Milk Consumption and Health (Food and Beverage Consumption and Health)

Food and Beverage Consumption and Health Series

MILK CONSUMPTION AND HEALTH

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FOOD AND BEVERAGE CONSUMPTION AND HEALTH SERIES Handbook of Green Tea and Health Research Helen McKinley and Mark Jamieson (Editors) 2009. ISBN: 978-1-60741-045-4 Marketing Food to Children and Adolescents Nicoletta A. Wilks 2009 ISBN: 978-1-60692-913-1 Food Labelling: The FDA's Role in the Selection of Healthy Foods Ethan C. Lefevre (Editor) 2009. ISBN: 78-1-60692-898-1 Fish Consumption and Health George P. Gagne and Richard H. Medrano (Editors) 2009 ISBN: 978-1-60741-151-2 Red Wine and Health Paul O'Byrne (Editor) 2009 ISBN: 978-1-60692-718-2 Milk Consumption and Health Ebbe Lange and Felix Vogel (Editors) 2009 ISBN: 978-1-60741-459-9

Food and Beverage Consumption and Health Series

MILK CONSUMPTION AND HEALTH

EBBE LANGE AND

FELIX VOGEL EDITORS

Nova Biomedical Books New York

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Lange, Ebbe. Milk consumption and health / Ebbe Lange and Felix Vogel. p. cm. Includes index. ISBN 978-1-61728-540-0 (E-Book) 1. Milk in human nutrition. I. Vogel, Felix. II. Title. QP144.M54L36 2009 613.2'6--dc22 2009018832

Published by Nova Science Publishers, Inc.  New York

Contents

Preface Chapter I

vii Plant Sterols and Plant Stanols in Milk Products Used as Functional Foods: Effects on Cardiovascular Risk Diseases Prevention Fernando Ramos and David Saraiva

Chapter II

Kefir and Health: A Perception Zaheer Ahmed and Yanping Wang

Chapter III

Fouling Reduction during Milk Processing Using Equipment Surface Modification Sundar Balasubramanian and Virendra M. Puri

Chapter IV

Milk Fat/Sunflower Oil Blends as Trans Fat Replacers Roberto J. Candal and María L. Herrera

Chapter V

Probiotic Bacteria Isolated from Breast Milk for the Development of New Functional Foods G. Vinderola, A. Binetti and J. Reinheimer

1 43

71 87

115

Chapter VI

Probiotics in Maternal and Early Infant Nutrition Yolanda Sanz

125

Chapter VII

Epilactose: Potential for Use as a Prebiotic Susumu Ito, Jun Watanabe, Megumi Nishimukai, Hidenori Taguchi, Hirokazu Matsui, Shigeki Hamada and Shigeaki Ito

153

Chapter VIII

Lactoferrin as an Added-value Whey Component and a Healthy Additive in Nutraceutical Drinks Palmiro Poltronieri, Carla Vetrugno, Antonella Muscella, Santo Marsigliante

163

vi Chapter IX

Chapter X

Index

Contents Conjugated Linoleic Acid: An Anticancer Fatty Acid Found in Milk and Meat T. R. Dhiman, A. L. Ure and J. L. Walters

175

Beneficial effects of Human Milk and Prebiotic-Like Fermented Infant Formulas on the Intestinal Microflora and Immune system Catherine J. Mullié, Daniel Izard and Marie-Bénédicte Romond

215 249

Preface Although there is no official definition of functional foods, it is generally considered that they are a group of foods which provide physiological benefits beyond those traditionally expected from food. Milk proteins have a great potential use as functional foods. Healthy foods, nutraceuticals and food for specified human use, are one of the fields in constant growth in the food industry, as well as an emerging field of medical interest. Many mainstream health and nutrition organizations worldwide recommend daily consumption of dairy products for optimal health. Nevertheless, the last decade or so has seen an increase in the number and variety of claims made against the inclusion of milk and/or its products in the diet. A single supplement cannot address all such matters, but the purpose of this book is to address in a scientific and objective manner the validity of some of these concerns. This book presents the views of some of the world's top nutrition scientists on this food that has served mankind for over 10,000 years. Milk is not a one-nutrient food, nor is its impact restricted to one condition such as osteoporosis. Its many bioactive components are only just beginning to be defined and explained. This new important book presents the latest research from around the world in this field. Chapter 1 - The early development of cardiovascular diseases (CVD), one of the major death causes in Europe, is clearly associated with high plasmatic cholesterol levels. Recently, it has been suggested that the ingestion of plant sterols and/or stanols could reduce cholesterolemia, and thereby contributing to the reduction of the CVD development. Vegetable oils, followed by cereal grains and their by-products and dry fruits, are the main sources of plant sterols/stanols. However, daily estimated consumption, even by eating referred sources, is very inferior to the recommended daily dose of 2g. Consequently, plant sterols/stanols enrichment was used by food industry to reach recommended dose. Thus, on this chapter, a brief presentation on plant sterols and stanols (nomenclature, chemical structures and properties; consumption and natural sources) was given, followed by a more detailed review on milk and other dairy products enriched with plant sterols/stanols (regulations; technological aspects; methods of analysis; consumption; mechanisms of action; prevention of cardiovascular diseases). Finally, along with the final remarks, some perspectives about future health research based on milk and other dairy products enriched with plant sterols/stanols were made Chapter 2 - Kefir is a fermented milk drink produced by the actions of bacteria and yeasts contained in kefir grains, and is reported to have a unique taste and properties. Kefir, the self-

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carbonated beverage, possesses nutritional attributes due to its content of vitamins, protein and minerals and therapeutic attributes contributed by its antibacterial spectrum, gastrointestinal proliferation, hypocholesterolemic effect, anti carcinogenic effect, lactic acid content, b-galactosidase activity and bacterial colonization, improves immune system and is also remedy for Helicobacter pylori infection which is only the property of kefir. Moreover, on one side kefir is good dietetic beverage, and of particular interest of athletes, and on other side the whole kefir is good for feeding premature infants because of good tolerance, and adequate weight gain. Lots of work has been done on kefir from a health point of view, this chapter summarizes all the data that has been completed to date. By reviewing the literature the chemical, microbiological, nutritional and therapeutic characteristics of kefir have been highlighted to justify its consumption as a healthy milk food. Chapter 3 - Fouling of equipment surfaces during milk processing is a phenomenon that needs to be immediately addressed due to the increased energy utilization and production costs encountered. Modifying the equipment surface is one method of reducing the incidence of fouling. Research was carried out at the Pennsylvania State University using different food-grade surface coatings to modify plate heat exchanger surface, and was tested for their ability to reduce fouling during skim milk pasteurization. The results were compared with traditional stainless steel 316 plate heat exchanger (PHE) surfaces typically used in the food industry. Results after 6 h continuous testing using a pilot scale PHE unit indicate that there was greater than 85% reduction in fouling when the three coated surfaces (AMC 148-18, NiP-PTFE and LectrofluorTM-641) were used. Chemical analyses of the foulants indicate that the coating integrity did not appear to be compromised for the LectrofluorTM-641 coatings. However, there were trace amounts of fluorine present in the foulants adhering to the other two coating types (AMC148-18 and Ni-P-PTFE). A preliminary cost estimate on the thermal energy savings when using the coated surfaces indicate that there is substantial savings in energy, further justifying the use of these coated surfaces, and making them more attractive for possible implementation in the food industry. Chapter 4 - As a body of evidence suggests that dietary trans fatty acids raise blood cholesterol levels, thereby increasing the risk of coronary heart disease, on July 11, 2003, FDA issued a final rule requiring the mandatory declaration in the nutrition label of the amount of trans fat present in foods, including dietary supplements. The agency required that the declaration of trans fat be on a separate line immediately under the declaration for saturated fat. Since there was no scientific basis for establishing a DV for trans fat, the final rule did not require the listing of a % DV as is required for some of the other mandatory nutrients, such as saturated fat. However, a report from the World Health Organization (WHO) and the Food and Agricultural Organization (FAO) of the United Nations has recommended a very low intake of TFA, less than 1% of daily energy intake. Therefore, efforts have been made and are ongoing to decrease TFA in the food supply both in the U.S. and globally. There are many challenges that food manufacturers have faced during the development of new trans fat alternatives. Any replacement ingredient must provide the functional characteristics of the material being replaced. In other words, the alternative ingredient must provide the functionality of flakiness, firmess of texture, crispness or desired appearance in the finished product or it is likely to be rejected by the consumer. The stability or shelf life of the finished product must also be maintained to ensure consumer acceptability.

Preface

ix

In some applications, like baked goods, a certain amount of solids is crucial. Consumer concerns associated with the atherogenic effect of trans fatty acids limit the future of the hydrogenation process as a way of modifying the solid-to-liquid ratio in vegetable oils/fats. As an alternative to hydrogenated vegetable oils, modification of high melting point stearins by blending with vegetable oils is becoming important, since shortenings with appropriate physicochemical properties and good nutritional characteristics that are free of trans fatty acids and rich in PUFA can be obtained. Thus, it is of interest to discuss the potential of blends of a stearin such as a high-melting fraction of milk fat with a vegetable oil as trans fat replacer. In this chapter the physical chemical properties of milk fat-sunflower oil low-trans blends, that is, crystallization behavior, polymorphism, microstructure and the effect of addition of emulsifiers in bulk systems will be reviewed. Chapter 5 - Baby’s intestine is (or was said to be) sterile at birth and gut microbiota development is a gradual process after delivery. Quantitative and qualitative differences in bifidobacterial and lactic acid bacteria levels and species composition have been shown between breastfed and formula-fed infants, bifidobacteria being the most dominant microorganisms in the former group. Establishment of the gut microbiota is a stepwise process which provides the earliest and most massive source of microbial stimuli for the normal maturation of the gut mucosal immune system, contributing to its development in infancy and to the control of the gut-associated immunological homeostasis later in life. Probiotic intervention in the neonatal period has attracted scientific interest after recent demonstrations showing that specific strains reduce the symptoms and risk of allergic and infectious diseases or improve feeding tolerance. However, no all early interventions in children reported rendered positive results. The question of the right dose and the specific pathologies that probiotic administration, to infants less than 6 month of age, could be helpful for is still under a vigorous debate. Breast milk contains several factors, including nutrients, antimicrobial agents, IgA antibodies and TGF-β, which contribute beneficially to the immunologic maturation and well-being of the infant as well as factors that promote the growth of bifidobacteria in the infant’s intestine. Additionally, healthy breast milk contains significant numbers of bacteria. In 2003 it was reported the isolation of lactobacilli from breast milk as potential probiotics. Breast milk seems to be a natural source of probiotic bacteria for infants. In this context, supplementation of infant formulas with these kinds of probiotics might beneficially alter the composition of the microflora of formula-fed infants in such a way that it resembles that of breast-fed infants. However, to date there is no available information concerning the technological potential of these strains for their industrialization (growth in milk, resistance to lactic acid, freezing or spray-drying, among others) if they are thought to be included in dairy products or in formulas for infants. Chapter 6 - During pregnancy fetal development is entirely dependent on the mother. Epidemiologic and clinical studies suggest that immunologic and metabolic profiles of the pregnant uterus are responsive to mother’s diet. This evidence supports the hypothesis that maternal nutrition may influence fetal programming and disease risk in the offspring. After birth, the gastrointestinal tract undergoes vast structural and functional adaptations under the stimulation of the microbiota and the diet that make possible handle with antigens and digest milk and latter solid food. The intestinal colonization process implies the activation of diverse metabolic functions either triggered by host-microbe interactions or directly encoded

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by the genome of the microbiota (microbiome). Moreover, microbial exposure through colonization process of the newborn intestine is essential to regulate epithelial permeability and immune function, with long-term consequences on host’s health. Bacterial composition and succession during the intestinal colonization process have been shown to determine susceptibility to infections and sensitization to dietary antigens. In this context, mammals seem to have a developmental window within the perinatal and postnatal period, in which the host-gut microbiota interactions are more influential in favoring later health. Probiotic and prebiotic administration has been demonstrated to be a dietary strategy that at least temporary modulates the microbiota composition and may favor a healthy status. These strategies have demonstrated moderate efficacy to reduce the risk of infections and allergic diseases in early life. In recent years, the administration of probiotics to pregnant and lactating mothers in addition to their newborns, along or not with prebiotics, has also been evaluated to extend their applications and improve effectiveness by acting in these critical developmental stages. This type of intervention has shown that specific probiotic strains influence gut growth and immune function in the offspring of animal models. Other studies have suggested that this dietary strategy may help to reduce the risk of atopy, infections, and metabolic disorders in humans. The current knowledge of the effectiveness and mechanisms by which the administration of probiotics to mothers and infants could positively affect early stages of development, favoring latter heath is review. Chapter 7 - Prebiotics are nondigestible food components that affect the host by stimulating the growth and/or activity of health-promoting bacteria in the colon and thus contribute to host health and well-being. Epilactose is the C2-epimer of lactose that is found in heat- and alkali-treated milk. We found that a cellobiose 2-epimerase of Ruminococcus albus isolated from cow rumen efficiently converts lactose in milk and whey to epilactose. The enzymatic synthesis of epilactose has the advantage over chemical synthetic protocols reported to date of producing byproducts. A dietary intervention study showed that epilactose has potential for use as a prebiotic or prebiotic foodstuff. In the colon of rats fed epilactose, 1) growth of health-promoting lactobacilli and bifidobacteria was enhanced, 2) rates of mineral absorption were increased, 3) levels of plasma total cholesterol and non-high-densitylipoprotein cholesterols were lowered, and 4) conversion of primary bile acids to secondary bile acids was suppressed. Therefore, the conversion of lactose to epilactose may increase the nutritional value of milk and whey. Chapter 8 - Lactoferrin (Lf) is a whey protein with potential food applications to sustain human health. Lf is already added to infant formula milk powder so that, like breastmilk, it contains Lf to help build resistance to disease. One yogurt is added with Lf and produced by the Morinaga factory in The Nederlands. Lf binds iron, and can deliver it to increase iron availability. This ability seems to affect also microbes and fungi, although iron-bound lactoferricin peptide seems to be as effective as the full protein. In this work it is shown the effect of Lf on MCF-7 cultured cells, i.e. the induction of apoptosis in the presence of sustained cell cycling driven by angiotensin-II growth factor. We thus show that Lf may have antiproliferative activity on selected cell types. Further work is needed to individuate the proteins interacting with Lf, and the downstream signalling that end in the shutting off of cell cycle effectors.

Preface

xi

We found that Lf-based emulsions storage with good stability up to 12 months. A milk or soy-milk beverage may be a convenient vehicle for delivery of Lf-based nutraceuticals. Chapter 9 - Conjugated linoleic acid (CLA) has been intensively studied recently, mainly because of its potential in protecting against cancer, atherogenesis, and diabetes. Conjugated linoleic acid is a collective term for a series of conjugated dienoic positional and geometrical isomers of linoleic acid, which among common human foods are found naturally in relative abundance in the milk and meat fat of ruminants. The cis-9, trans-11 isomer is the principle dietary form of CLA found in ruminant products, and is produced by partial ruminal biohydrogenation of linoleic acid or by endogenous synthesis in the tissues themselves. The CLA content in milk and meat is affected by several factors, such as an animal’s breed, age, diet, and management factors related to feed supplements affecting the diet. Conjugated linoleic acid in milk or meat has been shown to be a stable compound under normal cooking and storage conditions. Total CLA content in milk or dairy products ranges from 0.34 to 1.07% of total fat. Total CLA content in raw or processed beef ranges from 0.12 to 0.68% of total fat. It is currently estimated that the intake of the average adult consuming western diets is only one-third to one-half of the amount of CLA that has been shown to reduce cancer in animal studies. For this reason, increasing the CLA content of milk and meat has the potential to raise the nutritive and therapeutic values of dairy products and meat. Growing evidence suggests that consuming dairy products and meat enriched with CLA has beneficial effects on human health. Chapter 10 - Mother’s milk remains the gold standard for the nutrition of human neonates. Thanks to its adaptable biochemical and immunological composition, mother’s milk allows for an optimal development of the intestinal microflora, especially by promoting the implantation and growth of some of the so-called health beneficial bacteria: bifidobacteria. When bifidobacteria are dominant in the intestinal flora, they are thought to help preventing gastrointestinal disorders, repress a potentially harmful proliferation of other intestinal bacteria and stimulate the priming of the neonate’s intestinal immune system. This is why, among other research trends, the latest infant formulas are attempting to reproduce this bifidogenic effect of mother’s milk through various ways such as the addition of exogenous bifidobacteria and/or of prebiotics (specific carbohydrate substrates promoting the growth of indigenous intestinal bifidobacteria). We will first review the beneficial effects of mother's milk and those putatively related to indigenous bacteria. The probiotic (feeding of live bifidobacteria) and prebiotic (feeding of specific carbohydrates) approaches to increase intestinal bifidobacteria will also be defined. Then, we will focus on prebiotics and on a novel approach to promote indigenous intestinal bifidobacteria: the use of an infant formula containing products of milk fermentation by Bifidobacterium breve strain C50. These fermentation products have previously been shown to have a bifidogenic effect on indigenous bifidobacteria, thus acting like prebiotics. We will compare the effect of this formula on the intestinal microflora establishment to the ones of mother’s milk and of a standard formula. We will also deal with the issue of specifically stimulating the growth of certain species of indigenous bifidobacteria, as some bacterial species belonging to this genus (e.g., Bifidobacterium adolescentis) have been shown to be linked with immunological conditions in neonates and young children such as atopic dermatitis.

In: Milk Consumption and Health Editors: E. Lange and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter I

Plant Sterols and Plant Stanols in Milk Products Used as Functional Foods: Effects on Cardiovascular Risk Diseases Prevention Fernando Ramos* and David Saraiva Group of Bromatology, Center of Pharmaceutical Studies, University of Coimbra, Polo III, Azinhaga de Stª Comba, 3000-548, Coimbra, Portugal

Abstract The early development of cardiovascular diseases (CVD), one of the major death causes in Europe, is clearly associated with high plasmatic cholesterol levels. Recently, it has been suggested that the ingestion of plant sterols and/or stanols could reduce cholesterolemia, and thereby contributing to the reduction of the CVD development. Vegetable oils, followed by cereal grains and their by-products and dry fruits, are the main sources of plant sterols/stanols. However, daily estimated consumption, even by eating referred sources, is very inferior to the recommended daily dose of 2g. Consequently, plant sterols/stanols enrichment was used by food industry to reach recommended dose. Thus, on this chapter, a brief presentation on plant sterols and stanols (nomenclature, chemical structures and properties; consumption and natural sources) was given, followed by a more detailed review on milk and other dairy products enriched with plant sterols/stanols (regulations; technological aspects; methods of analysis; consumption; mechanisms of action; prevention of cardiovascular diseases). Finally, along with the final remarks, some perspectives about future health research based on milk and other dairy products enriched with plant sterols/stanols were made *

Corresponding author. E-mail: [email protected]

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Fernando Ramos and David Saraiva

1. Introduction The importance of cholesterol for human life is well ascertained. Besides its essential role in eukaryotes as a membrane component, indispensable for cell maintenance and permeability, cholesterol is used as a precursor of essential molecules for mammals, such as steroid hormones, the active form of vitamin D or biliary acids. However, and as it is also known, a high concentration of blood cholesterol is an added risk factor for the development of cardiovascular diseases. Nowadays, cardiovascular diseases are the main cause of death in developed countries, a tendency that is spreading to developing countries. This means that cardiovascular diseases are responsible for 30% of all deaths – or about 17.5 million people – in 2005. Among males, almost 50% of the excess mortality was due to cardiovascular diseases. For females, almost 80% of the difference in life expectancy was due to excess mortality from cardiovascular diseases (Leif & Gotto, 2006; WHO, 2008). Nevertheless, it has been demonstrated that a 10% decrease in total cholesterol could be associated with a reduction of 20% of coronary heart diseases in 70 years old individuals, and of 50% in 40 years old individuals. So, in order to improve life quality and life expectancy, all blood cholesterol reduction strategies are very important (Law et al., 1994). In the seventies of last century, phytosterols were commercialised as pharmacological medicines with hypocholesterolemic properties (Trautwein et al., 2003; Kritchevsky & Chen, 2005). However, due to their unpleasant taste, their weak solubility and their administration difficulties, the referred compounds had some difficulties to be considered ideal drugs to carry out the purposed field (Miettinen & Gylling, 1999; Miettinen, 2001; Moreau et al., 2002). Consequently, phytosterols (used in this chapter to refer plant sterols and their saturated counterparts, plant stanols) were substituted by a new more efficient medicine group, statins. However, some statins have been causing some side effects, like severe muscle weakness and toxicity (Clark, 2003; Maggini et al., 2004). So, alternative and/or complementary procedures for blood cholesterol reduction are welcome. Thus, and due to that phytosterols could be used as part of a normal human diet, as well as to the discovery of sitostanol’s effectiveness in cholesterol reduction in relatively low doses (1.5 g/day) (Heinemann et al., 1986), the interest for these compounds was reborn. In fact, this has been shown by Katan and co-workers (2003) in that phytosterol lower LDL-C (low-density lipoproteins cholesterol) by about 10% for a 2 g/d dose, on average. Consequently, phytosterols food enrichment is a subject of particular interest in health nutrition activities. Phytosterols esterification by fatty acids, developed in the beginning of the ninth decade of last century, was an innovation that had allowed its incorporation and solubility in fat foods, without any interference on their sensorial properties (Vanhanen et al., 1994; Gylling & Miettinen, 2000). Several other formulations (Moreau et al., 2002) have been subsequently developed in order to reduce technological limitations and to increase phytosterols food enrichment (Corliss et al., 2000; Akashe & Miller, 2001; Christiansen et al., 2001a; Engel & Knorr, 2004). So, foods with high fat content, like margarines, were considered to be ideal foods for phytosterols enrichment, due to their strong hydrophobic qualities (Mattson et al., 1982). However, this type of food does not conform to actual recommendations for a healthy diet lifestyle.

Plant Sterols and Plant Stanols in Milk Products Used As Functional Foods

3

For that reason, the scientific community has been exploring the incorporation of these compounds in foods of low fat level, such as milk and other dairy products (St-Onge & Jones, 2003). So, when Pollak (1953) had finished his article writing that "this preliminary report should open the new avenue of research", he was more than right for the future. His theory, that appropriate amounts of sitosterol ingestion could reduce cholesterol intestinal absorption and, consequently, lower blood cholesterol levels, was, undoubtedly, one of the steps for the expansion of the actual markets of phytosterols enriched foods. In this chapter of the entitled book “Milk Consumption and Health”, a brief presentation on plant sterols/stanols (nomenclature, chemical structures and properties; consumption and natural sources) was given, followed by a more detailed review on milk and other dairy products enriched with phytosterols (regulations; technological aspects; methods of analysis; consumption; mechanisms of action; prevention of cardiovascular diseases). Finally, a few words classified as conclusions finish this chapter, as well as some perspectives about future health research based on milk and other dairy products enriched with phytosterols.

2. Plant Sterols and Plant Stanols 2.1. Nomenclature, Chemical Structures and Properties Plant sterols and plant stanols, here referred to as phytosterols, are natural constituents of plants that are structurally similar to cholesterol (Pollak & Kritchevsky, 1981). Phytosterols have many essential functions in vegetable cells. Fluidity and permeability regulation of cellular membranes and its properties as compound biogenic precursors involved in plant growth (e.g. brassinosteroids) are very well known. Additionally, they are substrates for the synthesis of numerous secondary vegetable metabolites, as glycoalcaloids or saponins (Hartmann, 1998). Like cholesterol, they are bio-synthetically derived from squalen and they belong to isoprenoid group (Piironen et al., 2000a). The most common are constituted by a steroid nucleus, with a hydroxyl group in the 3β position and a double bond between carbons 5-6. While cholesterol lateral chain (in the C17 carbon) is constituted by 8 atoms of carbon, most of the phytosterols are characterized by one or two extra carbons bonded to C24 (Figure 1). Phytosterols can be classified according to their structure and biosynthesis, in 4desmethyl sterols, 4α-monomethyl sterols and 4,4-dimethyl sterols. The 4,4-dimethyl sterols (e.g. cicloartenol) and the 4α-methyl sterols (e.g. gramisterol) are less abundant in nature and they are 4-demethyl sterols precursors (Akihisa et al., 1991; Hartmann & Benveniste, 1987; Moreau et al., 2002). These last ones, more abundant in nature, include phytosterols with 28 or 29 carbon atoms in its structure. 4-dimethyl sterols differ from cholesterol in their lateral chains, presenting a methyl or an extra ethyl group in the C24 position (this kind of alkylation’s is a characteristic of plants), while some other introduce an additional double bond in the lateral chain, as can be observed on Figure 1 (Moreau et al., 2002).

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Fernando Ramos and David Saraiva

According to the number and double bound localizations, 4-dimethy sterols can be classified in Δ5 sterols (double bond between C5-C6), Δ7 sterols (double bond between C7C8) and Δ5,7 sterols (one double bond between C5-C6 and another one between C7-C8), as presented on Figure 1 (Piironen et al., 2000a). In spite of more than 250 phytosterols and related compounds having already been identified in several types of plants and algae, the most representative are β-sitosterol (24α-ethylcolest-5-en-3β-ol), campesterol (24αmethylcolest-5-en-3β-ol) and stigmasterol (24α-ethylcolest-5,22-en-3β -ol) (Figure 1) (Piironen et al., 2000a; Moreau et al., 2002). Saturated plant sterols, without double bounds in their structure, are designated as plant stanols. They are less abundant in nature than plant sterols. Sitostanol (24α-ethylcholest-3β ol) and campestanol (24α-metilcholest-3β-ol) are the most common in higher plants (Hartmann & Benveniste, 1987; Akihisa et al., 1991; Hallikainen, 2001). Plant stanols are currently produced by hydrogenating the plant sterols. However, such chemical modifications enhance manufacturing final product costs. Therefore, modification of plant sterols to plant stanols in plant, due to the activity of the 3-hydroxysteroid oxidase enzyme introduced into the transgenic plants could be more economical (Venkatramesh et al, 2003). 2.2. Natural Sources of Phytosterols Cholesterol can be found in animals mainly in its free form (as an alcohol, with a hydroxyl free group) and esterified by long chain fatty acids in smaller quantities (Ostlund, 2002). Phytosterols are not synthesized by animals, contrary to cholesterol, since they are plant exclusive (Ratnayake & Vavasour, 2004). However, in plants, phytosterols, besides their free form, can be found as conjugated, like esterified to fatty acids, steryl glycosides or acylated steryl glycosides (Wojciechowski, 1991; Soupas, 2006). Corn seeds, rice and other grains also contain esterified phytosterols by hydroxycinnamic, ferrulic or p-cumaric acids (Moreau et al., 2002; Moreau, 2005). Phytosterol are natural components of human diet and their concentrations in the different foods of plant origin are very different. Phytosterols are in significant amounts in seeds, nuts, cereals, fruits and vegetables; however, the richest source is the vegetable oils (Piironen et al., 2000a; Ostlund, 2002). In raw vegetable oils, phytosterol content ranges from 70 to 1600 mg/100g of oil. Rapeseed and corn oils are the richest sources, while olive and the palm oils are the ones that present smaller amount of phytosterols (Piironen et al., 2000a and b). Some special oils, like wheat germ oil, can have amounts of phytosterols up to 4240mg/100g of oil (Schwartz et al., 2008) or corn fiber oil with about 10,000mg/100g of oil (Moreau, 2005; Soupas, 2006). Cereals and derived products, like bread, have a lesser phytosterol content, comparatively to vegetable oils. Nevertheless, cereals and derived products, especially from rye, given its high consumption, are the main phytosterol suppliers in human diet (Valsta et al., 2004).

HO

HO

Gramisterol

5

4,4-Dimethylsterol

4-monomethylsterol

Plant Sterols and Plant Stanols in Milk Products Used As Functional Foods

Cycloartanol

21 20 12

24 17

11 19

13

1 2

22

18 23

25 26

16

9 14

10

8

27 15

7

HO 3 4

5

HO

6

HO

Sitosterol

HO

HO

Stigmasterol 4-desmethylsterols

Campesterol

HO

Δ5,7 desmethylsterols

Sitostanol

Campestanol

Δ7 desmethylsterols

Δ5 desmethylsterols

Cholesterol

HO

HO

Ergosterol

Δ7 - Avenasterol

Figure 1. Representative figure of structures of 4,4-dimethylsterols, 4 –methylsterols and 4-desmethylsterols (the major plant sterols/stanols).

Fruits and vegetables contain, usually, more reduced concentrations than alimentary oils or cereals and derived products. However, their contribution for phytosterols intake is not irrelevant, due to their average daily ingestion (Valsta et al., 2004).

Fernando Ramos and David Saraiva

6

Table I. Phytosterols in cereals and derived products, mg/100 g edible portion (Adapted from Piironen et al., 2000a and b and Normén et al., 2002).

Cereal grains Barley * Corn

*

Oats

*

Total phytosterols 59-83 178 33-52

Rye *

91-110

Wheat *

60-69

Cereal products

*

Cornflour

52

Rice flour

23

Rye flour

86

Wheat flour

28

Corn flakes, normal

26

Musli without sugar added

35

Special K

40

Oat bran

46

Wheat bran

200

Rye bread

51

Wheat bread

54

Wholemeal bread

86

mg/100g of fresh weight.

In the tables I to V, phytosterol content is presented for some foods: cereals and derived products (Table I), fruits (Table II), vegetables (Table III), vegetable oils (Table IV), and nuts and seeds (Table V) (Normén et al., 1999; 2002; 2007; Piironen et al., 2000a and b). Based on the methodology used for its determination, phytosterol concentrations could have slightly variations from the same foods. However and as previously referred, vegetable oils and correspondent by-products always contain high levels of phytosterols, comparatively to the other products of plant origin (Piironen et al., 2000a and b; Valsta et al., 2004; Piironen & Lampi, 2004; Normén et al, 2007). Nonetheless, plant phytosterol content is not constant. Many factors, as genetic, crop conditions or harvest period of the plant, as well as food processing, significantly influence phytosterol concentration in the final product (Piironen et al., 2000a). For instance, vegetable oils processing, depending on the oil type and the carried out operations (neutralization, deodorization, bleaching, deacidifying, steam distillation), can contribute to a decrease from 10 to 70% of the initial phytosterols concentration present in the raw material (Piironen et al., 2000a)

Plant Sterols and Plant Stanols in Milk Products Used As Functional Foods Table II. Phytosterols in fruit, mg/100 g edible portion (Adapted from Normén et al., 1999).

Fruit Apple Banana Clementine Fig Grapefruit Honeydew melon Kiwi Lemon Orange Passion-fruit Peach Pear Pineapple Watermelon

Total phytosterols 13 14 16 22 18 1.8 9.1 18 24 44 15 12 17 1.3

Table III. Phytosterols in vegetables, mg/100 g edible portion (Adapted from Normén et al., 1999).

Vegetables Broccoli Brussels sprouts Carrot Cauliflower Celeriac Celery Chinese cabbage Fennel Kale Leek Mushrooms Olives. green Olives. black Onion Parsnip Pepper. green Potato Radish Sauerkraut Swedish turnip Tomato White cabbage

Total phytosterols 39 43 16 40 20 17 8.5 9.8 8.8 8.1 18 35 50 8.4 27 7.2 3.8 9.0 15 17 4.7 13

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Table IV. Phytosterols in vegetable oils, mg/100 g edible portion (Adapted from Piironen et al., 2000a and Normén et al., 2007).

Vegetable oils Corn oil Crude Corn oil Refined Cottonseed crude Cottonseed refined Olive Extra virgin Olive Pomace Palm Crude Palm refined Rapeseed Crude Rapeseed refined Rice bran Crude Rice bran Refined Soybean Crude Soybean Refined Sunflower Crude Wheat germ Sesame seed Linseed Oat Peanut Walnut

Total phytosterols 809-1557 715-952 431-539 327-397 144-154 261-282 71-117 39-61 513-979 250-773 3225 1055 229-459 221-328 374-725 967 472 471 534 251-315 193

Table V. Phytosterols in nuts and seeds, mg/100 g edible portion (Adapted from Piironen et al., 2000a and Normén et al., 2007).

Nuts and seeds

Total phytosterols

Almonds Brazil nuts Cashew nuts Coconut rasps Hazelnuts Linseeds Peanuts Pistachio nuts Pumpkin seeds Sesame seed Sunflower seeds Walnuts

143-208 131 151-158 68 138 213 116-220 297 94 404 322 128

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2.3. Estimated Average Intakes of Phytosterols An average human daily intake of plant sterols is estimated between 150-400mg (3-6 mg/kg body weight), 65% corresponding to β-sitosterol, 30% to campesterol and 5% to stigmasterol (de Vries et al., 1997; Ostlund, 2002; Trautwein et al., 2003). As can be certified above, the average intake of phytosterols depends on the food type: some vegetarians can have almost an intake of 1 g/day of plant sterols, whereas others may consume even less than the non-vegetarian population (Piironen et al., 2000a). Regarding plant stanols, the average daily intake corresponds approximately to 10% of the respective plant sterols ingested, due to them being the least abundant in nature (Ostlund, 2002).

