be installed in the oral environment for a longer duration. At this point, ideal vehicles of probiotic installation should be determined. Effects Of Probiotics On Blood Cholesterol Agerbaek et al (1995) tested the effect of commercially available yogurt GAIO® (containing a specific bacterial culture, CAUSIDO® [consisting of Enterococcus faecium and Streptococcus thermophilus, and has been shown to have hypocholesterolemic properties when tested on animals]) against identical yogurt that had been chemically fermented with an organic acid (-glucolactone). Fifty-eight middleaged men with moderately raised cholesterol levels (5.0-6.5 mmol/l) were fed 200 ml per day of yogurt for a 6-wk period. They observed a 9.8% reduction in LDL cholesterol levels (P < 0.001) for the live yogurt group. The mechanism of action of probiotics on cholesterol reduction is unclear, although a number of possible mechanisms have been proposed. These include the physiological actions of fermentation end products (short chain fatty acids), deconjugation of bile acids (which could reduce cholesterol by co-precipitation at acidic pH or by increasing excretion of bile acids, thereby increasing the amount of cholesterol required for de novo synthesis in the liver, or a combination of both these mechanisms), cholesterol assimilation, and cholesterol binding to bacterial cell walls. It has been well documented that microbial metabolism of bile acid is a peculiar probiotic effect involved in the therapeutic role of some bacteria. The deconjugation reaction is catalysed by a conjugated bile acid hydrolase enzyme, which is produced exclusively by bacteria. Deconjugation is widely found in many intestinal bacteria including genera such as Enterococcus, Peptostreptococcus, Bifidobacterium, Fusobacterium, Clostridium, Bacteroides and Lactobacillus. This reaction liberates an amino acid moiety and a deconjugated bile acid, thereby reducing cholesterol re-absorption by increasing fecal excretion of the deconjugated bile acids. Many in vitro studies have investigated the ability of various bacteria to deconjugate a variety of different bile

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acids. Grill et al. (1995) reported Bifidobacterium longum as being the most efficient bacterium when tested against six different bile salts. Studies performed on in vitro responses are useful, although in vivo studies in animals and humans are required to more fully determine the contribution of bile acid deconjugation to cholesterol reduction. Intervention studies on animals and ileostomy patients have shown that oral administration of certain bacterial species can lead to an increased excretion of free and secondary bile salts (De Smet et al. 1998). There is also in vitro evidence to support the hypothesis that some bacteria can assimilate (take up) cholesterol. It has been reported that Lactobacillus acidophilus (Gilliland et al., 1985) and Bifidobacterium bifidum (Rasic et al., 1992) have the ability to assimilate cholesterol during in vitro studies, but only in the presence of bile salts and under anaerobic conditions. However, despite such reports, there is uncertainty about whether the bacteria are assimilating cholesterol or whether cholesterol is co-precipitating with the bile salts. Studies have been performed to address this question. Klaver and Van der Meer (1993) concluded that removal of cholesterol from the medium in which Lactobacillus acidophilus and Bifidobacterium were growing was not due to assimilation, but due to bacterial bile salt deconjugase activity. Cholesterol binding to bacterial cell walls has also been suggested as a possible mechanism for the hypocholesterolemic effects of probiotics. Hosono and Tono-oka (1995) reported that Lactococcus lactis subsp. lactis biovar. diacetylactis R-43 had the highest binding capacity for cholesterol for a range of bacteria tested. It was speculated that differences in binding of the bacteria were due to chemical and structural properties of their cell walls, and that even non-viable cells may have the ability to bind cholesterol in the host intestinal tract. The mechanism of action of probiotics on cholesterol reduction could include one or all of the above mechanisms, with an ability of different bacterial species to have varying effects on cholesterol lowering. Sources Of Probiotics Kefir, a traditional fermented milk drink originating from the Bulkans, cultured buttermilks, yogurt products, fermented whey-based drinks, some cheeses, fermented juices, some fermented vegetable products, symbiotic beverages, and fermented soy products are the major sources of probiotics.

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SYMBIOTICS Recently it has been suggested that a combination of prebiotics and probiotics, the so-called symbiotics might be more active than the individual components on the colon. Symbiotic is defined as “a mixture of prebiotics and probiotics that beneficially effects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract by selectively stimulating the growth and/or by activating the metabolism of one or a limited number of healthpromoting bacteria, and thus improving the health of the host” (Gibson and Roberfroid, 1995). Accordingly, Rowland et al (1998) showed that concomitant administration of inulin and Bifidobacteria to rats resulted in a more potent inhibition of AOM-induced ACF than the administration of the two separately. Research in the author’s laboratory is going on to develop symbiotic foods and to study their effects on health. There are studies where oats and probiotics are combined to prepare symbiotic products (Gokavi et al., 2005). But human feeding trials are required to prove their health promoting effects. Beneficial Effects Of Symbiotics On Lipid Metabolism The use of symbiotics as functional food ingredients is a new and developing area; very few human studies have been performed which look at their effect on risk factors for CHD. In one study, the effect of a fermented milk product with and without the addition of Lactobacillus acidophilus and fructooligosaccharides was examined in healthy men (Schaafsma et al., 1998). The design of the study was a randomized placebo controlled crossover form, in which there were two treatment periods of 3 weeks, with a 1 week washout period. The authors reported a significant reduction in total and LDL cholesterol following ingestion of the fermented milk product containing both the probiotic and prebiotic, compared to the placebo fermented milk. Other research has concentrated on the composition of the gut microflora. In one study of healthy subjects, a fermented milk product containing Bifidobacterium spp. with or without 18 g of inulin was given daily for 12 days. The authors concluded that administration of the fermented milk product (probiotic) substantially increased the proportion of bifidobacteria in the gut, but that this increase was not enhanced with the addition of inulin. The composition of the gut microflora was then assessed 2 weeks

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after completion of the supplementation period; it was found that subjects who received the fermented milk product with inulin maintained their gut bifidobacterial population compared to subjects receiving the fermented milk product only. Although a synergistic effect on the bifidobacterial population in the gut was not observed with the symbiotic, these results suggest that either there was better implantation of the probiotic and/or there was a separate prebiotic effect on bifidobacteria already present in the gut. The maintenance of high numbers of bifidobacteria in the gut flora may be beneficial in terms of maintaining healthy intestinal function, however, its effect on blood lipid reduction remains to be determined. A more recent study has shown that a lower dose of prebiotic (2.75 g) added to a Lactobacillus-fermented milk was able to significantly increase numbers of bifidobacteria when fed over a 7 week period in healthy human subjects (Roberfroid, 1998). If this effect was a result of the symbiotic product used in the study, the use of lower doses of prebiotics in symbiotic preparations will help to reduce the gastrointestinal complaints observed with prebiotics alone and will improve the acceptability of these types of products by the general public. Examples Of Symbiotic Foods Commonly available symbiotic foods include yogurt and yogurt beverage made with cow milk, goat milk and soy milk. The process of yogurt making is an ancient craft which dates back thousands of years. Fortunately, the process has still survived through the ages which can be attributed to the fact that the scale of manufacture is very small which was handed down from parents to children. Yogurt is made by fermenting milk with lactic cultures which belong to a category of microorganisms that can digest the milk sugar lactose and convert it into lactic acid. For the cells to utilize lactose, deriving carbon and energy from it, they must also possess the enzymes needed to break lactose into two simple sugars: glucose and galactose. Some representative strains are Streptococcus lactis, S. cremoris, thermophilus, Lactobacillus bulgaricus, L. acidophilus, and L. plantarum. Yogurt is defined as the product resulting from the culturing of a mixture of milk and cream products with the lactic acid producing bacteria L. bulgaricus and S. thermophilus. Yogurt contains not less than 3.25 percent milk fat and 8.25 percent solids-not-fat. Commercial yogurt production is composed of the following steps: pretreatment of milk, homogenization, heat treatment, cooling to incubation temperature, inoculation with starter, fermentation, cooling,

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FIGURE 4.9 — Process For The Preparation Of Symbiotic Yogurt And Yogurt Beverage

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post-fermentation treatment (flavoring, fruit addition, pasteurization), refrigeration, and packaging. For set yogurt, the packaging into individual containers is carried out before fermentation. A good strain of starter culture not only affects the flavor and aroma, it can also speed up the process and thus reduces the production costs. Yogurt containing prebiotics and probiotics is a good synergistic fit and a true functional food. Research on health benefits of prebiotics and probiotics has driven attention towards development of products containing both. Thus developed products are symbiotic foods. Yogurt is the most suitable and common vehicle for this purpose. Most common symbiotic foods found in the market are milk based and soy based. Milk based foods include those made from cow milk, goat milk and buffalo milk. For soy based products, soy milk is the base. Soy based foods are good for people who are lactose intolerant. The process of making these products is outlined in Figure 4.9. SUMMARY This chapter is a review of the scientific data on beneficial effects of prebiotics, probiotics and symbiotics on human health which may lead them to be classified as functional foods in the near future. Intestinal flora is comprised of different types of bacteria living in symbiosis with the host. The stable composition of the flora is one of the factors responsible for a balanced ecosystem and good health. The composition and the activity of intestinal flora are influenced by some dietary factors which are nothing but prebiotics, probiotics and symbiotics. All prebiotics are mostly carbohydrates including sugar alcohols, disaccharides, oligosaccharides and polysaccharides which are neither hydrolysed nor absorbed in the upper part of the gastrointestinal tract and are selective substrate for beneficial bacteria in the large intestine. Types of prebiotics include fructans, fiber gums, isomaltooligosaccharides, lactitol, lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, transgalacto oligosaccharides, and xylooligosaccharides. Probiotics are live microorganisms which when consumed in sufficient numbers influence the microbial environment of the host in a beneficial way. The commonly known probiotics include L. acidophilus, B. bifidum, L. casei, and many others. Symbiotics are foods that contain both. The best strategy to cure chronic diseases is to prevent them, and to prevent them it requires modification of day-to-day diet. The scientific research so far indicates that if a sufficient amount of prebiotics, probiotics and symbitoics are included in the diet, incidence of diseases

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could be reduced to a great extent.The consumer of today is health conscious and demands foods which are tasty as well as low in fat and calories, with additional health benefits. In present day society, the leading health concerns are heart disease, cancer, high cholesterol and diabetes. The chapter discusses how prebiotics and probiotics could be a possible dietary treatment for these chronic diseases. With the limited information available from scientific studies, the use of prebiotics, probiotics and symbiotics for improving human health holds promise. References Abdelali, H., Cassand, P., Soussotte, V., Daubeze, M., Bouley, C. and Narbonne, J. F. 1995. Effect of dairy products on initiation of precursor lesions of colon cancer in rats. Nutr. Cancer 24:121-132. Agerbaek, M., Gerdes, L.U. and Richelsen, B. 1995. Hypocholesterolaemic effects of a new product in healthy middle-aged men. Eur. J. Clin. Nutr. 49:346-352. Ahola, A. J., Yli-Knuuttila, H., Suomalainen, T., Ahlström, A., Meurman, J. and Korpela, R. 2002. Short term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch. Oral Biol. 47:799–804. Aiba, Y., Suzuki, N., Kabir, A. M., Takagi, A., Koga, Y. 1998. Lactic acidmediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model. Am. J. Gastroenterol. 93:2097–2101. Ayebo, A. D., Angelo, I. A. and Shahani, K. M. 1980. Effect of ingesting Lactobacillus acidophilus milk upon fecal flora and enzyme activity in humans. Milchwissen. 35:730-733. Baricault, L., Denariaz, G., Houri, J. J., Bouley, C., Sapin, C. and Trugnan, G. 1995. Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation. Carcinogeneis 16(2):245-252. Biasco, G., Paganelli, G., Brandi, G., Brillianti, S. and Lami, F. 1991. Effect of Lactobacillus acidophilus and Bifidobacterium bifidum on rectal cell kinetics and fecal pH. Italian J. Gastroenterol. 23:142. Biffi, A., Coradini, D., Larsen, R., Riva, L. and Di Fronzo, G. 1997. Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line. Nutr. Cancer 28:93-99. Bleichner, G., Blehaut, H., Mentec, H. and Moyse, D. 1997. Saccharomyces boulardii prevents diarrhea in critically ill tube-fed patients. A multicenter, randomized, doubleblind placebo-controlled trial. Intensive Care Med. 23:517–523. Bogdanov, I. G., Velichkov, V.T., Gurevich, A. I., Dalev, P. G., Kolosov, A. M. N., Mal’kova, V. P., Sorokina, I. B., Khristova, L. N. 1978. Antitumor action of glycopeptides from the cell wall of Lactobacillus bulgaricus. Bull. Exptl. Biol. Med. 84:1750-1753. Born, P., Lersch, C., Zimmerhackl, B. and Classen, M. 1993. The Saccharomyces boulardii therapy of HIV-associated diarrhea (letter). Dtsch. Med. Wochenschr. 118:765. Brady, L. J., Gallaher, D. D. and Busta, F. K. 2000. The role of probiotic cultures in the prevention of colon cancer. J. Nutr. 130:S410-S414. Buddington, K. K., Donahoo, J. B. and Buddington, R. K. 2002. Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumor inducers. J. Nutr. 132:472-477.

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Buddington, R. K., Williams, C. H., Chen, S. C. and Witherly, S. A. 1996. Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. Am. J. Clin. Nutr. 63:709-716. Caglar, E., Kargul, B. and Tanboga, I. 2005. Bacteriotherapy and probiotics’ role on oral health. Oral Dis. 11:131-137. Campbell, J. M., Fahey, G. C. and Wolf, B. W. 1997. Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats. J. Nutr. 127:130-136. Challa, A., Rao, D. R., Chawan, C. B. and Shakelford L. 1997. Bifidobacterium longum and lactulose suppress azoxymethane-induced colonic aberrant crypt foci in rats. Carcinogenesis 18(3):517-521. Comelli, E. M., Guggenheim, B., Stingele, F. and Neeser, J. R. 2002. Selection of dairy bacterial trains as probiotics for oral health. Eur. J. Oral Sci. 110:218–224. De Leenheer, L. and Hoebregs, H. 1994. Progress in the elucidation of the composition of chicory inulin. Starch 46:193-196. De Smet, I., De Boever, P. and Verstraete, W. 1998. Cholesterol lowering in pigs through enhanced bacterial bile salt hydrolase activity. Br. J. Nutr. 79:185-194. Delzenne, N. M. and Kok, N. 1998. Effect of non-digestible fermentable carbohydrates on hepatic fatty acid metabolism. Biochem. Soc. Transactions 26:228-230. Demigne, C., Morand, C., Levrat, M. A., Besson, C., Moundras, C. and Rémésey, C. 1995. Effect of propionate on fatty acid and cholesterol synthesis and on acetate metabolism in isolated rat hepatocytes. Br. J. Nutr. 74:209-219. Englyst, H. N., Wiggins, H. S. and Cummings, J. H. 1992. Classification and measurement of nutritionally important starch fractions. Eur. J. Clin. Nutr. 46:S33-S50. Fekety, R. and Shah, A. B. 1993. Diagnosis and treatment of Clostridium difficile colitis. J. Am. Med. Assoc. 269:71–75. Fekety, R., Silva, J., Kauffman, C., Buggy, B. & Deery, H. G. 1989. Treatment of antibioticassociated Clostridium difficile colitis with oral vancomycin: comparison of two dosage regimens. Am. J. Med. 86:15–19. Fiordaliso, M. F., Kok, N., Desager, J. P., Goethals, F., Deboyser, D., Roberfroid, R.M. and Delzenne, N. 1995. Dietary oligofructose lowers triglycerides, phospholipids and cholesterol in serum and very low density lipoproteins in rats. Lipids 30:163-167. Free, R. H., Busscher, H. J., Elving, G. J., Van der Mei, H. C., Van Weissenbruch, R., Albers, F. W. 2001. Biofilm formation on voice prostheses: in vitro influence of probiotics. Ann. Otol. Rhinol. Laryngol. 110:946-951. Gibson, G. R. 1998. Dietary modulation of the human gut microflora using probiotics. Br. J. Nutr. 80(Suppl. 2):209–212. Gibson, G. R., Beatty, E. B., Wang, X., Cummings, J. H. 1995. Selective stimulation of Bifidobacterium in the human colon by oligofructose and inulin. Gastroenterol. 108:975-982. Gibson, G. R. and Roberfroid, M. B. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125:1401–1412. Gibson, G.R. and McCartney, A.L. 1998. Modification of gut flora by dietary means. Biochem. Soc. Transactions 26:222- 228. Gokavi, S., Zhang, L., Huang, M. K., Zhao, X. and Guo, M. 2005. Oats based symbiotic beverage fermented by Lactobacillus plantarum, Lactobacillus paracasei ssp. casei and Lactobacillus acidophilus. J. Food Sci. 70(4):M216-M223. Goldin, B. R. and Gorbach, S. L. 1984. The effect of milk and lactobacillus feeding on human intestinal bacterial enzyme activity. Am. J. Clin. Nutr. 39:756-761. Grill, J. P., Manginot-Durr, C., Schneider, F. and Ballongue, J. 1995. Bifidobacteria and probiotic effects: action of Bifidobacterium species on conjugated bile salts. Current Microbiol. 31:23-27.

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Grudianov, A. I., Dmitrieva, N. A. and Fomenko, E. V. 2002. Use of probiotics Bifidumbacterin and Acilact in tablets in therapy of periodontal inflammations. Stomatologiia Mosk 81:39–43. Harms, H. K., Bertele-Harms, R. M. and Bruer-Kleis, D. 1987. Enzyme-substitution therapy with the yeast Saccharomyces cerevisiae in congenital sucrase-isomaltase deficiency. N. Engl. J. Med. 316:1306–1309. Hayatsu, H. and Hayatsu, T. 1993. Suppressing effect of Lactobacillus casei administration on the urinary mutagenecity arising from ingestion of fried ground beef in the human. Cancer Lett. 73:173-179. Hendry, G. A. F. and Wallace, R. K. 1993. The origin, distribution, and evolutionary significance of fructans, in Science and Technology of Fructans. Suzuki, M., Chatterton, N. J., Eds., CRC Press, Boca Raton, FL, pp. 119-139. Hilton, E., Kolakowski, P., Singer, C. and Smith, M. 1997. Efficacy of Lactobacillus GG as a diarrheal preventive in travelers. J. Travel Med. 4:41–43. Hosono, A. and Tono-oka, T. 1995. Binding of cholesterol with lactic acid bacterial cells. Milchwissen. 50:556-560. Hsu, C. K., Liao, J. W., Chung, Y. C., Hsieh, C. and Chan, Y. C. 2004. Xylooligosaccharides and fructooligosaccharides affect the intestinal microbiota and precancerous colonic lesion development in rats. J. Nutr. 134:1523-1528. Hughes, R. and Rowland, I. R. 2001. Stimulation of apoptosis by two prebiotic chicory fructans in the rat colon. Carcinogenesis 22(1):43-47. Isolauri, E., Juntunen, M., Rautanen, T., Sillanaukee, P. and Koivula, T. 1991. A human Lactobacillus strain (Lactobacillus casei sp strain GG) promotes recovery from acute diarrhea in children. Pediat. 88:90–97. Klaver, F.A.M. and Van Der Meer, R. 1993. The assumed assimilation of cholesterol by lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity. App.Environ. Microbiol. 59:1120-1124. Klurfeld, D. M. 1997. Fiber and cancer protection-mechanisms. Adv Exp Med Biol. 427:249-257. Kok, N.N., Morgan, L.M., Williams, C.M., Roberfroid, M.B., Thissen, J.-P. and Delzenne, N.M. 1998. Insulin, glucagon-like peptide 1, glucose dependent insulinotropic polypeptide and insulin-like growth factor 1 as putative mediators of the hypolipidemic effect of oligofructose in rats. J. Nutr. 128:1099-1103. Lidbeck, A., Nord, C. E., Gustafsson, J. A. and Rafter, J. 1992. Lactobacilli, anticarcinogenic activities and human intestinal microflora. Eur. J. Cancer Prev. 1:341–353. Marchetti, C., Migliorati, G., Moraca, R., Riccardi, C., Nicoletti, I., Fabiani, R., Mastrandrea, V. and Morozzi, G. 1997. Deoxycholic acid and SCFA-induced apoptosis in the human tumor cell-line HT-29 and possible mechanisms. Cancer Lett. 114:97-99. Marteau, P., Pochart, P., Flourie, B., Pellier, P., Santos, L., Desjeux, J. F. and Rambaud, J. C. 1990. Effect of chronic ingestion of a fermented dairy product containing Lactobacillus acidophilus and Bifidobacterium bifidum on metabolic activities of the colonic flora in humans. Am. J. Clin. Nutr. 52:685–688. Matsuzaki, T. 1998. Immunomodulation by treatment with Lactobacillus casei strain. Shirota. Int. J. Food Microbiol. 41(2):133-140. McFarland, L. V., Surawicz, C. M., Greenberg, R. N., Elmer, G. W., Moyer, K. A., Melcher, S. A., Bowen, K. E. and Cox, J. L. 1995. Prevention of beta-lactam associated diarrhea by Saccharomyces boulardii compared with placebo. Am. J. Gastroenterol. 90:439–448. McFarland, L. V., Surawicz, C. M., Greenberg, R. N., Fekety, R., Elmer, G. W., Moyer, K. A., Melcher, S. A., Bowen, K. E., Cox, J. L., and Noorani, Z. 1994. A randomized placebo-controlled trial of Saccharomyces boulardii in combination with standard antibiotics for Clostridium difficile disease. J. Am. Med. Assoc. 271:1913–1918.

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Meurman, J. H., Antila, H., Korhonen, A. and Salminen, S. 1995. Effect of Lactobacillus rhamnosus strain GG (ATCC 53103) on the growth of Streptococcus sobrinus in vitro. Eur. J. Oral Sci. 103:253–258. Moddler, G. W., McKellar, R. C. and Yaguchi, M. 1990. Bifidobacteria and bifiodgenic factors. Canadian Inst. Food Sci. Technol. J. 23:29-41. Montalto, M., Vastola, M., Marigo, L., Covino, M., Graziosetto, R., Curigliano, V., Santoro, L., Cuoco, L., Manna, R. and Gasbarrini, G. 2004. Probiotic treatment increases salivary counts of lactobacilli: a double-blind randomized, controlled study. Digestion 69:53–56. Niedzielin, K. and Kordecki, H. 1996. Therapeutic usefulness of “ProViva” solution in the treatment of irritable bowel syndrome and hemorrhoids. Presented at the Symposium of Gastroenterology, Heiligenstadt, Germany. Orrhage, K., Sillerstrom, E., Gustafsson, J. A., Nord, C. E. and Rafter, J. 1994. Binding of mutagenic heterocyclic amines by intestinal and lactic acid bacteria. Mutat. Res. 311:239–248. Pool-Zobel, B. L., Neudecker, C., Domizlaff, I., Ji, S., Schillinger, U., Rumney,C. J., Moretti, M., Villarini, M., Scassellatti-Sforzolini, G. and Rowland, I. R. 1996. Lactobacillus- and Bifidobacterium-mediated antigenotoxicity in colon cells of rats: prevention of carcinogen-induced damage in vivo and elucidation of involved mechanisms. Nutr. Cancer 26:365–380. Rafter, J. 2002. Lactic acid bacteria and cancer: mechanistic perspective. Br. J. Nutr. 88(1S):S89-S94. Rasic, J. L. J., Vujicic, I. F., Skrinjar, M. and Vulic, M. 1992. Assimilation of cholesterol by some cutures of lactic acid bacteria and bifidobacteria. Biotechnology Lett. 14:3944. Reddy, B. S., Hamid, R. and Rao, C. V. 1997. Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition. Carcinogenesis 18:1371-1374. Reddy, B. S. 1998. Prevention of colon cancer by pre- and probiotics: evidence from laboratory studies. Br. J. Nutr. 80:S219-S223. Risio, M., Lipkin, M., Newmark, H. L., Yang, K., Rossini, F. P., Steele, V. E., Boone, C. W. and Kelloff, G. J. 1996. Apoptosis, cell replication, and Western-style diet-induced tumorigenesis in mouse colon. Cancer Res. 56:4910–4916. Roberfroid, M. 2004. Inulin: A Fructan in Inulin-type fructans. Functional food ingredients. CRC series in Modern Nutrition. CRC Press, New York, NY. pp.39-60. Roberfroid, M. B. 1998. Prebiotics and symbiotics: concepts and nutritional properties. Br. J. Nutr. 80(supp. 2):S197-S202. Rolfe, R. D. 2000. The role of probiotic cultures in the control of gastrointestinal health. J. Nutr. 130:S396- S402. Rowland, I. R., Rumney, C. J., Coutts, J. T. and Lievense, L.C. 1998. Effect of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogeninduced aberrant-crypt foci in rats. Carcinogenesis 19(2):281-285. Ruseler-Van Embden, J. G. H., Hazenberg, M. P., Van Lieshout, L. M. C. and Schouten, W. R. 1995. Instability of the pouch flora: cause of pouchitis. Microecol. Ther. 23:81– 88. Scevola, D., Zambelli, A., Concia, E., Perversi, L. and Candiani, C. 1989. Lactitol and neomycin: monotherapy or combined therapy in the prevention and treatment of hepatic encephalopathy. Clin. Ther. 129:105–111. Schaafsma, G., Meuling, W. J. A., van Dokkum, W. and Bouley, C. 1998. Effects of a milk product, fermented with Lactobacillus acidophilus and with fructooligosaccharides added, on blood lipids in male volunteers. Eur. J. Clin. Nutr. 52:436440.

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Schiffrin, E. J., Rochat, F., Link-Amster, H., Aeschlimann, J. M. and Donnet-Hughes. A. 1995. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J. Dairy Sci. 78:491–497. Schrezenmeir, J. and deVrese, M. 2001. Probiotics, prebiotics, and symbioticsapproaching a definition. Am. J. Clin. Nutr. 73:361S-364S. Segal, I., Hassan, H., Walker, A. R. P., Becker, P. and Braganza, J. 1995. Faecal short chain fatty acids in South African urban and whites. Dis. Colon Rectum 338:732– 734. Sekine, K., Kawashima, T. and Hashimoto, Y. 1994. Comparison of the TNF-a levels induced by human-derived Bifidobacterium longum and rat-derived Bifidobacterium animalis in mouse peritoneal cells. Bifidobacteria Microflora 13:79-89. Sekine, K., Toida, T., Saito, M., Kuboyama, M. and Kawashima, T. 1985. A new morphologically characterized cell wall preparation (whole peptidoglycan) from Bifidobacterium infantis with a higher efficacy on the regression of an established tumor in mice. Cancer Res. 45:1300–1307. Singh, J., Rivenson, A., Tomita, M., Shimamura, S., Ishibashi, N. and Reddy, B. S. 1997. Bifidobacterium longum, a lactic acid-producing intestinal bacterium inhibits colon cancer and modulates the intermediate biomarkers of colon carcinogenesis. Carcinogenesis 18(4):833-841. Surawicz, C. M., Elmer, G. W., Speelman, P., McFarland, L. V., Chinn, J. and van Belle, G. 1989. Prevention of antibiotic-associated diarrhea by Saccharomyces boulardii: a prospective study. Gastroenterol.96:981–988 Suzuki, M. 1993. History of fructan research: Rose to Edelman. in Science and Technology of Fructans, Suzuki, M., Chatterton, N. J., Eds., CRC Press, Boca Raton, FL, pp. 21-39. Tanabe, T., Suyama, K. and Hosono, A. 1994. Effect of pepsin, trypsin or bile acid on the binding of tryptophane pyrolysates by Lactococcus lactis subsp. lactis T-80. Milchwissen. 49:438-441. Tanboga, I., Caglar, E. and Kargul, B. 2003. Campaign of probiotic food consumption in Turkish children, oral perspectives - Probiotics for your child. Int. J. Pediatr. Dent. 13(Suppl. 1):59. Taper, H. S. and Roberfroid, M. 1999. Influence of inulin and oligofructose on breast cancer and tumor growth. J. Nutr. 129:1488S-1491S. Todesco, T., Rao, A. V., Bosello, O. and Jenkins, D. J. A. 1991. Propionate lowers blood glucose and alters lipid metabolism in healthy subjects. Am. J. Clin. Nutr. 54:860865. Treptow-van Lishaut, S., Rechkemmer, G., Rowland, I. R., Dolara, P. and Pool-Zobel, B. L. 1999. The carbohydrate crystalean and colonic microflora modulate expression of glutathione S-transferase subunits in colon of rats. Eur. J. Nutr. 38:76–83. Uchiyama, T. 1993. Metabolism in microorganisms Part II. Biosynthesis and degradation of fructans by microbial enzymes other than levansucrase. in Science and Technology of Fructans, Suzuki, M., Chatterton, N. J., Eds., CRC Press, Bocan Raton, FL, pp.169190. Van Loo, J., Coussement, P., De Leenheer, L., Hoebregs, H. and Smits, G. 1995. On the presence of inulin and oligofructose as natural ingredients in the Western diet. Critic. Rev. Food Sci. Nutr. 35:525-552. Van Loo, J., Cummings, J., Delzenne, N., Englyst, H., Franck, A., Hopkins, M., Kok, N., Macfarlane, G., Newton, D., Quigley, M., Roberfroid, M., van Vliet, T. and van den Heuvel, E. 1999. Functional food properties of non-digestible oligosaccharides : a consensus report from the ENDO project (DGXII AIRII-CT94- 1095). Br. J. Nutr. 81:121-132.

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Verghese, M., Rao, D. R., Chawan, C. B. and Shackelford, L. 2002. Dietary Inulin suppresses Azoxymethane-induced preneoplastic aberrant crypt foci in mature fisher 344 rats. Nutr. Cancer 132:2804-2808. Wollowski, I., Ji, S. T., Bakalinsky, A. T., Neudecker, C. and Pool-Zobel, B. L. 1999. Bacteria used for the production of yogurt inactivate carcinogens and prevent DNA damage in the colon of rats. J. Nutr. 129:77-82. Wollowski, I., Rechkemmer, G. and Pool-Zobel, B. L. 2001. Protective role of probiotics and prebiotics in colon cancer. Am. J. Clin. Nutr. 73:S451-S455. (Guo, M.R., Gokavi, S.)

Chapter 5 LIPIDS AND LIPID RELATED FUNCTIONAL FOODS “Lipids” is a word derived from the Greek “lipos” meaning fat. However, there is no widely accepted definition of lipids. Over the years, the term lipids have been used interchangeably to describe a group of naturally occurring compounds that are soluble in organic solvents (e.g., hexane, chloroform, ether, alcohol) and insoluble in aqueous media. However, lipids also includes a series of other compounds that have little similarities either in structure or in function such as carotenoids, terpenes, steroids and their naturally occurring derivatives. Many of these naturally occurring compounds are partially soluble in water as well. The definition of lipids simply based on solubility is not justifiable and should be widened to include compounds that are related closely to fatty acid derivatives through biosynthetic pathways or by their biochemical or functional properties. Lipids serve as structural components of biological membranes and provide energy in the form of triacylglycerol (TAG) also called as triglycerides. Lipids are considered in relation to excess energy balance, obesity, and as a dietary factor in the development of cardiovascular disease and many other harmful disorders. However, not all lipids are bad and damaging to human health. In fact some play a unique role in maintaining good health and providing much of the flavor and texture to foods. The changing trend in our food supply and the industrial revolution have in fact jeopardized both the quantity and balance of these nutrients. The polyunsaturated fatty acids (PUFA), for example, are very beneficial and should be consumed on a regular basis. These include the n-3 and n-6 (also referred as omega3 or
-3; and omega-6 or
-6) PUFA, also known as essential fatty acids (EFA). EFA have been part of our diet since the beginning of human life and current estimates in Western diets suggest a higher ratio of n-6 and n-3 PUFA which can dramatically change the lipid composition of many parts of the body. Humans can’t produce EFA itself and the only way to receive these nutrients is through diet and supplementation.

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There is overwhelming evidence that
-3 acids are important for health and protecting against disease including lowering triglycerides, general heart benefits, improving brain functioning, helping with diabetes, strokes, depression and many others. The purpose of this chapter is to provide basic knowledge about the origin of these fatty acids, their nomenclature, sources and how these necessary components play a vital role in daily life, including the reference of conjugated linoleic acid (CLA), another health beneficial PUFA, trans fatty acids (TFA) and their health implications. Finally, at the end a brief description of olive and fish oils, phytosterols, is provided as an important source of healthy diet and their health beneficial effects. CHEMISTRY AND NOMENCLATURE The major component of most fatty foods and oils are triglycerides, whereas, mono- and diglycerides, free fatty acids, phosphatides, sterols and fatty alcohols constitute the minor components. A triglyceride is composed of glycerol and three fatty acids whereas, mono- and diglycerides are mono- and diesters of fatty acids and glycerol (Figure 5.1), where R1, R2 and R3 represent identical or different fatty acids with even/odd numbers of carbon atoms. The mono- and diglycerides are frequently used in foods as emulsifiers. They are prepared commercially by the reaction of glycerol and triglycerides or by the esterification of glycerol and fatty acids. Mono- and diglycerides are also formed in the intestinal tract as a result of the normal digestion of triglycerides. Both the physical and chemical characteristics of fats are influenced greatly by the kinds and proportions of the component fatty acids and the way in which these are positioned on the glycerol molecule. When all of the fatty acids in a triglyceride are identical, it is termed a “simple” triglyceride. However the more common forms are the mixed triglycerides in which two or three kinds of fatty acids are present in the molecule. Fats are the major source of energy which supply about 9 calories per gram whereas, proteins and carbohydrates each supply about 4 calories per gram. The classification of fatty acids, however, is based according to their degree of saturation, the length of the carbon chain (short, medium, or long); the number of double bonds (unsaturated, mono-, or polyunsaturated); or essentiality in the diet (essential or non-essential). Saturated fats contain no double bond (example, stearic acid), monounsaturated fats contain one double bond (example, oleic acid) and PUFA contain greater than one double bond (example, linoleic acid). PUFA are subdivided into two classes based on the location of the first

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163

FIGURE 5.1 — Chemical Structure Of Triglyceride, Monoglyceride And Diglyceride

double bond proximal to the methyl end of the carbon chain such as n6 and n-3 PUFA. In general the carbon atoms in a fatty acid chain are numbered consecutively from the end of the chain (example: with the carbon of the carboxyl group being considered number one). This system of nomenclature is widely used by chemists. However, Dr. Ralph Holman and others suggest numbering the unsaturated fatty acids from the far terminal methyl end (CH3-) of the molecule and called such a notation the omega (
, a Greek alphabet), a nomenclature favored mostly by biochemist and nutritionist (1998). In the omega or “n minus” notation, the length of the fatty acid chain is the first number, followed by a colon with the second number denoting the number of double bonds. After the second number, n “minus” a third number may appear. The n minus notation indicates the position of the first double bond counting from the methyl end of the molecule. Thus, linoleic acid (LA, 18:2n-6 or 18:2
-6) denotes an 18-carbon fatty acid with two double bonds, the first of which occurs six carbons from the methylend of the fatty acid. We will be using the “n minus” notation throughout this chapter. The n-3 PUFA have their first double bond located at the third carbon atom (C-3) whereas, n-6 PUFA have their first double located at C-6 (Figure 5.3). Figure 5.2 represents the nomenclature of some commonly available n-3 PUFA.

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FIGURE 5.2 — Nomenclature Of Commonly Available n-3 PUFA Trivial name

IUPAC* name

n- reference

Abbreviation

Linolenic acid

9,12,15-octadecenoic acid/alpha-linolenic acid

18:3n-3

ALA/-LA/LNA/-LNA

Docosahexaenoic acid

4,8,12,15,19docsahexaenoic acid

22:6n-3

DHA

Docosapentaenoic acid

7,10,13,16,19docosapentaenoic acid

22:5n-3

DPA

Eicosapentaenoic acid

5,8,11,14,17eicosapentaenoic acid

20:5n-3

EPA

*IUPAC = International Union of Pure and Applied Chemistry

FIGURE 5.3 — Line Drawing Representation Of n-3 and n-6 PUFA Where Each Angle Represents A Carbon Atom

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165

The n-3 PUFA have their first double bond located at the third carbon atom (C-3) whereas, n-6 PUFA have their first double located at C-6 (Figure 5.3). Figure 5.2 represents the nomenclature of some commonly available n-3 PUFA. Polyunsaturated fatty acids can also be conjugated, as in the case of conjugated linoleic acid (CLA) a naturally occurring anticarcinogenic compound. The conjugated diene structure is not usual in fatty acids. The specific structure of CLA based on the location of the double in the carbon chain is crucial to the compound’s ability to fight cancer and many other harmful diseases. In LA in part of the molecule, a double bond is followed by two single bonds (- -) and then another double bond (=) whereas in CLA a double bond is followed by one single bond (-) and then another double bond (Figure 5.4). FIGURE 5.4 — Chemical Structure Of Linoleic Acid (LA) and Conjugated Linoleic Acid (CLA) Isomers (c9, t11- and t10, c12-CLA).

This alternation of double and single bonds is called “conjugation,” hence the term “conjugated linoleic acid.” CLA is a mixture of eighteen carbon fatty acids that is biologically produced through microbial isomerization in the rumen, and thus present in dairy products and red meat. The c9, t11 and t10, c12 isomers of CLA, are the most abundant ones in foods. Other C18 positional isomers with the conjugated double bonds at 6,8; 7,9; 8,10; 11,13 or 12,14 have also been reported in cheese (Kepler et al., 1966). A mixture of CLA isomers is also produced during food processing, by thermal isomerization and by some industrial

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processes by partial hydrogenation or alkali-isomerization of LA. However, only c9, t11- and t10, c12-CLA with one trans double are proven to be biologically active. The c9, t11-CLA isomer was the first intermediate product in the biohydrogenation of LA by the anaerobic rumen bacterium Butyrivibrio fibrisolvens. The reaction is catalyzed by the enzyme linoleate isomerase, which converts the c12 bond of free LA to a t11 bond. One reason why ruminants produce more c9, t11-CLA than non-ruminants is that hydrolysis of fat within the rumen provides more unesterified LA than is available to bacteria in non-ruminants. TFA, however, are another class of unsaturated fats which are widely present in a variety of foods; some are derived from natural sources such as dairy products but most come from products that contain commercially hydrogenated oils that are processed into a solid or more stable liquid form. Hydrogenation is a process by which vegetable oils are converted to solid fats simply by bubbling hydrogen through the fat at an elevated temperature in the presence of a metal catalyst such as nickel and in the absence of oxygen, thereby, reducing the number of double bonds. The levels and types of these fatty acids formed depend on the condition such as temperature, pressure, catalyst and duration. The most common configuration of double bonds in naturally occurring fatty acids is of cis configuration (hydrogen atoms are on the same side of the double bond). During partial hydrogenation which is an incomplete saturation of the double bonds, some double bonds remain but change from cis to trans configuration (hydrogen atoms are on the opposite side of the double bond). This shifting of the double bonds along the carbon chain of the fatty acid molecule results in positional isomers of the same acid. For example, fatty acids containing double bonds at C-9 and C-12 positions are changed to isomeric forms containing double bonds ranging from positions C-4 to C-16. Geometric isomers or cis/trans isomers are formed when the naturally occurring cis double bonds in vegetable oils are isomerized to the more thermodynamically stable trans configuration. For example, cis9 octadecenoic acid or oleic acid is transformed into trans9 octadecenoic acid or elaidic acid (Figure 5.5). The cis configuration creates a bend or kink in the fatty acid molecule making the molecule more flexible while trans configuration yields a straighter, more rigid molecule. Trans fatty acids, therefore, behaves more like saturated fats even though they have a double bond. For example, the melting point of oleic acid (cis configuration) is 13.4°C which is a liquid, whereas, elaidic acid (trans configuration) 46.5°C, is a solid unsaturated fat. The most prevalent TFA in partially hydrogenated vegetable oils (PHVO) is t10-18:1, and

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167

the major TFA isomer in dairy products is t11-18:1. The hydrogenation process improves the oxidative and thermal stability by adding hydrogen to the molecule. FIGURE 5.5 — Chemical Structure Of Oleic And Elaidic Fatty Acids

As a result, oils such as soybean, safflower and cottonseed, which are rich in PUFA, are converted to semi-solids and solids that are useful in margarines and vegetable shortenings. Trans-polyunsaturated (18:2 and 18:3) acids are also widely distributed in our diets, but in low amounts. However, both mono and polyunsaturated fatty acids are suspected as harmful components. DIETARY SOURCES n-3 PUFA Many commonly used oils, including safflower, sunflower, soy, and corn oil are an important plant-based source of LA (n-6), whereas, ALA (n-3) is present in canola and soybean oil. Grains and vegetable oils are considered to be the major source of ALA, however, certain fish can naturally provide the DHA and EPA in the recommended amount. Fresh seaweed is the only plant food that contains EPA or DHA. It is possible to get DHA and EPA from plant seed oils such as flaxseeds, since it contains ALA which is the precursor of DHA and EPA. Figures 5.6 &

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5.7 represent the major source of n-3 PUFA in certain selected fish and vegetable oils (Alaswad et al., 2002). EPA and DHA are found higher in Mackeral followed by Albacore tuna. Among the plant source of ALA, flaxseed contained a higher amount (20.0 g/100 g) followed by butter nuts (dried, 8.7 g/100g) and English walnut (6.8 g/100g). At the present time no specific intake of these fatty acids is available. However, recently the new recommendation from the American Heart Association (AHA) suggested that all adults eat fatty fish at least two times a week including plant derived n-3 PUFA found in tofu, soybeans, walnuts, flaxseeds and their oil, or canola oil (Kris-Etherton et al., 2003). FIGURE 5.6 — n-3 PUFA In Some Selected Fish1 Fish

ALA2

EPA3

DHA4

Mackeral Atlantic Herring Albacore Tuna Chinook Salmon Anchovy Coho Salmon Greenland Halibut Rainbow Trout Atlantic Cod Atlantic White Shrimp Catfish Northern Lobster Flounder

0.1 0.1 0.2 0.1 Trace 0.2 Trace 0.1 Trace Trace Trace 0 Trace

0.9 0.7 0.3 0.8 0.5 0.3 0.5 0.1 0.1 0.2 0.1 0.1 0.1

1.6 0.9 1.0 0.6 0.9 0.5 0.4 0.4 0.4 0.2 0.2 0.1 0.1

1

Given as g/100 of raw material Alhpa linoleic acid Eicosapentaenoic acid 4 Docosahexaenoic acid 2 3

The AHA also recommend for patients with documented CHD to consume ~ 1 g of EPA and DHA (combined) per day. Conjugated linoleic acid (CLA) Conjugated linoleic acid (CLA) is primarily a product of microbial metabolism in the digestive tract of ruminants which ultimately accumulates into milk, beef, and dairy products such as butter, yogurt and a variety of cheeses. Traces of CLA can also be found in chicken and pork due to the inclusion of meat meal or tallow in commercial diets and may not be notable sources of CLA. However, fats and meats from ruminant species are the richest natural source. CLA is also present in plant oils and selected sea foods but unlike in ruminant-derived foods

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FIGURE 5.7 — Plant Source Of Alpha Linoleic Acid (ALA)1

1

Source

ALA

Flaxseed Butternuts (dried) English walnuts Soybean (raw) Leeks Wheat germ Purslane Almonds Pinto beans Barley barn Kale Chickpeas Avocados Strawberries Peanuts

20.0 8.7 6.8 3.2 0.7 0.7 0.4 0.4 0.3 0.3 0.2 0.1 0.1 0.1 0.0

Given as g/100 of raw material

where c9, t11 is the major isomer, accounted for about 40% of total, and absent in some selected seafood lipids (Watkins et al., 2000). CLA content in food may vary widely. Representative concentration of CLA in a variety of food and dairy products is summarized in Figure 5.8. Concentrations are highest in beef, lamb and dairy products (3-7 mg/ g fat). CLA content of cows' milk ranges from 0.7-10.1 mg CLA/g fat. The amount of CLA found in dairy and beef is a direct reflection of the diet the animals are fed. CLA content of milk fat can be influenced by manipulating the type of dietary supplement fed to dairy animals. Supplementing the diet with polyunsaturated oils that contain either corn oil or sunflower oil increases CLA content of milk fat substantially. CLA contents of selected milk including humans are summarized in Figure 5.9. CLA concentration of human milk ranges from 3.1 to 8.5 mg/g fat among mothers eating conventional diets whereas, values of 9.7 to 12.5 mg/g fat among Hare Krishna mothers (Fogerty et al. 1988) suggesting that diet may influence human milk CLA concentration, because followers of the Hare Krishna faith consume large amounts of butter or ghee, as well as cheese. Currently, there exists no database which contains the CLA distribution of foods commonly consumed in the US. If modest CLA intake imparts benefits greater than no CLA consumption at all, then there are many advantages to producing foods such as milk and beef with enhanced levels of bioformed CLA because the consumption of these foods is high.

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FIGURE 5.8 — Concentration Of Conjugated Linoleic Acid (CLA) In Uncooked Foods.* Food Dairy products Homogenized milk Butter Sour cream Plain yogurt Ice cream Sharp cheddar cheese Mozzarella cheese Colby cheese Cottage cheese Reduced fat swiss Am. Proc.cheese Cheez WhizTM Meat Fresh ground beef Beef round Beef frank Beef smoked sausage Veal Lamb Pork Poultry Chicken Fresh ground turkey Seafood Salmon Lake trout Shrimp Vegetable oils Safflower Sunflower Canola Corn

CLA (mg/g fat)

5.5 4.7 4.6 4.8 3.6 3.6 4.9 6.1 4.5 6.7 5.0 5.0 4.3 2.9 3.3 3.8 2.7 5.6 0.6 0.9 2.5 0.3 0.5 0.6 0.7 0.4 0.5 0.2

* Adopted from Chin et al., 1992

FIGURE 5.9 — CLA Content In Milksa

a

Watkins et al., 2000.

Milk

CLA (mg/g fat)

Cow Goat Sheep Mare Sow Water buffalob Human

0.7-10.1 6.1-10.35 10.8-29.7 0.9 2.2 4.4-7.0 1.7-36.4

b

Guo et al., 2005

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171

Trans Fatty Acids (TFA) The primary source of TFA in food products such as margarines, shortenings, fast and processed foods is due to the use of hydrogenated vegetable oils. Small amounts of naturally occurring trans fat can be found in some animal products, such as milk, butter and tallow as a result of biohydrogenation in ruminants as a secondary source. Hence, TFA comes from two different sources: industrial, partial hydrogenation of edible oils containing unsaturated fatty acids, and bacterial transformation of unsaturated fatty acids in the rumen of ruminants. In the rumen of ruminants, principally trans vaccenic acid (t11-18:1) is formed, which accounts for over 60% of the trans fatty acid content of butter fat from cows. A number of plant species also contain small amounts of trans unsaturated fatty acids in their seeds and leaves. For example, vegetables such as leeks, peas, spinach, and lettuce contain trans-3-hexadecenoic acid (t3-16:1). Vegetable oils do not contain trans fatty acids, however, in order to reduce the potential of oxidation and rancidity, these oils are often lightly hydrogenated. For example, soybean oil sold for use as salad and cooking is often lightly hydrogenated to reduce the content of ALA and, thereby, contains some TFA. TFA content of hydrogenated salad and cooking oils ranged from 8 to 12% (Hunter, et al., 1986; Enig et al., 1983). However, these products contribute very little trans fatty acids to the current food supply in the United States and other countries. The TFA content of some commonly consumed foods are summarized in Figure 5.10. The highest content of TFA ranged between 1.4 to 4.2 g/serving in vegetable shortening, whereas, vegetable oils appeared to contain less TFA. TFA content of processed foods and fast foods may vary widely, depending upon the type of fat used in processing. Fast food items are a significant source of trans fatty acids in the diet especially in the US due to its large consumption. In humans TFA comprise 1-7% of total fatty acids. Humans do not produce TFA and, therefore, their presence in milk and adipose tissues directly reflects the trans content of the maternal diet consumed. Figure 5.11 summarizes the amount of TFA in human milk analyzed in several countries. The highest amount of TFA (ranged 0.1 to 17.2% ) was found in Canadian lactating mothers indicating that partially hydrogenated vegetable oils were the major source of TFA in the milk, whereas, contribution from dairy products appeared to be relatively minor. Infant formulas contain variable amounts of TFA, with values 0.1-4.5% of total fatty acids. However, in one of the brands, the highest amount of TFA was found to be 15.7% (Hanson and Kinsella, 1981).

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FIGURE 5.10 — Trans Fatty Acid Content (TFA, g/serving) In Some Commonly Consumed Foods. Food

TFA

Breakfast cereals Chocolate chip cookies Chocolate candies Doughnuts French fries Margarine (stick) Margarine (tub) Microwave popcorn Pound cake Salad dressings (regular) Snack crackers Snack chips Vanilla wafers Vegetable oils Vegetable shortening White bread

0.05-0.5 1.2-2.7 0.04-2.8 0.3-3.8 0.7-3.6 1.8-3.5 0.4-1.6 2.2 4.3 0.06-1.1 1.8-2.5 0-1.2 1.3 0.01-0.06 1.4-4.2 0.06-0.7

Source FDA, 1999.

FIGURE 5.11 — Trans Fatty Acid (TFA) Content Of Human Milk Reference Ratnayake et al., 1996 Jorgensen et al., 1995 Chardigny et al., 1995 Genzel-Boroviczeny et al., 1997 Chen et al., 1997 Koletzko et al., 1991 Boatella et al., 1993 Laryea et al., 1995 Crag-Schmidt et al., 1984 a b

Human milka

TFA (%)b

Canada (198) Denmark (11) France (10) Germany (38) Hong Kong Nigeria (10) Spain (38) Sudan (77) US (8)

7.2 2.2 2.27 1.13 0.88 1.20 1.2 0.61 4.76

Number of subjects appears in parentheses Values reported as average of total TFA

Current estimates of trans-fatty acids in the North American population are 4-11% of total fatty acids or 3-13 g/person/d, whereas, in Mediterranean countries in which olive oil is the primary fat and in Far Eastern countries in which little commercially hydrogenated fat is consumed, per capita consumption of trans-fatty acids is <1-2 g/d. Estimates of intake are based on availability or disappearance data (that which disappears from available supplies), food-questionnaire data, and analysis of self-selected diets.

LIPIDS

173

Metabolism The n-3 PUFA, ALA and the n-6 PUFA, LA are the predominant essential fatty acids in humans. Following ingestion, the body converts ALA and LA to a series of longer-chain, more unsaturated bioactive metabolites. LA gets elongated and desaturated into AA and dihomogama-linolenic acid (DGLA) and ALA into two important n-3 PUFA, EPA and DHA through a series of alternating desaturation and elongation enzymes (Figure 5.12). FIGURE 5.12 — Metabolic Introversion Of n-3 and n-6 PUFA Into Their Longer Chain More Unsaturated Derived EFA.

During these metabolic processes the desaturation adds double bonds by removing hydrogens, while the elongation adds carbons to produce longer chain metabolites. Both the n-3 and n-6 pathways utilize the

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FUNCTIONAL FOODS

same enzymes and both compete for these enzymes in order to produce their end products. EPA and AA are further metabolized to eicosanoids (not shown in Figure 5.12). Ecosanoids are called prostanoids which includes prostaglandins, prostacyclins and leukotrienes. Prostaglandins are synthesized in the cells following the cyclooxygenase pathway or lipoxygenase pathway. The cyclooxygenase pathway produces thromboxane, prostacyclin and prostaglandin, whereas, the lipoxygenase pathway produces leukotrienes. The prostaglandins of series-3 (PGE3) and leukotirences of series-5 (LT5) are derived from EPA, a metabolite of n-3 fatty acid which is naturally present in fish oil, whereas, prostaglandins of series-2 (PGE2) and LT4 leukotrienes are derived from AA, a metabolite of n-6 fatty acids. Furthermore, GLA which is also a metabolite of n-6 fatty acid is a precursor of series-1 prostaglandin (PGE1). Thus, essential fatty acids (n-3 and n-6) are involved in the manufacture of prostaglandins which play a unique role in a number of body functions. For example, prostaglandins PGE1 and PGE3 are usually considered to have beneficial effects including dilating blood vessels, reducing clotting, lowering harmful LDL cholesterol levels, raising beneficial HDL cholesterol levels and having anti-inflammatory actions unlike prostaglandins PGE2 which have the opposite actions and are considered to have harmful effects since these prostaglandins promote an inflammatory response and increase platelet aggregation. The balance of prostaglandins in the body is affected by diet and can determine whether a person is at increased risk of disease. Therefore, higher intake of one family of EFA leads to the suppression of the metabolism of the other and a balance of n-3 and n-6 PUFA is essential for proper health. For example, appropriate intake of EPA and DHA decreases the production of PGE2 (unhealthy) metabolites and an increase of leukotriene B 5 (LTB 5 ), a weak inducer of inflammation and a weak chemotactic agent. Western diets contain an excess of LA (ratio of n-6 to n-3 PUFA 1520:1) instead of the recommended 4-5:1 (Wahle et al., 2004) which has a recognized cholesterol lowering effect. Recent studies suggest that excessive amounts of n-6 PUFA and a very high n-6/n-3 ratio promotes the pathogenesis of many diseases (e.g., heart disease, cancer, etc.), while balancing or reducing the ratio of n-6/n-3 fatty acids may decrease the risk of these diseases. Thus, for good health it is necessary to have a balance of n-6/n-3 fatty acids in the diet and in our bodies. The conversion of LA and ALA into long chain metabolites (EPA, DHA, AA) occurs slowly in a human and the regulation of these conversions is not well understood.

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175

Metabolism of CLA by rumen bacteria has not been fully studied. However, CLA were found to be intermediate products of the biohydrogenation of polyunsaturated fatty acid because of its structural similarities. Very little is known about the bacterial genes and enzymes involved in the different steps of the CLA metabolism. The only CLA isomers that have been shown to be metabolized are c9,t11 and t10,c12CLA which undergoes delta 6 desaturation, elongation and further delta 5 desaturation while maintaining the conjugated diene structure (Bani, 2002). During the process they form conjugated diene 18:3 (CD 18:3) by introducing a double bond at position 6, CD20:3 by adding two carbon atoms and CD20:4 by introducing a double bond at position 5. The lack of data on the metabolism of other CLA isomers is due to the unavailability of pure forms of these CLA isomers. There is only a limited quantity of CLA metabolites in humans. HEALTH IMPLICATION The positive role of n-3 PUFA and CLA in health and nutrition is paramount. The first association of n-3 PUFA and human health came as early as 1944 when Sinclair (1953) pointed out the rarity of coronary heart diseases (CHD) in Greenland Eskimos despite their consumption of a diet high in fat. The Eskimos' diets contain an enormous amount of fat from fish, seals and whales and these sources of fat are very high in n-3 PUFA. Subsequent investigation found that consumption of these fatty acids including CLA not only benefits the heart but also helps in reducing cancer, weight loss, rheumatoid arthritis, osteoporosis, diabetes and many other harmful disorders. However, TFA (t18:1 isomers) were found to have adverse effect on human health leading to cause CHD. We will be giving some examples on the positive/negative effects of these fatty acids on CHD, cancer, and diabetes which are considered to be the leading cause of death in most of the industrialized countries. Coronary Heart Diseases (CHD) CHD is caused by narrowing of the coronary arteries due to cholesterol and fat deposition, a process called atherosclerosis that prevents enough blood and oxygen to reach the heart. Overweight, high blood pressure, diabetes, and high cholesterol may also lead to CHD. CHD can stem from making unhealthy choices such as smoking, or eating a high-fat diet. Several epidemiological studies including animal and in vitro experiments, suggest that n-3 PUFA may be protective in patients suffering from CHD. In a randomized, placebo-controlled trial (Lemaitre, 2003) when patients with suspected acute myocardial

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infarction (AMI) consumed fish oil (EPA, 1.08 g/day, n = 122), mustard oil (ALA, 2.9 g/d, n = 120) and a placebo (n = 118) for 1 year. The total cardiac events were found significantly less in fish and mustard oil groups compared with the placebo group suggesting a protective effect provided by n-3 PUFA present in the fish and mustard oil against the AMI. Similarly, in GISSI-Prevenzione study (1999), when patients who had myocardial infarction (MI) were randomly assigned supplements of n-3 PUFA (1 g/d, n = 2836), vitamin E (300 mg/d, n = 28300, both (n = 2830) or none (control, n = 2828) for 3.5 years. Treatment with n-3 PUFA, but not vitamin E, significantly lowered the risk of primary end point (death). Dietary supplement with n-3 PUFA lead to a clinically important and statistically significant benefit. The ways that n-3 PUFA reduces CHD is still under investigation; however, it has been suggested that the following are the health beneficial effects of the n-3 PUFA if consumed in required amounts (i) it decreases risk for arrhythmias, which can lead to sudden cardiac death; (ii) it decreases the risk for thrombosis, which can lead to heart attack and stroke; (iii) it decreases triglyceride and remnant lipoprotein levels; (iv) it decreases the rate of growth of the atherosclerotic plaque; (v) it lowers blood pressure and (vi) it reduces inflammatory responses. Cancer Cancer is a disease in which the body’s cells become abnormal and divide rapidly without control, invading nearby tissues and finally spreading through the bloodstream and lymphatic system to other parts of the body. It is clearly a disease of alterations both in genetic structure and in genetic expression which can be affected by dietary fat. Numerous epidemiological studies have examined the effect of dietary fat on various types of cancer in animals including humans. While several studies have yielded mixed results, very few were found to have an association of cancer such as breast, colorectal and lung cancer with n3 PUFA. In animals, n-3 PUFA have slowed the growth of such cancers as lung, colon, mammary, and prostate. In addition, the efficacy of cancer chemotherapy drugs such as doxorubicin, epirubicin, CPT-11, 5fluorouracil, and tamoxifen, and of radiation therapy, has been improved when the diet included n-3 PUFA. In a population-based, case controlled study involving 414 breast cancer patients, examining the combined effect of carotenoids and EFA suggested that not any specific or total carotenoid, but a combined high intake of total carotenoids and DHA may have reduced the risk of breast cancer (Nkondjock et al., 2004). Another study (Maillard et al., 2002) showed a protective effect of n-3

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PUFA on breast cancer risk and supports the imperative balance of the n-6 to n-3 ratio as being important in the development of breast cancer. This has also been demonstrated in human lung cancer A549 cells suggesting a role for the ratio of n-6 to n-3 fatty acids in cancer prevention and treatment. Further research is still needed to understand the effect that n-3 PUFA may have on the prevention or treatment of breast cancer. Consuming significant amounts of foods rich in n-3 PUFA appears to reduce the risk of colorectal cancer. For example, Eskimos, who tend to follow a high fat diet but eat significant amounts of fish rich in n-3 PUFA, have a low rate of colorectal cancer. Animal studies and laboratory studies have found that n-3 PUFA prevents worsening of colon cancer while n-6 PUFA promotes the growth of colon tumors. Daily consumption of EPA and DHA also appeared to slow or even reverse the progression of colon cancer in people with early stages of the disease. Obesity Obesity and issues surronding being overweight are an important health issue in the U.S and in most of the industrialized world due to its relationship to increasing the risk of developing a number of health conditions including type II diabetes, hypertension, and CHD. Data on the effects of n-3 PUFA on adiposity in humans are scarce. However, recently research has been focused to investigate if n-3 PUFAs have positive effects on obesity. To investigate whether the substitution of fish oil (FO) for visible fats in a control diet influences body fat mass and substrate oxidation in healthy adults, Couet et al (1997) conducted an intervention trial where energy intake was measured over three weeks while the subjects (n = 6, healthy adults) consumed a control diet and later FO (6 g/ d) for three weeks. At the end of the study, the total energy was found unchanged. However, body fat mass decreased significantly with FO, also basal lipid oxidation increased with FO indicating a reduced body fat that stimulates lipid oxidation. Similarly, Mori et al. (1999) examined whether dietary fish enhances the effects of weight loss on serum lipids, glucose and insulin in overweight individuals. The results indicated that fish + weight loss- group showed the greatest improvement in lipids. TAG decreased by 38% and HDL cholesterol increased by 24% compared with the control group. This could not only help in reducing weight but also the CHD. Thus incorporating a daily fish meal into a weight-loss regimen will be more effective. In laboratory animals, n-3 PUFA have been reported to reduce the activities of certain enzymes (fatty acid synthetase and glucose-6-phosphate dehydrogenese) involved in fatty

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acid and triacylglycerol (TG) synthesis, and to stimulate fatty acid oxidation and thermogenesis in the liver, skeletal muscle and adipose tissue. Certain dietary oils (example fish oil), possibly because of their constituent fatty acids (example n-3 PUFA), beneficially affect lipid metabolism in various organs and thereby, obesity and diabetes. Ruzickova et al (2004) showed that a 60% n-3 concentrate, containing 50% DHA and 10% EPA increased oxidation of fat by activating genes that break down fat in the mitochondriae and peroxisomes. The fish oil concentrates not only caused weight reduction in the mice but they also appeared to stop the animals from gaining weight when given free access to food. Furthermore, the n-3 concentrate reduced the number of fat cells, especially in the abdominal region. These effects were increased in animals that were put on a 10 per cent calorie reduction regime. CLA Health Implication The potential for CLA to impact human health is strongly supported by a growing literature which suggests that CLA can influence carcinogenesis, glucose regulation, immune function in animal and cell culture models as well as being capable of retarding the initiation and progression of heart disease (atherosclerosis). Preliminary animal and test tube research suggests that CLA might reduce the risk of cancer at several sites, including breast, prostrate, colorectal, lung, skin, and stomach. Whether CLA will have a similar protective effect has yet to be demonstrated especially by supplementation and dietary intervention trials in humans. However, there are several epidemiological studies which were designed to assess the relationship between CLA intake or tissue concentration of CLA and risk of breast cancer. For example, Aro and colleagues (2000) studied dietary and serum CLA in Finnish subjects (n = 499) in a case control investigation. Data indicated that an 80% lower risk of cancer in women exhibiting the highest of serum CLA or its precursor trans-vaccenic acid indicating CLA and trans-vaccenic acid, might be involved in physiologic process inhibiting cancer initiation and /or growth in postmenopausal women. In contrast, in a similar, prospective, case control study in Netherlands, Voorrips et al. (2002) studied 2,539 women. The CLA intake was approximately 200 mg/g for each individual. When data were analyzed as risk ratios, it was found that the highest quintiles of both trans-vaccenic acid and CLA were associated with increased risks of breast cancer. Although there is some epidemiologic evidence that increased CLA intake might be related to decreased risk of breast cancer, the data are not consistent. However, there exists a large literature relating the effects of CLA consumption

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and nutrient portioning (body fat regulation). In a double-blind study, volunteers participating in an exercise program received 600 mg of CLA or a placebo three times per day for 12 weeks. Compared with the placebo, CLA significantly reduced percent of body fat, but did not significantly reduce body weight (Thom et al., 2001). In a double-blind study of obese men, supplementation with 4.2 grams of CLA per day for four weeks produced a small but statistically significant reduction in waist size. However, compared with the placebo, CLA did not promote weight loss. At present, there is not sufficient evidence to support the use of CLA as a treatment for obesity. Animal research suggests an effect of CLA supplementation on reducing body fat. Limited controlled human research found that CLA produced nonsignificant gains in muscle size and strength in experienced weight-training men. Animal research also suggests an effect of CLA supplementation on limiting food allergy reactions, preventing atherosclerosis and improving glucose tolerance. As with the cancer research, the effects of CLA on these conditions in humans remain unclear. TFA Health Implication Phasing-out of industrially produced TFA in food products is currently a great concern in the Western world and especially in the US due to their negative health impact. Concerns have been raised for several decades that consumption of trans fatty acids might have contributed to the 20th century epidemic of coronary heart disease. Metabolic studies have shown that trans fats have adverse effects on blood lipid levels increasing the low density lipoprotein (LDL) a “bad” cholesterol while decreasing high density lipoprotein (HDL) a “good” cholesterol. This combined effect on the ratio of LDL to HDL cholesterol is double that of saturated fatty acids. Both of these conditions are associated with insulin resistance which is linked to diabetes, hypertension, and cardiovascular disease. Based on the available metabolic studies, it is estimated that approximately 30,000 premature coronary heart disease deaths annually could be attributable to consumption of trans fatty acids (Willett et al., 1994). Studies have also suggested that the cholesterol raising effect of hydrogenated fat is somewhat lower than that of saturated fats. However, only recently attention has been given to the fact that although trans fatty acids increase LDL cholesterol to a similar degree as saturated fat, they decrease HDL cholesterol relative to both cis unsaturated or saturated fats. In a metabolic study, Mensink and Katan (1990) demonstrated that replacement of 10% of energy from oleic acid (the primary monounsaturated fat in diets) with trans 18:1

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fatty acids caused a 0.34 mmol/L increase in LDL cholesterol and a 0.17 mmol/L decrease in HDL cholesterol; whereas replacement of oleic acid with saturated fat caused a similar increase in LDL cholesterol, but virtually no change in HDL cholesterol. As a result, the LDL/HDL cholesterol ratio was significantly higher on the trans than on the saturated or oleic diets. These findings were soon confirmed in several investigations. Overall, trans fatty acids increased LDL cholesterol similarly to saturated fat, but, unlike saturated fat, they also decreased HDL cholesterol. As a result, the net effect of trans fat on the LDL/ HDL cholesterol ratio is approximately double that of saturated fat. Moreover, these effects of trans fat on the LDL/HDL cholesterol ratio are remarkably constant across studies. These results confirm the deleterious effects of trans fat on blood lipids and indicate that these may alter the beneficial effects of polyunsaturated fat. Another plasma lipoprotein thought to produce cardiovascular disease is lipoprotein(a). In addition to increasing the LDL/HDL cholesterol ratio, trans fatty acids also increase lipoprotein(a) level when substituted for saturated fat. A significant increase was reported in several trials. High blood levels of lipoprotein(a) have been associated in some studies with increased risk of CHD, independently of LDL or HDL cholesterol concentrations. However diet-induced variations in blood concentrations of lipoprotein(a) are modest relative to the genetic differences, and their quantitative impact on risk of CHD remains to be established. Another effect of trans fatty acids on blood lipids is on fasting triglyceride levels. A triglyceride-raising effect was also consistently seen in other studies that directly compared trans fatty acids with cis-unsaturated fatty acids. The increases ranged from 0.005 to 0.12 mg/ml, with an average of 0.15 mg/ml per 1% of energy intake. The effect on triglyceride levels of substituting saturated fatty acids for cis-unsaturated fatty acids is about zero (Mensink et al., 1992). Thus, trans fatty acids increase triglyceride levels when compared with other fatty acids. Eliminating 2% of energy trans fatty acid from the diet would lower triglyceride levels by about 0.03 mg/ml, the relation between triglycerides and risk of CHD is still uncertain, but the resulting benefit is probably modest. Potential effects of trans fat on LDL oxidation and coagulation and fibrinolytic factors have also been investigated, but so far there is no conclusive evidence of adverse effects. Fish Oil Fish oil is derived from the tissues of fatty fishes. Virtually every type of fish contains oil. However, the quantity and composition of the

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oil varies with the type of fish, the season of the year, the geographical location and the diet of the fish. The oil naturally contains the
-3 fatty acids EPA (20:5n-3) and DHA (22:6n-3) including a wide range of other fatty acids. Fish oils are produced in several countries for human consumption and when traded, they usually are described either by generic name of the fish or the name of the country in which the oil has been extracted. The major fish types which are caught commercially and processed for oil include, Anchovy, Capelin, Herring, Mackerel, Menhaden, Sardine and even some Shark. Some of the health benefits of
-3 has already been discussed previously. Marine sources of oil, while not very common in the United States, are frequently used to yield edible oils. The uniqueness of fish oils has been recognized for some time, but the pure health significance began only in the mid 1970s when Eskimos consuming a very rich, high fat diet, rarely suffered death. The reason for this remained a mystery until, it was found that their marine-based diet was very rich in
-3 polyunsaturated fatty acids. These have antiarrhythmic, endothelial protective, antiatherogenic, antithrombotic and antiplatelet effects in many observational studies, which have pointed to their potential role in secondary prevention post myocardial infarction. Fish oil is recommended for a healthy diet, and it is beneficial to eat fish once a week (or more) but care must be taken to avoid the fish species which contain the toxin mercury or other contaminants such as Chlordane. Health Beneficial Effects Of Fish Oil The two predominant PUFA in fish oils are EPA and DHA which have proven to possess beneficial health effects. For example, in hyperlipidemic subjects, feeding fish oil has been shown to have beneficial effects, especially in reducing the risk of heart disease. Fish oil concentrate K-85, containing 92% of total fatty acids as n-3 fatty acids, has been shown to lower serum triglycerides and very low density lipoprotein (VLDL) in nondiabetic hypertriglyceridemic subjects (Mackness et al., 1994). In diabetic subjects, the most consistent beneficial effect of dietary fish oils is the lowering of plasma triglyceride levels. The cholesterol/phospholipids ratio and the cholesterol/HDL cholesterol ratio, which is a measure of the atherogenic index were also found lower when fish oil was consumed. In a comparative study between n- PUFA from fish oil (EPA + DHA) with that from linseed oil (linolenic acid) on plasma triglyceride with diabetic patients, fish oil decreased plasma triglyceride but linseed oil was without effect indicating that preformed long-chain n-3 fatty acids are more effective

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in lowering the lipid levels than linolenic acid (Goh et al., 1997). n-3 PUFA are also more readily incorporated in brain and other tissues as compared to those from vegetable oils. Therefore, infant milk formulas now are being supplemented with fish oil for brain development and function (Clandinin et al., 1992). However, dietary fish oils have several deleterious effects on carbohydrate metabolism in diabetic subjects by increasing fasting and postprandial glucose. Owing to the increase in glucogenesis from glycerol, long-term feeding of fish oil is anticipated to deteriorate glucose control. Insulin secretion is also impaired by fish oil feeding but plasma insulin levels are generally not altered. From human studies, it is clear that in a diabetic subject, n-3 PUFA appear to have beneficial effects on lipid metabolism and may decrease the severity of cardiac disorders and lower the incidence of coronary artery disease. However, these long chain fatty acids have detrimental effects on carbohydrate metabolism. Olive Oil Olive oil is extracted from fresh or ripe fruits of the long lived ever green olive tree Olea europaea, which originated in the Mediterranean area. There are approximately 900 species of olives and most are familiar with the olive that is cultivated for its fruit also known as drupes. The oil is regarded as a healthy dietary oil because of its high content of monounsaturated fat. It is produced principally in Greece, Italy, Spain, France,Turkey, Portugal, Tunisia, Morocco, and California. Among global producers, Spain leads with more than 40% of world production, followed by Italy and Greece. It has been postulated that the lower incidence of coronary heart disease (CHD) in these countries is due to their Mediterranean diet which includes a large amount of olive oil. Main consuming countries are also the main olive oil producers. European Union accounts for 71% of world consumption. Mediterranean basin countries represent 77% of world consumption. Other consuming countries are United States, Canada, Australia and Japan. Chemical Composition The chemical composition of the olive depends upon several agronomic factors, including the variety, place, age of growth and harvesting season. In general the content of the olive is 47% water, 31% solids and 22% oil. Figure 5.13 represents the selected nutrient composition of one large olive (4.4 g). Olive oil contains a high percentage of the monounsaturated oleic acid (C18:1) as a major lipid constituent including some minor constituent as hydrocarbons, monoglyceride

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esters, tocopherols, flavenoids, anthocyanins, sterols and a number of simple and complex phenolic compounds such as oleouropein, hydroxytyrosol (3,4-dihydroxyphenyl) and tyrosol in higher concentration (Figure 5.13). FIGURE 5.13 — The Composition Of A Single Large Olive* Nutrient

Amount (g)

Water Energy Protein Total Lipid Carbohydrate Total dietary fiber Ash

3.52 5.05 kcal 0.037 0.47 0.28 0.14 0.10

Palmitic (C16:0) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2 n-6) Linolenic (C18:3 n-3)

0.05 0.01 0.34 0.04 0.003

Lipid

* Adopted from USDA nutrient database (1998)

FIGURE 5.14 — Chemical Structure Of Few Main Phenolic Constituent Of Olive Oil

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The most abundant phenolic compound in the drupe is oleuropein (glycoside) that contributes primarily a bitter test. On maturation oleuropein undergoes enzymatic and nonenzymatic hydrolysis and yields several simpler compounds (e.g, hydroxytyrosol, ligstroside) that builds up the full fruity taste. Hydroxytyrosol and tyrosol are structurally similar except that hydroxytyrosol possesses an extra hydroxyl group in the meta position. Oleuropein is an ester which consists of hydroxytyrosol and elenolic acid. The phenolic compounds present in olive oil are strong antioxidants and free radical scavengers. The greater the phenol content in olive oil, the better the oxidative stability. Hydroxytyrosol is a superior antioxidant and free radical scavenger to oleuropein and tyrosol. The level of these compounds varies between 50 to 800 mg/kg olive oil (Tuck et al., 2002). Olive oil also contains several tocopherols (example  -, b -,  -,). tocopherol being almost 88%. The taste of olive oil is attributed to a group of aroma compounds and trans-2-hexenal being the predominant component. It is unclear if the beneficial properties of olive oil are from its constituents or their metabolites. Olive Oil Extraction Traditionally, olive oil is produced by crushing the fruits in stone or wooden mortars or beam presses. Nowadays, olives are ground to tiny bits, obtaining a paste that is mixed with water and processed by centrifuge, which extracts the oil must from the paste, leaving behind the pomace (the residue). The oily must is further centrifuged to obtain the pure oil after filtration (Figure 5.15). Another process is by a coldpressing technique without using solvents, which does not alter the chemical nature of the oil. Olive oil comes in different varieties, depending on the amount of processing involved. The most common industrial processing method is a continuous extraction system with two centrifugations (first horizontal and then vertical). Vertical centrifugation may be in three phases obtaining oil, pomace and vegetable waters, or in two phases (in this case there is no water injection or little water) obtaining oil and a paste. The main disadvantages of this process are the huge amounts of water needed and, therefore, the production of vegetable fluids with the resulting pollution. However, there are new industrial techniques of continuous extraction. They reduce the production of vegetable waters by obtaining a much more humid pomace that can be moisturized and used later. This process does not need much water. It is being more and more widely used.

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FIGURE 5.15 — Extraction Of Olive Oil

TYPES OF OLIVE OIL Extra-Virgin Olive Oil Extra-virgin Olive oil is produced by the cold-pressing technique at room temperature with a maximum of 1% free fatty acids (must not be greater than 1 g per 100 g). It has a noticeable green color due to the presence of chlorophyll and pheophytin. The oil is considered best because of less processing and for possessing very high standards of aroma and flavor. There is no refined oil in extra-virgin olive oil.

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Virgin Olive Oil Virgin Olive oil is produced by second pressing in the same manner as extra-virgin oil. This oil has a slightly higher free fatty acid (2%) and possesses high standards of aroma and flavor. There is no refined oil in virgin olive oil. Standard Virgin Olive Oil It is also called commercial grade olive oil. It is mixed with refined oil to improve its taste. The oil undergoes some processing, such as filtering and refining. The acidity of this oil is around 3.3g per 100g of oil. Use And Storage Of Olive Oil When choosing an olive oil, it is imperative that one buys “extra virgin” olive oil, rather than “virgin” or the commercial grade so called pure olive oil. Extra virgin is the oil from the first pressing, it uses top grade olives, with less than one percent acidity, and has the highest nutritional value and the best taste. It is the only oil with which one can get the true benefits of olive oil. Olive oil may be used in dressings, marinades, and as cooking oil. It turns into a solid if refrigerated, so it is best stored as a liquid at room temperature. Using a dark bottle and corking it tightly can reduce the amount of oxidation. Health And Beneficial Effects Of Olive Oil The biological and therapeutic values of olive oil are due to its chemical structure. The triglyceride composition of olive oil is made up of 54 - 83% of monounsaturated fat (oleic acid). Monounsaturated fatty acids are much more stable than polyunsaturated ones in terms of the oxidation process that prevents rancidity. Secondly, olive oil’s beneficial properties lie in its minor components. The most common ones are the tocopherols, among them -tocopherol which acts as vitamin E and carotene as provitamin A, and the polyphenols. All of these components have a major antioxidant function and are closely connected with virgin olive oils because refining processes alter and partially remove them in the other types. Olive oil is very well tolerated by the stomach due to its high oleic acid content. Since ancient times olive oil has been described as having a beneficial effect on hyperchlorohydric gastritis and gastroduodenal ulcers, which is attributed to its protective function. Olive oil activates the secretion of bile and pancreatic enzymes much more naturally than prescribed drugs. Consequently, it lowers the incidence of gallstone formation. A roster of scientific studies have demonstrated that a balanced diet containing olive oil provides

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significant health benefits, including delaying the aging of cells by halting the excessive production of cells with oxidant substances, in reducing cholesterol levels, in the prevention of bone decalcification, and in the assimilation of calcium, iron, phosphorus, magnesium, zinc, so important in the growing process. It is well documented that monounsaturated fatty acid may lower blood cholesterol levels and may increase HDL cholesterol levels, therefore, linking olive oil consumption and the lower incidence of CHD and even cancer, particularly breast cancer, in cultures where a “Mediterranean diet” is consumed. “Mediterranean diet” is characterized by high content of oleic acid, fruits, vegetables, grains, legumes and low meat. Olive oil is clearly one of the good oils. Most people do quite well with it since it does not upset the critical n-6 to n-3 ratio. Phytosterols Phytosterols, also known as a plant sterol are widely distributed in the plant kingdom. These plant lipid-like components are chemically similar to the dietary and endogenously secreted cholesterol and exist in all foods of plant origin, as monomers, glycosides, esters, or glucosylated esters. The major common plant sterols are β-sitosterol, stigmasterol and compesterol which constitute the majority in normal foods, whereas, avenasterol and brassicasterol are minor components. They differ from cholesterol only in the identity of one side chain or the presence of the extra double bond (Figure 5.16). The different chemical forms of phytosterols exist in different compartments of the plant cell. For example, free phytosterols are mainly found in the plant membrane wall to give structural properties while phytosterol glucosides and esters mainly are found in the cytosol and endoplasmic reticulum. Vegetable oils are the major source of free phytosterols. Most crude vegetable oils contain 1-5 g kg -1 of total phytosterols. The major phytosterol components of some of the commonly consumed vegetable oils (crude and refined) are listed in Figure 5.17. Sitosterol is the major sterol in vegetable oil especially in refined/ crude rapeseed oil while sistostenol, the saturated derivative of sitosterol, occurs at negligible levels in plant lipids. Refining of oils lowers the phytosterol levels (Figure 5.17). Phytosterols are partly removed with other components of crude oils in vegetable oil refining. They may also react by atmospheric oxygen and undergo isomerization and other intermolecular transformation reactions. Campesterol and stigmasterol are more labile than sitosterol.

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FIGURE 5.16 — Structure Of Some Common Phytosterols Including Cholesterol

FIGURE 5.17. Major Phytosterols In Crude And Refined Vegetable Oils (g kg-1)* Oils

Campesterol

Sitosterol

Stigmasterol

Corn, crude Corn, refined Olive, extra virgin Olive, cold pressed Palm, crude Palm, refined Peanut, refined Rapeseed, crude Rapeseed, refined Soybean, crude Soybean, refined Sunflower, refined

1.69-2.01 1.23-1.64 0.045-0.050 0.02-0.05 0.14-0.20 0.02-0.05 0.24-3.8 2.93 1.64-3.00 0.57-0.71 0.34-0.82 0.27-0.55

5.41-6.46 4.54-5.43 1.18-1.33 1.22-1.30 0.43-0.52 0.35-0.41 1.15-1.69 4.20 3.58-3.95 1.73-1.84 1.24-1.73 1.94-2.57

0.58-0.68 0.46-0.59 0.009-0.013 nd1-0.03 0.07-0.10 0.07-0.10 0.12-0.22 nd nd-0.16 0.58-0.61 0.37-0.64 0.18-0.32

* Adapted from Piironen at al (2004)

nd = not detected

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In margarine the phytosterol content depend on their fat content, the oil and the fats used. The total phytosterol content of margarines with 80% fat ranged between 1.36 and 5.86 g kg-1 (Weihrauch and Gardner, 1978). According to the food composition database, phytosterols ranged for hard regular margarines from 1.36 to 5.71 g kg-1 and for soft margarines from 1.44 to 4.83 g kg-1 in the U.S (USDA, 2000). Cereals are generally considered as a good source of phytosterols depending on the dietary pattern and the way in which they are consumed. The total phytosterol contents of various cereals range mainly from about 350 to 1200 mg kg-1 fresh weight. The total sterol contents for rye, barley, wheat and oats were reported 71.2, 35.6, 42.0 and 12.1 mg g -1 , respectively (Dutta et al, 1996). Sitosterol is the major phytosterol of cereals accounting for 49% to 64% in wheat, rye, barley and oats (Piironen et al., 2002). Phytosterols in cereals are found as free sterols (FSs), esters with fatty acids (SEs), and phenolic acids (SPHEs), glycosides (SGs) and acylated glycosides (ASGs) and varies between different cereals and in various parts of the kernel. In bakery products the phytosterol contents vary between 410 to 824 mg kg-1 and is highest in bread baked mainly with whole-meal flour. In vegetables the phytosterol contents vary from low (38-51 mg kg-1), to moderate (160 mg kg-1). Broccoli, Brussels sprouts, cauliflower and dill are the best source of phytosterols. Their total phytosterol contents were more than 300 mg kg-1 fresh weight (Normén et al, 1999; Piironen et al, 2003). The content was less than 100 mg kg-1 in potted lettuce, onion, potato and tomato; within the range of 100-200 mg kg-1 in the carrot, Chinese cabbage, leek, red beet and white cabbage; and in the range of 200-300 mg kg-1 in the pea, sweet pepper and parsley (Piironen et al., 2003). Sitosterol is generally the main phytosterol in vegetables and contributed 43-86% to the total phytosterols of about 20 analyzed vegetables (Piironen et al, 2003). The phytosterol contents of some fruits ranged from 13 (watermelon) to 440 mg kg-1 (passion fruit), with a median content of 160 mg kg-1 for 14 analyzed fruits (Normén et al, 1999). Among the analyzed fruits, avocado contained significantly more sterols (752 mg kg-1). The phytosterol content in fresh berries ranged from 60 (red currant) to 279 mg kg-1 (lingonberry). Sitosterol is the main sterol both in fruits and berries. Its proportion ranged in fruits between 72% and 86% and in berries between 61% and 93% (Piironen et al., 2003). Campesterol and stigmasterol were the two other major sterols. Various peanuts and almonds are also rich in phytosterols. The total phytosterols contents in raw peanuts with skin ranged between 600 and 1608 mg kg-1 fresh weight and those of shelled peanuts ranged

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between 551and 1269 mg kg-1. Sitosterol contributed about 80% of sterols. Both heredity and growing conditions affect phytosterol contents and composition. Furthermore, the planting location and temperature affects the phytosterol accumulation in vegetable oils and fruits. For example, the total phytosterol levels of canola were markedly affected by genetic modification (Abidi et al., 1999). Similarly, the total sterols ranged from 1.76 to 3.48 g kg-1 and sitosterol from 0.93 to 1.71 g kg-1 of oil in genetically modified soybeans differing in their fatty acid compositions. Dietary intake of phytosterols ranges from 250 to 500 mg/d with about 65% of intake as β-sitosterol, 30% as compasterol, and 5% as stigmasterol and low amount of other sterols. Health Implication Of Phytosterol Phytosterols are non-nutritive components of foods having several health benefits. There exists a longstanding interest in the hypocholesterolemic effects of dietary phytosterols. Because of its close resemblance to cholesterol, they actually block food-based cholesterol from being absorbed into the bloodstream and inhibit the reabsorption of cholesterol from bile acids in the digestive process. In contrast to cholesterol, phytosterols are poorly absorbed (5-10% of cholesterol) and therefore the intestines are occupied by phytosterols for extended periods of time which makes the blocking process even more effective. The increased levels of cholesterol in chickens caused by cholesterol feeding was prevented after including 1% soybean sterols in the diet (Peterson, 1951). Since then, numerous studies have confirmed a hypocholesterolemic action of plant sterols, especially sitosterol. Sitostanol, prepared by hydrogenation of sitosterol, reduces the intestinal absorption of cholesterol and lowers serum cholesterol more effectively than sitosterol (Heinmann et al, 1986). Also, sitostanol reduced 33% LDL cholesterol in children having severe familial hypercholesterolemia in 3-months time (Becker et al., 1993). Currently, a great renewal has occurred in the use of sitosterols for the inhibition of cholesterol absorption. For example, plant sterols are first saturated to stanols and then further esterify stanols to a more fat-soluble form. Miettinen and Gylling (1999) have developed a sitostenol ester margarine that includes sitostanol in soluble ester form. Serum campesterol, a dietary plant sterol whose levels reflect cholesterol absorption, was decreased by 36% with the diet containing sitosterol ester margarine. Some phytosterols stimulate insulin secretion and may contribute to better sugar control. A combination of pravastatin and

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sitostenol ester (sitostenol ester margarine) was evaluated for control of mild hypercholesterolemia in men with non-insulin-dependent diabetes mellitus (Gylling et al., 1996). Serum total and LDL cholesterol were lowered 35% and 44% respectively, compared to levels observed with a control dietary margarine. In a randomized double-blind placebocontrolled study with margarine fortified with sterol esters from soybean or rice bran oil. Plasma total and LDL cholesterol were reduced by 8% to 13% for margarines enriched in soybean oil sterol esters or sitosterol esters compared to control margarine (Westrate and Meijer, 1998). It was concluded that a margarine with sterol esters from soybean oil, has mainly esters from sitosterol. Campesterol, and stimasterol, was as effective as margarine with sitostenol esters in lowering blood total and LDL cholesterol levels. Phytosterols do not appear to lower triglycerides or to raise the levels of HDL, the good cholesterol. Besides cholesterol lowering properties, phytosterols may also have effects on cancer development which is the largest killer of men and women in Western societies. There have been several studies on cancer prevention with phytosterol supplements in animals, however, the bioavailability of phytosterols is fulfilled only by unsaturated sterols and therefore they may have direct or indirect effects on endogenous cancer prevention. Furthermore, regulatory proteins which control cell proliferation and growth are the major site for cancer development, these sites also constitute the molecular targets for phytochemicals where these dietary constituents may work alone or in combination to prevent adverse effects (Gescher et al, 2001). The preventive effect of phytosterols on colon cancer was first observed by Nair et al. (1984) when Seventh Day Adventists experienced lower rates of colon cancer and had at the same time higher dietary intakes of phytosterols than the general population. As bile acids are well-known tumor promoters the lower incidence observed in Seventh Day Adventists was hypothesized to be due to the decreased bile acid excretion. Effects of phytosterols on breast cancer are very limited. In one study (Awad et al. 2000), female SCID mice were inoculated in the right inguinal mammary fat pad with cultured breast cancer cells (MDA-MB-231 type). The animals were randomized into two groups being fed a control diet + 0.2% cholic acid + 2% cholesterol, and a control diet + 0.2% cholic acid + 2% phytosterols. After 8 weeks of tumor growth, the tumor area and weight was 33% smaller in the phytosterol group compared to the control group. In a similar model and to test the effect of phytosterols on prostate cancer development, SCID mice having cultured prostate cancer cells (PC-3 type) were fed phytosterols. The phytosterol-treated animals had

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28% smaller tumors and also had only about one-half the rate of metastasis compared to the control diet (Awad et al., 2001). Overall phytosterols have an important function in the human body which has led to the introduction and commercialization of phytosterol-enriched food products in most of the developed countries. SUMMARY This chapter has summarized valuable information on omega fatty acids including CLA, TFA, and their presence in various food items. Omega-3 fatty acid, and CLA possess healthy appeals, whereas, TFA are considered harmful for humans. Omega-3 fatty acids are required for proper growth, and development. However, intake of these fatty acids are not a solution for the chronic diseases, but a healthy life style, regular exercise, and a choice of appropriate food may lead to better nutrition and good health. Although omega-6 fatty acids are essential, it is also essential that the ratio of omega-6 to omega-3 fatty acids be balanced for proper metabolism. The bioactive molecules such as phytosterols from plants, Omega-3 PUFA from fish oil and the phenolic components of olive oil have many important roles in promoting health and preventing diseases. Although, these components have not yet been considered essentials they already have proven to be beneficial to the health. Their major benefit to consumers is indirect. Inclusion of olive oil, fish and phytosterols in the daily diet may be prudent. It is apparent that beta sitosterol is a useful dietary supplement for the lowering of plasma cholesterol. Nevertheless, beta sitosterol should be used with caution in certain individuals who have a higher absorption rate of beta sitosterol. Since interest in these bioactive molecules as a functional food ingredient with health promoting properties has been more intense in recent years, further research needs to be completed to more fully elucidate the health implications. More studies with healthy populations and populations with acute and chronic disease states are also needed.

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References Abidi, S.L., List, G.R., and Rennick, K.A. 1999. Effect of genetic modification on the distribution of minor constituents in canola oil. J. Am. Oil Chem. Soc. 76:463-467. Almendingen, K., Jordal, O., Kierulf, P., Sandstad, B., and Pedersen, J.I. 1995. Effects of partially hydrogenated fish oil, partially hydrogenated soybean oil, and butter on serum lipoproteins and Lp[a] in men. J. Lipid Res. 36:1370-1384. Alswad, K., Lavie, C.J., Milani, R.V., and O’Keefe, J.H. 2002. Fish oil in cardiovascular prevention. The Ochsner J. 4:83-91. Aro, A., Mannisto, S., Salminen, I., Ovaskainen, M. L., Kataja, V., and Uusitupa, M. 2000. Inverse association between dietary and serum conjugated linoleic acid and risk of breast cancer in postmenopausal women. Nutr. Cancer, 38:151-157. Awad, A.B., Downie, A., Fink, C.S., Kim, U. 2000. Dietary phytosterol inhibits the growth and metastasis of MDA-MB-231- human breast cancer cells grown in SCID mice. Anticancer Res. 20(2A):821-824. Awad, A.B., Fink, C.S., Williums, H., Kim, U. 2001. In vitro and in vivo (SCDI mice) effects of phytosterols on the growth and dissemination of human prostrate cancer PC-3 cells. Eur. J. Cancer Pre. 10(6):507-513. Bani, S. 2002. Conjugated linoleic acid metabolism. Current opinion in Lipidology, 18:261-266. Becker, M., Staab, D., and von Bergmann, K. 1993. Treatment of severe familial hypercholesterolemia in childhood with sistosterol and sitostanol. J. Pediatr. 122:292296. Boatella, J., Rafecas, M., Codony, R., Gibert, A., Rivero, M., Tormo, R., Infante, D., and Sanchez-Valverde, F. 1993. Trans fatty acid content of human milk in Spain. J. Pediatr. Gastroenterol. Nutr. 16:432-434. Chardigny, J.M., Wolff, R.L., Mager, E., Sebedio, J.L., Martine, L., and Juaneda, P. 1995. Trans mono- and poly-unsaturated fatty acids in human milk. Eur. J. Clin. Nutr. 42:49-56. Chen, Z.Y., Kwan, K.Y., Tong, K.K., Ratnayake, W.M.N., Li, H.Q., and Leung, S.S.F. 1997. Breast milk fatty acid composition: a comparative study between Hong Kong and Chongqing Chinese. Lipids, 32:1061-1067. Chin, S.F., Liu, W., Storkson, J.M., Ha, Y.L., and Pariza, M.W. 1992. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J. Food Comp. Anal. 5:185-197. Clandinin, M.T., Grag, M.L., Parrot, A., VanAerde, J.V., Hevada, A., and Lien, E. 1992. Addition of long chain polyunsaturated fatty acids to formula for very low birth weight infant. Lipids, 27:896-899. Couet, C., Delarue, J., Ritz, P., Antoine, J.M., and Lamisse, F. 1997. Effects of dietary fish oil on body fat mass and basal fat oxidation in healthy adults. Int. J. obesity, 21:637-643. Craig-Schmidt, M.C., Weete, J.D., Faircloth, S.A., Wickwire, M.A., and Livant, E.J. 1984. The effect of hydrogenation fat in the diet of nursing mothers on lipid composition and prostaglandin content of human milk. Am. J. Clin. Nutr. 39:778786. Dutta, P.C., and Appelqvist, L-Å. 1996. Saturated sterols (stanols) in unhydrogenated and hydrogenated edible vegetable oils and in cereal lipids. J. Sci. Food Agric. 71:383391. Enig, M.G., Pallansch, L.A., Sampugna, J., and Keeney, M. 1983. Fatty acid composition of the fat in selected food items with emphasis on trans components. J. Am. Oil Chemists’ Soc. 60:1788-1795.

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Fogerty, A.C., Ford, G.L., and Svoronos, D. 1988. Octadeca-9,11-dienoic acid in foodstuffs and in the lipids of human blood and breast milk. Nutr. Rept. Intl. 38, 937-944. Food and Drug Administration (FDA). 1999. Food labeling: Trans fatty acids in nutrition labeling, nutrient claims and health claims. Fed. Reg. 64:62745-62825. Genzel-Boroviczeny, O., Wahle, J., Koletzko, B. 1997. Fatty acid composition of human milk during the 1st month after term and preterm delivery. Eur. J. Pediatr. 156:142-147. Gescher, A.J., Sharma, R.A., Steward, W.P. 2001. Cancer chemoprevention by dietary constituents: a tale of failure and promise. Lancet Oncol. 2(6):371-379. GISSI-Prevenzione Investigators. 1999. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E in 11,324 patients with myocardial infarction: results of the GISSI-Prevenzione trial. Lancet 354:447-55. Goh, Y.K., Jumpsen, J.A., Ryan, E.A., and Clandinin, M.T. 1997. Effects of omega 3 fatty acid on plasma lipids, cholesterol and lipoprotein fatty acid content in NIDDM patients. Diabetologia, 40:45-52 Gylling, H., Miettinen, T.A. 1996. Effects of inhibiting cholesterol absorption and synthesis in cholesterol and lipoprotein metabolism in hypercholesterolemic noninsulin-dependen diabetic men. J. Lipid. Res. 37:1776-1785. Hanson, J.M., and Kinsella, J.E. 1981. Fatty acid content and composition of infant formulas and cereals. J. Am. Dietet. Assoc. 78:250-255. Heinmann, T., Leiss, O., and von Bergmann, K. 1986. Effect of low-dose sitostanol on serum cholesterol in patients with hypercholesterolemia. Atherosclerosis 61:219-223. Holman, R.T. 1998. The slow discovery of the importance of
3 essential fatty acids in human health. J. Nutr. 128:427S-433S. Hunter, J.E., and Applewhite, T.H. 1986. Isomeric fatty acid in US diet: levels and health prospective. Am. J. Clin. Nutr. 44:707-717. Jorgensen, M.H., Lassen, A., and Michaelsen, K.F. 1995. Fatty acid composition in Danish infant formula compared to human milk. Scan. J. Nutr./Näringsforskning, 39:50-54. Kepler, C.R., Hirons, K.P., McNiell, J.J., and Toves, S.B. 1966. Intermediates and products of the biohydrogenation of linoelic fatty acid by Butyrivibrio fibrisolvens. J.Biol. Chem. 241:1350-1354. Keys, A., Menotti, A., and Karvonen, M.J. 1986. The diet and 15-year death rate in the seven countries study. Am. J. Epidemiol. 124: 903-915. Koletzko, B., Theil, I., and Abiodun, P.O. 1991. Fatty acid composition of mature milk in Nigeria. Z. Ernahrungswiss, 30:289-297. Kris-Etherton, P.M., Harris, W.S., and Appel, L.J. 2003. Omega-3 fatty acids and cardiovascular disease. New recommendation from the American heart association. Arterioscler Thromb. Vasc. Biol. 23:151-152. Laryea, M.D., Leichsenring, M., Mrotzek, M., El-Amin, E.O., El Kharib, A.O., Ahmed, H.M., and Bremer, H.J. 1995. Fatty acid composition of the milk of well-nourished Sudanese women. Int. J. Food Sci. Nutr. 46:205-214. Lee, F., Alam, M.S., Li, J., and M.R. Guo. 2005. Seasonal Changes in Chemical Composition and Conjugated Linoleic Acid (CLA) Content of Water Buffalo milk. (Unpublished data). Lee, K.N, Kritchevsky, D., and Pariza, M.W. 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19–25. Lemaitre, R.N., King, I.B., Mozaffarian, D., Kuller, L.H., Tracy, R.P., and Siscovic, D.S. 2003. n-3 polyunsaturated fatty acids, fatal ischemic heart disease, and nonfatal myocardial infarction in older adults: the Cardiovascular Health Study. Am. J. Clin. Nutr.77:320-5.

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Ling, W.H., and Jones, P.J.H. 1995. Dietary phytosterols; a review of metabolism, benefits and side effects. Life Sci. 57:195-206. Mackness, M.I., Bhatnagar, D., Durrington, P.N., Paris, H., Haynes, B., Morgan, J., and Borthwick, L. 1994. Effects of a new fish oil concentrate on plasma lipids and lipoproteins in patients with hypertriglyceridaemia. Eur. J. Clin. Nutr. 48:859-865. Maillard, V., Bougnoux, P., Ferrari, P., Jourdan, M.L., Pinault, M., Lavillonniére, F., Body, G., Floch, O.L and Chajés. 2002. n-3 and n-6 fatty acids in breast adipose tissue and relative risk of breast cancer in a case-control study in tours. France, Int. J. Cancer, 98:78-83. Mensink, R.P., and Katan, M.B. 1992. Effect of dietary fatty acids on serum lipids and lipoproteins: a meta-analysis of 27 trials. Arteriosclerosis and Thrombosis, 12:911919. Mensink, R.P.M., and Katan, M.B. 1990. Effect of dietary trans fatty acids on highdensity and low-density lipoprotein cholesterol levels in healthy subjects. N. Engl. J. Med. 323:439-445. Miettinen, T.A., and Gylling, H. 1999. Regulation of cholesterol metabolism by dietary plant sterols. Curr. Opin. Lipidol.10:9-14. Mori, T.A., Bao, D.Q., Burke, V., Puddey, I.B., Watts, G.F and Beilin, L.J. 1999. Dietary fish as a major component of a weight-loss diet: effect on serum lipids, glucose and insulin metabolism in overweight hypertensive subjects. Am. J. Clin. Nutr. 70:81725. Mounts, T.L., Abidi, S.L., and Rennick, K.A. 1996. Effect of genetic modification on the content and composition of bioactive constituent in soybean oil. J. Am. Oil Chem. So., 73:581-586. Nair, P.P., Turjman, N., Kessie, G., Calkins, B, Goodman, G.T, Davidovitz, H., and Nimmagadda, G. 1984. Diet, nutrition intake, and metabolism in population at high and low risk for colon cancer. Dietray cholesterol, beta-sitisterol, and stigmasterol. Am. J. Clin. Nutr. 40(4suppl):927-930. Nkondjock, A., and Ghadirian, P. 2004. Intake of specific carotenoids and essential fatty acids and breast cancer risk in Monteral, Canada. Am. Soc. Clin. Nutr. 79:857-864. Normén, L., Johnsson, M., Andersson, H., van Gameren, Y., and Dutta, P. 1999. Plant sterols in vegetables and fruits commonly consumed in Sweden. Eur. J. Nutr. 38:8489. Peterson, D.W. 1951. Effects of soybean sterols in the diet on plasma and liver cholesterol in chicks. Proc. Soc. Expt. Biol. Med. 78:143-148. Piironen, V., and Lampi, A.M. 2004. Occurrence and Levels of phytosterols in foods. In: Phytosterols as functional food components and nutraceuticals. P.C. Dutta (ed), Pp132, Marcel Dekker, Inc., New York, NY. Piironen, V., Toivo, J., and Lampi, A-M. 2002. Plant sterols in cereals and cereal products. Cereal Chem. 79:148-154. Piironen, V., Toivo, J., Puupponen-Pimiän, R., and Lampi, A-M. 2003. Plant sterols in vegetables, fruits and berries. J. Sci. Food Agric. 83:330-337. Ratnayake, W.M., Chen, Z.Y. 1996. Trans, n-3, and n-6 fatty acids in Canadian human milk. Lipids, 31 (Suppl):S279-S282. Ruzickova, J., Rossmeisl, M., Prazak, T., Flachs, P., Sponarova, J., Vecka, Marek., Tvrzicka, E., Bryhn, M., and Kopecky, J. 2004. Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue. Lipids, 39:1177-1185. Sinclair, H.M. 1953. The diet of Canadian Indians and Eskimos. Proc. Nutr. Soc. 12:6982.

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Thom, E., Wadstein, J., and Gudmundsen, O. 2001. Conjugated linoleic acid reduces body fat in healthy exercising humans. J. Int. Med. Res. 29:392–396. Thompson, H., Zhu, Z., and Banni, S., Darcy, K., Loftus, T., and Ip, C. 1997. Morphological and biochemical status of the mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk. Cancer Res. 57:5067–5072. Tuck, K.L., and Hayball, P.J. 2002. Major phenolic compounds in olive oil: metabolism and health effects. J. Nutritional Biochem. 13:636-644. U.S. Depatment of Agriculture. USDA Nutrient Data Base Release 13. Washington, DC, 2000. USDA nutrient database for standard reference, release 12, U.S. Department of Agriculture, Agriculture research service, nutrient data laboratory home page, 1998, available at: http://www.nal.usda.gov/fnic/foodcopm van den Reek, M.M., Craig-Schmidt, M.C, Clark, A.J. 1986. Use of published analyses of food items to determine dietary trans octadecenoic acid. J. Am. Diet Assoc. 86:13911394. Voorrips, L.E., Brants, H.A.M., Kardinaal, A.F.M., Hiddink, G., and Brant, P.A. van den. 2002. Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer:the Netherlands cohort study on diet and cancer. Am. J. Clin. Nutr. 76:873-882. Watkins, B.A., and Li, Y. 2000. Conjugated linoleic acid: The present state of knowledge In : Hand book of Nutraceuticals and Functional Foods. Wilman, R.E.C (ed), pp 445-476, CRC Press, Boca Raton, Florida. Weihrauch, J.L., and Gardner, J.M. 1978. Sterol content of foods of plant origin. J. Am. Dietet. Assoc. 73:39-47. Whale, K.W.J., Caruso, D., Ochoa, J.H.J., and Quiles, J.L. 2004. Olive oil and modulation of cell signaling in disease prevention. Lipids, 39:1223-1231. Willett, W.C., and Ascherio, A. 1994. Trans fatty acids: Are the effects only marginal? Am. J. Public Health, 84:722-724. (Guo, M. R., Alam, M.)

Chapter 5 LIPIDS AND LIPID RELATED FUNCTIONAL FOODS “Lipids” is a word derived from the Greek “lipos” meaning fat. However, there is no widely accepted definition of lipids. Over the years, the term lipids have been used interchangeably to describe a group of naturally occurring compounds that are soluble in organic solvents (e.g., hexane, chloroform, ether, alcohol) and insoluble in aqueous media. However, lipids also includes a series of other compounds that have little similarities either in structure or in function such as carotenoids, terpenes, steroids and their naturally occurring derivatives. Many of these naturally occurring compounds are partially soluble in water as well. The definition of lipids simply based on solubility is not justifiable and should be widened to include compounds that are related closely to fatty acid derivatives through biosynthetic pathways or by their biochemical or functional properties. Lipids serve as structural components of biological membranes and provide energy in the form of triacylglycerol (TAG) also called as triglycerides. Lipids are considered in relation to excess energy balance, obesity, and as a dietary factor in the development of cardiovascular disease and many other harmful disorders. However, not all lipids are bad and damaging to human health. In fact some play a unique role in maintaining good health and providing much of the flavor and texture to foods. The changing trend in our food supply and the industrial revolution have in fact jeopardized both the quantity and balance of these nutrients. The polyunsaturated fatty acids (PUFA), for example, are very beneficial and should be consumed on a regular basis. These include the n-3 and n-6 (also referred as omega3 or
-3; and omega-6 or
-6) PUFA, also known as essential fatty acids (EFA). EFA have been part of our diet since the beginning of human life and current estimates in Western diets suggest a higher ratio of n-6 and n-3 PUFA which can dramatically change the lipid composition of many parts of the body. Humans can’t produce EFA itself and the only way to receive these nutrients is through diet and supplementation.

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There is overwhelming evidence that
-3 acids are important for health and protecting against disease including lowering triglycerides, general heart benefits, improving brain functioning, helping with diabetes, strokes, depression and many others. The purpose of this chapter is to provide basic knowledge about the origin of these fatty acids, their nomenclature, sources and how these necessary components play a vital role in daily life, including the reference of conjugated linoleic acid (CLA), another health beneficial PUFA, trans fatty acids (TFA) and their health implications. Finally, at the end a brief description of olive and fish oils, phytosterols, is provided as an important source of healthy diet and their health beneficial effects. CHEMISTRY AND NOMENCLATURE The major component of most fatty foods and oils are triglycerides, whereas, mono- and diglycerides, free fatty acids, phosphatides, sterols and fatty alcohols constitute the minor components. A triglyceride is composed of glycerol and three fatty acids whereas, mono- and diglycerides are mono- and diesters of fatty acids and glycerol (Figure 5.1), where R1, R2 and R3 represent identical or different fatty acids with even/odd numbers of carbon atoms. The mono- and diglycerides are frequently used in foods as emulsifiers. They are prepared commercially by the reaction of glycerol and triglycerides or by the esterification of glycerol and fatty acids. Mono- and diglycerides are also formed in the intestinal tract as a result of the normal digestion of triglycerides. Both the physical and chemical characteristics of fats are influenced greatly by the kinds and proportions of the component fatty acids and the way in which these are positioned on the glycerol molecule. When all of the fatty acids in a triglyceride are identical, it is termed a “simple” triglyceride. However the more common forms are the mixed triglycerides in which two or three kinds of fatty acids are present in the molecule. Fats are the major source of energy which supply about 9 calories per gram whereas, proteins and carbohydrates each supply about 4 calories per gram. The classification of fatty acids, however, is based according to their degree of saturation, the length of the carbon chain (short, medium, or long); the number of double bonds (unsaturated, mono-, or polyunsaturated); or essentiality in the diet (essential or non-essential). Saturated fats contain no double bond (example, stearic acid), monounsaturated fats contain one double bond (example, oleic acid) and PUFA contain greater than one double bond (example, linoleic acid). PUFA are subdivided into two classes based on the location of the first

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FIGURE 5.1 — Chemical Structure Of Triglyceride, Monoglyceride And Diglyceride

double bond proximal to the methyl end of the carbon chain such as n6 and n-3 PUFA. In general the carbon atoms in a fatty acid chain are numbered consecutively from the end of the chain (example: with the carbon of the carboxyl group being considered number one). This system of nomenclature is widely used by chemists. However, Dr. Ralph Holman and others suggest numbering the unsaturated fatty acids from the far terminal methyl end (CH3-) of the molecule and called such a notation the omega (
, a Greek alphabet), a nomenclature favored mostly by biochemist and nutritionist (1998). In the omega or “n minus” notation, the length of the fatty acid chain is the first number, followed by a colon with the second number denoting the number of double bonds. After the second number, n “minus” a third number may appear. The n minus notation indicates the position of the first double bond counting from the methyl end of the molecule. Thus, linoleic acid (LA, 18:2n-6 or 18:2
-6) denotes an 18-carbon fatty acid with two double bonds, the first of which occurs six carbons from the methylend of the fatty acid. We will be using the “n minus” notation throughout this chapter. The n-3 PUFA have their first double bond located at the third carbon atom (C-3) whereas, n-6 PUFA have their first double located at C-6 (Figure 5.3). Figure 5.2 represents the nomenclature of some commonly available n-3 PUFA.

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FIGURE 5.2 — Nomenclature Of Commonly Available n-3 PUFA Trivial name

IUPAC* name

n- reference

Abbreviation

Linolenic acid

9,12,15-octadecenoic acid/alpha-linolenic acid

18:3n-3

ALA/-LA/LNA/-LNA

Docosahexaenoic acid

4,8,12,15,19docsahexaenoic acid

22:6n-3

DHA

Docosapentaenoic acid

7,10,13,16,19docosapentaenoic acid

22:5n-3

DPA

Eicosapentaenoic acid

5,8,11,14,17eicosapentaenoic acid

20:5n-3

EPA

*IUPAC = International Union of Pure and Applied Chemistry

FIGURE 5.3 — Line Drawing Representation Of n-3 and n-6 PUFA Where Each Angle Represents A Carbon Atom

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The n-3 PUFA have their first double bond located at the third carbon atom (C-3) whereas, n-6 PUFA have their first double located at C-6 (Figure 5.3). Figure 5.2 represents the nomenclature of some commonly available n-3 PUFA. Polyunsaturated fatty acids can also be conjugated, as in the case of conjugated linoleic acid (CLA) a naturally occurring anticarcinogenic compound. The conjugated diene structure is not usual in fatty acids. The specific structure of CLA based on the location of the double in the carbon chain is crucial to the compound’s ability to fight cancer and many other harmful diseases. In LA in part of the molecule, a double bond is followed by two single bonds (- -) and then another double bond (=) whereas in CLA a double bond is followed by one single bond (-) and then another double bond (Figure 5.4). FIGURE 5.4 — Chemical Structure Of Linoleic Acid (LA) and Conjugated Linoleic Acid (CLA) Isomers (c9, t11- and t10, c12-CLA).