2.4. Prevention of Cardiovascular Diseases Phytosterols are known to have various bioactive properties, which may have an impact on human health, and as such boosted interest in phytosterols in the past decade. The most important benefit is their blood cholesterol-lowering effect. In fact, phytosterols hypocholesterolemic activity was known since 1950, firstly by a research with chickens (Peterson, 1951) but later observed in humans by Pollak (1953). The link between phytosterols and cholesterol lowering effect was thus established and was confirmed later on by several human studies which showed their beneficial effect on total and LDL-C concentrations. Katan et al. (2003), as well as AbuMweis and collaborators (2008) are meta-analyses which combined outcomes from dozens of clinical trials that clearly show the hypocholesterolemic effect of phytosterols. Due to their hypocholesterolemic properties, phytosterols are believed to contribute to reduction of cardiovascular disease risk (CVD). Katan et al (2003) show an about 10 % LDL-C decrease for a 2 g/day dose of phytosterols (both plant sterols and/or stanols) that could be positively estimated as an equal percentage of CVD risk reduction. Also, as referred by Trautwein and Demonty (2007), over than 30 studies have investigated the effect of phytosterols on experimental atherosclerosis models in different animals. A prevention/regression of atherosclerotic plaque development was proven, clearly suggesting a beneficial impact on CVD risk (Moghadasian et al. 1997, 1999; Volger et al., 2001, Ntanios et al 2003, Plat et al 2006,). In addition, Awad and co-workers (2001b), on an in vitro study, have shown that phytosterols may prevent vascular smooth muscle cells hyperproliferation, which could play a beneficial role against atherosclerosis development, too. Besides these findings, a protection against LDL-oxidation was observed by Homma and co-workers (2003) and could also contribute to the anti-atherosclerotic properties attributed to phytosterols. Several international guidelines recommend the consumption of 2g/day phytosterols to lower LDL-C blood levels (NCEP ATPII, 2001; IAS, 2003; JBS, 2005; NHF, 2007; EFSA 2008a and b). Indeed, phytosterols daily intake equivalent to 2 g in an appropriate food could reduce LDL-C blood levels between 5 to 15% (Berger et al., 2004) with an average of 10%

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quoted in most relevant reported studies (Katan et al. 2003, Normen et al., 2004, AbuMweis et al., 2008). 2.4.1. Mechanisms of Cholesterolemia Reduction The main physiologic response to phytosterols intake is the reduction of intestinal absorption of both cholesterol from the diet and endogenously produced cholesterol (Law, 2000; Moreau et al., 2002; Ostlund et al., 2002). The mechanism by which plant sterols/stanols reduce cholesterol absorption is not completely elucidated, but some hypotheses were proposed. Most usually admitted are briefly described below (Trautwein et al, 2003; Rozner & Garti, 2006). 2.4.1.1. Competition between Cholesterol and Phytosterols for Mixed Micelles Solubilization Cholesterol, a lypophilic molecule, needs to be solubilized inside dietary mixed micelles (DMM), before reaching the absorption sites, in order to be absorbed into the blood stream. DMM are formed by bile acid salts, monoacylglycerols, free fatty acids, lysophospholipids, phospholipids and free cholesterol (Trautwein et al., 2003; Rozner & Garti, 2006). DMM, as any amphiphilic aggregate, have a limited capacity for the solubilization of hydrophobic molecules. So, phytosterols from diet give rise to a competition between these and cholesterol for solubilization in DMM. Furthemore, in vitro and in vivo studies suggest that phytosterols affinity for the micelles is higher, moving the cholesterol, or even substituting it in the mixed micelles, which could explain the decrease of cholesterol absorption (Ikeda & Sugano, 1983; Mel´nikov et al., 2003b; Trautwein et al., 2003; Rozner & Garti, 2006). 2.4.1.2. Phytosterols and Cholesterol Co-crystallization Phytosterols and cholesterol co-crystallization in the gastrointestinal tract, resulting in mixtures of crystals of difficult solubilization, could be another mechanism for lowering cholesterol intestinal absorption as pointed out by some authors (Christiansen et al., 2001b; Christiansen et al., 2003; Trautwein et al., 2003; Rozner & Garti, 2006). Cholesterol, like phytosterols’ free forms have little solubility in oil (3g/100ml at 37ºC in presence of water) and are practically insoluble in water (approximately 0.2mg/100ml) (Trautwein et al., 2003). Already in the 1950’s, Davis (1955) mentioned that cholesterol and β-sitosterol made a new crystal form when precipitated in methanol. In that sense, this was a mechanism which was believed to contribute to the reduction in cholesterol absorption, since the solubility of the new crystal is considerably inferior to that of the cholesterol itself (Davis, 1955). However, recently Mel´nikov et al. (2003a and b) have concluded that it is unlikely that mixed crystals formation could significantly affect in vivo cholesterol intestinal absorption, due to the high solubility of cholesterol, phytosterols in fat lipolysis products. 2.4.1.3. Reducing Cholesterol Absorption via Competition with Cholesterol Transporters Cholesterol intestinal absorption is regulated by transporters, which are located in the intestinal brush-border membrane (Kramer et al., 2000; Trautwein et al, 2003). A specific class of transporters for sterols is the ABC transporters - adenosine triphosphate (ATP)

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binding cassette, like ABCG5, ABCG8 and ABCA1, membrane integral proteins involved in cholesterol efflux from intestinal cells to the intestinal lumen, using ATP as an energy source (Trautwein et al, 2003; Rozner &Garti, 2006). These transporters, mainly ABC1 transporters, don't distinguish between phytosterols and cholesterol. Thus they are not selective. As such, phytosterols stimulation can promote sterol efflux, including cholesterol, to the intestinal lumen (Plat & Mensink, 2002; Rozner & Garti, 2006). Additionally, phytosterols can stimulate ABC-transporters in intestinal cells (particularly ABC1), which results in a cholesterol secretion increase from enterocytes to the intestinal lumen (Plat & Mensink, 2002; Trautwein et al, 2003; Rozner & Garti, 2006). Recently it has been recognized that other transporters also participate in the sterols absorption process. An example is the Nieman Pick C1 L1 (NPC1L1) transport systems that perform a fundamental role in the regulation of cholesterol influx to the enterocytes. However, these transport systems are unable to distinguish cholesterol from phytosterols, and both compete for the transporters. As such, an increment in intestinal phytosterols results in an enterocyte cholesterol reduction and, consequently, in a blood stream cholesterol decrease (von Bergmann et al., 2005; Gylling & Miettinen, 2005). In short, phytosterols could interfere in cholesterol membrane transporters activity, since these are not selective, resulting either in a cholesterol influx decrease to the enterocytes or in a cholesterol efflux increment to the intestinal lumen (Trautwein et al, 2003; Rozner & Garti, 2006). 2.4.1.4. Inhibition of Enzymes Involved in Phytosterols Absorption Process Cholesterol absorption can be divided in two steps, one corresponding to hepatocytes cholesterol ingress and the other to cholesterol passage from hepatocytes to the blood stream. Inhibiting lipases and esterases that promote cholesterol esters hydrolysis in the first step, and acyl-coenzym A cholesterol acyltransferase (ACAT) that participate in second step, a decrease in cholesterol absorption will be the outcome. However, phytosterols action seems to be markedly with this last enzyme (Trautwein et al, 2003; Rozner & Garti, 2006). So, another proposed mechanism to explain cholesterol reduction by phytosterols, is the possible esterification cholesterol rate diminution inside enterocytes by ACAT inhibition (Chen, 2001; de Jong et al., 2003; Trautwein et al., 2003; Rozner & Garti, 2006). This enzyme reduces intracellular free cholesterol concentration, transforming it in cholesterol ester. Phytosterols can suppress ACAT activity and reduce cholesterol absorption. Thus, approximately 80% of chylomicrons (QM) incorporated cholesterol is in the esterified form. Inhibition of this enzyme substantially reduces cholesterol incorporation in QM. Consequently, since cholesterol must be incorporated in QM before being transported by lymph, a decrease of cholesterol in the bloodstream is the final result (Ikeda et al., 1988; Dawson & Rudel, 1999).

2.5. Hypocholesterolemic Comparison between Plant Sterols and Stanols Whether plant sterols or stanols have a larger hypocholesterolemic effect is subject of discussion since the first studies on this topic (Sugano et al., 1977; Ikeda et al., 1981). Notwithstanding, O'Neill and co-workers (2004 and 2005), have suggested that the

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cholesterol-lowering efficacy of plant sterols diminished over time (comparing plasma cholesterol levels after 1 and 2 months) while plant stanols maintained their cholesterollowering efficacy (O'Neill et al., 2004, 2005). As a possible reason for this observation it is speculated that the increase in plasma plant sterol levels suppressed bile acid synthesis. Based on that, a reduction of cholesterol elimination under biliary acid forms will not be done and correspondent reduction of total- and LDL-cholesterol will not be, of course, so effective (O'Neill et al., 2004). However not only the paper of O'Neill and co-workers (2004) presents some flaws, because there were no significant differences in lowering total (3-7%) and LDLcholesterol (4-8%) seen between the 3 treatments (1.6g/d plant sterols, 1.6 g/d plant stanols or 2.6 g/d plant stanols) (O'Neill et al., 2004), but also a recent study (de Jong et al., 2008) has shown that markers of bile acid synthesis are unchanged with plant sterol consumption and this is not different from what is observed with stanols In fact, scientific evidence about efficacy of plant sterols and stanols has proven that it was similar. Not only Law (2000) but also the most quoted meta-analysis of Katan and coworkerrs (2003) have shown that the LDL-C lowering effect of plant sterols and stanols is the same. Katan and co-workers (2003) have calculated the effects for sterols and stanols separately, and they showed that the mean reduction in LDL-cholesterol was 10.1% (95% CI 8.9-11.3%) in 27 trials testing plant stanols (mean dose 2.5 g/d) and 9.7% (95% CI 8.510.8%) in 21 trials testing plant sterols (mean dose 2.3 g/d). It is concluded that the difference was not significant and that these trials cannot support a claim that either is better than the other (Katan et al., 2003). Moreover, human studies comparing side-by-side plant sterol- and stanol-enriched foods have further demonstrated the absence of a difference in efficacy between plant sterols and stanols (Westrate & Meijer, 1998; Hallikainen et al., 2000; Jones et al., 2000; Noakes et al., 2002). In the same way, regarding the capacity of plant sterols and stanols for cholesterol reduction in their free or esterified forms, it was demonstrated that both compounds have identical efficiency, both in free and esterified forms (Jones et al., 1999, Christiansen et al., 2001a and b, Nestel et al., 2001, Moreau et al., 2002). Overall, the different results between plant sterols and stanols for cholesterol reduction, isn ‘t because one is more efficiency than other, but because the influences of others factors, like food carrier, frequency and time of intake, as well as subjects baseline characteristics on cholesterol lowering action of plant sterols and stanols (AbuMweis et al., 2008).

2.6. Phytosterols Safety Use The actual opinion about phytosterols use is that they are safe when added to human diet as they are part of natural foods (von Bonsdorff-Nikander, 2005). For more than half a century phytosterols have been used for cholesterol plasmatic reduction levels and, until now, no marked adverse effect was observed (Ling & Jones, 1995; Baker et al., 1999; WaalkensBerendsen et al., 1999; Weststrate et al., 1999; Ayesh et al., 1999; Hepburn et al., 1999; Wolfreys & Hepburn, 2002; Katan et al., 2003; Berger et al., 2004; Kritchevsky, 2004; Gylling & Miettinen, 2005; Kritchevsky & Chen, 2005; Plat & Mensink, 2005; Salo et al.

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2005; Gonçalves et al., 2007). Most of the available safety alimentary information of phytosterols was about the respective esterified forms. Information concerning phytosterols in the free forms is scarce. Since phytosterols are also used in esterified forms, released free phytosterols in the gut due to early hydrolysis of the esters, it becomes relevant that safety information of phytosterols in esterified forms can also be considered for the free forms (SCF, 2003b; Fahy et al., 2004). It was demonstrated by a study by Delaney and co-workers (2004) that safety profiles for both esterified and free phytosterols forms are similar. In a recent review by Patel & Thompson, (2006), is mentioned that “the possibility that phytosterols are a CAD risk factor is speculative”. In fact, there is no consistent evidence for a relationship between elevated plasma plant sterol concentrations and increased CVD risk. Moreover, recent epidemiological studies have shown no such relationship (Wilund et al, 2004; Pinedo et al, 2007, Fassbender et al, 2008, Windler et al, 2009; Silbernagel et al, 2009). In fact, elevated plasma plant sterols are a marker of cholesterol absorption, and increased cholesterol absorption has been shown to be related to increased CVD risk. Therefore, increase plasma plant sterol concentrations would be more a marker than a causative factor. The findings from the LURIC study are in agreement with previous studies demonstrating that high absorption and low synthesis of cholesterol is associated with CHD. Therefore, a positive correlation of plasma plant sterols with CHD risk may be due to the atherogenic effects of increased intestinal cholesterol absorption (Silbernagel et al, 2009). Nevertheless, in humans that suffer of sitosterolemia the risk is greater (Patel & Thompson, 2006). Sitosterolemia, also called phytosterolemia, is a very rare autosomal recessive disorder (1 in 5 million people) in which plasmatic phytosterols, particularly sitosterol, concentrations are extremely higher (>30-fold) (Kwiterovich et al., 2003). This hereditary pathology is marked by an increase of sitosterol absorption accompanied by a decrease in the respective elimination rate, which probably happens due to inhibition of CYP7A and hepatic sterol 27-hydroxylase, the rate-limiting enzymes in bile acid metabolism (Lütjohann et al., 1996, Salen et al., 2002; Patel & Thompson, 2006). In heterozygous sitosterolemic individuals, the plant sterols do not increase pathologically when plant sterol rich foods are consumed; which is different from what is observed in homozygous subjects, in which plant sterol consumption is contra-indicated. Kwiterovich et al, 2003 have shown that obligate heterozygote relatives of patients with sitosterolemia and controls without critical mutations in ABCG5 and ABCG8 responded similarly to a diet enriched in plant sterols. Specifically, plasma cholesterol concentrations were lowered to a similar degree and the increase in plasma sitosterol and campesterol concentrations was of a similar magnitude in heterozygous sitosterolemics as seen in other studies with normal or modestly hypercholesterolemic subjects. Similar findings have been reported from other studies demonstrating that in heterozygotes plasma plant sterol concentrations are in a similar range as those of the general population, even after consumption of plant sterol enriched foods (Stalenhoef et al, 2001; Kratz et al, 2007). One of the last steps, but fundamental, in phytosterols safety evaluation was the definition of a dose for which no adverse effects are observed (NOAEL). Hepburn and coauthors (1999) have concluded for a NOAEL of 4.1g/Kg body weight. It means, when extrapolated to a 60kg individual, a daily intake of 246g of phytosterols, which is over and over above to the recommended 2g of phytosterols daily intake for lowering LDL-C.

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Finally, supplementary phytosterols safety information collected during, at least, 5 years in Finland and 2 years in the United States of America (USA), didn't shown any evidence of severe adverse effects (Katan et al., 2003). Lea and Hepburn (2006), in an innovative study, alert for the need of adverse effects monitoring of phytosterols enriched foods, through post-market surveillance programs. They showed however that there was no evidence of occurrence of unexpected or adverse health effects with long term use of plant sterols. The absence of severe adverse effects has allowed the commercialization of foods enriched with phytosterols since the beginning of the 1990’s. Safety guidelines fulfilment, European Food Safety Authority (EFSA) favourable opinion from its scientific committee of foods (SCF) and the classification of GRAS (generally recognized as safe) by USA FDA (Food and Drug Administration), clearly certificate their safety. However, given the importance of phytosterols market development, by constant introduction of new enriched foods and the consequent increase of consumer numbers, probabilities of occurrence of rare adverse effects have increased. So, implementation and reinforcement of epidemiological surveillance must be done.

3. Milk and other Dairy Products Enriched with Phytosterols Beneficial effects on blood cholesterol reduction by phytosterols enriched foods have lead to development of this kind of food industry (Lagarda et al., 2006). Foods that provide benefits for health, besides the basic nutrition, are classified as functional foods, in accordance with scientific concepts consensually accepted in Europe, as well as in other parts of the world (Roberfroid, 1999 and 2000 Jones & Jew, 2007; Sibbel, 2007). Evolution of concepts surrounding plant sterols in relation to disease prevention are one example of the positive aspects of functional foods which have contributed to the wellness and to the quality of life improvement of populations (Katan et al., 2003, AbuMweis et al., 2008; Trautwein & Demonty, 2007) . According to the meta-analysis of Katan et al (2003), phytosterols enriched foods constitute a type of functional food that with a 2g/d dose, lowers LDL-C by about 10%. As above mentioned, in a Western diet, the daily consumption of phytosterols from natural sources was estimated in a range of 150-400 mg (Ostlund, 2002; Trautwein et al., 2003; Rodrigues et al., 2007; EFSA, 2008a). So, it is obvious that for ingesting about 2g of phytosterols only from "conventional foods", the amount of food to be consumed on a daily basis would be quite considerable (i.e., broccolis – 4.8kg; nuts – 1.5kg; sesame seeds – 500g) (Phillips et al., 2005; Lecerf, 2007). Consequently, since most individuals do not consume daily these referred exorbitant amounts of foods, phytosterols incorporation in foods, like dairy products, seems to be an appropriate answer.

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3.1. Legislation In the USA, Food and Drug Administration (FDA) has granted statute (GRAS) to phytosterols and phytosterols esters for several alimentary applications, which means an implicit health claim for those ingredients. In the European Union (EU), however, the regulatory process for phytosterol-enriched foods has limited the number of food types available. As an example, from a total of 53 applications made between May of 1997 and May of 2004, only 14 new foods were approved for commercialization (EC, 2008b). It is therefore important to include a short synopsis about European legislation on this subject and, more specifically, on phytosterols enriched products. The EC/258/97 regulation is the key document for novel foods in the European Union (EC, 1997). Nevertheless, plant stanols enriched foods do not need a novel food authorization in view of the fact that they were already used as food in the EU before the introduction of this legislation (EC, 1997; EFSA, 2008a and b). Contrarily, plant sterols need a novel food authorization, since it was only after July 2000, that they received, by the EU decision No 258/97, the approval as novel foods, after which they were first commercialized in EU as plant sterol-enriched spreads. So the introduction of a novel food or ingredient in the EU market requires a specific authorization involving a safety evaluation procedure. Previous decisions of authorization or prohibition of market introduction were made by European Commission, based on opinion of member state experts. If necessary, European Food Safety Authority could be called to participate in the process, supplying additional scientific information (EFSA, 2003 and 2005). Table VI. Specifications of phytosterols for the addition into milk products (Adapted from European Commission decisions 2004/333/EC, 2004/334/EC, 2004/335/EC, 2004/336/EC and 2004/845/EC). Phytosterols ß-sitosterol ß-sitostanol Campesterol Campestanol Stigmasterol Brassicasterol Other sterols/stanols Total sterols/stanols

Percentage (%) < 80 < 15 < 40 <5 < 30 <3 <3 99

Still novel foods or ingredients could follow a simplified procedure, only requiring a simple notification by the respective company. For this, they must be considered, in at least one member state as "substantially equivalent" (composition, nutritional value, metabolism, fixed use and level of undesirable substances) to pre-existent foods or ingredients (EFSA, 2003; 2005). In 2004, for the specific case of phytosterol and phytosterol esters enriched dairy products European Commission adopted the 608/2004/EC Decision (EC, 2004f). Accordingly, several enriched phytosterols dairy products can be introduced on the European market, namely middle- and low-fat dairy products (with or without added fruits and/or

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cereals) and fermented dairy products, like yogurts and cheese type products, with fat content < 12g per 100g) and in which milk fat or proteins have been partial or totally substituted by fat and/or plant proteins. Additional EU regulations 2004/333/EC, 2004/334/EC, 2004/335/EC, 2004/336/EC and 2004/845/EC (EC 2004 a,b,c,d and e) specify the plant sterols and stanol profiles addition on milk products, and are briefly presented on the Table VI. Furthermore, these regulations define that plant sterols and stanols extracted from sources other than vegetable oil proper for foods, should be free of contaminants, with purity higher than 99% of phytosterols (EC, 2004 a,b,c and d). In the cases of Australia and New Zealand, the regulation of phytosterols enriched foods is under the responsibility of a common organisation, Food Standards Australia New Zealand (FSANZ). This organisation rules the incorporation of the referred compounds in foods according its origin. Hence vegetable oils derived phytosterol esters can be incorporated in milk if the following conditions are observed (FSANZ, 2006): • • •

milk present no more than 1.5g of total fat per 100g; milk is commercialised in a maximum of 1L packing volume; phytosterols are present in a concentration between 3.2 and 4.0g/L of milk .

They can also be incorporate in yogurt if the following conditions are observed (FSANZ, 2006): • with a concentration that do not exceed 1.5g/100g; • the commercialization packing does not surpass a 200g capacity; • the total amount of added phytosterol ester is in the range of 1.3-1.6g.

3.2. Market of Phytosterols Enriched Foods 3.2.1. Authorized Foods Since the launching of a phytostanol esters enriched margarine in 1965, in Finland, the global market of phytosterols enriched foods has been quickly expanded and diversified (Salo et al., 2005). In fact, food type matrices are gradually developing. It is observed a gradual substitution of high fat content foods for healthier alternatives, with a special attention to foods that could be easier incorporated in the daily diet. Spreads and fermented milk type products, like yoghurt and yoghurt drinks, were most commonly available (EFSA, 2008a). Phytosterol enriched products that are or could be marketed outside the EU include juices, ice creams, snack bars, white or whole-grain breads and buns, cereals, confectionery products and cooking oils. In addition, GRAS status was recently given to phytosterol esters for use as an ingredient in ground roasted coffee (FDA, 2005), to phytosterols and phytosterol esters for use in pasta, noodles, soups, and puddings (FDA, 2006a), and to phytosterols for use in different egg products (FDA, 2006b). Tables VII and VIII, displays a list of phytosterol enriched foods and ingredients that were authorized for commercialization in Europe and in the USA.

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Currently, dairy products are the area that presents the higher growth in this kind food industry/market. The first enriched dairy food, a yogurt enriched with phytostanol esters, was introduced in United Kingdom, in 1999 (Salo et al., 2002). Dairy products with low fat content are a suitable way to obtain phytosterols recommended daily dose, because they can be considered as healthy foods and are easily included in the diet. The possibility to produce a single dose that contains the optimal phytosterol daily amounts is another advantage of this type of foods, since it facilitates the calculation of the estimated daily intake estimation (Salo et al., 2002 and 2005). Table VII. Phytosterol-enriched novel foods available on EU marketup to 2008 (Adapted from EC, 2008b). Food type Yellow fat spreads Milk-based fruit drinks Milk-type products Fermented milk-type products Cheese-type products Yoghurt-type products Spicy sauces (including ketchup, mustard, marinades, dips, etc.) Salad dressings (including mayonnaise) Soya drinks Milk-based beverages Bakery products (rye bread) Oil Rice drinks

Status EC, 2000 EC, 2004d EC, 2004a,b and c EC, 2004a EC, 2004a and d EC, 2004b,c and d EC, 2004b EC, 2004a EC, 2004a EC, 2004e EC, 2006a and b EC, 2007 EC, 2008a

3.2.2. Market Characterization In these days, the functional foods market is very small. In 2005, the European phytosterols market was evaluated in 145million euros and could rise up to 311million euros in 2012 ($US 184.6 million in 2005 to $US 395.2 million by 2012) (Frost & Sullivan, 2006b; Jones & Jew, 2007). According to EFSA, it was commercialized in Europe, during 2007, approximately 9,500 tons of phytosterols, what corresponds to 160 million euros. Of these, 80% were used in food products (EFSA, 2008a). Margarines are the most common products in the market, corresponding to 75% of phytosterol enriched foods used in 2005 (EFSA, 2008a). Although plant stanols enriched products do not require previous approval as novel food, they just correspond to 1/3 of the market, while plant sterols enriched products represent the remaining 2/3 (EFSA, 2008a). In the United Kingdom, consumer inquiries demonstrated that phytosterols enriched products constitute a maximum of 12% of the total market of products to spread on, 15% of the market of yogurts and yogurt drinks, and 6% of the milk total market (EFSA, 2008a).

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American current market of phytosterols enriched foods is, also, slightly developed ($US 103.9 million or 81.9 million euros) in 2005 and estimates to rise to $US 196.7 million (155 million euros) in 2012. This fact could be justified by the American consumer's typical behaviour that associates the maintenance of a healthy life style more to the consumption of dietary supplements rather than of functional foods (Frost & Sullivan, 2006a) Table VIII. Phytosterol-enriched foods and GRAS status in U.S.A up to 2008 (Adapted from http://www.cfsan.fda.gov/~rdb/opa-gras.html). Food type Vegetable oil spread Vegetable oil spreads, dressings for salads, bars, and yogurt. Vegetable oil, baking, frying, and salad dressings Vegetable oil spreads, dressings for salad, health drinks, health bars, and yogurt-type products Margarine and vegetable-based spreads (margarine-like); yogurt and yogurtlike products; milk-based juice beverages; ice cream and non-standardized ice cream products; cream cheese and cream cheese-like products; snack bars (health bars); salad dressing, mayonnaise, French dressing, and dressings for salads; and white breads, white rolls and buns, and comparable nonstandardized white bread products Ground roasted coffee Margarines and vegetable oil spreads, dressings for salads, beverages, snack bars, dairy analogs (including soy milk, ice cream and cream substitutes), cheese and cream, baked foods, ready-to-eat breakfast cereals, mayonnaise, pasta and noodles, sauces, salty snacks, processed soups, puddings, yogurt, confections, vegetarian meat analogs, fruit/vegetable juices, edible vegetable oils, including diacylglycerol oil. Egg products, including egg whites and egg substitutes Baked goods and baking mixes; fats and oils; frozen dairy desserts and mixes; gelatins, puddings, and fillings; grain products and pastas; gravies and sauces; hard candy; milk; milk products; soft candy; soups and soup mixes; and snack foods

Status FDA, 2000a FDA, 2000b FDA, 2000c FDA, 2001

FDA, 2003

FDA, 2005

FDA, 2006a

FDA, 2006b FDA, 2006c

3.3. Labelling According to EFSA Scientific Committee on Food (SCF) and many independent studies, it is recommended an intake of not more than 3g/d, mainly based on the fact that with intakes higher than 2.5-3.0g/d, little additional benefit in LDL-C lowering is observed. (Ling & Jones, 1995; Katan et al 2003; Berger et al., 2004; Kritchevsky, 2004; Gylling & Miettinen, 2005; Kritchevsky & Chen, 2005; Plat & Mensink, 2005; Salo et al. 2005; Trautwein & Demonty, 2007).

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Following the SCF recommendations, the European Commission adopted specific regulations for the labelling of phytosterols and respective esters enriched foods and ingredients (EC, 2004f). Label must contain: 1. a statement "with plant sterol/stanol addition" on the same vision field where the commercial brand is displayed; 2. the amount of added phytosterols or respective esters (expressed in % or g of free plant sterols/stanols per 100 g or 100 ml of the foodstuff); 3. the information that the product is exclusively for people that intend to reduce blood cholesterol levels; 4. the information that patients under reducing blood cholesterol medication, should only consume the product under medical surveillance; 5. an easily visible and readable warning that the product, from the nutritious point of view, may not be appropriated for pregnant or suckle women, and children under five years old; 6. the advice that consumption should be integrated in a balanced and varied diet, including fruits and horticultural products in order to maintain carotenoids blood levels; 7. on the same vision field that displays the information pointed out on 3., should be present the indication that a daily consumption higher than 3g of plant sterols/stanols must be avoided; 8. a dose definition of the food or ingredient (preferably in g or ml) with phytosterols amount contained in each dose. Additionally, when phytosterols enriched foods are authorized, special rules should be applied in these foods presentation, namely (EFSA, 2008a): • • •

the easiness to be divided in portions (each one containing a maximum of 1g (3 portions/day) or a maximum of 3g of phytosterols (1 portion/day); drink packages should not contain more than 3g of phytosterols; the obligation of packing certain foods in single portions.

3.4. Intake Recommendations Milk and dairy products daily consumption and the respective phytosterols suggested intakes by manufacturers are shown on Table IX. It is clear that manufacturers follow the SCF recommended phytosterols daily intake of 1.5-3.0g for an individual adult of medium characteristics (SCF, 2002), as well as NCEP ATPIII (2001) and IAS (2003) which both recommend 2 g/day to lower elevated LDL-C concentrations. For some products as milk (1000ml) and cheeses, each portion supplies about a third of the phytosterols recommended daily intake. So, the manufacturers suggest that this specific product should be consumed 3 times a day or, alternatively, that other phytosterols enriched foods should be additionally consumed.

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Fermented milk derived products, as yogurts, are usually the most available enriched dairy food, as displayed by Table X, with a list of the available European market dairy products at the end of 2007. Table IX. Phytosterol concentrations in some dairy products and the recommended daily consumption level (Adapted from EFSA, 2008a). Product category Milk type products Yoghurt type products Cheese type products Cream cheese Milk-based soft drink

Common packing size 1000ml 65-125ml 125g 200g 1000ml

Recommended consumption 2-3x250ml 1-2x65-125ml 3x30g 40-60g 350ml

Phytosterol concentration 0.3 g/100ml 0.6-3.1 g/100ml 2.2 g/100g 5.0 g/100g 0.5 g/100ml

Daily intake 1.5-2.4g 1.5-2.0g 2.1g 2-3g 1.8g

Table X. Phytosterol-enriched dairy products available in EU at the end of 2007 (Adapted from EFSA, 2008a). Member State Austria Belgium Bulgaria Cyprus Czech Republic Denmark Estonia Finland France Germany Greece Hungary Ireland Italy Latvia Lithuania Luxembourg Malta Netherlands Poland Portugal Romania Slovakia Slovenia Spain Sweden United Kingdom

Milk products Yoghurt drink Yoghurt, yoghurt drink, milk, cheese spread Not known Milk Yoghurt, yoghurt drink, milk Yoghurt, yoghurt drink, milk Not known Milk, buttermilk, yoghurt, yoghurt drink Yoghurt, yoghurt drink, milk Yoghurt drink, milk, cheese Yoghurt, cheese spread Not known Yoghurt, yoghurt drink, cheese spread Yoghurt drink, milk, cheese spread Not known Yoghurt drink Cheese spread, yoghurt, yoghurt drink Yoghurt drink Yoghurt, yoghurt drink Yoghurt drink Yoghurt, yoghurt drink, milk Not known Not known Yoghurt drink Yoghurt, yoghurt drink, milk Milk Yoghurt, yoghurt drink, milk, cheese

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3.5. Technological Aspects Initially, phytosterols were administered in the form of powder. However, great amounts were needed to obtain an effective reduction in cholesterol blood levels. This was due to phytosterols solubility, namely their water insolubility and weak solubility in fats and oils that hinder its administration. These physical properties limited its applicability in foods and in several pharmaceutical oral preparations. They were therefore considered, for a long time, to have little practical health interest (Wester, 1999; Salo & Wester, 2005) The incorporation of phytosterols in food to become easily available for the consumer turned out to be an attention-grabbing challenge. This objective involves the production of phytosterols with higher solubility in the intestinal tract, thus allowing a more efficient cholesterol blood reduction (Rozner & Garti, 2006). Until now, commercialised plant sterols and stanols have been obtained from by-products of vegetable oil refinement and wood industrial processing (SCF, 2000; 2003a and b; Salo et al., 2002; Moreau, 2004). Vegetable oil phytosterols are usually isolated from vegetable oils, such as soy, corn, and rapeseed oils, as well as from palm, cottonseed and peanut oils, during refinement deodorising step (SCF, 2000; 2003a and b; Salo et al., 2002; Moreau, 2004), that results in a distillate with circa 15–30% of phytosterols (Moreau, 2004). The sterol fraction is then purified through solvents by a crystallization process (Salo et al., 2002; SCF, 2003a). In wood processing industry, phytosterols are extracted from conifer trees. Wood extracts are placed in an alkaline aqueous solution and phytosterols are then recovered as saponifiable fraction, esterified phytosterols by fat and resinic acids and as many other neutral substances. Phytosterols are then separated by liquid extraction in the case of acid esters or crystallization, in the case of neutral substances. Saponifiable fraction is, subsequently processed by a series of distillations. Phytosterols can also be recovered from the tall oil pitch (tall oil sterols), which is the initial distillation residue. Plant sterols extracted from this oil pitch and are, afterwards, crystallized (Salo et al., 2002; SCF, 2003a and b). Phytosterols concentration in tall oil soap is 3-4% and in tall oil pitches about 10% (Salo et al., 2002). The mixture composition between alimentary oils derived or wood derived plant sterols and stanols are different. For instance, the relative concentration of sitosterol, campesterol, stigmasterol and sitostanol is approximately 72%, 8.2%, 0.3%, and 15.3% in derived wood processing products, and 45%, 26.8%, 19.3% and 2.1% in derived vegetable oils products, respectively (Salo et al., 2002). Purified phytosterols make stable crystals and hold limited water and oil solubility. At room temperature, free form phytosterols solubility has been described as 0.01% in water (Engel & Knorr, 2004) and 3.5-4.0% (w/w) in oil (Christiansen et al., 2002). At the same temperature, phytostanols solubility in oils and fats is inferior to 1% (w/w) (Wester, 2000). Due to this low solubility, phytosterols free form can take several days to dissolve in biliary salt solutions (Armstrong & Carey, 1987; Ostlund et al., 1999). Thus, an option is to promote a formulation that grants them a higher solubility in the emulsified lipidic part of the alimentary digestion, as a way to improve an effective cholesterol blood reduction.

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3.5.1. Phytosterols Formulations 3.5.1.1. Esterified Phytosterols Formulations The first alimentary products successfully used were fatty acids plant stanols esters, in a strategy only later applied to plant sterols (Wester, 2000, Moreau et al., 2002). Phytosterols esterification by fatty acids increases its oil solubility in about 10 times -oil solubility of 2030% (Jandacek et al., 1977; Mattson et al., 1982). Consequently, phytosterols incorporation in alimentary oils or fats (i.e. margarine, butter, and fat spreads function the phytosterol esters carriers) is the actual formulations state of the art (Wester, 2000; Trautwein et al., 2003). The required dose of crystalline phytosterol to attain a 10% cholesterol reduction is 1020g/day, while for esterified phytosterols a similar effect could be attained with only 2-3 g/day. Besides, as esterified phytosterols are fat soluble, they could be evenly distributed in the lipidic phase of the food mass (Salo et al., 2002). Esterification is carried out through alimentary fatty acids esterification of the phytosterols free forms. Chemical reaction is made at high temperatures under moderate vacuum, using a catalytic agent (SCF, 2003c; Salo et al., 2005). When this reaction is complete, a wash procedure is performed to inactivate the catalytic agent by lixiviation and deodorization procedures as the final steps, just as in the case of alimentary oils (Salo et al., 2005). Also noteworthy, is the possibility of adjustment of the phytosterols esters physical properties according to fatty acids composition. Depending on the alimentary matrix to be enriched, esters can be adjusted for liquid or solid state at room temperature, and, besides, they can, also, be modified in order to achieve more nutritionally beneficial effects than the fat that they substitute (Wester, 2000; Salo et al., 2005). Recently, a new approach on the fatty acids esters involves the use of esterified plant sterols with oil fish enriched with long chain poly-unsaturated fatty acids (n-3) (Ewart et al., 2001) and have been shown to be effective in humans (Demonty et al., 2006). Phytosterols fatty acid esters composition can vary without decreasing its cholesterol blood reduction capacity. This fact was instantly observed in the earliest studies, when esterified phytosterols with small, medium or long chain fatty acids have presented similar effectiveness (Mattson et al., 1977). 3.5.1.2. Free Phytosterols Formulations Until now, and besides esterification, several formulations have been developed to reduce technological limitations of free phytosterols and to increase the possibility of incorporation in food. To achieve the best possible formulation has been a permanent challenge, especially since the observation that phytosterols effectiveness was dependent on its physical properties, on its alimentary matrix solubility, and on the food fat content in which they are incorporated. A new formulation approach to increase the efficiency of phytosterols involves the use of a microcrystalline suspension, which is prepared by heating a mixture of oils and phytosterols at 100-110ºC, followed by a cooling at 90ºC. At this temperature, it is added water and the resulting suspension is then agitated until it reaches room temperature. Using this crystallization method, more than 30% of phytosterols can be added to fats and oils without

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any food additive, because it acts as an emulsification agent. In the final suspension, phytosterols appear in both dissolved and crystalline forms (Christiansen et al., 2001a; 2002). Other formulations include, for instance, combinations of phytosterols and mineral salts, by which an air jet micronizer forms with less than 20μm size (SCF, 2003c). Phytosterols free form bioavailability can also be increased through its incorporation in liposomes (Engel & Knorr, 2004). The use of mesophase-stabilized compositions for free phytosterols incorporation in food products, as well as phytosterol-protein complex, were patented by Kraft Foods, Inc. and Monsanto Co., respectively (Corliss et al., 2000; Akashe & Miller, 2001). In the phytosterolprotein complex, free phytosterols were first dissolved in vegetable oil and, only after, proteins were added to act as transporters. The resulting complex is ready for use or could be dehydrated (Corliss et al., 2000). Another promising formulation seems to be phytosterols emulsification by lecithin. Lecithin disperses in water forming micelles that include phytosterols, thus rendering them water soluble. This formulation is compatible with foods without fat, just requiring small amounts of phospholipids, and it is believed that it is more efficient in the solubility of phytosterols than of triacilglycerols (Ostlund et al., 1999; Spilburg et al., 2003). Phytosterols can also be dissolved in diacilglycerol. In fact, its solubility in diacilglycerols is higher than in triacilglycerols, i.e. 6.0% and 1.3%, respectively. Diacilglycerols are less abundant natural components in alimentary oils and fats, and are currently used as emulsifier agents in foods. Up to now, diacilglycerol oil enriched in phytosterols is commercialized in Japan (Salo et al., 2002), USA (FDA, 2006a) and EU (EC, 2007).

3.6. Phytosterols Alimentary Matrices Margarines were the first commercial applications of phytosterols enriched foods. Mattson and collaborators (1977; 1982), had earlier considered alimentary fat as a good matrix for phytosterols, since cholesterol was also vehiculated by this kind of alimentary matrix. Given this, vegetable oil spreads were used in most studies of phytosterols enriched foods (Berger et al., 2004). But on the other hand, the incorporation of phytosterols in high fat foods is not compliant with current dietary health recommendations (St-Onge & Jones, 2003). Furthermore, some alimentary cultures do not include margarines in daily diets (Quilez et al., 2003; Winter, 2004). As a result, margarines are not the ideal alimentary matrices for phytosterols recommended daily intake, due to their high fat content as well as their uncommon use in some daily diet cultures (Salo et al., 2002). Several new types of foods, including alternatives with low fat content or, even, without fat are under research for market introduction as phytosterols enriched foods. However, some limitations, in terms of effectiveness, have been reported, namely with no fat liquid alimentary matrix. A key point in phytosterols action is its solubility level in alimentary lipidic emulsified portion that is, is necessary to present a good distribution along the whole lipidic portion in order to be efficiently incorporate in mixed micelles (Salo & Wester, 2005).

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In the case of fat soluble phytosterols esters, solubility is not a problem; instead, the decisive step is the ester’s hydrolysis by the non specific enzyme, cholesterol pancreatic esterase. This enzyme secretion is stimulated by dietary emulsions that enter in the intestinal lumen. In other words, low fat foods should be consumed together with the meal to reach a greater effectiveness (Salo et al., 2005; Doornbos et al., 2006). In the case of free phytosterols, solubility step represents a problem. Therefore, free sterols should be incorporated in fat foods, unless they are activated by an appropriate procedure, like lecithin emulsification (Ostlund, 2004). Contrarily to its esterified form, free phytosterols efficiency, appears to be more dependent on the alimentary matrix. Doornbos et al. (2006) investigated single dose (2.83.2g) effectiveness of phytosterol esters enriched liquid yogurt with a total fat content of 2.2% or 3.3%, and consumed in different occasions (with or without meal). Their data indicated that the effect of those products in cholesterol reduction was not dependent of the fat content used. However, intake occasion was important. LDL-cholesterol reduction was 6.0% without meal and 9.4% with meal. Noakes et al. (2005) evaluated low fat (0.54%) yogurts enriched with phytosterol esters (1.7-1.8g/day) consumed as part of a normal diet. A LDL-cholesterol reduction of 5-6% was observed. This reduction was considerably lower than data reported by Mensink et al. (2002). Noakes et al. (2005) concluded that, in spite of existing differences between phytosterols doses in both studies, the phytosterols intake along with meal in Mensink et al. (2002) study was the reason for the improved effectiveness reported. Low fat milk has also been studied as matrix for free and esterified phytosterols. Thomsen et al. (2004) observed that daily intake of phytosterols (1.2-1.6g) enriched milk containing 1.2% of fat, reduced blood LDL-cholesterol in 7-10% when consumed as part of a typical Danish diet. Clifton et al. (2004) investigated phytosterols (1.6g/day) esters enriched milk containing 2% of fat and consumed in at least two meals. They observed a LDL-cholesterol reduction of 16%. On this study, observed cholesterol reduction was superior to other matrices, such as yogurt or cereals. Authors suggest that phytosterols are probably incorporated in the membrane of milk globules, and thus becoming more easily available to be transferred to micelle membranes. In other low fat foods, phytosterols can be kept in small lipidic drops core, remaining unavailable until fat digestion (Clifton et al., 2004). The most controversial data in respect to phytosterol effects in the reduction of LDLcholesterol have been reported in liquid matrices without fat. Jones et al. (2003) observed that free phytosterols (1.8g/day) enriched drinks with (up to 1 %) or without fat, consumed along with meals did not resulted in superior reductions in LDL-cholesterol, comparatively to a control diet. However, in a latter study by Devaraj et al.(2004), the consumption of free phytosterols enriched orange juice without fat (2g/day), with breakfast and dinner, a reduction of 12.4% in LDL-cholesterol was observed. The same authors considered that in respect to the alimentary matrix, the food vehicle used should be fatter to allow sufficient solubilization of phytosterols and better micellar absorption. Furthermore, the authors concluded that solid alimentary matrices would be more effective, owing to the increase of the contact time period between

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the phytosterols and the gastrointestinal content, which favours the access to mixed micelles (Jones et al., 2003). When analysing the same results, Ostlund (2004) concluded that the lack of phytosterols effectiveness was probably due to the absence of an appropriate free phytosterols formulation.

3.7. Phytosterols Analytical Methodologies Phytosterols reduction cholesterol properties contributed to a growing interest in its determination. As a consequence, several analytical methods have been developed for its quantification (Lagarda et al., 2006). Qualitative and quantitative phytosterols analyses in a given alimentary sample are generally used to evaluate its contribution on the global diet. For this purpose, phytosterols analytical data in alimentary products were usually obtained using methodologies where these compounds were determined as free sterols/tanols (Aitzetmüller et al., 1998). Phytosterols analyses could, also, be used in the control or monitoring of other purposes like, for instance, to detect adulterations in vegetable oils (Mandl et al., 1999). According to a revision done by Piironen and co-workers (2000a), most of the methods used so far in phytosterols analysis are based on cholesterol determinations. Initially, phytosterols were determined using enzymatic and spectrophotometric methods. However, these methods have presented some problems with interferences and lack of specificity. Thus, it could only be determined the total amount of sterols. Likewise, many of the chromatographic methods used led to serious quantification errors, due to sample inadequate preparation. So, to minimize phytosterols methodology variations, a validated methodology should be used (Piironen et al., 2000a). An important help on different evaluation methodologies would be the possibility to use certified materials. The existence of reference samples would allow the use of the same sample in different laboratories to compare their analytical performance. Presently, the only reference materials available for phytosterols determination are a mixture of alimentary oils and of animal fat from the European Union Bureau of Community's Reference (BCR) Agency (Piironen et al., 2000a). Phytosterols determination is usually done by gas chromatography (GC) using a capillary column with flame ionization detection (FID) or with mass spectrometry (MS) to confirm peak identification, although high performance liquid chromatography (HPLC) could also be used. However, sterol sylil derivatives gas chromatography coupled to mass spectrometry (GC-MS) is the methodology that gives the most effective resolution, identification and quantification (Volin, 2001). Typically, free form phytosterols analyses include lipidic extraction after sample homogenization, alkaline (saponification) and/or acid hydrolyse, extraction of unsaponifiable compounds, sterols derivatization and gas chromatographic analysis (Piironen et al., 2000a; Laakso, 2005; Lagarda et al., 2006).

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3.7.1. Sample Preparation One of the most critical steps in phytosterols analysis in different matrices is sample preparation. Sterol oxidation prevention must be carried out by oxygen elimination. This oxygen removal is usually done by using a nitrogen atmosphere (Lagarda et al., 2006). 3.7.1.1. Solvent Extraction Applied extraction solvent depends of the matrix nature, of the physical state (liquid or solid), and also of the free, esterified or glicosylated form in which phytosterols are present in the matrix. Phytosterols presented in plant tissues or in seed oils could be extracted by chloroformmethanol, chloroform-methanol-water, hexane, methylene chloride or acetone. Solid phase extraction (SPE) and supercritical fluid extraction (SFE) methodologies could also be used to extract free and esterified phytosterols from oils or from fats (Lagarda et al., 2006). 3.7.1.2. Saponification As it was already referred, phytosterols are usually determined as free sterols (Aitzetmuller et al., 1998; Piironen et al., 2000a). Lipidic extracts cannot include conjugated polar phytosterols like esteril glycosids, because they are not soluble in apolar lipidic phases. In this case, an alkaline hydrolyse is mandatory. After this saponification step, that release sterols/stanols from their esters, the respective free forms could be extracted as unsaponifiable fraction. According Piironen and collaborators (2000a), with this method, it is possible to determine the great majority of the phytosterols. Alkaline hydrolyse by ethanolic potassium hydroxide could be made at room or warm temperatures. Nevertheless, hot saponification, with internal standard addition, is adequate for the majority of enriched phytosterols matrices like, for instance, milk and yogurts (Laakso, 2005; Ahmida et al., 2006; Santos et al., 2007). However, phytosterols glycoside conjugates could not be hydrolysed by alkaline saponification. Consequently, this type of phytosterols quantification through direct saponification methodology is not efficient. So, the total phytosterols concentration determined is lower than the real value. Toivo and coworkers (2000, 2001), suggested a previous step of acid hydrolyse (with HCl), before the alkaline hydrolyse, as an alternative to release the phytosterols from the glycides. According the same authors, combination of acid and alkaline hydrolyses gives better results than alkaline hydrolyse, when used separately. Therefore, to obtain sterols totality (free, esterified and glycosilated), in some natural sources, such as cereals, an acid hydrolyse must be applied to the lipidic extract (Normén et al., 1999). Notwithstanding, acid hydrolyse application is limited for countless factors. Firstly, given the variety of alimentary products processed, not all the natural sources that have glycoside phytosterols conjugated are part of common diet. Refined alimentary oils are an example, since phytosterols are only presented in free and esterified forms. If glycoside esterified forms are originally present, they are removed during refinement oil process (Toivo et al., 2001). Besides, acid hydrolysis procedures are quite laborious and slowly for routine analyses. On the other hand, its use in unstable sterols abundant matrices like, for instance 7-

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phytosterols (7-avenasterol, 7-stigmasterol, etc.) and D5-avenasterol becomes quite problematic (Lagarda et al., 2006). 3.7.1.3. Unsaponifiable Fraction Extraction Usually, in alimentary matrices, as milk and yogurts, and in biological samples, as plasma, unsapnifiable phytosterols fraction was extracted by apolar organic solvents, like nhexane (Ahmida et al., 2006; Santos et al., 2007). However, unsaponifiable phytosterols fraction could also be extracted by other solvents, such as dichloromethane (Louter, 2004), chloroform-methanol (Conchillo et al., 2005), chloroform-methanol-water (Piironen et al., 2000a) or acetone (Claasen et al., 2000). 3.7.2. Determination 3.7.2.1. Gas Chromatography Analysis Phytosterols GC separation is not possible without previous derivatization (Piironen et al., 2000a; Lagarda et al., 2006). The phytosterols derivatization procedure usually used is a transformation, by chemical reaction, on its silyl (or acetyl) derivatives which provide high thermo-stability, low polarity and very well defined peaks with a more easily GC separation and detection (Laakso, 2005). Derivatization reactives commonly used are N-methyl-Ntrimethylsylil-trifluoroacetamide (MSTFA) in pyridine and bis-(trimethyl silyl)trifluoroacetamide (BSTFA) containing 1% of trimethylchlorosilane (TMCS) which are added to the dry sample residue (Lagarda et al., 2006). As for chromatographic columns, in most laboratories, the packed ones have been replaced by capillary columns. The latter columns offer short times of analysis, less peak interferences; resolution improvement and high thermal stability, when compared with packed ones (Abidi, 2001). GC methods with capillary column are also capable to separate stanols from the correspondent sterols (Phillips et al., 1999). This excellent separation occurs with common stationary phases, like 14% of cyano-propyl-phenyl-methyl-poli-siloxane (for instance, DB-1701) and like 5% diphenyl-95% dimethyl-poli-siloxane (for instance, OV-5) (Lagarda et al., 2006). As mobile phase, helium is usually used (Grandgirard et al., 2004). Sterols/stanols quantification is, usually, done by internal standard (IS) methodology. In this kind of methodology, IS should be added to the sample as soon as possible, to compensate the losses that occur during extraction, transference, evaporation, and derivatization steps, and thus correcting the analyte final quantification results. The most common IS proposed in literature for sterols/stanols determinations include betuline, cholestan, 5 β-cholestan-3 β-ol (coprostanol) and 5 α-cholestan-3 β-ol (cholestanol) (Lagarda et al., 2006). An IS should be similar to the analyte, but IS right choice could be problematic. Cholestan is a sterol hydrocarbon without the hydroxyl group in the position 3, while betuline has two hydroxyl groups and its structure and chemical properties still are more different than sterol ones. Cholestanol (5 α-colestan-3 β-ol) should not be used for stanol analyses because samples can contain cholestanol small amounts derived from cholesterol hydrogenation. Coprostanol (5 β-cholestan-3 β- ol) seems to be the appropriate IS for most of the matrices. It structure is similar to phytosterols, it is absent in alimentary samples and it FID

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detector response is identical as for the determined analytes. So, no correction factors are necessary for quantification (Laakso, 2005). When IS method is used, detector response factors and linearity, for each sterol/stanol, should be stabilized. Usually sterol/stanol amounts are calculated between peak area comparison of analysed sterol/stanol and of IS. Then, as IS concentration is known, these sterol (stanol)/IS ratios are used in calibration curve in order to easily quantify sample phytosterol concentration (Conchillo et al., 2005; Johnsson & Dutta, 2006). As referred previously, GC-FID or GC-MS are the selected methods for phytosterols determination. However, GC-MS present some advantages relatively to GC-FID in sample complex mixtures quantification (for instance, foods and biological samples), since only GCMS can confirm phytosterols identification as well as eluted peak purity evaluation (Piironen et al., 2000a). 3.7.2.2. Liquid Chromatography Analysis High performance liquid chromatography (HPLC) by ultraviolet (UV) detection can also used to determine the main phytosterols in foods. HPLC offer, in relation to GC method, an analyte no destructive alternative and no derivatization phytosterols are needed. However, HPLC methods have low selectivity to separate phytosterols from corresponding phytostanols (Piironen et al., 2000a). Nonetheless, and while an effective HPLC application is not developed, namely by mass detection, GC will continue to be the selected determination methodology for this type of compounds.

4. Conclusion Consumption of milk and dairy products enriched with phytosterols is a simple and safe strategy to reduce blood cholesterol and, consequently, to avoid premature development of cardiovascular diseases. Recent studies confirm that phytosterols daily intake of 2g reduces blood LDLcholesterol levels by 10% (Katan et al 2003, EFSA, 2008a and b; AbuMweis et al., 2008; Mannarino et al., 2009). However, no additional effect could be observed with an intake higher than 3g. So, this dose should be established as the maximum daily intake. Accordingly, dairy products seem to be a good matrix to obtain these phytosterols daily doses (1-3g). Additionally, phytosterols enriched milk and yogurts low fat content, are considered healthy foods with a high nutritional value and are easier to incorporate in any type of diet.

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5. Future Perspectives Phytosterol enriched food can be considered an "open book", because many aspects and health benefits are still under research (Trautwein & Demonty, 2007). In addition to their well-established cholesterol-lowering effect that was described above, other potential health benefits of phytosterols have been mentioned, namely immune stimulating (Calpe-Berdiel et al., 2007) and anti-inflammatory activities (Navarro et al., 2001; Bouic, 2002,), as well as beneficial effects against the development of different types of cancers, like breast, lung, stomach and colon (Bradford & Awad, 2007) oesophagus (de Stefani et al., 2000), ovarian (Mccann, et al., 2003) and prostate (Wilt et al., 1999; Berges et al., 2000) cancers, respectively. However, evidence for such promising effects is still at a rudimentary stage and more research is clearly needed to draw firm conclusions. Given that, milk and yoghurts, while privileged phytosterols enriched foods, could be considered as elected functional foods. Their nutritional allegations, today clearly proven, could be substituted, in an early future, for no less interesting health allegations in the most relevant subjects mentioned above.

References Abidi, S.L. (2001). Chromatographic analysis of plant sterols in foods and vegetable oils. Journal of Chromatography A, 935, 173-201. AbuMweis, S.S., Barake, R. & Jones, P.J.H. (2008). Plant sterols/stanols as cholesterol lowering agents: A meta-analysis of randomized controlled trials. Food & Nutrition Research, 52, DOI: 10.3402/fnr.v52i0.1811 Ahmida, M.H.S., Bertucci, P., Franzò, L., Massoud, R., Cortese, C., Lala, A. & Federici, G. (2006). Simultaneous determination of plasmatic phytosterols and cholesterol precursors using gas chromatography–mass spectrometry (GC–MS) with selective ion monitoring (SIM). Journal of Chromatography B, 842, 43–47. Aitzetmüller, K., Bruè, H.L.L. & Fiebig, H. J. (1998). Analysis of sterol content and composition in fats and oils by capillary gas-liquid chromatography using an internal standard. Comments on the German sterol method. Fett/Lipid, 100, 429-435. Akashe, A. & Miller, M. (2001). Use of mesophase-stabilized compositions for delivery of cholesterol-reducing sterols and stanols in food products. United States Patent 6274574, issued on August 14, 2001. Akihisa, T., Kokke, W. & Tamura, T. (1991). Naturally occurring sterols and related compounds from plants. In: G. W. Patterson, & W. D. Nes (Eds), Physiology and Biochemistry of Sterols (pp. 172-228). Champaign, IL: American Oil Chemists' Society. Armstrong, M. J. & Carey, M. C. (1987). Thermodynamic and molecular determinants of sterol solubilities in bile salt micelles. Journal of Lipid Research, 28, 1144-1155. Awad, A.B., Smith, A.J. & Fink, C.S. (2001). Plant sterols regulate rat vascular smooth mulscle cell growth and prostacyclin release in culture. Prostaglandins Leukot Essent Fatty Acids, 64, 323-230

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High Blood Cholesterol in Adults (Adult Treatment Panel III). Journal of American Medical Association, 285, 2486-2497. Nestel, P., Cehun, M., Pomeroy, S., Abbey, M. & Weldon, G. (2001). Cholesterol-lowering effects of plant sterol esters and non-esterified stanols in margarine, butter and low-fat foods. European Journal of Clinical Nutrition, 55, 1084-1090. NHF - Australian Heart Foundation (2007). Summary of the evidence on phytosterols/stanol enriched foods. http://www.heartfoundation.org.au/document/NHF/HF_Phytosterols_ Stanols_Summ of Evidence_2007_Aug_FINAL.pdf Noakes, M., Clifton, P., Ntanios,F., Shrapnel, W., Record,I. & McInerne, J. (2002). An increase in dietary carotenoids when consuming plant sterols or stanols is effective in maintaining plasma carotenoid concentrations. The American Journal of Clinical Nutrition, 75, 79-86. Noakes, M., Clifton, P.M., Doornbos, A.M.E. & Trautwein, E.A. (2005). Plant sterol esterenriched milk and yoghurt effectively reduce serum cholesterol in modestly hypercholesterolemic subjects. European Journal of Nutrition, 44, 214-222. Normén, L., Johnsson, M., Andersson, H., van Gameren, Y. & Dutta, P. (1999). Plant sterols in vegetables and fruits commonly consumed in Sweden. European Journal of Nutrition, 38, 84-89. Normen, L., Bryngelsson, S., Johnsson, M., Evheden, P., Ellegard, L., Brants, H., Andersson, H. & Dutta, P. (2002). The Phytosterol Content of Some Cereal Foods Commonly Consumed in Sweden and in the Netherlands. Journal of Food Composition and Analysis, 15, 693–704. Normén, L., Frohlich, J. & Trautwein, E. (2004). Role of plant sterols in cholesterol lowering. In P.C. Dutta (ed.), Phytosterols as Functional Food Components and Nutraceuticals (pp. 243-315). New York, NY: Marcel Dekker Inc. Normén, L., Ellega, L., Brants, H., Dutta, P. & Andersson, H. (2007). A phytosterol database: Fatty foods consumed in Sweden and the Netherlands. Journal of Food Composition and Analysis, 20, 193-201. Ntanios, F.Y., van de Kooij, A.J., de Deckere, E.A.M., Duchateau, G.S.M.J.E. & Trautwein, E.A. (2003). Effects of various amounts of dietary plant sterol esters on plasma and hepatic sterol concentration and aortic foam cell formation ofcholesterol-fed hamsters. Atherosclerosis 169, 41-50. O’Neill, F.H., Sanders, T.A.B. & Thompson, G.R. (2005). Comparison of efficacy of plant stanol ester and sterol ester: short-term and longer-term studies. The American Journal of Cardiology, 96, 29-36. O'Neill, F.H., Brynes, A., Mandeno, R., Rendell, N., Taylor, G., Seed, M. & Thompson, G.R. (2004). Comparison of the effects of dietary plant and sterol esters on lipid metabolism. Nutrition, Metabolism & Cardiovascular Diseases, 14, 133-142. Ostlund, R.E.Jr., Spilburg, C.A. & Stenson, W.F. (1999). Sitostanol administered in lecithin micelles potently reduces cholesterol absorption in humans. American Journal of Clinical Nutrition, 70, 826-831. Ostlund, R.E. (2002). Phytosterols in human nutrition. Annual Review of Nutrition, 22, 533549.

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Ostlund, R.E., Mcgill, J.B., Zeng, C.M., Covey, D.F., Stearns, J., Stenson, W.F. & Spilburg, C.A. (2002). Gastrointestinal absorption and plasma kinetics of soy Δ5 phytosterol and phytostanols in humans. American Journal of Physiology Endocrinology and Metabolism, 282, 911-916. Ostlund, R.E.Jr. (2004). Phytosterols and cholesterol metabolism. Current Opinion in Lipidolology, 15, 37-41. Patel, D.M. & Thompson, D.P. (2006). Phytosterols and vascular disease. Atherosclerosis, 186, 12–19. Peterson, D.W. (1951). Effect of soybean sterols in the diet on plasma and liver cholesterol in chicks. Proceedings of the Society for Experimantal Biology and Medicine, 78, 143-147. Phillips, K.M., Ruggio, D.M. & Bailey, J.A. (1999). Precise quantitative determination of phytosterols, stanols, and cholesterol metabolites in human serum by capillary gas-liquid chromatography. Journal of Chromatography B, 732, 17-29. Phillips, K.M., Ruggio, D.M. & Ashraf-Khorassani, M. (2005). Phytosterols composition of nuts and seeds in the United States. Journal of Agricultural and Food Chemistry, 53, 9436-9445. Piironen, V. & Lampi, A. M. (2004). Occurrence and levels of phytosterols in foods. In P.C. Dutta (ed.), Phytosterols as Functional Food Components and Nutraceuticals (pp. 1-32). New York, NY: Marcel Dekker Inc. Piironen, V., Lindsay, D.G., Miettinen, T.A. Toivo, J. & Lampi, A.M. (2000a). Plant sterols: biosynthesis, biological function and their importance to human nutrition. Journal of the Science of Food and Agriculture, 80, 939-966. Piironen, V., Toivo, J. & Lampi, A.M. (2000b). Natural sources of dietary plant sterols. Journal of Food Composition and Analysis, 13, 619-624. Pinedo, S., Vissers, M.N., von Bergmann, K., Elharchaoui, K. Lütjohann, D., Luben, R., Wareham, N.J., Kastelein,J.J.P.,, Khaw, K-T. & Boekholdt, S.M. (2007). Plasma levels of plant sterols and the risk of future coronary artery disease in apparently healthy men and women; the prospective EPIC-Norfolk population study. Journal of Lipid Research, 48,139-144. Plat, J. & Mensink, R.P. (2002) Increased intestinal ABCA1expression contributes to the decrease in cholesterol absorption after plant stanol consumption. FASEB Journal, 16, 1248-1253. Plat, J. & Mensink, R. P. (2005). Plant stanol and sterol esters in the control of blood cholesterol levels: mechanism and safety aspects. The American Journal of Cardiology, 96, 15-22. Plat, J., Beugels, I., Gijbels, M.J.J., de Winther, M.P.J. & Mensink, R.P. (2006). Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols. Journal of Lipid Research, 47, 2762-2771. Pollak, O.J. (1953). Reduction of blood cholesterol in man. Circulation, 7, 702-706. Pollak, O.J. & Kritchevsky, D. (1981). Sitosterol. In Beynen, A.C., Kritchevsky, D. & Pollak, O.J. (Eds.), Monographs on Atherosclerosis. Basel, Switzerland: S. Karger. Quílez, J., Rafecas, M., Brufau, G., García-Lorda, P., Megías, I., Bulló, M., Ruiz, J.A. & Salas-Salvadó, J. (2003). Bakery products enriched with phytosterol esters, α-tocopherol and β-carotene decrease plasma LDL-cholesterol and maintain plasma β-carotene

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concentrations in normocholesterolemic men and women. Journal of Nutrition, 133, 3103-3109. Ratnayake, W.M.N. & Vavasour, E.J. (2004). Potential health risks associated with large intakes of plant sterols. In Dutta, P.C. (ed.), Phytosterols as Functional Food Components and Nutraceuticals (pp. 365-395). New York, NY: Marcel Dekker Inc. Roberfroid, B.M. (1999). What is Beneficial for Health? The Concept of Functional Food. Food and Chemical Toxicology, 37, 1039-1041. Roberfroid, B.M. (2000). A European Consensus of Scientific Concepts of Functional Foods. Nutrition, 16, 7-8. Rodrigues, N.J., Torres, P.R., Mancini-Filho, J. & Gioielli, A. L.(2007). Physical and chemical properties of milk fat and phytosterol esters blends. Food Research International, 40, 748-755 Rozner, S. & Garti, N. (2006). The activity and absorption relationship of cholesterol and phytosterols. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 283, 435-456. Salen, G., Shefer, S., Nguygen, L., Ness, G.C., Tint, G.S. & Shore V. (1992). Sitosterolemia. Journal of Lipid Research, 33, 945-955. Salo, P., Wester, I. & Hopia, A. (2002). Phytosterols. In Gunstone, F.D. (ed.), Lipids for Functional Foods and Nutraceuticals (pp. 183-224). Bridgwater, Englandd: The Oily Press. Salo, P. & Wester, I. (2005). Low-fat formulations of plant stanols and sterols. The American Journal of Cardiology, 96, 51-54. Salo, P., Hopia, A., Ekblom, J., Lahtinen, R. & Laakso P. (2005). Plant stanol ester as a cholesterol lowering ingredient of Benecol® foods. In Akoh, C.A. & Lai O.M. (eds.), Healthful Lipids (pp. 699-730). Champaign, Il: AOCS Press. Santos, R., Limas, E., Sousa, M., Castilho, M.C., Ramos, F. & Silveira, M.I.N. (2007). Optimization of analytical procedures for GC–MS determination of phytosterols and phytostanols in enriched milk and yoghurt. Food Chemistry, 102, 113-117. SCF (2000). Opinion of the Scientific Committee on Food on a request for the safety assessment of the use of phytosterol esters in yellow fat spreads. SCF/CS/NF/DOS/1 Final. SCF (2002). General view on the long-term effects of the intake of elevated levels of phytosterols from multiple dietary sources with particular attention to the effects on ßcarotene. SCF/CS/NF/DOS/20 ADD 1 Final. SCF (2003a). Opinion of the Scientific Committee on Food on an application from ADM for approval of plant sterol-enriched foods. SCF/CS/NF/DOS/23 ADD2 Final. SCF (2003b). Opinion of the Scientific Committee on Food on applications for approval of a variety of plant sterol-enriched foods. SCF/CS/NF/DOS/15 ADD2 Final. SCF. (2003c). Opinion of the Scientific Committee on Food on an application from Multibene for approval of plant-sterol enriched foods. SCF/CS/NF/DOS/24 ADD2 Final. Schwartz, H., Ollilainen, V., Piironen, V., Lampi, A-M. (2008). Tocopherol, tocotrienol and plant sterol contents of vegetable oils and industrial fats. Journal of Food Composition and Analysis, 21, 152-161.

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Sibbel, A. (2007). The sustainability of functional foods, Social Science & Medicine, 64, 554561. Silbernagel, G., Fauler, G., Renner, W., Landl, E.M., Hoffmann, M.M., Winkelmann, B.R., Boehm, B.O. & März, W (2009). The relationship of cholesterol metabolism and plasma plant sterols with the severity of coronary artery disease. Journal of Lipid Research, 50, 334-341. Soupas, L. (2006). Oxidative stability of phytosterols in food models and foods. PhD Thesis, University of Helsinki publications, Finland. Spilburg, C.A., Goldberg, A.C., McGill, J.B., Stenson, W.F., Racette, S.B., Bateman, J., McPherson, T.B. & Ostlund, R.E.Jr. (2003). Fat-free foods supplemented with soy stanol-lecithin powder reduce cholesterol absorption and LDL cholesterol. Journal of the American Dietetic Association, 103, 577-581. Stalenhoef, A.F., Hectors, M., Demacker, P.N. (2001). Effect of plant sterol-enriched margarine on plasma lipids and sterols in subjects heterozygous for phytosterolaemia. Journal of Internal Medicine, 249,163-166. St-Onge, M.P. & Jones, P.J.H. (2003). Phytosterols and human lipid metabolism: efficacy, safety, and novel foods. Lipids, 38, 367-375. Sugano, M., Morioka, H. & Ikeda, I. (1977). A comparison of hypercholesterolemic activity of beta-sitosterol and beta-sitostanol in rats. Journal of Nutrition, 107, 2011-2019. Thomsen, A.B., Hansen, H.B., Christiansen, C., Green, H. & Berger, A. (2004). Effect of free plant sterols in low-fat milk on serum lipid profile in hypercholesterolemic subjects. European Journal of Clinical Nutrition, 58, 860-870. Toivo, J., Lampi, A.M., Aalto, S. & Piironen, V. (2000). Factors affecting sample preparation in the gas chromatographic determination of plant sterols in whole wheat flour, Food Chemistry, 68, 239-245. Toivo, J., Phillips, K., Lampi, A.M. & Piironen, V. (2001), Determination of sterols in foods: recovery of free, esterified and glycosidic sterols. Journal of Food Composition and Analysis, 14, 631–643. Trautwein, E.A., Duchateau, G.S.M.J.E., Lin, Y., Mel’nikov, S.M., Molhuizen, H.O.F., Ntanios, F.Y. (2003). Proposed mechanisms of cholesterol-lowering action of plant sterols. European Journal of Lipid Science andTechnology, 105, 171-185. Trautwein, E.A. & Demonty, I. (2007). Phytosterols: natural compounds with established and emerging health benefits. Oliéginaux, Corps Gras, Lipids, 14, 259-266 Valsta, L.M., Lemström, A., Ovaskainen, M.L., Lampi, A.M., Toivo, J., Korhonen, T. & Piironen, V. (2004). Estimation of plant sterol and cholesterol intake in Finland: quality of new values and their effect on intake. British Journal of Nutrition, 92, 671-678. Vanhanen, H.T., Kajander, J., Lehtovirta, H. & Miettinen, T. A. (1994). Serum levels, absorption efficiency, faecal elimination and synthesis of cholesterol during increasing doses of dietary sitostanol esters in hypercholesterolaemic subjects. Clinical Science, 87, 61-67. Venkatramesh, M., Karunanandaa, B., Sun, B., Gunter, C.A., Boddupalli, S. & Kishore G.M. (2003) Expression of a Streptomyces 3-hydroxysteroid oxidase gene in oilseeds for converting phytosterols to phytostanols. Phytochemistry, 62, 39-46.