This alternation of double and single bonds is called “conjugation,” hence the term “conjugated linoleic acid.” CLA is a mixture of eighteen carbon fatty acids that is biologically produced through microbial isomerization in the rumen, and thus present in dairy products and red meat. The c9, t11 and t10, c12 isomers of CLA, are the most abundant ones in foods. Other C18 positional isomers with the conjugated double bonds at 6,8; 7,9; 8,10; 11,13 or 12,14 have also been reported in cheese (Kepler et al., 1966). A mixture of CLA isomers is also produced during food processing, by thermal isomerization and by some industrial

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processes by partial hydrogenation or alkali-isomerization of LA. However, only c9, t11- and t10, c12-CLA with one trans double are proven to be biologically active. The c9, t11-CLA isomer was the first intermediate product in the biohydrogenation of LA by the anaerobic rumen bacterium Butyrivibrio fibrisolvens. The reaction is catalyzed by the enzyme linoleate isomerase, which converts the c12 bond of free LA to a t11 bond. One reason why ruminants produce more c9, t11-CLA than non-ruminants is that hydrolysis of fat within the rumen provides more unesterified LA than is available to bacteria in non-ruminants. TFA, however, are another class of unsaturated fats which are widely present in a variety of foods; some are derived from natural sources such as dairy products but most come from products that contain commercially hydrogenated oils that are processed into a solid or more stable liquid form. Hydrogenation is a process by which vegetable oils are converted to solid fats simply by bubbling hydrogen through the fat at an elevated temperature in the presence of a metal catalyst such as nickel and in the absence of oxygen, thereby, reducing the number of double bonds. The levels and types of these fatty acids formed depend on the condition such as temperature, pressure, catalyst and duration. The most common configuration of double bonds in naturally occurring fatty acids is of cis configuration (hydrogen atoms are on the same side of the double bond). During partial hydrogenation which is an incomplete saturation of the double bonds, some double bonds remain but change from cis to trans configuration (hydrogen atoms are on the opposite side of the double bond). This shifting of the double bonds along the carbon chain of the fatty acid molecule results in positional isomers of the same acid. For example, fatty acids containing double bonds at C-9 and C-12 positions are changed to isomeric forms containing double bonds ranging from positions C-4 to C-16. Geometric isomers or cis/trans isomers are formed when the naturally occurring cis double bonds in vegetable oils are isomerized to the more thermodynamically stable trans configuration. For example, cis9 octadecenoic acid or oleic acid is transformed into trans9 octadecenoic acid or elaidic acid (Figure 5.5). The cis configuration creates a bend or kink in the fatty acid molecule making the molecule more flexible while trans configuration yields a straighter, more rigid molecule. Trans fatty acids, therefore, behaves more like saturated fats even though they have a double bond. For example, the melting point of oleic acid (cis configuration) is 13.4°C which is a liquid, whereas, elaidic acid (trans configuration) 46.5°C, is a solid unsaturated fat. The most prevalent TFA in partially hydrogenated vegetable oils (PHVO) is t10-18:1, and

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the major TFA isomer in dairy products is t11-18:1. The hydrogenation process improves the oxidative and thermal stability by adding hydrogen to the molecule. FIGURE 5.5 — Chemical Structure Of Oleic And Elaidic Fatty Acids

As a result, oils such as soybean, safflower and cottonseed, which are rich in PUFA, are converted to semi-solids and solids that are useful in margarines and vegetable shortenings. Trans-polyunsaturated (18:2 and 18:3) acids are also widely distributed in our diets, but in low amounts. However, both mono and polyunsaturated fatty acids are suspected as harmful components. DIETARY SOURCES n-3 PUFA Many commonly used oils, including safflower, sunflower, soy, and corn oil are an important plant-based source of LA (n-6), whereas, ALA (n-3) is present in canola and soybean oil. Grains and vegetable oils are considered to be the major source of ALA, however, certain fish can naturally provide the DHA and EPA in the recommended amount. Fresh seaweed is the only plant food that contains EPA or DHA. It is possible to get DHA and EPA from plant seed oils such as flaxseeds, since it contains ALA which is the precursor of DHA and EPA. Figures 5.6 &

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5.7 represent the major source of n-3 PUFA in certain selected fish and vegetable oils (Alaswad et al., 2002). EPA and DHA are found higher in Mackeral followed by Albacore tuna. Among the plant source of ALA, flaxseed contained a higher amount (20.0 g/100 g) followed by butter nuts (dried, 8.7 g/100g) and English walnut (6.8 g/100g). At the present time no specific intake of these fatty acids is available. However, recently the new recommendation from the American Heart Association (AHA) suggested that all adults eat fatty fish at least two times a week including plant derived n-3 PUFA found in tofu, soybeans, walnuts, flaxseeds and their oil, or canola oil (Kris-Etherton et al., 2003). FIGURE 5.6 — n-3 PUFA In Some Selected Fish1 Fish

ALA2

EPA3

DHA4

Mackeral Atlantic Herring Albacore Tuna Chinook Salmon Anchovy Coho Salmon Greenland Halibut Rainbow Trout Atlantic Cod Atlantic White Shrimp Catfish Northern Lobster Flounder

0.1 0.1 0.2 0.1 Trace 0.2 Trace 0.1 Trace Trace Trace 0 Trace

0.9 0.7 0.3 0.8 0.5 0.3 0.5 0.1 0.1 0.2 0.1 0.1 0.1

1.6 0.9 1.0 0.6 0.9 0.5 0.4 0.4 0.4 0.2 0.2 0.1 0.1

1

Given as g/100 of raw material Alhpa linoleic acid Eicosapentaenoic acid 4 Docosahexaenoic acid 2 3

The AHA also recommend for patients with documented CHD to consume ~ 1 g of EPA and DHA (combined) per day. Conjugated linoleic acid (CLA) Conjugated linoleic acid (CLA) is primarily a product of microbial metabolism in the digestive tract of ruminants which ultimately accumulates into milk, beef, and dairy products such as butter, yogurt and a variety of cheeses. Traces of CLA can also be found in chicken and pork due to the inclusion of meat meal or tallow in commercial diets and may not be notable sources of CLA. However, fats and meats from ruminant species are the richest natural source. CLA is also present in plant oils and selected sea foods but unlike in ruminant-derived foods

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FIGURE 5.7 — Plant Source Of Alpha Linoleic Acid (ALA)1

1

Source

ALA

Flaxseed Butternuts (dried) English walnuts Soybean (raw) Leeks Wheat germ Purslane Almonds Pinto beans Barley barn Kale Chickpeas Avocados Strawberries Peanuts

20.0 8.7 6.8 3.2 0.7 0.7 0.4 0.4 0.3 0.3 0.2 0.1 0.1 0.1 0.0

Given as g/100 of raw material

where c9, t11 is the major isomer, accounted for about 40% of total, and absent in some selected seafood lipids (Watkins et al., 2000). CLA content in food may vary widely. Representative concentration of CLA in a variety of food and dairy products is summarized in Figure 5.8. Concentrations are highest in beef, lamb and dairy products (3-7 mg/ g fat). CLA content of cows' milk ranges from 0.7-10.1 mg CLA/g fat. The amount of CLA found in dairy and beef is a direct reflection of the diet the animals are fed. CLA content of milk fat can be influenced by manipulating the type of dietary supplement fed to dairy animals. Supplementing the diet with polyunsaturated oils that contain either corn oil or sunflower oil increases CLA content of milk fat substantially. CLA contents of selected milk including humans are summarized in Figure 5.9. CLA concentration of human milk ranges from 3.1 to 8.5 mg/g fat among mothers eating conventional diets whereas, values of 9.7 to 12.5 mg/g fat among Hare Krishna mothers (Fogerty et al. 1988) suggesting that diet may influence human milk CLA concentration, because followers of the Hare Krishna faith consume large amounts of butter or ghee, as well as cheese. Currently, there exists no database which contains the CLA distribution of foods commonly consumed in the US. If modest CLA intake imparts benefits greater than no CLA consumption at all, then there are many advantages to producing foods such as milk and beef with enhanced levels of bioformed CLA because the consumption of these foods is high.

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FIGURE 5.8 — Concentration Of Conjugated Linoleic Acid (CLA) In Uncooked Foods.* Food Dairy products Homogenized milk Butter Sour cream Plain yogurt Ice cream Sharp cheddar cheese Mozzarella cheese Colby cheese Cottage cheese Reduced fat swiss Am. Proc.cheese Cheez WhizTM Meat Fresh ground beef Beef round Beef frank Beef smoked sausage Veal Lamb Pork Poultry Chicken Fresh ground turkey Seafood Salmon Lake trout Shrimp Vegetable oils Safflower Sunflower Canola Corn

CLA (mg/g fat)

5.5 4.7 4.6 4.8 3.6 3.6 4.9 6.1 4.5 6.7 5.0 5.0 4.3 2.9 3.3 3.8 2.7 5.6 0.6 0.9 2.5 0.3 0.5 0.6 0.7 0.4 0.5 0.2

* Adopted from Chin et al., 1992

FIGURE 5.9 — CLA Content In Milksa

a

Watkins et al., 2000.

Milk

CLA (mg/g fat)

Cow Goat Sheep Mare Sow Water buffalob Human

0.7-10.1 6.1-10.35 10.8-29.7 0.9 2.2 4.4-7.0 1.7-36.4

b

Guo et al., 2005

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171

Trans Fatty Acids (TFA) The primary source of TFA in food products such as margarines, shortenings, fast and processed foods is due to the use of hydrogenated vegetable oils. Small amounts of naturally occurring trans fat can be found in some animal products, such as milk, butter and tallow as a result of biohydrogenation in ruminants as a secondary source. Hence, TFA comes from two different sources: industrial, partial hydrogenation of edible oils containing unsaturated fatty acids, and bacterial transformation of unsaturated fatty acids in the rumen of ruminants. In the rumen of ruminants, principally trans vaccenic acid (t11-18:1) is formed, which accounts for over 60% of the trans fatty acid content of butter fat from cows. A number of plant species also contain small amounts of trans unsaturated fatty acids in their seeds and leaves. For example, vegetables such as leeks, peas, spinach, and lettuce contain trans-3-hexadecenoic acid (t3-16:1). Vegetable oils do not contain trans fatty acids, however, in order to reduce the potential of oxidation and rancidity, these oils are often lightly hydrogenated. For example, soybean oil sold for use as salad and cooking is often lightly hydrogenated to reduce the content of ALA and, thereby, contains some TFA. TFA content of hydrogenated salad and cooking oils ranged from 8 to 12% (Hunter, et al., 1986; Enig et al., 1983). However, these products contribute very little trans fatty acids to the current food supply in the United States and other countries. The TFA content of some commonly consumed foods are summarized in Figure 5.10. The highest content of TFA ranged between 1.4 to 4.2 g/serving in vegetable shortening, whereas, vegetable oils appeared to contain less TFA. TFA content of processed foods and fast foods may vary widely, depending upon the type of fat used in processing. Fast food items are a significant source of trans fatty acids in the diet especially in the US due to its large consumption. In humans TFA comprise 1-7% of total fatty acids. Humans do not produce TFA and, therefore, their presence in milk and adipose tissues directly reflects the trans content of the maternal diet consumed. Figure 5.11 summarizes the amount of TFA in human milk analyzed in several countries. The highest amount of TFA (ranged 0.1 to 17.2% ) was found in Canadian lactating mothers indicating that partially hydrogenated vegetable oils were the major source of TFA in the milk, whereas, contribution from dairy products appeared to be relatively minor. Infant formulas contain variable amounts of TFA, with values 0.1-4.5% of total fatty acids. However, in one of the brands, the highest amount of TFA was found to be 15.7% (Hanson and Kinsella, 1981).

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FIGURE 5.10 — Trans Fatty Acid Content (TFA, g/serving) In Some Commonly Consumed Foods. Food

TFA

Breakfast cereals Chocolate chip cookies Chocolate candies Doughnuts French fries Margarine (stick) Margarine (tub) Microwave popcorn Pound cake Salad dressings (regular) Snack crackers Snack chips Vanilla wafers Vegetable oils Vegetable shortening White bread

0.05-0.5 1.2-2.7 0.04-2.8 0.3-3.8 0.7-3.6 1.8-3.5 0.4-1.6 2.2 4.3 0.06-1.1 1.8-2.5 0-1.2 1.3 0.01-0.06 1.4-4.2 0.06-0.7

Source FDA, 1999.

FIGURE 5.11 — Trans Fatty Acid (TFA) Content Of Human Milk Reference Ratnayake et al., 1996 Jorgensen et al., 1995 Chardigny et al., 1995 Genzel-Boroviczeny et al., 1997 Chen et al., 1997 Koletzko et al., 1991 Boatella et al., 1993 Laryea et al., 1995 Crag-Schmidt et al., 1984 a b

Human milka

TFA (%)b

Canada (198) Denmark (11) France (10) Germany (38) Hong Kong Nigeria (10) Spain (38) Sudan (77) US (8)

7.2 2.2 2.27 1.13 0.88 1.20 1.2 0.61 4.76

Number of subjects appears in parentheses Values reported as average of total TFA

Current estimates of trans-fatty acids in the North American population are 4-11% of total fatty acids or 3-13 g/person/d, whereas, in Mediterranean countries in which olive oil is the primary fat and in Far Eastern countries in which little commercially hydrogenated fat is consumed, per capita consumption of trans-fatty acids is <1-2 g/d. Estimates of intake are based on availability or disappearance data (that which disappears from available supplies), food-questionnaire data, and analysis of self-selected diets.

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Metabolism The n-3 PUFA, ALA and the n-6 PUFA, LA are the predominant essential fatty acids in humans. Following ingestion, the body converts ALA and LA to a series of longer-chain, more unsaturated bioactive metabolites. LA gets elongated and desaturated into AA and dihomogama-linolenic acid (DGLA) and ALA into two important n-3 PUFA, EPA and DHA through a series of alternating desaturation and elongation enzymes (Figure 5.12). FIGURE 5.12 — Metabolic Introversion Of n-3 and n-6 PUFA Into Their Longer Chain More Unsaturated Derived EFA.

During these metabolic processes the desaturation adds double bonds by removing hydrogens, while the elongation adds carbons to produce longer chain metabolites. Both the n-3 and n-6 pathways utilize the

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same enzymes and both compete for these enzymes in order to produce their end products. EPA and AA are further metabolized to eicosanoids (not shown in Figure 5.12). Ecosanoids are called prostanoids which includes prostaglandins, prostacyclins and leukotrienes. Prostaglandins are synthesized in the cells following the cyclooxygenase pathway or lipoxygenase pathway. The cyclooxygenase pathway produces thromboxane, prostacyclin and prostaglandin, whereas, the lipoxygenase pathway produces leukotrienes. The prostaglandins of series-3 (PGE3) and leukotirences of series-5 (LT5) are derived from EPA, a metabolite of n-3 fatty acid which is naturally present in fish oil, whereas, prostaglandins of series-2 (PGE2) and LT4 leukotrienes are derived from AA, a metabolite of n-6 fatty acids. Furthermore, GLA which is also a metabolite of n-6 fatty acid is a precursor of series-1 prostaglandin (PGE1). Thus, essential fatty acids (n-3 and n-6) are involved in the manufacture of prostaglandins which play a unique role in a number of body functions. For example, prostaglandins PGE1 and PGE3 are usually considered to have beneficial effects including dilating blood vessels, reducing clotting, lowering harmful LDL cholesterol levels, raising beneficial HDL cholesterol levels and having anti-inflammatory actions unlike prostaglandins PGE2 which have the opposite actions and are considered to have harmful effects since these prostaglandins promote an inflammatory response and increase platelet aggregation. The balance of prostaglandins in the body is affected by diet and can determine whether a person is at increased risk of disease. Therefore, higher intake of one family of EFA leads to the suppression of the metabolism of the other and a balance of n-3 and n-6 PUFA is essential for proper health. For example, appropriate intake of EPA and DHA decreases the production of PGE2 (unhealthy) metabolites and an increase of leukotriene B 5 (LTB 5 ), a weak inducer of inflammation and a weak chemotactic agent. Western diets contain an excess of LA (ratio of n-6 to n-3 PUFA 1520:1) instead of the recommended 4-5:1 (Wahle et al., 2004) which has a recognized cholesterol lowering effect. Recent studies suggest that excessive amounts of n-6 PUFA and a very high n-6/n-3 ratio promotes the pathogenesis of many diseases (e.g., heart disease, cancer, etc.), while balancing or reducing the ratio of n-6/n-3 fatty acids may decrease the risk of these diseases. Thus, for good health it is necessary to have a balance of n-6/n-3 fatty acids in the diet and in our bodies. The conversion of LA and ALA into long chain metabolites (EPA, DHA, AA) occurs slowly in a human and the regulation of these conversions is not well understood.

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175

Metabolism of CLA by rumen bacteria has not been fully studied. However, CLA were found to be intermediate products of the biohydrogenation of polyunsaturated fatty acid because of its structural similarities. Very little is known about the bacterial genes and enzymes involved in the different steps of the CLA metabolism. The only CLA isomers that have been shown to be metabolized are c9,t11 and t10,c12CLA which undergoes delta 6 desaturation, elongation and further delta 5 desaturation while maintaining the conjugated diene structure (Bani, 2002). During the process they form conjugated diene 18:3 (CD 18:3) by introducing a double bond at position 6, CD20:3 by adding two carbon atoms and CD20:4 by introducing a double bond at position 5. The lack of data on the metabolism of other CLA isomers is due to the unavailability of pure forms of these CLA isomers. There is only a limited quantity of CLA metabolites in humans. HEALTH IMPLICATION The positive role of n-3 PUFA and CLA in health and nutrition is paramount. The first association of n-3 PUFA and human health came as early as 1944 when Sinclair (1953) pointed out the rarity of coronary heart diseases (CHD) in Greenland Eskimos despite their consumption of a diet high in fat. The Eskimos' diets contain an enormous amount of fat from fish, seals and whales and these sources of fat are very high in n-3 PUFA. Subsequent investigation found that consumption of these fatty acids including CLA not only benefits the heart but also helps in reducing cancer, weight loss, rheumatoid arthritis, osteoporosis, diabetes and many other harmful disorders. However, TFA (t18:1 isomers) were found to have adverse effect on human health leading to cause CHD. We will be giving some examples on the positive/negative effects of these fatty acids on CHD, cancer, and diabetes which are considered to be the leading cause of death in most of the industrialized countries. Coronary Heart Diseases (CHD) CHD is caused by narrowing of the coronary arteries due to cholesterol and fat deposition, a process called atherosclerosis that prevents enough blood and oxygen to reach the heart. Overweight, high blood pressure, diabetes, and high cholesterol may also lead to CHD. CHD can stem from making unhealthy choices such as smoking, or eating a high-fat diet. Several epidemiological studies including animal and in vitro experiments, suggest that n-3 PUFA may be protective in patients suffering from CHD. In a randomized, placebo-controlled trial (Lemaitre, 2003) when patients with suspected acute myocardial

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infarction (AMI) consumed fish oil (EPA, 1.08 g/day, n = 122), mustard oil (ALA, 2.9 g/d, n = 120) and a placebo (n = 118) for 1 year. The total cardiac events were found significantly less in fish and mustard oil groups compared with the placebo group suggesting a protective effect provided by n-3 PUFA present in the fish and mustard oil against the AMI. Similarly, in GISSI-Prevenzione study (1999), when patients who had myocardial infarction (MI) were randomly assigned supplements of n-3 PUFA (1 g/d, n = 2836), vitamin E (300 mg/d, n = 28300, both (n = 2830) or none (control, n = 2828) for 3.5 years. Treatment with n-3 PUFA, but not vitamin E, significantly lowered the risk of primary end point (death). Dietary supplement with n-3 PUFA lead to a clinically important and statistically significant benefit. The ways that n-3 PUFA reduces CHD is still under investigation; however, it has been suggested that the following are the health beneficial effects of the n-3 PUFA if consumed in required amounts (i) it decreases risk for arrhythmias, which can lead to sudden cardiac death; (ii) it decreases the risk for thrombosis, which can lead to heart attack and stroke; (iii) it decreases triglyceride and remnant lipoprotein levels; (iv) it decreases the rate of growth of the atherosclerotic plaque; (v) it lowers blood pressure and (vi) it reduces inflammatory responses. Cancer Cancer is a disease in which the body’s cells become abnormal and divide rapidly without control, invading nearby tissues and finally spreading through the bloodstream and lymphatic system to other parts of the body. It is clearly a disease of alterations both in genetic structure and in genetic expression which can be affected by dietary fat. Numerous epidemiological studies have examined the effect of dietary fat on various types of cancer in animals including humans. While several studies have yielded mixed results, very few were found to have an association of cancer such as breast, colorectal and lung cancer with n3 PUFA. In animals, n-3 PUFA have slowed the growth of such cancers as lung, colon, mammary, and prostate. In addition, the efficacy of cancer chemotherapy drugs such as doxorubicin, epirubicin, CPT-11, 5fluorouracil, and tamoxifen, and of radiation therapy, has been improved when the diet included n-3 PUFA. In a population-based, case controlled study involving 414 breast cancer patients, examining the combined effect of carotenoids and EFA suggested that not any specific or total carotenoid, but a combined high intake of total carotenoids and DHA may have reduced the risk of breast cancer (Nkondjock et al., 2004). Another study (Maillard et al., 2002) showed a protective effect of n-3

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PUFA on breast cancer risk and supports the imperative balance of the n-6 to n-3 ratio as being important in the development of breast cancer. This has also been demonstrated in human lung cancer A549 cells suggesting a role for the ratio of n-6 to n-3 fatty acids in cancer prevention and treatment. Further research is still needed to understand the effect that n-3 PUFA may have on the prevention or treatment of breast cancer. Consuming significant amounts of foods rich in n-3 PUFA appears to reduce the risk of colorectal cancer. For example, Eskimos, who tend to follow a high fat diet but eat significant amounts of fish rich in n-3 PUFA, have a low rate of colorectal cancer. Animal studies and laboratory studies have found that n-3 PUFA prevents worsening of colon cancer while n-6 PUFA promotes the growth of colon tumors. Daily consumption of EPA and DHA also appeared to slow or even reverse the progression of colon cancer in people with early stages of the disease. Obesity Obesity and issues surronding being overweight are an important health issue in the U.S and in most of the industrialized world due to its relationship to increasing the risk of developing a number of health conditions including type II diabetes, hypertension, and CHD. Data on the effects of n-3 PUFA on adiposity in humans are scarce. However, recently research has been focused to investigate if n-3 PUFAs have positive effects on obesity. To investigate whether the substitution of fish oil (FO) for visible fats in a control diet influences body fat mass and substrate oxidation in healthy adults, Couet et al (1997) conducted an intervention trial where energy intake was measured over three weeks while the subjects (n = 6, healthy adults) consumed a control diet and later FO (6 g/ d) for three weeks. At the end of the study, the total energy was found unchanged. However, body fat mass decreased significantly with FO, also basal lipid oxidation increased with FO indicating a reduced body fat that stimulates lipid oxidation. Similarly, Mori et al. (1999) examined whether dietary fish enhances the effects of weight loss on serum lipids, glucose and insulin in overweight individuals. The results indicated that fish + weight loss- group showed the greatest improvement in lipids. TAG decreased by 38% and HDL cholesterol increased by 24% compared with the control group. This could not only help in reducing weight but also the CHD. Thus incorporating a daily fish meal into a weight-loss regimen will be more effective. In laboratory animals, n-3 PUFA have been reported to reduce the activities of certain enzymes (fatty acid synthetase and glucose-6-phosphate dehydrogenese) involved in fatty

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acid and triacylglycerol (TG) synthesis, and to stimulate fatty acid oxidation and thermogenesis in the liver, skeletal muscle and adipose tissue. Certain dietary oils (example fish oil), possibly because of their constituent fatty acids (example n-3 PUFA), beneficially affect lipid metabolism in various organs and thereby, obesity and diabetes. Ruzickova et al (2004) showed that a 60% n-3 concentrate, containing 50% DHA and 10% EPA increased oxidation of fat by activating genes that break down fat in the mitochondriae and peroxisomes. The fish oil concentrates not only caused weight reduction in the mice but they also appeared to stop the animals from gaining weight when given free access to food. Furthermore, the n-3 concentrate reduced the number of fat cells, especially in the abdominal region. These effects were increased in animals that were put on a 10 per cent calorie reduction regime. CLA Health Implication The potential for CLA to impact human health is strongly supported by a growing literature which suggests that CLA can influence carcinogenesis, glucose regulation, immune function in animal and cell culture models as well as being capable of retarding the initiation and progression of heart disease (atherosclerosis). Preliminary animal and test tube research suggests that CLA might reduce the risk of cancer at several sites, including breast, prostrate, colorectal, lung, skin, and stomach. Whether CLA will have a similar protective effect has yet to be demonstrated especially by supplementation and dietary intervention trials in humans. However, there are several epidemiological studies which were designed to assess the relationship between CLA intake or tissue concentration of CLA and risk of breast cancer. For example, Aro and colleagues (2000) studied dietary and serum CLA in Finnish subjects (n = 499) in a case control investigation. Data indicated that an 80% lower risk of cancer in women exhibiting the highest of serum CLA or its precursor trans-vaccenic acid indicating CLA and trans-vaccenic acid, might be involved in physiologic process inhibiting cancer initiation and /or growth in postmenopausal women. In contrast, in a similar, prospective, case control study in Netherlands, Voorrips et al. (2002) studied 2,539 women. The CLA intake was approximately 200 mg/g for each individual. When data were analyzed as risk ratios, it was found that the highest quintiles of both trans-vaccenic acid and CLA were associated with increased risks of breast cancer. Although there is some epidemiologic evidence that increased CLA intake might be related to decreased risk of breast cancer, the data are not consistent. However, there exists a large literature relating the effects of CLA consumption

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and nutrient portioning (body fat regulation). In a double-blind study, volunteers participating in an exercise program received 600 mg of CLA or a placebo three times per day for 12 weeks. Compared with the placebo, CLA significantly reduced percent of body fat, but did not significantly reduce body weight (Thom et al., 2001). In a double-blind study of obese men, supplementation with 4.2 grams of CLA per day for four weeks produced a small but statistically significant reduction in waist size. However, compared with the placebo, CLA did not promote weight loss. At present, there is not sufficient evidence to support the use of CLA as a treatment for obesity. Animal research suggests an effect of CLA supplementation on reducing body fat. Limited controlled human research found that CLA produced nonsignificant gains in muscle size and strength in experienced weight-training men. Animal research also suggests an effect of CLA supplementation on limiting food allergy reactions, preventing atherosclerosis and improving glucose tolerance. As with the cancer research, the effects of CLA on these conditions in humans remain unclear. TFA Health Implication Phasing-out of industrially produced TFA in food products is currently a great concern in the Western world and especially in the US due to their negative health impact. Concerns have been raised for several decades that consumption of trans fatty acids might have contributed to the 20th century epidemic of coronary heart disease. Metabolic studies have shown that trans fats have adverse effects on blood lipid levels increasing the low density lipoprotein (LDL) a “bad” cholesterol while decreasing high density lipoprotein (HDL) a “good” cholesterol. This combined effect on the ratio of LDL to HDL cholesterol is double that of saturated fatty acids. Both of these conditions are associated with insulin resistance which is linked to diabetes, hypertension, and cardiovascular disease. Based on the available metabolic studies, it is estimated that approximately 30,000 premature coronary heart disease deaths annually could be attributable to consumption of trans fatty acids (Willett et al., 1994). Studies have also suggested that the cholesterol raising effect of hydrogenated fat is somewhat lower than that of saturated fats. However, only recently attention has been given to the fact that although trans fatty acids increase LDL cholesterol to a similar degree as saturated fat, they decrease HDL cholesterol relative to both cis unsaturated or saturated fats. In a metabolic study, Mensink and Katan (1990) demonstrated that replacement of 10% of energy from oleic acid (the primary monounsaturated fat in diets) with trans 18:1

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fatty acids caused a 0.34 mmol/L increase in LDL cholesterol and a 0.17 mmol/L decrease in HDL cholesterol; whereas replacement of oleic acid with saturated fat caused a similar increase in LDL cholesterol, but virtually no change in HDL cholesterol. As a result, the LDL/HDL cholesterol ratio was significantly higher on the trans than on the saturated or oleic diets. These findings were soon confirmed in several investigations. Overall, trans fatty acids increased LDL cholesterol similarly to saturated fat, but, unlike saturated fat, they also decreased HDL cholesterol. As a result, the net effect of trans fat on the LDL/ HDL cholesterol ratio is approximately double that of saturated fat. Moreover, these effects of trans fat on the LDL/HDL cholesterol ratio are remarkably constant across studies. These results confirm the deleterious effects of trans fat on blood lipids and indicate that these may alter the beneficial effects of polyunsaturated fat. Another plasma lipoprotein thought to produce cardiovascular disease is lipoprotein(a). In addition to increasing the LDL/HDL cholesterol ratio, trans fatty acids also increase lipoprotein(a) level when substituted for saturated fat. A significant increase was reported in several trials. High blood levels of lipoprotein(a) have been associated in some studies with increased risk of CHD, independently of LDL or HDL cholesterol concentrations. However diet-induced variations in blood concentrations of lipoprotein(a) are modest relative to the genetic differences, and their quantitative impact on risk of CHD remains to be established. Another effect of trans fatty acids on blood lipids is on fasting triglyceride levels. A triglyceride-raising effect was also consistently seen in other studies that directly compared trans fatty acids with cis-unsaturated fatty acids. The increases ranged from 0.005 to 0.12 mg/ml, with an average of 0.15 mg/ml per 1% of energy intake. The effect on triglyceride levels of substituting saturated fatty acids for cis-unsaturated fatty acids is about zero (Mensink et al., 1992). Thus, trans fatty acids increase triglyceride levels when compared with other fatty acids. Eliminating 2% of energy trans fatty acid from the diet would lower triglyceride levels by about 0.03 mg/ml, the relation between triglycerides and risk of CHD is still uncertain, but the resulting benefit is probably modest. Potential effects of trans fat on LDL oxidation and coagulation and fibrinolytic factors have also been investigated, but so far there is no conclusive evidence of adverse effects. Fish Oil Fish oil is derived from the tissues of fatty fishes. Virtually every type of fish contains oil. However, the quantity and composition of the

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oil varies with the type of fish, the season of the year, the geographical location and the diet of the fish. The oil naturally contains the
-3 fatty acids EPA (20:5n-3) and DHA (22:6n-3) including a wide range of other fatty acids. Fish oils are produced in several countries for human consumption and when traded, they usually are described either by generic name of the fish or the name of the country in which the oil has been extracted. The major fish types which are caught commercially and processed for oil include, Anchovy, Capelin, Herring, Mackerel, Menhaden, Sardine and even some Shark. Some of the health benefits of
-3 has already been discussed previously. Marine sources of oil, while not very common in the United States, are frequently used to yield edible oils. The uniqueness of fish oils has been recognized for some time, but the pure health significance began only in the mid 1970s when Eskimos consuming a very rich, high fat diet, rarely suffered death. The reason for this remained a mystery until, it was found that their marine-based diet was very rich in
-3 polyunsaturated fatty acids. These have antiarrhythmic, endothelial protective, antiatherogenic, antithrombotic and antiplatelet effects in many observational studies, which have pointed to their potential role in secondary prevention post myocardial infarction. Fish oil is recommended for a healthy diet, and it is beneficial to eat fish once a week (or more) but care must be taken to avoid the fish species which contain the toxin mercury or other contaminants such as Chlordane. Health Beneficial Effects Of Fish Oil The two predominant PUFA in fish oils are EPA and DHA which have proven to possess beneficial health effects. For example, in hyperlipidemic subjects, feeding fish oil has been shown to have beneficial effects, especially in reducing the risk of heart disease. Fish oil concentrate K-85, containing 92% of total fatty acids as n-3 fatty acids, has been shown to lower serum triglycerides and very low density lipoprotein (VLDL) in nondiabetic hypertriglyceridemic subjects (Mackness et al., 1994). In diabetic subjects, the most consistent beneficial effect of dietary fish oils is the lowering of plasma triglyceride levels. The cholesterol/phospholipids ratio and the cholesterol/HDL cholesterol ratio, which is a measure of the atherogenic index were also found lower when fish oil was consumed. In a comparative study between n- PUFA from fish oil (EPA + DHA) with that from linseed oil (linolenic acid) on plasma triglyceride with diabetic patients, fish oil decreased plasma triglyceride but linseed oil was without effect indicating that preformed long-chain n-3 fatty acids are more effective

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in lowering the lipid levels than linolenic acid (Goh et al., 1997). n-3 PUFA are also more readily incorporated in brain and other tissues as compared to those from vegetable oils. Therefore, infant milk formulas now are being supplemented with fish oil for brain development and function (Clandinin et al., 1992). However, dietary fish oils have several deleterious effects on carbohydrate metabolism in diabetic subjects by increasing fasting and postprandial glucose. Owing to the increase in glucogenesis from glycerol, long-term feeding of fish oil is anticipated to deteriorate glucose control. Insulin secretion is also impaired by fish oil feeding but plasma insulin levels are generally not altered. From human studies, it is clear that in a diabetic subject, n-3 PUFA appear to have beneficial effects on lipid metabolism and may decrease the severity of cardiac disorders and lower the incidence of coronary artery disease. However, these long chain fatty acids have detrimental effects on carbohydrate metabolism. Olive Oil Olive oil is extracted from fresh or ripe fruits of the long lived ever green olive tree Olea europaea, which originated in the Mediterranean area. There are approximately 900 species of olives and most are familiar with the olive that is cultivated for its fruit also known as drupes. The oil is regarded as a healthy dietary oil because of its high content of monounsaturated fat. It is produced principally in Greece, Italy, Spain, France,Turkey, Portugal, Tunisia, Morocco, and California. Among global producers, Spain leads with more than 40% of world production, followed by Italy and Greece. It has been postulated that the lower incidence of coronary heart disease (CHD) in these countries is due to their Mediterranean diet which includes a large amount of olive oil. Main consuming countries are also the main olive oil producers. European Union accounts for 71% of world consumption. Mediterranean basin countries represent 77% of world consumption. Other consuming countries are United States, Canada, Australia and Japan. Chemical Composition The chemical composition of the olive depends upon several agronomic factors, including the variety, place, age of growth and harvesting season. In general the content of the olive is 47% water, 31% solids and 22% oil. Figure 5.13 represents the selected nutrient composition of one large olive (4.4 g). Olive oil contains a high percentage of the monounsaturated oleic acid (C18:1) as a major lipid constituent including some minor constituent as hydrocarbons, monoglyceride

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esters, tocopherols, flavenoids, anthocyanins, sterols and a number of simple and complex phenolic compounds such as oleouropein, hydroxytyrosol (3,4-dihydroxyphenyl) and tyrosol in higher concentration (Figure 5.13). FIGURE 5.13 — The Composition Of A Single Large Olive* Nutrient

Amount (g)

Water Energy Protein Total Lipid Carbohydrate Total dietary fiber Ash

3.52 5.05 kcal 0.037 0.47 0.28 0.14 0.10

Palmitic (C16:0) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2 n-6) Linolenic (C18:3 n-3)

0.05 0.01 0.34 0.04 0.003

Lipid

* Adopted from USDA nutrient database (1998)

FIGURE 5.14 — Chemical Structure Of Few Main Phenolic Constituent Of Olive Oil

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The most abundant phenolic compound in the drupe is oleuropein (glycoside) that contributes primarily a bitter test. On maturation oleuropein undergoes enzymatic and nonenzymatic hydrolysis and yields several simpler compounds (e.g, hydroxytyrosol, ligstroside) that builds up the full fruity taste. Hydroxytyrosol and tyrosol are structurally similar except that hydroxytyrosol possesses an extra hydroxyl group in the meta position. Oleuropein is an ester which consists of hydroxytyrosol and elenolic acid. The phenolic compounds present in olive oil are strong antioxidants and free radical scavengers. The greater the phenol content in olive oil, the better the oxidative stability. Hydroxytyrosol is a superior antioxidant and free radical scavenger to oleuropein and tyrosol. The level of these compounds varies between 50 to 800 mg/kg olive oil (Tuck et al., 2002). Olive oil also contains several tocopherols (example  -, b -,  -,). tocopherol being almost 88%. The taste of olive oil is attributed to a group of aroma compounds and trans-2-hexenal being the predominant component. It is unclear if the beneficial properties of olive oil are from its constituents or their metabolites. Olive Oil Extraction Traditionally, olive oil is produced by crushing the fruits in stone or wooden mortars or beam presses. Nowadays, olives are ground to tiny bits, obtaining a paste that is mixed with water and processed by centrifuge, which extracts the oil must from the paste, leaving behind the pomace (the residue). The oily must is further centrifuged to obtain the pure oil after filtration (Figure 5.15). Another process is by a coldpressing technique without using solvents, which does not alter the chemical nature of the oil. Olive oil comes in different varieties, depending on the amount of processing involved. The most common industrial processing method is a continuous extraction system with two centrifugations (first horizontal and then vertical). Vertical centrifugation may be in three phases obtaining oil, pomace and vegetable waters, or in two phases (in this case there is no water injection or little water) obtaining oil and a paste. The main disadvantages of this process are the huge amounts of water needed and, therefore, the production of vegetable fluids with the resulting pollution. However, there are new industrial techniques of continuous extraction. They reduce the production of vegetable waters by obtaining a much more humid pomace that can be moisturized and used later. This process does not need much water. It is being more and more widely used.

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FIGURE 5.15 — Extraction Of Olive Oil

TYPES OF OLIVE OIL Extra-Virgin Olive Oil Extra-virgin Olive oil is produced by the cold-pressing technique at room temperature with a maximum of 1% free fatty acids (must not be greater than 1 g per 100 g). It has a noticeable green color due to the presence of chlorophyll and pheophytin. The oil is considered best because of less processing and for possessing very high standards of aroma and flavor. There is no refined oil in extra-virgin olive oil.

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Virgin Olive Oil Virgin Olive oil is produced by second pressing in the same manner as extra-virgin oil. This oil has a slightly higher free fatty acid (2%) and possesses high standards of aroma and flavor. There is no refined oil in virgin olive oil. Standard Virgin Olive Oil It is also called commercial grade olive oil. It is mixed with refined oil to improve its taste. The oil undergoes some processing, such as filtering and refining. The acidity of this oil is around 3.3g per 100g of oil. Use And Storage Of Olive Oil When choosing an olive oil, it is imperative that one buys “extra virgin” olive oil, rather than “virgin” or the commercial grade so called pure olive oil. Extra virgin is the oil from the first pressing, it uses top grade olives, with less than one percent acidity, and has the highest nutritional value and the best taste. It is the only oil with which one can get the true benefits of olive oil. Olive oil may be used in dressings, marinades, and as cooking oil. It turns into a solid if refrigerated, so it is best stored as a liquid at room temperature. Using a dark bottle and corking it tightly can reduce the amount of oxidation. Health And Beneficial Effects Of Olive Oil The biological and therapeutic values of olive oil are due to its chemical structure. The triglyceride composition of olive oil is made up of 54 - 83% of monounsaturated fat (oleic acid). Monounsaturated fatty acids are much more stable than polyunsaturated ones in terms of the oxidation process that prevents rancidity. Secondly, olive oil’s beneficial properties lie in its minor components. The most common ones are the tocopherols, among them -tocopherol which acts as vitamin E and carotene as provitamin A, and the polyphenols. All of these components have a major antioxidant function and are closely connected with virgin olive oils because refining processes alter and partially remove them in the other types. Olive oil is very well tolerated by the stomach due to its high oleic acid content. Since ancient times olive oil has been described as having a beneficial effect on hyperchlorohydric gastritis and gastroduodenal ulcers, which is attributed to its protective function. Olive oil activates the secretion of bile and pancreatic enzymes much more naturally than prescribed drugs. Consequently, it lowers the incidence of gallstone formation. A roster of scientific studies have demonstrated that a balanced diet containing olive oil provides

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significant health benefits, including delaying the aging of cells by halting the excessive production of cells with oxidant substances, in reducing cholesterol levels, in the prevention of bone decalcification, and in the assimilation of calcium, iron, phosphorus, magnesium, zinc, so important in the growing process. It is well documented that monounsaturated fatty acid may lower blood cholesterol levels and may increase HDL cholesterol levels, therefore, linking olive oil consumption and the lower incidence of CHD and even cancer, particularly breast cancer, in cultures where a “Mediterranean diet” is consumed. “Mediterranean diet” is characterized by high content of oleic acid, fruits, vegetables, grains, legumes and low meat. Olive oil is clearly one of the good oils. Most people do quite well with it since it does not upset the critical n-6 to n-3 ratio. Phytosterols Phytosterols, also known as a plant sterol are widely distributed in the plant kingdom. These plant lipid-like components are chemically similar to the dietary and endogenously secreted cholesterol and exist in all foods of plant origin, as monomers, glycosides, esters, or glucosylated esters. The major common plant sterols are β-sitosterol, stigmasterol and compesterol which constitute the majority in normal foods, whereas, avenasterol and brassicasterol are minor components. They differ from cholesterol only in the identity of one side chain or the presence of the extra double bond (Figure 5.16). The different chemical forms of phytosterols exist in different compartments of the plant cell. For example, free phytosterols are mainly found in the plant membrane wall to give structural properties while phytosterol glucosides and esters mainly are found in the cytosol and endoplasmic reticulum. Vegetable oils are the major source of free phytosterols. Most crude vegetable oils contain 1-5 g kg -1 of total phytosterols. The major phytosterol components of some of the commonly consumed vegetable oils (crude and refined) are listed in Figure 5.17. Sitosterol is the major sterol in vegetable oil especially in refined/ crude rapeseed oil while sistostenol, the saturated derivative of sitosterol, occurs at negligible levels in plant lipids. Refining of oils lowers the phytosterol levels (Figure 5.17). Phytosterols are partly removed with other components of crude oils in vegetable oil refining. They may also react by atmospheric oxygen and undergo isomerization and other intermolecular transformation reactions. Campesterol and stigmasterol are more labile than sitosterol.

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FIGURE 5.16 — Structure Of Some Common Phytosterols Including Cholesterol

FIGURE 5.17. Major Phytosterols In Crude And Refined Vegetable Oils (g kg-1)* Oils

Campesterol

Sitosterol

Stigmasterol

Corn, crude Corn, refined Olive, extra virgin Olive, cold pressed Palm, crude Palm, refined Peanut, refined Rapeseed, crude Rapeseed, refined Soybean, crude Soybean, refined Sunflower, refined

1.69-2.01 1.23-1.64 0.045-0.050 0.02-0.05 0.14-0.20 0.02-0.05 0.24-3.8 2.93 1.64-3.00 0.57-0.71 0.34-0.82 0.27-0.55

5.41-6.46 4.54-5.43 1.18-1.33 1.22-1.30 0.43-0.52 0.35-0.41 1.15-1.69 4.20 3.58-3.95 1.73-1.84 1.24-1.73 1.94-2.57

0.58-0.68 0.46-0.59 0.009-0.013 nd1-0.03 0.07-0.10 0.07-0.10 0.12-0.22 nd nd-0.16 0.58-0.61 0.37-0.64 0.18-0.32

* Adapted from Piironen at al (2004)

nd = not detected

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In margarine the phytosterol content depend on their fat content, the oil and the fats used. The total phytosterol content of margarines with 80% fat ranged between 1.36 and 5.86 g kg-1 (Weihrauch and Gardner, 1978). According to the food composition database, phytosterols ranged for hard regular margarines from 1.36 to 5.71 g kg-1 and for soft margarines from 1.44 to 4.83 g kg-1 in the U.S (USDA, 2000). Cereals are generally considered as a good source of phytosterols depending on the dietary pattern and the way in which they are consumed. The total phytosterol contents of various cereals range mainly from about 350 to 1200 mg kg-1 fresh weight. The total sterol contents for rye, barley, wheat and oats were reported 71.2, 35.6, 42.0 and 12.1 mg g -1 , respectively (Dutta et al, 1996). Sitosterol is the major phytosterol of cereals accounting for 49% to 64% in wheat, rye, barley and oats (Piironen et al., 2002). Phytosterols in cereals are found as free sterols (FSs), esters with fatty acids (SEs), and phenolic acids (SPHEs), glycosides (SGs) and acylated glycosides (ASGs) and varies between different cereals and in various parts of the kernel. In bakery products the phytosterol contents vary between 410 to 824 mg kg-1 and is highest in bread baked mainly with whole-meal flour. In vegetables the phytosterol contents vary from low (38-51 mg kg-1), to moderate (160 mg kg-1). Broccoli, Brussels sprouts, cauliflower and dill are the best source of phytosterols. Their total phytosterol contents were more than 300 mg kg-1 fresh weight (Normén et al, 1999; Piironen et al, 2003). The content was less than 100 mg kg-1 in potted lettuce, onion, potato and tomato; within the range of 100-200 mg kg-1 in the carrot, Chinese cabbage, leek, red beet and white cabbage; and in the range of 200-300 mg kg-1 in the pea, sweet pepper and parsley (Piironen et al., 2003). Sitosterol is generally the main phytosterol in vegetables and contributed 43-86% to the total phytosterols of about 20 analyzed vegetables (Piironen et al, 2003). The phytosterol contents of some fruits ranged from 13 (watermelon) to 440 mg kg-1 (passion fruit), with a median content of 160 mg kg-1 for 14 analyzed fruits (Normén et al, 1999). Among the analyzed fruits, avocado contained significantly more sterols (752 mg kg-1). The phytosterol content in fresh berries ranged from 60 (red currant) to 279 mg kg-1 (lingonberry). Sitosterol is the main sterol both in fruits and berries. Its proportion ranged in fruits between 72% and 86% and in berries between 61% and 93% (Piironen et al., 2003). Campesterol and stigmasterol were the two other major sterols. Various peanuts and almonds are also rich in phytosterols. The total phytosterols contents in raw peanuts with skin ranged between 600 and 1608 mg kg-1 fresh weight and those of shelled peanuts ranged

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between 551and 1269 mg kg-1. Sitosterol contributed about 80% of sterols. Both heredity and growing conditions affect phytosterol contents and composition. Furthermore, the planting location and temperature affects the phytosterol accumulation in vegetable oils and fruits. For example, the total phytosterol levels of canola were markedly affected by genetic modification (Abidi et al., 1999). Similarly, the total sterols ranged from 1.76 to 3.48 g kg-1 and sitosterol from 0.93 to 1.71 g kg-1 of oil in genetically modified soybeans differing in their fatty acid compositions. Dietary intake of phytosterols ranges from 250 to 500 mg/d with about 65% of intake as β-sitosterol, 30% as compasterol, and 5% as stigmasterol and low amount of other sterols. Health Implication Of Phytosterol Phytosterols are non-nutritive components of foods having several health benefits. There exists a longstanding interest in the hypocholesterolemic effects of dietary phytosterols. Because of its close resemblance to cholesterol, they actually block food-based cholesterol from being absorbed into the bloodstream and inhibit the reabsorption of cholesterol from bile acids in the digestive process. In contrast to cholesterol, phytosterols are poorly absorbed (5-10% of cholesterol) and therefore the intestines are occupied by phytosterols for extended periods of time which makes the blocking process even more effective. The increased levels of cholesterol in chickens caused by cholesterol feeding was prevented after including 1% soybean sterols in the diet (Peterson, 1951). Since then, numerous studies have confirmed a hypocholesterolemic action of plant sterols, especially sitosterol. Sitostanol, prepared by hydrogenation of sitosterol, reduces the intestinal absorption of cholesterol and lowers serum cholesterol more effectively than sitosterol (Heinmann et al, 1986). Also, sitostanol reduced 33% LDL cholesterol in children having severe familial hypercholesterolemia in 3-months time (Becker et al., 1993). Currently, a great renewal has occurred in the use of sitosterols for the inhibition of cholesterol absorption. For example, plant sterols are first saturated to stanols and then further esterify stanols to a more fat-soluble form. Miettinen and Gylling (1999) have developed a sitostenol ester margarine that includes sitostanol in soluble ester form. Serum campesterol, a dietary plant sterol whose levels reflect cholesterol absorption, was decreased by 36% with the diet containing sitosterol ester margarine. Some phytosterols stimulate insulin secretion and may contribute to better sugar control. A combination of pravastatin and

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sitostenol ester (sitostenol ester margarine) was evaluated for control of mild hypercholesterolemia in men with non-insulin-dependent diabetes mellitus (Gylling et al., 1996). Serum total and LDL cholesterol were lowered 35% and 44% respectively, compared to levels observed with a control dietary margarine. In a randomized double-blind placebocontrolled study with margarine fortified with sterol esters from soybean or rice bran oil. Plasma total and LDL cholesterol were reduced by 8% to 13% for margarines enriched in soybean oil sterol esters or sitosterol esters compared to control margarine (Westrate and Meijer, 1998). It was concluded that a margarine with sterol esters from soybean oil, has mainly esters from sitosterol. Campesterol, and stimasterol, was as effective as margarine with sitostenol esters in lowering blood total and LDL cholesterol levels. Phytosterols do not appear to lower triglycerides or to raise the levels of HDL, the good cholesterol. Besides cholesterol lowering properties, phytosterols may also have effects on cancer development which is the largest killer of men and women in Western societies. There have been several studies on cancer prevention with phytosterol supplements in animals, however, the bioavailability of phytosterols is fulfilled only by unsaturated sterols and therefore they may have direct or indirect effects on endogenous cancer prevention. Furthermore, regulatory proteins which control cell proliferation and growth are the major site for cancer development, these sites also constitute the molecular targets for phytochemicals where these dietary constituents may work alone or in combination to prevent adverse effects (Gescher et al, 2001). The preventive effect of phytosterols on colon cancer was first observed by Nair et al. (1984) when Seventh Day Adventists experienced lower rates of colon cancer and had at the same time higher dietary intakes of phytosterols than the general population. As bile acids are well-known tumor promoters the lower incidence observed in Seventh Day Adventists was hypothesized to be due to the decreased bile acid excretion. Effects of phytosterols on breast cancer are very limited. In one study (Awad et al. 2000), female SCID mice were inoculated in the right inguinal mammary fat pad with cultured breast cancer cells (MDA-MB-231 type). The animals were randomized into two groups being fed a control diet + 0.2% cholic acid + 2% cholesterol, and a control diet + 0.2% cholic acid + 2% phytosterols. After 8 weeks of tumor growth, the tumor area and weight was 33% smaller in the phytosterol group compared to the control group. In a similar model and to test the effect of phytosterols on prostate cancer development, SCID mice having cultured prostate cancer cells (PC-3 type) were fed phytosterols. The phytosterol-treated animals had

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28% smaller tumors and also had only about one-half the rate of metastasis compared to the control diet (Awad et al., 2001). Overall phytosterols have an important function in the human body which has led to the introduction and commercialization of phytosterol-enriched food products in most of the developed countries. SUMMARY This chapter has summarized valuable information on omega fatty acids including CLA, TFA, and their presence in various food items. Omega-3 fatty acid, and CLA possess healthy appeals, whereas, TFA are considered harmful for humans. Omega-3 fatty acids are required for proper growth, and development. However, intake of these fatty acids are not a solution for the chronic diseases, but a healthy life style, regular exercise, and a choice of appropriate food may lead to better nutrition and good health. Although omega-6 fatty acids are essential, it is also essential that the ratio of omega-6 to omega-3 fatty acids be balanced for proper metabolism. The bioactive molecules such as phytosterols from plants, Omega-3 PUFA from fish oil and the phenolic components of olive oil have many important roles in promoting health and preventing diseases. Although, these components have not yet been considered essentials they already have proven to be beneficial to the health. Their major benefit to consumers is indirect. Inclusion of olive oil, fish and phytosterols in the daily diet may be prudent. It is apparent that beta sitosterol is a useful dietary supplement for the lowering of plasma cholesterol. Nevertheless, beta sitosterol should be used with caution in certain individuals who have a higher absorption rate of beta sitosterol. Since interest in these bioactive molecules as a functional food ingredient with health promoting properties has been more intense in recent years, further research needs to be completed to more fully elucidate the health implications. More studies with healthy populations and populations with acute and chronic disease states are also needed.

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Thom, E., Wadstein, J., and Gudmundsen, O. 2001. Conjugated linoleic acid reduces body fat in healthy exercising humans. J. Int. Med. Res. 29:392–396. Thompson, H., Zhu, Z., and Banni, S., Darcy, K., Loftus, T., and Ip, C. 1997. Morphological and biochemical status of the mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk. Cancer Res. 57:5067–5072. Tuck, K.L., and Hayball, P.J. 2002. Major phenolic compounds in olive oil: metabolism and health effects. J. Nutritional Biochem. 13:636-644. U.S. Depatment of Agriculture. USDA Nutrient Data Base Release 13. Washington, DC, 2000. USDA nutrient database for standard reference, release 12, U.S. Department of Agriculture, Agriculture research service, nutrient data laboratory home page, 1998, available at: http://www.nal.usda.gov/fnic/foodcopm van den Reek, M.M., Craig-Schmidt, M.C, Clark, A.J. 1986. Use of published analyses of food items to determine dietary trans octadecenoic acid. J. Am. Diet Assoc. 86:13911394. Voorrips, L.E., Brants, H.A.M., Kardinaal, A.F.M., Hiddink, G., and Brant, P.A. van den. 2002. Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer:the Netherlands cohort study on diet and cancer. Am. J. Clin. Nutr. 76:873-882. Watkins, B.A., and Li, Y. 2000. Conjugated linoleic acid: The present state of knowledge In : Hand book of Nutraceuticals and Functional Foods. Wilman, R.E.C (ed), pp 445-476, CRC Press, Boca Raton, Florida. Weihrauch, J.L., and Gardner, J.M. 1978. Sterol content of foods of plant origin. J. Am. Dietet. Assoc. 73:39-47. Whale, K.W.J., Caruso, D., Ochoa, J.H.J., and Quiles, J.L. 2004. Olive oil and modulation of cell signaling in disease prevention. Lipids, 39:1223-1231. Willett, W.C., and Ascherio, A. 1994. Trans fatty acids: Are the effects only marginal? Am. J. Public Health, 84:722-724. (Guo, M. R., Alam, M.)

Chapter 6 VITAMINS AND MINERALS AS FUNCTIONAL INGREDIENTS Vitamins and minerals are widely used in functional foods and supplements. The aim of this chapter is to provide the chemistry, dietary sources and the nutrient functional claims of some of the important vitamins and minerals that are considered essential for maintaining good health and in treating diseases of deficiency. Vitamins are complex organic compounds that are required for normal metabolism, growth and development, and regulation of cell function. All naturally occurring vitamins are organic food substances that are found only in living things (plants and animals). With a few notable exceptions, the human body cannot manufacture or synthesize vitamins on its own and, therefore, must be supplied in the diet or in dietary supplements. Vitamins and minerals that our bodies require in small quantities commonly function as essential coenzymes and cofactors for metabolic reactions and thus help support basic cellular reactions. Currently, these nutrients are added to a wide variety of food and supplements, such as cereals, flours, bread, milk, margarine, infant formulas, soy milk, orange juice, salt and formulated beverages. Most fortifying compounds are vitamins and minerals and in some cases essential amino acids, proteins and others. The use of vitamins, minerals and other complementary nutrition-based therapies has increased dramatically in the United States and in other Western countries. Dietary manipulation and food fortification is practiced to beneficially affect immunity in patients and can also potentially serve to decrease the risk of many chronic diseases in the general population.

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VITAMINS Vitamins are divided into two categories: fat-soluble vitamins (e.g., A, D, E and K) and water soluble vitamins (e.g., C, B1, B2, B6, B12, folic acid, pantothenic acid and biotin). They are also classified on the basis of toxicity but this classification is rather complex and is based on the level of toxicity of different vitamins. Toxic vitamins have a therapeutic index of 20 or less and include vitamin A (retinal, and retinoic acid) and vitamin D. Therapeutic index is the difference between the minimum effective dose and maximum tolerated dose of a vitamin. The larger the value is, the safer the vitamin. Non-toxic vitamins have a therapeutic index of 20 or more and include all vitamins other than A and D. Water soluble vitamins are not stored for very long in the body and, therefore, foods which supply these vitamins should be consumed in adequate amounts, whereas, fat-soluble vitamins are digested and absorbed with the help of fats occurring naturally in the diet. Fat soluble vitamins can be stored in the body for long periods of time. They are stored mostly in the fatty tissues and in the liver. Hence, supplementations are not required as frequently as with water-soluble vitamins. FAT SOLUBLE VITAMINS Vitamin A Chemistry: Vitamin A comes in two forms. The first is retinol, which is already pre-formed in animal foods. The other is pro-vitamin A, which is found in plant foods in the forms of compounds called carotenoids. Retinol refers to isoprenoid compounds that posses the biological activity of all-trans retinol. The retinol structure contains a substituted β-ionone ring with a side chain of three isoprenoid units linked at the 6-position of the β-inone ring (Figure 6.1). The human body makes trans-retinol from β-carotene available in carrots. Trans-retinol is converted to 11cis-retinal, which binds to the opsin protein to form rhodopsin. When light hits rhodopsin molecules in the retina of the eye, 11-cis-retinal breaks away from opsin and transforms to all-trans-retinal, causing the eye to send signals to the brain. The are many factors that influence the stability of vitamin A such as heat, oxygen, and pH (Figure 6.2). Analytical problems are mainly encountered during sample analysis which includes instability when isolated from the biological matrix, susceptibility to isomerization yielding the lower biologically active cisisomers. Light, acids, metals and heat processing can produce rapid isomerization of vitamin A.

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FIGURE 6.1 — Chemical Structure Of Trans-Retinol (Vitamin A)

FIGURE 6.2 — Stability Of Some Important Fat And Water Soluble Vitamins.a Effect of pH Neutral Acidic Alkaline pH 7 < pH 7 > pH 7 Vitamin A Vitamin D Vitamin K Vitamin E Vitamin C Vitamin B1 Vitamin B2 Vitamin B6 Vitamin B12 Folic Acid Biotin a

S S S S U U S S S U S

U U S S S S S S U S

S U U S U U U S S S S

Air/ Oxygen

Light

Heat

Maximum Cooking Losses (%)

U U S U U U S S U U S

U U U U U S U U U U S

U U S U U U U U S U U

40 40 5 55 100 80 75 40 10 100 60

S = stable; U = Unstable

Functions and deficiencies: Vitamin A plays an essential role in vision, growth and development, immune functions and reproduction. Vitamin A deficiency results in various disorders. The most common one is the dryness of the conjunctiva and later of the cornea (xerophthalmia) also the epithelial tissues such as skin and the mucous membranes lining the internal body surfaces. Vitamin A deficiency leads to night blindness and continued deficiency eventually results in loss of sight. Deficiency of vitamin A may also result in defective bone and teeth formation. Vitamin A deficiency is a common problem worldwide, particularly in developing countries due to famine or shortages of vitamin A-rich foods. In the United States it is found among the urban poor, the elderly, alcoholics, and patients with malabsorption.

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Recommended Dietary Allowances (RDA): The RDA is the measured amount of an essential nutrient that is needed on a daily average intake in the diet to meet the needs of almost all healthy people. The United States federal government (FDA) sets these levels. However, Japan has been leading the rest of the world in the research and development of functional foods and their functional claims. The RDA’s of the United States and Japan are different and so are their nutrient functional claims especially of the vitamins and minerals. Japan has more health claims for vitamins and minerals than the United States (discussed later). Following the lead of the United Sates FDA registration statement for dietary supplements, many European countries have established their own standards. The RDA for vitamin A in the United States is 1,000 retinol equivalents (RE) for boys and men and 800 RE for girls over 12 and adult women. No increase in intake is recommended during pregnancy; however, the RDA is increased by 500 RE during the first six months of lactation. Vitamin A is also measured in international units (IU). IU are defined by the relationship of 1 IU = 0.3 µg of all-trans-retinol or 0.6 µg of β-carotene. Preformed vitamin A (vitamin A acetate and palmitate) has well recognized toxicity when consumed at levels of 25,000 IU/d or higher. Food sources: Dietary sources of vitamin A include organ meats such as liver. Fish oils, butter, eggs, whole milk, fortified low fat milk, margarine and other dairy products are good sources of vitamin A. Pumpkin, sweet potatoes, spinach, butter squash, dandelion greens and cantaloupe, mangoes and turnip greens are also good sources. Provitamin A is found throughout the plant kingdom such as carrots and broccoli which supply carotenoids that can be converted into vitamin A by the body. Chemical forms as functional ingredients: The primary commercial forms of vitamin A are acetate (C22H32O2) and palmitate (C36H60O2) esters used by pharmaceutical and food industries. These ester forms of vitamin A, greatly stabilize the food products in relation to oxidation. In developed countries vitamin A fortification includes milk, dairy products, margarine, fat spreads and breakfast cereals. Nutrient functional claims: There are no approved health claims of vitamin A in the United States; however, Japan has the following: (1) Vitamin A helps maintain vision at night (2) helps to maintain healthy skin and mucosal membranes. Vitamin D Chemistry: Vitamin D was discovered in the 1930s following the discovery of rickets, a well known disease resulting from the deficiency

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of this vitamin. Vitamin D exists in several forms. However, only ergocalciferol (vitamin D 2) and cholecalciferol (vitamin D 3 ) are biologically active (Figure 6.3). The biologically active form of vitamin D is a steroid hormone and is prepared from respective 5,7-diene sterols. The A, B, C, and D rings of the vitamin are derived from the cyclopentanoperhydrophenanthrene ring structure with cholesterol serving as the parent compound. Furthermore, vitamin D is classified as a seco-steroid. Seco-steroids are those in which one of the rings has been broken, in vitamin D, the 9,10 carbon-carbon bond of ring B is broken, and it is indicated by the inclusion of “9,10-seco” in the nomenclature. Thus the IUPAC-IUB name for vitamin D2 is 9,10seco(5Z,7E)-5,7,10(19), 22-ergostatetraene-3β-ol and for vitamin D3, it is 9,10-seco(5Z,7E)-5,7,10(19)cholestatriene-3β-ol. Vitamins D4, D5, and D6 have also been prepared chemically, but they have a much lower biological activity. FIGURE 6.3 — Chemical Structure Of Vitamin D2 and D3

All vitamin D compounds are closely allied to that of the classical steroid hormones (e.g. cortisol, estradiol, progesterone etc.) and possess a common triene structure showing a characteristic broad UV spectrum with maximum absorption at 265 nm and a minimum of 228 nm. The triene system of vitamin D makes it labile to light-induced isomerization. In addition, it is easily protonated resulting into isotachy-sterol, which is devoid of biological activity. Vitamin D3 can be produced photo

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chemically by the action of sunlight or ultraviolet light from the precursor sterol 7-dehydrocholesterol which is present in the epidermis or skin of most higher animals and humans. The chief structural prerequisite of pro-vitamin D compounds which are abundant in plant and animal tissues is to contain a delta 5,7-diene conjugated system. The conjugated double bond system in this specific location of the molecule allows the absorption of light at certain wavelengths in the UV range initiating a complex series of transformations that ultimately results in the formation of vitamin D3. Vitamin D3 concentration in animal tissue is dependant on dietary intake and exposure of the animal to sunlight. Humans receive most of their vitamin D requirement through sunlight exposure. Vitamin D is stable in the absence of light, water, acidity and low temperature. The vitamin stands alkalinity and saponification and is less susceptible to oxidative loses. Vitamin D3 is more stable than vitamin D2. Functions and deficiencies: Vitamin D is the principal regulator of calcium homeostasis in the body. It is particularly important in skeletal development and bone mineralization. The active form of vitamin D is 1 alpha, 25-dihydroxyvitamin D or 1,25(OH2)D. The vitamin D hormone 1, 25 (OH2)D mediates its actions via binding to vitamin D receptors (VDRs) which are principally located in the nuclei of target cells. 1,25(OH2)D enhances the efficiency of calcium absorption and to a lesser extent phosphorus absorption, from the small intestine. Vitamin D supplementation usually repairs conditions caused by poor dietary intake. Vitamin D helps ensure that the body absorbs and retains calcium and phosphorus, both critical for building bone. Laboratory studies also show that vitamin D helps control cancer cells from growing and dividing (Holick et al., 2004). Prolonged deficiency of vitamin D results in changes in the bones of children and adults and possible hearing loss with aging. In addition, the lack of vitamin D promotes rickets (in children) and osteomalacia (in adults) where bones are malformed and weak from poor calcium and phosphorus deposition. Osteoporosis is due to a poor dietary vitamin D and calcium intake. Hearing loss from vitamin D deficiency may progress as the adult ages due to increased porosity of the cochlea bone in the inner ear. RDA: RDA ranges from 5 µg (200 IU)/d for adults, 10 µg (400 IU)/d for children, pregnant and lactating women may prevent osteomalacia in the absence of sunlight. However, more is needed to help prevent osteoporosis and secondary hyperparathyroidism. Total-body sun exposure easily provides the equivalent of 250 µg (1000 IU) of vitamin D/d suggesting a physiological limit (Vieth, 1996).

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Food sources: Food sources of vitamin D are very limited in nature but easily and cheaply synthesized. However, vitamin D is found naturally in animals and animal products in small amounts. Fruit and nuts contain no vitamin D at all. The richest sources include: fortified foods and beverages like milk, soy drinks and margarine. Fish liver oil, and egg yolks naturally contain vitamin D. Chemical forms of vitamin D as functional ingredients: Vitamin D3 (C27H44O) also known as cholecalciferol is the primary synthetic form of vitamin D used for food fortification and in pharmaceuticals. Fortified foods are the major dietary sources of vitamin D. In the United States milk is fortified with 10 micrograms (400 IU) of vitamin D per quart. Nutrient functional claims: There are no approved health claims of vitamin D in the United States, however, Japan has the following health claims: (1) Vitamin D promotes absorption of calcium in the intestine (2) It helps in development of bone. Vitamin E Chemistry: Vitamin E was discovered by Evan and Bishop in 1922, as food factor “X” which is necessary for the reproductive system and prevention of fetal death (Friedrich et al., 1988). In 1924 this new substance was named vitamin E and then tocopherol from the Greek term tocos which means “to birth” and “phero” which means “providing power.” Vitamin E exits in eight different forms and each form has its own biological activity. The parent compound is 2-methyl-2 (4',8',12'trimethyltridecyl)-chroman-6-ol. The homologues of vitamin E existing in nature are
-, b-, - and -tocopherol and
-, b-, - and -tocotrienol characterized by a saturated side chain consisting of three isoprenoid units (Figure 6.5, Figure 6.4). The difference in chemical structure between
-, b-, - and -comes from differences in the position of methyl groups located on the the chroman nucleus. Furthermore, the difference between tocotrienols and tocopherols originates from whether double bonds exist in the side chain. The four tocopherol homologues have 16carbon phytol side chain, whereas, tocotrienols have three double bonds on the side chain. The asymmetric carbon (chiral center) at 2' of the chroman ring and 4',8' carbons of the side chain are the main cause for its various isomeric forms. The chemical structure differs distinctively between the natural and synthetic forms. Synthetic vitamin E consists of a mixture of d- and l-form which are optical isomers of each other. Natural vitamin E consists of only the d-form (RRR-) and, therefore, it is easy to distinguish natural vitamin E from the synthetic one. The dl-

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-tocopherol, which is the synthetic form of
-tocopherol contains equal amounts of eight stereoisomers. The tocotrienols have lower bioactivity and thus lower nutritional value than the tocopherols. The most widespread and most active tocopherol is the
form and is called RRR
-tocopherol. Vitamin E is sensitive to heat, light, oxygen, alkali pH, and various metals (iron and copper). Under an oxygen free environment, tocoferols and tocotrienols are stable in heat and alkali conditions. Refining of edible oil results in some losses of vitamin E activity. However, refining removes pro-oxidant from the oil and makes the oil more stable towards oxidation. FIGURE 6.4 — Types Of Tocopherols And Tocotrienols. Trivial Name

Chemical Name

Abbreviation

Substitution R1

Tocol
-Tocopherol β-Tocopherol

5,7,8-Trimethyltocol 5,8-Dimethyltocol


- T β- T

-Tocopherol -Tocopherol

7,8-Dimethyltocol 8-Methyltocol

-T -T

Tocotrienol
-Tocotrienol β-Tocotrienol

5,7,8-Trimethyltocotrienol 5,8-Dimethyltocotrienol


- T3 β- T3

-Tocotrienol -Tocotrienol

7,8-Dimethyltocotrienol

-T3 -T3

8-Methyltocotrienol

R2

H H CH3 CH3 CH3 H

R3 H CH3 CH3

H

CH3

CH3

H

H

CH3

H H CH3 CH3 CH3 H

H CH3 CH3

H

CH3

CH3

H

H

CH3

FIGURE 6.5 — Chemical Structure Of Tocopherols and Tocotrienols

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Functions and deficiencies: Vitamin E is a well-known antioxidative agent which stops major fat soluble-chain reactions in the blood and tissue including protecting the unsaturated fatty acids, protein and DNA from oxidation, and it stabilizes the structure of the biomembrane by eliminating free radicals. Vitamin E also plays an important role in cell signal transduction. It may help prevent or delay coronary heart disease (Lonn and Yusuf, 1997). Evidence indicates that oxidative changes to LDL promote blockages in coronary arteries that may lead to heart attacks. Vitamin E may help prevent or delay coronary heart disease by limiting the oxidation of LDL-cholesterol (Jialal and Fuller, 1995). Vitamin E also may help prevent the formation of blood clots, which could lead to a heart attack. Observational studies have associated lower rates of heart disease with higher vitamin E intake. Vitamin E is believed to help protect cell membranes against the damaging effects of free radicals, which may contribute to the development of chronic diseases such as cancer. Cataracts are abnormal growths in the lens of the eye causing cloudy vision. They also increase the risk of disability and blindness in aging adults. Observational studies have also found that lens clarity, which is used to diagnose cataracts was better in regular users of vitamin E supplements and in persons with higher blood levels of vitamin E (Leske et al., 1998). Vitamin E deficiency is a very rare problem that results in damage to nerves and is almost always due to factors other than lack of dietary intake. Malabsorption results from pancreatic and liver abnormalities that lower fat absorption, abnormalities of the intestinal cell and length of the intestine. Vitamin E deficiency may cause cystic fibrosis (affects the lungs, digestive system, sweat glands and male fertility), pancreatitis (inflammation of the pancreas) and cholestasis (bile-flow obstruction). Premature infants may be at risk for vitamin E deficiency because they are born with low tissue levels of the vitamin, and they have a poorly developed capacity for absorbing dietary fats. Vitamin E deficiency in humans results in ataxia (poor muscle coordination with shaky movements), decreased sensation to vibration, and lack of reflexes and paralysis of eye muscles. RDA: The RDA for vitamin E was previously 8 mg/d for women and 10 mg/d for men. The RDA was revised by the Food and Nutrition Board of the Institute of Medicine in 2000, (Figure 6.6). This new recommendation was based largely on the results of studies done in the 1950s in men fed vitamin E deficient diets. The latest RDA for vitamin E continues to be based on the prevention of deficiency symptoms rather than on health promotion and the prevention of chronic disease.

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FIGURE 6.6 — RDA For RRR-
tocopherol. Life Stage Infants Infants Children Children Children Adolescents Adults Pregnancy Breastfeeding

Age

Males (mg/day)

Females (mg/day)

0-6 months 7-12 months 1-3 years 4-8 years 9-13 years 14-18 years 19 years and older All ages All ages

4 5 6 7 11 15 15 -

4 5 6 7 11 15 15 15 19

Food sources: Vitamin E is a plant product and widely distributed in vegetable oils, nuts, green leafy vegetables and fortified cereals which are common food sources in the United States. All eight forms of vitamin E (tocopherols and tocotrienols) occur naturally in foods, but in varying amounts. Vegetable oils are the major sources with good concentrations of the vitamin. For example, a tablespoon of wheat germ oil supplies 20.4 mg, while a tablespoon of sunflower oil (over 60% linoleic acid) has 5.6 mg (USDA 2004). Similarly, one tablespoon of safflower oil (70% oleic acid) supplies 4.6 mg of
-tocopherol while a tablespoon of corn oil (salad or vegetable oil) has 1.9 mg. Almonds are the most concentrated nut source with 13.5 mg in a one-third cup serving. Peanuts (dry roasted, 1 oz) have 2.2 mg. Ready-to-eat fortified cereals provide from 7 to 17 mg. Salad and cooking oils, margarine, salad dressings, mayonnaise, and shortening provide approximately 27% of the vitamin E in the U.S. diet. Animal fats, such as butter and lard, contain lower levels of the vitamin. Fish, eggs, and beef contain relatively low levels of the vitamin, with about 1 mg per 100 g food. Vitamin E is available in the acetate and free tocopherol forms as oil for use in soft gelatin capsules. Vitamin E acetate has a variety of applications in the fortification of beverages and dry premixes. Chemical forms as functional ingredients: The following chemical forms of vitamin E are commonly used as function ingredients: D-
-tocopherol, DL-
-tocopherol, D-
-tocopheryl acetate, D-
-tocopheryl succinate. Nutrient functional claims: Japan has the following health claims for vitamin E: (1) Vitamin E helps protect oxidation of fat in the body (2) It helps to maintain healthy cells (3) Vitamin E intake may prevent hardening of arteries and oxidation of LDL in the blood. Consumption of E may reduce the risk of certain cancers, however, the FDA has

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determined that this evidence is limited and not conclusive. Vitamin K Chemistry: Vitamin K was first isolated from alfalfa by Henrik Dam, a Danish biochemist and named vitamin K or a coagulation vitamin. Normally vitamin K is produced by bacteria in the intestines. However, its first synthesis was carried out by Doisy in 1939 and both Dam and Doisy received Nobel prizes for their work on the discovery of vitamin K and its synthesis. Vitamin K is a group name for a number of related compounds which have in common a methylated napthoquinone ring structure, which vary in the aliphatic side chain attached at the 3-position. Phylloquinone, is the most common form of vitamin K (also known as vitamin K1, Figure 6.7) and contains in its side chain four isoprenoid residues one of which is unsaturated. Vitamin K1 is produced by plants, whereas, vitamin K2 also called menaquinone, can be synthesized by bacteria in the intestine. Vitamin K3 (menadione) is a synthetic form of this vitamin which is man made. Menaquinones have side chains composed of a variable number of unsaturated isoprenoid residues. Generally they are designated as MK-n, where n specifies the number of isoprenoids. Hydrogenation of plant oils containing vitamin K1 gets converted into another form dihydro-vitamin K1 (dK) whose biological activity is not yet known. Vitamin K is soluble in lipid, ether, and other non-polar organic solvents. Newborns are often vitamin K deficient because they do not have bacteria that produce the vitamin in the gut. Vitamin K is stable to oxidation in most food processing processes. However, it is unstable to light and in alkaline condition which prohibits them from saponification and extraction procedures. Reducing agents also destroy the biological activity of vitamin K1. Isomerization of trans bond into cis causes problems since the cis form possesses no biological activities. FIGURE 6.7 — Chemical Structure Of Vitamin K1

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Functions and deficiencies: Vitamin K is used by the body to control blood clotting and is essential for synthesizing the liver protein that controls the clotting. Vitamin K is also involved in bone formation and repair. In the intestines it assists in converting glucose to glycogen which can then be stored in the liver. Deficiencies of vitamin K have been linked to: heavy menstrual bleeding, gastrointestinal bleeding, hematuria (blood in the urine), nosebleeds, gum bleeding and eye hemorrhages etc. Birth defects linked directly to vitamin K deficiencies includes underdevelopment of the nose, mouth and mid face, shortened fingers, cupped ears, flat nasal bridges etc. RDA: 0-12 months,10-20 µg/d, 1-10 years, 15-60 µg/d, 11-18 years, 100 µg/d, 18 years and plus,100 µg/d. Food sources: The predominant dietary form of vitamin K (phylloquinone; K1) occurs primarily in green leafy vegetables and in certain plant oils including soybean, canola and cottonseed. A substantial amount is also found in cheese and liver. It is also found in asparagus, coffee, bacon and green tea. Among the fast foods, including chicken products, hamburgers, burritos and nachos the K1 and dK contents ranged from 0.4 to 23.4 and non-detected (ND)-69.1 µg/100g, respectively. Crackers and potato chips had wide ranges in K1 (1.4-24.3 µg/100g) and dK content (ND-102 µg/100g) (Weizmann et al., 2004). The vitamin K content of nuts and fruits in the US diet has recently been investigated. With the exception of pine nuts and cashews, which contain 53.9 and 34.8 µg/100 gram per nut, respectively, nuts are not an important dietary source of vitamin K. Some berries and green fruits are the exception (Dismore et al., 2003). Chemical forms as functional ingredients: Phylloquinone (K1, C31H46O2) is synthesized commercially for use in infant formula, medical foods and pharmaceuticals. Several stabilized forms of menadione (K3, C11H8O2) are also available such as menadione sodium bisulfate and menadione dimethyl-pyrimidinol bisulfite. These chemical forms are water soluble and are more stable to the processing condition compared to the free menadione. Nutrient functional claims: Neither Japan nor the United States has approved health claims of vitamin K. WATER SOLUBLE VITAMINS Vitamin C Chemistry: Vitamin C is also called L-ascorbic acid. In 1747, Scottish naval surgeon James Lind discovered that a nutrient (now known to be

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vitamin C) in citrus foods prevented scurvy. Later, it was rediscovered by Norwegian scientists, A. Hoist and T. Froelich. Vitamin C was the first vitamin to be artificially synthesized in 1935, a process invented by Dr. Tadeusz Reichstein, of the Swiss Institute of Technology in Zurich. Vitamin C is an enolic form of 3-oxo-L-gulofuranolactone (Figure 6.8) and an antioxidant vitamin. It efficiently scavenges O2-, OH, peroxyl radicals and singlet oxygen. It can be chemically produced from glucose as well as extracted from plant sources such as rose hips, blackcurrants or citrus fruits such as oranges and vegetables, such as, berries, tomatoes, and leafy greens. It occurs as a white or slightly yellow crystal or powder with a slight acidic taste. Vitamin C is sparingly soluble in alcohol, insoluble in chloroform, ether, and benzene. At higher pH (7.4) most of the vitamin C (99.95%) is present as ascorbate(donar antioxidant) form thus the antioxidant chemistry of vitamin C represents the chemistry of ascorbate. Vitamin C is the least stable of vitamins and is very sensitive to oxygen. Its potency can be lost through exposure to light, heat and air which stimulate the activity of oxidative enzymes. FIGURE 6.8 — Chemical Structure Of Vitamin C

Functions and deficiencies: Vitamin C has various physiological functions. It is necessary for the prevention of scurvy. It inhibits the formation of nitrosamines (a suspected carcinogen) and is important for maintenance of bones, teeth, collagen and blood vessels (capillaries). It decreases glycosylation of albumin which can significantly reduce the risk of developing atherosclerosis. It can protect biomembranes and

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LDL from peroxidative damage. Epidemiological studies have shown that consumption of vitamin C correlates with a reduction in cancer incidence especially cancer of the stomach and esophagus. Vitamin C deficiency causes scurvy, listlessness, fatigue and weakness. RDA: Adults (male and female) need 100 mg/d, pregnant women 110 mg/d, lactating women, 140 mg/d, infants (0-5 yrs) 40 mg/d. The tolerable Upper Intake Levels (UL) are 2000 mg/d and lowest observed adverse effect level (LOAEL) is 3,000 mg/d. For Japan no LOAEL has been established. It is known that smoking reduces the plasma/leukocyte of vitamin C level, therefore, heavy smokers are recommended to take twice the vitamin C intake of non-smokers. Food sources: Good sources of vitamin C are broccoli, Brussels sprouts, cauliflower, cabbage, green leafy vegetables, red peppers, chilis, watercress, parsley, blackcurrants, strawberries, kiwi fruit, guavas, and citrus fruit. Chemical forms as functional ingredients: The following chemical forms are used as functional ingredients: L-ascorbic acid, L-ascorbyl palmitate, sodium L-ascorbate and calcium L-ascorbate. As an antioxidant (ascorbyl palmitate) is often used to prevent the formation of rancidity in stored lipid products and the phenolic browning of commodities such as dehydrated potatoes. It is also used as a flour improver in the Chorleywood bread process, where its oxidation product (dehydroascorbic acid) modifies the availability of glutathione in dough development, thereby shortening the period of fermentation. Nutrient functional claims: Vitamin C has an antioxidative effect and helps in protecting against oxidative agents at cellular level. Like vitamin E, vitamin C is not approved by FDA for claiming to reduce certain types of cancer. Japan has the following health claims: (1) Vitamin C may aid collagen and carnitine synthesis (2) May promote absorption of iron (3) Vitamin C is useful for preventing heart disease and useful for keeping the eyes healthy. Vitamin B1 Chemistry: Vitamin B1 is also known as thiamine, thiamin (no “e” at the end) and aneurin, isolated and characterized in 1926. However, thiamine is the currently accepted name for this vitamin in the United States whereas in Europe especially in the United Kingdom, aneurin is still widely used. The chemical name for vitamin B1 is 3-[(4'-amino2'-methyl-5'-pyrimidinyl)methyl]- 5-(2-hydroxyethyl)-4-methyl thiazole. Other forms of vitamin B 1 are thiamine monophosphate (TMP),

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thiamine diphosphate or thiamine pyrophophate (TPP) and thiamine triphosphate (TTP). The free form of thiamin occurs mainly in plasma whereas the coenzyme thiamine diphosphate (TDP) predominates intracellularly. All forms exist in animal and plant tissues. Thiamine consists of a pyrimidine ring and a thiazole ring connected by a one carbon link (Figure 6.9). The nitrogen in the thiazole ring has a charge of +1 and serves as an important electron link in thiamine pyrophosphate mediated reactions. Vitamin B1 is one of the most unstable vitamins. It is sensitive to heat, alkali, oxygen and radiation. Thiamine is least stable when the pH approaches neutral. Maximum stability in solution is between pH 2.0 to 4.0. Baking, pasteurization, or boiling of foods fortified with thiamine can reduce its content by up to 50 percent. The stability of thiamine during storage greatly depends on the moisture content of the food. FIGURE 6.9 — Chemical Structure Of Vitamin B1

Thiamine (base free) 3-[(4' amino-2'-methyl-5'pyrimidinyl)methyl)]5-(2-hydroxyethyl)-4-methyl-thiazole

Functions and deficiencies: Thiamin is important for the normal functioning of nerves. It is necessary for the synthesis of acetylcholine, a neurotransmitter which affects several brain functions including memory. It is vital for normal development, growth, reproduction, healthy skin, hair, blood production and immune function. Deficiency of vitamin B1 usually causes weight loss, cardiac abnormalities, and neuromuscular disorders. The thiamine deficiency syndrome in humans is beri-beri, most common in parts of Southeast Asia where polished rice was a dietary staple. Beri-beri is characterized by anorexia (loss of

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appetite) with subsequent weight loss, enlargement of the heart muscle weakness and foot and wrist droop. There are three main types of beriberi (1) dry beri-beri (muscle wasting with heart involvement, hypotension, sodium retention and pulmonary edema); (2) wet beriberi (also edematous or cardiac) and (3) infantile beri-beri. Dry beriberi usually inflicts older adults and affects mainly the peripheral nerves with little cardiac involvement. It is characterized by atrophy and peripheral neuritis of the legs and paraplegia. In contrast wet beriberi displays substantial cardiac involvement especially tachycardia (rapid heart beat) in addition to peripheral neuropathy. Edema progresses from the feet upwards to the heart causing congestive heart failure in severe cases. Infantile beri-beri is characterized by vomiting, convulsions, abdominal distention and anorexia. Another thiamine deficiency disease is Wernicke-Korsakoff Syndrome seen most often in alcoholics after long periods of alcohol intake. Wernicke-Korsakoff Syndrome is a severe deficiency characterized by mental disorder, including confusion, hallucinosis and psychosis. RDA: Babies 0 to 1 years need 0.2 mg/d, children 1 to 3 years 0.4 mg/ d, children 4 to 6 years 0.6 mg/d, children 7 to 9 years 0.8 mg/d, children 10 to 12 years, males 1.3 mg/d and women 1.1 mg/d. Food sources: The best source of vitamin B1 includes asparagus, romaine lettuce, mushrooms, spinach, sunflower seeds, pork, tuna, green peas, tomatoes, eggplant and Brussels sprouts. Chemical forms as functional ingredients: Thiamine hydrochloride (C 12 H 18 ON 4 SCl 2 ) and thiamine mononitrate (C12H17O4N5S) are commercially produced chemical forms used in pharmaceuticals and for functional food fortification. These two forms of thiamine differ in solubility, the later being used in dry blends, multivitamins, dry products and is less hydroscopic. Nutrient functional claims: There are no approved health claims by the FDA. However, Japan has the following health claims: Vitamin B1 helps to produce energy from carbohydrate and helps to maintain healthy skin and mucosal membranes. Vitamin B6 Chemistry: In 1934, Paul György a Hungarian-born physician, identified vitamin B6 as a curative factor for a dermatitis in rats and proposed the name vitamin B6. A few years later, György and several co-workers, and Richard Kuhn finally isolated the crystalline form from rice bran and duly named it vitamin B6 (or pyridoxine) (Figure 6.10). The chemical structure was determined in 1939 and synthesized by

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Harris and Karl Folkers. Vitamin B6 exists in six major chemical forms: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their phosphate derivatives: pyridoxal 5'-phosphate (PLP), pyridoxine 5'phosphate (PNP), and pridoxamine 5'-phospate (PMP). PLP is the active coenzyme form, and has the most importance in human metabolism. PN, PL, and PM are metabolically interconvertible and considered to be biologically active. PN is more stable than PL and PM. The stability of vitamin B6 greatly depends on the type of thermal processing. pH, light and temperature are main factors for its degradation. However, all forms are stable in acidic solutions if protected from light. PN is more stable than PL and PM. PM is the least stable. Processing and cooking conditions cause variable losses. For example high losses of B6 occur during sterilization of liquid infant formula, in contrast B6 in enriched flour and corn meal is resistant to baking temperatures. FIGURE 6.10 — Chemical Structure Of Vitamin B6

Functions and deficiencies: Pyridoxine is normally stored as pyridoxal-5-phosphate (PLP), the coenzyme form of the vitamin. It is needed for metabolism of amino acids, cellular metabolism of carbohydrate, protein and fat formation of neurotransmitters and production of nicotinic acid (vitamin B3). Pyridoxine is the cofactor for enzymes that convert L-tryptophan to serotonin and L-tyrosine to norepinephrine. It facilitates the conversion of amino acids from one to another and is necessary for the normal synthesis of hemoglobin and the normal function and growth of red blood cells. Vitamin B6 deficiency

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is rare, however, marginal vitamin B6 status is relatively common. Vitamin B6 deficiency can occur in individuals with poor quality diets that are deficient in many nutrients. Symptoms occur during later stages of deficiency when intake has been very low for an extended time. Signs of vitamin B6 deficiency include dermatitis, glossitis (a sore tongue), depression, confusion, and convulsions. Vitamin B6 deficiency also can cause anemia. RDA: Men: 2 mg/d; Women: 1.6 mg/d; Pregnant women: 2.2 mg/d. Food sources: Vitamin B6 is usually bound to protein, pyridoxol being the prominent form in plants and pyridoxamine in animal products. Major dietary sources of pyridoxine include: chicken, liver, yeast extract, fish (tuna, trout, herring, salmon), nuts, and whole grains. Chemical forms as functional ingredients: Pyridoxine hydrochloride (PN . HCl, C 8 H 12 ClO 3 ) is the commonly available commercial form used for food fortification. The PN.HCl salt is a white crystalline powder with a salty taste. PN.HCL is soluble in water, alcohol and propylene glycol and sparingly soluble in acetone and insoluble in diethyl ether and chloroform. However, pyradoxal hydrochloride . (C8H9NO3 HCl)) and pyridoxamine hydrochloride (C8H12ClNO3) are also being used for food fortification but are less stable. Nutrient functional claims: Dietary supplementation of vitamin B6, when a person maintains a well balanced diet that is low in saturated fat and cholesterol, may reduce the risk of vascular disease. However, FDA evaluated this claim and found it inconclusive. Japan has the following approved claims of vitamin B6: Vitamin B6 helps to produce energy from protein and maintain healthy skin and mucosal membranes similar to vitamin B1. Vitamin B12 Chemistry: Vitamin B12 is the largest and most complex of all the vitamins. It is available in several forms and is a collective name for cobalt-containing corrinoids with the biological activity of cyanocobalamin (CNCbl). Vitamin B12 is the only known bio-molecule with a stable carbon-metal bond. The core of the molecule is a corrin ring with various attached side groups. The ring consists of 4 pyrolle subunits, joined on opposite sides by a C-CH3 methylene link on one side by a C-H methylene link and with the two of the pyrroles joined directly. It is thus structurally similar to heme except it has one less
methene bridge and has cobalt in place of iron (Figure 6.11). The most common form is CNCbl which is tasteless, odorless with good water solubility. In CNCbl, the β-position of the cobalt atom is occupied by a

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cyano-ligand (CN ) which can also be occupied by OH , NO2 and SO3 to form hydroxocobalamin (OHCbl) nitrocobalamin (NO 2 Cbl) and deoxyadenosul (SO3CBl). Vitamin B12 is stable to heat but is sensitive to light, oxygen, acid and alkali and considered to be stable under most food processing operations. It is fairly stable at pH 4-6, even at higher temperatures. CNCBl is the most stable form of the vitamin. FIGURE 6.11 — Chemical Structure Of Vitamin B12

Functions and deficiencies: Vitamin B12 is needed for normal functioning of the stomach, pancreas and small intestine. Stomach acid and enzymes free vitamin B12 from food allowing it to bind to other proteins known as R protein. In the alkaline environment of the small intestine, R proteins are degraded by pancreatic enzymes freeing vitamin B12 to bind to intrinsic factor (IF), a protein secreted by specialized cells in the stomach. Receptors on the surface of the small intestine take up the IF-B12 complex only in the presence of calcium,

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which is also supplied by the pancreas. Symptoms of vitamin B12 deficiency may show after a prolonged period of poor dietary intake or inadequate secretion of intrinsic factor. The classic deficiency symptom of vitamin B12 is pernicious anemia which evolves through abnormal absorption of vitamin B12 resulting from inadequate digestion, lack of binding factors (Ca++) etc. The sign includes neurological involvement resulting from demyelination of the spinal cord, brain, optic and peripheral nerves. General symptoms include glossitis, weakness, loss of weight, lose of appetite, memory impairment and hallucinations. It can also cause impaired mental function that in the elderly mimics Alzheimer’s disease. Vitamin B12 deficiency is thought to be quite common in the elderly and is a major cause of depression in this age group. In addition to anemia and nervous system symptoms, vitamin B12 deficiency can also result in a smooth beefy red tongue and diarrhea. RDA: Vitamin B12 is necessary in only very small quantities. The RDA is 2 µg/d for adults, 2.2 µg/d for pregnant women and 2.6 µg/d during lactation. Vegetarian diets can produce deficiency, however, 1 to 5 µg/d may provide necessary requirement. Food sources: The richest food sources include liver, kidney, spleen, sea foods, eggs and dairy products. Fermented soy products, seaweeds and algae have all been proposed as possible sources of B12 at a lower level. Chemical forms as functional ingredients: Cyanocobalamin (CNCbl) and hydroxocobalamin (OHCbl) are the well known chemical forms of vitamin B12 available for food fortification and for medical uses. CNCbl is a tasteless, odorless and red crystalline substance with good water solubility. It has a better stability than OHCBl. Nutrient functional claims: Diets low in saturated fat and cholesterol may reduce the risk of vascular disease. This claim has not been approved by the FDA, and the FDA found the evidence in support of the above claim is inclusive. However, Japan has the following approved claim: Vitamin B12 aids in red blood cell formation. Folic Acid Chemistry: Folic acid and folate are interchangeable terms. Folic acid is the synthetic form of folate, which is found naturally in some foods. The term is used as a generic name (pteroylglutamate) with a series of derivatives with folic acid activity. The name folic acid was derived from the Latin word “folium” for leaf (Mitchell et al., 1941). Other names for folic acid are folacin, vitamin BC, vitamin B9 and Lactobacillus casei factor. The IUPAC name of folic acid is 2-amino-4-

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hydroxy-6-methyleneaminobenzoyl-L-glutamic acid pteridine and the parent compound is pteroic acid, 4-[(pteridin-6-ylmethyl)amino]benzoic acid (Figure 6.12). The foliates compounds are based on pteroic acid skeleton conjugated with one or more L-glutamate units linked through the -carboxyl of the amino acid. The salts and the acyl group derived from the acid are named pteroates and pteroyl, respectively. Although folic acid is not found in nature, it is the common and quite stable synthetic form used for food fortification and for formulation of pharmaceuticals. The reduced form of folic acid are dihydrofolate (H2 folate) and tetrahydrofolate (H4 folate), the later being the active coenzyme form of the vitamin. The stereochemistry of foliates is very complicated due to the number and diversity of biologically active forms. The variations in structure occurs due to the oxidation state of the pteridine ring, the non-carbon moiety carried by the specific folate and the number of conjugated glutamate residues on the specific folate. For these reasons the IUPAC-IUB commission on biochemical nomenclature has set certain rules for a systematic nomenclatures of the folate chemistry (Blakley, 1987). For example, pteroic acid conjugated with one or more L-glutamate units are named pteroylglutamate, pteroyldiglutamate, etc. The name “pteroylmonoglutamate” should not be used (IUPAC-IUB recommendation). Folic acid in food is unstable and considerable losses occur during short storage and cooking. However, it is stable to 100°C when protected from light at pH 5.0 to 12.0. The stability of folic acid is greater than naturally occurring folates in most foods. Folate is stable in dry products and in the absence of light and oxygen. Presence of ++ metal (F ) can increase folate loss (Day et al., 1983). FIGURE 6.12 — Chemical Structure Of Folic Acid

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Functions and deficiencies: Folic acid helps to prevent several major birth defects called neural tube defects (NTDs). It is also necessary for cell replication and growth as well as the synthesis of DNA and RNA. Folate helps prevent alterations to DNA that can lead to cancer. Both adults and children require folate to build normal red blood cells and prevent anemia. Supplementation with folic acid and vitamin B12 improves vascular endothelial function in patients with coronary heart disease (Chambers et al., 2000). The evidence in vitro demonstrate that 5-methyltetrahydrofolate, the main circulation metabolies of folic acid can increase nitric oxide production and directly scavenge super oxide radicals. These properties may account for some of its cardiovascular effects (Moat et al., 2004). While most folic acid studies have focused on heart health, some recent findings suggest that folic acid either has antidepressant properties or can act as an augmenting mediator for standard antidepressant treatment. Evidence suggests that elderly depressed patients have lower levels of folate than their non-depressed cohorts. Supplementing with folate may thus reduce the incidence of depression in the elderly people (Alpet et al., 2003). Folate deficiency is a common cause of anemia. The signs of folic acid deficiency can be subtle such as diarrhea, loss of appetite, weight loss as well as weakness, a sore tongue, headaches, heart palpitations, and irritability. Folic acid deficiency is one of the most common vitamin deficiencies in the United States, largely owing to its association with excessive alcohol intake. RDA: Lactating women need 400 µg/d and men 200 µg/d. Food sources: There are many food sources containing folic acid including the green leafy vegetables (broccoli, cauliflower), beans, liver, yeast extract, whole grains, egg yolk, milk and milk products, oranges and orange juice, beets and whole meal bread. Chemical forms as functional ingredients: Folic acid or pteroylmonoglutamic acid (C19H19N7O6) are commonly used names. The synthetic form is used for food fortification and pharmaceutical formulation. Nutrient functional claims: The FDA has approved health claims of folic acid and has ordered mandatory fortification with folic acid for cereal grain products in order to reduce the neural tube defects (NTDs). NTDs are serious defects of the spine (spina bifida) and brain (anencephaly) affecting large numbers of pregnancies each year in the United States. 0.8 mg folic acid as a dietary supplement may be more effective in reducing the risk of NTDs. However, Japan has several health claims including NTDs. A few are listed here: (1) folic acid keeps blood homocystein concentration normal and may reduce the risk of arteriosclerosis (2) folic acid may keep the heart healthy (3) folic acid

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may keep the metabolism of monoamine normal and maintain healthy neural/psychiatric status etc. Biotin Chemistry: Biotin acts as a coenzyme by assisting in making fatty acids and the oxidation of fatty acids and carbohydrates. The chemical structure is cis-hexahydro-2-oxo-1H-thieno [3,4-d] imidazole-4pentanoic acid. There are at least eight stereoisomers of biotin but only d (+) is biologically active (Figure 6.13). The bicyclic ring structure contains an uredo ring which is fused to a tetrahydrothiophene with a valeric acid side chain. Biotin is one of the safest vitamins which utilizes protein, folic acid, pantothenic acid, and vitamin B12 in the body. Biotin synthesis occurs mostly in the microflora, however, it was also isolated and crystallized from egg yolk. There is no known toxicity of biotin vitamin. The sulfur atom in biotin is prone to oxidation and provides a primary route for loss of biotin in processed foods. Biotin solution is quite stable at pH 4.0 to 9.0 and is commonly extracted by autoclaving biological samples in 2 N or 6 N sulfuric acid for two hours. UV light exposure also leads to loss of biotin activity. FIGURE 6.13 — Chemical Structure Of Biotin

Functions and deficiencies: In humans, biotin is involved in important metabolic pathways such as gluconogenesis, fatty acid synthesis, and amino acid catabolism. Biotin regulates the catabolic enzyme propionyl-CoA carboxylase at the posttranscriptional level,

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whereas, the holo-carboxylase synthetase is regulated at the transcriptional level. Biotin functions as a cofactor that aids in the transfer of CO2 groups to various target macromolecules. Although biotin deficiency is very rare, however, the first symptoms to develop in biotin deficiency are associated with the dry skin and hair losses (alopecia). Others include dry scaly dermatitis, rashes and an increase in serum cholesterol and bile pigments. Although human deficiency of biotin is rare, it may occur if raw eggs are consumed for a long period of time. RDA: Although biotin is necessary for the body, only exceedingly small quantities are needed. The RDA has not been set for biotin. The estimated safe and adequate daily intake for adults is 30 to 100 mg. Food sources: Good sources of biotin include egg yolks, kidney, liver, tomatoes, and yeast. Vegetables such as lettuce, green peppers, cauliflower contain higher amounts of biotin. Chemical forms as functional ingredients: The United States Pharmacopeia (USP) standard is d-biotin. The food and pharmaceutical industries use the crystalline biotin with diluents such as dicalcium phosphate to aid in dispersibility and ease of binding. Nutrient functional claims: No health claims are approved by the FDA. However, Japan has approved the claim that biotin helps maintain healthy skin and mucosal membranes. MINERALS Minerals are found in rocks, metals, soil and water, though they may be in slightly different forms. While each mineral plays a unique role, collectively they support the body’s enzyme systems and keep blood and other body fluids balanced and healthy. Minerals also help regulate blood pressure and heart muscle contraction, heal wounds and conduct nerve impulses. A minimum of at least 60 trace minerals has been demonstrated to be vital to health and well-being. Macrominerals that are needed in relatively large amounts include calcium, phosphorus, magnesium, sodium, potassium, chloride and sulfur; and trace minerals that are needed in smaller amounts consist of iron, zinc, selenium, chromium, copper, fluoride, iodine, molybdenum and manganese. Although the body can not produce any minerals of its own, minerals are found in a large variety of fruits, vegetables, beans, grains, meats and dairy products.

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Macrominerals Calcium Chemistry: Calcium was first isolated in its metallic form by Sir Humphrey Davy in 1808 through the electrolysis of a mixture of calcium oxide and mercury oxide. It is a bivalent cation found in bones, teeth and body tissues. More than 99% of the body’s calcium is found in bones. The total amount of calcium in the body is about 1,500g. It is abundant in the skeleton and considered essential because of its importance in building and maintaining bones. In addition, calcium found in plasma and cells is important for regulatory mechanisms such as chemical and electric neuromuscular transmission systems, cellular secretion and blood coagulation. The amount of calcium in the human body is regulated by parathyroid hormone. Low calcium intake triggers parathyroid hormone which then send signals for bone breaking and ultimately releases calcium into the blood stream. Diets with adequate calcium intake produce less parathyroid hormone and thus help in restoring more calcium in the bones. Calcium is best absorbed when it is taken with food at a dose not exceeding 500 milligrams. High calcium intake (> 2000 mg/d) may cause constipation and kidney stones and may inhibit zinc and iron absorption. Functions and deficiencies: Calcium is used for building bones and teeth and in maintaining bone strength. The major deficiency symptoms of calcium are skeletal abnormalities. Osteomalacia, osteoporosis and rickets may all be caused by calcium deficiency. RDA: The daily intake levels set by the Consensus Development Conference of the National Institutes of Health in Bethesda, Maryland are given below. Infants, up to age 6 months need 400 mg/d, infants, ages 6 to 11 months 600 mg/d, children, ages 1 to 10 years 800 to 12,00 mg/d, adolescents and young adults, ages 11 to 24 years 1,200 to 1,500 mg/d, men, ages 25 to 65 1,000 mg/d, pregnant and nursing women 1,200 to 1,500 mg and men and women over age 65 years 1,500 mg/d. Food sources: Milk and dairy products such as skim milk, nonfat yogurt, and cheeses, are primary sources of calcium. In addition, a variety of other foods are excellent sources of calcium such as dark green vegetables (spinach, broccoli, turnip greens etc.). Foods with added calcium such as fortified orange juice, corn tortillas processed with lime can be a good source of calcium. Salmon and sardines with bones are also good sources of calcium.

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Chemical forms as functional ingredient: Calcium lactate (D-, DL-), calcium gluconate, calcium carbonate, calcium phosphate, calcium chloride, calcium citrate and calcium glycerophosphate, calcium hydroxide and calcium oxide are used as food additives. Nutrient functional claims: The FDA has approved the health claim of calcium that a healthy diet with enough calcium helps maintain good bone health and may reduce the risk of osteoporosis later in life. Similarly, Japan also has approved health claims of calcium such as calcium is necessary for the development of bone and teeth. Phosphorus (P) Chemistry: Phosphorus is a multivalent, nonmetal of the nitrogen group and commonly found in inorganic phosphate rocks and in all living cells. Due its important role in biological processes, phosphorous is one of the most dispersed elements in nature. It is highly reactive and never found as a free element in nature. It emits a faint glow upon exposure to oxygen. The most important commercial use of phosphorus is in the production of fertilizers. It is also widely used in explosives, friction matches, fireworks, pesticides, toothpaste, and detergents. Functions and deficiencies: Phosphorus is of vital importance in the growth and health of plants and animals. It is an important constituent of teeth and bones. As triphosphate adenosine (ATP) and other organic phosphates play an indispensable role in biological reactions. All the biological mechanisms use phosphorus as orthophosphate form or as polyphosphate which by hydrolysis becomes orthophosphate. Examples of these processes are photosynthesis, fermentation, and metabolism, etc. In living animals, phosphorus is also a constituent element of the nervous tissues as well as of the cellular plasma. Phosphorus deficiency can result in anorexia, impaired growth, osteomalacia, skeletal demineralization, weakness, cardiac arrhythmias, respiratory insufficiency, increased erythrocyte, lymphocyte dysfunction and nervous system disorders. Phosphate salts are used in the treatment of phosphorus deficiency. RDA: The RDA for phosphorus is based on the maintenance of normal serum phosphate levels in adults. The following RDA is recommended: infants 0-12 months 100-275 mg/d, children 1-13 years 460-1250 mg/d, adults (19 years and older) 700 mg/d, breast feeding mom 1250 mg/d. Food sources: It is found in most foods, especially asparagus, bran, corn, dairy products, eggs, fish, dried fruit, garlic, sunflower, pumpkin seeds, meats, poultry, salmon, soda. Wheat bran and whole grains are

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particularly rich sources of phosphorus. Phosphorus is also a component of many polyphosphate food additives, and is present in most soft drinks as phosphoric acid. Chemical forms as functional ingredients: Trisodium phosphate is used as a food additive, phosphoric acid used in soft drinks, and mono-calcium phosphate is employed in baking powder. Nutrient functional claims: Neither the United States nor Japan has approved any health claims of phosphorous. Magnesium (Mg) Chemistry: Magnesium is the eighth most abundant element and constitutes about 2% of the earth’s crust by weight. It is the third most common element and is dissolved in seawater as a positive ion and is found in the terrestrial crust in magnesite form (MgCO3), dolomite (CaCO3, MgCO3) and many common silicates, as asbestos, talc and olivine. In 1808, Sir Humphrey Davy isolated the metal and called it “magnium”. At the time, the terms “magnesium” and “manganese” were used to denominate the manganese, obtained from the mineral pyrolusite. Magnesium is an essential mineral for human nutrition. In the body magnesium serves several important metabolic functions including an important role in enzymatic catalysis reactions involving the phosphate group, which are associated to the energy transfer and the stimulus at the muscular level. It plays a role in the production and transport of energy. It is also important for the contraction and relaxation of muscles. In plants the photosynthetic activity is based on the activity of chlorophyll, whose pigments have a rich composition of magnesium. Functions and deficiencies: Magnesium possesses a tremendous healing effect on a wide range of diseases. The relationship between serum magnesium levels and the risk of coronary heart disease has been found to have an inverse correlation (Ford, 1999). Also studies have found an inverse correlation between serum magnesium levels and blood pressure. Similarly, there is evidence that a positive correlation between the intake of dietary magnesium and increased bone mineral density (Tucker et al. 1999). Magnesium deficiency can cause numerous psychological changes, including depression. The symptoms of magnesium deficiency are nonspecific and include poor attention, memory loss, fear, restlessness, insomnia, cramps and dizziness. The lack of magnesium in the human body can induce diarrhea or vomiting as well as hyperirritability or a slight tissue calcification. In extreme cases, this deficiency causes tremors, disorientation or even convulsions eventually leading to death.

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RDA: The RDA for men is 350 mg/d, women 280 mg/d. Dietary surveys have shown that many Americans fail to achieve the recommended dietary allowance for magnesium. As a result, subtle magnesium deficiency may be common in the United States. Food sources: Although magnesium is present in many foods, it usually occurs in small amounts. Green vegetables such as spinach, broccoli, and beans provide magnesium. Nuts, seeds and some whole grains are also good sources of magnesium. Water can also provide magnesium but the amount varies according to the water supply. Using “hard” water consumption to estimate magnesium intake from water may lead to underestimate total magnesium intake and its variability. Following are some foods and the amount of magnesium in them: spinach (1/2 cup) = 80 mg, peanut butter (2 tablespoons) = 50 mg, blackeyed peas (1/2 cup) = 45 mg, milk, low fat (1 cup) = 40 mg. Chemical forms as functional ingredients: Magnesium is used as magnesium chloride, magnesium oxide, magnesium carbonate, magnesium sulfate, magnesium salt and magnesium yeast etc. In Japan magnesium oxide and magnesium carbonate have limitations in their use as food additives. Magnesium for food fortification is used as magnesium acetate, magnesium lactate, magnesium citrate, magnesium gluconate, magnesium glycerophosphate and magnesium protein hydrolysate. Nutrient functional claims: Japan has approved health claims of magnesium such as keeping the heart healthy, reducing high blood pressure, and reducing stress etc. However, there are no approved health claims of magnesium in the United States. Sodium (Na) Chemistry: Sodium was first isolated in 1807 by Sir Humphrey Davy, who made it by the electrolysis of dry molten sodium hydroxide (NaOH). The symbol “Na” came from the neo-Latin name for a common sodium compound named natrium, derived from the Greek nitron, a kind of natural salt. Sodium is a soft, waxy, silvery metal and is abundant in natural compounds. It is highly reactive, burns with a yellow flame, reacts violently with water and oxidizes in air. Sodium makes up about 2.6% of the weight of the Earth’s crust, making it the fourth most abundant element. Sodium chloride or common salt is the most common compound of sodium, however, sodium occurs in many other minerals such as amphibole, cryolite, halite, soda niter, zeolite etc. Sodium compounds are important to the chemical, glass, metal, paper, petroleum, soap and textile industries.

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Functions and deficiencies: Sodium is the primary electrolyte that regulates the extracellular fluid levels in the body. Sodium is essential for hydration. In addition to maintaining water balance, sodium is necessary for osmotic equilibrium, acid-base balance and regulation of plasma volume, nerve impulses and muscle contractions. A sodium deficiency frequently results during treatment with drugs called diuretics since they cause loss of sodium from the body. Diuretics can lead to sodium deficiency, resulting in low plasma sodium levels. RDA: Total daily sodium intake should not exceed 2400 mg/d. The RDA for sodium for adults (both men and women) is 500 mg/d, children 400 mg/d, and infants 120-200 mg/d. Food sources: Sodium is found naturally in many foods and is added to the prepared foods as sodium salt. Good sources of sodium are cheeses, most meat especially ham and bacon, canned soups, canned vegetables, baked goods, pickles, and sauces etc. Chemical forms as functional ingredients: The sodium compounds that are most important to the food industry are common salt (NaCl), baking soda (NaHCO3), food grade caustic soda (NaOH), di- and tri-sodium phosphates, sodium benzoate and sodium metabisulfite, sodium lactate and sodium malate. Nutrient functional claims: The FDA has approved the health claims of sodium intake. For example, diets low in sodium may reduce the risk of high blood pressure. Therefore, foods must meet criteria for low sodium. Potassium (K) Chemistry: Potassium was discovered in 1807 by Sir Humphrey Davy, who derived the metal from caustic potash (KOH). Potassium was the first metal that was isolated by electrolysis. The name “potassium” came from the word potash. Potassium is a soft silverywhite metallic alkali metal that occurs naturally bound to other elements in seawater and many minerals. It oxidizes rapidly in air, and is very reactive especially in water, and it resembles sodium chemically. With a density less than that of water, potassium is the second lightest metal after lithium. It is a soft solid that can easily be cut with a knife. When in water, it may catch fire. Potassium makes up about 2.40% of the weight of the earth’s crust and is the seventh most abundant element. Functions and deficiencies: Potassium assists in muscle contraction and in maintaining fluid and electrolyte balance in body cells. Potassium is also important in sending nerve impulses as well

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as releasing energy from protein, fat and carbohydrates during metabolism. A shortage of potassium can cause a potentially fatal condition known as hypokalemia. Deficiency of potassium results in hypokalemia which refers to possessing abnormally low plasma potassium concentration. Hypokalemia is most commonly due to excessive loss of potassium from prolonged vomiting, use of some diuretics or due to metabolism disturbances. The symptoms of hypokalemia are related to alterations in membrane potential and cellular metabolism. They include fatigue, muscle weakness and cramps and intestinal paralysis which may lead to bloating, constipation and abdominal pain. Severe hypokalemia may result in muscular paralysis or abnormal heart rhythms (cardiac arrhythmias) that can be fatal. RDA: The RDA of potassium for an adult is 4.7 g/d. For children ages 1 to 3, it is 3.0 g/d, for children ages 4-8, 3.8 g/d and for children ages 9 to 13, 4.0 g/d. Food sources: Potassium is found in potatoes, dried fruits, bananas, legumes, raw vegetables, avocados, citrus fruits and mushrooms. It is found also in lean meat, milk and fish. Chemical forms as functional ingredients: Potassium metabisulfite, potassium acetate (preservative), potassium chloride, potassium citrate, potassium benzoate, potassium gluconate are commonly used in the food industry. Nutrient functional claims: No health claims have been made either by the United States or Japan. Sulfur (S) Chemistry: Sulfur is a non-metallic element that occurs in both combined and free states and is distributed widely over the earth’s surface. It is tasteless, odorless, and insoluble in water, and often occurs in yellow crystals or masses. It is one of the most abundant elements found in a pure crystalline form. It displays three allotropic forms: orthorhombic, monoclinic and amorphous. The orthorhombic form is the most stable form of sulfur. Monoclinic sulfur exists between the temperatures of 96°C and 119°C and reverts back to the orthorhombic form when cooled. Functions and deficiencies: As a part of amino acids, sulfur performs a number of functions in enzyme reactions and protein synthesis. It is necessary for formation of collagen. Sulfur is also present in keratin, which is necessary for the maintenance of the skin, hair and nails helping to give strength, shape and hardness to these protein tissues. Sulfur as cystine and methionine is a part of other important

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body chemicals such as insulin, which helps regulate carbohydrate metabolism and heparin (an anticoagulant). There is minimal risk of sulfur deficiency/toxicity in the body. No clearly defined symptoms exist with either state. Sulfur deficiency is more common when foods are grown in sulfur-depleted soil, with low-protein diets or with a lack of intestinal bacteria though none of these seems to cause any problems in regard to sulfur functions and metabolism. RDA: Sulfur is so widely available from foods, water and air that there is no established RDA for this element. Our needs are usually easily met through diet. About 850 mg/d, are thought to be needed for basic turnover of sulfur in the body. Food sources: Sulfur residue foods are commonly recognized by their characteristic spicy, heating effect such as garlic, onions, mustard, and horseradish. The following are sulfur residue foods: red hot peppers, radishes, mustard leaves, cabbage etc. Apart from vegetables, sulfur is also readily available in protein foods-meats, fish, poultry, eggs, and milk. Egg yolks are one of the better sources of sulfur. Chemical forms used as functional ingredients: Sulphur dioxide and sulfite are used as fruit preservatives. Nutrient functional claims: There are no approved health claims either by Japan or the United States. Trace Minerals Iron (Fe) Chemistry: It is ranked after aluminium, making it the second most abundant metallic element in the earth's crust. Iron plays a vital role in many enzymes involved in oxidation and amino acid metabolism (examples: per-oxidase, catalase, hydroxylases); hence, it is an essential ingredient of the daily diet. Iron comes in two forms: heme-iron which is found in red meats and is better-absorbed (20-30%) than non-heme iron, which is found in enriched cereals, and leafy green vegetables like spinach and lettuce. The absorption of iron is much less from liver (6.3%) and fish (5.9%). In humans, the major amount of iron is in the porphyrin complexes hemoglobolin (blood) and myglobin (muscle tissue) and in various heme containing enzymes. The remainder is stored in the soluble form, ferritin, and insoluble non-reactive form hemosiderin. Humans get most of their iron needs from the heme iron source. In the brain, iron is present as heme and non-heme iron. The human body normally contains 3 to 4 g of iron, more than half of which is utilized to form hemoglobin which transports oxygen from the lungs to the tissues. The body’s iron balance

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varies mainly according to dietary intake, as losses from the body are generally small. Women lose iron during menstruation and pregnancy. Functions and deficiencies: Iron is incorporated in a number of body constituents, such as cytochromes, myoglobin and hemoglobin. Ribonucleotide reductase is an iron-dependent enzyme that is required for DNA synthesis (Fairbanks, 1999). Thus, iron is required for a number of vital functions, including growth, reproduction, healing and immune function. Iron may improve learning ability and growth. Lack of iron causes iron deficiency anemia (IDA) which occurs when the blood does not have enough red blood cells that carry oxygen from the lungs to all parts of the body. IDA is the most common type in children. This happens when the body does not have enough iron in it to make red blood cells. IDA can occur at any age, but most often it is seen in toddlers and adolescent females. Infants, toddlers and adolescents all have high iron needs because they are growing relatively fast compared with other times in their lives. While infants tend to get enough iron with breast milk and iron-fortified formula, toddlers often have diets with very little iron-rich foods. RDA: The RDA has been set at 15 mg/d for women of 19 to 50 years of age and 10 mg/d for men 25 to 50 years of age. Food sources: Meat, especially red meat and organ meats are the richest sources. Shellfish, tuna, salmon, and eggs (egg yolks) are also good sources of iron. Other sources include whole wheat products, nuts, dried fruits, like raisins, and dark leafy green vegetables, such as broccoli etc. It is unlikely that iron toxicity can develop from an increased dietary intake of iron alone. However, iron supplements can cause side effects such as nausea, vomiting, constipation, diarrhea, dark-colored stools and abdominal pain. Chemical form as functional ingredient: Ferric chloride/citrate/ sulphate/carbonate, sodium ferrous citrate, ferrous lactate/ pyrophoispahte, and ferrous ascorbate are the compounds used as food ingredients. Nutrient functional claims: Japan has approved health claims of iron as being necessary for red blood cell formation and the prevention of iron deficiency anemia. Zinc (Zn) Chemistry: Zinc is a metallic chemical element, which is less abundant in nature; however, it has great commercial importance. Zinc has a white color with a bluish tinge and a high resistance to atmospheric corrosion. The melting point of zinc is 419°C. It is used principally for galvanizing iron, but is also important in the preparation

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of certain alloys, e.g., brass. It is brittle and crystalline at ordinary temperatures, but when heated to between 110°C and 150°C it becomes ductile and malleable which then can be rolled into sheets. It is a fairly reactive metal. Zinc compounds are numerous and are widely used. Zinc is essential to the growth of many kinds of organisms, both plant and animal. It is a constituent of insulin, which is used in the treatment of diabetes. Also zinc is a constituent of many enzymes that permit chemical reactions to proceed at normal rates. In addition it is involved in the transmission and expression of genetic information and in protein synthesis. Zinc has the least toxicity among the essential trace elements in the body. Functions and deficiencies: Zinc has a range of functions. It plays a crucial role in growth and cell division where it is required for protein and DNA synthesis, in insulin activity, and in the metabolism of the ovaries and testes. As a component of many enzymes, zinc is involved in the metabolism of proteins, carbohydrates and lipids. Deficiency of zinc is associated with short stature, anemia, increased pigmentation of skin (hyperpigmentation), an enlarged liver and spleen (hepatosplenomegaly), impaired gonadal function (hypogonadism), impaired wound healing, and immune deficiency. In addition zinc deficiency causes significant delay in growth and dysfunction of sexual glands. Zinc deficiency in agricultural soils is also a major problem affecting both crop yield and quality. Severe soil zinc deficiency can cause complete crop failure. Losses of up to 30% can occur in the yield of cereal grains in crops such as wheat, rice and maize as a result of even mild deficiencies. RDA: The RDA is as follows: adults 15-30 mg/d, pregnant women 15 mg/d, infants 5 mg/d, male children over 10, 15 mg/d and female children over 10, 12 mg/d. Food sources: Food sources of zinc include meat, liver, seafood, eggs, nuts, and cereal grains. In general, meat, eggs and dairy products contain more zinc than plants. Liver is a particularly rich source of zinc and high zinc levels are also found in wheat, rye, yeast and oysters. White sugar and citrus fruits have some of the lowest zinc levels. Chemical forms as functional ingredients: The following zinc compounds are used as functional ingredients: zinc oxide, zinc sulfate, zinc glucuronate, zinc lactate, zinc citrate and zinc carbonate. Nutrient functional claims: Japan has the following approved health claims of zinc: It may help to maintain normal growth of infants, may reduce the risk of gastric ulcers and may reduce the risk of reproductive function failure. The United States has none.