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Volger,O.L., Mensink, R.P., Plat, J., Hornstra, G., Havekes, L.M., & Princen, H.M.G. (2001). Dietary vegetable oil and wood derived plant stanol esters reduce atherosclerotic lesion size and severity in apoE3-Leiden transgenic mice. Atherosclerosis, 157, 375-381. Volin, P. (2001). Analysis of steroidal lipids by gas and liquid chromatography. Journal of Chromatogaphy A , 935, 125-140. von Bergmann, K., Sudhop, T. & Lütjohann, D. (2005). Cholesterol and plant sterol absorption: recent insights. The American Journal of Cardiology, 96, 10-14. von Bonsdorff-Nikander, A.K. (2005). Studies on a cholesterol-lowering microcrystalline phytosterol suspension in oil. Academic dissertation, University of Helsinki, Finland. Waalkens-Berendsen, D.H., Wolterbeek, A.P.M., Wijnands, M.V.W., Richold, M. & Hepburn, P.A. (1999). Safety evaluation of phytosterol esters. Part 3. Two-generation reproduction study in rats with phytosterol esters – a novel functional food. Food and Chemical Toxicology, 37, 683-696. Wester, I. (1999) Dose responsiveness to plant stanol esters. European Heart Journal Suppl, 1(suppl S), S104 –S108. Wester, I. (2000). Cholesterol-lowering effect of plant sterols. European Journal of Lipid Science and Technology, 102, 37-44. Weststrate, J.A. & Meijer G.W. (1998). Plant sterol-enriched margarines and reduction of plasma total and LDL-cholesterol concentrations in normocholesterolaemic and mildly hypercholesterolaemic subjects. European Journal of Clinical Nutrition, 52, 334-343. WHO (2008). World Health Statistics. Geneva: Switzerland, WHO press. Wilt, T.J., Macdonald, R. & Ishani, A. (1999). Beta-sitosterol for the treatment of benign prostatic hyperplasia: a systematic review. BJU International, 83, 976-983. Wilund, K.R., Yu,L., Xu, F., Vega,G.L., Grundy, S.M., Cohen, J.C., & Hobbs, H.H. (2004). No Association Between Plasma Levels of Plant Sterols and Atherosclerosis in Mice and Men. Arteriosclerosis Thrombosis and Vascular Biology, 24, 2326-2332. Windler, E., Zyriaz, B-C., Kuipers, F, Linseisen, J., Boeing, H. (2009). Association of plasma phytosterol concentrations with incident coronary heart disease. Data from the CORA study, a case-control study of coronary artery disease in women. Atherosclerosis, 203, 284-290.. Winter, J. (2004) Sterols: the key to heart health. Functional Foods & Nutraceuticals,. [Online]. [Cited 2008.10.01]. Available from: www.ffnmag.com. Wojciechowski, Z.A. (1991). Biochemistry of phytosterol conjugates. In G.W. Patterson & W.D. Nes (eds.), Physiology and Biochemistry of Sterols (pp. 361-395). Champaign, Il: AOCS. Wolfreys, A.M. & Hepburn, P.A. (2002). Safety evaluation of phytosterol esters. Part 7. Assessment of mutagenic activity of phytosterols, phytosterol esters and the cholesterol derivative, 4-cholesten-3-one. Food and Chemical Toxicology, 40, 461-470.

In: Milk Consumption and Health Editors: E. Lange and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter II

Kefir and Health: A Perception Zaheer Ahmed2 and Yanping Wang*1 *1

School of Food Engineering and Biotechonolgy, Tianjin University of Science & Technology, Tianjin 300457 China 2 Faculty of Sciences. Department of Home and Health Sciences. Allama Iqbal Open University. H-8, Islamabad Pakistan

Abstract Kefir is a fermented milk drink produced by the actions of bacteria and yeasts contained in kefir grains, and is reported to have a unique taste and properties. Kefir, the self-carbonated beverage, possesses nutritional attributes due to its content of vitamins, protein and minerals and therapeutic attributes contributed by its antibacterial spectrum, gastro-intestinal proliferation, hypocholesterolemic effect, anti carcinogenic effect, lactic acid content, b-galactosidase activity and bacterial colonization, improves immune system and is also remedy for Helicobacter pylori infection which is only the property of kefir. Moreover, on one side kefir is good dietetic beverage, and of particular interest of athletes, and on other side the whole kefir is good for feeding premature infants because of good tolerance, and adequate weight gain. Lots of work has been done on kefir from a health point of view, this chapter summarizes all the data that has been completed to date. By reviewing the literature the chemical, microbiological, nutritional and therapeutic characteristics of kefir have been highlighted to justify its consumption as a healthy milk food.

*

[email protected]

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Zaheer Ahmed and Yanping Wang

Introduction The benefits of probiotics have been recognized and explored for over a century. Research in probiotics concentrates on modes of action, host-microbe interactions and such specific questions as the influences on the human or animal immune system. Recently, consumers’ growing preference for food with enhanced nutritional and therapeutic properties has led to the inclusion of various cultured milk products as part of their diet based upon their dietetic features. The increasing cost of health care, the steady increase in life expectancy and the desire of the elderly for improved quality of their lives are driving factors for research and development in the area of functional foods. Among a number of functional compounds recognized so far, bioactive components from fermented foods and probiotics certainly take the center stage due to their long tradition of safe use, and established and postulated beneficial effects. Probiotics occur naturally in fermented food products such as yoghurt, kefir, sauerkraut, cabbage kimchee, and soybean- based miso and natto (Sarkar 2008). Among them kefir which is defined as the yogurt of the 21st century (Gorski 1994), has got considerable attention of food scientists because of its unique and complex probiotic properties. Kefir is self-carbonated refreshing fermented milk with slight acidic taste, made from kefir grains, a complex and specific mixture of bacteria and yeast held together by a polysaccharide matrix. Kefir means “feel good” in Turkish and falls under the category of a mixed lactic acid and ethanol fermented beverage (Kurmann, 1984). Kefir is also known as Kefyr, Kephir, Kefer, Kiaphur, Knapon, Kepi and Kippi. Kefir distinguishes itself from the traditional fermented milks (yogurt) and other fermented dairy products in that it is the product of fermentation of milk in the presence of a mixed group of microflora confined to a matrix of discrete ‘kefir grains’, which are recovered subsequent to fermentation (Malbaša et al. 2009, Marshall & Cole, 1985). Kefir grains resemble small cauliflower florets or cooked rice: measure 1–3 cm in length, are lobed, irregularly shaped, white to yellow-white in colour, and have a slimy but firm texture (Plessas et al. 2007, Loretan et al. 2003). These grains contain lactic acid bacteria (lactobacilli, lactococci, leuconostocs), acetic acid bacteria and yeast mixture coupled together with casein and complex sugars by a matrix of polyssacharide. Various lactic acid bacteria and yeasts have been identified as being present in kefir grains, including L. brevis, L.helveticus, L. kefir, Leuconostoc mesenteroides, Kluyveromyces lactis, K. marxianus and Pichia fermentans (Angulo et al. 1993, Lin et al. 1999). The micro-organisms contained within the kefir grains typically produce lactic acid, antibiotics and several kinds of bactericide, such products inhibiting the proliferation of both degrading and pathogenic microorganisms in kefir milk (Angulo et al. 1993). Kefir occupies an important place in the human diet in many parts of the world including Southwest Asia, Eastern and Northern Europe, North America, Japan (Otles and Cagindi, 2003), the Middle East, North Africa and Russia (Koroleva, 1982, IDF, 1988) due to its nutritional and therapeutic significance. Especially in Soviet countries, kefir has, anecdotally, been recommended for consumption by healthy people in order to lower the risk of chronic diseases, and has also been provided to certain patients for the clinical treatment of a number of gastrointestinal and metabolic diseases, hypertension, IHD and allergy (St-Onge et al. 2002; Farnworth & Mainville, 2003). Kefir has also been recommended for infants over the

Kefir and Health: A Perception

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age of 6 months as mixed or artificial diet (Ivanova et al. 1980) and bifidokefir, containing physiologically active cells of Bifidobacterium bifidum proved to be more efficacious than ordinary kefir in eliminating intestinal infection in children (Murashova et al. 1997).

Production of Kefir Kefir was first made from goat milk with kefir grains (Figure 1) in goat skin bag by hanging in the house during winter and outside during summer (Ozer and Ozer, 1999). Traditionally, kefir was produced by inoculating milk with kefir grains (Pijanowski, 1980) or by the widely adopted European method, which involved use of bulk milk culture obtained by kefir grains for milk inoculation (Puhan and Vogt, 1985). Kurmann (1984) classified kefir under the class of mixed lactic acid and ethanol fermented milk and can be further subclassified as kefir obtained using kefir grains and artificial kefir obtained without kefir grains. The traditional and industrial process of making kefir is presented in Figure 2a and Figure 2b respectively. Kefir produced from pure cultures did not receive high sensory evaluation scores in Canada unless it was sweetened (Duitschaever et al. 1987, 1991); Duitschaever et al. (1987) also showed that only about 40% of people tasting natural kefir for the first time gave it a positive taste rating. Addition of peach flavour, or modification of the fermentation process (e.g. addition of lactococci, lactobacilli or yeasts) increased the acceptability of kefir, compared to traditionally made kefir (Duitschaever et al. 1991; Muir et al. 1999). To overcome these problems some biotechnological innovations in kefir production has bee made (Sarkar 2008). Tratnik et al. (2006) recommended supplementation of goat milk with whey protein concentrate at a level of 60.0-60.5 g/100 g proteins for enhancement in ethanol production (0.35 ml/100 ml). To comply with the consumers demand for more healthy foods, soya milk could be a suitable substitute for kefir production owing to its low saturated fat and cholesterol (Berry, 2000), higher polyunsaturated fatty acids, lecithin, linolenic acid, magnesium, iron, folic acid and vitamin E (Hermann, 1991). At the present refreshing beverahge of kefir can be made by with fruit juice, molasses, sugar, and any kind of milk such as cow, goat, sheep, camel, buffalo, or soy milk (Santos et al. 2003, Harta et al. 2004). Fermentation of milk by traditional methods employing kefir grains resulted in disparity in product quality due to diverse microflora and uncontrolled fermentation. Two methods have been suggested to overcome the drawbacks of traditional methods of kefir production, kefir can be produced either by simultaneous (Tamai et al. 1996) or consecutive lactic acid and yeast fermentation (Beshkova et al., 2002). Probiotic kefir capable of exhibiting antimicrobial properties could be obtained employing L. acidophilus, Lactobacillus kefiranofuctens and Lactobacillus kefiranofaciens (Santos et al. 2003) or Bifidobacterium bifidum (Murashova et al. 1997). Introduction of Candida kefyr during kefir production may be advantageous as it did not disappear completely at pH 2.0, retains 97.2 per cent viability in presence of 1per cent bile salts and not inhibited by most antibiotics including tetracycline (You et al. 2006). In order to meet the consumer’s demand for healthful foods in the current era of self-care and complementary medicine kefir with enhanced dietetic properties could be obtained by co-inoculation of soya milk with yeasts (Sacch. cerevesiae, Candida kefir), lactic acid bacteria (Streptococcus thermophilus,

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Lactobacillus bulgaricus) and probiotic cultures (Lactobacillus acidophilus, Bifidobacterium bifidum) (Sarkar 2008).

Figure 1 Kefir grains.

Chemical Composition of Kefir The composition of kefir is variable and not well defined (Zubillaga et al. 2001). It depends on the source and the fat content of milk, the composition of the grains or cultures and the technological process of kefir (Kneifel and Mayer 1991, Bottazzi et al. 1994). Liut Kevicius and Sarkinas (2004) reported kefir grains to contain 86.3 percent moisture, 4.5 percent protein, 1.2 percent ash and 0.03 percent fat. A typical kefir contains 89-90 percent moisture, 0.2 percent lipids, 3.0 percent protein, 6.0 percent sugar, 0.7 percent ash (Ozer and Ozer, 1999) and 1.0 percent each of lactic acid and alcohol (Webb et al., 1987). Kefir has been reported to contain 1.98 g/l carbon dioxide and 0.48 percent alcohol (Beshkova et al., 2002) and the content of carbon dioxide increased (201.7-277.0 ml/l) with increased concentration (10-100 g/l) of kefir grains (Garrote et al., 1998). The major products formed during fermentation are lactic acid, CO2 and alcohol. Diacetyl and acetaldehyde ( aromatic compounds) (Zourari and Anifantakis, 1988, Beshkova et al. 2003), pyruvic acid, hoppuric acid,acetic acid, propionic acid and butyric acid (Guzel-Seydim et al. 2000) are also present in kefir. Diacetyl is produced by Str. Lactis subsp. diacetylactis and Leuconostoc sp. (Libudzisz and Piatkiewicz, 1990). Commercial kefir contains half as much ortic acid, twice as much pyruvic acid, nine time as much acetic acid, and about equal amount of uric acid as does in commercial yoghurt (Dousset and Caillet 1993). Storage of kefir at 48 °C for 21 days induced abatement in concentrations of ethanol to 0.08 percent, acetaldehyde to 11mg/g and a decline in acetoin to 16 ppm, however diacetyl was not detected during either fermentation or storage (Guzel-Seydim et al. 2000). Kefir also contains vitamins, macro and micro elements (Liut Kevicius and Sarkinas, 2004). Klyavinya

Kefir and Health: A Perception

47

(1980) suggested a composition of 3.2-3.4 percent fat, 8.0 percentsolids-not-fats and 5.0 percent sucrose for kefir intended for infant feeding. The chemical composition of kefir is given in Table 1. Boiling of raw milk

↓ Cooling at 20-25 °C

↓ Incubation at 20-25 °C ← Kefir grain

↓ Fermentation at 20-25 °C, 18-24h

↓ Separation → Kefir grain

↓ Maturation and cooling at 4 °C

↓ Stored at 4 °C Figure 2a. The traditional process of kefir. (Otles and Cagindi, 2003)

Raw milk

↓ Homogenization

↓ Pasteurization at 90-95 °C 5-10 min

↓ Cooling at 18-24 °C

↓ Incubation at 18-24 °C ← Kefir culture 2-8 %

↓ Fermentation 18-24 °C, 18-24 h

↓ Separation the coagulum

↓ Distribution in bottles

↓ Maturation 12-14/ 3-10 °C, 24 h

↓ Stored 4 °C Figure 2b. The industrial process of kefir. (Otles and Cagindi, 2003)

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Table 1. The chemical composition and nutritional values of kefir Components Energy Fat (%) Protein (%) Lactose (%) Water (%)

100 g 65 kcal 3.5 3.3 4.0 87.5

Milk acid (g) Ethyl alcohol (g) Lactic acid (g) Cholestrol (mg) Phosphatateds (mg)

0.8 0.9 1 13 40

Essential amino acids (g) Tryptophan Phenylalanin+tyrosine Leucine Isoleucine Threonine Methionine+cystine Lysine Valine Vitamins (mg) A Carotene B1 B2

Components Mineral content (g) Calcium Phosphorus Magnesium Potassium Sodium Chloride

0.12 0.10 12 0.15 0.05 0.10

Trace elements Iron (mg) Copper (μg) Molybdenum (μg) Manganese (μg) Zinc (mg)

0.05 12 5.5 5 0.36

0.05 0.35 0.34 0.21 0.17 0.12 0.27 0.22

Aromatic-compounds Acetaldehyde Diacetyl Acetoin

0.06 0.02 0.04 0.17

B12 Niacin C D

100 g

0.5 0.09 1 0.08

Source: Sarkar (2008), Liut Kevicius and Sarkinas (2004).

Microbiological Characteristics Microflora of kefir grain and its approximate count in presented in Table 2. Microfora of kefir grain is complex and not always constant, consisting of undefined species of bacteria and yeasts (IDF, 1991). The microbial population found in kefir grains has been used as an example of a symbiotic community (Margulis 1995); this symbiotic nature has made identification and study of the constituent microorganisms within kefir grains difficult. Abraham and Antoni (1999) reported that approximately 0.9 percent of the total weight of wet kefir is represented by its microflora. Contents of bacteria in kefir varied from 6.4 ×1048.5 × 108 cfu/g, and yeasts from 1.5 × 105-3.7 × 108 cfu/g (Witthuhm et al. 2004). Irigoyen et al. (2005) reported that besides a viable population of 108 cfu/ml of lactobacilli and lactococci and 105 cfu/ml yeasts, kefir also had 106 cfu/ml acetic acid bacteria after 24 h of fermentation.

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Table 2. Microflora of kefir grains, kefir starter and kefir beverage Microbial constituents Thermophilic lactobacilli Mesophiilic lactobacilli Lactococci Leuconostoc Acetic acid bacteria Yeasts

Kefir grains (cfu/g) 108 106-109 106 106 108 106-108

Kefir starter (cfu/ml) 105 102-103 108-109 107-108 105-106 105-106

Kefir beverage (cfu/ml) 10-108 109 107-108 104-105 104-105

Source: Bottazzi et al. (1994); Rea et al. (1996).

Bacterial cultures identified in kefir were Lactobacilli, Leuconostoc and Lactococcus (Witthuhm et al., 2004) and Wang et al. (2004) reported it to be consisted of Lactobacillus delbrueckii subsp. delbrueckii (8), Lactobacillus delbrueckii subsp. bulgaricus (6), Lactobacillus Kefir (2), Lactobacillus acidophilus (1), Enterococcus faecalis (7), Enterococcus faecium (2), Streptococcus thermoplilus (1) and Lactococcus lactis subsp. cremoris (3). Identified yeasts were Zygosaccharomyces, candida and Saccharomyces (Witthuhm et al., 2004), Klyveromyces matxianus, Klyveromyces lactis and Saccharomyces cerevesiae as the dominant flora and Saccharomyces unisporus, Zygosaccharomyces rouxii, Torulaspora delbrus, Zygosaccharomyces rouxii, Torulaspora delbrueckii and Debaryomyces hansenii (Loretan et al. 2003).

Nutritional Composition Beyond its inherent high nutritional value as a source of proteins and calcium, kefir has a long tradition of being regarded as good for health in countries where it is a staple in the diet (Vinderola 2004). The nutritional attributes of kefir (Table 1) are due to its chemical ingredients such as vitamins, protein and minerals and fermentation induced further enhancement in its nutritional profiles. Limited information available regarding nutritional characteristics of kefir are delineated underneath.

Vitamin Content Kneifel and Mayer (1991) found that appreciable amounts of pyridoxine, vitamin B12, folic acid and biotin were synthesized during kefir production, depending on the source of kefir grains used, while thiamine and riboflavin levels were reduced. Several investigation have, measured the quantity of kefir to determine whether fermentation changed levels of vitamin compared to milk, but the results have not often been consistent. An early study of the vitamin B12 content of kefir indicated that both during the fermentation and maturation stages, the vitamin B12 content went down (Guzel-Seydim et al. 2000). Alm (1982) used commercially available grains to produce kefir that had increased folic acid content compared

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to starting milk. Kefir contained vitamin B1, vitamin B2, vitamin B5 (Liut Keviciusand and Sarkinas, 2004), vitamin B1, Vitamin B12, folic acid, vitamin K (Otles & Cadingi, 2003) and vitamin C (Khamnaeva et al. 2000). The vitamin content of kefir is influenced by the type of milk as well as by supplementing flora. An abatement in vitamin B12 (34-37 percent) due to the inclusion of Propionibacterium peterssoni and Propionibacterium pituitosum (Roczniakova et al. 1974) and vitamin B12 (30 times) and folic acid (five times) with a slight increase in pantothenic acid and vitamin B6 due to Propionibacterium freudenreichii subsp. shermanii as supplementing organism (Cerna and Hrabova, 1982) were noted during the manufacture of kefir.

Protein Content Knowledge of grain proteins is limited (Abraham and Antoni 1999). A higher protein content was found in kefir, when the grains were cultured in whey (Fil’ Chakova and Koroleva, 1997) or in soy milk (Abraham and Antoni, 1999) than those cultured in milk. Yuksekdag et al. (2004a) demonstrated the proteolytic activity of lactococci (13/21 strains) isolated from kefir. Otles & Cadingi (2003) reported that during kefir fermentation process, along with vitamin production, amion acids were also produced.Molokeev et al. (1998) narrated that bifidofefir containing 2x107 cfu/ml bifidobacteria had higher contents of glutamic acid and threonine than normal kefir. During fermentation of milk, there is a change in amino acid profiles and higher amounts of threonine, serine, alanine, lysine and ammonia are produced in kefir than in milk (Guzel-Seydim et al., 2003). Liut Kevicius and Sarkinas (2004) reported amino acid profiles of kefir showing the presence of valine, isoleucine, methionine, lysine, threonine, phenylalanine and tryptophan.

Sugar Contents A typical kefir contains 6% sugar [Ozer & Ozer 1999], a major portion of the gelatinous matrix containing kefir microflora. Sugar present in kefir is known as kefiran, a heteropolysaccharide which is glucogalactan in nature. Kefiran improves viscosity and viscoelastic properties of acid milk gels [Rimada et al. 2006] and itself forms gels at low temperatures. Kefiran had been reported to form films isolated from LAB with low water vapor permeability and extra ordinary flexibility, even higher than those corresponding to low density polyethylene [Piermaria et al. 2009]. In addition, polysaccharides of kefir have several health promoting properties such as inhibitory effects on rotavirus [Song et al. 2007], immunomodulation or epithelium protection.

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Mineral Content Liut Kevicius and Sarkinas (2004) reported the presence of macro-elements such as potassium, calcium, magnesium, phosphorus and micro-elements such as copper, zinc, iron, manganese, cobalt, molybdenum in kefir.

Therapeutic Characteristics Kefir exhibits varied therapeutic attributes due to the possession of different components (Table 3), which are discussed below.

Anticarcinogenic Effect Epidemiological studies have indicated a reduced risk of breast cancer in women who consumed bovine fermented milk products (Redyy et al. 1983, Veer et al 1989). The bioactive anticancer components present in fermented bovine milk include proteins and peptides, which have been shown to have anti-tumorigenic activities in feeding trials both in animal models of cancer and in cultured tumor cells (Svensson et al. 1999). The anticarcinogenic role of fermented milk products may be broadly classified into prevention of cancer initiation and suppression of an initiated tumour by retarding the activity of enzymes that convert procarcinogen to carcinogen or by activating the immune system (Kneating, 1985). Anticarcinogenic effect of kefir and kefir extract has been studied extensively. Encouraging results regarding antitumor activities of kefir and isolated kefir extracts in animal studies have been reported (Shiomi et al. 1982, Cevikbas et al. 1994, Furukawa et al 1990, Kubo et al 1992). Shiomi et al. (1982) showed that polysaccharides extracted from kefir grains had antitumor activity in mice. Also, oral doses of 100 or 500 mg/kg of kefir fed to mice with transplanted solid tumors of E-ascites carcinoma were shown to cause a significant reduction in transplanted tumor size and activate the immunosuppressive activity of the spleen (Kubo et al 1992). It has been shown that isolated strains of Streptococcus, Lactobacillus and Leuconostoc (Hosono et al., 1990) and Streptococcus lactis subsp. cremoris (Miyamoto et al., 1991) from kefir were able bind mutagens. Ingestion of kefir (2 g/Kg body weight) for nine days proved to be more effective for tumour inhibition than yogurt (Furukawa et al., 1990), with better inhibition being noted (70.9 and 64.8 percent, respectively) for soy milk Kefir than milk kefir (Liu et al., 2002). Kefiran, which is a watersoluble glucogalactan, either isolated from kefir grain or produced by L.kefiranofaciens, a strain isolated from kefir, (Wang et al. 2008) also have antitumor activity. However water soluble polysaccharides containing kefir grain microflora are more efficacious than water soluble polysaccharides for the suppression of tumors (Furukawa et al., 2000) and higher dosages may be more effective if administered after the establishment of tumours (Murofushi et al., 1983). It has been concluded that antitumour properties of kefir may be either due to the microorganisms or polysaccharides produced during fermentation (Liu et al. 2002), especially

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lactobacillus (Santos et al. 2003). Guzel-Seydim et al. (2003) registered that based upon previous research, milk protein and especially those with high concentration of sulphur containing amino acids are important for anticarcinogenicity. De Moreno De Leblanc et al. (2007) studied the involvment of immune cells in the antitumor effect of kefir in a murine breast cancer model. He found that immune response in mammary gland played an important role to avoid tumor growth. Chen et al (2007) studied the effect of kefir extract on suppression of estrogen-dependent human breast cancer cells. He concluded that extracts from early-stage kefir fermentation (kefir mother culture) and the final kefir product demonstrated antiproliferative effects that were specific to breast cancer cells. Although the bioactive component(s) and the mechanism of the antitumor activity were not clear, but his study suggested the potential of kefir fermentation produced new peptides or other bioactive compounds, which could have anticancer activities. Figure 3 exhibits proposed sites and modes of action of Kefir throughout tumor cycle.

Antibacterial Spectrum There are data to show that many lactobacilli are capable of producing a wide range of antimicrobial compounds, including organic acids (lactic and acetic acids), carbon dioxide, hydrogen peroxide, ethanol, diacetyl and peptides (bacteriocins) that may be beneficial not only in the reduction of foodborne pathogens and spoilage bacteria during food production and storage, but also in the treatment and prevention of gastrointestinal disorders and vaginal infections (Zamfir et al. 1999 Zhou et al., 2008, Liu et al 2008, Simova et al. 2009 ).The beneficial action of kefir can be partially attributed to the inhibition of pathogenic microorganisms by metabolic products such as organic acids produced by kefir microflora (Golowczyc et al 2008). The antibacterial activity of fermented milk products could be attributed to metabolic end products present and/or antibacterial compounds produced by starter cultures. Kefir has a higher bacteriostatic effect against Gram-negative organisms but a better bactericidal effect against Gram positive organisms (Czamanski et al., 2004). The antagonistic behaviour of kefir against Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica (Gulmez and Guven, 2003), Listeria innocua (Morgan et al., 2000), Salmonella enteritidis (Czamanski et al., 2004) and Staphylococcus aureus (ATCC 29213), Bacillus cereus (ATCC 11778), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (ATCC 7644) and Escherichia coli (ATCC 8739) has been mentioned (Ulusoy et al. 2007). Silva et al. (2008) studied the effect of antimicrobial activity of broth (added with different sugars) with kefir grains and he found that kefir grains promoted the hydrolysis of non-reducing sugars, which were converted into organic acids and substances capable of producing inhibition halos in experiments with pathogenic microorganisms. The inhibited species included: Candida albicans, Salmonella typhi, Shigella sonnei, Staphylococcus aureus, and Escherichia coli.

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Figure 3. Tumor formation in body and proposed sites of action of kefir.

In an in vitro study conducted by Rodrigues et al (2005a), kefir inhibited the growth of Streptococcus pyogenes and Candida albicans. Santos et al. (2003) noted the antagonistic behavior of isolated strains of lactobacilli from kefir grains against E. coli (43/58 strains), L. monocytogenes (28/58 strains), Salmonella typhimurium (10/58 strains), S. enteritidis (22/58 strains), Shigella flexneri (36/58 strains) and Y. enterocolitica (47/58 strains). In another study, strains of Lactococcus cremoris, Lc. lactis, Str. thermophilus and Str. durans, isolated from kefir inhibited the growth of S. aureus (Yuksekdag et al 2004a). In the same study, two strains of Lc. lactis and a strain of Lc. Cremoris inhibited the growth of E. coli and Pseudomonas aeruginosa (Yuksekdag et al., 2004a). Witthuhn et al. (2004) also described a strain of Str. thermophilus active against P. aeruginosa. The antibacterial activity of kefir depends on the extent of fermentation. The populations of E. coli, L. monocytogenes and Y. enterocolitica increase in kefir fermented for one day but only E. coli increased when fermented for two days (Gulmez and Guven, 2003). The antibacterial activity of kefir against various pathogens may be attributed to organic acids and specific antibodies produced by acetic acid bacteria and yeasts (Koroleva, 1988), undissociated lactic and acetic acid produced during fermentation (Garrote et al. 2000) or hydrogen peroxide produced by LAB (Yuksekdag et al. 2004a, 2004b).

Effect on Immune System Nutritional status has a major impact on the immune system (Vinderola et al. 2006a). Numerous studies have demonstrated the beneficial effects of lactic acid bacteria (LAB) and fermented dairy products in boosting specific or nonspecific immune responses (Gill, 1998; Isolauri et al. 2004, Matar et al. 2001, Perdigon et al. 2001, Vinderola et al. 2004). Probiotic microorganisms can exert their beneficial properties mainly through two mechanisms: direct effects of the live microbial cells (probiotics) or indirect effects via metabolites of these cells

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(biogenics). Biogenics are defined as food components derived from microbial activity which provide health benefits without involving the intestinal microflora (Takano, 2002) while irregularity in gut microflora functions could potentially contribute to a wide range of diseases [Mai et al. 2009]. Vinderola et al (2005b) demonstrated the immunomodulating capacity of kefir in a murine model, showing the importance of the dose and cell viability to obtain a Th2 or Th1 response. Kefir can increase the phagocytic activity of peritoneal and pulmonary macrophages and can modulate the mucosal response at distant sites. In the latter study they observed the effects of kefir microflora and its non-bacterial fraction on cytokine production by cells of the innate immune system (peritoneal macrophages and adherent cells derived from Peyer’s patches). Moreover they noticed a higher capacity of the products derived from milk fermentation by kefir microflora (PMFKM), compared with kefir microflora itself, of inducing the proliferation of IL-10 producing cells among adherent cells derived from Peyer’s patches of mice(Vinderola et al. 2006a).In the most recent study, Vinderola et al. (2006b) demonstrated the immunomodulating capacity of kefir in a murine model, aimed at studying the immunomodulating capacity in vivo of the products derived from milk fermentation by kefir microflora (PMFKM) on the gut. They observed that the PMFKM induced a mucosal response and it was able to up and down regulate it for protective immunity, maintaining the intestinal homeostasis, enhancing the IgA production at both the small and large intestine level. Table 3. Therapeutic attributes of kefir Therapeutic attributes Anti-carcinogenic effect Antimutagenicity Antioxidant Scavenging activity Mucositis Cholestrol lowering effect

Immunomodulating capacity Anti-allergenic properties Antibacterial spectrum

Gastrointestinal proliferation b-galactosidase activity Bacterial colonization Antiflammatory

Therapeutic components Polysaccharide Peptide Peptide

Polysaccharide Cholesterol degrading enzyme Kefir as whole kefir and soymilk kefir Hydrogenperoxide Lactic acid Acetic acid Bacteriocin Lactic acid bacteria b-galactosidase enzyme S-layer protein Polysaccharide

References Furukawa et al. (2000) Liu et al. (2005) Liu et al. (2005) Liu et al. (2005) Nagira et al.(1999a) Topuz et al. (2008) Liu et al. (2006b) Vujicic et al. (1992) Vinderola et al. (2005a) Liu et al. (2006) Yuksekdag et al. (2004b) Garrote et al. (2000) Garrote et al. (2000) Santos et al. (2003) Marquina et al. (2002) De Vrese et al. (1992) Garrote et al. (2004) Moreira et al (2008)

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Thoreux and Schmucker (2001) fed kefir produced from grains to young (6 months) and old (26 months) rats and found an enhanced mucosal immune response in the young animals, as shown by a higher anti-cholera toxin (CT) IgA response compared to controls. Both young and old rats had significantly increased total non-specific IgGblood levels, and a decreased systemic IgG response to CT. Taken together, Thoreux and Schmucker concluded that kefir, like other probiotics, was exerting an adjuvant effect on the mucosal immune system, perhaps produced by bacterial cell wall components. Stimulation of the immune system may also occur due to the action of exopolysaccharides found in kefir grains. Murofushi et al. (1983) used the method of La Riviere et al. (1967) for the extraction of kefiran from kefir grains to produce a water-soluble polysaccharide fraction that they fed to mice. The reduction in tumour growth that they observed was linked to a cell-mediated response, and it appeared that the total dose of the polysaccharide determined its effectiveness. Furukawa et al. (1992) have also shown that a water-soluble fraction of kefir grains may act as a modulator of the immune response. Vinderola et al (2006c) also observed the effects of kefir microflora and the non-bacterial fraction on cytokine production by cells of the innate immunity-adherent populations of Peyer’s patches and the peritoneal macrophages. Since kefir drink contains kefiran, it was of interest to determine the effect of this exopolysaccharide on immune function. It was found that the exopolysaccharide induced a gut mucosal response and it was able to up and down regulate it for protective immunity, maintaining intestinal homeostasis, enhancing the IgA production at both the small and large intestine level and influencing the systemic immunity through the cytokines released to the circulating blood. The effect of kefir exopolysaccharides on the immune system may be dependent on whether the host is healthy or has developed any tumours. Furukawa et al. (1996) incubated kefir grain polysaccharides with Peyer’s Patch (PP) cells from tumour-bearing mice and found that the supernatant of this mixture enhanced proliferation of splenocytes from normal mice and increased the mitogenic activities of lipopolysaccharides (LPS) and phytohaemagglutinin- P (PHA-P) in splenocytes. They concluded that the polysaccharide stimulated PP cells, causing them to secrete water-soluble factors that, in turn, enhanced the mitogenic response of thymocytes and splenocytes in normal mice.

Anti-inflammatory Rodrigues and coworkers (2005b) demonstrated the anti-inflammatory properties of kefir and its polysaccharide extract. The results showed significant inhibition in the formation of granuloma tissue for all the test groups, as compared to the blank group. Kefir suspensions in molasses presented an inhibition of 41 ± 3% for the inflammatory process, fermented milk prepared from kefir showed 44 ± 6% inhibition and kefiran extract 34 ± 15%. In order to investigate the pharmacological effects of kefir, Lee and coworker (2007) used a mouse asthma model, in which airway inflammation and airway remodeling was produced by ovalbumin sensitization and challenge. BALB/c mice sensitized and challenged to ovalbumin, were treated with kefir (50 mg/kg administered by intra-gastric mode) 1 h before

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the ovalbumin challenge. Histological studies demonstrated that kefir substantially inhibited ovalbumin-induced eosinophilia in lung tissue and mucus hypersecretion by goblet cells in the airway. Kefir displayed anti-inflammatory and anti-allergic effects in a mouse asthma model and may possess new therapeutic potential for the treatment of allergic bronchial asthma. Allergic asthma is a complex chronic airway disorder that is characterized by airway inflammation, lung eosinophilia, mucus hypersecretion by goblet cells, an elevated serum IgE level, and airway hyperresponsiveness (AHR) (Elias et al. 2003). An orally administered L. plantarum isolated from kefir, had also been reported to exert anti-inflammatory effects in a mouse model (Lee et al. 2007).