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Selenium (Se) Chemistry: Selenium was discovered by Jöns Jacob Berzelius in 1817, who found the element associated with tellurium. This is a toxic nonmetal that is chemically related to sulfur and tellurium. It occurs in several different forms but one of these is a stable gray metal like form that conducts electricity better in the light than in the dark and is used in photocells. This element is found in sulfide ores such as pyrite. Selenium is an essential micronutrient in all known forms of life. It is a component of the unusual amino acid selenocystein. Functions and deficiencies: Selenium acts as an antioxidant and protects cells against damage by eliminating free radicals and other antioxidant enzymes. It binds with toxic substances such as arsenic, cadmium and mercury to make them less harmful. In humans, like other animals, selenium supplementation has appeared to offer some anticancer protection. Selenium deficiency in western regions of China has been found to be associated with Keshan diseases, a cardiomyopathy found only in the People’s Republic of China (Keshan Disease Research Group, 1979). The addition of selenium to salt significantly reduced the incidence of liver cancer in a Chinese population. It was shown that five years of supplementation with selenium, vitamin E, and carotene significantly reduced the incidence of stomach and esophageal cancer in a Chinese population. Epidemiological and animal experiments suggest that low selenium intakes and low plasma selenium concentrations increase the risk of coronary heart disease. There are no clear symptoms of selenium deficiency. However, it can occur in patients with severely compromised intestinal function or those undergoing total parenteral nutrition. Lake of selenium can result in the degeneration of skeletal muscles. RDA: A small amount is needed to maintain good heath. 55 µg/d for women and 70 µg/d for men is recommended. More than 400 µg/d can lead to toxicity (selenosis). Food sources: Brazil nuts are the riches source of selenium. It is also found in whole-grain cereals, fish, lobster, meat and dairy products. Chemical forms as functional ingredients: Selenomethionine. Nutrient function claims: There are no approved health claims by Japan or the United States. Chromium (Cr) Chemistry: Chromium was discovered by Louis-Nicholas Vauquelin while experimenting with a material known as Siberian red lead, also known as the mineral crocoite (PbCrO4), in 1797. He produced chromium

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oxide (CrO3) by mixing crocoite with hydrochloric acid (HCl). Today, chromium is primarily obtained by heating the mineral chromite (FeCr2O4) in the presence of aluminum or silicon. Chromium is a bluewhite metal that is hard, brittle and very corrosion resistant. Chromium can be polished to form a very shiny surface and is often plated to other metals to form a protective and attractive covering. Chromium is added to steel to harden it and to form stainless steel, a steel alloy that contains at least 10% chromium. Other chromium-steel alloys are used to make armor plate, safes, ball bearings and cutting tools. Functions and deficiencies: Chromium metabolizes carbohydrate and helps to raise HDL cholesterol which may prevent high cholesterol and atherosclerosis. Signs of chromium deficiency include diabetes-like symptoms of high blood cholesterol and problems with insulin levels. RDA: There are none. 50 to 200 µg/d is suggested. Food sources: It is found in brewer’s yeast, broccoli, ham, grape juice, and whole wheat grains. Chemical forms as functional ingredients: Chromium picolinate, chromium chloride, chromium niacin amino acid and chromium nicotinate are also used as food additives. Nutrient functional claims: There are no approved health claims by Japan or the United States. Copper (Cu) Chemistry: People discovered methods for extracting copper from ore at least 7,000 years ago. The Roman Empire obtained most of its copper from the island of Cyprus, which is where copper’s name originated. Today, copper is primarily obtained from the ores cuprite (CuO2), tenorite (CuO), malachite (CuO3·Cu(OH)2), chalcocite (Cu2S), covellite (CuS) and bornite (Cu6FeS4). Large deposits of copper ore are located in the United States, Chile, Zambia, Zaire, Peru and Canada. Copper is used in large amounts by the electrical industry in the form of wire and is second only to silver in electrical conductance. It resists corrosion from the air, moisture and seawater, therefore, it has been widely used in coins. Hydrated copper sulfate (CuSO4·H2O), also known as blue vitrol, is the best known copper compound. It is used as an agricultural poison, as an algicide in water purification and as a blue pigment for inks. Cupric chloride (CuCl2), another copper compound, is used to fix dyes to fabrics. Functions and deficiencies: Copper plays an important role in the production of neurochemicals in the brain and in the function of muscles, nerves and the immune system. It helps in the cross-linkage

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of collagen and elastin the two important connective tissues which are used throughout the body. It builds bones, red blood cells and hemoglobin, and metabolizes iron. Deficiency of copper is uncommon, but is sometimes found in combination with iron deficiency especially with iron deficiency anemia. Fatigue, paleness, skin sores, edema, slowed growth, hair loss, anorexia, diarrhea and dermatitis can be the symptoms of copper insufficiency. RDA: None; 2 to 3 mg/d is the suggested amount. Copper is toxic in large amounts (likely to cause vomiting). Food sources: It is found in shellfish, nuts, seeds, cocoa powder, beans, whole grains, and mushrooms. Chemical forms as functional ingredients: Copper gluconate, copper sulfate, cupric acetate. Nutrient functional claims: There are no approved health claims either by Japan or the United States. Fluoride Chemistry: Fluoride is the form of fluorine that normally exists in nature. Fluoride is added to most drinking water supplies. It is considered a beneficial nutrient and is present in trace amounts in the body. Fluoride is important for the integrity of bones and teeth. About 99% of the fluoride in the body is in the hard tissues. Fluoride is consumed in optimal amounts from water and food. It is in toothpastes, mouth rinses, and professionally applied office treatments. Functions and deficiencies: Fluoride increases tooth mineralization and bone density, and reduces the risk and prevalence of dental caries (decay). Fluoride deficiency may appear in the form of increased incidence of dental caries and unstable bones and teeth. RDA: Adults need 1.5 to 4 mg/d, children up to six months, 0.1 to 0.5 mg/d, ages six to 11 months, 0.2 to I mg/d and ages one to three years, 0.5 to 1.5 gm/d. Food sources: It can be found in fluoridated water, tea, coffee, soybeans, marine fish with bones such as canned salmon and mackerel, Chemical forms as functional ingredients: Sodium fluoride, sodium fluorophosphate and hexafluorosilicic acid are commonly used as food additives. Nutrient functional claims: There are no approved health claims by Japan or the Uinted States. Iodine (I) Chemistry: Iodine was discovered by the French chemist Barnard Courtois in 1811, which he isolated from treating seaweed ash with

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sulphuric acid while recovering sodium and potassium compounds. Iodine is an insoluble element and is a required trace element for living organisms. Chemically, iodine is the least reactive of the halogens and the most electropositive metallic halogen. It is primarily used in medicine, photography and in dyes. Iodine is a bluish-black, lustrous solid that sublimes at standard temperatures into a blue-violet gas that has an irritating odor. Iodine dissolves easily in chloroform, carbon tetrachloride or carbon disulfide to form purple solutions. The deep blue color with starch solution is characteristic of the free element. Functions and deficiencies: Iodine is part of thyroxin, a hormone produced by the thyroid gland that controls the body’s rate of physical and mental development. Adequate iodine intake during pregnancy is crucial to normal fetal development. Lack of iodine can cause a goiter (swelling of the thyroid gland). Iodine is added to salt (iodized salt) to prevent these diseases. RDA: Trace amounts of iodine are required by the human body. The RDA is 150 µg/d. Food sources: Iodized salts, lobster, shellfish, sea kelp, seaweed, mushrooms, sesame seeds, soybeans, spinach (cooked) and turnip greens are its food sources. Chemical forms as functional ingredients: Potassium iodide(KI) is added to table salt to make it iodized. Potassium iodate for food fortification is another form. Nutrient functional claims: There are no approved functional claims either by Japan or the United States. Molybdenum (Mo) Chemistry: Molybdenum is a silvery-white, hard, transition metal. Scheele discovered it in 1778. Molybdenum is used in alloys, electrodes and catalysts. It is an essential trace mineral in animal and human nutrition which is not found in free state. The pure metal is very hard and has one of the highest melting points of all pure elements (m.p. 2623°C). Functions and deficiencies: It functions as a cofactor for a number of enzymes that catalyze important chemical transformations in the global carbon, nitrogen, and sulfur cycles. Thus, molybdenum-dependent enzymes are not only required for the health of the people, but for the health of its ecosystems as well. In spite of its low abundance, molybdenum deficiency in humans has been observed. For example, patients suffering from longterm total parenteral nutrition (TPN) condition develop a syndrome characterized by hypouricemia, hypermethioninemia, low urinary sulfate

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excretion, tachycardia, tachypnea and mental and visual disturbances. These symptoms are improved when molybdenum in the form of ammonium molybdate, is added to the TPN. The deleterious effects of molybdenum deficiency are primarily due to the accumulation of sulfite coming from the catabolism of L-cysteine. Sulfite is toxic to the nervous system and molybdenum is necessary for its metabolism to a nontoxic form. However, the dietary molybdenum deficiency has never been observed in healthy people. RDA: 150-500 mg/d is the recommendation. Food sources: It is found in legumes, whole-grain cereals, liver, kidney and dairy products. Chemical forms as functional ingredients: Tetrathiomolybdate, ammonium molybdate are normally used as food additives. Nutrient functional claims: There are no approved health claims by Japan or the United States Manganese (Mn) Chemistry: Manganese was isolated by Gahn in 1774 after reducing the dioxide MnO2, the mineral pyrolusite with charcoal. Manganese is a grey-white metal, resembling iron and is very brittle, is fusible with difficulty and is easily oxidized. It becomes ferromagnetic (a material with high magnetic permeability) only after special treatment. Functions and deficiencies: Manganese is an essential trace nutrient for all forms of life. It is involved in reproductive processes, sex hormone formation and is essential for normal brain function and bone development. The classes of enzymes that have manganese cofactors are very broad such as oxidoreductase, transferases, hydrolyases, lyases, isomerases, ligases, lectins and integrins. Manganese deficiency has been observed in a number of animal species. Signs of manganese deficiency include impaired growth, impaired reproductive function, skeletal abnormalities, impaired glucose tolerance and altered carbohydrate and lipid metabolism. In humans, demonstration of a manganese deficiency syndrome has been less clear. Women fed a manganese-poor diet developed mildly abnormal glucose tolerance in response to an intravenous infusion of glucose. RDA: 2.5-7 mg/day is recommended. Food sources: It is found in canned pineapple juice, wheat bran, wheat germ, whole grains, seeds, nuts, cocoa, tea, oats and rice. Chemical forms as functional ingredients: Manganese chloride, manganese gluconate, manganese glycerophosphate, manganese sulfate are the manganese salts used in functional foods.

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Nutrient functional claims: There are no approved health claims either by Japan or the the United States. SUMMARY Vitamins are noncaloric, organic nutrients that are essential for life. Although we need only small amounts of vitamins, the roles they play both independently and synergistically are life-giving. They are integral helpers in all cell functions. Extra vitamins will not treat anxiety, depression or lack of adequate rest, bad interpersonal relationships or unhappiness. Most people can do their best by eating well and exercising regularly. Persons at risk may require an additional supplement of vitamins and minerals; however, one shouldn’t exceed the recommended daily values. As science continues to uncover the many roles for all of these vitamins and minerals, scientists are also finding exciting solutions to several disorders that may be successfully treated by using these nutrients. Unless we begin replacing these minerals early on in life, we put ourselves at risk for many diseases of mineral deficiency that are becoming more and more prevalent in society today. One should keep in mind that excessive intake of these ingredients may either lead to illness directly or indirectly because of the competitive nature between mineral levels in the body. REFERENCES Alpet, M.M. Silva, R.R., and Pouget, E.R. 2003. Prediction of treatment response in geriatric depression from baseline folate level: interaction with an SSRI or a tricyclic antidepressant. J. Clin. Psychopharmacol, 23(3):309-13. Blakley, R.L., 1987. IUPAC-IUB joint commission on biochemical nomenclature (JCBN). Nomanclature and symbols for folic acid and related compounds. Recommendations 1986. Eur. J. Biochem. 168, 251. Chambers, J.C., Ueland, P.M., and Obeid, O.A. 2000. Improved vascular endothelial function after oral B vitamins: An effect mediated through reduced concentrations of free plasmahomocystein. Circulation, 102(20):2479-83. Day, B.P.F., and Gregory, J.F. 111. 1983. Thermal stability of folic acid and 5methyltetrahydrofolic acid in liquid model food systems. J. Food Sci. 48, 581. Dismore, M.L., Haytowitz, D.B., Gebhardt, S.E., Peterson, J.W., Booth, S.L. 2003. Vitamin K content of nuts and fruits in the US diet. J. Am. Diet. Assoc. 103:16501652. Ellenbogen, L., and Cooper, B.A., Vitamin B12, in Handbook of Vitamins, 2nd ed., Machlin, L.J., Ed., Marcel Dekker, New York, 1991, chap.13. Fairbanks, V.F. Iron in Medicine and Nutrition. In: Shils, M., Olson, J.A, Shike, M.R AC, eds. Nutrition in Health and Disease. 9th ed. Baltimore: Williams and Wilkins; 1999:223-239. Food and Nutrition Board, Institute of Medicine.Vitamin E. Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids. Washington D.C.: National Academy Press; 2000:186-283. (National Academy Press).

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Ford, E.S. 1999. Serum magnesium and ischaemic heart disease: Findings from a national sample of US adults. Int. J. Epidem. 28:654-651. Gregory, J.F. 111 and Kirk, J.R. 1977. Interaction of pyridoxal and pyridoxal phosphate with peptides in a model food system during thermal processing. J. Food Sci, 42, 1554. Gregory, J.F. 111., Ink, S.L., and Sartain, D.B. J.R. 1986. Degradation and binding to food proteins of vitamin B6 compounds during thermal processing. J. Food Sci., 51, 1345, 1986. Holick, M.F. 2004. Vitamin D: importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Am. J. Clin. Nutr. 79:362-71. Jialal, I., and Fuller, C.J. 1995. Effect of Vitamin E, vitamin C and beta-carotene on LDL. Oxidation and atherosclerosis. Can. J. Cardiol, 11 Suppl. G:97G-103G. Keshan Disease Research Group. 1979. Epidemiological studies on the etiological relationships of selenium and Keshan disease. Clin. Med. J. 92:477-482. Leske, M.M., Chylack, L.T. Jr., He, Q., Wu, S.Y., Schoenfeld E, Friend, J., and Wolfe, J. 1998. Antioxidant vitamins and nuclear opacities: The longitudinal study of cataract. Ophthalmology, 105:831-6. Lonn, E.M., and Yusuf, S. 1997. Is there a role for antioxidant vitamins in the prevention of cardiovascular disease? An update on epidemiological and clinical trials data. Can. J. Cardiol, 13:957-65. Mitchell, H.K., Snell, E.E., and Williums, R.J. 1941. The concentration of folic acid. J. Am. Chem. Soc. 63:2284. Moat, S.J., Lang, D., and McDowell, I.F. 2004. Folate, homocystein, endothelial function and cardiovascular disease. J. Nutr. Biohem. 15(2):64:79. Leklem, J.E. Vitamin B6. In: Shils, M.E, Olson, J.A, Shike, M. Ross AC, ed. Modern, Nutrition in Health and Disease. 9th ed. Baltimore: Williams and Wilkins, 1999: 413-421. Piironen, V., Syväoja, E.L., Varo, P., Salminen, K., and Koivistoinen, P. 1985. Tocopherols and tocotrienols in Finnish foods:meat and meat products. J. Agric. Food Chem., 33, 1215-1218. The heart outcomes prevention evaluation study investigators. 2000. Vitamin E supplementation and cardiovascular events in high-risk patients. N. Engl. Med. 342:154-60. Tucker, K.L., Hannan, M.T., Chen, H., Cupples, L.A., Wilson, P.W.F and Kiel, D.P. Am. J. Clin.Nutr. 69:727736. U.S. Department of Agriculture, Agricultural Research Services. 2004. USDA National Nutrient Database for Standard Reference, Release 16-1. Nutrient Data Laboratory Home Page, http://www.nal.usda.gov/fnic/foodcomp. Vieth, Reinhold. 1999. Vitamin D supplementation, 25-hydroxyvitamin D concentrations and Safety. Am. J. Clin. Nutr. 69:842-26. Weizmann, N., Peterson, J.W., Haytowitz, D., Pehrsson, P.R., de Jesus, V.P., and Booth, S.L. 2004. Vitamin K content of fast foods and snack foods in the US diet. J. Food Comp. and Anal. 17:379-384. (Guo, M. R., Alam, M.)

CHAPTER 7

SOY FOOD PRODUCTS AND THEIR HEALTH BENEFITS The soybean [Glycine max (L.) Merrill], a native of China, has been used in various forms as one of the most important sources of dietary protein and oil. So this little old bean has been called “yellow jewel”, “great treasure”, “nature’s miracle protein”, and “meat of the field”. Most recently, in the Western world, the soybean has been touted as a possible weapon against chronic diseases. Soybean, combines in one crop both the dominant world supply of edible vegetable oil, and the dominant supply of high-protein feed supplements for livestock. Other fractions and derivatives of the seed have substantial economic importance in a wide range of industrial, food, pharmaceutical, and agricultural products (Figure 7.1). History Of The Soybean The soybean first emerged as a domestic crop in the eastern half of North China, around the 11th century B.C. of Zhou dynasty. The soybean, then known as ‘shu’ is repeatedly mentioned in later records and was considered one of the five sacred grains along with rice, wheat, barley, and millet essential for Chinese civilization. Later, ‘shu’ was found inscribed on tortoise shells from the Shang dynasty (from about the 16th to the 11th century B.C.). Soybean seeds have been discovered several times in relics unearthed in archaeological studies. In 1959, large amounts of yellow seeded soybeans weighing 18-20 g and dating back 2300 years were found in Shanxi Province. Soybean cultivation spread into Japan, Korea, and throughout Asia from China. The soybean was first introduced to Europe in about 1712 by a German botanist, Engelbert Kaempfer, out of curiosity. Later Carl von Linne, a Swedish botanist, gave a genetic name Glycine max, to soybeans. Glycine

FIGURE 7.1 — Utilization Of Soybean Products

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is a Greek word meaning “sweet”, and it applies to all the groundnut species of legumes. The word max means “large,” referring to the large nodules on the soybean plant. The early introduction of soybeans into the United States dates back to the mid-eighteenth century. A gentleman named Samuel Bowen sailed on a British ship and reached Canton, China in 1759, and he stayed there for several years. In 1764, Bowen immigrated to Savannah, Georgia, and apparently brought soybean samples with him. The soybean was planted in the local plantation the next year. However, the large-scale official introduction did not occur until the early 1900s. By the late 1920s, William Morse brought in a number of new varieties mostly from China. He played a main role in forming the American Soybean Association and became its first president. Meanwhile, there were breakthroughs in harvesting and processing and as a result large scale production had begun. Until 1954 China led the world in soybean production and export. After 1954, The United States became the world leader (Figure 7.2 and Figure 7.3). FIGURE 7.2 — World Soybean Production 2003 In Million Metric Tons

FIGURE 7.3 — Annual Soybean Production In The United States And The Whole World (Soya Bluebook 2004)

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Morphology Of Soybean The soybean is a papilionoid legume (family Fabaceae, subfamily Faboideae), and a member of the tribe Phaseoleae, subtribe Glycininae. The soybean plant is a branched, non-frost tolerant, annual about one meter above ground level and two meters below ground level. The stem tissues are mostly primary, although the basal and more mature portions of the stems develop secondary vascular tissues during later development. The foliage leaves are alternate, pinnately trifoliolate, with pulvini, stipels, and stipules. The soybean flower is a standard papilionaceous flower with calyx of five united sepals; zygomorphic corolla of carina, alae, and vexillum; androecium of ten diadelphous 9+1 stamens; and gynoecium of a single carpel. Two to four seeds develop in the pods. The seeds have two large cotyledons and scant endosperm. The mature seeds are made of three basic parts: the seed coat, the embryo, and one or more food storage structures. Chemical Composition Of The Soybean Dry soybeans are constituted of 60% of oil and protein together. Among cereal and other legumes, soybeans have the highest protein content (around 40%); other legumes have protein content between 20% and 30%, whereas cereals have protein content in the range of 8-15%. They also contain 20% lipids, the second highest content among all other legumes. The remaining dry matter is composed of 35% carbohydrates and 5% ash (Figure 7.4). Other valuable components found in soybeans include phospholipids (0.2-0.6%), phenolic acids (0.03%), isoflavones (0.2% in flour), saponins (0.5% in flour), phytic acid (1.7% in flour), and vitamins. FIGURE 7.4 — Chemical Composition Of Soybeans Chemical composition (% dry matter) Protein Lipid Phospholipids Carbohydrate Soluble Sucrose Raffinose Stachyose Insoluble Ash

Soybean 40 20 0.2-0.6 35 13.2 2.5-8.2 0.1-0.9 1.4-4.1 21.8 5.0

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PROTEIN Soybeans contain approximately 40% protein on dry weight basis. Soybean proteins are classified based on different criteria such as biological function in plants and solubility patterns. Classification Of Soy Proteins Based On Biological Function Metabolic proteins: Metabolic proteins include enzymatic and structural proteins. They play a role in normal cellular activities, including the synthesis of the storage proteins. Examples include hemagglutinin, trypsin inhibitors, and lipoxygenases. Storage proteins: Storage proteins are synthesized during soybean seed development together with reserves of oils. Following seed germination they provide a source of nitrogen and carbon skeletons for the developing seedling. Storage proteins constitute 80% of total protein in soybean. Examples of storage proteins are glycinin and conglycinin which will be discussed in detail in a later section because they constitute a major portion of the total protein in the soybean. Classification Based On Solubility Pattern Albumins: Albumins are soluble in water. Globulins: Globulins are soluble in a salt solution. Most soy protein is globulin. Globulins in most legume species are further classified into two distinct types: legumin and vicilin. Legumins have larger molecular size, less solubility in salt solutions, and higher thermal stability compared with vicilins. They constitute a major part of the seed globulins. In soybeans, legumins and vicilins are commonly known as glycinin and conglycinin, respectively. These common names are apparently derived from the genus name of the soybean plant, Glycine. Classification Based On Sedimentation Coefficient As each protein is associated with other proteins, there is no assurance that a single pure protein would be extracted based on difference in solubility. A more precise means of identifying proteins has been based on sedimentation coefficients using ultracentrifugation to separate seed proteins. Soy protein exhibits four fractions after centrifugation which are designated as 2, 7, 11, and 15S. Here S stands for Svedburg unit. It is computed as the rate of sedimentation per unit field of centrifugal strength based on the equation S=(dc/dt) w2c where as c is the distance from the centre of centrifuge, t is time, and w is angular velocity. The value for S ranges between 1 and 200, with a unit of 10-13 sec.

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2S fraction: This accounts for about 20% of the extractable protein and includes the Kunitz and Bowman-Birk trypsin inhibitors and cytochrome c. 7S fraction: This accounts for 35% of the extractable protein and consists of conglycinin,
-amylase, lipoxygenase and hemagglutinin. 11S fraction: This fraction is the soybean glycinin and accounts for 35% of extractable protein. 15S fraction: This is thought to be a polymer of glycinin and accounts for about 10% of extractable protein. However, this classification based on sedimentation coefficient is only for the purpose of classification and identification. By no means do they imply that sedimentation constants are always these exact whole numbers among different studies. In fact, these constants as well as separation patterns of different fractions depend largely on conditions of buffer composition, pH, and other factors. Glycinin and Conglycinin: These are two major soybean globulins which differ in both nutritional quality and functional properties. The 11S globulin contains 3-4 times more methionine and cysteine per unit protein than 7S protein. The 11S protein becomes more valuable from a nutritional point of view because soybean protein in total is deficient in these sulphur containing amino acids. The two globulins also show considerable differences in key functional properties, including gelling ability, thermal stability, and emulsifying capacity. In general, the 11S protein has a better gel formation ability than the 7S globulin. On the other hand, the 7S protein has a greater emulsifying capacity and emulsion stability than 11S globulin. Furthermore, the presence or absence of the A5A4B3 subunit in glycinin has been shown to exert significant effects on the gelling properties of soymilk and tofu gel hardness; it is easier to make nigari tofu with a smoother and more uniform gel using the soymilk lacking the subunit. Both 11S and 7S proteins form gels when induced by heat and/or a coagulant as in tofu making. In the heat-induced gel formation, the 7S gels were harder than 11S gels when heated at 80°C for 30 min. However, the denaturation temperature of 7S protein is lower than that of 11S. In other words, the 11S fraction requires a higher heating temperature to form a gel than 7S. In the presence of calcium sulfate, 11S protein coagulates faster and forms larger aggregates than the 7S fraction. More important is that the 11S gel is harder than the 7S gel. It also has a higher water-holding capacity and higher tensile values, expands more

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on heating, and is more sensitive to the softening effect of phytic acid, as compared with the 7S gel. Relationships between the structure of a protein and its functional properties include; • Heat instability of the constituent subunits of protein, such as glycinin, is related to the heat-induced gel-forming ability. • Hydrophobicity is an important factor in the emulsifying properties. • The surface properties of a protein depend on the conformational stability—the more unstable, the higher the emulsifying properties. Disulfide linkages play an important role in the formation of heatinduced gel. The number and topology of free sulfhydryl residue are closely related to heat-induced gel-forming ability and the properties of gel but not to its emulsifying properties. Trypsin Inhibitors Trypsin inhibitors are protease inhibitors which when added to a mixture of protease (such as trypsin and chymotrypsin) and a substrate, bind to the enzyme and produce a decrease in rate of substrate cleavage. Trypsin inhibitors isolated from soybean are of two types: the Kunitz trypsin inhibitor (TI) and the Bowman-Birk (BB) inhibitor. They are associated with the storage proteins in the seed. The Kunitz inhibitor was first isolated and crystallized by Kunitz by extracting soybeans with water and precipitating the inhibitor with alcohol. It has a molecular weight between 20 and 25 kDa, with a specificity directed primarily toward trypsin. The inhibitor was shown to combine tightly with trypsin in a stoichiometric fashion i.e., 1 mole of the inhibitor inactivates 1 mole of trypsin. The amino acid sequence shows it has 181 amino acid residues and two disulfide bonds, with a reactive site at residues Arg63 and Ile64. The soybean BB inhibitor was first isolated by extracting beans with 60% alcohol solution and precipitating the inhibitor with acetone. It is an acetone insoluble fraction in contrast to the alcohol-insoluble Kunitz inhibitor. It has a molecular weight of about 8 kDa. The amino acid sequence showed that it is a single polypeptide chain of 71 amino acids including seven disulfide bonds. The BB inhibitor is capable of inhibiting both the trypsin and chymotrypsin at independent reactive sites, the trypsin reactive site being at residues Lys 16 and Ser 17 and the chymotrypsin reactive site being at Leu44 and Ser45. The conformation (secondary structure) of BB inhibitor has 61% β-sheet, 38% unordered form, 1% β-turn, and 0%
-helical form suggesting that it has a stable conformation even after disulfide bonds are broken by heating.

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Lectin The lectins, also known as hemagglutinins are proteins that possess a specific affinity for certain sugar molecules. Since carbohydrate moieties exist in most animal cell membranes, they may attach themselves to these so-called receptor groups if the specific structure of the latter is suitable. As indicated by their names, hemagglutinins or lectins can be characterized and detected by their action on red blood cells: the ability to agglutinate the blood cells. Lectins are characterized by a relatively high content of 4hydroxyproline. Their ability to agglutinate cells results from their ability to bind specifically to saccharides on the cell membranes and act as bridges between cells. Because of this feature, lectins have provided a new tool for cell biologists to investigate the architecture of cell surfaces. Seed lectins are primarily localized in the protein bodies of the cotyledon cells. Soy lectin settles down with the 7S fraction during ultracentrifugation indicating it has sedimentation coefficient of 7S. It is a glycoprotein containing 5 glucosamine and 37 mannose residues per mole and has a molecular weight of approximately 120 kDa with four identical subunits each of which has a molecular weight of 30 kDa. Lunasin Lunasin is a unique 43 amino acid soybean peptide, whose carboxyl end contains nine ASP (D) residues, an Arg-Gly-Asp (RGD) cell adhesion motif, and a helix with structural homology to a conserved region of chromatin binding proteins. Lipoxygenases Lipoxygenase is an iron-containing dioxygenase that catalyzes the oxidation of certain polyunsaturated fatty acids, producing conjugated unsaturated fatty acid hydroperoxides. The iron atom in lipoxygenases is essential for enzymatic activity due to its reduction potential for the reaction. The enzyme also has an ability to form free radicals, which can attack other constituents. Soybean lipoxygenase is the most studied, and four types of the enzyme have been isolated and identified as L-1, L-2, L-3a and L-3b. All isozymes are monomeric proteins with a molecular weight in the range of 100,000 and contain one atom of tightly bound nonheme iron per molecule. L-1, the best characterized enzyme among the isozymes, differs from the others in being heat stable, having a pH optimum of approximately 9, and preferring anionic substrates e.g., linoleic and linolenic acids. L-2 and L-3 are less heat stable, prefer esterified

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substrates and have optimal pH close to neutrality. With linoleate as a substrate, the isozymes also differ in their product regiospecificity; L-1 shows a preference for the 13 position as the site for hydroperoxidation, whereas, L-2 and L-3 use either position 9 or 13. FAT Soybean contains about 20% fat which is mainly in the form of triglycerides, in an organelle known as oil bodies or lipid bodies or spherosomes or oleosomes or lipid-containing vesicles. Triglycerides Triglycerides constitute more than 99% of refined soybean oil. Any given natural fat or oil has a unique fatty acid composition regardless of its origin. Most fatty acids in soybean are unsaturated like many other oils of plant origin. The highest percentage of fatty acids in soybean oil is linoleic acid (53.2%), followed in a decreasing order by oleic (23.4%), palmitic (11.0%), linolenic (7.8%), and stearic acid (4.0%). It also contains some minor fatty acids, including arachidic (0.3%), behenic (0.1%), palmitoleic (0.1%) and myristic acid (0.1%). There is a large genetic variation in the fatty acid composition of soybean oil, mainly resulting from plant breeding. Lipids exhibit a difference in physical properties as well as oxidative stability during storage and food application due to a difference in their fatty acid composition. One of the physical properties, such as melting point, is higher for the fats containing fatty acids with greater chain length. Oils containing a high percentage of saturated fatty acids have a high melting point, giving a semisolid or solid appearance, whereas, those containing a high percentage of unsaturated fatty acids have a low melting point, thus giving a liquid appearance as in the case of soybean oil. The presence of a double bond in unsaturated fatty acid also makes it susceptible to oxidation, leading to the development of an off flavor. The more double bonds present, the less stable the fatty acid. The chemical stability of soybean oil has been a problem because it contains relatively high proportions of both linoleic and linolenic acids which contain 2 and 3 double bonds, respectively. To increase the melting point as well as the oxidative stability of soy oil, hydrogenation becomes necessary. Another quality factor is the distribution of fatty acids at the glycerol molecule of a triglyceride. The orderly fatty acid distribution in soybean oil as well as other fats and oils can be altered to a random pattern by an industrial process known as interesterification. A general chemical

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interesterification involves heating oil to drive away water residue, mixing the oil with a catalyst, normally sodium methylate or sodium ethylate, breaking the emulsion after completion of the reaction, and separating and drying the oil layer. Although it does not change the fatty acid composition, interesterification generally increases crystallization tendencies (melting point) of fats and oils. Phospholipids Phospholipids together with proteins are building blocks of biological membranes. Hence, they invariably occur in all foods of animal and plant origin. Phospholipids constitute 1-3% of crude soybean oil. Lecithin (phosphatidylcholine) is a major component constituting about 35-40% of the total phospholipids and the rest being about 25% phosphatidylethanolamine, about 15% phosphatidyl inositol, 5-10% phosphatidic acid and the rest is a composite of all the minor phospholipids compounds. Phospholipids contain glycerol, two fatty acids, a phosphate and a basic component. Phosphatidic acid, the parent molecule of phospholipids, is formed by the joining of 3-glycerol-phosphate and a diglyceride; then the base (choline or other) is linked to the phosphate group of phosphatidic acid to form phosphatidyl choline (or other phospholipids). The other phospholipids have structures like phosphatidylcholine’s except for the difference in their base: choline versus serine, ethanolamine, or inositol. Free Fatty Acids Soybean oil contains about 0.3-0.7% free fatty acids which are formed when the enzyme lipase in the soybean acts on the fatty acids in the triglyceride molecule. Plant Sterols Plant sterols represent a class of non-nutrient molecules that are consumed in large amounts because of their ubiquitous presence in plant cell membranes, but they have no known function. Their major benefit to mankind is indirect as their consumption from plant foods increases, the intake of cholesterol decreases because of the reduced consumption of animal products (for the information about health benefits of plant sterols, see Chapter 2). Refined soybean oil contains plant sterols namely βsitosterol, campestrol, and stigmasterol, which constitute almost 100% of the membrane sterols of soybeans, serve a role in plants much like that of cholesterol in animal membranes. These sterols are in the ratio of 2.5:1:1, in which the total sterols represent 221 mg/100 g of oil.

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CARBOHYDRATES Carbohydrates constitute about 35% of the dry weight of soybeans which make them the second largest component in soybeans. The major components are polysaccharides (amyloses and amylopectins) and indigestible fiber, but the flatulence-producing disaccharides raffinose and stachyose also exist in soybeans. Soluble Carbohydrates Soybeans contain trace amounts of monosaccharides, such as glucose and arabinose, and measurable amounts of di- and oligosaccharides, with sucrose in the range of 2.5-8.2%; raffinose 0.1-0.9%; and stachyose 1.4-4.1%. Oligosaccharides in soybeans are nonreducing sugars, containing fructose, glucose, and galactose as two or more units, linked by β-fructosidic and
-galactosidic linkages. Insoluble Carbohydrates The insoluble carbohydrates in soybeans include cellulose, hemicellulose, pectin, and trace amounts of starch. They are structural components found mainly in cell walls. The seed coat makes up about 8% of the whole soybean by dry weight and contains about 86% complex carbohydrates. Therefore, it contributes a noticeable amount of insoluble carbohydrates to the whole bean. Soy cell walls contain about 30% pectins, 50% hemicellulose and 20% cellulose. Therefore, most soybean carbohydrates fall into a category known as dietary fiber. MINERALS Soybeans have an ash content of approximately 5%. The oxygen content of the ash accounts for much of its weight since the major forms of minerals in ash are sulfates, phosphates, and carbonates. Among the major mineral components in soybeans, potassium is found in the highest concentration, followed by phosphorous, magnesium, sulphur, calcium, chloride and sodium. The contents of these minerals range from 0.2 to 2.1 g/100 g on average values (dry weight basis). The minor minerals present in soybeans and soy products include silicon, iron, zinc, manganese, copper, molybdenum, fluorine, chromium, selenium, cobalt, cadmium, lead, arsenic, mercury and iodine. The contents of these minor minerals range from 0.001 mg/100g to 14 mg/100 g. Like other components, minerals in soybeans are also influenced by agronomical conditions and genetic variation.

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PHENOLIC ACIDS The term ‘phenolic acids’ covers a large group of organic molecules found naturally in soybean and its derivative products but only in small quantities of approximately 1-10 mg/100 g. The phenolic acids, simple molecules such as the organic solvent benzoic acid, are included in the broad class of polyphenols because of diverse phenol groups within them. Also, typically each of these phenol groups contains one and sometimes two hydroxyl groups, which increase their solubility in aqueous solutions. They are also found in the soybean spatially associated with proteins. All of the phenolic molecules are thought to be derived from amino acid phenylalanine. Isoflavones The basic structural feature of flavonoid compounds is the flavone nucleus, which comprises two benzene rings (A and B) linked through a heterocyclic pyrane C ring (Figure 7.5). The position of the benzenoid B ring divides the flavonoid class into flavonoids (2position) and isoflavonoids (3-position). The primary isoflavones of soybeans are genistein (4’,5,7-trihydroxy-isoflavone) and daidzein (4’,7-dihydroxyisoflavone), and their respective beta-glycosides, genistin and diadzin (sugars being attached at the 7 position of the A ring). Typically, there is more genist(e)in than diadz(e)in in soybeans and soyfoods. There is also a small amount of other isoflavones, glycitein (7,4’-dihydroxy-6-methoxyisoflavone) and its glycoside glycitin. In total there are 12 different soybean isoflavone isomers; in addition to the six described above, each of the isoflavone glycosides can have attached, an acetyl or malonyl group at carbon 6 of the glucose molecule. In non-fermented soyfoods, the isoflavones appear mostly as the conjugate, whereas, in fermented soy products such as miso, the aglycones dominate. In addition to the isoflavones in soybeans, the intestinal microflora can convert diadzein into several different isoflavonoid products, including equol (7-hydroxyisoflavan), dihydrodaidzein and O-desmethyl-angolensin. The majority of isoflavones is associated with proteins. Soybeans and soy products contain roughly 1-3 mg isoflavones per gram protein, one serving of traditional soyfoods (i.e., ½ a cup of tofu or 1 cup of soymilk) containing about 30 mg isoflavones, expressed as the aglycone form.

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FIGURE 7.5 — Structure Of The Primary Isoflavones In Soybeans

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Saponins Saponins are compounds consisting of triterpenoidal or steroidal aglycones with various carbohydrate moieties that are found in many plants. Soy saponins have been purified and classified by their structure into 3 groups: A, B, and E. Because of the presence of both hydrophilic and hydrophobic regions, saponins are excellent emulsifiers and foaming agents, and provide functional roles in foods. The ability of saponins to form emulsions in the intestine have lead to the investigation into their role for lowering serum cholesterol in humans. Phytic Acid Phytic acid, also known as myo-inositol hexaphosphate, is abundant in soybeans and soy products, especially soy flour. The common phytic acid is a hexaphosphate. Other inositol phosphates may contain from one to five phosphate groups on the inositol ring. Each of these phosphate groups is capable of binding one monovalent or divalent cation, but typically the number of cations bound is less only three to five cations per phytic acid. The phytate content of soybean ranges from 1.00-1.47% on a dry matter basis. This value represents 51.4-57.1% of the total phosphorous in seeds. VITAMINS Soybeans contain both water-soluble and fat-soluble vitamins. The water-soluble vitamins present in soybeans mainly include thiamin, riboflavin, niacin, pantothenic acid, and folic acid. The whole soy flour contains 6.26 to 6.85 mg/g and 0.92 to 1.19 mg/g of thiamin and riboflavin, respectively. The oil-soluble vitamins present in soybeans are vitamins A and E, with no vitamins D and K. Vitamin A exists mainly as the provitamin β-carotene but its content is negligible in mature seeds but measurable in immature and germinated seeds. Tocopherols (Vitamin E) play an important role as the major fat soluble antioxidants that protect our bodies against free radical damage. Of the four different forms, alpha tocopherol is the most potent and has the greatest nutritional and biological value. Crude soy oil contains 9-12 mg of
-tocopherol/g, 74-102 mg of -tocopherol/g, and 24-30 mg of -tocopherol/g. In fact, tocopherol is considered as an important constituent of soy oil partly because it is the most effective natural antioxidant and partly because it is good for human nutrition.

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Nutritional And Physiological Functions Of The Soybean The nutrients of soybean discussed above have one or more physiological functions. The most studied nutrients in relation to health are soy protein and isoflavones. NUTRITIONAL AND PHYSIOLOGICAL PROPERTIES Soy Protein The quality of soybean proteins has actually been undervalued until recently, because the protein efficiency ratio was based upon the growth of laboratory rats. A new official method, the protein digestibility corrected amino acid score (PDCAAS) method for evaluating protein quality adopted by the World Health Organization (WHO) and the United States Food and Drug Administration (FDA) was used to evaluate the protein quality of soybean. Results showed that soybean proteins have a PDCAAS of 1.0, indicating that it is able to meet the protein needs of children and adults when consumed as the sole source of protein at the recommended level protein intake of 0.6 g/kg body weight. It is now concluded that the quality of soybean proteins is comparable to that of animal protein sources such as milk and beef. In addition to playing a role as traditional nutrients, soy proteins were found to have a hypocholesterolemic effect in the later half of the 1970s. The serum cholesterol is lowered markedly when the animal proteins in the diet are exchanged with soy proteins. Since then numerous investigations on this effect of soy protein have been carried out. Lovati et al., 1992 found that soybean storage proteins possess the hypocholesterolemic effect, because the plasma total cholesterol of the rats fed casein-cholesterol diets was reduced by 35 and 34% by the administration of purely isolated β-conglycinin and glycinin, respectively. In a meta-analysis of 38 separate studies involving 743 subjects, the consumption of soy protein resulted in significant reduction in total cholesterol (9.3%), LDL cholesterol (12.9%), and triglycerides (10.5%), with a small but insignificant increase (2.4%) in HDL cholesterol (Anderson et al., 1995). In linear regression analysis, the threshold level of soy intake, at which the effects on blood lipids became significant, was 25 g. Thus, soy protein represents a safe, viable, and practical nonpharmacologic approach to lowering cholesterol. The exact mechanism by which soy protein reduces cholesterol is not yet known

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clearly. But some studies suggest that cholesterol absorption and/or bile acid reabsorption is impaired when soybean proteins are fed, while others propose that changes in endocrine status such as alteration in insulin to glucagon ratio and thyroid hormone concentrations are responsible. In addition to the cholesterol-lowering effects, soybean proteins suppress the lipogenic enzyme gene expression in the livers of genetically fatty rats, indicating that dietary soybean proteins are useful for the reduction of body fats. Peptide Fragments From Soybean Proteins Peptide fragments from soybean proteins are found to have hypocholesterolemic, anticarcinogenic, hypotensive, immunostimulating and/or antioxidant effects (Figure 7.6). The hydrophobic peptide fragments which appeared through the proteinase digestion of soybean proteins are responsible for their plasma-cholesterol lowering action. Since the hydrophobic peptides bind well to bile acids, the fecal excretion of bile acids is increased. Consequently, the bile acid synthesis in the liver is stimulated, resulting in the reduction of serum cholesterol. Soybean protein digests have the highest hydrophobicity among commonly used food protein sources and give the lowest cholesterol level. Major hydrophobic peptides to bind to bile acids are A1a and A2, which are the acidic peptides of glycinin subunits, A1aB1b and A2B1a, respectively (Minami et al., 1990). The region comprising residues 114-161 (48 amino acid residues) represents the most hydrophobic area of the A 1a subunit. This hydrophobic region is also highly conserved in the A2 subunit. Most recently, Yoshikawa et al (2000) found that Leu-Pro-Tyr-Pro-Arg, the low molecular weight peptide fragment derived from soybean glycinin, also reduced serum cholesterol in mice after oral administration. This may be a different mechanism from that of high molecular weight fraction, because there was no increase in the excretion of the fecal cholesterol and bile acids. It is known that bile acid is an intrinsic promoter of colon cancer. Azuma et al (2000) found that high molecular weight fraction (HMF) described above suppresses the tumorigenesis in the liver and colon in rats through the inhibitory effect on the reabsorption of bile acids in the intestine. Another low molecular weight peptide fragment Met-LeuPro-Ser-Tyr-Ser-Pro-Tyr derived from soybean proteins has anticarcinogenic properties Kim et al (2000).

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FIGURE 7.6 — Physiologically Active Peptide Fragments From Soybean Proteins* Peptide fragments

Physiological activity

Protein source

High molecular weight fraction

Hypocholesterolemic and anticarcinogenic

Soybean proteins

LPYPR

Hypocholesterolemic

Soybean glycinin

MLPSYSPY

Anticarcinogenic

Soybean proteins

Peptide fraction

Hypotensive through ACEinhibition

Soybean proteins

MITLAIPVNKPGR

Phagocytosis-stimulating

β-conglycinin
-subunit

MITLAIPVN

Phagocytosis-stimulating

β-conglycinin
-subunit

MITL

Phagocytosis-stimulating and protection from hair loss

β-conglycinin
-subunit

HCQRPR

Phagocytosis-stimulating

Glycinin A1a-subunit

QRPR

Phagocytosis-stimulating

Glycinin A1a-subunit

VNPHDHQN

Antioxidant

β-conglycinin

LVNPHDHQN

Antioxidant

β-conglycinin

LLPHH

Antioxidant

β-conglycinin

LLPHHADADY

Antioxidant

β-conglycinin

VIPAGYP

Antioxidant

β-conglycinin

LQSGDALRVPSGTTYY

Antioxidant

β-conglycinin

*Fukushima, 2004

Immunostimulating peptides are expected to improve senile immunodeficiency. Yoshikawa et al (2000) isolated a peptide stimulating phagocytosis by human polymorphonuclear leukocytes from soybean proteins. It is Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg which was derived from the
subunit of β-conglycinin and named soymetide. Soymetide-4, the tetrapeptide at the amino terminus, that is, Met-Ile-Thr-Leu, is the shortest peptide stimulating phagocytosis. Soymetide-9 (Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn) is the most active in stimulating phagocytosis in vitro. Besides these, soymetide-4 prevents hair loss induced by a cancer chemotherapy agent. The peptides derived from soybean glycinin A1a subunit, Gln-Arg-Pro-Arg and His-Cys-GlnArg-Pro-Arg, also stimulated phagocytic activity of human polymorphonuclear leukocytes, but their activities are weaker than those of soymetide described above. Chen et al (1995) isolated six antioxidative peptides against peroxidation of linoleic acid from the protease hydrolysates of soybean β-conglycinin. They are 1) Val-AsnPro-His-Asp-His-Gln-Asn, 2) Leu-Val-Asn-Pro-His-Asp-His-Gln-Asn, 3) Leu-Leu-Pro-His-His, 4) Leu-Leu-Pro-His-His-Ala-Asp-Ala-Asp-Tyr, 5)

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Val-Ile-Pro-Ala-Gly-Tyr-Pro and 6) Leu-Gln-Ser-Gly-Asp-Ala-Leu-ArgVal-Pro-Ser-Gly-Thr-Thr-Tyr-Tyr. These peptides are characterized by the hydrophobic amino acids such as valine or leucine at the N-terminal positions and proline, histidine, or tyrosine in the sequences. Yokomizo et al (2002) also isolated four antioxidative peptides from the protease hydrolysates of the water-insoluble residues of soybeans. The amino acid sequences were 1) Ala-Tyr, 2) Ser-Asp-Phe, 3) Ala-Asp-Phe and 4) Gly-Tyr-Tyr. These peptides possess aromatic amino acid at the Cterminal end. Gly-Tyr-Tyr has the strongest antioxidative activity among these four peptides, which is nearly equal to that of carnosine. It should be noted that the molecular weights of these four peptides are much lower than those of the other antioxidative peptides previously isolated from soybean proteins (Chen et al., 1995). Minor Components Some minor components may also have important physiological functions (Figure 7.7). Although most of these minor components are not proteins, they coexist more or less with soy protein products as a food ingredient. Hitherto, these minor components, such as isoflavones, saponins, trypsin inhibitors, phytic acid, lectin, etc., were thought to be antinutritional factors, but now they are recognized to have preventative effects on cancer. Among these, isoflavones (mainly genistein and diadzein) are particularly noteworthy, because soybeans are the only significant dietary source of these compounds. Isoflavones seem to have not only anticarcinogenic activities, but also preventative effects on osteoporosis and the alleviation of menopausal symptoms. FIGURE 7.7 — Physiological Functions Of Minor Components Of Soybeans* Minor Components

Physiological Functions

Isoflavones

Anticarcinogenic activities, prevention of cardiovascular diseases, prevention of osteoporosis, antioxidant activities, and alleviation of menopausal symptoms.

Saponins

Anticarcinogenic activities, hypocholesterolemic effects, inhibition of platelet aggregation, HIV preventing effects (group B saponin), and antioxidant activities (DDMP saponin)

Phytosterol

Anticarcinogenic activities

Phytic acid

Anticarcinogenic activities

Lectin (Hemagglutinin)

Activation of lymphocytes (T cell) and aggregating action of tumor cells

Nicotianamine

Inhibitor of angiotensin-converting enzymes

Protease inhibitors

Anticarcinogenic activities

*Fukushima, 2004

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ROLE OF SOY PRODUCTS IN PREVENTION OF CHRONIC DISEASES Cardiovascular Diseases Cardiovascular disease (CVD) is the leading cause of death in the United States. Diet has a major impact on several modifiable risk factors such as hypercholesterolemia, hypertriglyceridemia, elevated LDL cholesterol, low HDL cholesterol, hypertension and obesity for heart disease. It has been known for ~60 years that replacement of animal protein in the diet with soy protein reduces hyperlipoproteinemia and atherosclerosis. The increased level of research intensity over the past 10-12 years has resulted from evidence that soy consumption might improve cardiovascular health. Dietary soy protein has well-documented beneficial effects on plasma lipid and lipoprotein concentrations. The effects in human subjects are reductions in LDL cholesterol of ~13%; reductions in plasma triglycerides of ~10%; and increases in HDL cholesterol, greater in some subjects than others, with average increases of ~2% (Anderson et al., 1995). These beneficial effects of soy protein on plasma lipoprotein concentrations culminated recently in the U.S. Food and Drug Administration’s approval of a health claim that “25 g of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease”. Mechanisms Of Cholesterol Reduction By Soy Components Several components of soy have been implicated in lowering cholesterol: trypsin inhibitors, phytic acid, saponins and soy protein. 1. Trypsin Inhibitors All soy products are heat treated, which destroys most of the activity of trypsin inhibitors. Small amounts of heat stable Bowman-Birk inhibitor may exert a hypocholesterolemic effect by increasing the secretion of cholecystokinin. This would then stimulate bile acid synthesis from cholesterol and thus help to eliminate cholesterol through the gastrointestinal tract (Erdman, 2000). 2. Phytic Acid Phytic acid, myoinositol hexaphosphate, is found in all nonfermented soy protein products and is very stable during heating. Phytic acid chelates zinc strongly in the intestinal tract, thus decreasing its absorption. A copper deficiency or a high ratio of zinc to copper results in a rise in blood cholesterol. The hypothesis advanced is that soy foods contain both copper and phytic acid and, therefore, may lower cholesterol levels by decreasing the ratio of zinc to copper (Zhou and Erdman, 1995).

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3. Saponins These compounds may contribute to cholesterol lowering by increasing bile excretion (Sidhu and Oakenfull, 1986). 4. Soy Protein Effects Early researchers noted in animal studies that the amino acids lysine and methionine tend to raise cholesterol levels, whereas arginine has the opposite effect (Kurowska and Carroll, 1994). Soy protein, compared with animal protein sources, has a higher ratio of arginine to lysine and methionine. Interestingly, animal studies found that a mixture of L-amino acids equivalent to the pattern of soy protein had an intermediate cholesterol-lowering effect that was not as pronounced as that of hydrolyzed whole soy protein (Tasker and Potter, 1993). Thus, some other component in the whole soy protein may have a beneficial effect beyond that of the protein alone. The higher arginine-to-lysine ratio of soy protein may decrease insulin and glucagon secretion, which would then inhibit lipogenesis. Soybean contains 2 types of storage proteins, the globulins 11S and 7S. Cell culture studies suggest that these globulins stimulate LDL receptor activity (Lovati et al., 1992). On the basis of several clinical studies, it is suggested that consumption of soy protein upregulates LDL receptors in humans. Soy protein treated with proteases forms 2 distinct fractions: an insoluble high-molecular-weight fraction and a soluble low-molecularweight fraction. The insoluble fraction when fed to rats, lowered blood cholesterol levels by increasing fecal excretion of sterols. The theory that soy protein lowers cholesterol by enhanced bile excretion has been explored extensively. Cholesterol lost from the body in the form of bile shifts the liver toward providing more cholesterol for increased bile acid synthesis and increases LDL receptor activity. Thus, the end result is increased LDL removal from the blood. 5. Isoflavones Isoflavones have weak estrogenic effects in both animals and humans. The beneficial effects of estrogen include lower LDL cholesterol and increased HDL cholesterol. Phytoestrogens presumably work in a similar manner, although less potent. Soy protein containing isoflavones lowered cholesterol significantly more than soy protein without isoflavones in humans (Crouse et al., 1999). Soy protein with isoflavones (20% of diet) also inhibits formation of atherosclerotic lesions in primates (Anthony et al., 1997). Genistein is known to inhibit tyrosine kinase, an enzyme involved in the cascade of events leading to formation of thrombi and lesions. Isoflavones also act as antioxidants and can inhibit LDL oxidation (Kapiotis et al., 1997).

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Soyfoods And Cancer The theory that phytoestrogen could have a protective effect against cancer due to their similarity in structure to estrogen was first postulated in the 1980s. Soy isoflavones bind to mammalian estrogen receptors (ER) and generate estrogenic responses in vitro and in vivo. It is generally accepted that these compounds have low binding affinity for both ER alpha and beta but preferentially bind to ER-β. There are a number of hormone-related risk factors for breast cancer; for example, early onset of menarche, late onset of menopause, delayed age of first pregnancy and elevated free oestradiol concentrations in post-menopausal women. In addition, environmental factors, especially diet, are also thought to play a major role in cancer risk. This is mainly due to the fact that breast cancer incidence is much higher in Western populations in comparison with Asian populations, a finding which has been associated with the consumption of a traditional low fat, highfiber, high-soy diet among Asian populations. These studies suggest that early exposure to phytoestrogen is extremely important in order to gain from their cancer preventive effects. Wu et al (1996) found increased tofu consumption being associated with a decreased breast cancer risk in a case-control study of pre- and post-menopausal Asian American women. In a soya-feeding (154+8.4 mg total isoflavones consumed/d) intervention study, circulating levels of 17β-estradiol were found to be reduced by 25% in premenopausal women, implicating a protective effect against breast cancer (Lu et al., 2000). What has become increasingly apparent is that the time of exposure to the test compound is of the utmost importance. For example, rats treated with genistein neonatally or prepubertally have a longer latency before the appearance of chemically induced mammary tumors and a marked reduction in tumor number whereas rats treated after 35 days of age have smaller alterations in breast cancer risk (Barnes, 1997). These findings suggest that early exposure to soybean products is vital in breast cancer prevention and may explain why protection against breast cancer is lost in Asian immigrants after a few generations. It is postulated that genistein may exert its chemoprotective effects in animal models by enhancing mammary cell maturation and lobularalveolar development, thus reducing cell proliferation in the mammary gland. 1. Possible mechanisms of effects of phytoestrogen on breast cancer Mechanisms of action of phytoestrogen appear to be of two types.

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One is estrogen-dependent and the other is estrogen-independent. Estrogen-dependent mechanisms are: • Estrogen receptor
- and β-mediated mechanisms • Effects on endogenous hormones and growth factors • pS2 expression Estrogen-independent mechanisms are: • Protein tyrosine kinase and topo-isomerase II inhibition • Free radical scavenging • Role in metastasis 2. Effects of isoflavones on cancer development Proliferation Many in vitro studies have examined the effects of phytoestrogens on the proliferation of both Estrogen receptor (+) (mainly MCF-7) and Estrogen receptor (-) breast cancer cell lines. Genistein exerts biphasic effects on the proliferation of Estrogen receptor (+) cell lines, stimulating growth at concentrations up to 10 M and potently inhibiting cell proliferation at >10 M (Le Bail et al., 2000). Zava and Duwe (1997) have shown that stimulation of the Estrogen receptor (+) cell lines MCF-7 and T47D by genistein and equol correlates with the binding affinities of these compounds to the Estrogen receptor. These studies suggest differential mechanisms of action for phytoestrogens on cell proliferation; at low concentrations they appear to act via an Estrogen receptor-mediated mechanism whereas at higher concentrations a different mechanism of action is exerted on the cells as both Estrogen receptor (+) and Estrogen receptor (-) cell growth is inhibited. Cell Cycle And Apoptosis Genistein reduces the risk of breast cancer by influencing the cell cycle and apoptosis. Genistein at concentration of 10 M causes a reversible G2/M arrest in MCF-7 cell cycle progression whereas doses > 50 M result in a marked fall in S-phase cell percentage associated with a persistent arrest in the G2/M phase. In addition, exposure of MCF-7 cells to genistein for >48 h induced apoptosis. Genistein blocks G2/M cell-cycle progression in non-neoplastic human mammary epithelial cells. G2/M cell cycle arrest induced by genistein in breast cancer cells is associated with an increased expression of the cell-cycle inhibitor p21WAF/CIP1 followed by an increase in apoptosis. Thus, the anti-tumor effects genistein may be modulated by the compound’s ability to arrest two critical points in the control of the cell cycle and by the induction of apoptosis (Frey et al., 2001).

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Invasion And Metastasis The development of clinical metastasis is a significant cause of morbidity and mortality from cancer. Metastasis is the movement or spreading of cancer cells from one organ or tissue to another. Cancer cells usually spread via the bloodstream, or lymph system. An important step in metastasis is tumor invasion and any agent that can inhibit this process may have potential therapeutic value. Genistein has been shown to inhibit the invasion of MCF-7 cells and also the estrogen receptor (-) cell lines MDA-MB-231 and MDA-MB-468 (Shao et al., 1998). Scholar and Toews (1994) have postulated that the ability of genistein to inhibit tumor cell invasion is due to its potent inhibitory action on tyrosine kinases and have supported this theory with preliminary studies which demonstrate that other tyrosine kinase inhibitors for example, methyl 2,5-dihydroxy-cinnamate and herbimycin, also inhibit tumor invasion. Angiogenesis Angiogenesis refers to the process by which new blood vessels are formed within the body. When tissues need more oxygen, for example, they release molecules that encourage blood vessels to grow. The ability to inhibit angiogenesis and turn off the blood supply to tumors could potentially lead to a new generation of cancer therapies. Tumors require a blood supply to develop and grow. They take over existing blood vessels and stimulate the production of new vessels from these; a process termed angiogenesis. Phytoestrogens can inhibit angiogenesis, both in vitro and in vivo. Fotsis et al (1995) have shown that genistein can inhibit the proliferation and in vitro angiogenesis of vascular endothelial cells at half-maximal concentrations of 5 and 150 M, respectively. The ability of genistein to inhibit capillary formation in vivo has been demonstrated in both mouse xenografts of various cancer cells (Zhou et al., 1998) and in animal models of experimentally induced angiogenesis (Hayashi et al., 1997). In rats, genistein administered as an eye drop (5 mg/ml), prevented extensive neovascularisation of the cornea induced by chemical cauterization (Hayashi et al., 1997). These findings may explain one of the mechanisms of action by which phytoestrogens exert their protective effects against cancer metastasis, as the angiogenic process is a key mechanism in tumor growth, progression and metastatic dissemination. 3. Free-radical scavenging effects The antioxidative effects of soy were the focus of much of the early research on how soy prevents cancer. The powerful free-radical scavenging effects of soy compounds and how they impact cancer continue to emerge.

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Soy has an additive effect with vitamin E; it lowers estrogen levels in women and androgen levels in men (Jenkins et al. 2000). Damage to DNA caused by certain types of free radicals is strongly inhibited by genistein and other soy compounds. This helps prevent cancer. The effects of genistein against the activation of epidermal growth factor receptor (EGFR) by free radicals were demonstrated. Genistein reversed the free-radical activation of EGFR in normal cells. The benefits of genistein against oxidative stress are evident from a study on brain cells exposed to hydrogen peroxide. Free radicals generated by hydrogen peroxide degrade phospholipids and activate enzymes, which are crucial for memory and other brain functions. Genistein, through its ability to inhibit a tyrosine kinase enzyme that sets off the reaction, rescues cells from damage (Servitja et al. 2000). Osteoporosis Osteoporosis is a disease that primarily affects older women in which the bones become porous and fracture easily. Japanese women, who generally consume soy products, have half the rate of hip fractures as U.S. women. Isoflavones consumption has been shown to reduce bone loss and slow calcium loss in an animal model of osteoporosis, suggesting a possible beneficial role in preventing osteoporosis in humans. In addition, certain soy products such as tofu contain relatively high calcium content. It is also interesting to note that soy protein seems to cause less loss of calcium from the body compared to other dietary sources of protein which may promote calcium loss and bone breakdown at high levels. Ipriflavone, a synthetic isoflavone drug prescribed in Europe, is metabolized in the body into diadzein, and has potent effects on reducing bone resorption in post-menopausal women. It is reasonable to suggest that soy or its isoflavones enhance bone formation due to 1) soy isoflavones stimulate osteoblastic activity through activation of estrogen receptors, and 2) soy or its isoflavones promote insulin-like growth factor-I (IGF-I) production. One factor that is thought to adversely affect bone health is dietary protein and high protein intake could lead to osteoporosis by increasing urinary calcium excretion. The hypercalciuric effect of protein is generally attributed to the sulfur amino acids, methionine and cysteine, which are metabolized to sulfate and hydrogen resulting in an acid ash. Because the skeletal system is the major source of alkali, in response to acid conditions, calcium is leached from the bones resulting in an increase in calcium excretion. Methionine supplementation results

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in a lowering of pH and an increase in urinary calcium excretion. Calcium excretion is very much related to bone health. Heaney (1994), in a series of balance studies in over 500 women, found that urinary calcium excretion accounts for more than 50% of the variation in calcium balance, whereas calcium intake accounted for only about 10%. Thus reducing calcium excretion is important for optimizing bone calcium retention. The lower sulphur amino acid content of soy protein may help to reduce calcium excretion in comparison to consuming animal protein. On a per gram protein basis, soy protein contains lower amounts of sulphur amino acids (29.6 mg/100 g protein) than either milk or beef protein (33.8 and 34.2 mg/100 g protein, respectively). Consistent with this hypothesis are results from Anderson et al (1987) who found that the urinary calcium:creatinine ratio increased by 45% (relative to water) 4 hours following the consumption of the meal containing milk whey (2.8 g methionine/100 g) as the primary protein source. In contrast, in response to a meal containing soy protein (1.3 g methionine/100 g), the calcium:creatinine ratio increased by only 3%. Menopause Soy foods which contain isoflavones may help in the treatment of menopause symptoms. In women who are producing little estrogen, phytoestrogens may produce enough estrogenic activity to relieve symptoms such as hot flashes. From an epidemiological point of view, it is interesting that in Japan, where soy consumption is very high, menopause symptoms of any kind are rarely reported. Kidney Disease The “soy protein hypothesis” suggests that substitution of soy protein for animal protein in diabetic individuals results in less hyperfiltration and glomerular hypertension; therefore, protecting against diabetic nephropathy. It is also thought that incorporating soy into the diet will have therapeutic benefits in diabetic nephropathy by slowing deterioration of renal function and decreasing proteinuria. Available data indicates that substitution of soy protein for animal protein is associated with less postprandial hyperfiltration and albuminuria (Anderson et al., 1998). Clinical trials on human subjects have found that not only the quantity of protein, but also the types of protein have important implications in renal disease. Short-term incorporation of soy protein in the diet (three weeks) has been associated with lower renal plasma flow, glomerular filtration rate and fractional clearance of albumin.

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The long-term effects of soy protein have yet to be fully understood. However, animal studies indicate that chronic soy protein intake preserves the function of damaged kidneys better than animal protein. Obesity Obesity is thought to be a basic preclinical status of life-style-related diseases such as hyperlipidemia, diabetes, hypertension, coronary heart disease, and stroke. Various methods are recommended for the treatment of obesity. The first choice for controlling obesity is to reduce energy consumption by reducing food intake and to increase energy expenditure by exercise. Intake of protein produces significantly greater postprandial energy expenditure than intake of the same number of calories from glucose and fat in humans. Several studies in obese humans and animals suggest that soy as a source of dietary protein has significant antiobesity effects. In genetically obese mice, it is reported that soy-protein isolate and its hydrolysate were more effective than was whey-protein isolate and its hydrolysate in weight reduction and acts by lowering the perirenal fat pad weight and plasma glucose concentrations. This effect may be due to an active tetrapeptide present in soy. The tetrapeptide from soy also decreased visceral fat weight in mice during a swimming exercise. The reduction in body fat by soy-protein isolate and its hydrolysate compared with casein was also observed in genetically obese yellow KK mice and in rats made obese by being fed a high-fat diet; plasma glucose decreased more with the soy-protein isolate and its hydrolysate than with casein. The antiobesity effect of soybean peptides is thought to involve the increase of lipid and carbohydrate metabolism. However, because the soybean peptides used by researchers are a mixture of hydrolysates of soybean protein, it is possible that a particular peptide or the amino acid composition of the peptides causes the effect. Further examination will be necessary to determine this. Soy Products There are many types of soy foods available throughout the world today. Some are produced through the use of modern processing techniques in large processing plants, whereas others are produced in more traditional ways, owing their history to oriental processing techniques. These are the foods that are usually referred to as traditional soyfoods. These soyfoods are typically divided into two categories: nonfermented and fermented. Traditional nonfermented soyfoods

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include fresh green soybeans, whole dry soybeans, soynuts, soysprouts, whole soyflour, defatted soy flour, soy milk, okara and yuba. Traditional fermented soyfoods include tempeh, miso, soy sauces, natto and fermented tofu and soy yogurt. The most popular soyfoods in the United States now are tofu, soy milk, soy sauce, miso and tempeh. Americans known for their ability to adapt foreign foods to their own tastes, have developed a whole new class of “second generation” soyfoods, which includes such products as soy hot dogs, soy ice cream, veggie burgers, tempeh burgers, soy yogurt, soy cheeses, soy flour pancake mix and a myriad of other prepared Americanized soyfoods. Soymilk Soymilk is an aqueous extraction of whole soybeans. Soymilk is used as a base in a wide variety of products including tofu, soy yogurt and soy-based cheeses. The chemical composition of soy milk is given in Figure 7.8. Soy milk production technology is shown in Figure 7.9. Further, soymilk is used to prepare symbiotic soy yogurt (Figure 7.10) and symbiotic soy yogurt beverage (Figure 7.11). FIGURE 7.8 — Chemical Composition Of Soymilk And Cow’s Milk (per 100g) Nutrient Calorie Water (g) Protein (g) Fat (g) Carbohydrates (g) Ash (g) Minerals (mg) Calcium Phosphorus Sodium Iron Vitamins (mg) Thiamine Riboflavin Niacin Saturated fatty acids (%) Unsaturated fatty acids (%) Cholesterol (mg)

Soy milk

Cow’s milk

44 90.8 3.6 2.0 2.9 0.5

59 88.6 2.9 3.3 4.5 0.7

15 49 2 1.2

100 90 36 0.1

0.03 0.02 0.5 40-48 52-60 0

0.04 0.15 0.2 60-70 30-40 9.24-9.9

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FIGURE 7.9 — Preparation Of Soymilk

FIGURE 7.10 — Preparation Of Symbiotic Soy Yogurt

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FIGURE 7.11 — Preparation Of Symbiotic Soy Yogurt Beverage

FIGURE 7.12 — Preparation Of Tofu

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Tofu Tofu is a curd that is made directly from soybeans and resembles a soft white cheese or a very firm yogurt (Figure 7.12). Tofu is waterextracted and salt- or acid-coagulated soy protein gel with water, soy lipids, and other constituents trapped in its network. It is inexpensive, nutritious, and versatile. On a wet basis, a typical pressed tofu with moisture content in the range of 85% contains about 7.8% protein, 4.2% lipid, and 200 mg calcium/100 g. On the dry matter basis, it contains about 50% protein and 20% oil; the remaining components are carbohydrates and minerals. Tofu can be served as a meat or cheese substitute. It is cholesterol free, lactose free and lower in saturated fat. Tempeh Tempeh is a fermented soyfood and is unique in its texture, flavor and versatility. It originated in Indonesia, where today it is still the most popular soy food. Tempeh, while not as popular as tofu in the United States, lends itself easily to being used as a meat alternative because of its chewy texture and distinct flavor. As a result, a wide variety of tempeh-based meat analogues are available. Tempeh is a cake of cooked and fermented soybeans held together by the mycelium of Rhizopus oligosporus. The production technology of tempeh is given in Figure 7.13. Miso Miso, a white, brown or reddish-brown soybean paste, is another fermented soyfood. It’s a traditional food in Japan, with a history going back about 1300 years. Made from fermented soybeans, and sometimes in combination with wheat, barley or rice, this salty paste is a treasured soup base and flavouring ingredient used throughout Japan, Korea, Taiwan, Indonesia, and China (Figure 7.14). Soy Sauce Soy sauce is probably man’s oldest prepared seasoning. Processed similarly to miso except that the paste produced is pressed to yield a liquid, this savory seasoning sauce is widely used in both Oriental and American cuisine. There are two basic types of soy sauce: fermented soy sauce and soy sauce made from hydrolysed vegetable proteins (HVP). Within the naturally fermented category, there are many types of soy sauce, with shoyu and tamari being the most popular. For the most part, defatted soybean meal or grits are used to produce soy sauce,

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FIGURE 7.13 — Preparation Of Tempeh

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FIGURE 7.14 — Preparation Of Miso

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FIGURE 7.15 — Preparation Of Soy Sauce

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with some specialty products being made from whole soybeans. HVP soy sauce is made from soy proteins hydrolyzed into amino acids by using acid hydrolysis and blended with sugars, color and other flavoring ingredients into a sauce that somewhat resembles naturally fermented soy sauce. The production technology of soy sauce is shown in Figure 7.15. Tofuyo Tofuyo is a unique soybean fermented food that has long been a tradition in Okinawa. Ordinary tofu is first produced from soybeans. The tofu is dried at room temperature and is pickled in a mixture containing yeast and awamori (a distilled liquor produced in Okinawa) for maturation. The Aspergillus oryzae including Monascus and Aspergillus bacteria are the microorganisms used in the fermentation process. Less salty than miso or shoyu, there is a certain sweetness to the taste of tofuyo, which has an elastic feel and a smooth texture like that of soft cheese. Natto Natto is a traditional fermented soy food also known as Itohiki-natto. It originated in the northern part of Japan about 1000 years ago. It is one of the few products in which bacteria predominate during fermentation. Properly prepared natto has a slimy appearance, sweet taste, and a characteristic aroma. The production technology of natto is shown in Figure 7.16. Soy Protein Products Soy protein products include defatted soy flakes, soy meal, soy flour and grits, soy concentrates, soy isolates, texturized soy proteins, fullfat soy flour, and enzyme active soy flour (see Figure 7.1). Defatted Soy Flakes Soybeans are first dried, cleaned, cracked, and dehulled. Dehulled beans are then conditioned to 10-11% moisture at 63-74°C and flaked using smooth rolls. The flakes are defatted using hexane extraction. These flakes contain about 30-35% residual hexane. Therefore, they need to be desolventized before being processed into meals and subsequently into various protein products. Soy Meal Soy meal is produced by grinding defatted and desolventized flakes, containing a little over 50% protein. The other major component in the

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FIGURE 7.16 — Preparation Of Natto

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meal is carbohydrate, which accounts for about 30-35% of the meal. Soy meal usually contains less than 1% lipids. The remaining minor components include ash and moisture. Defatted Soy Grits Grits are obtained by coarsely grinding the defatted flakes followed by screening. They are classified as coarse (10-20 mesh), medium (2040 mesh) or fine (40-80 mesh) grits according to particle size. Defatted Soy Flour Soy flour is produced by grinding the soy flakes to very fine particles, so that 97% of the product passes through 100-mesh screen. Soy flour or grits can be used as an ingredient in a variety of food products, including soup, stews, beverages, desserts, bakery goods, breakfast cereals, and meat products. Soy Protein Concentrates Soy protein concentrates are prepared by removing soluble carbohydrate fraction as well as some flavor compounds from defatted meal. Three basic processes are used for carbohydrate removal: 1. acid leaching (isoelectric pH 4.5), 2. aqueous ethanol (60-80%) extraction, and 3. moist heat-water leaching. In all of these treatments proteins become insolubilized while a portion of the carbohydrates remain soluble so that their separation becomes possible by centrifugation. Solids containing mainly proteins and insoluble carbohydrates are then dispersed in water, neutralized to pH 7.0 if necessary, and spray-dried to produce soy concentrates. Most commercial soy concentrates are made by the aqueous alcohol extraction or acid leaching process. Soy Protein Isolates Soy protein isolates are traditionally prepared from defatted soy meal using aqueous or mild alkali extraction (pH 7-10) of proteins and soluble carbohydrates. The insoluble residue, mostly carbohydrate, is thus removed by centrifugation, followed by precipitation of soy protein at its isoelectric point (pH in the range of 4.5). The precipitated protein is separated by mechanical decanting, washed, and neutralized to a pH about 6.8 and then spray-dried. The resulting product is a highly purified proteinate form of soy protein with minimal beany flavor. Alternatively, the final precipitate may be washed and dried without neutralization to give an isoelectric form of soy isolates.

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Toasted Full-Fat Soy Flour For preparing toasted full-fat flour, soybeans are first steamed under light pressure to inactivate lipoxygenase that catalyzes lipid oxidation that leads to off-flavor formation. The beans are then dried, cracked, dehulled, and finely ground to obtain full-fat soy flour. Because the product is rather difficult to screen, it is usually prepared by two steps of grinding with separation of coarse from fine particles by air classification between the steps. Full-fat flour contains all the lipids (18-20%) originally present in the raw soybeans. Enzyme Active Full-Fat Soy Flour Enzyme active soy flour is produced by a procedure similar to that described for toasted soy flour except the initial steam or heat treatment is omitted. The beans may be dehulled prior to grinding. The resulting product is widely used in food industries for bleaching wheat flour and conditioning doughs in Western type breads. Soy lipoxygenases are responsible for its bleaching action, whereas the improved texture of the bread is attributed to the fact that soy β−amylases remain active longer during the initial stages of baking than those of wheat or barley, leading to the reduction in the starch viscosity. In the United States, enzyme active soy flour is also available in defatted form. Textured Soy Protein Products Soy flour and concentrates may be further processed by thermoplastic extrusion to impart meat like texture to these products. The flour or concentrates are mixed with water and additives to form dough and extruded under high temperature and pressure to obtain fibrous texture. Similarly, soy isolates may also be textured by a spinning process that involves solubilizing soy isolate in alkali and then forcing it through a spinneret into an acid bath to coagulate the proteins. The fibers formed are stretched and combined into bundles or tow. The tows are then used to produce meat analogs. SUMMARY For more than 2,000 years people throughout East Asia have consumed soybeans in the form of traditional soy foods, such as cooked whole beans, soy milk, tofu, soy sauce, etc. In Western countries, soybeans have attracted people’s attention since the 1960s as an economical and high-quality vegetable protein source for humans. In the United States, new soy protein products were developed, such as soy flour, soy protein concentrates, soy protein isolates, and their texturized products.

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The soybean is a high-protein food and a good source of nitrogen for humans because the amino acid composition of protein in the soybean has the equivalent nutritional value as animal protein. Soybeans also contain dietary fiber and oligosaccharides such as sucrose, raffinose, and stachyose. Soybean oil contains abundant essential fatty acids such as linoleic acid and linolenic acid. Also, the soybean contains functional minor components such as isoflavone, saponin, lecithin, and phytosterol. Soyfoods and soybean constituents have been widely investigated for their preventive role in chronic disease which is attributed to their major physiological functions such as cholesterol lowering, antiobesity, antihypertensive, immunity regulation, lipid lowering, anticarcinogenic, anticoagulant, antiosteoporosis and antioxidant. Furthermore, the FDA confirmed the ‘Soy Protein Health Claim’ on 26 October, 1999, that 25 grams of soy protein a day may reduce the risk of heart disease. The market is very much responsive to this health claim. Therefore, taking this opportunity, soy foods will penetrate rapidly into Western cultures and diets. In the public health area, we know that relatively minor substitution or addition of soy to the conventional diet can have healthful consequences. References Anderson, J. J. B., Thomsen, K. and Christiansen, C. 1987. High protein meals, insular hormones and urinary calcium excretion in human subjects. Ch 1. In Osteoporosis 1987, C. Christiansen, J. S. Johansen and B. J. Riis (Ed.), pp.240-245. Nrrhaven A/ S, Viborg, Denmark. Anderson, J. W., Blake, J. E., Turner, J. and Smith, B. M. 1998. Effects of soy protein on renal function and proteinuria in patients with type 2 diabetes. Am. J. Clin. Nutr. 68(suppl):1347–1353. Anderson, J. W., Johnstone, B. J. and Cook-Newell, M. E. 1995. Meta-analysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 333: 276-282. Anthony, M. S., Clarkson T. B., Bullock, B. C. and Wagner, J. D. 1997. Soy protein versus soy phytoestrogens in prevention of diet-induced coronary artery atherosclerosis of male cynomolgus monkeys. Arterioscler. Thromb. Vasc. Biol. 17:2524 –2531. Azuma, N., Suda, H., Iwasaki, H., Yamagata, N., Saeki, T., Kanamoto, R. and Iwami, K. 2000. Antitumorigenic effects of several food proteins in a rat model with colon cancer and their reverse correlation with plasma bile acid concentration. J. Nutr. Sci. Vitaminol. 46, 91-96. Barnes, S. 1997. The chemopreventive properties of soy isoflavonoids in animal models of breast cancer. Breast Cancer Res. Treat. 46:169–179. Chen, H. M., Muramoto, K. and Yamauchi, F. 1995. Structural analysis of antioxidative peptides from soybean β-conglycinin. J. Agric. Food Chem. 43, 574-578. Crouse, J. R. III., Morgan, T., Terry, J. G., Ellis, J., Vitolins, M. and Burke, G. L. 1999. A randomized trial comparing the effect of casein with that of soy protein containing varying amounts of isoflavones on plasma concentrations of lipids and lipoproteins. Arch. Intern. Med. 159, 2070 –2076.

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Erdman, J. W. 2000. Soy protein and cardiovascular disease. A statement for healthcare professionals from the Nutrition Committee of the AHA. Circulation 102, 2555-2559. Fotsis, T., Pepper, M., Adlercreutz, H., Hase, T., Montesano, R. and Schweigerer, L. 1995. Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis. J. Nutr. 125, 790S – 797S. Frey, R., Li, J. and Singletary, K. 2001. Effects of genistein on cell proliferation and cell cycle arrest in nonneoplastic human mammary epithelial cells: involvement of Cdc2, p21waf/cip1, p27kip1 and Cdc25C expression. Biochem. Pharmacol. 61, 979 – 989. Fukushima, D. 2004. Soy proteins. In Proteins in food processing. Yada, R. Y. (Ed.). Woodhead Publishing Ltd. Cambridge, England. Pp.123-140. Gunstone, F. D. 2001. Soybeans pace boost in oilseed production. Inform 11: 1287– 1289. Hayashi, A., Popovich, K., Kim, H. and de Juan, E. 1997. Role of protein tyrosine phosphorylation in rat corneal neovascularization. Graefes Arch. Clin. Exp. Opthalmol. 235, 460 – 467. Heaney, R. P. 1994. Cofactors influencing the calcium requirement. – other nutrients. NIH Consensus Development Conference on Optimal Calcium Intake. NIH Consensus Development Conference, Program and Abstracts, pp.71-77. June 6-8. Jenkins, D. J., Kendall, C. W., Garsetti, M., Rosenberg-Zand, R. S., Jackson, C. J., Agarwal, S., Rao, A. V., Diamandis, E. P., Parker, T., Faulkner, D., Vuksan, V. and Vidgen, E. 2000. Effect of soy protein foods on low-density lipoprotein oxidation and ex vivo sex hormone receptor activity—a controlled crossover trial. Metabolism 49(4). 537-543. Kapiotis, S., Hermann, M., Held, I., Seelos, C., Ehringer, H. and Gmeiner, B. M. K. 1997. Genistein, the dietary-derived angiogenesis inhibitor, prevents LDL oxidation and protects endothelial cells from damage by atherogenic LDL. Arterioscler. Thromb. Vasc. Biol. 17, 2868 –2874. Kim, S. E., Kim, H. H., Kim, J. Y., Kang, Y. I., Woo, H. J. and Lee, S. E. 2000. Anticancer activity of hydrophobic peptides from soy proteins. Biofactors 12, 151-155. Le Bail, J. C., Champavier, Y., Chulia, A. J. and Habrioux, G. 2000. Effects of phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer cells. Life Sciences 66(14):1281-1291. Lovati, M. R., Manzoni, C., Corsini, A., Granata, A., Frattini, R., Fumagalli, R. and Sirtori, C. R. 1992. Low density lipoprotein receptor activity is modulated by soybean globulins in cell culture. J. Nutr. 122, 1971-1978. Lu, L., Anderson, K., Grady, J., Kohen, F. and Nagamani, M. 2000. Decreased ovarian hormones during a soya diet: implications for breast cancer prevention. Cancer Res. 60, 4112-4121. Minami, K., Moriyama, R., Kitagawa, K. and Makino, S. 1990. Identification of soybean protein components that modulate the action of insulin in vitro. Agric. Biol. Chem. 54, 511-517. Scholar, E. and Toews, M. 1994. Inhibition of invasion of murine mammary carcinoma cells by the tyrosine kinase inhibitor genistein. Cancer Lett. 87, 159 – 162. Servitja, J. M., Masgrau, R., Pardo, R., Sarri, E. and Picatoste, F. 2000. Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices. J Neurochem. 75(2):788-794. Shao, Z., Wu, J., Shen, Z. and Barsky, S. 1998. Genistein inhibits both constitutive and EGF-stimulated invasion in ER-negative human breast carcinoma cell lines. Anticancer Res. 18, 1435 – 1440. Sidhu, G. S. and Oakenfull, D. G. 1986. A mechanism for the hypocholesterolemic activity of saponins. Br. J. Nutr. 55, 643-649. Soya Bluebook, 2004. Soyatech, Inc. Bar Harbor, ME.

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Tasker, T. and Potter, S. M. 1993. Influence of dietary proteins and amino acid variation on plasma lipids, HMG CoA reductase activity, and reduced glutathione concentrations in inbred versus outbred gerbils. J. Nutr. Biochem. 4, 458-462. Wu, A. H., Ziegler, R. G., Hornross, P. L., Nomura, A. M. Y., West, D. W., Kolonel, L. N., Rosenthal, J. F., Hoover, R. N. and Pike, M. C. 1996. Tofu and risk of breast cancer in Asian-Americans. Cancer Epidemiol. Biomark Prev. 5: 901-906. Yokomizo, A., Takenaka, Y. and Takenaka, T. 2002. Antioxidative activity of peptides prepared from okara protein. Food Sci. Technol. Res. 8, 357-359. Yoshikawa, M., Fujita, H., Matoba, N., Takenaka, Y., Yamamoto, T., Yamauchi, R., Tsuruki, H. and Takahata, K. 2000. Bioactive peptides derived from food proteins preventing lifestyle-related diseases. BioFactors. 12, 143-146. Zava, D. T. and Duwe, G., 1997. Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutrition and Cancer 27: 31-40. Zhou, J. R., Mukherjee, P., Gugger, E. T., Tanaka, T., Blackburn, G. L. and Clinton, S. K. 1998. Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis and angiogenesis. Cancer Res. 58, 5231 – 5238. Zhou, J. R., Erdman, J. W. Jr. 1995. Phytic acid in health and disease. Crit. Rev. Food Sci. Nutr. 35, 495-508. (Guo, M. R., Gokavi, S.)

Chapter 8 SPORTS DRINKS History And Background In the United States there is no standard of identity, or definition, for sports drinks. However, sports drinks, also referred to as isotonic beverages and fluid replacement beverages, are generally accepted as beverages formulated to provide quick replacement of fluids, electrolytes, and carbohydrate fuel for working muscles. Sports drinks may be designed to be consumed before, during, and after exercise. Ideally, sports drinks should taste good and provide all necessary nutrients, electrolytes, and fluid requirements that are lost during exercise in order to rehydrate the body, and enhance performance. When looking into the history of sports drinks, it should be noted that in 1939 Christensen and Hansen reported that pre-competition diets rich in carbohydrates greatly enhanced endurance during sporting activities (Ford, 2002). However, this concept did not materialize via the sports drink phenomena until the mid 1960’s when “Dynamo”, a sports drink formulated to provide high amounts of carbohydrate and a mixture of electrolytes commonly lost in sweating was introduced into the American market (Ford, 2002). Dynamo was not an isotonic beverage (osmotically balanced with the body’s fluids), but rather a high carbohydrate concentrated drink. Isotonic beverages, more like today’s common sports drink, were originally developed for use by college football teams. The first sports drink introduced into college football was “Bengal Punch”, a drink given to the Louisiana State football team by Dr. Martin Broussard. However, the drink that won national attention was developed for the Gators based on work by Cade et al (1972) which demonstrated that loss of volume and compositional changes that occur in body fluids during vigorous exercise could be prevented by the consumption of a glucose electrolyte drink. The drink given to the Gators’ did improve their performance. This was apparent when they won the Orange Bowl in 1967. The drink responsible for their superior

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performance was none other than “Gatorade”. Gatorade® (PepsiCo, Inc) was introduced into the American market in 1969. It was the first product to be marketed as a “sports drink”. It became the official sports drink of the NFL (which it still is today), and its popularity spawned what is now a multi-billion dollar industry around the world. Sports Drinks Market Sports drinks are a fast growing segment of the worldwide beverage market. In the United States, sports drinks sales increased more than 25% in 2005, followed by the bottled water sector with a 16.5% increase (Sloan, 2006), during which time, both carbonated beverage and bottled juice sales declined. Since entering the market in 1969, Gatorade® has controlled the predominant share in the global market, with over 80% of sales in the United States. However, Powerade® (Coca-Cola Company), introduced in 1998, is growing fast taking a 13% and 12% share of the American and European markets, respectively. Gatorade® and Powerade® are the most popular sports drinks in the United States; but the market is also shared with other smaller competitors, notably Capri Sun Sport® (Kraft Foods) and Allsport® of PepsiCo, Inc. (Holay, 2005). Just 30 years after the introduction of sports drinks into the American market, sales have reached over $2.2 billion per year, with per capita consumption exceeding 8 liters. This is small in comparison to carbonated drinks, which are still averaging $14 billion per year (Murray and Stofan, 2001), but nonetheless, sports drink sales and consumption are on the rise. FIGURE 8.1 — Sports Drink Market In Terms Of Volume Sold

Asian countries are also experiencing a boom in the sports drink market. Japan has the largest and most established sports drink market in the world, with per capita consumption higher than 11 liters and total volume sales of over 2.4 billion liters per year (Hilliam, 2002). The

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United States and Japan together share 83% of the total market volume, leaving 17% for Australia and the major European markets (Figure 8.1). In Europe, Germany remains the leading market for sports drinks with a 26% volume share, followed by Italy (19%) and the United Kingdom (15%). Spain has around 11%, while the Netherlands have 9% of the market, but the highest consumption per person—2.8 liters, over twice the European average. Greece, with 0.4 liters per capita, lags well behind the overall average of 1.2 liters, beating only France and Portugal, where per capita consumption is only 0.2 liters (Zenith, 2003). The dominant form of sports drinks is ready to drink liquids (88%). However, there are powders and liquid concentrates (11% and 1%, respectively) on the market, which require mixing by the individual who plans to consume them (Figure 8.2) (Ford, 2002). These are popular among sports teams, where mixing can be performed in bulk. These alternatives can also be appealing to the average consumer, because they eliminate the need to transport heavy or bulky bottles of liquid. FIGURE 8.2 — Distribution Of Different Forms Of Sports Drinks

The fact remains, that even if scientifically formulated to enhance performance, sports drinks are still a relatively small part of the mainstream market. This means taste, packaging, and convenience play as much a role in formulation as physiological consideration. There is a balance to be maintained between a nutritionally superior product, and a product that will be appealing to the senses of the general population. Exercise And Nutrient Requirements Dehydration and substrate depletion are significant factors in fatigue during prolonged exercise. Below et al (1993) showed that provision of

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carbohydrate and fluids have independent and additive effects on performance. Engaging in regular exercise results in additional nutrient requirements, in order to meet the energy demand imposed by increased energy expenditure. Failure to maintain an energy balance will result in decreased body mass, a loss of active tissue (Maughan, 2001), and chronic fatigue. If body mass and performance levels are to be maintained, a high rate of energy expenditure must be matched by an equally high energy intake. The energy required for exercise is generated by the oxidation of lipid and carbohydrate in the body. Protein is also oxidized, but only serves about 5% of energy needs (Maughan, 2001). At moderate intensity levels up to 50% of maximum oxygen uptake (VO2 max), lipid oxidation plays the dominant role in energy generated. However, as intensity increases up to about 75% VO2 max, carbohydrate becomes the major fuel source. If carbohydrate is not available for oxidation, or is only available in limited amounts, then the intensity of exercise must be reduced to a level where lipid oxidation can again be the major source of energy (Maughan, 2001). The typical energy expenditures (kcal/min) of selected activities are listed in Figure 8.3. FIGURE 8.3 — Typical Energy Expenditure Of Selected Activities Activity

Kcal/min

Jogging Rapid Walking Running Cycling Swimming Golfing Gymnastics

7-8 5-7 16 5-11 5-14 2.5-5 5-7.5

(Modified from Ford, 2002)

This poses an interesting situation, because glycogen stores of carbohydrate in the body are relatively small (ranging from 300-500 g based on exercise and intake of carbohydrate), and the amount that is there, is also used to fuel the brain and red blood cells (RBC). The brain and RBCs rely exclusively on carbohydrate as an energy source, so once muscle glycogen is depleted during exercise, the competition for energy is against one’s own brain and blood (Figure 8.4). There is, however, an easy solution to this problem. If muscle glycogen stores are rebuilt between training sessions, the athlete may continue

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to train just as hard as before, if not harder. This is where maintaining an adequate amount of energy, in the form of carbohydrate, comes into play. Recovery of muscle and liver glycogen stores after exercise normally takes at least 24 to 48 hours (Maughan, 2001). Ivy (1998) reported that the rate of glycogen resynthesis after exercise is largely determined by the amount, not type, of carbohydrate supplied by the diet. Glycogen synthesis is most rapid immediately after exercise, so consuming carbohydrate as soon as possible after working out is recommended. FIGURE 8.4 — Effect Of Sports Drinks On Glycogen Balance

An athletic diet should have 60% or more to total energy intake coming from carbohydrate (Maughan, 2001). The requirement for vitamins, most minerals, and protein may be slightly increased by exercise, but maintaining a healthy diet will adequately meet the body’s needs. Carbohydrate is the major factor in maintaining a healthy diet for an athletic lifestyle.

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There is no evidence suggesting that minerals need to be supplemented during exercise. Deficiencies are not common among athletes. However, many minerals are lost during exercise, mainly due to sweating. Sodium, in particular is lost in sweat, as well as potassium, magnesium, iron, and zinc. However, the amount lost relative to total daily requirements is small, and with exception to sodium, these minerals have not been proven, in small excesses or deficiencies, to affect performance in any way (Murray and Stofan, 2001). Sodium, however, plays an integral role in stimulating voluntary fluid uptake and promoting fluid absorption, maintaining plasma volume, and assuring rapid and complete rehydration. Figure 8.5 shows the amount of major electrolytes present in body fluids. Although mineral deficiencies are not normally associated with athletes or exercise, it is important to note that working out in warmer climates will result in excess sweating, and the subsequent loss of electrolytes. FIGURE 8.5 — Concentration (mmol/l) Of Major Electrolytes Present In Body Fluids Electrolytes

Plasma

Sweat

Intracellular

Sodium Potassium Calcium Magnesium Chloride

137-144 3.5-4.9 4.4-5.2 1.5-2.1 100-108

40-80 4-8 3-4 1-4 30-70

10 148 0-2 30-40 2

Fluid balance and thermoregulation are likely factors associated with fatigue, especially if exercising in a warmer climate. Typically, the amount of water lost in a day without excessive exercise, is equal to the amount taken in. Fluid balance is easily achieved through normal diet and bodily function. However, if exercising in a warm climate, the amount of water lost in just a few hours can be equal to that which is usually lost in one day, mostly as a result of excess sweating. Consuming fluids during exercise is essential in maintaining physiological homeostasis, and, therefore, sustained physical activity. Even the slightest dehydration will result in decreased performance, and dehydration is not something that the body can adapt to. In fact, severe dehydration can be fatal. Considerations In The Formulation Of Sports Drinks A properly formulated sports drink should encourage voluntary fluid consumption, stimulate rapid fluid absorption, supply carbohydrate for improved performance, augment physiological response, and speed

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rehydration. It should also be isotonic, meaning in balance with the body’s fluids, therefore containing the same number of osmotically active particles as plasma (280-300 mOsmol/kg). Voluntary fluid consumption is a complex objective. How does a food scientist know that the product they are developing will be one that people reach for, and drink a lot of? Factors that come into play when choosing a sports drink go beyond thirst, and include appearance, taste, packaging, and physiological response. The desire to consume fluids comes from the feeling of thirst, which is triggered only after the body is somewhat fluid deficient (1-2% body mass lost). Dehydration triggers thirst by triggering osmoreceptors and baroreceptors that respond to increased plasma osmolality and decreased circulating blood volume from sweating (Murray and Stofan, 2001). Fluid consumption restores normal osmolality and circulating blood volume, which then relieves thirst. Our bodies respond very quickly to the ingestion of fluids. If plasma osmolality levels decline below threshold, before complete rehydration takes place, a person may stop drinking due to the disappearance of thirst (Murray and Stofan, 2001). This is why sports drinks can offer benefits that plain water alone cannot. Sports drinks have the appropriate balance of energy and electrolytes that will encourage rehydration to happen quickly, and are less likely to be discarded prematurely. Appearance and taste also comes into play when it comes to commercial products. It is well-known that during exercise, people prefer beverages that are lightly sweetened; citrus flavored, and moderately tart (Murray and Stofan, 2001). However, it is not known exactly why. What is known is that before tasting these flavors, there are certain cues that persuade the consumer to buy them. Color is an important part of this concept. Cherry sports drinks should be red. Bright orange and yellow sports drinks suggest a citrus, fresh flavor. For the young athletes, colors like blue, purple, and green suggest vibrant activity. Based on these visual cues, consumers pick the sports drinks that relate best with the taste they are looking for. And taste preference is important. A consumer study found that nearly 60% of adults and 75% of teens in the United States consume sports drinks as an “any time” drink rather than just for exercise (Ohr, 2003). This indicates that although designed to maximize performance, sports drinks are also viewed by consumers as just another beverage choice on the market. This means taste plays just as much a role in formulation as nutritional superiority. A factor that contributes to the taste of sports drinks, but is also an essential factor in the efficacy of the beverages, is carbohydrate. Both

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the type and amount of carbohydrate used to formulate a sports drink will have dramatic effects on the final product. There are three objectives when adding carbohydrate to sports drinks; achieve a good taste, provide adequate amounts of energy, and maximize intestinal water absorption (Murray and Stofan, 2001). Types of carbohydrate that should be used in sports drinks are sucrose, glucose, maltose, maltose dextrins, and to a lesser extent corn syrup solids, because high levels of fructose can slow absorption (Murray and Stofan, 2001). However, a good formulation will have a small amount of fructose added (less than half the total carbohydrate), because in low levels, fructose in combination with glucose or sucrose can optimize fluid absorption. A solution with two transport sugars will enhance solute and water absorption when compared with a solution with only one type of transport sugar (Murray and Stofan, 2001). These sugars are sweet, but sweetness can be easily controlled. Also, they are not complex carbohydrates, so an acceptable mouth-feel can be maintained. The concentration of carbohydrate used should be adequate to support a range of physical activities. Ideally, every athlete would have a sports drink specifically formulated for their particular activity. But, in a global market, sports drinks have to be efficient in replenishing the energy stores and satisfying the needs of a number of different people and activities. Therefore, carbohydrate should make up at least 4-6% of a sports drink, which is enough to provide adequate amounts of energy that will be absorbed quickly, but not too much to over sweeten the product or delay absorption of nutrients. Increasing the concentration of carbohydrate above 6% (w/v) has been shown to significantly decrease the rate of fluid absorption (Murray and Stofan, 2001). Sodium, without a doubt, plays an integral part in the formulation of sports drinks. Sodium improves taste, promotes voluntary fluid intake, speeds rehydration, aids in absorption, helps maintain plasma volume, and effectively rehydrates the active individual to complete hydration. However, despite all of these qualities, levels of sodium in sports drinks are relatively low (10-30 mmol/l). This is because the amount of sodium lost in sweat is relatively low in comparison to amounts in the body. This varies though, according to duration of exercise and climate in which exercise is being done. Therefore, sodium levels can theoretically be quite high in a sports drink, and still be very good for the individual. Other electrolytes that are often added to sports drinks include potassium and zinc. Potassium has long been touted as the mineral of

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choice to relieve and prevent muscle cramping, and zinc has been recognized as an immune booster. However, potassium and zinc added to sports drinks have not been shown to have any effect on performance. Adding these minerals, however, has not been shown to have any detrimental effects on athletes. Water is the most abundant ingredient in any sports drink. Because of this, the water used to formulate sports drinks should be of the highest quality. Depending on the source, treated, filtered water will not have any off flavors, hardness, or softness. It will serve as a blank palette for the flavor blends that can be introduced by carbohydrates, sodium salts, fruit juices and flavors, and other components that will be added to ensure optimum formulation. Isotonicity, or balance based upon osmolality, is also of essential importance. Plasma osmolality is between 280-300 mOsmol/kg. Ideally, a sports drink should be the same. Technically, the beverage should be called isosmotic, since measurements are by the number of solute particles, but the term isotonic is widely used to describe sports drinks in the marketplace, and so it is used in this chapter as well. The isotonicity of sports drinks is essential, because cells that come into contact with solutions of the same osmolality, do not gain or lose water. The cell remains the same due to the impermeability of the cell membrane. This keeps the body in equilibrium, while providing fluid, energy, and electrolytes to aid in rehydration and repletion to energy stores. FIGURE 8.6 —

Chemical Composition And Osmolality Of Selected Sports Drinks And Other Beverages

Beverage Gatorade® (Quaker Oats Co.)

Carbohydrate (w/v)

Na (mmol/l)

K (mmol/l)

Osmolality (mOsm/kg)

6

20

3

280 (powder) 325-380 (liquid)

8-9 (varies with flavor)

10

5

516

8

5

3

381

Perform® (Powerbar)

6.6

20

4

599

Coca Cola Classic® (Coca-Cola Co.)

11

-

-

700

10.8

-

49

663

AllSport® (Pepsico) Powerade® (Coca-Cola Co.)

Orange Juice (Tropicana)

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Sports drinks may be hypotonic or hypertonic (<50-250 mOsmol/kg or 600-700 mOsmol/kg, respectively) and still be effective (Ford, 2002). These drinks will allow the cell to gain or lose water, but are formulated to an amount that will minimize gains and losses to appropriate proportions for which the body can easily compensate. Hypotonic drinks will result in water losses that will maximize fluid uptake from the intestinal lumen, while hypertonic drinks will result in water gains that will delay absorption until fluid has been withdrawn from body fluids. Although the body is equipped to cope with such variances, an isotonic formulated beverage is ideal to complete hydration and energy repletion. Figure 8.6 lists the chemical composition and osmolality of some commercial sports drinks. The following equations are used to calculate osmolality: Osmolality (Osmol/kg) = k n molality k = constant for non-ideality n = number of particles 1 molal solution of sucrose = 342 g of sucrose into 1000 g H2O Displacement of water with 342 g sucrose = 212 ml H2O Therefore, g of sucrose associated with 1000 g H2O = (342 x 1000)/788 = 434 g Molality of a 1 molar solution of sucrose = 434/342 = 1.27. Now, using the above equation (osmolality = k n molality), osmolality by each additional ingredient in the formulation can be calculated. A 0.2 molal solution of sodium chloride (k = 0.93) is: Osmolality = 0.93 x 2 x 0.2 = 0.372 Osmol/kg or 372 mOsmol/kg. However, for sports drinks, salts are normally considered as dilute solutions. Therefore, the constant, k, can be ignored, or equal to one, making Osmolality equal to n molality. For the example above: A 0.2 molal solution of sodium chloride (k=1.00) is Osmolality = 1.00 x 2 x 0.2 = 0.4 Osmol/kg or 400 mOsmol/kg

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In real practice, electrolyte concentrations in sports drinks are normally in a low range (less than 0.05 molal. At such concentrations, the volume displaced by the solute is negligible and molal concentration equals to molar concentration. The following equation is an example of how one could calculate osmolality (sodium chloride) in a sports drink containing 117 mg salt per kg. 117 mg NaCl in 1 kg = 0.117 g/kg Molality = 0.002 (Based on NaCl = 58.5g/mole) n=2, k=1 1 x 2 x 0.002 = 0.004 Osmol/kg Or, 4 mOsmol/kg. Carbohydrate components greatly affect Osmolality. These nonelectrolytes do not dissociate and n=1. Assuming ideal behavior, and by substituting the appropriate values in the expression above, a molal solution of carbohydrate will have a value of 1 Osmol/kg or 1000 mOsmol/kg. For example, if there is 54 g of glucose (molecular weight=180) in 1 kg or liter of a sports drink, its molal concentration equals 0.3 and will have 0.3 Osmol/kg or 300 mOsmol/kg. The variety and availability of carbohydrate sources are also essential in new product development. In order to formulate products without compromising osmolality, total energy, or taste; food technologists often work with mixtures of sugars to manipulate their products. Sports Drinks Processing Technology Purified water is the major ingredient in commercial sports drinks, comprising more than 90% of the total. Sports drinks contain carbohydrates as an important energy source. Small quantities of simple carbohydrates, such as glucose, sucrose, fructose, or maltodextrins, aid water absorption, but larger quantities interfere. Electrolytes are also present to facilitate water absorption and to restore electrolytes after exercise. Sodium and chloride are the principal electrolytes; others are potassium, magnesium, calcium, iron, phosphates and carbonates. Electrolytes are usually stabilized in ionic form by co-addition of citric acid or malic acid. The basic taste of electrolytes is unpleasant and it is common practice to add some type of fruit or milk-based flavoring. Vitamin mixtures, especially C, B-complex and E, are present in some beverages formulated for post-exercise use. More recent formulations,

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especially in the US, include chromium and creatine to place emphasis on enhancing performance. Colorants are added to stimulate and optimize voluntary fluid consumption. The final product is either heat treated and aseptically packed or a preservative is added. The manufacture of sports drinks is primarily a matter of blending ingredients. Once an optimized formulation has been developed, preparation of the beverage usually involves mixing of the measured ingredients in stainless steel tanks fitted with mechanical agitators. The process must be protected against microbial contamination and is usually carried out in a separate clean room, ideally equipped with air filtration equipment and maintained at a slight positive air pressure. It is now common practice to pasteurize the final product using a plate heat exchanger. Water Treatment In addition to its functional role, water plays a critical role in the quality characteristics of sports drinks. The quality of water of sports drinks has a major role on the organoleptic and microbiological quality of the final product as well as its life on the shelves. Even though, in most of the cases the source water used as a raw ingredient is potable, it still needs to be treated to accomplish rigorous quality tests. The main factors are the microbial indicators of contamination such as coliform or Pseudomonas aeruginosa, but also unstable components like dissolved iron or possible changes in its physico-chemical composition due to seasonal and environmental conditions. Water treatment includes removal of the following elements from the raw water supplied: 1. 2.

3.

4.

5.

Undesirable biological elements. This includes, on decreasing size order, protozoa, mold, bacteria and viruses. Undesirable chemical elements. The source of these chemical elements may be due to natural conditions, such as the geological characteristics of the soil, or to human contamination. Undissolved elements. Undissolved elements can be in particulate or colloidal form and may originate from the water source or during the manufacturing process. Iron and manganese. In the underground, these two elements are dissolved in their stable form in the water; but in contact with the air they oxidize and ultimately precipitate. Arsenic and Fluoride. Both of them identified as “chemical of health significance” by World Health Organization (WHO). Arsenic is naturally found in underground water from volcanic

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soils and fluoride is associated with highly mineralized and naturally carbonated water. 6. Volatile organic compounds and pesticides. Both groups of substances are identified as “chemical of health significance” by WHO. They usually result from contamination by human activities. 7. Ammonium. Even though ammonia is not identified as a “chemical of health significance” by WHO, it has to be strictly controlled due to its ability to be oxidized into nitrite which is recognized to be toxic 8. Organic matter. Although it is not a toxic element by itself, organic water may develop toxic-byproducts when oxidized with ozone or chloride and is a possible source of biological growth in the process that may lead to biomass accumulation and off-flavor. Water treatment plant design and level of technology involved should be based on a series of factors regarding the quality of raw water supplied to the sports drink manufacturer, which include: • Confidence in the source water treatment process • Consistency in quality • Standard levels of adverse raw quality factors (e.g., microbiology, suspended matter, dissolved minerals, disinfectant residues, alkalinity, etc) • Protection from external contamination at the source as well as throughout the distribution system • Seasonal variations in source water composition and characteristics. The more favorable these factors are, the more simple the water plant design and operation will be. Below is the basic water treatment process: • Raw water • Enhanced filtration • Disinfection • Activated carbon purification • Polishing filtration • Treated water INGREDIENTS Commercial sports drinks in the U.S. are generally composed of varying types of carbohydrates such as monosaccharides, disaccharides, and sometimes maltodextrins, in concentrations ranging from 6% to

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9% weight/volume. These beverages typically contain a small amount of minerals (electrolytes) such as sodium, potassium, chloride, and phosphate, and are available in a number of fruit-related flavors. For the overall purpose of maintaining physiological homeostasis and of sustaining the capacity for continued physical activity, sports drinks are generally formulated to supply fuel for working muscles, to maintain or enhance performance, and to provide water to replace the amount lost in sweat. Carbohydrates Carbohydrate type and concentration influence the rate of water, carbohydrate, and electrolyte absorption of a sports drink. The proper combination of carbohydrates optimizes sweetness and flavor characteristics, maximizes intestinal water flux, and guarantees adequate energy provision. Commercially available carbohydrate sources typically include glucose, sucrose, fructose, corn syrup solids, maltose, and maltodextrin. Sports drinks rarely contain complex carbohydrates because of the low sweetness and unacceptably thick mouthfeel characteristics that such carbohydrates impart. Each type of carbohydrate has a different sweetness profile, as characterized by the perceived intensity of the sweetness and by its onset and duration. There may be benefits in using several different carbohydrates, including free glucose, sucrose, and maltodextrin. Electrolytes Sodium plays an important role in improving beverage taste, stimulating voluntary fluid intake, promoting fluid absorption, maintaining plasma volume, and assuring rapid and complete rehydration. Sports drinks usually contain 10 to 30 mmol/L sodium, which is usually added as sodium chloride. Other forms of sodium used in sports drinks include sodium citrate and sodium acetate, and each possesses different flavor and functional characteristics. The presence of sodium chloride is a critical ingredient in a sports drink. Sodium stimulates sugar and water uptake in the small intestine and will help to maintain extracellular fluid volume. Therefore, ingesting sodium during exercise helps to restore sweat that is lost during exercise, and stimulates further drinking. However, excess sodium restores the extracellular fluid space too rapidly, removing the volume-dependent drive to continue drinking.

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Consuming too much or too little sodium both discourages further drinking and impedes complete rehydration. It is now quite clear that some sodium is necessary and sodium-containing fluid provides distinct advantages over plain water and other sodium-free drinks. The addition of a small amount of sodium chloride to a sports drink can markedly influence drinking behavior. Potassium is normally present in commercial sports drinks in concentrations of 3-6 mmol/L and is added as a means of replacing mineral losses in sweat and urine. An increase in the circulating potassium concentration is the normal response to exercise and increasing this further by ingestion of potassium has not been proven useful. However, potassium deficiency stimulates thirst and might be implicated in the relationship between thirst and fluid intake and seems to be linked to muscle cramping. Replacement of losses will normally be achieved after exercise from the potassium present in foods. Acidulants The incorporation of acidulants is considered important in determining the sensory quality of commercial sports drinks. Electrolytes are often stabilized in ionic form in the presence of acidulants in a beverage system. A number of acidulants are permitted in sports drinks, of which citric acid is the most widely used, followed by malic acid, and vitamin C. Citric acid is a weak organic acid found in citrus fruits but is most concentrated in lemons and limes, where it can comprise as much as 8% of the dry weight of the fruit. It is a natural preservative and is also used to add an acidic (sour) taste to sports drinks. Citric acid has light and fruity properties, which are highly acceptable in sports drinks. The production technique, which is still the major industrial route to citric acid used today, cultures of Aspergillus niger are fed on sucrose to produce citric acid. After the mold is filtered out of the resulting solution, citric acid is isolated by precipitating it with lime (calcium hydroxide) to yield calcium citrate salt, from which citric acid is regenerated by treatment with sulfuric acid. Malic acid is a tart-tasting organic dicarboxylic acid that plays a role in many sour or tart foods and beverages. It has a slightly stronger flavor than citric acid with a more pronounced fruitiness characteristic. Vitamin C or ascorbic acid is now also being used for sports drinks formulation as a functional ingredient. The level of ascorbic acid in sports drinks should be around 50 to 100 mg per serving.