Hypocholesterolemic Effect Since the early studies of Mann & Spoerry (1974), there appears to have been an increasing interest in the hypocholesterolaemic activity of fermented dairy products. A number of studies have been performed with experimental animals, and also human subjects, in order to elucidate the effect of fermented dairy products on serum cholesterol (Anderson & Gilliland, 1999, St-Onge et al. 2000; Xiao et al. 2003). Although some contradictory results have been obtained (Nakajima et al. 1992; de Roos et al. 1998), the majority of results from these reports indicate that fermented dairy products, possibly including kefir, possess hypocholesterolaemic properties. Considerable attention has been given to the cholesterol level of foods due to its public health significance as high levels are associated with greater risk of cardiovascular disease. A higher population of LAB in kefir and their ability to bind up to 33.9 percent cholesterol led to a direct reduction of cholesterol by cultures in the intestine (Hosono and Tanako, 1995). Culturing of milk with kefir cultures at 248°C for 24 h induced an assimilation of cholesterol by 28-65 percent and the corresponding figure reached to 41-84 percent after 48 h of incubation (Vujicic et al. 1992). Application of invertase deficient yeasts such as Saccharomyces cerevesiae (Tamai et al. 1996) was reported to exert a hypocholesterolemic effect to consumers (Noh et al., 1997). Wojtowski et al. (2003) mentioned that sheep milk kefir can be considered to be more advantageous for health effects than kefirs from cow or goat milk due to the presence of higher levels of linoleic and/or linolenic acids. Hydroxymethylglutaric and/or orotic acids presumably inhibit a rate-limiting enzyme in cholesterol synthesis (Shahani and Chandan, 1979). Ozer and Ozer (1999) reported that the hypocholesterolemic effect of kefir in humans may be attributed to a loss of orotic acids during kefir fermentation, which are known to cause fat accumulation in the liver. Maeda et al. (2004) reported that an exopolysaccharide produced by L. kefiranofaciens reduced serum cholesterol level in rats when they consumed excessive dietary cholesterol. Liu et al (2006) studied the effect of milk kefir and soya-milk kefir in cholesterol-fed hamsters. He also found that the soyamilk, milk-kefir and soyamilk-kefir diets all tended towards a lowering of serum triacylglycerol and total cholesterol concentrations, and a reduction of cholesterol accumulation in the liver, the decrease in serum cholesterol concentration being mainly in the non-HDL fraction. These findings demonstrate that kefir or its component may be considered

Kefir and Health: A Perception to be among the more promising hypocholesterolaemic action. β-galactosidase Activity

food

components

57 in

terms

of

preventing

Lactose maldigestion is the inability to completely digest lactose, the major carbohydrate in virtually all mammalian milks. Lactose maldigestion affects approximately 75% of the world’s adult population and occurs most often as the result of a genetically programmed decrease in intestinal lactose activity after the age of 3 to 5 years. The use of fermented dairy foods has long been employed as a strategy for overcoming lactose intolerance (Kolars et al 1984). This appears to be related to the presence of β-galactosidase in the yogurt starter culture bacteria (Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) (Hertzler and Clancy 2003). Reduction in lactose content and the presence of β-galactosidase activity in cultured milk products render it suitable for consumption by persons classified as lactose-intolerant, attributed to better absorption of lactose than other sources of sugar and autodigestion of lactose by its endogenous microorganisms. Kefir grains possess β-galactosidase activity (De Vrese et al., 1992) and kefir may be equally effective as yogurt for reducing breath hydrogen in lactose-intolerant adults (Hertzler and Clancy, 2003). Kefir contains less lactose than milk (Dies, 2000) and have 60% higher β-galactosidase than plain yoghurt and its ingestion improves lactose digestion and reduced the perceived severity of flatulenceby 54-71 percent in contrast to milk (Hertzler and Clancy, 2003).

Gastrointestinal Proliferation Gastrointestinal tract constitutes the largest interface between animals and their external environment. As a barrier it prevents the penetration of harmful entities such as food antigens (Umeda et al 2005). Fermented milk products are capable of restoring the normal lactic intestinal flora by inhibiting undesirable flora and the antibacterial activity is influenced by the culture activity, temperature and time of storage and the initial level of contamination. Intake of kefir induced an abatement in LAB by ten-fold and a decline in levels of sulphite reducing Clostridia by 100-fold in mice (Marquina et al., 2002), benefited protein digestion and reduced glycemic index in female rats prevented (Urdaneta et al 2007), Campylobacter jejuni colonization in the caecum of chicks (Zacconi et al., 2003), affected the intestinal mucosal and systemic immune response in young rats (Troreux and Schmucker, 2001) and induced mucosal resistance to gastrointestinal infection in mice (Liu et al., 2002). Kefir is helpful in the treatment of post-operative patients or patients with gastrointestinal disorders (Fil’ Chakova and Koroleva, 1997). Murashova et al. (1997) noted that infants with severe intestinal infection receiving bifidokefir (kefir containing physiological cells of Bifidobacterium bifidum) induced rapid inhibition of Salmonella and Shigella within 7-11 days of illness and the faecal flora reverted to normal after 4.8 ±0.8 days, whereas the corresponding figures for the group of infants not receiving bifidokefir were 12-18 and 6.6 ± 0.9 days, respectively. Bifidokefir containing 5x107 Bifidobacteria/ml had a positive effect on intestinal flora in humans and normalized intestinal Bifidobacteria: Lactobacilli balance

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accompanied by a decline in the population of pathogens and abatement in lactic acid bacteria within 7-14 days (Molokeev et al., 1998). Bacterial Colonization Lactic acid bacteria in food can transiently colonize the intestine and exert beneficial effects (probiotic). Survival during intestinal transit or adhesion to epithelium or both seem to be important for modifying the host’s immune reactivity. Because Successful seeding of bacteria depends on their bile salt tolerance and ability to withstand the conditions prevailing in the intestine (Schiffrin et al 1995). It has been observed that 85 percent of the isolated species of Lactobacillus from kefir grains can resist oxgall and many of them are able adhere to enterocyte-like cells (Santos et al., 2003). Similarly, yeasts like Kluyveromyces lactis and Kluyveromyces ladderae are more acid tolerant and have the ability to adhere to human intestine due to the proteinaceous factor (Kumura et al., 2004). Garrote et al. (2004) also mentioned that the presence of S-layer protein in Lactobacillus kefir and Lactobacillus parakefir was responsible for the adhesive properties to CaCo 2 cells, auto aggregation and haemagglutination. Both specific attachment and multiplication on the surface of the membrane of the gastro-intestinal epithelial cells are critical mechanisms in the establishment of these organisms in the intestinal tract. Adhesion is not a necessary requirement for the successful colonization of the intestine, but those organisms that do adhere may have more effect on the physiological functioning of the intestinal tract (Cole and Fuller, 1984).

Anti-Diabetic Effect Kwon and coworkers (2006) studied anti-diabetes functionality of Kefir culture-mediated fermented soymilk supplemented with Rhodiola extracts. Their results indicated that Kefir culture-mediated fermentation of soymilk supplemented with Rhodiola extracts resulted in mobilization of total phenolics, which could be effectively designed as complimentary therapies for postprandial hyperglycemia. Water-soluble or chloroform/ methanol-extracted fractions from Kefram-Kefir were examined to evaluate the glucose uptake ability of L6 myotubes. As a result, the water-soluble fraction augmented the uptake of glucose in L6 myotubes both in the presence and absence of insulin stimulation. Estimation of intracellular ROS level revealed that the water-soluble fraction of Kefram-Kefir reduced the intracellular ROS level on both the undifferentiated and differentiated L6 cells linked to Type II diabetes management. So it was suggested that the water-soluble fraction of Kefram-Kefir activates PI 3-kinase or other upstream molecules in the insulin signaling pathway, which resulted in the augmentation of glucose uptake and its specific inhibition by wortmannin (Teruya et al 2002). Later on Maeda et al (2004) also found that kefiran supplementation demonstrated the ability to significantly lower blood glucose in KKAy mice.

Antiallergic Properties

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Food allergy is now recognized as a worldwide problem, and like other atopic disorders its incidence appears to be increasing. Consumption of milk kefir and soymilk kefir suppressed the IgE and IgG1 responses and altered the intestinal microflora in our supplemented group, suggesting that milk kefir and soymilk kefir may be considered among the more promising food components in terms of preventing food allergy and enhancement of mucosal resistance to gastrointestinal pathogen infection (Liu et al 2006). Another histological studies demonstrated that kefir substantially inhibited ovalbumin-induced eosinophilia in lung tissue and mucus hypersecretion by goblet cells in the airway. Kefir displayed anti-inflammatory and anti-allergic effects in a mouse asthma model and may possess new therapeutic potential for the treatment of allergic bronchial asthma (Lee et al 2007).

Antioxidative Properties Dietary constituents of antioxidative vitamins and other nutrients may play an important role in protecting the body against oxidative damage. Guven et al (2003) carried out study to investigate the protective effect of kefir against oxidative damage of CCl4 in mice, compared with the well-known antioxidant vitamin E. Three-week-old Swiss Albino mice, weighing 22–26 g were used for the experiment. At the end of the microbiological analysis of kefir, the averages of the total mesophilic aerobic colony counts, lactic acid bacteria, lactic streptococci, enterococci, and yeasts were found to be 1.04 × 109, 9.87 × 108, 4.38 × 108, 7.80 × 104 and 1.26 ×105 CFU/ml, respectively. While both vitamin E and kefir were found to have a protective effect aganist CCl4-induced damage, kefir was more protective (Guven et al 2003).

Effect on Lipid and Blood Pressure Level A study on the effects of kefiran (polysaccharide present in kefir) in animals demonstrated that kefiran significantly suppressed increase of blood pressure and reduced the serum cholesterol levels in SHRSP/Hos rats when subjects consumed excessive dietary cholesterol. These results suggested that kefiran could be used as a functional food to prevent some commonly occurring diseases (Maeda et al. 2004).

Protection against Apoptosis UV irradiation produces reactive oxygen species from in skin cells and damage melanocytes and other skin cells, causing liver spots, freckles, wrinkles and skin cancer. The exposure of the intestine to ionizing radiation results apoptotic death of the stem cells. Nagira and coworkers (1999a) studied the studied the scavenging effect of the Kefran-Kefir against superoxide radicals and the protective effect of the Kefran–Kefir extract on UV damage of human melanoma HMV-1 cells. When HMV-1 cells were treated with the Kefran-Kefir

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extracts before, during or after UV irradiation, the alive cell number was remarkably increased compared to the control values, proving that Kefran–Kefir can protect human melanomas from UV damage. In another study they found that apoptosis of HMV-1 cells caused by UV irradiation was also suppressed by the Kefir extract, suggesting that DNA repair enhancing factors in the Kefir extract could decrease DNA damage by UV irradiation and suppress apoptosis (Nagira et al 1999b). To evaluate the effect of fermented milk kefir on X-ray-induced apoptosis in the colon of rats, Matsuu and colleague (2003) examined the apoptotic index, the mean number of apoptotic cells detected by H&E staining per crypt in the colon, in control rats and kefir-pretreated rats drinking kefir for 12 days before irradiation. They demonstrated that kefir protected the colonic epithelial stem cell region, and is extremely important for regeneration following radiation-induced apoptosis; this antiapoptic effect of kefir was mediated through the inhibition of caspase-3 activation. Kefir treatment may have possibilities to diminish side effects in the intestinal epithelium of patients undergoing irradiation therapy for malignancy.

Conclusion Recently, consumers’ growing preference for food with enhanced nutritional and therapeutic properties has led to the inclusion of various cultured milk products as part of their diet based upon their dietetic features. Among them kefir can attained a significantly high ranking due to its long list of therapeutic properties. The self carbonated beverage, kefir may be recommended as a dietary beverage owing to its nutritional attributes due to its vitamin, protein and mineral content and therapeutic attributes, namely its antibacterial spectrum, gastrointestinal proliferation, hypocholesterolemic effect, anticarcinogenic effect, L(+) lactic acid content, β-galactosidase activity, protection against apoptosis, Effect on lipid and blood pressure level antiallergic properties, antidibetic properties, anti-inflammatory and bacterial colonization. Kefir is able to normalize intestinal microflora and is highly suitable for consumption by normal and sick adults as well as infants.

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Vinderola, C.G., Duarte, J., Thangavel, D., Perdigon, G., Farnworth, E. & Matar, C. (2005b). Remote-site stimulation and duration of the immune response by kefir. European Journal of Inflammation, 3, 63–73. Vinderola, C.G., Perdigon, G., Duarte, J., Farnworth, E. & Matar, C. (2006a). Effects of kefir fractions on innate immunity. Immunobiology 211, 149–156. Vinderola, G., Perdigon G., Duarte, J., Farnworth, E. and Matar, C. (2006b). Effects of the oral administration of the products derived from milk fermentation by kefir microflora on immune stimulation. Journal of Dairy Research, 73, 472–479. Vinderola, G., Perdigon, G., Duarte J., Farnworth, E. & Matar, C. (2006c). Effects of the oral administration of the exopolysaccharide produced by Lactobacillus kefiranofaciens on the gut mucosal immunity. Cytokine, 36, 254–260. Vinderola, C.G., Medici, M., Perdigon, G., (2004). Relationships between interaction sites in the gut, hydrophobicity, mucosal immunomodulating capacities and cell-wall protein profiles in lactic acid bacteria. Journal of Applied Microbiology, 96, 230–243. Vujicic, L.F., Vulic, M. and Komyves, T. (1992). Assimilation of cholesterol in milk by kefir cultures. Biotechnology Letters, 14, 847-850. Ulusoy, B.H., Çolak, H., Hampikyan, H. and Erkan, M.E. (2007). An in vitro study on the antibacterial effect of kefir against some food-borne pathogens. Türk Mikrobiyoloji Cemiyeti Dergisi, 37(2), 103-107. Wang, Y., Ahmed, Z., Feng, W., Li, C. and Song, S. (2008). Physicochemical properties of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir. International Journal of Biological Macromolecules, 43, 283–288. Wang, Y.F., Huo, G.C. and Liu, L.B. (2004). Isolation and identification of the lactic acid bacteria from Kefir grains. China Dairy Industry, 32,17-19. Webb, B.H. Johnson, A.H. and Alford, J.A. (1987). Composition of milk products. Fundamentals of Dairy Chemistry, p. 64. Delhi, CBS Publishers & Distributors. Witthuhn, R..C., Schoeman, T., & Britz, T.J. (2004). Isolation and characterisation of the microbial population of different South African kefir grains. International Journal of Dairy Technology, 57, 33–37. Wojtowski, J., Dankow, R., Skrzypek, R. and Fahr, R.D. (2003). The fatty acid profiles in Kefirs from sheep, goat and cow milk. Milchwiss, 58, 633-636. Xiao, J.Z., Kondo, S., Takahashi, N., Miyaji, K., Oshida, K., Hiramatsu, A., vIwatsuki, K., Kokubo, S. & Hosono, A. (2003). Effects of milk products fermented by Bifidobacterium longum on blood lipidsvin rats and healthy adult male volunteers. Journal of Dairy Science, 86, 2452–2461. You, S.J., Cho, J.K., Ha, C.G., Kim, C.H. and Heo, K.C. (2006). Probiotic properties of the Candida kefyr isolated from kefir. Journal of Animal Science and Technology, 48, 307314. Yuksekdag, Z.N., Beyath, Y. and Aslim, B. (2004). Metabolic activities of Lactobacillus spp. strains isolated from kefir. Nahrung/Fd., 48, 218-220. Yuksekdag, Z.N., Beyatli, Y. and Aslim, B. (2004b). Determination of some characteristics coccoid forms of lactic acid bacteria isolated from Turkish kefirs with natural probiotic. Lebensmittel-Wissenschaft und-Technologie, 37, 663-667.

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Zacconi, C., Scolari, G., Vescova, M. and Sarra, P.G. (2003). Competitive exclusion of Campylobacter jejuni by Kefir fermented milk. Annals.of Microbiology, 53, 179-87. Zamfir, M., Callewaert, R., Cornea, P.C., Savu, L., Vatafu, I. and De Vuyst, L. (1999). Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801. Journal of Applied Microbiology, 87, 923-931. Zhou, K., Zhou, W., Li, P. Liu, G. R., Zhang, J., Dai, Y. (2008). Mode of action of pentocin 31- 1: an anti-Listeria bacteriocin produced by Lactobacillus pentosus from Chinese traditional ham. Food Control, 19, 817–822. Zourari, A. and Anifantakis, E.M. (1988). Le kir. Caractes physico- chimiques, microbiologiques et nutritionnels. Technologie de production. Une revue. Le Lait 68, 373-392. Zubillaga, M., Weill, R., Postaire, E., Goldman, C., Caro R. and Boccio, J. (2001). Effect of probiotics and functional foods and their use in different diseases. Nutrition Research, 21, 569-579.

In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter III

Fouling Reduction during Milk Processing Using Equipment Surface Modification Sundar Balasubramanian1,* and Virendra M. Puri2 1

Department of Biological and Agricultural Engineering, Louisiana State University AgCenter, 149 E.B. Doran Building, Baton Rouge, LA 70803, USA 2 Department of Agricultural and Biological Engineering, 229 Agricultural Engineering Building, Pennsylvania State University, University Park, PA 16802, USA

Abstract Fouling of equipment surfaces during milk processing is a phenomenon that needs to be immediately addressed due to the increased energy utilization and production costs encountered. Modifying the equipment surface is one method of reducing the incidence of fouling. Research was carried out at the Pennsylvania State University using different food-grade surface coatings to modify plate heat exchanger surface, and was tested for their ability to reduce fouling during skim milk pasteurization. The results were compared with traditional stainless steel 316 plate heat exchanger (PHE) surfaces typically used in the food industry. Results after 6 h continuous testing using a pilot scale PHE unit indicate that there was greater than 85% reduction in fouling when the three coated surfaces (AMC 148-18, Ni-P-PTFE and LectrofluorTM-641) were used. Chemical analyses of the foulants indicate that the coating integrity did not appear to be compromised for the LectrofluorTM-641 coatings. However, there were trace amounts of fluorine present in the foulants adhering to the other two coating types (AMC148-18 and Ni-P-PTFE). A preliminary cost estimate on the thermal energy savings when using the coated surfaces indicate that there is substantial savings in energy, further justifying the

*

Corresponding Author. E-mail: [email protected]; Tel: (225)-578-1072 (office).

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Sundar Balasubramanian and Virendra M. Puri use of these coated surfaces, and making them more attractive for possible implementation in the food industry.

Introduction Fouling is an undesirable side effect of thermal processing that results in an increase in electrical and thermal energy usages due to the decrease in heat transfer coefficient and the increase in pressure drop across the heat exchanger unit; thereby lowering the overall system performance. Fouling occurs during thermal processing of foods rich in proteins; milk and milk-based products. Unfolding of protein chains, commonly known as protein denaturation, occurs when protein-rich foods are treated with heat. When these unfolded proteins agglomerate and adhere to the food processing equipment surface, the phenomena is termed as fouling. The resultant fouling layer is a low thermal conductivity layer and results in an increase in the input thermal energy. Frequent cleaning of the fouling layer is required which results in further increased energy and water usage. Further ramifications on the food product quality and food safety aspects due to microbiological hazards, over heating or under heating of foods cannot be ignored as a result of fouling. Fouling of processing lines particularly plate heat exchangers has a profound economic impact on the operating costs. A study estimates that about 80% of the total operating costs involved in a typical dairy plant are attributed towards the effects of fouling (Bansal and Chen, 2006). In dairy plants, the frequency of cleaning the fouled processing equipment is higher than in other industries where fouling is prevalent. Cleaning the fouled equipment is a time-consuming and energy-intensive process, consuming large quantities of water and chemicals. For a typical dairy processing plant handling 283.91 m3 of milk per day an average of approximately 416,395.3 m3 of water per year is required for cleaning (Rausch and Powell, 1997). If the equipment is severely fouled or if the foulants adhere strongly with the equipment surface, then the water and chemical requirement for cleaning the dairy equipment further increases. On an average the CIP (cleaning-in-place) process accounts for 9.5% of the primary energy demand (energy consumption) in the Dutch dairy industry and accounts for 0.14 to 0.30 MJ/cycle of the thermal energy requirement for milk pasteurization (Ramirez et al., 2006). To add to this high energy requirement, the incidence of fouling causes about 21% increase in the total energy consumption related to the operation and cleaning of milk pasteurization units (Sandu and Singh, 1991). Thus, prolonging the processing operation and minimizing the frequency of cleaning the foulants can be achieved by minimizing or delaying the process of equipment fouling. This results in substantial energy and cost savings. Equipment surface properties like roughness, wettability, charge, and charge density influence the protein adsorption (fouling). Hence, modification of the processing equipment surface properties could reduce the rate of protein adsorption by minimizing the surface free energy. This has led to an interest in investigating the role of modified surfaces on fouling reduction. Primarily, two methods are used to modify the surface properties of equipment: chemical methods and mechanical methods (Zettler et al., 2005). Machining, sand blasting, and pulsed plasma combined with photolithographic techniques are some of the mechanical

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means of altering surface roughness, thus altering the coefficient of friction and the frictional forces acting on the food product flowing over the equipment surface (Pier Francesco et al., 2006; Muller et al., 2006; Bretagnol et al., 2007). In the chemical approach, use of anti-fouling coatings, ion implantation techniques, and acid etching are some of the methods employed to modify the surface characteristics (Rosmaninho and Melo, 2006; Ban et al., 2006; Braceras et al., 2007). There is significant literature available on the use of non-stick anti-fouling coatings like Ni-P-PTFE (nickel phosphorus polytetrafluroethylene) for reduction of fouling during processing of food products (e.g., Zhao et al., 2005; Rosmaninho and Melo, 2006; Zhao and Liu, 2006). However, there could be some concerns on the use of non-stick coatings in food processing equipment owing to the possibility of coating degradation due to the processing conditions and food components that come in touch of the coating material. This could lead to the possibility of coating incursion into the processed food. Some studies have pointed out the lower wear resistance and lower adhesion property of Ni-P-PTFE coatings on stainless steel (Zhao and Liu, 2006; Wu et al., 2006). Hence, it is imperative to find a suitable commercially available coating material that will have good binding properties with stainless steel as well as be able to provide the necessary surface conditions that could reduce or prevent fouling. A research effort was undertaken at the Department of Agricultural and Biological Engineering, Pennsylvania State University to investigate the proof-of-concept of using various food-grade coatings in reducing fouling during skim milk pasteurization. Successful implementation of this technique will have a significant impact on reduction of energy consumption in the food industry, improve food quality, reduce the extensive use of cleaning chemicals, and reduce the use of valuable natural resources such as water. This chapter summarizes the results obtained during the course of that study.

Materials and Methods Plate Heat Exchanger Set-up The plate heat exchanger (PHE) system for skim milk pasteurization has been described by Balasubramanian and Puri (2008a) in detail. The PHE system was designed and installed in-house at the Pilot Plant Facility of the Department of Agricultural and Biological Engineering, the Pennsylvania State University. Briefly, the system consists of a PHE unit (Model M6-MBase, Alfa Laval USA, Glen Allen, VA, USA) with heating and cooling sections that were fed with hot water by heating and cooling sub-systems, and a data acquisition unit. To accelerate the occurrence of fouling no re-generation section was used in the study. This was in accordance to the findings of researchers who indicate that pre-heating milk reduces the extent of fouling (de Jong 1997; Visser and Jeurnink 1997). The PHE unit was operated in a single pass counter-current flow path. The temperature of the fluids entering and leaving the PHE system was recorded using Ttype thermocouples (type T) installed at various locations in the process line including the nine locations shown in Figure 1 to record the time-temperature history along the middle flow channel. In addition, the thermocouple CT monitored the exiting fluid milk temperature,

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which served to control the heater by turning it on or off. This control thermocouple ensured that the temperature of the fluid milk leaving the heating channels to be 72±2oC. The timetemperature data were collected every 2 s using LabView software (Version 6i, National Instruments, Austin, TX, USA) via a data acquisition system.

Figure 1. Plate arrangement and milk flow direction in the heating section of the PHE system (top). The bottom figure shows the plate with the thermocouples in 9 locations on the plate surface (denoted by dots) and the control thermocouple in the flow channel (Recreated from Balasubramanian and Puri, 2008a).

Table 1. Physical properties and calculated overall heat transfer values for the control (SS-316) and SS-316 coated surfaces in the PHE system (Balasubramanian and Puri, 2009) Surface type

Thermal conductivity 1

Control / SS-316 Ni-P-PTFE Lectrofluor-641TM AMC148-18 1 2

W/mk 16.3 5.44 0.245 1.4

Continuous usage temperature1, o C NA 316 260 229

Coefficient of friction1

Calculated overall heat transfer coefficient2, W/m2K

0.15-0.25 < 0.1 0.1-0.15 0.2-0.3

244.3 244.0 238.2 243.2

Values obtained from the coating manufacturers. Calculated using the equations available for determining the Nusselt, Prandtl, and Reynolds numbers. The calculations were based on coating material of thickness of 13 microns (0.0005”) applied uniformly onto the stainless steel plates of thickness 3 mm.

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Food Grade Surface Coatings After carefully studying the various properties (chemical resistance, thermal properties, mechanical strength, coefficient of friction and cost of coating the material) of different foodgrade coating materials available in the market, three coating materials (Table 1) were selected for this study. The various types of coatings applied are briefly discussed below. Ni-P-PTFE Coatings The final coating contained a blend of hard materials (like nickel and phosphorus) mixed with finely suspended particles of a soft material, Teflon® (about 4 microns in size). The coating was applied by an electroless nickel procedure. The strong carbon-fluorine bond in Teflon® imparts chemical inertness and non-stick qualities to the surface. The phosphorus increases the hardness of the surface and enhances its corrosion resistance property while nickel helps the coated surface to maintain an appreciable thermal conductivity value. Although the precise composition is not known due to the proprietary nature, Ni-P-PTFE coatings usually contains about 15-25% Teflon,® 60-65% nickel and 10-12% phosphorus. The TM-117P Ni-P-PTFE coating (Techmetals Inc., Dayton, OH, USA ) was applied uniformly all over stainless steel-316 PHE plates (both faces) to a thickness of 20-25 µm, yielding thermal conductivity of 5.44 Wm-1K-1 (Techmetals, 2008). LectrofluorTM-641 Coatings The polymer-based LectrofluorTM 641 coatings (General Magnaplate Corporation, Linden, NJ, USA) have a lower thermal conductivity (0.25 Wm-1K-1) value than that of Ni-PPTFE coatings (General Magnaplate, 2007). The advantage of using polymer-based coatings is that they are more resistant to the cleaning chemicals than Ni-P-PTFE coatings. Since these coatings are already being used in the bio-medical industry (in surgical instruments and in implants) they anticipated to have minimum interaction with the food products during contact. These coatings are known to be deposited on the substrate either by standard spraying method or electrostatic spraying method (the exact procedure is proprietary). LectrofluorTM-641 – USDA, FDA, and AgriCanada code compliant – was applied uniformly to one of the SS-316 PHE plates (both faces) to a thickness of 13-25 µm. AMC148-18 Coatings The AMC148-18 coatings (Advanced Materials Components Express, AMCX, Lemont, PA, USA) was applied onto the cleaned SS-316 surface in a two-step process. In the first step, the AMC-18 layer was reactively bonded to the SS-316 substrate through an intermediate layer (complementary metal-oxide or nitride semiconductor) which was in turn bonded to the SS-316 surface. Once the AMC-18 layer was formed, the AMC-148 coating was applied to form a robust, low critical surface energy surface. The AMC-148 layer is FDA approved to be GRAS (generally regarded as safe) and is approved for full contact. More information regarding this coating is described elsewhere (Balasubramanian and Puri, 2008a). The overall thickness of the coating was about 0.4 to 0.5 μm.

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Fouling Experimentation The fouling experiments were conducted using skim milk obtained from the Penn State Creamery at two flow rates, namely 0.162 m3h-1channel-1 (typical flow rate in dairy industry) and 0.144 m3h-1channel-1 (performance evaluation at reduced flow rate attributed to foulant build-up). The system was optimized by a series of trial runs so that the temperatures of the incoming skim milk and pasteurized skim milk entering the cooling section of the PHE were maintained at 15 ± 3oC and 72 ± 2oC, respectively. A total of three experimental replications were conducted at the nominal flow rate (0.162 m3h-1channel-1) for the control and the three surface coatings forming a total of eighteen experimental trials. At the other flow rate, there were three replications conducted for the control and two types of coated surfaces (Ni-PPTFE and LectrofluorTM 641). The same coated plate was used for all the replications and flow rate condition tested. At the end of each experimental run, the PHE unit was disassembled and the experimental plate (control or coated plate) was removed and visually inspected for any defects. The weight of the plate was recorded prior to and following the experiment (16-h after plates were removed from the PHE unit and air dried). During the cleaning procedure, it was ensured that the coated plates were not in the PHE unit. This is because of the uncertainty of the coated plate’s reaction to strong acid and alkali solutions which are typically used during cleaning. The PHE unit was again assembled with SS-316 plates alone and the entire unit was cleaned thoroughly using alkali and acid based solutions as per the cleaning protocols outlined by the Penn State Creamery (Balasubramanian and Puri, 2008b).

Statistical Analysis One way analysis of variance (ANOVA) of the collected data was performed using the PROC ANOVA procedure available in SAS (version 9.1, SAS Institute Inc., Cary, NC, USA). The test was performed on the mean product pasteurization temperature values of the control and coated plate experiment trials. One way ANOVA was also performed on the weights of foulants collected from the different surfaces after the experiments (at the different flow rates) to ascertain if there was any significant difference (at α=0.05) between the fouling produced on the different surfaces. To analyze the difference in the total thermal energy utilized by the pilot-scale system containing the different coated surfaces and the control surface, Duncan multiple comparison test was performed in one way ANOVA. Duncan multiple comparisons was also conducted to determine if there was any difference between the average hot water temperatures entering and leaving the PHE system due to the use of the different coated surfaces and the control surface.

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Analytical Characterization of Foulants The foulants deposited on the plate surfaces were carefully removed and examined using x-ray photo electron spectroscopy (XPS) to determine if the coating material entered the processed food product. The procedure for analysis of the foulants by XPS is outlined by Balasubramanian and Puri (2008a). The detection limit using this technique was in the range of 0.1- 1.0 atom%.

Results Visual Inspection of Fouled Plated Surface Analysis of the average milk pasteurization temperatures showed no significant differences (p>0.05) during the entire 6-h duration of testing with the coated and control plates at the two different flow rates. This clearly indicates that similar pasteurization temperatures were obtained during the course of the experiments using the different surfaces. The fouling deposits on the test plates after the 6-h experiments under normal product flow conditions are shown in Figure 2. There was a region on the top left corner of the plates were similar fouling pattern was observed in all the plates types. This region of the plate was where the milk flowing on the left side of the plate surface changes direction towards the exit, and hence, encounters a reduction in velocity. Other than that region, the fouling patterns observed in all the plate types were not similar. For SS-316 and graded Ni-P-PTFE (TM117P) coated plates, the region containing the most dense fouling occurred on the left hand side of the plates, i.e., away from the product entry point. These regions of most fouling are adjacent to the side where hot water enters the heating section of the PHE on the backside of the plate. Owing to the higher thermal conductivity of SS-316 and Ni-P-PTFE (Table 1) these regions are exposed to higher temperature gradients than on the right hand side of the plates resulting in more fouling. Balasubramanian and Puri (2008 a and b) have shown the temperature gradients occurring across the plate surface during milk and tomato juice pasteurization. Jun et al. (2004) indicate that the temperature difference between the different regions of the plate (left, right, top, middle and bottom) can sometimes be as large as 12oC. For the LectrofluorTM-641 and the AMC148-18 plates, the regions of excess fouling are interestingly near the center and the upper right hand side (farther to the entrance of the product) section of the plate. In addition, the region of fouling observed on the AMC148-18 plates was more widespread and concentrated more towards the upper middle portion the plate surface. The velocity and corresponding momentum of the milk flowing up the ridged plate is suspected to influence the fouling deposition and the exact reasons for observing these conflicting fouling patterns are being investigated in detail.

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Figure 2. Milk fouling deposits occurring on the test plate, A. Stainless steel 316 B. LectrofluorTM-641 C.TM-117P graded Teflon® D. AMC-148-18. The arrows represent the product flow direction. Pictures were taken the next day (Balasubramanian and Puri, 2008a and 2008c).

The foulants deposited on SS-316 and AMC148-18 plates were held tightly to the surface requiring considerable abrasive (shear) force to dislodge them from the surface. On the other hand the foulants deposited on the coated surfaces appeared to be loosely held and could be easily dislodged with lesser (shear) force similar to the observation of Rosmaninho et al. (2007) on their investigation of calcium phosphate fouling using Ni-P-PTFE coating. Most foulants adhering to these coated surfaces could be removed by wiping it with a piece of cheese cloth indicating possible lesser time for cleaning and lesser use of cleaning chemicals. Inspection of the coatings after the experiments indicated that the LectrofluorTM-641 and AMC148-18 coatings did not discolor and there were no signs of peeling of the coating from the SS-316 surface. On the other hand, Ni-P-PTFE (TM-117P) coated surfaces exhibited blackening at certain spots all over the plate; though the coating did not appear to peel off. Oxidation of the nickel in the coating could be a reason for observing the discoloration, and further chemical analysis will help to reveal more information. Ni-P-PTFE coatings are believed to have low adhesion strength (Wu et al. 2006) and are known to form cracks as a result of its larger differential thermal expansion coefficients between the PTFE coating, the metal substrate and the dispersed Ni particles (Liu et al., 2007). Providing a nickel strike prior to coating the surface with Ni-P-PTFE or by using graded type Ni-P-PTFE coatings could minimize the problem of low adhesion to the metal substrate and improve the coating integrity (Huang et al., 2004; Valova et al., 2005). Another major concern on the use of Ni-PPTFE coatings is that they are susceptible to stripping by some acids particularly nitric acid (Balaraju et al., 2003), which is commonly used in CIP cleaning processes. Hence, if Ni-PPTFE coatings are used; nitric acid should be used with caution. The advantage of using polymer-based coatings like LectrofluorTM-641 is that they are more resistant to common cleaning chemicals than Ni-P-PTFE coatings.

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Amount of Foulants Deposited The amount of foulants adhering to each test plate and the relative amount of reduction in fouling occurring on the coated surfaces in comparison with standard SS-316 surface for skim milk pasteurization obtained during our experiments are shown (Table 2). At a given flow rate, there was a significant difference (p≤0.0001) between the amount of foulants deposited on the four different surfaces. Comparison of percentage reduction in the amount of foulants for control (SS-316) vs. food-grade surface coatings showed substantial decrease (greater than 85% reduction), Table 2. This result was noticed at both the flow rates. For the plates coated with LectrofluorTM-641, the extent of decrease in fouling when compared with SS-316 plates was as high as 94%. At the nominal (higher) flow rate (0.162 m3h-1channel-1), the amount of skim milk foulants deposited on the control plates was significantly less (p=0.013) than the amount of foulants deposited at a lower flow rate (Balasubramanian and Puri, 2009). Lower flow rates increase the residence time of the product within the PHE equipment resulting in higher incidence of fouling. At higher flow rates, the higher Reynolds numbers result in greater hydrodynamic forces increasing the shear forces acting upon the already deposited foulants. These forces in the long run could wear down the adhesive and cohesive forces between the foulants and the surface resulting in the possible dislodging of the deposited foulants. However, for the coated plates there was no significant difference observed between the amounts of foulants deposited at the two flow rates (p-value for the foulants deposited on the LectrofluorTM-641 and Ni-P-PTFE plates being 0.072 and 0.146, respectively). Hence, the amount of foulants deposited on the coated test plates did not vary at the tested flow rates. The preliminary test results obtained are encouraging since percentage decrease of fouling noticed due to the use of surface coatings could translate into substantial reduction in electrical and thermal energy usage. Also, with lesser amount of foulants, less time and resources for cleaning the surfaces will be required. These results are for a single coated channel in a single-pass countercurrent flow heat exchanger set-up. In actual practice the entire cooling and heating sections need to be coated. Since the thermal conductivity of the coatings used are a fraction lesser than that of SS-316 (Table 1), a loss in overall heat transfer is expected. However, when comparing the energy savings obtained through reduction in fouling, this loss in heat transfer could be overlooked since a net positive savings is expected in the long run.