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FUNCTIONAL FOODS

Flavorings And Colorants Flavor is an important factor on the acceptability of sports drinks that ultimately will determine voluntary fluid intake. Flavors are the ingredients used to give sports drinks their distinctive taste and smell properties, even though other ingredients such as electrolyte concentration, acidulant, and sweetener also contribute to the overall flavor profile of the product. Most sports drink flavoring components are used in liquid form, but powdered spray-dried flavors can also be used, especially for home made sports beverage preparation. Overall, citrus flavors are the most popular flavors in the sports drink industry; although in the US the new trends involve very innovative flavors such as passion fruit or papaya, mainly created to satisfy the acceptability of the growing Hispanic sector. Coloring is often used to reinforce consumer perception of flavor, especially on sports drinks bottle packaging. In some cases the color is actually of greater importance than taste in the overall impression made on the consumer: reds invoke the flavor associated with berry fruits, orange and yellow invoke citrus flavors while green and blues are associated with fruit and peppermint flavors (Varnam and Sutherland, 1994). Overall a wide range of colors is permitted, although there is some variation from country to country. Coloring agents must have good performance in the presence of light, acid pH, and other formulation agents like flavoring and preservatives. Colors in use in the beverage industry have a high tinctorial strength and consequently the concentrations required are very low, i.e., 20-70 mg/l (Varnam and Sutherland, 1994). Functional Ingredients As mentioned earlier, there are a number of ingredients that, despite a lack of supporting evidence, have gained popular attention as immune boosters, energy boosters, or fatigue fighters. Some of these ingredients have been suggested or actually incorporated into sports drinks. However, there tends to be some confusion in regards to supplements that are intended to boost energy, and whether the solution they are sold as is a sports drink or an energy drink. A difference between sports drinks and energy drinks does exist. As has already been discussed, characteristics of sports drinks include rapid and complete rehydration, adequate energy, and enhanced performance. Energy drinks, however, have different functional aspects and should be differentiated into their own category. Energy drinks can possess a number of ingredients, such as vitamins, minerals, and

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legal stimulants that are intended to give the user a mental and physical energy boost. Regardless of whether specific ingredients are better suited for energy drinks as opposed to sports drinks, many have still been suggested as additions to the sports drink formulation. These include, but are not limited to; taurine, choline, caffeine, carnitine, sodium bicarbonate, branched chain amino acids (BCAA), and antioxidants. Taurine is a derivative of cysteine, which can be conjugated in the body with cholic acid to produce bile salts. Bile salts are essential in the absorption of lipids, and it is therefore speculated that taurine and choline added to a beverage will enhance energy via the oxidation of fat. However, this has not been substantiated in sports drinks by any scientific data. Choline, besides the role it plays with taurine, has been suggested to combat fatigue, because of its role in sustaining muscle performance by increasing the rate of acetylcholine synthesis. It has been shown that choline levels can be maintained, and elevated, by oral administration (Spector, 1995). Caffeine has been found to enhance mental concentration and cognitive function. It has, therefore, been added to many commercial energy drinks. However, to add it to a sports drink could introduce some detrimental effects. Caffeine is a diuretic, and can therefore lead to a loss of fluids, rather than replenishing them. And the proposed benefits of caffeine do not outweigh the known benefits of hydration to performance. It has been reported that during exercise, hormones may override the effects of caffeine in an attempt to maintain fluid levels in the body (Wemple, 1997), but no scientific studies have concluded that caffeine added to sports drinks has beneficial effects. Carnitine is a nitrogenous compound that serves to transport lipids over the mitochondrial membrane, for lipid oxidation. Theory suggests that supplementation with carnitine will increase lipid oxidation, and reserve energy stores in the form of muscle glycogen. However, there is no evidence to support this claim. Evidence does show that carnitine prevents lactate build up in blood, which could be useful in prevention of cramping muscles, but research has also shown that additional carnitine through diet does not increase muscle carnitine concentrations at all. The addition of sodium bicarbonate to sports drinks is a means to control pH. Exercise promotes a lowering of pH due to the build up of lactic acid in the muscles. Sodium salts help to prevent this by acting as buffers. The extra hydrogen ions out of skeletal muscle serve to reduce

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FUNCTIONAL FOODS

the accumulation of protons that impair enzyme activity. Sodium bicarbonate has been proven effective in balancing pH and forming cross bridges between contractile proteins. However, it has also been shown to produce side effects such as gastrointestinal distress, cramping, and diarrhea. This is because sodium bicarbonate can elevate osmolality in the lumen, and decrease absorption of fluids. This is where careful formulation is essential. Sodium bicarbonate should only be added at low levels, or supplemented instead by sodium citrate, which has also been shown to have a similar buffering effect, but without gastrointestinal side effects (Ford, 1995). It is proposed that BCAA provide a substrate for oxidation when glycogen stores are low, either from low carbohydrate intake or prolonged exercise. BCAA are also thought to bind to the plasma transport protein albumin, which is responsible for the uptake of tryptophan into the brain. If BCAA bind to albumin, and tryptophan is displaced, less serotonin is released into the body. This is important because serotonin is thought to induce fatigue. It is true that BCAA are oxidized when glycogen is low, however research has not shown that this reduces fatigue during exercise. So, although the oxidation theory is substantiated mechanistically, it still has not been proven to have application to sports drinks. The same is true for the tryptophan displacement theory. Although this may happen, it has not been shown to reduce fatigue during exercise. Therefore, adding BCAA to sports drinks has not been clinically shown to be effective, but may prove to be a valuable formulation tool in the future. Antioxidants may start receiving the most attention when it comes to sports drinks supplementation, because there is clinical evidence to show that they effectively decrease the rate of lipid peroxidation and damage to skeletal muscle after exercise. Antioxidants scavenge oxygen free radicals that are formed with high rates of energy metabolism and repetitive contractions that disrupt muscle integrity. Clinical trials have shown that muscles loaded with vitamin E had less decrease in fatty acid concentrations (a measure of peroxidation) and less oxidative damage when compared to muscles without vitamin E (Horswill, 2001). It has also been well established that vitamin C is an antioxidant, responsible for scavenging free radicals in the body. However, vitamin E and C have not yet been clinically proven to decrease muscle soreness and damage from exercise when consumed in a sports drink. These antioxidants have been tested time and again in other forms, and results are consistent that they do in fact positively impact the body. With further research, specifically aimed at sports drinks, the future could

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hold exciting breakthroughs for antioxidants in the world of sports drinks. Water, carbohydrate, and electrolytes are standard ingredients for sports drinks. There are many claims attached to added functional ingredients, many of which are unsubstantiated by laboratory research. This, however, should not be a discouraging thought, for it opens the door to many future research endeavors. The general processing technology of sports drinks is shown as follow: • Sugar and salt preparation and water treatment • Mixing • Add flavors, and colorants • Preservatives/heat treatment/bacteria inactivation • Blending • Bottling • Packaging • Storage • Distribution Future Developments In Sports Drinks A United States consumer report found that children 10 years of age and below were a demographic sector for sales growth of the sports drink industry (Holay, 2005). Their study also found that children establish their preferences toward sports drinks early, and parents provide support for their choice over soft drinks. This information may suggest that sports drinks will be marketed to children in the future. This could mean new flavors and colors, different packaging, perhaps with cartoon celebrity endorsements, and different plastic bottles that are more child friendly. Almost 50% of adults are watching what they eat, 25% are dieting, 28% think about the calories in what they eat, and 18% normally count calories. So, low calorie foods are a hot commodity. In 2005, sales of low carbohydrate foods fell by 33.7%, while sales of light foods excluding low carbohydrate foods grew by 2.5%. This is good news for the sports drink industry (Sloan, 2006). Manufacturers can keep the carbohydrates essential to their product in sports drinks, but will most likely be incorporating lower calorie or light beverages into the market. In summary, in just 40 years sports drinks have been scientifically proven effective in quickly replacing energy and fluids to the body, therefore enhancing performance. They have grown from being the secret weapon behind a gold star team of athletes to being a multibillion dollar industry, available to the global market. With an emphasis

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on health and wellbeing in the global marketplace, sports drinks will continue to be a popular choice among Americans and others around the world. Future research will determine what changes will come about in this industry, whether they will be in taste and flavor development, packaging, marketing, or health promotion. One thing is for certain; the basic components of water, carbohydrates, and electrolytes will always be the essential factors in the formulation of sports drinks. References Below, P., Mora-Rodriguez R., Gonzalez-Alonzo J., and Coyle E.F. 1993. Fluid and carbohydrate ingestion independently improve performance during 1h of intense cycling. Medicine and Science in Sports and Exercise, 27, 200-210. Cade, R., Spooner, G., Schlein, E., Pickering, M., and Dean R. 1972. Effect of fluid, electrolyte, and glucose replacement during exercise on performance, body temperature, rate of sweat loss, and compositional changes of extracellular fluid. Journal of Sports Medicine and Physical Fitness, 12, 150-156. Ford, M. A. 2002. The formulation of sports drinks. In: Production and Packaging of Non-Carbonated Fruit Juices and Fruit Beverages. pp 310-330. 2nd edn. P.R. Ashurst (ed.) Blackie Academic & Professional, New York, USA. Hilliam, M. 2002. Sports beverages: new functional ingredients and health claims are driving the segment. Food Ingredients First.com. CNS Media BV, Arnhem, The Netherlands. Holay, A. 2005. Good sports, sports and energy beverages. Prepared Foods, Development Trends & Technologies for Formulators & Marketers. 174 (No. 9), 13-24. Horswill, C. 2001. Other ingredients: role in the nutrition of athletes. In: Sports Drinks: Basic Science and Practical Aspects. pp 225-256. I. Wolinsky (ed.) CRC Press LLC, Boca Raton, USA. Ivy J. L. 1998. Glycogen resynthesis after exercise: effect of carbohydrate intake. International Journal of Sports Medicine, 19, S142-S145. Maughan, R.J. 2001. Fundamentals of sports nutrition: application to sports drinks. In: Sports Drinks: Basic Science and Practical Aspects. pp 1-28. I. Wolinsky (ed.) CRC Press LLC, Boca Raton, USA. Murray, R. and Stofan, J. 2001. Formulating carbohydrate-electrolyte drinks for optimal efficacy. In: Sports Drinks: Basic Science and Practical Aspects. pp 197-224. I. Wolinsky (ed.) CRC Press LLC, Boca Raton, USA. Ohr, M.L. 2003. More for the sport. Food Technology. 57 (2), 63-68. Sloan, E. A. 2006. Top 10 functional food trends. Food Technology. 60 (4), 23-40. Spector, S.A., Jackman, M.R., Sabounjian, L.A., Sakkas, C., Landers, D.M., and Willis W.T. 1995. Effect of choline supplementation on fatigue in trained cyclists. Medicine and Science in Sports and Exercise, 27, 668. Varnam, A. H. and Sutherland, J. P. 1994. Soft Drinks. In: Beverages: Technology, Chemistry and Microbiology. pp 73-125. Chapman & Hall, London, UK. Wemple, R.D., Morocco, T.S., and Mack, G.W. 1997. Influence of sodium replacement on fluid ingestion following exercise-induced dehydration. International Journal of Sports Nutrition, 7, 104-116. Zenith Reports International. 2004. International functional soft drink report 2003. Zenith Reports International, Decision New Media: www.foodanddrinkeurope.com. (Guo, M.R. Lee, F.L., Rice, B.)

Chapter 9 HUMAN MILK AND INFANT FORMULA Finding the perfect alternative to Mother’s milk has proven to be a very complicated task that continues today with an ever-growing assortment of modified and specialized formulas. If you were born in the 1930’s and 40’s and not breastfed as an infant, there is a good chance that you were fed a formula created by mixing 13 oz of unsweetened evaporated milk with 19 oz of water and two tablespoons of either corn syrup or table sugar. Every day, parents prepared a day’s worth of this formula, transferred it to bottles that they had sterilized in a pan of boiling water, and stored it in a refrigerator until used. In addition to this formula, infants received supplemental vitamins and iron. Supplemental infant nutrition has a fascinating history that began long before pediatricians recommended evaporated milk formula as alternatives to breastfeeding. Prior to the use of evaporated milk as a substitute for breast milk, wet nursing was the method of choice; breastfeeding was the preferred method of feeding infants, just as it is today. But if a mother’s milk supply was inadequate or she chose not to nurse, the family often employed a “wet nurse” to nourish infants. This practice was common in Europe during the 18th century and in America during the colonial period. Families would hire a wet nurse to reside in the home or sometimes send the infant to live in the wet nurse’s home and retrieve the baby after he or she had been weaned, perhaps three to six months later. Wet nurses were selected with the utmost care, because it was believed the quality of milk the baby received determined his or her future “disposition”. Brunette wet nurses were preferred to redheads or blondes because their breast milk was thought to be more nutritious and their disposition more “balanced”. This is perhaps the genesis of all those bad blonde jokes we still hear today.

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In 18th century Europe, the demand for wet nurses was so great that bureaus were established where wet nurses could register and reside until their services were needed. The British governments regulated the bureaus very strictly; passing laws mandating that wet nurses undergo routine health examinations and prohibiting them from nursing more than one infant at a time. Eventually, wet nursing fell out of favor and attention turned to finding an adequate substitute for mother’s milk. The practice of feeding human babies milk from animals; “dry nursing”, began to flourish in the 19th century and milk from a variety of animals was tried: goats, cows, mares, and donkeys. Cow’s milk quickly became the most widely used because of its ready availability, although donkey’s milk was thought to be healthier because its appearance more closely resembled that of human milk. Physicians at the time argued about the best way to prepare the milk. Some promoted giving fresh raw milk to the infant. Others recommended that it be warmed or boiled first, and still others suggested diluting it with water and adding sugar or honey. When baby bottles were adopted during the Industrial Revolution, many popular designs evolved. Prior to that, the milk was spoon fed to infants or given via an improvised feeding device such as a hollowed out cow’s horn fitted with a leather nipple. Rubber nipples became widely available and very popular after their invention by Elijah Pratt in 1845. After weaning the infant from breast milk or a substitute animal’s milk, he or she was given an infant food called pap which consisted of boiled milk or water, thickened with baked wheat flour and sometimes egg yolk. A more elaborate infant food, called panada, was made from bread, flour, and cereals cooked in a milk or water-based broth. Detailed recipes for various kinds of infant paps and panadas have been published in cookbooks throughout history. In 1930, three Canadian doctors; Frederick Tisdall, Theodore Drake, and Alan Brown developed Pablum at the Hospital for Sick Children in Toronto. During the 1920s and 1930s, considerable time and effort were spent studying the science of artificial feeding. Society seemed to welcome the scientific approach to infant feeding and food and bought products that advertised increased nutritional value for their children. In 1931, Pablum, an infant cereal fortified with minerals and vitamins, became commercially available in Canada and the United States. The food was heralded as an excellent cereal addition to the infant’s diet, and it was a major commercial success. Modern infant formula is an industrially produced milk substitute designed for infant consumption. Usually based on either cow or soy

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milk, infant formula strives to duplicate the nutrient content of natural human breast milk. Since the exact chemical and biological properties of breast milk are still not fully understood, ‘formula’ is necessarily an imperfect approximation. Besides breast milk, infant formula is the only other infant milk which the medical community considers nutritionally acceptable for infants under the age of one year. However, its use particularly in the third world is somewhat contentious. Cow’s milk is not recommended because of its high protein and electrolyte (e.g., sodium) contents which may put a strain on an infant’s immature kidneys. Evaporated milk, although perhaps easier to digest due to denaturing of the proteins, is still nutritionally inadequate. Further, the biological benefits of human milk is a potent defense mechanism in breast-fed infants, particularly in third world countries where it helps to control severe diarrhea which is often lethal (Hendricks and Guo, 2006). Human Milk Chemistry Human milk is the “gold” reference standard for infant nutrition and is recognized as the preferred food for infants due to the nutrient balance, immunological protection, and other growth-promoting substances. Infant formula formulation is a mimic of human milk chemistry. Gross Composition The gross composition of human milk, cow’s milk and infant formula is shown in Figure 9.1. The protein content of human milk is approximately 1.0%, with approximately 70% of the protein being provided by whey proteins. Human milk has the highest level of lactose (7.0%) among mammals, providing 40% of human milk’s total energy. Fat provides approximately 50% of the gross energy of human milk, with an average content of 3.8%. Ash content in human milk is only 0.2% compared with 0.7% in bovine milk. Water content in human milk is similar to bovine milk at about 87%. Infants need approximately 125 ml of water /kg body weight per day supplied from either breast milk or a commercial formula (water loss is estimated at 20 ml/kg body weight per day). As lactation progresses, the chemical composition of human milk changes as a result of physiological and external factors. Some external factors may contribute negatively to the quality of human milk, for example, some environmental pollutants, such as heavy metals, can be detected in human milk, as well as many drugs. It is difficult to fully measure the impact of maternal diet on milk composition. Maternal

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malnutrition plays a role in many developing countries where the food supply is limited, infections are common due to poor hygiene, and economic situations do not allow for the choice of properly used infant formulas. Dehydration can affect water fluxes in the body and thus, reduce the volume of milk produced (Hendricks, 2001). FIGURE 9.1 — Composition Of Human Milk, Bovine Milk And Infant Formula (%) Human Milk Protein CN:WP Fat Lactose Total Solids Ash

1.00 30:70 3.80 7.00 12.40 0.20

Bovine Milk 3.40 80:20 3.50 5.00 12.50 0.70

Formula 1.50 40:60 3.80 7.20 13.0 0.30

Proteins In Human Milk The level of total protein in milk is approximately 0.9 - 1.2%, of which approximately 70% is whey protein and 30% is casein along with small amount of proteins associated with the milk fat globules. There is no blactoglobulin in human milk. The primary whey proteins are
-lactalbumin, lactoferrin, and secretory IgA (SIgA). The whey proteins human milk lacks are
-caseins, b-casein and -casein (Figure 9.2).
-Lactalbumin is one of the major whey proteins and is required for the biosynthesis of lactose. Human
-lactalbumin can bind both Ca and Zn. However, only a small part of the total calcium found in human milk is bound to
-lactalbumin. It is possible that
-lactalbumin may generate peptides that facilitate the absorption of divalent cations, thus exerting a positive effect on mineral absorption. Supplementation of infant formula with bovine
-lactalbumin may increase the absorption of iron and zinc. Lactoferrin tightly binds iron, presumably limiting the availability of iron to potentially pathogenic microflora. SIgA can bind specific antigens in the infant gastrointestinal tract, preventing infection. Lysozyme is another human milk protein that plays a specific role in host protection, by lysing the cell walls of potential pathogens, preventing infection. Caseins contribute to the amino acid pattern of human milk, and they are also highly digestible. Functionally, their most important property is the ability to form stable aggregates that include calcium and phosphorus, which allows for greater concentrations of these minerals in human milk than is possible by

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their solubility alone. Non-protein nitrogen components consist of urea, peptides, nucleotides, nucleosides, and free amino acids, and remain after milk protein has been precipitated with 12% trichloroacetic acid. Casein exists as micelles in colloidal dispersion. Micelles of human milk range from 20 to 55 nm in size compared with those from 100 to 150 nm of bovine milk. FIGURE 9.2 — Protein Components Of Human And Bovine Milk (%)

Total Caseins
S1-Casein
S2-Casein β-Casein -Casein Micelle Size (nm) Whey Proteins
-Lactalbumin β-Lactoglobulin Lactoferrin Serum albumin Lysozyme Immunoglobulins Others

Human Milk

Bovine Milk

0.3 g/100 g — — 85 15 50 0.7g/100g 26 26 10 10 16 (IgA) 12

2.6 g/100 g 40 8 38 12 150 0.8g/100g 17 43 trace 5 trace 10 (IgG) 24

Proteins in human milk provide an important source of amino acids to the growing infant, and also play a very important role in facilitating the digestion and uptake of many other components in human milk. Lactoferrin, β-casein, and haptocorrin may enhance the absorption of iron, calcium, and vitamin B12, respectively. Other activities of human milk proteins include immune function enhancement, defense against pathogenic bacteria, viruses and yeasts, and gut development and function (Lonnerdal, 2003). The contents of casein and whey proteins change profoundly in the early stages of lactation; whey protein concentration is very high and casein is virtually undetectable during the initiation of lactation. As lactation progresses, casein synthesis in the mammary gland and milk casein concentration increases, while the concentration of whey proteins decreases, in part due to a larger volume of milk produced. Therefore, the ratio of whey: casein is not constant, but fluctuates between 70:30 or 80:20 in early lactation, to 50:50 in late lactation (Lonnerdal, 2003). The amino acid profile of caseins and whey proteins are different, thus, the amino acid profile of human milk varies during lactation.

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Historically, the protein content of human milk was overestimated due to the large proportion of non-protein nitrogen (NPN) in human milk relative to the milk of other species. In the milk of most species, NPN makes up a small fraction (~5%) of the total nitrogen (TN); therefore, it is fairly accurate to estimate the protein content by total nitrogen analysis. In cases such as this, the true milk protein content is estimated by multiplying the nitrogen content of the milk by a conversion factor of 6.38, which takes into account the fraction of NPN in dairy products. In human milk, NPN accounts for approximately 20 - 25% of TN, thus, the use of the 6.38 conversion factor with the total nitrogen in milk yields an overestimate of total protein (Lonnerdal, 2003). To obtain a more accurate estimate, it is best to determine the TN content, subtract the NPN, then multiply the remaining nitrogen by the conventional Kjeldahl factor of 6.25. The protein content in human milk ranges from 1.4 – 1.6 g/100 ml during early lactation, 0.8-1.0g/100ml by 3 - 4 months of lactation, and 0.7-0.8 g/100 ml after 6 months. The levels of protein and corresponding intakes may not accurately represent the amount of utilizable amount of amino acids supplied to infants, as intact breast-milk proteins have been found in the stool of the breastfed infant, indicating that they are incompletely digested, and that available amino acids do not represent utilized amino acids. Undigested, biologically active proteins may have physiological benefits for the breastfed infant, therefore, the nutritional loss of the amino acids in these proteins may be insignificant, depending on the quantity lost. It is commonly understood that nutrients in human milk are exceptionally well utilized by the breastfed infant. Human milk proteins play many roles in the absorption of these nutrients. Proteins bind essential nutrients, aid in maintaining their solubility, and facilitate their uptake by intestinal mucosa. Protease inhibitors may assist in this process by limiting proteolytic enzyme activity, which can preserve the physiological function of some relatively stable binding proteins. In addition, some enzymes in human milk affect the digestion and utilization of some micronutrients. Human milk proteins involved in digestive function include bile saltstimulated lipase, amylase, and
1-antitrypsin (Lonnerdal, 2003). Bile salt-stimulated lipase may aid in the digestion of lipids in newborns, especially in premature infants, who experience reduced lipase activity and poor lipid utilization. Bile salt-stimulated lipase hydrolyzes di- and triacylglycerols, cholesterol esters, diacylphosphatidylglycerols, and micellar and water-soluble substrates. Human milk has a significant

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amount of
-amylase, although there is not a substrate for amylase in human milk. It has been suggested that the amylase in human milk may compensate for low salivary and pancreatic amylase activity in newborns, and may aid in the digestion of complex carbohydrates when complementary foods are fed close to the breastfeeding session. The protease inhibitors
1-antitrypsin and antichymotrypsin are both present in human milk. It is thought that they may collaborate to limit the activity of pancreatic enzymes in breast fed infants. Proteins such as
1-antitrypsin may escape complete digestion, and can be found in the stool of breastfed infants. In in vitro experiments, the addition of
1-antitrypsin results in a larger amount of lactoferrin resisting proteolytic degradation, although data on total nitrogen balance of breastfed infants are not substantially affected. This suggests that the protease inhibitor effect of
1-antitrypsin and antichymotrypsin may simply delay breakdown of these proteins, rather than preventing it completely (Lonnerdal, 2003). β-Casein is the major constituent of caseins in human milk, and it is a highly phosphorylated protein. Phosphopeptides formed during digestion have been shown to keep Ca soluble, thus enhancing calcium absorption. Clusters of phosphorylated threonine and serine residues are located close to the N-terminal end of β-casein, and can complex Ca ions. Thus, phosphopeptides formed from β-casein contribute to the high bioavailability of calcium in breast milk (Lonnerdal, 2003). Casein phosphopeptides may also affect the absorption of zinc and other divalent cations. Lactoferrin, a major iron-binding protein capable of binding two ferric irons, binds a major portion of the iron in human milk. It facilitates human intestinal cell iron uptake in cultured cells, which is most likely mediated by a specific enterocyte lactoferrin receptor (Suzuki, et al, 2002). Studies investigating the addition of bovine lactoferrin to infant formula have not revealed an enhancing effect on either iron uptake or iron status. Therefore, it appears that bovine lactoferrin does not bind to a human lactoferrin receptor, or that lactoferrin only exerts a benefit in human milk, and that when added to infant formula, other constituents interfere with iron utilization from lactoferrin. Heat treatment processing in formula after lactoferrin is added may contribute to the lack of effect observed when lactoferrin is added to human milk (Lonnerdal, 2003). Haptocorrin, previously known as vitamin B12 binding protein, binds nearly all the vitamin B12 found in human milk. Haptocorrin exists at a much higher level than vitamin B12 in human milk, which results in this protein being found most commonly in the unsaturated form. This

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may have important antimicrobial benefits, as research indicates that haptocorrin may inhibit bacterial growth. Holohaptocorrin, the complex of vitamin B12 and haptocorrin, appears to bind in a saturable manner to human intestinal brush border membranes, and human intestinal cells in culture take up haptocorrin-associated vitamin B12 (Adkins & Lonnerdal, 2002). This collectively suggests a role for haptocorrin in vitamin B12 absorption early in life. Intrinsic factor is a substance secreted by the gastric mucosa that facilitates vitamin B12 absorption. Although intrinsic factor is present in the stool of breastfed infants at a young age, it may not be present in amounts adequate to facilitate the uptake of vitamin B 12 via the intrinsic factor receptor, therefore, haptocorrin is the main route for vitamin B12 absorption (Adkins & Lonnerdal, 2002). Folate-binding protein (FBP) has been found in human milk, both in particulate and soluble forms. When found as the soluble form, FBP is ~22% glycosylated which may aid this protein in resisting proteolytic digestion. In newborn goats, FBP has been found to resist proteolysis and tolerate low gastric pH, and it is possible that it behaves in a similar manner in human infants (Salter & Mowlem, 1983). Experiments performed with rat intestinal cells observed an increased uptake of folate when it was complexed with FBP than when it was provided in the free form (Colman, Hettiarachchy, & Herbert, 1981). It has been theorized that FBP may slow the release of folate in the small intestine, allowing for a slow absorption of folate, which could increase tissue use. Insulin-like growth factors (IGFs) I and II are also present in human milk, most commonly associated with IGF-binding proteins. These IGFbinding proteins may protect against IGF being digested, prolong their half-life, and control their interaction with intestinal receptors. Lipids In Human Milk Lipids play a diverse role in human nutrition and development (e.g., energy source, energy storage, vehicles for the absorption and transport of fat-soluble compounds). Fat is the most variable component of human milk and although the fat content in human breast milk is markedly influenced by lactational stage, fatty acid composition remains relatively stable. Normal growth and weight gain of infants is dependent on an adequate supply of fats in the diet. Especially the essential fatty acids, a group of naturally occurring unsaturated fatty acids with chain lengths of 18, 20, and 22 carbon atoms and containing between two and six methylene interrupting double bonds (Hendricks and Guo, 2006). Of

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these, Oleic (18:1), Palmitic (16:0), Linoleic (18:2 -6), and
-linolenic acid (18:3 -3) are most abundant in mature breast milk. With the latter two generally recognized as dietary essential fatty acids because of the inability of tissues to introduce the necessary double bonds in the carbon chains before carbon 9. Human milk contains about 3 to 5% total lipid, existing as emulsified globules, 1-4 µm in diameter, covered with a phospholipid-protein membrane derived from the mammary cells that line ducts of the glands and are released with the milk during lactation. The main function of the phospholipids in milk is as emulsifying agents and stabilizers of the milk fat globule membrane. They readily bind cations like calcium, sodium and magnesium, and possibly interact with digestive enzymes. Bovine milk contains substantial quantities of C4:0 to C10:0 short chain, saturated fatty acids, about 2% (w/w of fat) C18:2 (linoleic), and almost no other long-chain polyunsaturated fatty acids (Figure 9.3). The fatty acid composition is not altered by ordinary changes in diet. In contrast, human milk contains very little short chain fatty acids (C4:0 to C10:0), 10 to 14% (w/w of fat) linoleic (18:2 -6), and small quantities of other polyunsaturated fatty acids. The triacylglycerol structure differs as well, with much of the sn-2 position in human milk lipids occupied by C16:0 (palmitic). Human milk also contains the long chain polyunsaturated fatty acids docosahexanoic (DHA) (22:6 -3) and eicosapentaenoic (EPA) (20:5 -3) which have been shown to be important in the development of retinal and brain tissue. The major sterol in both human and bovine milk is cholesterol. Trace amounts of other sterols are present also, e.g., lanosterol in bovine milk and desmosterol and some phytosterols in human milk. The amount of cholesterol present in human milk is 10 to 15 µg/100ml. Since the role of dietary cholesterol is still not fully defined, an intake similar to that obtained through breast feeding is generally recommended. EPA and DHA are the predominant long chain polyunsaturated fatty acids in human milk, and are known to be essential to normal development of infants. These fatty acids may also be formed from precursors, even in preterm infants. The studies indicated that DHA supplementation increased DHA levels and there are correlations between DHA levels in maternal plasma and human milk, and between milk and infant plasma phospholipids. Until recently, infant formulas did not contain any significant levels of DHA, even though it is present in human milk. DHA can be synthesized from linoleic acid, but high intakes of linoleic acid can also inhibit this process. Thus, a preformed source of DHA in

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the infant diet may be more efficient in assuring the supply of an adequate amount (Lonnerdal, 1986). FIGURE 9.3 — Fatty Acid Profiles Of Human And Bovine Milks (%, w/w) Human

Bovine

1.4 6.2 7.8 22.1 6.7 44.2

3.5 1.9 1.3 2.5 2.8 10.7 27.8 12.6 63.1

Monounsaturated Palmitoleic (16:1) Oleic (18:1) Gadoleic (20:1) Cetoleic (22:1) Total

3.1 35.5 0.96 Trace 39.8

2.5 26.5 Trace Trace 30.3

Polyunsaturated Lineoleic (18:2) Linolenic (18:3) Parinaric (18:4) Arachidonic (20:4) Eicosapentenoic (20:5) Total

8.9 1.2 0.72 Trace 10.82

2.9 1.6 Trace Trace Trace 4.5

Saturated Butyric (4:0) Caproic (6:0) Caprylic (8:0) Capric (10:0) Lauric (12:0) Myristic (14:0) Palmitic (16:0) Stearic (18:0) Total

Carbohydrates In Human Milk Carbohydrate in human milk is comprised of monosaccharides, such as glucose and galactose; disaccharides, such as lactose and lactulose; oligosaccharides; and some more complex carbohydrates, such as glycoproteins. Lactose is the primary carbohydrate in human milk and most likely to contribute to malabsorption and intolerance syndromes resulting from metabolic disturbances, such as lactose intolerance, lactose malabsorption, and galactosemia. Monosaccharides in milk are primarily made up of glucose and galactose, and are found at levels of approximately 100 mg/100ml in human milk. Lactose is the nutrient least likely to be affected by maternal nutrition, including malnutrition or energy supplementation. The concentration of lactose in human milk is relatively stable at about 7%. Total oligosaccharide levels comprise of up to 10% of total carbohydrates.

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Lactose together with mineral constituents, is crucial to maintaining a constant osmotic pressure in milk. An exclusively breastfed baby receives approximately 10 - 14 g lactose per day/ kg body weight. Lactose in human milk has been reported to exert a beneficial effect on the absorption of minerals, most notably calcium, which is most likely due to its conversion to lactic acid by intestinal flora, which lowers the pH, causing increased solubility of calcium salts. This is possible because human milk has a low buffering capacity and a low content of protein and phosphorus. Neither lactose nor lactulose are hydrolyzed in the upper GI tract, and only to a very small extent in the proximal intestinal tract, but are hydrolyzed in the distal intestines. Lactulose, a disaccharide of galactose and fructose, is a growth promoting factor and energy source to Lactobacillus bifidus and Lactobacillus acidophilus. In addition, the production of lactic acid has a slight laxative effect. Oligosaccharides in human milk, ranged from tri- to octasaccharides at levels of 0.8-1.4%. At least 21 different types of oligosaccharides have been identified in human milk, composed of many different molecules, including simple sugars and sugar derivatives such as uronic acid. These can be acidic, neutral, linear, or branched. Oligosaccharides in human milk have been divided into nitrogen-free oligosaccharides, or oligosaccharides containing either N-acetylglucosamine or Nacetylneuraminic acid (sialic acid). Small oligosaccharides are common in human milk, as well as a high content of complex and fucosylated and sialylated oligosaccharides. More than 130 components have been characterized in human milk. Some components are thought to be involved with the immune system, while others may be involved with the development of a specific intestinal microflora. The oligosaccharide component of human milk is thought to be the main energy source for the intestinal flora of the breast-fed infant, which is rich in bifidobacteria and lactobacilli. Lactobacilli ferment lactose to lactic acid which, along with a low pH, promotes the growth of Lactobacillus bifidus, as well as the bifidus factors lactulose, oligosaccharides, glycoproteins, and glycopeptides. The bifidus factor is most likely found in the nitrogencontaining oligosaccharides. Oligosaccharides added to cow’s milk based infant formula include galacto-oligosaccharides and inulin, and have been shown to stimulate the growth of bifidi and lactobacilli. Vitamins In Human Milk All water-soluble and fat-soluble vitamins are found in human milk. Human milk contains more vitamin A, E, C, nicotinic acid, and inositol than bovine milk, however, it has a lower content of vitamins B1, B2,

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B6, B12, K, biotin, pantothenic acid, and choline (Figure 9.4). Human milk appears to contain adequate amounts of most vitamins to support normal infant growth, with the exception of vitamin D and possibly, vitamin K. Exclusively breastfed infants of mothers on a total vegetarian diet may require vitamin B12 supplementation to prevent a deficiency, which results in severe and permanent neurologic damage. FIGURE 9.4 — Vitamin Content In Human And Bovine Milk (mg/L) Human Milk Vitamin A Carotene Cholecalciferol (D) Tocopherol (E) Vitamin K Thiamin (B1) Riboflavin (B2) Pyridoxine (B6) Cobalamin (B12) Niacin Folic acid Ascorbic acid (C) Biotin Pantothenic acid Inositol

0.53 0.24 0.001 5.4 0.015 0.15 0.37 0.10 0.0003 1.7 0.043 47 0.007 2.1 300

Bovine Milk 0.37 0.21 0.0008 1.1 0.03 0.42 1.72 0.48 0.0045 0.92 0.053 18 0.036 3.6 160

Fat-Soluble Vitamins Vitamin A is comprised of a family of compounds in which the basic constituent is all-trans-retinol. Vitamin A is required for a large number of life processes and a deficiency has been associated with clinical disorders unique to infants. Compounds with vitamin A activity present in human milk include retinyl esters, retinol, and β-carotene. When maternal nutritional status is good, human milk supplies adequate amounts of vitamin A. Although vitamin A content of milk decreases as lactation progresses, milk ingestion volumes increase; therefore, the infant receives adequate amounts of vitamin A. Poor maternal nutritional status results in milk with a low vitamin A content, which can place the infant at risk for deficiency. Mechanisms regulating storage, mobilization, and secretion of retinoids from mammary cells have yet to be determined; although, there is indication that the concentration of retinal binding protein in serum determines the amount of retinol delivered to milk. Research indicates that vitamin A supplementation just preceding or following parturition can

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significantly increase vitamin A levels in human milk, especially in situations of low intake levels. Vitamin D plays an essential role in bone metabolism and may also be implicated in immune system regulation. The serum concentration of 25-OH-D (25-OH cholecalciferol), the active metabolite of vitamin D, is generally used to measure vitamin D status. Dietary ergocalciferol (D2) and cholecalciferol (D3) are converted to the active metabolite, 25OH-D, in the body. Infants can synthesize vitamin D endogenously in the epidermis upon exposure to sunlight, or they can receive it through dietary intake. Seasonal variations in vitamin D synthesis in infants have been observed, and light-skinned infants are more likely to benefit from sunlight exposure than dark-skinned infants. The level of 25-OHD in human milk is low, corresponding with both maternal serum 25OH-D levels and maternal dietary vitamin D intake, and can also be affected by race, season, and latitude. Infants who are exclusively breastfed receive below the minium recommended intake of vitamin D, and much lower than the recommended dietary intake, and as such, are at risk for deficiency, rickets, and improper bone mineralization, especially if sunlight exposure is poor. Normal vitamin D stores present at birth are depleted within 8 weeks, and formula-fed infants have higher serum concentrations of vitamin D metabolites than breast fed infants. Maternal supplementation with 400 - 2000 IU of vitamin D daily increases the vitamin D content of human milk, however, only the 2000 IU dose achieves satisfactory levels of 25-OH-D in the infant. Adequate sunlight exposure levels have not been clearly established, and due to the low level of vitamin D in human milk, vitamin D supplementation is recommended for breast-fed infants in Europe and the northern United States. Vitamin E is comprised of a group of compounds with different degrees of biological activity, with the most active being
-tocopherol. Vitamin E is an antioxidant, acting as a free radical scavenger and protecting against PUFA peroxidation in cell membranes. The transport of vitamin E across the placenta is limited, thus, neonatal tissues have low levels of vitamin E. Hemolytic anemia can result in neonates with a vitamin E deficiency. The vitamin E content of human milk is adequate for a term infant, but may not be sufficient for preterm infants, that have even lower levels of vitamin E at birth than the term infants. Decreased vitamin E levels in preterm infants may be related to PUFA, iron, and selenium concentrations, and hemolytic anemia is observed more frequently in preterm infants than term infants, presumably due

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to lower vitamin E levels in infants fed formula supplemented with PUFAs and iron. Therefore, preterm infants who are not breastfed should receive formula enriched with vitamin E as well as LC-PUFA, although these fatty acids may provoke early postnatal decreases in both serum vitamin E and total lipids ratio. The vitamin E content of human milk is dependent on many factors, including individual variation, stage of lactation, and large amounts of dietary vitamin E. However, maternal supplementation with vitamin E has not been shown to affect the vitamin E content of human milk in mothers with moderate vitamin E intake. In addition, in populations with low vitamin E status, adequate vitamin E content in human milk has been observed, suggesting that maternal stores of vitamin E can be mobilized during lactation to ensure adequate supply in human milk. Vitamin K activity is provided by several different naturally occurring compounds, including vitamin K1, which is provided in the diet as phylloquinone, and vitamin K 2, menaquinones, which are synthesized by bacteria in the gastrointestinal tract. Vitamin K is essential to the proteins involved in blood coagulation, and some plasma proteins and organs have been shown to be dependent on vitamin K, including proteins that are involved in the maintenance of bone structure. The transfer of vitamin K across the placenta is very limited, thus, newborn infants generally exhibit extremely low concentrations of vitamin K. However, vitamin K levels remain constant in human milk over 6 months of lactation. Vitamin K is localized in the lipid core of the milk fat globule, and not the membrane. Even in situations where maternal vitamin K consumption exceeds the recommendations, exclusively breastfed infants do not receive the recommended dietary intake, and their plasma concentrations are low compared with formula-fed infants. In addition, breast-fed infants more frequently report the development of hemorrhagic disease. Due to the low content of vitamin K in human milk, and the low concentration of vitamin K in neonates, vitamin K supplementation is recommended after birth. Studies investigating the relationship between maternal vitamin K intake and content in human milk have mixed results, as some indicate no correlation, while another observed that maternal supplementation with vitamin K appeared to increase maternal plasma and breast milk concentrations, unless the supplemental dose of vitamin K was low. Preterm infants may require vitamin K supplementation as they tend to develop deficiencies more easily than term infants.

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Water-Soluble Vitamins Water-soluble vitamins are not effectively stored; therefore, it might be expected that maternal dietary intake would affect the contents of water-soluble vitamins in human milk more readily than fat-soluble vitamins. Thiamin content in human milk is average 0.15 mg/L. Six weeks of supplementation with thiamin from 1.3 to 3.4 mg/day did not increase milk thiamin levels in women from the United States who were adequately nourished. Urinary excretion of thiamin was higher in supplemented women, compared to unsupplemented women, suggesting that a limit exists in the transfer of this vitamin into human milk. Early studies indicated that a maternal thiamin deficiency could lead to low levels of thiamin in milk. Low maternal intake of riboflavin can produce low concentrations of riboflavin in breast milk. Supplementation with a modest amount of riboflavin (2 mg/day) increased milk riboflavin levels. A maternal intake of 2.5 mg/day was considered sufficient to maintain riboflavin status during lactation. The concentration of biotin in human milk from women in the U.S. was reported to be between 5-12 mg per liter. Supplementation with high levels of other B vitamins does not appear to affect biotin levels in milk. Supplementation with biotin increases biotin level in milk when its level is low, and has no effect when biotin levels are in the normal range. Vitamin B6 concentration in milk of mothers with vitamin B6 intakes around the RDA (2.5 mg/day) appears to be approximately 210 µg/L. Vitamin B6 level in the milk of U.S. women with low socioeconomic status and low vitamin B6 intake was 120 µg/L. Supplementation of vitamin B6 at levels above the RDA (5.3 mg/day) did not alter the vitamin B6 level in milk. However, it is important to note that supplementation of vitamin B6 at high levels to lactating women should be avoided as this can suppress lactation. Folate concentration in human milk increases with lactation time, ranging from 15 – 20 g/L in early lactation, to 40 - 70 g/L in mature milk. Supplementation of 0.8 g/L per day of folate to well-nourished U.S. women did not change milk folate concentration. However, when women of lower socioeconomic status and concomitant low folate intake (60% of the RDA) were supplemented with folate, the folate level in their milk was increased. Low intakes of vitamin B12, cyanocobalamin, are most likely reflected in lower milk concentration of this vitamin. Mean vitamin B12 levels in

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well nourished U.S. women range between 0.97 - 1.10 µg/mL, and women of low socioeconomic status averaged 0.55 µg/mL. Maternal supplementation for 40 days with additional vitamin B12 raised the milk levels to only 0.79 µg/mL in the women of lower socioeconomic status, suggesting that long-term impaired maternal vitamin B12 status may not be completely alleviated in this time period. Supplementation of well-nourished women with additional vitamin B12 does not appear to augment milk concentration. Vitamin B12 levels can be low in the milk of U.S. women, especially when the mother follows a vegetarian diet, and can cause a vitamin B12 deficiency in the infant. Vitamin B12 in human milk is found as a protein-bound vitamin. Vitamin C level in the milk of women from the United States is about 50 mg/L. In well-nourished U.S. women, neither short-term nor longterm (6 months) supplementation of high levels (800 mg/day) of vitamin C affected milk concentration of this vitamin. Therefore, there appears to be an upper limit on the transfer of vitamin C into human milk, past which additional supplementation will not further augment levels in human milk. However, women with a niacin level of 1.96 mg/L with an intake between 15 - 23 mg niacin/day, showed a substantial increase in their milk to 3.9 mg/L with 120 mg niacin/day for 6-14 days. Pantothenic acid level in human milk appears to be influenced by maternal daily dietary intake. Milk pantothenic acid level correlated strongly with the maternal intake of pantothenic acid over the previous 24 hours. Minerals In Human Milk Minerals exist in the body in several chemical forms, including inorganic ions and salts, or as constituents of other organic molecules, including proteins, fats, and nucleic acids. They contribute to a variety of physiological functions, including structural components of body tissues to essential parts of many enzymes and biologically important molecules. Sodium, potassium, chloride, calcium, magnesium, phosphorus, and sulfate make up the macrominerals found in human milk. Citrate is not a mineral, but it is found as a water-soluble portion of human milk that can bind some minerals. The primary determinant of macromineral concentration in human milk is duration of lactation, during which sodium and chloride decrease, and potassium, calcium, magnesium, and free phosphate increase over time. However, the mineral content of human milk is also influenced by nutritional status of the mother and environmental and other factors. The concentrations

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of macro- and microelements in human milk and bovine milk are compared in Figure 9.5. FIGURE 9.5 — Mineral Composition Of Mature Human And Bovine Milk (Per Liter)

Sodium (mg) Potassium (mg) Chloride (mg) Calcium (mg) Magnesium (mg) Phosphorus (mg) Iron (mg) Zinc (mg) Copper (mg) Manganese (g) Iodine (g) Fluoride (g) g Selenium (g) Cobalt (g) Chromium (g) Molybdenum (g)

Human Milk

Bovine Milk

150 600 430 300 30 130 0.3 1.5 0.3 12 70 16 16 0.1 0.3 3

500 1500 950 1200 120 950 0.5 3.5 0.1 30 260 20 12 0.5 3 73

(Adapted from Guo, 1990; Flynn, 1992)

Microelements Sodium is the main cation of extracellular fluid, and it is also the main controller of extracellular volume. It is involved in the regulation of osmolarity, acid-base balance, active transport across cells, and the membrane potential across cells. Potassium is the primary intracellular cation, as its concentration is 30 times greater concentration inside the cell than in the extracellular fluid. Potassium in the extracellular fluid is involved in the transmission of nerve impulses, maintenance of blood pressure, and control of skeletal muscle contraction. Chloride is also essential in the maintenance of fluid and electrolyte balance, as it is the principal extracellular anion (Flynn, 1992). Under normal circumstances, a dietary deficiency of sodium, potassium, or chloride does not occur. However, depletion of sodium and chloride can occur during extreme conditions, such as chronic diarrhea, heavy perspiration, or renal disease, and depletion of potassium can occur in situations where there are large alimentary or renal losses. The concentrations of sodium, potassium, and chloride in breast milk decrease with duration of lactation, from a reported 480, 740, and 850 mg/L in colostrum,

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respectively, to 160, 530, and 400 mg/L respectively. No relationship has been identified between maternal intake and concentrations of these electrolytes in milk. Sodium, potassium, and chloride in human milk are present almost entirely as free ions. Calcium comprises about 1.5 - 2% of body weight in an adult, which has accrued approximately 1200 g of calcium. About 99% of this calcium is found in teeth and bones, providing structure and strength as calcium phosphate. The remaining calcium is found in extracellular fluids, intracellular structures, and cell membranes, and is involved in several regulatory functions, such as maintenance of a normal heart beat, hormone secretion, blood coagulation, nerve conduction, muscle contraction, activation of enzymes, and integrity of membranes. Human milk supplies approximately 200 mg of calcium in an average daily milk secretion of 750 ml, which appears to be sufficient for the term infant, but may not be adequate for a preterm infant. Supplementation of 1000 mg calcium/day in lactating women does not affect milk calcium or lactation-associated bone mineral changes. The calcium content of human milk increases in early lactation, from 250 at day 1, to 320 mg/ L by day 5, and remains constant at approximately 300 mg/L up to day 36 of lactation. Studies on calcium concentration during lactation reveal an approximate 30% decrease between the first and ninth months of lactation. There is no correlation between maternal dietary consumption of calcium and its concentration in human milk. Calcium binds with phosphate and casein in human milk to produce calcium phosphate linkages in casein micelle subunits, and it can also bind to citrate, or be found in the ionized form. The calcium in human milk is more available for absorption. Magnesium plays an essential role in a variety of physiological processes, including neuromuscular transmission, muscle contraction, protein and nucleic acid metabolism, and as a cofactor for many enzymes. Magnesium, along with calcium and phosphate, supports skeletal growth. A deficiency of magnesium is not common except in conditions of severe malnutrition and certain disease states. Mature human milk contains magnesium at a concentration of approximately 30-35 mg/L. With a normal range of dietary magnesium intake, there is no relationship between maternal magnesium consumption and concentration of magnesium in human milk. It has been reported to be about 30% higher in colostrum than in mature milk. Some magnesium associates with phosphate and caseins in human milk. Phosphorus is a nutrient essential to humans, as it serves a number of important biological functions. It exists as organic and inorganic

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phosphates in all tissues and fluids, and is essential to many body components, including lipids, proteins, carbohydrates, and nucleic acids, and also plays an important role in metabolism. It is an important part of calcium phosphate, a major structural component of teeth and bones. Dietary deficiencies of phosphorus do not usually occur as phosphorus is contained in a wide variety of foods of plant and animal origin. In human milk, phosphorus content increases from 100 mg/L on day 1 to 170 mg/L by day 8, and decreases to 130 mg/L by day 36 of lactation. Trace Elements Trace elements, also known as microminerals, are substances that make up less than 0.01% of the body mass. In human milk these include iron, zinc, copper, manganese, selenium, iodine, fluorine, molybdenum, cobalt, chromium, and nickel. Iron is an essential component of heme in hemoglobin, myoglobin, cytochromes, and other proteins; therefore, it plays a role in the transport, storage, and utilization of oxygen. Iron deficiency anemia affects about 30% of the world’s population, including Western and underdeveloped countries. The mean iron concentration in human milk is 0.3 mg/L. The iron content of human milk decreases over the duration of lactation; colostrum iron level is about 1 mg/L, and decreases to 0.3 - 0.6 mg/L in mature milk. Dietary intake of iron has no relationship with iron concentration in human milk, and supplementation with iron at levels up to 30 mg/day does not affect milk iron concentration. Human milk iron is bound to three main components: lactoferrin, a low molecular weight compound, and a component of the milk fat globule membrane. Lactoferrin is the primary iron-binding protein in human milk, possessing a high affinity for the ferric ions, which bind two sites together with bicarbonate or carbonate ions. Lactoferrin concentration in human milk is much higher than iron concentration, so although one-third of iron is bound to lactoferrin, only 3-5% of lactoferrin is saturated with iron. However, iron released from other components during digestion may become bound to lactoferrin, especially when bicarbonate from pancreatic fluid is present. Citrate in the low molecular weight fraction and xanthine oxidase in the fat globule membrane may be among these other iron-binding components. Very little iron in human milk is bound to casein (Lonnerdal, 1989). Zinc is essential to proper growth and development, sexual maturation, wound healing, and it may play a role in immune system function and other physiological processes. Zinc assists several hormones involved in reproduction, is required for DNA, RNA, and

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protein synthesis, and is a cofactor for many enzymes involved in most major metabolic processes (Flynn, 1992). Human zinc deficiency was first reported in the 1960s in the Middle East, resulting in dwarfism, impaired sexual development, and anemia. It is difficult to detect mild deficiencies of zinc, although they have been shown to occur in Western countries, especially in infants and children, and give rise to suboptimal growth, poor appetite, impaired taste acuity, and low hair zinc levels. Mean zinc concentration in mature human milk during the first six months of lactation is about 2 mg/L, although large variations in zinc have been reported at 0.65 - 5.3 mg/L. Dietary zinc intake has no correlation to zinc content of human milk, and zinc supplementation of a zinc adequate diet does not significantly affect human milk zinc concentration. Zinc in human milk is found in three major components: serum albumin and citrate in the whey, and in alkaline phosphatase in the fat globule membrane. Copper is required for iron utilization and is a cofactor for enzymes involved in glucose metabolism, as well as the synthesis of hemoglobin, phospholipids, and connective tissue. Copper deficiency is rare except in conditions of severe malnutrition. Mature human milk contains copper at a concentration of 0.3 mg/L. Copper concentration decreases with advancing lactation, from 0.6 mg/L in weeks one and two of lactation, to 0.36 mg/L by 6 - 8 weeks, and 0.21 - 0.25 mg/L by 20 weeks of lactation. No significant correlation exists between milk copper concentrations and dietary copper intake. Copper in human milk is bound to serum albumin and citrate. Copper has also been found in the fat globule membrane, however, the ligand has not yet been identified. Manganese is a cofactor for glycosyl transferases, which play a role in mucopolysaccharide synthesis, and is a nonspecific cofactor for many other enzymes. Two manganese metalloenzymes have been identified: mitochondrial superoxide dismutase and pyruvate carboxylase (Hurley & Keen, 1987). As manganese is widely distributed in foods, a dietary deficiency is not known to occur in humans (Flynn, 1992). In mature human milk, the mean concentration of manganese is approximately 10 µg/L and manganese is known to decrease with duration of lactation. No cases of manganese deficiency in human infants have been reported, thus, fully breastfed infants appear to receive adequate manganese (Lonnerdal et al., 1983). Manganese in human milk is mainly bound to lactoferrin, however, it exists at such a low concentration that approximately 2000 times more iron is bound to lactoferrin than manganese. Therefore, very little of the metal-binding capacity of lactoferrin is occupied by manganese (Lonnerdal, 1989).

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Selenium is an important component of the enzyme glutathione peroxidase. Glutathione peroxidase is present in many tissues, where it works with vitamin E, catalase, and superoxide dismutase as an antioxidant, protecting cells against oxidative damage. Selenium concentration in the milk of U.S. women is approximately 16 µg/L. Selenium concentration is higher in colostrum, at 41 µg/L, than in mature milk, 16 µg/L. A correlation was observed between human milk selenium content and both maternal plasma selenium concentration and plasma glutathione peroxidase activity, suggesting that milk selenium content is influenced by maternal selenium status (Levander et al., 1987). The average selenium content of milk of North American women is considered more than sufficient for breastfed infants. Iodine is essential to the thyroid hormones, thyroxine and triiodothyronine, which play an important role in the regulation of basal energy metabolism and reproduction. Iodine deficiency causes the thyroid gland to enlarge and form a goiter, while excess iodine in the diet reduces uptake of iodine by the thyroid gland, which yields signs of thyroid deficiency. In the United States, mean iodine concentrations in human milk have been reported as 142 µg/L (range: 21 – 281 µg/L). A correlation between milk iodine concentration and dietary iodine intake has been observed, therefore, the use of iodized salt can augment milk iodine content (AAP, 1981). North American women have an elevated iodine intake, and thus, the amounts of iodine in their milk are adequate. Molybdenum is a crucial component of several enzymes, including aldehyde oxidase, xanthine oxidase, and sulfite oxidase, where it exists in the prosthetic group molybdopterin. It has yet to be determined whether the human requirement is specifically for molybdenum, or whether it is for molybdopterin or a precursor. Dietary deficiency has not been observed in humans, except for a patient on long-term total parenteral nutrition. Molybdenum content of human milk is strongly correlated with the stage of lactation, decreasing from 15 µg/L on day 1, to 4.5 µg/L by day 14, and finally to a concentration of approximately 2 µg/L by one month and thereafter. Chromium is considered essential to human health, and the earliest sign of a deficiency is impaired glucose tolerance. Chromium deficiency has been observed exclusively in patients receiving long-term total parenteral nutrition. These patients respond to intravenous trivalent chromium with amelioration of glucose intolerance. The mean chromium content of mature human milk is 0.27 µg/L.