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Figure 3. Comparison of the XPS spectrum obtained from the skim milk foulants adhering to the different surfaces (Balasubramanian and Puri, 2008a and 2008c).

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Table 2. Comparison of the foulants deposited on the various surfaces during a 6-hour experimental period of skim milk pasteurization (average of three replicates) (Balasubramanian and Puri, 2008a and 2008c) Coating

Stainless steel Ni-P-PTFE (graded) Lectrofluor-641TM AMC-148-18

Flow rate of 0.162 m3h-1channel-1 Percent decrease Weight of compared with foulants the next day (g) control next day (%) 8.4 ± 0.51 0.0 1.0 ± 0.15 87.5 ± 0.03

Flow rate of 0.144 m3h-1channel-1 Percent decrease Weight of compared with foulants the control next day next day (g) (%) 13.3 ± 1.93 0.0 1.9 ± 0.82 85.9 ± 0.04

0.4 ± 0.23 1.0 ± 0.31

0.8 ± 0.06 Not Applicable

94.7 ± 0.03 87.6 ± 0.04

94.2 ± 0.02 Not Applicable

Chemical Analysis of the Foulants A major constraint encountered during the present study was the limited information on the type of coating material and coating process (since it was deemed proprietary information). With this constraint, our investigation on the leeching/migration of the coating material into the processed product was limited to looking for known sources. Discussion with the coating manufacturers revealed that fluorine was an element present in all the coating materials investigated, and incursion of fluorine into the processed product is an undesirable contaminant. Hence, analyzing the extent of fluorine in the foulants will form a basis of understanding the coating material integrity. The spectra obtained from the foulants by XPS is shown in Figure 3. The binding energy for each element is unique and once that information was obtained it could be used to investigate if fluorine or any other desired/undesired element was present in the samples. From Figure 3 it can be seen that except for the plates coated with graded Ni-P-PTFE and AMC148-18, the foulants from the other two surfaces did not contain any fluorine. The extent of fluorine present in the foulants adhering to the Ni-P-PTFE coated plates was lower than that present in the AMC148-18 plates. This is indicated in Figure 3 at a binding energy level of 689.4 eV region (Yfantis et al., 1999) indicating the presence of fluorine in the milk foulant samples. The results are encouraging showing that the Lectrolfuor-641TM coating could withstand the process conditions without showing signs of peeling off. A detailed study on the foulants and the mechanism of fouling will need to be carried out to understand the anti-fouling properties of the applied coatings. This is beyond the scope of the present study which was primarily focused on studying the feasibility of using the novel coatings in controlling/minimizing fouling under pilot plant trial runs at continuous operation (6-h).

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Table 3. Approximate thermal energy savings incurred during skim milk pasteurization at 0.162 m3h-1channel-1 with the pilot-scale PHE set-up (Balasubramanian and Puri, 2009) Parameter

Mean hot water temperature, oC (T1) Ambient temperature, o C (T2) Mean returning hot water temperature, oC (T3) Heat required (Q1) for heating water in the tank from T2 to T1, MJ 1 Heat required (Q2) to sustain T1 for 6 h, MJ Total heat required (Q), MJ Total energy required, kWh Average percent change in energy over the control, %

Surface type SS-316 control

Ni-P-PTFE coated

LectrofluorTM -641 coated

AMC148-18 coated

78.7±0.5

81.9±2.6

80.6±0.6

76.8±0.2

20.5±0.9

21.4±0.2

23.5±0.4

19.7±0.9

59.9±0.5

66.4±3.4

65.1±0.4

59.4±0.3

37.28±0.88

38.79±1.84

36.56±0.09

36.54±0.47

306.32±16.10

253.36±13.00

252.55±2.41

282.16±1.72

343.59±16.98

292.15±11.16

289.11±2.51

318.70±2.19

95.44±4.72

81.15±3.10

80.30±0.70

88.52±0.61

2

-14.97

-15.86

-7.25

NA

1

Heat required to raise the temperature from T3 to T1. Not applicable.

2

Thermal Energy Savings during Skim Milk Pasteurization Although, the average hot water temperatures entering the PHE system (Table 3) had no significant difference (p > 0.0685) there was a significant difference (p < 0.0355) observed in the temperatures of hot water leaving the PHE system when the different coated surfaces were used. This change in temperatures will result in differences in the thermal energy requirements of the system. This was obvious while analyzing the thermal energy requirements during pasteurization of skim milk (at industry comparable flow rates) which indicated a significant difference in the energy requirements between the control and coated surfaces (p < 0.0174, R2 value of 0.902). Duncan multiple range test comparison also indicates that there was not a significant difference in the energy requirement while using the control and AMC148-18 coated surfaces. However, there was a maximum of 15-16% decrease in the total energy required while using the plates coated with Lectrofluor-641TM and graded Ni-P-PTFE (Table 3) when

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compared with the control. This is interesting since Lectrofluor-641TM has the least thermal conductivity value (Table 1) when compared with SS-316 and other coating materials.

Conclusion The three food-grade coated surfaces tested could reduce milk fouling by about 85-95% compared to the control SS-316 surfaces at the two product flow rates tested. These preliminary test results are encouraging by paving a step in the right direction on employing surface alteration techniques to minimize fouling. LectrofluorTM-641-coated plates did not appear to discolor during the experimental trials, reduced fouling by more than 94% and indicated 15.86% less thermal energy requirement when compared with the control surface. On the other hand, Ni-P-PTFE -coated plates and AMC148-18 showed some presence of fluorine in the milk foulants adhering to these surfaces after experimentation, and reduced fouling to a lower extent than LectrofluorTM-641-coated plates (about 85% when compared with control). However, the use of these two coating types also resulted in reduction in the thermal energy requirement utilized when compared with the control surface. The results are encouraging and demonstrate the ability of the modified surfaces to reduce fouling during milk pasteurization.

Acknowledgments The authors also would like to express their sincere gratitude and appreciation to the California Energy Commission’s Public Interest Energy Research (PIER) program for their critical financial support to undertake this timely research project.

Disclaimer The mention of a product or company name does not imply any endorsement, recommendation, exclusion, or any other type of implication by any of the authors or their affiliated entities.

Referentes Balaraju, J. N., Sankara Narayanan, T. S. N. & Seshadri, S. K. (2003). Electroless Ni-P composite coatings. Journal of Applied Electrochemistry, 33, 807-816. Balasubramanian, S. & Puri, V. M. (2008a). Fouling mitigation during product processing using a modified plate heat exchanger surface. Transactions of the ASABE, 51(2): 629639.

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Balasubramanian, S. & Puri, V. M. (2008b). Reduction of fouling during tomato juice pasteurization in plate heat exchanger system using food-grade surface coating. Food Manufacturing Efficiency, 2(1), 1-13. Balasubramanian, S. & Puri, V. M. (2008c). Reduction in milk fouling in a plate heat exchanger system using food-grade surface coating. Personal Communication, September 16, 2008. Balasubramanian, S. & Puri, V. M. (2009). Thermal energy savings in pilot-scale plate heat exchanger system during product processing using modified surfaces. Journal of Food Engineering, 91(4), 608-611. Ban, S., Iwaya, Y., Kono, H. & Sato, H. (2006). Surface modification of titanium by etching in concentrated sulfuric acid. Dental Materials, 22(12), 1115-1120. Bansal, B. & Chen, X. D. (2006). A critical review of milk fouling in heat exchangers. Comprehensive Reviews in Food Science and Food Safety, 5, 27-33. Braceras, I., Alava, J. I., Goikoetxea, L., de Maeztu, M. A. & Onate, J. I. (2007). Interaction of engineered surfaces with the living world: ion implantation vs. osseointegration. Surface and Coatings Technology, 201(19-20), 8091-8098. Bretagnol, F., Kylian, O., Hasiwa, M., Ceriotti, L., Rauscher, H., Ceccone, G., Gilliland, D., Colpo, P., & Rossi, F. (2007). Micro-patterned surfaces based on plasma modification of PEO-like coating for biological applications. Sensors and Actuators B: Chemical, 123(1), 283-292. de Jong, P. (1997). Impact and control of fouling in milk processing. Trends in Food Science and Technology, 8, 401-405. General Magnaplate. (2007). Magnaplate coatings – LectrofluorTM. General Magnaplate Corporation, Linden, New Jersey, USA. Available at: http://www.magnaplate.com/ coatings/lectrofluor.html. Accessed 28 September 2008. Huang, Y. S., Zeng, X. T., Hu, X. F. & Liu, F. M. (2004). Corrosion resistance properties of electroless nickel composite coatings. Electrochimica Acta, 49, 4313-4319. Jun, S., Puri, V. M. & Roberts, R. F. (2004). A dynamic 2D model for thermal performance of plate heat exchangers. Transactions of the ASAE, 47(1), 213-222. Liu, Z., Jayasinghe, S., Gao, W. & Farid, M. M. (2007). Corrosion mechanism of electrodes in ohmic cooking. Asia-Pacific Journal of Chemical Engineering, 2, 487-492. Muller, R., Hiller, K. A., Schmalz, G.., & Ruhl, S. (2006). Chemiluminescence-based detection and comparison of protein amounts adsorbed on differently modified silica surfaces. Analytical Biochemistry, 359(2), 194-202. Pier-Fransesco, A., Adams, R. J., Waters, M. G. J. & Williams, D. W. (2006). Titanium surface modification and its effect on the adherence of Porphyromonas gingivalis: an invitro study. Clinical Oral Implants Research, 17, 633-637. Ramirez, C. A., Patel, M., & Blok, K. (2006). From fluid milk to milk powder: energy use and energy efficiency in the European dairy industry. Energy, 31, 1984-2004. Rausch, K. D., & G. M. Powell. (1997). Dairy processing methods to reduce water use and liquid waste load. Department of Agricultural and Biological Engineering Report # MF2071, Cooperative Extension Service, Kansas State University, Manhattan, Kansas.

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Rosmaninho, R. & Melo, L. F. (2006). Calcium phosphate deposition from simulated milk ultrafiltrate on different stainless steel-based surfaces. International Dairy Journal, 16, 81-87. Rosmaninho, R., Santos, O., Nylander, T., Paulsson, M., Beuf, M., Benezech, T., Yiantsios, S., Andritsos, N., Karabelas, A., Rizzo, G., Muller-Steinhagen, H. & Melo, L. F. (2007). Modified stainless steel surfaces targeted to reduce fouling – Evaluation of fouling by milk components. Journal of Food Engineering, 80, 1176-1187. Techmetals. (2008). Engineered finishes. Techmetals Incorporated, Dayton, Ohio, USA. Available at: http://www.techmetals.com/EngineeredFinishes.asp. Accessed 25 September 2008. Valova, E., Dille, J., Armyanov, S., Georgieva, S., Tatchev, D., Marinov, M., Delplancke, J. L., Steenhaut, O. & Hubin, O. (2005). Interface between electroless amorphous Ni-Cu-P coatings and Al substrate. Surface and Coatings Technology, 190, 336-344. Visser, J. & Jeurnink, Th., J. M. (1997). Fouling of heat exchangers in the dairy industry. Experimental Thermal and Fluid Science, 14, 407-424. Wu, Y., Liu, H., Shen, B., Liu, L. & Hu, W. (2006). The friction and wear of electroless Ni-P matrix with PTFE and/or SiC particles composite. Tribology International, 39(6), 553559. Yfantis, A., Appel, G., Schmeiber, D. & Yfantis, D. (1999). Polypyrrole doped with fluorometal complexes: thermal stability and structural properties. Synthetic Metals, 106, 187195. Zettler, H. U., Weib, M., Zhao, Q., & Muller-Steinhagen, H. (2005). Influence of surface properties and characteristics on fouling in plate heat exchangers. Heat Transfer Engineering, 26, 3-17. Zhao, Q., Liu, Y., Wang, C., Wang, S. & Muller-Steinhagen, H. (2005). Effect of surface free energy on the adhesion of biofouling and crystalline fouling. Chemical Engineering Science, 60 (17), 4858-4865. Zhao, Q. & Liu, Y. (2006). Modification of stainless steel surfaces by electroless Ni-P and small amount of PTFE to minimize bacterial adhesion. Journal of Food Engineering, 72 (3), 266-272.

In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter IV

Milk Fat/Sunflower Oil Blends as Trans Fat Replacers Roberto J. Candal1 and María L. Herrera2 1

University of Buenos Aires, Faculty of Exact and Natural Sciences, INQUIMAE, Ciudad Universitaria, Buenos Aires, Argentina 2 University of Buenos Aires, Faculty of Exact and Natural Sciences, Organic Chemistry Department, Ciudad Universitaria, Buenos Aires, Argentina

Abstract As a body of evidence suggests that dietary trans fatty acids raise blood cholesterol levels, thereby increasing the risk of coronary heart disease, on July 11, 2003, FDA issued a final rule requiring the mandatory declaration in the nutrition label of the amount of trans fat present in foods, including dietary supplements. The agency required that the declaration of trans fat be on a separate line immediately under the declaration for saturated fat. Since there was no scientific basis for establishing a DV for trans fat, the final rule did not require the listing of a % DV as is required for some of the other mandatory nutrients, such as saturated fat. However, a report from the World Health Organization (WHO) and the Food and Agricultural Organization (FAO) of the United Nations has recommended a very low intake of TFA, less than 1% of daily energy intake. Therefore, efforts have been made and are ongoing to decrease TFA in the food supply both in the U.S. and globally. There are many challenges that food manufacturers have faced during the development of new trans fat alternatives. Any replacement ingredient must provide the functional characteristics of the material being replaced. In other words, the alternative ingredient must provide the functionality of flakiness, firmess of texture, crispness or desired appearance in the finished product or it is likely to be rejected by the consumer. The stability or shelf life of the finished product must also be maintained to ensure consumer acceptability. In some applications, like baked goods, a certain amount of solids is crucial. Consumer concerns associated with the atherogenic effect of trans

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Roberto J. Candal and María L. Herrera fatty acids limit the future of the hydrogenation process as a way of modifying the solidto-liquid ratio in vegetable oils/fats. As an alternative to hydrogenated vegetable oils, modification of high melting point stearins by blending with vegetable oils is becoming important, since shortenings with appropriate physicochemical properties and good nutritional characteristics that are free of trans fatty acids and rich in PUFA can be obtained. Thus, it is of interest to discuss the potential of blends of a stearin such as a high-melting fraction of milk fat with a vegetable oil as trans fat replacer. In this chapter the physical chemical properties of milk fat-sunflower oil low-trans blends, that is, crystallization behavior, polymorphism, microstructure and the effect of addition of emulsifiers in bulk systems will be reviewed.

Introduction As a body of evidence suggests that dietary trans fatty acids (TFA) raise blood cholesterol levels, thereby increasing the risk of coronary heart disease (CHD), on July 11, 2003, the Food and Drug Administration (FDA) issued a final rule requiring the mandatory declaration in the nutrition label of the amount of trans fat present in foods, including dietary supplements (DHHS/FDA, 2003). The agency required that the declaration of trans fat be on a separate line immediately under the declaration for saturated fat. It was anticipated that the declaration of this nutrient on a separate line will help consumers understand that trans fat is chemically distinct from saturated fat and will assist them in making dietary choices that aid in maintaining healthy dietary practices. For the purpose of nutrition labeling, trans fats are defined as the sum of all unsaturated fatty acids that contain one or more isolated (i.e., nonconjugated) double bonds in a trans configuration. Under FDA´s definition, conjugated linoleic acid would be excluded from the category of TFA (Schrimpf and Wilkening, 2005). Since there was no scientific basis for establishing a Daily Value (DV) for trans fat, the final rule did not require the listing of a % DV as is required for some of the other mandatory nutrients, such as saturated fat. However, a report from the World Health Organization (WHO) and the Food and Agricultural Organization (FAO) of the United Nations has recommended the traditional target intake of saturated fatty acids (for most people), less than 10% of daily energy intake, and less than 7% for high risk groups. A very low intake of TFA, less than 1% of daily energy intake, also was recommended. WHO/FAO considers myristic and palmitic acids and TFA to increase the risk of developing CHD (Anon, 2003; Hunter, 2005). Therefore, efforts have been made and are ongoing to decrease TFA in the food supply both in the U.S. and globally. There are many challenges that food manufacturers have faced during the development of new trans fat alternatives. Any replacement ingredient must provide the functional characteristics of the material being replaced. In other words, the alternative ingredient must provide the functionality of flakiness, firmess of texture, crispness or desired appearance in the finished product or it is likely to be rejected by the consumer. The stability or shelf life of the finished product must also be maintained to ensure consumer acceptability. Another major factor involved in the development of trans fat alternatives is the assurance that such products will be available in adequate commercial quantities. In some cases, this may mean very large quantities. There are several sources of trans fat alternatives:

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naturally stable oils/fats, interestirified oils, “modified” partially hydrogenated oils, traitenhanced oils from newer varietes, fractionation and blending of hard and soft feed stocks. These techniques may be used singly or in combination with each other (List and Reeves, 2005). Most native vegetable oils have only limited applications in their original forms due to their specific chemical composition. To widen their use, vegetable oils are modified either chemically by hydrogenation or interesterification or physically by factionation. In some applications, like baked goods, a certain amount of solids is crucial. However, consumer concerns associated with the atherogenic effect of trans fatty acids limit the future of the hydrogenation process as a way of modifying the solid-to-liquid ratio in vegetable oils/fats since during partial hydrogenation part of the cis double bonds are isomerized into their trans form. To produce a zero-trans solid fat with the physical properties and functionality of commercial fats, Petrauskaite et al. (1998) interesterified fat blends formulated by mixing a highly saturated fat (palm stearin or fully hydrogenated soybean oil) with a native vegetable oil (soybean oil) in different ratios from 10:90 to 75:25 (wt%). They concluded that chemical randomization of 20-50% highly saturated fat with a soft vegetable oil can be used as an alternative to partial hydrogenation to produce a plastic fat phase that is suitable for manufacture of shortenings, stick or tube-type margarines, and confectionary fats. The final products had comparable physical properties and acceptable fatty acid compositions. In addition, interesterified hard stocks can be further fractionated to obtain the required products with low to zero trans isomer contents. As an alternative to hydrogenated vegetable oils, modification of high melting point stearins by blending with vegetable oils is becoming important, since shortenings with appropriate physicochemical properties and good nutritional characteristics that are free of trans fatty acids and rich in PUFA can be obtained. Nor Aini et al. (1999) formulated a vanaspati, a vegetable oil-based product which is an alternative to Indian Ghee, by mixing palm oil solid fraction (palm stearin) with palm oil liquid fraction (palm olein) and/or palm kernel olein. These formulations were based on direct blending, thus there were no trans fatty acids. Some of them were suitable for the Malaysian market with slip melting point (SMP) not exceeding 44°C while others were recommended for Yemen market with SMP between 41 and 46°C. Pal et al. (2001) modified butter stearins, obtained by dry and solvent techniques of fractionation by blending and lipase-catalyzed interesterification process techniques. Liquid oils rich in polyunsaturated fatty acids were chosen for making fats with desired physical properties and fatty acid composition and therefore suitable for utilization in a variety of food products. On the basis of slip point and SFC data, these authors stated that the interesterified products were suitable in formulating melange products and spread fats with almost zero trans fatty acid content and with reasonable content of polyunsaturated fatty acids. Butter stearin fractions, on blending with liquid oils like sunflower oil and soybean oil in different proportions, offer nutritionally important fat products with enriched content of essential fatty acids like C18:2 and C18:3. Yella Reddy and Jeyarani (2001) prepared three types of bakery shortenings for cakes, biscuits and puff pastry by blending the fractions of mango kernel and mahua fats. Mahua trees, found in several parts of India, have green-colored egg-size fruits consisting of about 75% concave kernels that contain about 50% pale yellow semisolid fat. The fat is edible and can be used to prepare value-added products. Mango seeds, constituting 8-22% of the fruit,

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contain 45-73% kernel. The fat content in kernels ranges between 8 and 14%. The trans free formulations thus prepared had melting and crystallization characteristics, especially the onset and enthalpy, similar to those of commercial hydrogenated shortenings, although they showed delayed crystallization to the stable forms. Bakery shortenings without any trans fatty acids were prepared from mango and mahua fats. These processes can be utilized by shortening manufacturers. Although hydrogenated shortenings are cheaper than fractionated and blended shortenings the latter should be preferred because of their health benefits. Jeyarani and Yella Reddy (2003) prepared a series of plastic fats containing no TFA and having varying melting or plastic ranges, suitable for use in bakery, margarines, and for cooking purposes as vanaspati from palm oil. These fats were prepared by fractionation and blending and had physical chemical properties similar to hydrogenated fats. In this way palm oil could be utilized to the maximum extent. Mayamol et al. (2004) studied the effects of processing conditions such as rate of agitation, crystallization temperature, and composition of the blends on the crystal structure of shortenings formulated with a blend of palm stearinrice bran oil. The products were evaluated for their physical chemical characteristics using differential scanning calorimetry (DSC), X-ray diffraction (XRD), high performance liquid chromatography (HPLC), and Fourier transformed infrared spectroscopy (FTIR) techniques. The formulation containing 50% palm stearin and 50% rice bran oil showed melting and cooling characteristics similar to those of hydrogenated commercial “vanaspati” samples. Analysis of the fatty acid composition revealed that the formulated shortenings contained 1519% C18:2 polyunsaturated fatty acids (PUFA). Tocopherol and tocotrienol contents of the experimental shortenings were in the range of 850-1000 ppm with oryzanol content up to 0.6%. XRD studies demonstrated that the crystal form in the shortenings was predominantly the β’ form, and there was less of the undesirable β form. Zhang et al. (2005) produced margarine hardstocks from two enzymatically interesterified fats at conversion degrees of 80 and 100%, a chemically randomized fat and a physically mixed fat. These four hardstocks were blended with 50% of sunflower oil in a pilot plant. Margarines from the enzymatically interesterified fats were compared to the margarines produced by conventional methods and to selected commercial products. The margarine produced from interesterified fats had good physical properties. Khatoon et al. (2005) prepared plastic fats for use in bakery and as vanaspati by interesterification of blends of palm hard fraction with mahua and mango fats at various proportions. The blends containing palm stearin/mango (1:1) showed improvement in plasticity after interesterification, whereas those containing palm stearin/mango (2:1) were hard and showed high solid contents at higher temperature and hence may not be suitable for bakery or as vanaspati. The blends with palm and mahua oils were softer and may be suitable for margarine-type products. Farmani et al. (2006, 2008) studied the utilization of palm olein in the production of zero-trans Iranian vanaspati through enzymatic interesterification. A comparison between the solid fat content (SFC) at 20-30°C of the final products and those of a commercial low-trans Iranian vanaspati was 37.2% for directly interesterified blends and 28.8% for fats prepared by blending interesterified palm olein with liquids oils. ReyesHernández et al. (2007) prepared three vegetable oil blends, intended for formulation of high melting temperature confectionary coatings, by mixing different proportions of coconut oil, palm stearin, and either partially hydrogenated soybean oil or native soybean oil. Overall, all trans-free blends showed lower SFC and heat of crystallization than the ones obtained with

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partially hydrogenated soybean oil (PH-SBO). At particular crystallization temperature some trans-free formulations provided crystallization systems with rheological properties that would result in softer textures than the ones obtained with PH-SBO blends. In addition of these systems, it is also of interest to discuss the potential of blends of a stearin such as a high-melting fraction of milk fat (HMF) with a vegetable oil as trans fat replacer. In this chapter the physical chemical properties of milk fat-sunflower oil low-trans blends, that is, crystallization behavior, polymorphism, microstructure and the effect of addition of emulsifiers will be reviewed.

Milk Fat Stearin Milk fat contains the most complex lipid composition of the natural fats. Triacylglycerols (TAG) comprise by far the greatest proportion of lipids in milk fat, making up 97-98% of the total lipid. The other components included are diacylglycerols (DSG), monoacylglycerols (MAG), free fatty acids, free sterols, and phospholipids (Swaisgood, 1985). Due to its complex composition, the melting range of milk fat is broad, spanning from about -40 to 40°C. Furthermore, the composition changes with season, region, and diet. To extend the use of milk fat in food, pharmaceutical, and cosmetic applications, fractionation may be performed to produce components with specific properties (e.g., melting point). Several applications in which fractionated milk fat peforms better than unmodified milkfat have been studied: •

• •

•

Improved flavor and performance by high-melting milkfat fractions when used as roll-in pastry fats for croissants and Danish (Baker, 1970; Deffense, 1989; Humphries, 1971; Munro and Illingworth, 1986; Pedersen, 1988; Schaap, 1982; Tolboe, 1984). The inhibition of bloom by high-melting milkfat fractions when used in chocolate manufacture (Baker, 1970; Timms and Parekh, 1980; Yi, 1993). Cold-spreadability provided by combinations of low-melting and high-melting fractions in the manufacture of butter (Bumbalough, 1989; Deffense, 1987; Dolby, 1970; Jamotte and Guyot, 1980; Kaylegian, 1991; Kaylegian and Lindsay, 1992; Makhlouf et al., 1987; Munro et al., 1978). Increased foam stability by the addition of high-melting fractions to whipped creams (Bratland, 1983; Tucker, 1974).

To fractionate milk fat three major methods have been employed (Kaylegian and Lindsay, 1995): crystallization from melted milk fat or dry fractionation, crystallization of milk fat dissolved in a solvent solution and supercritical fluid extraction. Dry fractionation is the most common method employed since it uses no additives and it is relatively simple and inexpensive. It is a temperature-based process in which the milk fat is held at a given temperature to allow a portion of the milk fat to crystallize, and then the crystals are physically separated of the liquid fraction. The HMF of milk fat used in this study was produced using a commercial anhydrous milk fat (AMF) made from sweet cream. AMF was

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dry fractionated using the Tirtiaux process. The milk fat was heated until fully melted, cooled under controlled conditions to 35°C, and pressure filtered to separate the fraction. The fraction was not further processed after fractionation. Milk fat fractions are also blended to give a manufacturer greater flexibility to tailor milk fat as an ingredient to specific functional requirements than could be accomplished with fractionation alone (Kaylegian and Lindsay, 1995). Chemical functionality relies in part on the radio of saturated and unsaturated fatty acids, which has nutritional as well as flavor implications. Physically functionality includes the crystallization properties and melting properties. One of the most important concerns in the blending of fats is that the crystallization properties of the glycerides in the mixture remains compatible. Incompatible glycerides in fats can cause retardation or complete lack of crystallization, due to intersolubility effects and formation of eutectic mixtures. To improve chemical composition of HMF, three blends were prepared by mixing 10, 20, and 40% of sunflower seed oil (SFO) with HMF. Dropping points (the temperature at which a solid fat just begins to flow under controlled conditions) of the samples were determined with the Mettler FP 80 Dropping Point Apparatus, using a heating rate of 1°C/min. Acyl carbon profile of samples was determined by gas chromatography (GC) using a Hewlett-Packard 5890 unit equipped with a flame ionization detector (FID) and on-column injector. Chemical composition and Mettler Dropping Points of the blends and starting materials (HMF and SFO) are reported in Table 1. Milk fat usually contains high proportions of TAG with carbon numbers 4 and 10 (C4 y C10). When AMF is fractionated the resulting HMF is enriched in TAG with longer chains as noticed from Table 1. SFO had 73.1% of TAG with carbon numer 54. Addition of SFO significantly increased the C54 fraction which is mostly composed by unsaturated fatty acids. The melting point measured as MDP of the HMF was 40.2°C and addition of 10% SFO had no effect on MDP. Addition of 20% SFO decreased MDP by 1.4°C, and addition of 40% SFO decreased the MDP of HMF by less than 3°C. The melting points of all samples were similar to the ones reported for the hydrogenated sunflower seed oils used to formulate margarine before 2006 (Herrera et al. 1998).

Equilibrium Solid Fat Content Solid fat contents (SFC) of the fully-crystallized samples were measured by pulsed nuclear magnetic resonance (p-NMR) in a Minispec PC/120 series NMR analyzer Bruker. HMF and its blends with 10, 20 and 40% SFO were tempered according to the AOCS temperature treatment (AOCS Official Method) to ensure full crystallization. Despite the small changes in Tm due to addition of SFO, the SFC curves of the blends decreased substantially as SFO content increased (Figure 1). At 40% addition of SFO to HMF, the melting point only decreased by a few degrees, but the SFC decreased by nearly 50% at all temperatures. Thus, the SFO caused a substantial dilution of the crystalline content of HMF, but had only a small effect on the final melting temperature of the highest-melting TAG.

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Table 1. Chemical Composition and Mettler Dropping Points of Starting Materials and its Blends Acyl Carbon Number

Chemical Composition in Weight% of Starting Materials SFO HMF 10-90% 20-80% 40-60% 0.3 0.5 0.5 0.4 0.4 0.0 0.5 0.5 0.4 0.4 0.0 1.0 0.9 0.8 0.7 0.0 2.1 2.0 1.8 1.4 0.0 4.8 4.4 3.9 3.2 0.3 8.6 7.9 7.2 6.3 3.9 13.2 12.4 11.4 7.8 0.0 8.0 7.3 6.6 7.2 0.0 7.0 6.1 5.5 4.2 0.0 7.5 6.5 5.8 4.8 0.0 9.0 6.6 6.9 5.8 0.1 11.0 10.0 8.3 6.6 2.2 13.2 12.5 10.3 8.8 20.1 9.2 10.0 9.9 13.1 73.1 4.3 12.5 20.6 29.3 0.8 0.3 1.4 0.3 0.5 0.0 1.0 0.8 0.5 0.5 65.6 1.5 8.3 17.2 24.0 6.7 1.5 2.0 2.6 4.3 40.2 40.4 38.8 37.4

C26 C28 C30 C32 C34 C36 C38 C40 C42 C44 C46 C48 C50 C52 C54 C54 (18:0) C54 (18:1 trans) C54 (18:1cis) C54 (18:2) MDP (°C)

80 70

SFC (%)

60 50 40 30 20 10 0 0

5

10

15

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25

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35

40

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Figure 1. Solid fat content (SFC), as measured by pulsed NMR, of mixtures of high-melting milk fat fraction (HMF) with sunflower oil (SFO).

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HMF had values of SFC that typically correspond to bakery margarine while fractions had SFC values similar to kitchen, wrapper or tub margarines (Bockisch 1998).

Thermal Behavior of HMF and the Blends Crystallization behavior and thermal properties of fats are related to its chemical composition. From the relatively complex polymorphism of TAG, especially those with mixed chains, it is expected that, the mixtures of TAG demonstrate a very complex behavior when a fat crystallizes since TAG interact to each other. It is known that TAG similar in melting points form solid solutions over extensive, but not usually complete, ranges of composition. This leads to several types of phase behavior, eutectic formation being the most common, although peritectics and monotectics are also formed. Calorimetric analysis of samples, performed after 24 h at 4°C, demonstrated relevant differences among them which indicated that SFO modified the DSC profile of HMF (Figure 2). Peak temperatures and total melting enthalpies are summarized in Table 2. Addition of SFO significantly diminished peak temperature of melting peaks as well as total melting enthalpies. However, the number of endotherms remains the same as in HMF. This indicated that fat were compatible and although the TAG of SFO have different chain length and are more unsaturated they were incorporated into solid solutions formed by the TAG of HMF. 0

-1

-2

dQ/dt

-3

-4

-5

-6

-7

-8 -40 -30 -20 -10

0

10

20 30

40

50 60

70

80 90 100

Temperature (°C)

Figure 2. Differential scanning calorimetry (DSC) melting diagrams of high-melting milk fat fraction (HMF) and its blends with sunflower oil (SFO). Program: heating from -40°C to 100°C at 10°C/min.

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Table 2. Peak Temperature of Melting Endotherms and Total Melting Enthalpies Corresponding to the Diagrams in Figure 2 Sample HMF 10% SFO in HMF 20% SFO in HMF 40% SFO in HMF a b

Shoulder (°Ca) 6.35 4.73 -0.30 -6.95

First Endotherm (°Ca) 16.21 14.62 12.90 11.26

Second Endotherm (°Ca) 39.34 37.77 37.62 36.02

ΔH (Jg-1b) 148.54 79.88 76.47 56.30

Standard deviation for all values were < ± 0.5°C. Standard deviation for all values were < ± 1 Jg-1.

Polymorphism of HMF and its Blends with SFO Polymorphism of a fat originates from different molecular conformations and packings, resulting in different 3D unit cell structures (Aquilano and Sgualdino 2001). There are two important phenomena that are closely related to the thermal behavior, microstructure and rheological properties of natural fats: polymorphism and intersolubility. Physical deterioration of fat products, such as margarine, shortening and chocolate, depends on size, morphology and polymorphic structure of fat crystals. Cocoa butter and other fats, such as palm oil, have been reported as examples of polymorphic fats. Under proper crystallization and aging conditions, different crystal forms are obtained with characteristic melting points and x-ray diffraction patterns. The polymorphic form required for a fat depends on the product. For both all- purpose and emulsified shortenings, it is essential that the solids of the fat crystallize in the β’ form, whereas β crystals are desirable in salad dressings because their physical dimensions prevent the crystals from settling. A major problem in many fat based food products is the polymorphic transition of fat crystals during storage. Margarine and chocolate are well-known examples in which transitions to the most stable crystal form lead to unacceptable product qualities. Undesirable physical properties of the stable polymorph such as excessively high melting points, excessively large crystals and unpleasant texture should be avoided. Figure 3 shows X-ray diffraction patterns of first crystals for HMF and the blend with 40% SFO crystallized at 35°C and a cooling rate of 5.5°C/min. HMF (a) showed the characteristic pattern of the β’ form with two strong signals at 4.3 and 3.9 Å. This form was stable at different crystallization temperatures and with time. Crystallization behavior and thermal properties of hydrogenated sunflower oil have been related to its chemical composition (Herrera et al. 1991, Herrera and Añón 1991). When the hydrogenated oil is crystallized from the melted state, the β’ polymorphic form was observed at a wide range of cooling rates. Neither α nor β crystallization was found. Intersolubility of TAG has also been shown to be important in the understanding of their thermal behavior (Herrera et al. 1992). Besides, the β’ form is stable and transforms to β in different times, depending on the storage temperature and cooling rate selected. Addition of SFO to HMF (b) did not modify substantially polymorphic behavior since the blends mostly crystallized in the β’ form. However, a small shoulder at 4.6 Å, which indicated the presence of trace amounts of the β form, was also present in X-ray patterns.