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The only function of cobalt identified in humans is its presence as an essential part of vitamin B12. Vitamin B12 is synthesized from bacteria. Therefore, inorganic cobalt is essential for all animals that rely completely on their bacterial flora for vitamin B12 supply. Mature human milk contains cobalt at a concentration of approximately 0.1 µg/L. Dietary supplementation of cobalt increases the vitamin B12 level of human milk only when the maternal diet is cobalt deficient. Fluoride is considered a beneficial element, rather than an essential element, to human health, as it protects against dental caries and accumulates in bones and teeth. However, excessive fluoride intake leads to fluorosis, which causes mottling of the teeth, and also affects bone health and kidney function. In mature human milk, the mean fluoride content is about 16 µg/L. Infants who are breast fed or consuming concentrated or powdered formula prepared with nonfluorinated water have a low fluoride intake, and should receive fluoride supplements (NRC, 1989). Substantial evidence exists to establish the necessity of nickel, silicon, arsenic, and boron in animals, and it is most likely that these trace elements are also essential to humans. However, the nutritional functions of these elements are yet to be determined (NRC, 1989). Nickel is found in mature human milk at a level of 1.2 µg/L, silicon is found at 700 µg/L, arsenic is found at 0.2 - 0.6 µg/L (Renner, 1983). Biological Functions Of Human Milk A main function of some important human milk proteins is to provide antimicrobial activity against pathogenic bacteria, viruses, and fungi. The major immunoglobulin (>90%) in human milk is secretory immunoglobulin A (SlgA), which is a dimer linked to a secretory component and a joining chain. This molecular arrangement allows the molecule to resist intestinal proteolysis, which is confirmed by the detection of modest amounts of SlgA in the stool of breastfed infants. SlgA binds to bacteria and viruses in the intestine and prevents attachment to mucosal epithelial cells, limiting infection and colonization. Concentrations of SlgA in human milk range from 1-2 g/L in early lactation, and remain steady at 0.5 - 1 g/L up to the late stage of lactation (Goldman, 1993). Maternal immunity against many general pathogens can be transferred to the infant in the form of SlgA in the milk, mediated via the enteromammary pathway. Antibodies against bacterial pathogens including Escherichia coli, Vibrio cholera, Streptococcus pneumonia, Clostridium difficile, Haemophilus influenzae, and Salmonella; against rotavirus, cytomegalovirus, HIV,

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respiratory syncytial virus, and influenza virus; and against yeasts such as Candida albicans, have been found in human milk, demonstrating the extent of this defense (Goldman, 1993). Lactoferrin may be responsible for many antimicrobial activities of human milk. It was originally thought that lactoferrin, which is largely unsaturated with iron yet has a high affinity for it, could withhold iron from iron-requiring pathogens, thereby exerting a bactericidal activity against pathogens (Lonnerdal, 2003). This may be possible, however, studies have observed a strong bactericidal activity of lactoferrin without dependence on iron saturation. This activity may be related to the production of lactoferricin, a potent bactericidal peptide formed during lactoferrin digestion. Recent research revealed that lactoferricin inhibits enteropathogenic Escherichia coli from attaching to intestinal cells. There appear to be several defense contributions by lactoferrin against bacterial infection (Lonnerdal, 2003). In vitro, lactoferrin has been shown to have activity against viruses, including HIV, and fungi, such as Candida albicans, however, the mechanism of these activities is not known. In vitro digestion of human milk produced two bifidogenic peptides that originated from lactoferrin. These peptides were stable against further digestion with pepsin, trypsin, and chymotrypsin, were active at low concentrations, and possessed a bifidogenic effect approximately 100 times stronger than N-acetyl-glucosamine (Liepke et al., 2002). Advantages of the bifidogenic effect include potentially decreasing the allergenicity of non-digestible proteins, decreasing the incidence of rotavirus-induced diarrhea, antibacterial activity, and increased production of short chain fatty acids in the colon. Lysozyme is a major enzymatic component of human milk that can degrade the outer cell wall of gram-positive bacteria. In synergistic action with lactoferrin, lysozyme has also been shown to kill gramnegative bacteria in vitro. This is accomplished when lactoferrin binds to the lipopolysaccharide and removes it from the outer cell membrane of bacteria, allowing lysozyme to access and degrade the inner proteoglycan matrix of the membrane, which destroys the bacteria. Lactoperoxidase in human milk may contribute to the defense against infection in the mouth and the upper GI tract (Lonnerdal, 2003). In the presence of hydrogen peroxide, lactoperoxidase catalyzes the oxidation of thiocyanate to hypothiocyanate, which can render inactive both grampositive and gram-negative bacteria. Hydrogen peroxide is produced in small quantities by cells, and thiocyanate is provided by saliva. Lactoperoxidase in cow’s milk has been used in developing countries for many years to maintain microbial quality, and although human milk

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does contain lactoperoxidase, the physiological significance is not yet determined. Exposure of
-lactalbumin to intestinal tract proteases yields peptide fractions, three of which have been implicated in antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, Klebsiella pneumonia, Streptococci, and Candida albicans. These findings may explain the inhibitory effect of
-lactalbumin-supplemented infant formula on diarrhea, caused by enteropathogenic Escherichia coli, in infant rhesus monkeys (Kelleher et al., 2003). Breastfeeding appears to provide protection against Helicobacter pylori infection in young children (Stromquist, et al., 1995). The heavily glycosylated -casein molecule in human milk, has been shown to inhibit the adhesion of Helicobacter pylori to human gastric mucosa (Stromquist et al., 1995). The mechanism by which -casein prevents attachment is that it acts as a receptor analogue, thus halting bacterial attachment to the mucosal lining. Human milk proteins are also involved in the immune system function of breastfed infants. Human milk contains many cytokines, including tumor necrosis factor a, transforming growth factor b, and interleukins (IL) 1 b, IL-6, IL-8, and IL-10. All of these cytokines are immunomodulatory, and most of them are anti-inflammatory, which may mitigate the effect of infections. The cytokines are found in free form, and also may be released from cells in breast milk (Lonnerdal, 2003). Human milk also contains lactoferrin, which has been shown to increase the production and release of cytokines such as IL-1, IL-8, tumor necrosis factor a, nitric oxide, and granulocyte-macrophage colony stimulating factor, which may also affect the immune system (Kelleher & Lonnerdal, 2001). When lactoferrin binds to its receptor in the small intestine, this may either cause signaling events that affect cytokine production downstream, or it is possible that the internalized lactoferrin can bind to the nucleus, which could affect nuclear transcription factor B, and subsequently, cytokine expression. Lactoferrin was recently shown to activate the transcription of IL-1b in mammalian cells, which indicates that lactoferrin may interact directly with the nucleus. Several proteins are also implicated in the development of the infant gut and its functionality, including growth factors, lactoferrin, and casein-derived peptides. Research has shown that IGF-I and IGF-II stimulate DNA synthesis and promote the growth of many types of cells in culture; therefore, they may play a role in the development of the infant gastrointestinal tract.

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Several peptides that possess physiological activity have been generated from human casein, and especially from β-casein (Lonnerdal, 2003). Although these proteins have been generated in vitro, they have also been detected from intestinal contents, suggesting that they are formed in vivo as well (Lonnerdal, 2003). Weight gain has been higher in infants who are fed formula supplemented with bovine lactoferrin than unsupplemented formula (Hernell & Lonnerdal, 2002). In support of this theory, administration of lactoferrin has been shown to enhance cell proliferation in the small intestine of experimental animals, and also to affect crypt cell development. The rapid development of intestinal mucosa in suckling newborns has been hypothesized to be in part due to the mitogenic effect of lactoferrin. Breastfed premature infants excrete intact lactoferrin in their urine, demonstrating that intact lactoferrin is absorbed by the infant gut (Goldman, 1989). Infant Formula Human milk is the best reference standard by which all infant formula is compared, and it has always been considered a speciesspecific food. In addition to nutritional components, human milk also contains immunoglobulin SlgA, peptide and non-peptide hormones, growth factors, proteins, peptides, lipids, and milk membrane fractions. Each discovery regarding infant formula, including formulation and processing, allows for the improvement of a product that continues to be increasingly similar to human milk. Although much is still unknown about human milk, and how to produce the optimum infant formula, new information is constantly being discovered. Some of the recent progress made in infant formula formulation and processing includes fortification with arachidonic and docosahexaenioc acids, nucleotides, and ingredients that promote healthy colonic microflora; effect of removal of phytate on soy formulas; trace mineral solubility and availability; component distribution and interactions; addition of whey peptides fractions. Ingredient Selection For Infant Formula Infant formula is designed to substitute for breast milk when mothers are not able to breast-feed their infants. It is most commonly prepared with cow’s milk, whose composition is modified to be more similar to human milk. Milk-based infant formulas include ingredients such as milk and whey protein, and soy formulas are based on soy protein isolate. Protein

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hydrolysate formulas include protein that has been hydrolyzed to peptides and amino acids. Milk-free formulas are used in exceptional cases of intolerance to cow’s milk, and they exclude cow’s milk components. Medical formulations of infant formula exist for infants with special needs, including those caused by congestive heart failure, inborn errors of metabolism, and steatorrhea. Soy-based formulas contain soy protein isolate, in which methionine is a limiting amino acid, therefore, supplemental methionine must be added to achieve a more appropriate amino acid profile. Milk-based formulas are based on 0.6% casein and 0.9% whey proteins, yielding a 40:60 ratio of casein to whey proteins. Protein levels in milk-based infant formula are approximately 15 g/ L, providing 10% of total energy. Protein in milk-based formula is provided by non-fat milk and whey protein products. Protein in soybased formula provides approximately 11-13% of total energy, at 18 21 g/L. Soy-based formulas also contain additional L-methionine, Lcarnitine, and taurine. In special use formulas, protein is provided by casein hydrolysates, whey, and skim milk. It is contained at levels of 15 - 30 g/L accounting for 9 -12 % of the total energy. The carbohydrate content of milk-based infant formulas ranges from 70 - 72 g/L providing 40% of total kilocalories. In soy-based infant formula, carbohydrate levels average 67 - 69 g/L, providing about 40% of energy. Special use formulas contain carbohydrates at levels of 70 109 g/L, accounting for 40 - 45% of the total energy. Lactose is the major carbohydrate source in milk-based infant formulas, whereas carbohydrate in soy-based and special use formulas is provided by sucrose, corn syrup solids, dextrins, hydrolyzed corn starch, glucose, and glucose polymers. Fat levels in milk-based formulas range from 36 - 38 g/L providing about 50% of total energy. In milk-based formulas, fat is provided by oleo, coconut, soy, palm, sunflower, and safflower oils. In soy-based formulas, fat content ranges from 36 - 38 g/L, providing 47 - 49 % of the total energy. Soy-based formulas contain fat from oleo, soybean, safflower, coconut, palmolein, and high oleic safflower oil. Special use formulas contain fats from corn, safflower, soy, palm, coconut, sunflower, and high oleic safflower oils, and medium chain triglycerides. These fats are contained at levels of 34 - 49 g/L, accounting for 44 - 50 % of the total energy (Feldhausen et al., 1996). Formulation Aspects Of Infant Formula Infant formula is a complex and balanced food for infants. In the

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quest to produce an infant formula that closely mimics human milk, there are many steps to consider. The formulation of infant formula is a complicated task, with many details regarding composition, physicochemical properties, and shelf stability. Infant formula does vary in composition, but within narrow and precise limits. Formula should provide protein of an appropriate biological quality at levels of 10 -15% of calories, fat at 45 - 50% of calories, linoleic acid at 2 - 3% of total calories, and carbohydrate should make up the remaining calories. Milk, or milk and whey-based formulas must take into account the need to alter the natural composition of bovine milk. The changes that must be employed include lowering protein content, while maintaining biological quality, raising carbohydrate content, changing fat composition, and lowering the mineral content. In general, the guidelines for infant formula formulation are listed as follows: • • • • • • • • • •

All ingredients proven by FDA regulations Protein: fat: CHO ≈ 1: 2: 4 C18:2 accoutanting for 2-3% of total energy CN: WP = 40: 60 Ca: P = 1.5: 1 Minerals and Vitamins fortified Functional nutrients: -3, carnitine, nucleotides, prebiotics pH ≈ 7.0-7.2 Osmolarity ≈ 270 mOsm/L Processing damages to nutrients

Osmolarity is the measure of osmotically active substances, including sugars, amino acids, and mineral contents per liter of a solution. The osmolarity of human milk ranges between 270 – 290 mOsm/L, while bovine milk and infant formulas range between 200 – 400 mOsm/L, although osmolarities above 350 mOsm/L, should be avoided, as they can stress the newborn kidney and increase water loss. The osmolarity of commercial infant formula is approximately 230 - 270 mOsm/L, or 300 mOsm/kg water. Osmolarity, along with renal solute load, plays an important role on the efficacy of the food. The renal solute load is defined as the sum of solutes that must be excreted by the kidney. The renal solute load is most commonly expressed in mOsm/day, and the concentration in urine is expressed as osmolality (mOsm/kg water). If the renal solute load is too high, hypernatremic dehydration can occur, and if the renal solute load is too low, hyponatremia can occur (Fomon, 1993). Osmolarity has a relationship with renal solute load in that it relates to the amount of osmotically active substance that is contributed

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by the infant formula. In a typical formula containing 67 kcal and 7 grams of lactose per 100 mL, lactose contributes approximately 200 mOsm/kg water, which is approximately 70% of solutes. By replacing lactose with glucose, the osmotic solute load would be doubled (Fomon, 1993). This type of substitution is important when formulating infant formula, as the osmolarity range is narrow. Therefore, when formulating infant formula it is crucial to keep the osmolarity within an appropriate range. Components in milk, including proteins, carbohydrates, fats, and mineral salts, which provide pH buffering properties. Buffering groups in milk include protein bound residues and salts. Protein bound residues include aspartic acid, glutamic acid, histidine, tyrosine, lysine, esterphosphate, N-acetyl neuraminic acid, and terminal groups. The salts native to milk that possess buffering capabilities include phosphate, phosphate esters, citrate, carbonate, various carboxylic acids, various amines, and lactic acid. Human milk has an average pH of 7.00 -7.25, which is higher than bovine milk. Therefore, infant formula is modeled on human milk, and the optimal pH for infant formula would be similar to that of human milk. When bovine milk products are used as a base for infant formula, careful consideration of ingredients and mineral salts must be employed to produce a product that follows all the necessary guidelines and achieves the proper pH. Bovine milk-based infant formulas are prepared using skim milk powder, demineralized whey, lactose, vegetable oils, essential fatty acids, lecithin, nucleotides, vitamins, and minerals. The American Academy of Pediatrics Committee on Nutrition 1982 Task Force and 1987 FDA Recommendations established recommended nutrition levels of infant formulas per 100 kcal (Figure 9.6). The recommended global standard for the composition of infant formula is shown in Figure 9.7. These guidelines provide the acceptable and safe ranges for the composition of infant formula, including energy-contributing and non-energy containing nutritive ingredients (Committee on Nutrition, American Academy of Pediatrics, 1998). Some nutrients have wide ranges that are considered acceptable. For example, sodium, potassium, and chloride, where the maximum limit can be approximately three times as high as the minimum limit. Other nutrients, such as selenium, have a very narrow safe range. Nitrate salts are not usually added to infant formula, due to the potential to cause methemoglobinemia. In regards to trace elements, such as iron, copper, and zinc, sulfate forms have been traditionally used in commercial production. The process of infant formula formulation begins with determination of the target nutrient

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levels that will make up the composition of the formula. The ingredients are then selected, and calculations are performed to determine the amount of each ingredient that will contribute to the gross energy composition of the formula, including protein, fat, and carbohydrate. Once these main ingredients of the formula are calculated, the composition of each ingredient is examined to determine the contribution of minerals and vitamins provided by the main ingredients, and vitamins and mineral salts can be added as necessary to achieve the target nutrient profile. FIGURE 9.6 — Nutrient Specifications For Infant Formulas

Protein (g) Fat (g) Linoleic acid (g) Vitamin A (IU) Vitamin D (IU) Vitamin E (IU) Vitamin K (g) Thiamin (g) Riboflavin (g) Vitamin B6 (g) Vitamin B12 (g) Niacin (g) Folic acid (g) Pantothenic acid (g) Biotin (g) Vitamin C (mg) Choline (mg) Inositol (mg) Calcium (mg) Phosphorus (mg) Magnesium (mg) Iron (mg) Zinc (mg) Manganese (g) Copper (g) Iodine (g) Sodium (mg) Potassium (mg) Chloried (mg) (Fomon, 1993)

Minimum

Maximum

1.8 3.3 0.3 250 40 0.7 4 40 60 35 0.15 250 4 300 1.5 8 7 4 60 30 6 0.15 0.5 5 60 5 20 80 55

4.5 6.0 750 100 3.0 75 60 200 150

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FIGURE 9.7 — Proposed Compositional Requirements Of Infant Formula (Koletzko, et al, 2005)

Energy Proteins Cows’ milk protein Soy protein isolates Hydrolyzed cow’s milk protein Lipids Total fat Linoleic acid
-linolenic acid Ratio linoleic acid/
-linolenic acid Lauric + myristic acids Trans fatty acids Erucic acid Carbohydrates Total carbohydrates1 Vitamins Vitamin A Vitamin D3 Vitamin E Vitamin K Thiamin Riboflavin Niacin2 Vitamin B6 Vitamin B12 Pantothenic acid Folic acid Vitamin C Biotin Minerals and trace elements Iron - (formula based on cows’ milk protein and protein hydrolysate) Iron (formula based on soy protein isolate) Calcium Phosphorus (formula based on cows’ milk protein and protein hydrolysate) Phosphorus (formula based on soy protein isolate) Ratio calcium/phosphorous

Unit

Minimum

Maximum

kcal/100mL

60

70

g/100 kcal g/100 kcal g/100 kcal

1.8* 2.25 1.8†

3 3 3

g/100 kcal g/100 kcal mg/100 kcal % of fat % of fat % of fat

4.4 0.3 50 5:1 NS NS NS

6.0 1.2 NS 15:1 20 3 1

g/100 kcal

9.0

14.0

g RE/100 kcal‡ g/100 kcal mg
-TE/100 kcal2 g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal mg/100 kcal g/100 kcal

60 1 0.5¶ 4 60 80 300 35 0.1 400 10 10 1.5

180 2.5 5 25 300 400 1500 175 0.5 2000 50 30 7.5

mg/100 kcal

0.3**

1.3

mg/100 kcal mg/100 kcal

0.45 50

2.0 140

mg/100 kcal

25

90

mg/100 kcal mg/mg

30 1:1

100 2:1

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FIGURE 9.7 — Proposed Compositional Requirements Of Infant Formula (Koletzko, et al, 2005) - Continued Unit

Minimum

Maximum

Minerals and trace elements - continued Magnesium mg/100 kcal Sodium mg/100 kcal Chloride mg/100 kcal Potassium mg/100 kcal Manganese g/100 kcal Fluoride g/100 kcal Iodine g/100 kcal Selenium g/100 kcal Copper g/100 kcal Zinc mg/100 kcal

5 20 50 60 1 NS 10 1 35 0.5

15 60 160 160 50 60 50 9 80 1.5

Other substances Choline Myo-inositol L-carnitine

7 4 1.2

50 40 NS

mg/100 kcal mg/100 kcal mg/100 kcal

*

The determination of the protein content of formulae based on non-hydrolyzed cows’ milk protein with a protein content between 1.8 and 2.0 g/100 kal should be based on measurement of true protein ([total N minus NPN] ×6.25). † Formula based on hydrolyzed milk protein with a protein content less than 2.25 g/ 100kcal should be clinically tested. 1

Sucrose (saccharose) and fructose should not be added to infant formula.

‡

1g RE (retinol equivalent) = 1g all-trans retinol = 3.33 IU vitamin A. Retinol contents shall be provided by performed retinol, while any contents of carotenoids should not be included in the calculation and declaration of vitamin A activity. 2

1mg
-TE (
-tocopherol equivalent) = 1 mg d-
-tocopherol.

¶Vitamin E content shall be at least 0.5 mg
-TE per g PUFA, using the following factors of equivalence to adapt the minimal vitamin E content to the number of fatty acid double bonds in the formula: 0.5 mg
-TE/g linoleic acid (18:2n-6); 0.75 mg
-TE/g
-linoleic acid (18:3n-3); 1.0 mg
-TE/g arachidonic acid (20:4n-6); 1.25 mg
-TE/g eicosapentaenoic acid (20:5n-3); 1.5 mg
-TE/g docosahexaenoic acid (22:6n-3). 2

Niacin refers to performed niacin.

**

In populations where infants are at risk of iron deficiency, iron contents higher than the minimum level of 0.3 mg/100 kcal may be appropriate and recommended at a national level. NS = not specified.

In order to achieve the proper pH, different mineral salts should be used. Some mineral salts contribute to a pH that is more acidic, while other mineral salts contribute to a more neutral or basic pH. For example, when the pH of infant formula is lower than optimal (i.e., 6.8 - 7.0), mineral salts that include bicarbonates (i.e., sodium bicarbonate) and oxides (i.e., magnesium oxide) can be substituted to increase pH to an optimal level, and chlorides (i.e., magnesium chloride) and citrates

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FUNCTIONAL FOODS

(i.e., potassium citrate) can be used to reduce a higher pH, or in a formula where the pH is already acceptable (Smith, 2004). As skim milk powder and whey protein products are important functional ingredients of milk-based infant formulas, their physical and chemical composition also contributes to the properties that are observed in infant formulas. Bovine milk proteins play an important role in infant formula. Casein micelles will irreversibly aggregate at temperatures above the boiling point, and heating also results in precipitation of proteins onto the fat globule surface. Acid causes casein micelles to destabilize and/or aggregate due to decreased electric charge around that of the isoelectric point. Acid also increases the solubility of minerals so that organic calcium and phosphorus within the micelle slowly become more soluble. Heat causes whey proteins to adsorb onto the surface of the casein micelle. The buffering capacity of milk salts change with heating, releasing carbon dioxide, producing organic acids, and precipitating tricalcium phosphate and casein phosphate with the subsequent release of hydrogen ions. Clearly, pH and heat treatment are two important areas to consider when formulating and processing infant formula, as they can both exert considerable impacts on the properties of milk proteins. In infant formulas and milk-based nutritional supplements, dairy ingredients are used extensively to build a nutritional base, and are used in conjunction with additional proteins, lipids, carbohydrates, vitamins, minerals, and other ingredients, when merited, to achieve the desired nutrient profile. Clearly, dairy ingredients possess many functional characteristics in addition to nutrition, which is important during both processing and reconstitution. Lactose, a reducing sugar, can also contribute to Maillard reactions in sterilized products. In addition to carbohydrate, protein is a very important part of infant formula and other milk-based nutritional supplements. Some infant formulas are based solely on bovine milk proteins, with an approximate whey protein: casein ratio of 60:40, which is more similar to human milk (80:20), although the whey protein systems in human and bovine milks are markedly different. The addition of whey protein to infant formula creates additional stability issues, such as the heat stability of the final product. The main objective in manufacturing heat-stable infant formulas with added whey proteins is to control the formulation and processing variables that can prevent whey protein self-aggregation. It is most effective to begin with a whey protein source that contains low levels of heat-denatured protein, and to employ factors such as processing (heat treatment) and formulation

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(salt balance) in order to produce the best heat stable final product. To form a successful protein-stabilized emulsion with optimum heat stability, solubilization of the protein is essential. Heat treatment alters the solubility of the calcium phosphate in milk, and calcium salts play a role in protein aggregation at pH > 6.5, which is the pH range for infant formula. Casein enhances the protein-stabilized emulsion in milkbased products, while viscosity can be controlled to prevent creaming during storage, which results from milk protein reactivity. Another factor to consider is that divalent cations may contribute to the instability of milk protein systems during heating. This effect can be controlled by proper selection of the minerals used in fortification, and by balancing the divalent cations with other mineral salts, such as citrates or phosphates. Clearly, many factors come into play when formulating a milk-based formula or nutritional supplement. Once the proper considerations and preparations have been made, the infant formula should yield the desirable properties. Infant Formula Processing Infant formula is processed as ready-to-feed and concentrated liquids, and as dried powder. Liquid products must be sterilized in order to prevent spoilage during long-term storage. For commercial liquid infant formula, this would be classified as a retort product, which is sterilized inside the container. Sterilization is accomplished by heating in a commercial pressure cooker (retort) at temperatures of 115 - 123°C for 12 - 20 minutes. The sterile liquid formula may then be stored for 6-12 months without spoiling or exhibiting textural changes. However, it is difficult to control the textural changes in sterile liquid infant formula, as sediment and gelation may occur. Even though microbial spoilage may not occur, textural and chemical changes may render the formula less usable or less nutritious. In order to prevent these changes, appropriate formulation, homogenization, and heat treatment must be employed. In processing both liquid and dry ingredients, processes include intense preheating conditions, nonfat dry milk or condensed milks should be the “high-heat” type, indicating that heat applied during the manufacture of these ingredients was sufficient to denature most whey proteins. If ingredients used under these conditions are not “high-heat”, then they may contribute to sediment formation during storage of the finished product. When fresh milk is used to produce evaporated products, vitamin and mineral addition should be delayed until after

332

FUNCTIONAL FOODS

evaporation, and before sterilization, in order to mitigate loss of vitamins. Two procedures exist for the processing portion of infant formula manufacturing, the “dry procedure”, which consists of ingredients being blended in the dry form, and the “wet procedure”, in which liquid ingredients are mixed and then dried. The dry procedure consists of blending all ingredients in dry form, producing a homogeneous blend, which is its most favorable feature. This is accomplished by completing the entire mixing in a batch plant, with precise dosing and filling in a continuous plant. The wet procedure follows a specific chain of events in which liquid ingredients are mixed prior to drying. In the wet method, the procedure consists of selection and reception of raw materials (skim milk), clarification, deaeration, separation, pasteurization, evaporation, blending with oil and other components (including fat-soluble vitamins, emulsifiers, and stabilizers), mixing, homogenization, addition of water soluble vitamins and minerals, and drying. There is also a combined method of these two processes that has the advantages of both and is more commonly applied. The combined method involves adding watersoluble components to milk prior to drying, and adding less soluble components in a dry form to the blend after drying. The wet procedure allows for optimal mixing, while the dry procedure is less costly for operation and investment. From a nutritional standpoint, ultra-high-temperature (UHT) is preferred for heat treatment of liquid infant formula as nutrient and vitamin loss is minimized, as well as the browning that takes place between reducing sugars and amino groups of protein, which can lower protein quality. Pretreatment of the mix at an intense preheating temperature prior to UHT processing may mitigate the likelihood of gelation, which can be caused by enzymes associated with bacteria found in the original milk ingredients. Powdered infant formula is produced either by blending dry ingredients or by drying a mixture of liquid ingredients. Microbiological quality is easiest to control when the infant formula powder is produced by drying the liquid mixture. Fresh milk can be used, and it is filtered, clarified, deaerated, separated into skim milk and cream, and pasteurized (74 - 77°C, 15 - 20 seconds). Then these ingredients, or dried milk products, can be combined with warmed vegetable oils, followed by emulsifiers, stabilizers, and possible fat-soluble vitamins. Vitamin and mineral fortification can be delayed until the product is dried and cooled, although this is not ideal as thorough mixing is difficult (Packard, 1982). When vitamins and minerals are added prior to drying,

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some overdosages of heat-labile vitamins are required to account for processing losses. Ideally, fat-soluble vitamins are added prior to evaporation, while water-soluble vitamins and minerals are added after evaporation and before drying. Concentrated liquid formula is prepared by blending the ingredients to the desired solids level, or by evaporating excess liquid. Most infant formulas contain high carbohydrate content, which has the potential to stick to the dryer walls at high temperatures and moisture levels. Thus, low inlet air temperature, low solids in-feed, heating the concentrated mix prior to in-feed, and using an insulated or cooledwall drying system is recommended. In general, a batch mix of 45% or less solids and an in-feed temperature of approximately 70°C is ideal. Two-stage drying can also be performed, producing a moist powder that is then dried on a surface dryer to yield the desired moisture content. Following the drying process, the product is cooled and bagged in bulk or into consumer size units. RECENT DEVELOPMENTS IN INFANT FORMULA FORMULATION Essential Fatty Acids Human milk contains small amounts of arachidonic acid (AA) and docosahexaenoic acid (DHA). DHA is a long-chain polyunsaturated fatty acid that plays a major structural role in the grey matter of the brain and in the retina of the eyes. AA, another long-chain polyunsaturated fatty acid, is the principal omega-6 fatty acid of the brain. It is important in brain development and growth in infants, and is a precursor to eicosanoids, which are involved in the regulation of immunity, blood clotting, and other functions in the body and the precursor to the prostaglandin hormones. Infants were fed either infant formula, infant formula fortified with these fatty acids, or human milk exclusively for 17 weeks. Visual acuity was measured at 6, 17, 26, and 52 weeks, and electroretinography was used to measure retinal maturity at 17 and 52 weeks. Blood levels of DHA and AA were measured and correlated with the results from the visual and developmental tests. The results from this study indicate that infants fed infant formula fortified with the fatty acids had more mature retinal function and improved visual function at 6 and 17 weeks, respectively. When followed at one year, the supplemented groups maintained higher levels of visual function than unsupplemented groups (Hoffman et al., 2000).

334

FUNCTIONAL FOODS

In another study by Birch, Garfield, Hoffman, & Uauy (2000), supplementation of term infant formula with 0.36% DHA and 0.72% AA (weight percent of fat) during the first four months of life was associated with a mean increase of 7 points on the Mental Development Index of the Bayley scores at 18 months of age compared with control formula infants. Based on studies such as this, companies in the US have produced commercial infant formulas that include added DHA and AA. The levels of DHA are approximately 0.32% (weight percent of fat), and the levels of AA are approximately 0.64% (weight percent of fat). These natural DHA and AA are extracted from the algae Crypthecodinium cohnii and the fungal source Mortierella alpina, respectively. Nucleotides Nucleotides are one component of human milk identified as having an effect on immune function. The effect of human milk followed by infant formula, and infant formula fortified with nucleotides were compared with respect to their effect on response to immunizations as an indicator of immune development. The level of nucleotides (72 mg/ L) and ratio of individual nucleotides were patterned after those found in human milk. Results showed that infant formula fortified with nucleotides enhanced H influenza type b and diphtheria humoral antibody responses post vaccination. The consumption of human milk also enhanced antibody response to oral polio virus (Pickering et al., 1998). These results indicated that infant formula supplemented with nucleotides enhanced immune function in infants compared to the control infant formula. Prebiotic Compounds Infants who consume breast milk have gastrointestinal flora that are richer in bifidobacteria and lactobacilli than infants who consume bovine milk-based formula, and both of these species are considered to be potentially beneficial to the health of the host. The absence of oligosaccharides from infant formula, another major component in human milk, may be responsible for the differences in colonic flora. The addition of two oligosaccharides, galacto-oligosaccharides and inulin, to bovine milk-based infant formula has been shown to stimulate the growth of bifidi and lactobacilli, and to have a bifidogenic effect (Vandenplas, 2002). Therefore, the addition of oligosaccharides to infant formula could improve the colonic balance of microflora, and possibly,

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the health of the infant host (Vandenplas, 2002). The addition of oligosaccharides to bovine milk-based infant formula is one more improvement that brings infant formula one step closer to the gold standard of human milk. However, prebiotic oligosaccharides are presently not recommended to be supplemented in infant formulas according to ESPGHAN Committee on Nutrition and FDA due to inadequate information. It is clear that progress is still being made in the way of infant formula formulation and improvement. However, there are still many areas that merit further research in the quest to formulate and produce an infant formula that really mimics human milk. Component interactions that occur during processing should be considered since such interactions could lead to the loss of nutritional value in the final product. Summary Human milk is the best reference standard by which all infant formula is compared, and it has always been considered a speciesspecific food. Modern infant formulas are designed for infants based on our knowledge of human milk. There are numerous differences in chemical and biological properties between human milk and infant formula since we still do not fully understand chemical and biological properties of human milk. In addition to nutritional components, human milk also contains immunoglobulin SlgA, lactoferrin, peptide and nonpeptide hormones, growth factors, peptides, lipids, and other fractions. It is in fact a living tissue much like blood or plasma. Each advance in infant formula, including formulation and processing, allows for the improvement of a product that continues to be increasingly similar to human milk. Although much is still unknown about human milk, and how to produce the optimum infant formula, new information is constantly being discovered. Some of the recent progress made in infant formula formulation and processing includes fortification with -6 fatty acids such as arachidonic and -3 fatty acids including docosahexaenioc acid and eicosapentaenoic acid, nucleotides, and ingredients that promote healthy colonic microflora; effect of removal of phytate on soy formulas; trace mineral solubility and availability; component distribution and interactions; modification of whey protein profile and addition of bioactive peptide fractions.

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References AAP (American Academy of Pediatrics) 1981. Nutrition and lactation. Pediatrics, 68, 435-443. Adkins, Y. & Lonnerdal, B. 2002. Mechanisms of vitamin B12 absorption in breast fed infants. J. of Pediatric Gastroenterology and Nutrition, 35, 192-198. Agostoni, C., Axelsson, I., Goulet, O., Koletzko, B., Michaelsen, K. F., Puntis, J. W. L., Rigo, J., Shamir, R., Szajewska, H., and Turck, D. 2005. Prebiotic oligosaccharides in dietetic products for infants: A commentary by the ESPGHAN Committee on Nutrition. J. of Pediatric Gastroenterology and Nutri. 39, 465-473. Colman, N., Hettiarachchy, N., and Herbert, V. 1981. Detection of a milk factor that facilitates folate uptake by intestinal cells. Science, 211, 1427-1428. Committee on Nutrition, American Academy of Pediatrics 1998. Iron fortified infant formulas. Pediatrics, 84, 1114-1115. Feldhausen, J., Thomson, C., Dunca, B. and Taren, D. 1996. Pediatric Nutrition Handbook. Chapman & Hall. NY. USA. Flynn, A. 1992. Minerals and trace elements in human milk. Advances in Food & Nutrition Research, 36, 209-252. Fomon, S.J. 1993. Nutrition of Normal Infants. Boston: Mosby-Yearbook, Inc. St. Louis, USA. Goldman, A.S., and Goldblum, R.M. 1989. Immunoglobulins in human milk. In: Protein and Non-Protein Nitrogen in Human Milk. S.A. Atkinson and B. Lonnerdal (Eds), pp 44-51. CRC Press Inc, Boca Raton, Forida. Goldman, A. 1993. The immune system of human milk: antimicrobial, anti-inflammatory, and immunomodulating properties. Pediatric Infectious Disease J. 12, 664-672. Guo, M.R. 1990 Heat-Induced Modification of Milk Protein. Ph.D. Thesis. The National University of Ireland, Ireland. Hendricks, G. M. 2001. Solubility and Relative Absorption of Copper, Iron, and Zinc in Infant Formulae. Ph.D. Thesis. University of Vermont, USA. Hendricks, G.M. and Guo, M. 2006. The significance of milk fat in infant formula. Advanced Dairy Chemistry, Volume 2: Lipids, 3rd edition. pp 467-479.Spring, New York. Hernell, O. & Lonnerdal, B. 2002. Iron status of infants fed low iron formula: no effect of added bovine lactoferrin or nucleotides. Am. J. of Clini. Nutri., 76, 858-864. Hoffman, D.R., Birch, E.E., Brich, D.G., Uauy, R, Castaneda, Y.S., Lapus, M.G. & Wheaton, D.H. 2000. Impact of early dietary intake and blood lipid composition of long-chain polyunsaturated fatty acids on later visual development. J. of Pediatric Gastroenterology and Nutri, 31 (5), 540-553. Hurley, L.S. & Keen, C.L. 1987. Manganese. In W. Mertz, Trace elements in human and animal nutrition, 5th Ed, Vol. 1 pp. 185-223. Academic Press. San Diego, USA. Kelleher, S.L. & Lonnerdal, B. 2001. Immunological activities associated with milk. In: B. Woodward & H.H. Draper, Advances in nutritional research. Immunological properties of milk, Vol. 10, pp. 39-65. Plenum Press, New York, USA. Kelleher, S.L.,Chatterton, D, Neilsen, K., and Lonnerdal, B. 2003. Glycomacropeptide and
-lactalbumin supplementation of infant formula affects growth and nutritional status in infant rhesus monkeys. Am. J. of Clini. Nutri, 77, 126-128. Koletzko, B., Baker, S., Cleghorn, G., Neto, U. F., Gopalan S., Hernell O., Hock Q. S., Jirapinyo P., Lonnerdal, B., Pencharz, P., Pzyrembel, H., Ramirez-Mayans, J., Shamir, R., Turck, D., Yamashiro, Y., and Z. Ding. 2005. Golbal standard for the composition of infant formula: Recommendations of an ESPGHAN coordinated international expert group. J. of Pediatric Gastroenterology and Nutri 41:584-599.

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Levander, O.A.; Moser, P.B.; & Morris, V.C. 1987. Dietary selenium intake and selenium concentrations of plasma, erythrocytes and breast milk in pregnant and postpartum lactating and nonlactating women. Am. J. of Clini. Nutri., 46, 694-698. Liepke, C.; Adermann, K.; Raida, M.; Magert, H-J.; Forssmann, W-G.; & Zucht, H-D. 2002. Human milk provides peptides highly stimulating the growth of bifidobacteria. Europ. J. of Biochem., 269, 712-718. Lonnerdal, B.; Keen, C.L.; Ohtake, M.; & Tamura, T. 1983. Iron, zinc, copper, and manganese in infant formulas. Am. J. of Dis. of Children, 137, 433-437. Lonnerdal, B. 1986. Effect of maternal dietary intake on human milk consumption. J. of Nutri., 116, 499-513. Lonnerdal, B. 1989. Trace element nutrition in infants. Ann. Rev. Nutri. 9, 109-125. Lonnerdal, B. 2003. Nutritional and physiological significance of human milk proteins. Am. J. of Clini. Nutri., 77 (Suppl), 1537S-43S. NRC National Research Council. 1989. Recommended daily allowances. 10th ed. National Academy of Science, National Research Council. Washington, DC. Packard, V.S. 1982. Human Milk and Infant Formula. Academic Press, New York, USA. Pickering, L.K.; Granoff, D.M.; Erickson, J.R.; Masor, M.L.; Cordle, C.T.; Schaller, J.P.; Winship, T.R.; Paule, C.L. & Hilty, M.D. 1998. Modulation of the immune system by human milk and infant formula containing nucleotides. Pediatrics, 101 (2), 242249. Renner, E. 1983. Milk and Dairy Products in Human Nutrition. Volkswirtschaftlicher Verlag, Munich, Germany. Smith, C. R. 2004. Solubility and Relative Bioavailability of Iron and Zinc in Whey Protein Dominated Infant Formulas. Ph. D. Thesis. University of Vermont. Stromquist, M.; Falk, P.; Bergstrom, S. et al. 1995. Human milk -casein and inhibition of Helicobacter pylori adhesion to human gastric mucosa. J. of Pediatric Gastroenterology and Nutri., 21, 288-296. Suzuki, Y.A.; Shin, K.; & Lonnerdal, B. 2002. Molecular cloning and functional expression of a human intestinal lactoferrin receptor. Biochem., 40, 15771-15779. Vandenlas, Y. 2002. Oligosaccharides in infant formula. Brit. J. of Nutri, 87 (Suppl. 2): S293-S296. (Guo, M. R. Hendricks, G.M.)

INDEX

AACC, 64, 67, 77-78, 105 Absorption electrolyte, 292 ACF, 129-131 Acids cis-unsaturated fatty, 180 linoleic, 162-163, 165, 193 n-6 fatty, 174, 195 total fatty, 171-173, 181 unsaturated fatty, 163, 171 Acidulants, 293-294 Allicin, 43-44, 48, 58-59 Allicin, garlic homogenate, 45 Allicin-derived garlic compounds, 50 Alpha-tocopherol equivalent, 328-329 Anderson, 252, 256, 262, 275-276 Anderson, 86, 109 Anemia, iron deficiency, 228, 232 Antibiotic therapy, 136-138 Antibiotics, 115, 128, 135-138 Antioxidant vitamins, 209, 236 Antioxidants, 3, 6, 295-297 Antioxidants, dietary, 19-20, 58 Antioxidative peptides, 254-255, 275 Apoptosis, 259, 277 Arsenic, 290 Ascorbic acid, 293 Atherosclerosis, 175, 178, 194 Bacteria, anaerobic, 114 Bacteroides, 114, 123, 130, 149 Basal diet, 130-131 BCAA, 295-296 B-casein, 302, 303, 305, 323 Beri-beri, infantile, 212 Beta-glucan, 101, 105, 109 BHA, 57, 58 BHT, 57, 58 Bifidobacteria growth of, 127-128, 130 intestinal, 134

Bifidobacterium, 126, 137, 139-140, 149 bifidum, 135, 150, 157 longum, 143, 156, 158-159 human-derived, 159 species, 126, 128 growth of, 127, 156 Bile acids, 115, 143, 149, 159, 253 Biotin, 12, 198-199, 219-220, 310, 313, 327, 328 deficiency, 220 Body fluids, 279, 286, 288 Bone health, promotion of, 2 Boulardii, 137-141 Bovine milk, 301-303, 307-310, 315, 325-326, 330 milk-based infant formulas, 326, 334-335 Breast cancer, 258, 275 risk of, 258-259, 277 Caffeine, 295 Calcium, 2, 4, 86, 101-102, 111, 202-203, 215, 220-222, 248, 261-262, 264, 267 absorption, 202-203 oxide, 221-222 excretion, 261-262 Campesterol, 187, 189, 191 Cancer, 3, 4 Cancer, colon, 177, 191, 195 Cancers, stomach, 50-51, 56 Carbohydrates, 307, 317, 324-328, 330 components, 298 concentration of, 286 ingestion, 298 intake, 282, 298 Carcinogenesis, 129-130, 156-159 Cardiovascular diseases, 255-256, 276 Caries, dental, 146-148 Carnitine, 295 Casei Shirota, 142-143, 145 Catechins, 20-21, 29, 32-33, 54

354

FUNCTIONAL FOODS

Chicory inulin, 121, 123, 125, 156 China, 237, 239, 267 Chloride, 284, 289, 291-292 Cholecalciferol, 201, 203 Cholesterol, 72, 86, 91, 94, 104, 107, 132, 149-150, 156-158, 175, 179, 181, 190-191, 194, 247, 253, 256-257, 259, 267, 275 absorption, 190 reduction, 149-150 Choline, 295 Chromium, 215, 220-221 Chymotrypsin, 244 Citric acid, 289, 293 CLA isomers, 165, 175 Coca-Cola Co., 287 Colon, 5, 6 Colon cancer, 128-129, 136, 142-143, 146, 155, 158, 160 Commercial sports drinks, 289-290, 292, 294 Composition of infant formula, 326, 336 Conglycinin, 242-243 Conjugated linoleic acid, 162, 165, 168, 170, 193-194, 196 Connexin, 43 Copper, 204, 215, 221-226 Cow’s milk, 300, 301 Cranberries, 23-29, 31, 58-60 Dairy products, 91, 93, 98, 165-171 Davy, Sir Humphrey, 221, 223-225 Dehydration, 281, 284-285 Demigne, 86, 110-111 Designer foods, 1 D-galactopyranosyl, 94-95 D-glucopyranosyl, 92-94, 119 D-glucose, 91-94 DHA, 167-168, 173-174, 176-178, 181, 307, 333-334 Diabetes, 3, 162, 175, 177-179 Diarrhea, 128, 136-141, 155 rotavirus, 137-139 Dietary cholesterol, 195 fatty acids, 195 fish oils, 181-182, 193 trans fatty acids, 195 Dietary supplements, 126-127, 136, 151

Difficile disease, 138 Digestive system, 2 Diglycerides, 162-163 Dynamo, 279 Electrolyte concentrations, 289, 294 Electrolytes, 279, 284-289, 292-293, 297-298 Endogenous antioxidants, 20, 23 Energy, 282-283, 285-287, 294, 295 balance, 282, 285 drinks, 294-295 expenditures, 282 source, 282, 289 total, 301, 324-325 Enzymatic-Gravimetric Method, 77 Enzymes, bacterial, 142 EPA, 167-168, 173-174, 176-177, 181 Epicatechin, 21, 29, 32-33, 54 Erdman, 246, 276-277 Estrogen receptor, 259-261 Eubacteria, 114 European markets, 280-281 Exo-inulinase, 123 Fat, dietary, 173 Fat-soluble vitamins, 309, 310, 313, 323-333 Fatty acids, 7, 11, 13, 246-247, 275 composition, 246-247 unsaturated, 246, 264 Fekety, 137-138, 156-157 Fermentation, colonic, 80-81, 90 Fermented milk product, 144, 151, 152 Fiber, 2, 4-6 dietary, 7 supplemented foods, 111 Fiber types, 71-72, 82, 86, 89, 108 Fish oil, 162, 174, 177-178, 180-182, 192-193, 195 Flavonols, 21, 32-33, 37, 39-40, 60 source of, 39-40 Fluid absorption, 284, 286, 292, 296 extracellular, 298 intake, 293 voluntary, 286, 292, 294 replacement beverages, 279 requirements, 279

INDEX

Fluoride, 221 Folate, 216-218, 236 Folic acid, 198-199, 216-219, 235-236 deficiency, 218 Formulation, 323-325, 330-331, 335 Free radicals, 9-15, 17, 22, 49, 296 Fructans, 117-121, 131, 154, 157, 159 bacterial, 120 prebiotic chicory, 157 Fructooligosaccharides, 73, 86, 123, 126, 127, 129-130, 132-133 Fructose, 118, 121, 123, 126-127, 286, 289, 292 Functional foods, 113, 126-127, 136, 154, 197, 200 food benefits, 2 development, 4 market, 1 Galactose, 308-309 Garlic, 43, 48-53, 58, 60-61, 119-120, 122-123, 128 cloves, 49-51 extract, 50-53, 59 Garlic - continued fresh, 49-51 oil, 50, 52 Gastrointestinal tract, upper, 126-128 Gatorade, 280, 287 Genistein, 249, 255, 257-261, 276-277 ability of, 260 Globulins, 242-243, 257 Glucose, 118, 119, 121, 126-127, 133, 152, 157, 286, 289, 292 electrolyte drink, 279 Glycerol, 162, 182 Glycinin, 242-244, 252 Glycoproteins, 308-309 Grapes, 23, 29, 37, 58, 60-61 Green tea, 21, 54, 56-67, 59 Gylling, 190-191, 194-195 Haptocorrin, 303, 305, 306 Hayatsu, 143, 157 HDL cholesterol, 177, 179, 180 levels, 174, 187 ratio, 180-181 Heart disease. 2 Hemagglutinins, 242-243, 245, 255

355

Hemicelluloses, 71-72, 75 Hepatic encephalopathy, 126-127, 139, 158 Hormone, parathyroid, 221 Howell, 29-30, 59-60 Human milk, 169, 171-172, 193-195 chemistry, 301 consumption, 334, 337 iron, 317 lipids, 307 proteins, 302-304, 320, 322, 337 zinc concentration, 318 Hydrophobic peptides, 253, 276 Hydroxytyrosol, 183-184 Hypocholesterolemic effect, 252, 255-256 Hypokalemia, 226 Iced teas, 6 IGF-binding proteins, 306 Infant formula, 7, 134 commercial, 325 formulation, 301, 323, 325-326, 333, 335 formula-fed, 311-312 liquid, 332 milk-based, 323-324, 330 soy-based, 324 Intestinal bacteria, 114-115, 126, 143, 149 flora, 113-116, 137, 142, 154 Inulin-type fructans, 120, 134, 158 Iodine, 220, 232-233 Ionic strength, 101-102 Iron, 204, 214, 227-228, 234, 236 binding protein, 305 concentration, 317 non-heme, 227 Isoflavones, 241, 249, 252, 255, 257, 259, 261-262, 275 primary, 249-250 Isotonic beverages, 279 Isotonicity, 287 Isozymes, 245-246 Japan, 126-128, 237, 262, 267, 272 Kaempferol, 20-21, 41-43 Lactation, duration of, 315-316, 317, 318 Lactic acid bacteria, 135, 143, 145, 158-159 Lactobacilli, 309, 334

356

FUNCTIONAL FOODS

Lactobacillus acidophilus, 135, 150-151, 156, 158 Lactobacillus GG, 139-141 Lactoferrin, 302, 303, 305, 318, 321-323, 335 Lactoperoxidase, 321-322 Lactose, 94, 99-100 Lactulose, 126-128, 154 Lawson 50, 53, 60 LDL, 20, 49, 56-57, 179-180 cholesterol, 179-180, 190-191 Lectins, 245, 255 Legumes, 73-74, 94 Lignin, 64-67, 72-73, 75-76, 102, 107 Linoleic acid, 307, 325, 327-329 Lipids, 9-13, 17, 19-20, 50, 53, 58, 161, 177, 183, 193-196, 282, 295 hydroperoxides, 11-12, 14 oxidation, 10-11, 13, 18 peroxidation, 13, 17, 19-20 serum, 177, 195 Lipoproteins, 180-195 Lipoxygenases, 242-243, 245 Lycopene, 38-43, 58 Magnesium, 220, 223-224, 284, 289 carbonate, 224 deficiency, 223-224 Malic acid, 289, 293 Maltodextrins, 289, 291-292 Manganese, 220, 223, 234 Margarines, 167, 171-172, 189, 191 Market, worldwide beverage, 280 Maternal supplementation, 311-312, 314 Maughan, 280-281, 298 McFarland, 137-138, 157, 159 Medical foods, 1 Mediterranean diet, 182, 187 Metastasis, 259-260 Methionine, 243, 257, 261-266 Microbes, 5 Milk, 133-134, 136, 142, 144, 152, 154, 156, 168-169, 171, 194 calcium, 316 iodine concentration, 319 iron concentration, 317 pantothenic acid level, 314 protein, 303, 328-330, 336 bovine, 330

Milk - Continued riboflavin levels, increased, 313 thiamin levels, 313 Milk-based formulas, 324, 331 bovine, 334 Milk-free formulas, 324 Minerals, 7 Modern infant formulas, 300, 336 Molybdenum, 220, 233-234 Monounsaturated fatty acids, 186-187 Murray, 280, 284-286, 298 Muscle carnitine concentrations, 295 N-3 fatty acids, 174, 177, 181 N-3 PUFA, 161, 163, 165, 167-168, 173-178, 182, 192 N-6 PUFA, 163-165, 173-174, 177 Natural antioxidants, 10, 20-21, 23 Nondigestible carbohydrates, 67, 111 Non-electrolytes, 289 Nonstarch polysaccharides, 64-65, 72-73, 109, 111 Oat bran, 86, 104-105, 109 Obesity, 256, 263 Obesity, 3 Oleic acid, 162, 166, 179-180, 186-187 Oligofructose, 119, 121, 123, 125, 127-131, 156-157, 159 Oligosaccharides, 64, 67, 72-73, 75, 84, 86, 92, 94, 96-101, 117, 121, 127-128, 133-134, 154, 308-309, 334-335, 337 human milk, 134 soy, 127, 154 Olive oil, 172, 182-187, 192, 196 Omega fatty acid, 192 Oolong teas, 54 Osmolality, 287-289 Osmolarity, 315, 325-326 Osteomalacia, 202, 221-222 Osteoporosis, 202, 221-222, 236, 255, 261, 275 Oxidative damage, 9-10, 17, 19-20 Oxygen, 9, 11-12, 14 Peptides, 302, 303, 321, 323-324, 335, 337 Phagocytosis, 254 Phenolic acids, 24, 26-28, 31-32, 58, 241, 249

INDEX

Phosphatidic acid, 247 Phospholipids, 241, 257 Phosphoric acids, 223 Phosphorus, 202, 220, 222-223 deficiency, 217 Phylloquinone, 207-208 Phytic acid, 241, 244, 251, 255-267, 275 Phytoestrogens, 257-259 Phytosterols, 162, 187-193, 195 contents, total, 189 dietary, 190, 195 free, 187 total, 187, 189 Piironen, 188-189, 195 Plant inulin, 120-122 Plant sterols, 187, 190, 195 dietary, 190, 195 Polydextrose, 64, 72-74, 78, 92 Polyphenols, 21, 23, 32, 41, 54 Polyunsaturated fatty acids, 307, 333, 336 Pool-Zobel, 145-146, 158-160 Potassium, 220, 225-226, 284, 286-287, 289, 282-283 Powdered infant formula, 332 Powerade, 280, 287 Preterm infants, 307, 311-312, 316 Proanthocyanidins, 24, 29-32, 58 Probiotics Bifidumbacterin, 157 Probiotics, 2, 5-7 bacteria, 127, 135-136 treatment, 138, 158 use, 138-139 Prosky, 65, 110 Prostaglandins, 174 Prostate, 24, 40-41 cancer, 40-43 risk, 40-41, 59 Proteins intact breast-milk, 304 soy, 323-324, 328 vitamin B12 binding, 305 whey, 106-107 Protein content of human milk, 301, 304 Pro-vitamin, 198, 202 Psyllium, 71, 86, 97 PUFA, 161-162, 164, 167, 177 Pylori, 31, 50-51, 53 Pyridoxine, 212-214 RDA, 200, 202, 205-208, 210, 212, 214, 216, 218, 220-222, 224-234

357

Rehydration, 284-285, 287, 292-294 Renal solute load, 325 Resistant starch, 64, 66, 72, 74, 76-77, 79, 84, 87, 90 Resveratrol, 24, 28, 37 Riboflavin, 310, 313, 327, 328 Saponins, 241, 251, 255-257, 275-276 Saturated fat, 104, 107, 162, 166, 179, 180 Sekine, 144-145, 159 Selenium, 220, 230, 236 deficiency, 230 Serotonin, 296 Serum cholesterol, 252-253 Sitosterol, 187, 189-191 beta, 192 Sitostanol, 190, 193 Sodium, 220, 224-225 acetate, 292 bicarbonate, 295-296 chloride, 289, 292-293 citrate, 292, 296 deficiency, 225 levels, 286 replacement, 298 Soy meal, 271, 273 milk, 264, 274 products, 1, 7 protein products, 255, 271, 274 sauce, 264, 267, 270-271, 274 fermented, 267, 271 yogurt, 264 Soybean beta-conglycinin, 254, 275 globulins, 243, 253 glycinin, 243, 253 isoflavone isomers, 249 oil, 246-247, 275 peptides, 263 proteins, 242-243, 252-255, 263 quality of, 252 Soy Protein Health Claim, 275 Soy Protein Isolates, 273 Soy-based formulas, 324 Soymilk, 243, 249, 264-265 Sports beverage preparation, 294 drinks, 7 industry, 294, 297

358

FUNCTIONAL FOODS

Sports - Continued market, 280 nutrition, 298 Sterols, plant, 247 Stigmasterol, 187, 189-190, 195 Stofan, 280, 284, 286, 298 Storage proteins, 242, 244, 257 Sucrose, 117, 120-121, 125-126, 140, 286, 289-299, 292-295 Sulfur, 220, 226-227, 230 deficiency, 227 residue foods, 227 Supplementation of infant formula, 302 Sutherland, 294, 298 Symbiotics, 116, 151-152, 154-155, 158 Synergy, 123, 125 Synthetic antioxidants, 10, 57-58 Tablets, garlic powder, 50 Takenaka, 277 Taurine, 295 TBHQ, 57-58 Tea, 29, 53-56, 58 Tempeh, 264, 267-268 Therapeutic index, 198 Thiamin, 210-211, 310, 313, 327, 328 Tocopherols, 203-204, 206, 236 Tocotrienols, 203-204, 206, 236 Tofu, 7, 243, 249, 261, 264, 266-267, 271, 274, 277 Tomato, 38-40, 43, 42, 58-60 juice, 39-40, 42 powder, 39, 41 products, 38, 40, 43 Total dietary fiber, 77, 103, 105 Toxins, 5 Trans fat, 171, 179-180 fatty acid content, 171-172 human milk, 193 acids, 162, 166, 171-172, 179, 180, 194, 196 consumption of, 179 Triglycerides, 161-163, 176, 180, 246, 253 Trypsin inhibitors, 242, 244, 255, 256 Tryptophan, 296

United States, 126-128, 239, 240, 256, 264, 267, 274 Unsaturated lipids, 10-13 Urinary tract, 24, 28-29 UTIs, 24-25, 28-29 Symptomatic, 25, 28-29 Varnam, 294, 289 Vicilins, 242 Vitamin, 1, 2, 6, 7, 11, 17-18, 24, 38, 40, 241, 251, 261, 264 biotin, 219 fat soluble, 198 Vitamin - continued protein-bound, 314 water soluble, 198-199, 208, 313, 333 Vitamin A-rich foods, 199 Vitamin B1, 199, 210-212 Vitamin B12, 303, 305, 306, 313-314, 320, 327-328 Vitamin B6, 199, 212-214, 236 Vitamin B6, 313, 327-328 Vitamin B12, 199, 214-216, 235 deficiency, 216 Vitamin D2, 201-202 Vitamin D3, 201-202 Vitamin K1, 207 VO2 max, 282 Water absorption, 286, 289 binding capacity, 102 holding capacity, 80, 82, 102 treatment, 290, 297 Weight loss, 211-212, 216, 218 Western diets, 161, 174 Whey proteins, 301-303, 323-326, 330-331 Wolinsky, 298 Wollowski, 141, 144-145, 160 Yogurt, 126, 136, 149, 152, 154 Younes, 86-87, 110-111 Zinc, 220, 228-229, 284, 286-287, 336 deficiency, 229