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HMF

40% SFO

b

Intensity

Intensity

a

10

15

20 2θ (°)

25

30

10

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2θ (°)

Figure 3. Short spacings of (a) high melting fraction of milk fat (HMF) and (b) the 40% sunflower oil (SFO) blend.

Rheological Properties of HMF and its Blends with SFO Rheological measurements of fats can be performed at low or high deformation. In the latter, the fat crystal network undergoes irreversible deformation, whereas in the former, viscoelasticity is measured below the yield point, and is reversible. For practical applications of solidified fat, mechanical properties related to large deformations are usually more important. These properties can be characterized by measurable quantities such as yield strain, yield stress, and apparent viscosity. An often-used and convenient method to characterize the firmness of semi-solid substances is penetrometry (Haighton 1959), which gives an apparent yield stress. It is called an “apparent” yield stress because during the measurement, the sample is strongly deformed locally. It is often found that apparent yield stress is proportional to other measures for firmness, such as apparent Young modulus measured by uniaxial compression (Kloek 1998) or an apparent elastic shear modulus (Narine and Marangoni 1999). Fat samples were analyzed by means of a TA-XT2i Texture Analyzer which measures force exerted on a probe as it penetrates the sample. Samples were penetrated to 75% of their original height (15 mm) with a stainless steel needle probe, 3 mm diameter, at a constant velocity of 1 mm/s. For each determination, 7 fat samples were used, and average values were calculated. The 1st peak appearing in the force-time curves is attributed to yield point. Penetration force corresponding to the yield point was determined from the force-time curves. Table 3 shows the effects of blending with SFO on penetration force when samples

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were crystallized at 35°C for 90 min with agitation and slow cooling rate (0.1°C/min) followed by quiescent cooling and storage for 24 h at 10°C. The firmness depends in a complicated way on three main parameters: SFC, interaction between crystals, and structure of crystal network. These three parameters depend on the crystallization process, which is in turn affected by the conditions during crystallization (Van Aken and Visser 2000). Addition of SFO significantly diminished penetration force in agreement with the decrease in SFC. These results indicate the relevance of TAG composition and interactions among TAG on crystallization and rheological properties of fats. Table 3. Penetration Force (in Newton) Corresponding to the Yield Point for HMF and its Blends with SFO Sample HMF 10% SFO in HMF 20% SFO in HMF 40% SFO in HMF

Penetration Force (N) 17 ± 3 7±2 4±1 2±1

Crystallization of a Fat Crystallization can generally be classified in two steps: nucleation and growth. Nucleation involves the formation of molecular aggregates that exceed a critical size and therefore are stable. Once nuclei have formed, they grow and develop into crystals. The nucleation process depends on supersaturation or supercooling. Growth rate, however, depends on thermodynamic (supersaturation) and kinetic factors (solvent, impurities, agitation rate, viscosity; Boistelle 1988). Avrami (1940) stated that an overwhelming amount of evidence points to the conclusion that a phase is nucleated by tiny germ nuclei. The number of germ nuclei per unit region at time t decreases from the initial number because some of them are swallowed by the growing grains of the new phase. Clearly, the number of crystals that form with time does not necessarily correlate with the number of nuclei. Nuclei size is strongly dependent on supercooling but when we say “nuclei,” we are referring to molecular aggregates of nanosizes in most fat systems. Distinguishing between nucleation and growth constitutes a major challenge in lipid crystallization studies. According to Hartel (2001) induction times (τ) measure the time required for the first detectable evidence of nuclei formation. Effectively, induction times indicate a combination of the true time required for nuclei to form plus a measurement time, depending on the sensitivity of the experiment. τ measurements can be broken into 2 terms: τn is the true induction time for nuclei formation and τg is the time required for a nucleus to grow to sufficient size to be detected. Therefore, techniques to describe the nucleation process must be very sensitive to disregard growth. The induction time of crystallization (τ), the reciprocal of nucleation rate (J), is the time statistically required to obtain 1 nucleus per unit volume (Boistelle 1988). Practically speaking τ is a kinetic parameter that is usually defined as the time interval between the

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moment the crystallization temperature (Tc) is reached and the start of crystallization (Sato 1988). The 1st stage of crystallization in an undercooled liquid is the formation of solid embryos (Rousset 2002). Transformation of the liquid into a solid generates a decrease in the Gibbs free energy per unit volume, ΔGv. This creation of solid in the liquid also generates a solid/liquid interface associated with a change in the surface-free energy, ΔGs, with ΔGv = σ, the surface energy. The Gibbs free energy due to the creation of a solid embryo ΔGhom is given by a combination of both ΔGv and ΔGs. A plot of ΔGhom as a function of the radius of the embryo shows that there is a critical radius r* (corresponding to n* molecules composing the embryo) where ΔGhom is maximum. Growth of the embryo does not induce a decrease in the free energy up to this critical radius r*. Therefore, below this radius the embryo is unstable. It becomes a stable nucleus only above r*. The activation-free energy is a function of supercooling. At high supercooling, this energy barrier tends to zero (Boistelle 1988). Experimentally, crystallization temperature selection should take into account the melting temperature of the fat system, because when a very low temperature is selected, there is no measurable induction period; therefore, the fat system crystallizes before it reaches crystallization temperature. In general when enough supercooling is generated in a fat system, it can remain as a liquid for a noticeable time interval at temperatures no more than 10 °C below the melting point. Turbidimetry is a more sensitive technique for the study of the early stages of a crystallization process than pNMR (Wright and others 2000). In this method the attenuation of the intensity of the light beam after its passage through a sample is measured. Figure 4 shows a typical absorbance with time curve from which the induction time of crystallization was calculated as the interval between the moment crystallization temperature was reached (zero time in the graph) and the start of crystallization (absorbance increase). By using turbidimetry, Herrera and others (1999) successfully described the effects of minor components on induction times for crystallization in a milk fat model system. 2.5

Absorbance

2

1.5

1

0.5

0 0

100

200

300

400

500

600

700

800

Time (s) Figure 4. Typical absorbance with time curve from which the induction time of crystallization (τ) was calculated. The example corresponds to a 20% SFO in HMF blend cooled at 5.5°C/min to 30°C with an agitation rate of 150 rpm.

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Induction Times of Crystallization Supersaturation given by the variation in chemical potencial (Δμ) is defined as:

Δμ =

ΔHm ------- (Tm - Tc) Tm

(1)

melting enthalpy (ΔHm) divided by melting temperature (Tm) and multiplied by supercooling, that is, the difference between melting and crystallization (Tc) temperatures. No kinetics parameters are involved in this definition. However, the manner by which the thermodynamic driving force for crystallization is achieved and the rate of development of this driving force determine the rates of formation and growth of crystals. In particular, the rate of cooling can substantially influence crystallization rate. Figure 5 shows the induction times obtained when samples were crystallized at two cooling rates: 5.5°C/min and 0.1°C/min. Despite the small differences in melting points (measured as Mettler Dropping Points, MDP) for different ratios of SFO to HMF (Table 1), induction times were significantly different at p<0.05 between samples. At slow cooling rates, there was a significant difference (p<0.05) in induction times for HMF and 10% SFO blends and for 10% and 20% SFO blends at all Tc; and a significant difference (p<0.01) for 20% and 40% SFO samples. Induction times were shorter at slow cooling rate for the same supercooling, which is somewhat surprising. In general, when a fat is crystallized at fast cooling rate (80 °C/min to a temperature below Tm of the α-polymorph), the α-polymorph can be expected, whereas at the rates used in this study (i.e., 0.1°C/min), the β’ or β polymorph is expected (Sato 1988). When palm oil was cooled at 0.1°C/min to crystallization temperatures close to the melting point, the β’-form had shorter induction time than the β-form. For the three main polymorphic forms of fats, induction times increase in the order α, β’ and β (Chong 1993). A slower cooling rate might be expected to promote the formation of more stable forms (β-polymorph) which have longer induction times. However, the results obtained in this study were reversed, with faster cooling rate giving longer induction times. The thermodynamic driving force was the same in both cases, but rapidlycooled samples (5.5°C/min) took longer at each temperature to crystallize. Similar results were also found for hydrogenated sunflower seed oil, which has very different chemical characteristics (rich in elaidic acid TAG, Herrera 1994). In nucleation, molecules need sufficient time to organize and align with their neighbors to form stable nuclei. In the rapidlycooled sample, that organization took place primarily at crystallization temperature, whereas the slower cooling process allowed reorganization to occur at warmer temperatures as the sample was cooling to crystallization temperature. The end result was that the sample that was cooled slowly needed less time at crystallization temperature to nucleate since much of the molecular organization had already taken place by the time it reached crystallization temperature.

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Induction Times (min)

140 120

HMFSC

100

HMFFC 10%SFOSC

80

10%SFOFC

60

20%SFOSC 20%SFOFC

40

40%SFOSC

20

40%SFOFC

0 32

34

36

38

40

Temperature (°C)

Figure 5. Induction times of crystallization for slow cooling (solid symbols) and fast cooling (open symbols).

Actual Solid Fat Content Figure 6 shows the increase in SFC with time for the 20% SFO in HMF blend crystallized at slow rate (0.1°C/min, a) and fast rate (5.5°C/min, b). Zero time for these graphs was the moment at which the samples reached crystallization temperature.

60

SFC (%)

50 40 30 20 10 0 0

10 20 30 40 50 60 70 80 90 Tim e (m in) a)

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60

SFC (%)

50 40 30 20 10 0 0

10 20 30 40 50 60 70 80 90 Tim e (m in)

b)

Figure 6. SFC with time for the 20% SFO in HMF blend crystallized at slow cooling rate (0.1°C/min, a) and at fast cooling rate (5.5°C/min, b). Symbols: ■, □, ▲, Δ, ●, ○ ,♦ at 5, 10, 20, 25, 30, and 35°C, respectively.

The 20% blend crystallized slowly (a) at temperatures of 5, 10, 15 and 20°C had an initial solid content of at least 90% of the final solid content (after 200 min), indicating that most of the crystallization took place before the temperature reached the designated crystallization temperature. At 30 and 35°C, a short induction period was necessary to start crystallization. A 25°C, initial SFC was 50% of the final solid content. As expected, the final SFC decreased as crystallization temperature increased, indicating the decrease in crystalline phase volume as temperature increased. Crystallization with fast cooling (b) showed a different behavior from that for slow cooling. Most of the crystallization process took place at crystallization temperature. Below 25°C, there was no induction time of crystallization, and curves showed a hyperbolic shape. However, a slight plateau in SFC was visible in all curves. The SFC at the plateau decreased steadily both in rate and height as crystallization temperature was increased. The second step of crystallization, above the plateau, was sigmoidal in shape. For low supercoolings (crystallization temperatures above 25°C), curves had sigmoidal shapes. At the beginning, there was an induction time when no fat crystallized, which was followed by a period of rapid crystallization. The thermodynamic driving force is one factor that influences the rate of crystallization. However, other processing factors, such as heat and mass transfer during processing, also can have significant effects on the rate of crystallization. For example, the manner by which the thermodynamic driving force for crystallization is achieved and the rate of development of this driving force determine the rate of formation and growth of crystals (Hartel 2001). In particular, the rate of cooling can substantially influence crystallization rates since at slow cooling rates, molecular organization takes place as the sample is slowly being cooled to crystallization temperature. In contrast, rapid cooling forces the molecules to organized into crystals under conditions farther from equilibrium. Faster cooling generally results in more compound crystals formation (lower purity) and higher SFC compared with slower cooling.

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Comparison of Figures 6 a and b, particularly at crystallization temperatures below 25°C, shows that there is a higher SFC for fast than for slow cooling. At higher crystallization temperatures (lower supercooling), the SFC is about the same for both cooling rates. Crystallization properties of SFO/HMF blends remains compatible since crystallization kinetics is fast and no lack of crystallization was found in any case. SFC values were comparable to that obtained for hydrogenated sunflower seed oil (Herrera et al. 1999).

Microstructure Many factors influence lipid crystallization, among them processing conditions and composition of fat are especially relevant (Hartel 2001). Cooling rate, initial and final temperatures, and agitation rate strongly modified crystallization kinetics. TAG organization, fatty acid composition, minor lipids, and emulsifiers also determine crystallization rates and induction times for crystallization. Crystal structure is mainly affected by all these factors. In many applications of edible fats, the morphology and number of glyceride crystals determine the suitability of the fat for a given purpose. For example, the morphology of TAG crystals is related to the possibility of network formation to give a plastic fat. Rheological properties such as hardness, spreadability, viscoelastic properties, flavor release and mouthfeel depend in a complicated way on three main parameters: SFC, interaction between crystals, and structure of the crystal network. These three parameters depend on crystallization process, which is in turn affected by the conditions during crystallization. The rate of cooling, for example, can substantially influence crystallization rates. Light microscopy is a well-developed and increasingly used technique for studying the microstructure of food systems in relation to their physical properties and processing behavior. Figure 7 shows the effect of cooling rate on crystal morphology for the 10% SFO sample crystallized without agitation on a microscope slide and kept isothermally for 30 min at 30°C. At 30°C, the 10% SFO blend crystallized with high supercooling, as evidenced by the small crystal size that appeared when samples were crystallized with the fast cooling rate (a). These crystals were more transparent than the ones obtained with slow cooling (b) and had lower amount of solids in each crystal. For the slow cooling rate, denser and larger crystals, with a more regular boundary, were found. Typically, fewer crystals of higher purity are obtained with slow cooling. To better understand the processes of nucleation and growth under dynamic conditions, we took samples from the glass cell periodically, beginning when the cell temperature reached crystallization temperature and continuing for 3 h. As an example, Figure 8 shows the morphology of crystals with time obtained when the mixture with 40% SFO in HMF was crystallized isothermally at 35°C with fast (5.5°C/min, a) or slow rate (0.1°C/min), with an agitation rate of 100 rpm. A few large crystals were formed under these processing conditions. The photographs showed that they were not single crystals but grew by aggregation. These crystals had a spherical shape consisting of small needle crystals.

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Figure 7. Effect of cooling rate on crystal size and morphology for the 10% SFO in HMF blend cooled isothermally without agitation at 30°C for 30 min. a) cooling rate 5.5°C/min, b) cooling rate 0.1°C/min.

a)

b) Figure 8. Images of crystals corresponding to a 40% SFO in HMF blend crystallized isothermally at 35°C in dynamic conditions. a) cooled at 5.5°C/min, 90 min, b) cooled at 0.1°C/min, 70 min. Scale as in Figure 7.

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a)

b) Figure 9. Images of crystals corresponding to a 10% SFO in HMF blend crystallized isothermally at fast cooling rate without agitation: a) 25°C b) 35°C. Scale as in Figure 7.

Figure 9 shows the effect of temperature on crystal structure for the 10% SFO blend crystallized at a fast cooling rate without agitation. Crystal size increased dramatically, whereas crystal number decreased significantly with temperature. This effect was found in all cases. At higher temperatures, fewer initial crystals were formed and their growth was favored. These conditions favor crystal growth over nucleation (Hartel 2001). On the contrary, when supercooling was higher (25°C), nucleation was favored and smaller crystals appeared.

Effects of Emulsifiers Emulsifiers are useful functional additives without which many food products would be impossible to make. Emulsifiers typically act in multiphase systems in two main ways. The first is as an emulsifying agent to enable two distinct phases to be combined in a stable quasihomogeneous state for an indefinite length of time. The second function of an emulsifier is

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often to modify the behavior of the continuous phase of a food product so as to bring about a specific effect or benefit, for example, the use of lecithin in chocolate to reduce the viscosity of the product and improve the ease of handing and processability. In food, several circumstances for controlling crystallization can be distinguished. In the first case, control of size, distribution, shape and polymorphism of crystals is necessary to provide the desired physical characteristics or quality. Here, the kinetics of nucleation and growth must be controlled through formulation and processing conditions. In other products, controlling crystallization means preventing crystallization from occurring even though the product is thermodynamically prone to crystallize (Hartel 2001). Altering the physical properties of fats and controlling the polymorphic transformation of TAG and their rheological behavior have been important subjects for technologists and scientists. For example, the kinetics of β’ to β transition is important from a technological point of view. It is clear that retarding polymorphic transformation in solid fats can help, at least for some time, to delay loss in quality. Emulsifiers, such as lecithin and monoglycerides, are used as both viscosity controllers and as antibloom agents (Garti 1988). Some vegetable fats, such as partially hydrogenated sunflower seed oil and low-erucic rapessed oil, have a strong tendency to form β- crystals and cause sandiness in margarine. Several food emulsifiers, such as saturated and unsaturated fatty acid monoglycerides, act as modifiers of crystal structure, thus helping to prevent this unwanted phenomenon. The addition of 0.3% sorbitan tristearate inhibits the β’ to β transition in margarine (Madsen and Als !998). Probably the most important step for controlling crystallization is nucleation. As a main strategy, the use of emulsifiers has drawn attention for years. Emulsifiers alter the properties of the fat surface and the fat crystallization process, resulting in an altered solid fat content and crystal size. In fat systems, some emulsifiers act as heteronuclei (seeds or impurities that promote heterogeneous nucleation). Nucleation of fat crystals is accelerated through the catalytic actions of such impurities. Other emulsifiers affect the formation of critical nuclei and elongate induction times (Garti and Yano 2001). Sucrose esters can be used in food as emulsifiers because they are nontoxic, tasteless, odorless, and are digested to sucrose and fatty acids in the stomach. Sucrose esters can also be used in pharmaceuticals, cosmetics, foods, and in other products where a nonionic, nontoxic, biodegradable emulsifier is required (Gupta et al. 1983). In addition to their major function of producing and stabilizing emulsions, sucrose esters contribute to numerous other functional roles as texturizer and film former. Typical applications are baked goods, fruit coatings, and confectionary (Hasenhuettl 1997). Sucrose esters have a common feature that makes them suitable as emulsifying agents: they are ambiphillic, possessing both lipophilic and hydrophilic properties. The nature of this property is often expressed as the hydrophilic/lipophilic balance (HLB) on a scale of 0 to 20, with low numbers indicative of the oil-like tendency (Weyland 1997). The polyglycerol esters of fatty acids include a large group of closely related compounds of complex composition. However, the individual components are found as normal constituents of the human diet, i.e. glycerol, glycerol mono-, di- and tri-fatty acid esters and individual fatty acids. Hydrolysis of the tri- and polyglycerol esters in vitro with fresh pancreatic juice plus bile showed that 89 to 98% of oleate esters were hydrolyzed. As a result of their multifunctional properties and harmless nature, polyglycerol esters are widely used in many applications in the food and cosmetic industries.

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They notably function as emulsifiers, dispersants, thickeners, solubilizers, spreading agents, or emollients. More recently, new industrial applications based on polyglycerol esters have been developed. This includes their utilization as antifogging and antistatic additives, lubricants, or plasticizers. The following data shows the effect of highly hydrophobic food emulsifiers with different chemical structures on nucleation behavior. The selected emulsifiers were: a palmitic sucrose ester with HLB = 1, a MDP of 58.0 and monoester content of 1 wt%, with di-, tri-, and polyesters comprising 99 wt% and a polyglycerol stearic acid ester, DAS 7S, with HLB = 3.7 and a melting point of 58.0°C. The sucrose head of P-170 has no chemical similarity with the TAG structure, while polyglycerol alcohols are the result of glycerol polymerization. The selected emulsifiers allow studying the effect of the hydrophilic heads. Esters were added at concentrations of 0.1 wt% to HMF and the blends with SFO. The selected concentration was within the concentrations usually employed in foods for these esters.

Effect of Emulsifiers on Induction Times Figure 10 shows values of induction times of nucleation vs. temperature for HMF and the 40% SFO blend, with and without addition of 0.1% of P-170 or DAS 7S, based on polarized laser light turbidimetry. All samples showed similar behavior. A continuous curve can be drawn, which means that when samples were crystallized at these temperatures, only one polymorphic form was obtained (Ng 1990). A t-test at a confidence interval of 95% was performed on the average induction times at every temperature for all systems. For HMF, no significant differences were found at this level between the values of means below 313 K; however, above this temperature, differences were significant, being notorious for low supercooling (temperatures close to the melting point). For the blends with 40% SFO, the same behavior was found, but differences were significant above 312 K. Sucrose ester P-170 elongated the induction times of all samples, and therefore delayed nucleation. On the contrary, polyglycerol DAS 7S caused acceleration of nucleation. Shorter induction times were found in all cases.

Effect of Emulsifiers on Polymorphism Figure 11 shows X-ray diffraction patterns for HMF and the 40% SFO blend crystallized with and without addition of emulsifiers at 35°C and a cooling rate of 5.5°C/min. HMF showed the characteristics pattern of the β’ form with two strong signals at 4.3 and 3.9 Å. Addition of emulsifiers did not modified the polymorphic behavior as may be noticed from the patterns (a). As may be expected from the β tendency of SFO, HMF with addition of 40% SFO had a shoulder at 4.6Å, indicating a trace amount of the β form (b). Addition of DAS 7S increased the β’ tendency of the blend. This pattern did not show traces of the β form. Even at the low concentration selected in this study (0.1%), the emulsifiers were able to modify the polymorphic behavior of these systems. In agreement with these results, when polyglycerol

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behenic acid esters were added to palm oil, they promoted the crystallization of the β’ form from the melt, preventing the formation of granular crystals (Sakamoto 2003). These results are important regarding technological applications, since different products require different polymorphic forms to achieve the necessary macroscopic properties. 80

a

70 60

t (min)

50 40 30 20 10 0 310

312

314

316

318

320

T (°K) 80

b

70 60

t (min)

50 40 30 20 10 0 310

312

314

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318

320

T (°K)

Figure 10. Induction times of crystallization vs. temperature for all samples: (a) HMF ( ♦ ) and with addition of DAS 7S ( ○ ), and P-170 ( ■ ), (b) the 40% SFO blend ( ♦ ) and with addition of DAS 7S ( ○ ) and P-170 ( ■ ). Data points are the average of three runs. Error bars are standard deviations.

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Intensity

a

HMF

DAS 7S

P-170 10

15

20

25

30

2θ (°)

b

Intensity

40% SFO

DAS 7S

P-170 10

15

20

25

30

2θ (°)

Figure 11. Short spacings of (a) high melting fraction of milk fat (HMF) and with addition of emulsifiers, and (b) the 40% sunflower oil (SFO) blend and with addition of emulsifiers.

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109

b)

c) Figure 12. Images of crystals corresponding to a 40% SFO in HMF blend crystallized isothermally at 35°C for 60 min with a cooling rate of 5.5°C/min and an agitation rate of 100 rpm: a) 40% SFO blend, b) 40% SFO blend with 0.1% of sucrose ester P-170, and c) 40% SFO blend with 0.1% of polyglycerol DAS-7S.

Effect of Emulsifiers on Microstructure Figure 12 shows representative PLM images of the 40% SFO blend (a), with addition of 0.1% of P-170 (b), and with addition of 0.1% of DAS 7S (c), crystallized at 35°C, 100 rpm and 5.5°C/min. The 40% SFO blend showed spherical agglomerated crystals that grew by accumulation of small needle crystals separated by the liquid phase (a). Crystals were

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brighter which indicated that they were form by more solid material. When P-170 was added to HMF (b), crystals showed a different morphology. Spherical crystals with a less dense crystal structure (related to the brightness of the crystals) were present. P-170 also diminished crystal size, showing that P-170 affected not only nucleation but growth rate of the crystals as well. These results were in agreement with the elongation of induction times found when P170 was added to the 40% SFO blend. It was reported that any factor that delays nucleation produces this effect on crystal morphology (Herrera and Hartel 2000). Addition of DAS 7S to the 40% SFO blend (c) accelerated crystallization. Images showed many small crystals covering the field together with big spherical agglomerates. In agreement with our results, Sakamoto et al. studying the isothermal behavior of palm oil by PLM found that polyglycerol behenic acid esters accelerated nucleation. Palm oil crystals were smaller and their number larger with the additives.

Effect of Emulsifiers on Rheology Table 4 shows the effect of emulsifier addition on penetration force of HMF and its blends with SFO crystallized at 35°C with agitation. As previously reported (Martini et al. 2002), primary crystals were formed under agitation at 35°C. After 90 min, samples were cooled to 10°C, where crystallization continued without agitation. Confocal images of these samples showed larger primary crystals formed at crystallization temperature, surrounded by small and somewhat diffuse crystals in the background, which most likely were formed during storage (Martini et al. 2002). Addition of sucrose esters significantly decreased penetration force for all samples. After 24 h at 10°C, the SFC of all samples, both with and without the addition of sucrose esters, was within the experimental error of the method (1%). However, the average size of the primary crystals decreased significantly with addition of P170 and the microstructure of the final products were quite different (Martini and others 2002). DAS 7S promoted nucleation and therefore many small crystals were formed at crystallization temperature. This polyglycerol ester increased penetration force. Although emulsifiers did not affect the final SFC of the fats they had significant effect on the nature of the crystalline microstructure. Therefore, they affected rheological properties. The effect of emulsifiers on microstructure was related to their effects on crystallization kinetics. Table 4. Effect of Emulsifier Addition on Penetration Force for HMF and its Blends with SFO Sample HMF 10% SFO in HMF 20% SFO in HMF 40% SFO in HMF

Without emulsifiers 17 ± 3 7±2 4±1 2±1

Penetration force (N) With 0.1% P-170 9±2 4±1 2±1 1±1

With 0.1% DAS 7S 20 ± 4 10 ± 2 7±3 4±2

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Conclusion Nucleation process was modified adding sucrose esters and polyglycerol esters. The effect on induction times of nucleation (delay or acceleration) was related to the molecular structure of emulsifiers and, more specifically, to the hydrophilic head. Both families of emulsifiers seemed to follow a simple mechanism of incorporation into the crystal. However, hydrophilic heads with no chemical similarity inhibited nucleation while heads with chemical similarity promoted it. The emulsifiers selected in this study were also able to modify the polymorphic behavior and crystalline microstructure of the low trans HMF/SFO blends. A deep understanding of the role of sucrose and polyglycerols esters is a necessity to chocolate producers, ice cream makers and food technologists.

References Anon, Joint WHO/FAO Expert Consultation on Diet, Nutrition and the Prevention of Chronic Diseases. Diet, Nutrition and the Prevention of Chronic Diseases, pp. 87-89, WHO/FAO, Geneva, Switzerland (2003). AOCS. Official Methods of the American Oil Chemists’ Society. 4th Edition. Method Cd 1681 (1989). Aquilano, D., Sgualdino, G. Fundamental Aspects of Equilibrium and Crystallization Kinetics. In: Crystallization Processes in Fats and Lipid Systems, Nissim Garti, Kiyotaka Sato, editors, Marcel Dekker, New York, pp. 1-51 (2001). Avrami M. Kinetics of phase change II. Transformation-time relations for random distribution of nuclei. J Chem Phys 8:212–24 (1940). Baker, B.C. Use of Butterfat Fractions for Special Purposes. In: Proceeding of the XVIII International Dairy Congress, Sydnay, Australia, vol. 1E, p. 244 (1970). Bockisch, M. Fats as or in Food. In: Fats and Oil Handbook, M. bockisch, editor, AOCS Press, Champaign, IL, p. 750 (1998). Boistelle R. Fundamentals of nucleation and crystal growth. In: Garti N, Sato, K, editors. Crystallization and polymorphism of fats and fatty acids. New York: Marcel Dekker. p 189–226 (1988). Bratland, A. Production of a dairy emulsion, European Patent 0095001 (1983). Bumbalough, J.E., Spreadable Product Having an Anhydrous Milk Fat Component, U.S. Patent 4,839,190 (1989). Chong, C.L.; Sato, K. Kinetic Study of Palm Oil Cristallization. Inform 4:537 (1993). Deffense, E. Fractionated Milk Fat Products in Bakery Products. In: Proceeding New Uses for Milk Fat, The Dairy Sciences Research Center, Laval University, Quebec, Canada, p. 79 (1989). Deffense, E. Multi-step Butteroil Fractionation and Spreadable Butter. Sonderdruck aus Fett Wiss. Technol. – Fat Sci. Technol. 13,1 (1987). DHHS/FDA. Food Labeling: Trans fatty acids in nutrition labeling; nutrient content claims, and health claims: Final Rule. Vol 68 Federal Register, p. 41433, July 11, 2003.

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Dolby, R.M. Properties of Recombined Butter Made from Fractionated Fats. In: Proc. XVIII Int. Dairy Congr., Sydnay, Australia, vol. 1E, p.243 (1970). Farmani, J., Hamedi, M., Safari, M. Production of Zero trans Iranian Vanaspati Using Chemical Transesterification and Blending Techniques from Palm Olein, Rapeseed and Sunflower Oils. International Journal of Food Science and Technology 43:393-399 (2008). Farmani, J., Safari, M., Hamedi, M. Application of Palm Olein in the Production of Zerotrans Iranian Vanaspati through Enzymatic Interesterification. European Journal of Lipid Science and Technology 108:636-643 (2006). Garti, N. Crystallization and Polymorphism of Fats and Fatty Acids, Marcel Dekker, Inc., New York, p.280 (1988). Garti, N., Yano, J. The Roles of Emulsifiers in Fat Crystallization. In: Crystallization Processes in Fats and Lipids Systems. N. Garti and K. Sato Eds. Marcel Dekker, New York, USA, pp. 211-250 (2001). Gupta , R.K., James, K., Smith, F.J. Sucrose Esters and Sucrose Esters/Glyceride Blends as emulsifiers. J. Am. Oil Chem. Soc. 60:862-869 (1983). Haighton, A. The measurement of the hardness of margarine and fats with the cone penetrometer. J. Am. Oil Chem. Soc. 36:345-349 (1959). Hartel RW. Nucleation. In: Crystallization in foods. Gaithersburg, Md.: Aspen Publishers, Inc. p 145–91 (2001). Hasenhuettl, G.L. Overview of Food Emulsifiers. In: Food Emulsifiers and Their Applications. G.L. Hasenhuettl, R.W. Hartel, editors. New York: Chapman & Hall, pp.19 (1997). Herrera, M.L. Crystallization behavior of hydrogenated sunflowerseed oil: kinetics and polymorphism. J. Am. Chem. Soc. 71, 1255-1260 (1994). Herrera, M.L., Segura, J.A., Añón, M.C. J. Am. Oil Chem. Soc. 68:793-798 (1991). Herrera, M.L., Añón, M.C. J. Am. Oil Chem. Soc. 68:799-803 (1991). Herrera, M.L., Segura, J.A., Rivarola, G.J., Añón, M.C. J. Am. Oil Chem. Soc. 69:898-905 (1992). Herrera, M.L., Falabella, C., Melgarejo, M., Añón, M.C. Isothermal Crystallization of Hydrogenated Sunflower Oil: I- Nucleation. J. Am. Oil Chem. Soc. 75:1273-1280 (1998). Herrera, M.L., Falabella, C., Melgarejo, M., Añón, M.C. Isothermal Crystallization of Hydrogenated Sinflower Oil: II. Growth and Solid Fat Content. J. Am. Oil Chem. Soc. 76:1-6 (1999). Herrera ML, Hartel RW. Effect of processing conditions on crystallization kinetics of a milk fat model system. J Am Oil Chem Soc 77:1177–87 (2000). Humphries, M.A. Used of Fractionated Milk Fat in Bakery Products. N.Z.J. Dairy Sci. Technol. 6:28 (1971). Hunter, J.E. Nutritional Considerations of trans fatty acid. In: Trans fat alternatives. D.R. Kodali and G.R. List, eds, AOCS Press, Champaign, IL, USA, pp. 34-46 (2005). Jamotte, P., Guyot, A. Final Report on Research Dealing with the Preparation and Use of New Types of Butter Fats Modified by Fractional Crystallization in the Absence of Solvents, Ministry of Agric., Dairy Stn., Chaussee de Namur, Belgium (1980).

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Jeyarani, T, Yella Reddy, S. Preparation of Plastic Fats with Zero trans FA from Palm Oil. J. Am. Oil Chem. Soc. 80:1107-1113 (2003). Kaylegian, K.E. Physical and Chemical Properties of Milk Fat Fractions, M.S. Thesis, University of Wisconsin-Madison (1991). Kaylegian, K.E., Lindsay, R.C. Performance of Selected Milk Fat Fractions in ColdSpreadable Butter. J. Dairy Sci. 75:3307 (1992). Khatoon, S., Reddy, S., Reddy, Y. Plastic Fats with Zero trans Fatty Acids by Interesterification of Mango, Mahua and Palm Oils. European Journal of Lipid Science and Technology 107:786-791 (2005). Kloek, W. Mechanical properties of fats in relation to their crystallization (Ph. D. Thesis). Wageningen Agricultural Univ., The Netherlands (1998). List, G.R., Reeves, R. Dietary Guidelines, Processing, and Reformulation for trans Reduction. In: Trans fat alternatives. D.R. Kodali and G.R. List, eds, AOCS Press, Champaign, IL, USA, pp. 71-86 (2005). Madsen, J., Als, G. Sandiness in Table Margarine. In: Crystallization and Polymorphism of Fats and Fatty Acids, edited by N. Garti and K. Sato, Marcel Dekker, Inc., New York, 1988, results from the 9th International Society for Fat Research Congress, Vlaardingen, The Netherlands, hkeld Sept. 16-21, pp. 11-17 (1968). Makhlouf, J., Arul, J., Boudreau, A., Verret, P., Sahasrabudhe, M.R. Fractionnement de la matière grass laitière par cristallisation simple et son utilisation dans la fabrication de beurres mous. Can. Inst. Food Sci. Technol. 20, 236 (1987). Martini, S., Puppo M.C., Herrera, M.L., Hartel, R.W. Effect of sucrose Esters Addition on microstructure of Milk Fat Fractions Sunflower Oil Blends. J. Food Sci. 67:3412-3418 (2002). Mayamol, P.N., Samuel, T., Balachandran, C., Sundaresan, A., Arumughan, C. Zero-trans Shortening Using Palm Stearin and Rice Bran Oil. J. Am. Oil Chem. Soc. 81:407-413 (2004). Munro, D.S., and Illingworth, D. Milk Fat Based Food Ingredients: Present and Potential Products. Food Technol. Aust. 38:335 (1986). Munro, S.D., Bissell, T.G., Jebson, R.S., Norris, R., Taylor, M.W. Fat. In: Proc. XX Int. Dairy Congr., Paris, France, P. 862 (1978). Narine, S.S., Marangoni, A.G. Fractal nature of fat crystal networks. Phys. Rev. E59:19081920 (1999). Ng, W.L. A Study of the Kinetics of Nucleation in a Palm Oil Melt. J. Am. Oil Che. Soc. 67:879-882 (1990). Nor Aini, I., Che Maimon, C.H., Hanirah, H., Zawiah, S., Che Man, Y.B. Trans-Free Vanaspati Containing Ternary Blends of Palm Oil-Palm Stearin-Palm Kernel Olein. J. Am. Oil Chem. Soc. 76:643-648 (1999). Pal, P.K., Bhattacharyya, D.K., Ghosh, S. Modifications of Butter Stearin by Blending and Interesterification for Better Utilization in Edible Fat Products. J. Am. Oil Chem. Soc. 78:31-36 (2001). Pedersen, A. Puff Pastry Butter- a New Product in the Dairy Industry. Special Issue of: Danish Dairy and Food Industry (1988).

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Petrauskaite, V., De Grey, W., Kellens, M., Huyghebaert, A. Physical and Chemical Porperties of trans-Free Fats Produced by Chemical Interesterification of Vegetable Oil Blends. J. Am. Oil Chem. Soc. 75:489-493 (1998). Reyes-Hernández, J., Dibildox-Alvarado, E., Charó-Alonso, M.A., Toro-Vázquez, J.F. Physicochemical and Rheological Properties of Crystallized Blends Containing transfree and Partially Hydrogenated Soybean Oil. Journal of American Oil Chemists Society 84:1081-1093 (2007). Rousset P. Modeling crystallization kinetics of triacylglycerols. In: Marangoni AG, Narine SS, editors. Physical properties of lipids. New York: Marcel Dekker. p 1–36 (2002). Sakamoto, M., Maruo, K., Kuriyama, J., Kouno, M., Ueno, S., Sato, K. Effects of adding Polyglycerol Behenic Acid Esters on the Crystallization of Palm Oil. J. Oleo Sci. 52:639645 (2003). Sato K. Crystallization of fats and fatty acids. In: Garti N, Sato K, editors. Crystallization and polymorphism of fats and fatty acids. New York: Marcel Dekker. p 254–259 (1988). Schaap, J.E., Applications of Fractionated Milk Fat as Shortening in Flour Confectionery Products. In: Dairy Ingredients in Foods, Int. Dairy Fed., Doc. 147, p.51 (1982). Schrimpf, J., Wilkening, V. Trans Fats- New FDA Regulations. In: Trans fat alternatives. D.R. Kodali and G.R. List, eds, AOCS Press, Champaign, IL, USA, pp. 26-33 (2005). Swaisgood, H.E. Characteristics of edible fluids of animal origin: milk. In Food Chemistry, Fennema, O.R., Ed.; Marcel Dekker: New York, pp791-828 (1985). Timms, R.E., Parekh, J.V. The Possibilities for Using Hydrogenated, Fractionated or Interesterified Milk Fat in Chocolate. Lebensm.-Wiss.u.-Technol. 13:177 (1980). Tolboe, O. Physical Characteristics of Butter Fat and Their Influence on the Quality of Danish Pastry and Cookies. In: Milkfat and Its Modifications Contributions at a LIPIDFORUM Symposium (1984). Tucker, V.C. Uses for Hard Milk Fat Fraction. In: Proc. XIX Int. Dairy Congr., New Delhi, India, vol E, p. 762 (1974). Van Aken, G.A., Visser, K.A. Firmness and Crystallization of milk fat in relation to processing conditions. J. Dairy Sci. 83:1919-1932 (2000). Weyland, M. Emulsifiers in Confectionary. In: Food Emulsifiers and Their Applications. G.L. Hasenhuettl, R.W. Hartel, editors. New York: Chapman & Hall, pp.235-254 (1997). Wright AJ, Narine SS, Marangoni AG. Comparison of experimental techniques used in lipid crystallization studies. J Am Oil Chem Soc 77:1239–42 (2000). Yella Reddy, S., Jeyarani, T. Trans-Free Bakery Shortenings from Mango Kernel and Mahua Fats by Fractionation and Blending. J. Am. Oil Chem. Soc. 78:635-640 (2001). Yi, M. Effects of Milk Fat on Chocolate Bloom, M.S. Thesis, University of WisconsinMadison (1993). Zhang, H., Jacobsen, Ch., Adler-Nissen, J. Storage Stability Study of Margarines Produced from Enzymatically Interesterified Fats Compared to Margarines Produced by Conventional Methods. I. Physical Properties. European Journal of Lipid Science and Technology 107:530-539 (2005).

In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter V

Probiotic Bacteria Isolated from Breast Milk for the Development of New Functional Foods G. Vinderola, A. Binetti, and J. Reinheimer Instituto de Lactología Industrial (INLAIN, UNL-CONICET), Facultad de Ingeniería Química, Universidad Nacional del Litoral. 1º de Mayo 3250, Santa Fe (3000), Argentina

Abstract Baby’s intestine is (or was said to be) sterile at birth and gut microbiota development is a gradual process after delivery. Quantitative and qualitative differences in bifidobacterial and lactic acid bacteria levels and species composition have been shown between breastfed and formula-fed infants, bifidobacteria being the most dominant microorganisms in the former group. Establishment of the gut microbiota is a stepwise process which provides the earliest and most massive source of microbial stimuli for the normal maturation of the gut mucosal immune system, contributing to its development in infancy and to the control of the gut-associated immunological homeostasis later in life. Probiotic intervention in the neonatal period has attracted scientific interest after recent demonstrations showing that specific strains reduce the symptoms and risk of allergic and infectious diseases or improve feeding tolerance. However, no all early interventions in children reported rendered positive results. The question of the right dose and the specific pathologies that probiotic administration, to infants less than 6 month of age, could be helpful for is still under a vigorous debate. Breast milk contains several factors, including nutrients, antimicrobial agents, IgA antibodies and TGF-β, which contribute beneficially to the immunologic maturation and well-being of the infant as well as factors that promote the growth of bifidobacteria in the infant’s intestine. Additionally, healthy breast milk contains significant numbers of bacteria. In 2003 it was reported the isolation of lactobacilli from breast milk as potential probiotics. Breast milk seems to be a natural

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G. Vinderola, A. Binetti and J. Reinheimer source of probiotic bacteria for infants. In this context, supplementation of infant formulas with these kinds of probiotics might beneficially alter the composition of the microflora of formula-fed infants in such a way that it resembles that of breast-fed infants. However, to date there is no available information concerning the technological potential of these strains for their industrialization (growth in milk, resistance to lactic acid, freezing or spray-drying, among others) if they are thought to be included in dairy products or in formulas for infants.

Short Communication Traditional Ideas Revised For years, microbiological studies of breast milk have been restricted to the screening of the presence of potential pathogenic bacteria, mainly related to clinical cases of mastitis. Then, there is a limited quantity of reports about the isolation and analysis of commensal or potential beneficial bacteria from breast milk of healthy women (Martín et al., 2003). Moreover, the knowledge of the bacterial diversity in breast milk is very limited, and is almost exclusively based on the use of culture media. The presence of non-cultivable bacterial species might be overlooked if culture-independent molecular techniques are not used (Martín et al., 2007a). Tissier (1900) introduced the idea that fetus is sterile in utero and that bacterial colonization of the newborn intestinal tract begins during the transit through the labour channel due to cross-contamination with vaginal and faecal bacteria of the maternal microflora (Isolauri et al., 2001). Since then, these ideas have been widely accepted. However, there is emerging and surprising evidence that unveil ways of mother-to-child transmission of commensal microflora, during lactation and even before birth. Maybe one of the first signs of the presence beneficial bacteria in breast milk was given in 1979 when West et al. (1979) reported the isolation, among pathogenic species, of a Lactobacillus plantarum strain from human milk. However, by that time, those finding were assumed to be a contamination occurring during sample extraction (Gavin and Ostorv, 1977). Later on, RAPD and PFGE analysis permitted to conclude that a strain of Lactobacillus salivarius, previously isolated from feces of a one-month-old- breast-fed infant, could be detected in the breast milk of his mother (Martín et al., 2006), suggesting that gut microbiota of breast-fed infants reflects that of maternal breast milk. Recently, the presence of commensal bacteria was traced back and they were found to occur in the umbilical cord blood and meconium of healthy neonates and in murine amniotic fluid, suggesting that term fetuses are not completely sterile and that a prenatal mother-to-child efflux of commensal bacteria may exist (Jiménez et al., 2008a). This emerging evidence let us think that the traditional idea of a newborn with a sterile intestine acquiring his gut intestine microflora exclusively from exogenous sources should be put into test under the light of new molecular techniques indicating the presence of commensal bacteria passing from the mother to the child via breast milk and even before during the intrauterine life.

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Probiotics for Infants Baby’s intestine, sterile at birth or having a low charge of mother’s commensal bacteria (Jiménez et al., 2008a), is massively colonized by a gradual process and evolves quickly over the first days of life, but quantity and species vary markedly over the first 2 years of life (Kliger et al., 2007). The first colonizers are enterobacteria, streptococci and staphylococci, followed by bifidobacteria (Rotimi and Duerden, 1981). Quantitative and qualitative differences in bifidobacterial and lactic acid bacteria (LAB) concentrations and species composition have been reported between breastfed and formula-fed infants, bifidobacteria being the most dominant microorganisms in the former group (Harmsen et al., 2000). Breastfed infants are colonized predominantly by bifidobacteria and lactobacilli, whereas the microflora of formula-fed infants is composed by a smaller proportion of bifidobacteria and lactobacilli, dominating the anaerobic microorganisms, such as bacteroides and clostridia (Harmsen et al., 2000; Kalliomaki et al., 2001; Penders et al., 2006). Establishment of the gut microbiota is a stepwise process which provides the earliest and most massive source of microbial stimuli (Rautava et al., 2004) for the normal maturation of the gut mucosal immune system, contributing to its development in infancy and to control of the gut-associated immunological homeostasis later in life (Rautava and Isolauri, 2002). It has been shown that microbiota aberrancies precede the development of some atopic diseases (Kalliomaki et al., 2001). Probiotic intervention in the neonatal period has attracted scientific interest after recent demonstrations showing that specific strains reduce the symptoms and risk of allergic and infectious diseases (Isolauri et al., 2001). Stratiki et al. (2007) demonstrated that the administration of bovine milk supplemented with bifidobacteria for 7 days to preterm infants decreased intestinal permeability and lead to increased head growth. Lee et al (2007) reported that administration of probiotics to preterm infants led to Lactobacillus colonization in 27 out of 73 patients and improved their feeding tolerance. Even if continuously administered during the first 6 months after birth, it seems that no permanent establishment of a probiotic strain is achieved (Gueimonde et al., 2006), but only a transient colonization of the gut leading to a reduced prevalence of atopic eczema later in life. On the contrary, other studies showed no effects on atopic dermatitis (Brouwer et al., 2006) or rectal bleeding (Szajewska et al., 2007) after the administration of Lactobacillus GG to infant of less than 6 months of age. Even undesired effects were observed followed the administration of L. casei Shirota during lactation in an animal model (Ezendam and van Loveren, 2008). In other study where infants were given daily 109 cells of L. acidophilus LAVRI-A1 during their 6 first months of life, authors concluded that probiotic supplementation did not alter early innate immune responses in this population at high risk of developing allergic disease (Taylor et al., 2006). Other pathologies in which probiotic bacteria had been used are the prevention of antibioticassociated diarrhea, community-acquired diarrhea, necrotizing enterocolitis in premature children, irritable bowel syndrome and constipation, infantile colic and atopic dermatitis (Kliger et al., 2007). Although complementary feeding is not advised before the age of 6 months (Chouraqui et al., 2008), many results from clinical trials suggest that specific probiotics might be useful in reducing the risk of certain pathologies but, which probiotics should be used? Which doses shall be used? Should they be administered continuously or cyclically (administration/withdrawal/administration)? Yet, a great number of issues need to

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be resolved before general guidelines regarding the use of probiotics in the neonatal period can be released (Rautava, 2007). The specific strain to be administered, its origin, its safety and functional dose, the frequency of administration and the age of the individuals to which it will be administered, seems to be crucial issues in order to warrant a safe and efficacious use in newborn or infants.

Isolation and Characterization of Probiotic Bacteria from Human Breast Milk Breast milk contains several factors, including nutrients, antimicrobial agents, prebiotic oligosaccharides, IgA antibodies, TGF-β, and the innate microbe receptor soluble (s)CD14, which contribute beneficially to the immunologic maturation and well-being of the infant as well as factors that promote the growth of bifidobacteria in the infant’s intestine (Rautava et al., 2006). Additionally, healthy breast milk, for instance, contains significant numbers of bacteria. These commensal bacteria include streptococci, lactobacilli, corynebacteria, micrococci and propionibacteria (O’Sullivan et al., 2005). Bacterial species generally isolated by agar plating breast milk samples include Staphylococcus epidermis, S. hominis, S. capitis, S. aureus, Streptococcus salivarius, S. mitis, S. parasanguis, S. peores, Lactobacillus gasseri, L. rhamnosus, L. acidophilus, L. plantarum, L. fermentum, L. salivarius, L. reuteri, Enterococcus faecium and E. faecalis (Lara-Villoslada et al., 2007a). However, it seems that breast milk microflora is much more diverse than suggested by culture-dependant methods if molecular tools are used. For instance, Martín et al. (2007b) and Delgado et al. (2008) reported isolates from breast milk belonging to genera as diverse as Weisella, Propionibacterium, Serratia, Acinetobacter, Gemella, Pseudomonas, Veillonella, Kocuria, Corynebacterium, Klebsiella, Rothia and Escherichia. In 2003, two European groups, one from Finland (Heikkila and Saris, 2003) and another one from Spain (Martín et al., 2003) simultaneously and independently reported the isolation of LAB from human breast milk. Heikkila & Saris (2003) studied the bacterial diversity in human milk with focus on the detection of bacteria with antimicrobial activity against Staphylococcus aureus, a known causative agent of infectious mastitis in women. In that study, LAB such as L. rhamnosus, L. crispatus, Lactococcus lactis and Leuconostoc mesenteroides were isolated from breast milk. As L. crispatus is a predominant vaginal species, authors hypothesized that the infant may have derived it from the vagina during delivery and then transmitted it to the maternal breast skin during nursing or even to breast milk while suckling. However, in another research (Martín et al., 2007b), the diversity of Lactobacillus in infant faeces, breast milk and vaginal swabs from mothers whose neonates were born by vaginal delivery or caesarean section was studied. Authors concluded that the bacterial composition of breast milk and infant faeces is not related to the delivery method and that the origin of the LAB present in breast milk and in the infant gut is still far from clear. None of the Lactobacillus species detected in vaginal samples were present in breast milk provided by the women whose neonates were born by vaginal delivery, a fact that suggests that transit through the vagina does not play a role in the establishment of lactobacilli found in breast milk (Martín et al., 2007b).

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Martín et al. (2003) isolated several L. gasseri strains from breast milk of 8 healthy women on the fourth day postpartum. They found no RAPD correlation between strains isolated from breast milk and strains isolated from breast skin or mammary areola, which suggests that most breast milk LAB have an endogenous origin and that cross-contamination with skin bacteria has, if any, a secondary role as LAB source for the infant gut (Martín et al., 2003). A possible mechanism for commensal bacteria delivery from gut to breast milk has been suggested. Dendritic cells can sample bacteria from the gut mucosa by penetration of their dendrites into the gut lumen while preserving the integrity of the epithelial barrier (Rescigno et al., 2001). Once inside dendritic cells, or any other antigen-presenting cell, bacteria could spread to other locations by lymphatic circulation. It is known that, during the lactation period, colonization of the mammary gland by cells of the immune system is a selective process regulated by hormones. In this colonization, prokaryotic cells might be involved as well. Some beneficial bacteria can have mechanisms to spread from the gut to other organs in healthy hosts without leading to a pathogenic condition and at least some of the LAB present in the maternal gut could reach the mammary gland through an endogenous route (Martín et al., 2004). The probiotic potential of strains isolated from breast milk was evaluated and authors concluded that it was similar, at least, to that of the strains commonly used in commercial probiotic products (Martín et al., 2005). Once evaluated and guaranteed the safety for oral use of these news strains (Lara-Villoslada et al., 2007b), some functional properties of strains of breast milk origin was determined. It was demonstrated their antimicrobial activity (Olivares et al., 2006), the enhancement of the intestinal function in healthy adults (Olivares et al., 2006), a differential capacity to modulate the gut immune response by inducing the production of Th1 cytokines or IL-10 by two different strains (Díaz-Ropero et al., 2007) and their potential to treat infectious mastitis if administered to mothers during lactation (Jiménez et al., 2008b). Finally, Puleva Food S.L. (www.puleva.es) has recently industrialized one of these lactobacilli strains and launched into the market Puleva Peques 2 Hereditum®, the first dried milk containing probiotic bacteria isolated from breast milk and recommended for infants from 6 moths onwards. However, no information about the specific strain used and the level of viable cells present in the commercial product were available on the web site. In this context, supplementation of infant formulas with these kinds of probiotics might beneficially alter the composition of the microflora of formula-fed infants in such a way that it resembles that of breast-fed infants (Ezendam and van Loveren, 2008). Cell viability of probiotic bacteria in the food matrix is a prerequisite in order to guarantee their claimed health effects (Galdeano and Perdigón, 2004; Ouwehand and Salminen, 1998). However, even when a strain isolated from human milk has been successfully included in a commercial dried milk, to date there is no available information concerning the technological performance of strains from this ecological niche (growth in milk, resistance to lactic acid or spray-drying), if they are thought to be included in dairy products or feed formulas for infants. Recently, 6 strains of Bifidobacterium lactis subsp. lactis have been isolated from breast milk at our Institute (Instituto de Lactología Industrial, INLAIN UNL-CONICET, Santa Fe, Argentina) (Zacarías et al., 2009). The samples were obtained from 16 different mothers between days 1 and 12 after delivery, plated on agar media (MRS-cysteine and MRS-cysteine

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acidified to pH 5.5 with acetic acid), and incubated anaerobically at 37ºC for 72h. In 5 of those 16 mothers, two samples were obtained in two different days after delivery. Total counts ranged from 3x101 to 1x105 CFU/ml in MRS-cysteine, and were predominantly cocci. It is important to highlight that no lactobacilli were isolated from any of the samples, contrarily to previous reports on the microbiological composition of breast milk (LaraVilloslada et al., 2007a; Martín et al., 2003; Martín et al., 2006). Bifidobacteria were detected in 6 samples, mainly after the 1st day postpartum, at levels from 1x101 to 7.3x103 CFU/ml (Figure 1). They were later identified by molecular tools. Isolates were preliminary characterized according to different technological criteria such as storage stability as frozen (20ºC and -70ºC) cultures (in 20% skim milk) or as cultures suspended in 10% skim milk at pH 6 or 4.5 for 4 weeks at 5ºC. Resistance to spray drying was evaluated as well. All the isolated strains showed a high resistance to frozen storage for at least 6 months at -20ºC and 70ºC. Strains also showed adequate stability during 4 weeks of refrigerated storage in milk at 5ºC, since cell viability losses were smaller than 0.5 log order CFU/ml at pH 6 or 4.5. Finally, cell suspensions (108 CFU/ml) were spray-dried (Büchi mini spray dryer model B-290, Flawil, Switzerland) and a very soluble white powder was obtained containing more than 109 CFU/ml and a moisture content less than 4% (w/w). Functional in vivo studies are presently carried out in our laboratory to assess the probiotic potential of these bifidobacteria strains in a murine model. This work represents the first report in Latinoamerica on the isolation and preliminary technological characterization of bifidobacteria from breast milk, demonstrating a satisfactory technological potential for their use in the elaboration of fermented milk products or as adjunct in infant formulas if any probiotic property is observed for them. 6

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1 LM ( 5) 1( 1 L M 2) 2( LM 5) 2( 1 LM 0) 3( LM 7) 3( 1 LM 0) 4( L M 2) 4 L M ( 6) 5( L M 5) 5( L M 7) 6( L M 1) 7( L M 1) 8( L M 1) 9( LM 1) 10 LM (1 ) 11 LM (1 ) 12 LM (1 ) 13 LM (1 ) 14 LM (1 ) 15 LM (1 ) 16 (1 )

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Figure 1. Total ( ) and bifidobacteria ( ) counts in MRS-cysteine agar (37ºC, 72h, anaerobiosis) of fresh breast milk samples (LM) of 16 healthy women, between day 1 and 12 postpartum (the day of sampling is indicated between brackets).

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Concluding Remarks Breast milk can be considered a natural symbiotic food containing diverse nutrients and factors as well as functional bacteria (Lactobacillus and Bifidobacterium) able to satisfy the nutritional and immunological needs of the neonate. Therefore, it is not surprising that the bacterial composition of the infant faecal flora reflects the bacterial composition of breast milk. The modulation of the intestinal microbiota by probiotic bacteria isolated from breast milk has been shown to regulate the gut immune functions and to enhance defences against intestinal pathogens and allergies. These facts open new perspectives for the isolation, functional and technological characterization and addition of breast milk probiotics to infant formulas, in order to get closer to the natural composition of breast milk, for children with limited breast feeding or in cases where exclusive maternal care is not possible.

References Brouwer, M.L., Wolt-Plompen, S.A., Dubois, A.E., van der Heide, S., Jansen, D.F., Hoijer, M.A., Kauffman, H.F. & Duiverman, E.J. (2006). No effects of probiotics on atopic dermatitis in infancy: a randomized placebo-controlled trial. Clinical and Experimental Allergy, 36, 899-906. Chouraqui, J.P., Dupont, C., Bocquet, A., Bresson, J.L., Briend, A., Darmaun, D., Frelut, M.L., Ghisolfi, J., Girardet, J.P., Goulet, O., Putet, G., Rieu, D., Rigo, J., Turck, D. & Vidailhet, M. (2008). Feeding during the first months of life and prevention of allergy. Archives of Pediatrics, 15, 431-42. Delgado, S., Arroyo, R., Martín, R. & Rodríguez J.M. (2008). PCR-DGGE assessment of the bacterial diversity of breast milk in women with lactational infectious mastitis. BMC Infectious Diseases, 8, 51-59. Ezendam, J. & van Loveren, H. (2008). Lactobacillus casei Shirota administered during lactation increases the duration of autoimmunity in rats and enhances lung inflammation in mice. British Journal of Nutrition, 99, 83-90. Galdeano, C.M. & Perdigón, G. (2004). Role of viability of probiotic strains in their persistence in the gut and in mucosal immune stimulation. Journal of Applied Microbiology, 97, 673-81. Gavin, A. & Ostovr, K. (1977). Microbiological characterization of human milk. Journal of Food Protection, 40, 614–616. Gueimonde, M., Kalliomäki, M.D., M., Isolauri, E. & Salminen, S. (2006). Probiotic intervention in neonates - Will Permanent Colonization Ensue? Journal of Pediatric Gastroenterology and Nutrition, 42, 604-606. Harmsen, H.J., Wildeboer-Veloo, A.C., Raangs, G.C., Wagendorp, A.A., Klijn, N., Bindels, J.G. & Welling, G.W. (2000). Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. Journal of Pediatrics Gastroenterology and Nutrition, 30, 61–67.

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Isolauri, E., Sutas, Y., Kankaanpaa, P., Arvilommi, H. & Salminen, S. (2001). Probiotics: effects on immunity. American Journal of Clinical Nutrition, 73S, 444S-450S. Jiménez, E., Fernández, L., Maldonado, A., Martín, R., Olivares, M., Xaus, J. & Rodríguez, J.M. (2008b). Oral administration of Lactobacillus strains isolated from breast milk as an alternative for the treatment of infectious mastitis during lactation. Applied and Environmental Microbiology, 74, 4650 – 4655. Jiménez, E., Marín, M.L., Martín, R., Odriozola, J.M., Olivares, M., Xaus, J., Fernández, L. & Rodríguez, J.M. (2008b). Is meconium from healthy newborns actually sterile? Research in Microbiology, 159, 187e193. Kalliomaki, M., Kirjavainen, P., Eerola, E., Kero, P., Salminen, S. & Isolauri, E. (2001). Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. Journal of Allergy and Clinical Immunology, 107, 129–134. Kligler, B., Hanaway, P. & Cohrssen, A. (2007). Probiotics in children. Pediatric clinics of North America, 54, 949–967. Lara-Villoslada, F., Olivares, M., Sierra, S., Rodríguez, J.M., Boza, J. & Xaus, J. (2007a). Beneficial effects of probiotic bacteria isolated from breast milk. British Journal of Nutrition, 98, S96–S100. Lara-Villoslada, F., Sierra, S., Díaz-Ropero, M.P., Olivares, M. & Xaus, J. (2007b). Safety assessment of the human milk-isolated probiotic Lactobacillus salivarius CECT5713. Journal of Dairy Science, 90, 3583–3589. Lee, S.J., Cho, S.J. & Park, E.A. (2007) Effects of probiotics on enteric flora and feeding tolerance in preterm infants. Neonatology, 91, 174-9. Martín R., Heilig, G.H.J., Zoetendal, E.G., Smidt, H., & Rodríguez, J.M. (2007b). Diversity of the Lactobacillus group in breast milk and vagina of healthy women and potential role in the colonization of the infant gut. Journal of Applied Microbiology, 103, 2638–2644. Martín, R., Heilig, H.G.H.J., Zoetendal, E.G., Jiménez, E, Fernández, L., Smidt, H. & Rodríguez, J.M. (2007a). Cultivation-independent assessment of the bacterial diversity of breast milk among healthy women. Research in Microbiology, 158, 31-37. Martín, R., Jiménez, E., Olivares, M., Marín, M.L., Fernández, L., Xaus, J. & Rodríguez, J.M. (2006). Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother–child pair. International Journal of Food Microbiology, 112, 35–43. Martín, R., Langa, S., Reviriego, C., Jiménez, E., Marín, M.L., Olivares, M., Boza, J., Jiménez, J., Fernández, L., Xaus, J. & Rodríguez, J.M. (2004). The commensal microflora of human milk: new perspectives for food bacteriotherapy and probiotics. Trends in Food Science & Technology, 15, 121–127. Martín, R., Langa, S., Reviriego, C., Jiménez, E., Marín, M.L., Xaus, J., Fernández, L. & Rodríguez, J.M. (2003). Human milk is a source of lactic acid bacteria for the infant. Journal of Pediatrics, 143, 754–758. Martín, R., Olivares, M., Marín, M.L., Fernández, L., Xaus, J. & Rodríguez, J.M. (2005). Probiotic potential of 3 lactobacilli strains isolated from breast milk. Journal of Human Lactation, 21, 8 -17.

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Olivares, M., Díaz-Ropero, M.P., Gómez, N., Lara-Villoslada, F., Sierra, S., Maldonado, J.A., Martín, R., López-Huertas, E., Rodríguez, J.M. & Xaus, J. (2006b). Oral administration of two probiotic strains, Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711, enhances the intestinal function of healthy adults. International Journal of Food Microbiology, 107, 104 – 111. Olivares, M., Díaz-Ropero, M.P., Martín, R., Rodríguez, J.M. & Xaus, J. (2006a). Antimicrobial potential of four Lactobacillus strains isolated from breast milk. Journal of Applied Microbiology, 101, 72–79. O'Sullivan, G.C., Kelly, P., O'Halloran, S., Collins, C., Collins, J.K., Dunne, C. & Shanahan, F. (2005). Probiotics: an emerging therapy. Current Pharmaceutical Design, 11, 3-10. Ouwehand, A.C. & Salminen, S.J. (1998). The health effects of cultured milk products with viable and non-viable bacteria. International Dairy Journal, 8, 749–758. Penders, J., Thijs, C., Vink, C., Stelma, F.F., Snijders, B., Kummeling, I., van den Brandt, P.A. & Stobberingh, E.E. (2006). Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics, 118, 511–521. Rautava, S. & Isolauri, E. (2002). The development of gut immune responses and gut microbiota: effects of probiotics in prevention and treatment of allergic disease. Current Issues in Intestinal Microbiology, 3, 15-22. Rautava, S. (2007). Potential uses of probiotics in the neonate. Seminars in Fetal and Neonatal Medicine, 12, 45-53. Rautava, S., Arvilommi, H. & Isolauri, E. (2006). Specific probiotics in enhancing maturation of IgA responses in formula-fed infants. Pediatric Research, 60, 221-224. Rautava, S., Ruuskanen, O., Ouwehand, A., Salinen, S. & Isolauri, E. (2004). The hygiene hypothesis of atopic disease – an extended version. Journal of Pediatrics Gastroenterology and Nutrition, 38, 378-88. Rescigno, M., Urbano, M., Valsazina, B., Francoloni, M., Rotta, G., Bonasio, R., Granucci, F., Kraehenbuhl, J. P. & Ricciardi-Castagnoli, P. (2001). Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature Immunology, 2, 361–367. Rotimi, V.O. & Duerden, B.I. (1981). The development of the bacterial flora in normal neonates. Journal of Medical Microbiology, 14, 51-62. Stratiki, Z., Costalos, C., Sevastiadou, S., Kastanidou, O., Skouroliakou, M., Giakoumatou, A. & Petrohilou, V. (2007). The effect of a bifidobacter supplemented bovine milk on intestinal permeability of preterm infants. Early Human Development, 83, 575-9. Szajewska, H., Gawronska, A., Wos, H., Banaszkiewicz, A. & Grzybowska-Chlebowczyk, U. (2007). Lack of effect of Lactobacillus GG in breast-fed infants with rectal bleeding: a pilot double-blind randomized controlled trial. Journal of Pediatric Gastroenterology and Nutrition, 45, 247-251. Taylor, A.L., Hale, J., Wiltschut, J., Lehmann, H., Dunstan, J.A. & Prescott, S.L. (2006). Effects of probiotic supplementation for the first 6 months of life on allergen- and vaccine-specific immune responses. Clinical and Experimental Allergy, 36, 1227-1235. Weizman Z. & Alsheikh, A. (2006). Safety and tolerance of a probiotic formula in early infancy comparing two probiotic agents: a pilot study. Journal of the American College of Nutrition, 25, 415–419.

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West, P.A., Hewit, J.H. & Murphy, O.M. (1979). The influence of methods of collection and storage on the bacteriology of human milk. Journal of Applied Bacteriology, 46, 269211. Zacarías, M.F., Binetti, A., Laco, M., Reinheimer, J. & Vinderola, G. (2009). Leche materna como fuente de bifidobacterias. Aislamiento, identificación y caracterización tecnológica preliminar de cepas. Proceedings of the III International Congress of Food Science and Technology, Córdoba, Argentina, April 15th -17th 2009. ISBN: 978-987-24620-1-7.

In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter VI

Probiotics in Maternal and Early Infant Nutrition Yolanda Sanz* Microbial Ecophysiology and Nutrition Group. Institute of Agrochemistry and Food Technology (IATA), Spanish National Research Council (CSIC), PO Box 73, 46100 Burjassot, Valencia, Spain

Abstract During pregnancy fetal development is entirely dependent on the mother. Epidemiologic and clinical studies suggest that immunologic and metabolic profiles of the pregnant uterus are responsive to mother’s diet. This evidence supports the hypothesis that maternal nutrition may influence fetal programming and disease risk in the offspring. After birth, the gastrointestinal tract undergoes vast structural and functional adaptations under the stimulation of the microbiota and the diet that make possible handle with antigens and digest milk and latter solid food. The intestinal colonization process implies the activation of diverse metabolic functions either triggered by host-microbe interactions or directly encoded by the genome of the microbiota (microbiome). Moreover, microbial exposure through colonization process of the newborn intestine is essential to regulate epithelial permeability and immune function, with long-term consequences on host’s health. Bacterial composition and succession during the intestinal colonization process have been shown to determine susceptibility to infections and sensitization to dietary antigens. In this context, mammals seem to have a developmental window within the perinatal and postnatal period, in which the host-gut microbiota interactions are more influential in favoring later health. Probiotic and prebiotic administration has been demonstrated to be a dietary strategy that at least temporary modulates the microbiota composition and may favor a healthy status. These

*

Corresponding author. Tel.: 34 96 390 00 22; Fax: 34 96 363 63 01. E-mail: [email protected]

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Yolanda Sanz strategies have demonstrated moderate efficacy to reduce the risk of infections and allergic diseases in early life. In recent years, the administration of probiotics to pregnant and lactating mothers in addition to their newborns, along or not with prebiotics, has also been evaluated to extend their applications and improve effectiveness by acting in these critical developmental stages. This type of intervention has shown that specific probiotic strains influence gut growth and immune function in the offspring of animal models. Other studies have suggested that this dietary strategy may help to reduce the risk of atopy, infections, and metabolic disorders in humans. The current knowledge of the effectiveness and mechanisms by which the administration of probiotics to mothers and infants could positively affect early stages of development, favoring latter heath is review.

1. Introduction The prevalence of metabolic diseases associated with obesity is increasing worldwide presumably as a result of societal changes and environmental influences. In addition, whereas hygienic conditions and medical advances have reduced the mortality from infectious diseases in most countries, immune-mediated diseases, such as allergies and autoimmune diseases, are becoming more prevalent worldwide. This has led to speculate that modern lifestyle has altered the relationship between predisposing genes and environmental factors so as to promote development of immune and metabolic disorders, which are becoming major causes of concern and mortality in developed countries. The “hygienic hypothesis” proposed by Strachan (1989) seems to explain reasonably well the increased prevalence of immunemediated diseases such as allergies. It is known that pregnancy favors Th2 lymphocyte development in the mother and the fetus to prevent the fetus from rejection. However, adequate exposure to microbes in early life is also essential to trigger Th1 lymphocyte development that will prevent form persistence of the Th2 biased responses in the newborn and thereby development of an allergic phenotype latter in life. Epidemiological and intervention studies also suggest that diet and exposure to microbes influence the metabolic and immunologic profiles of the pregnant uterus and the risk of immune and metabolic diseases development in the offspring (Yajnik et al., 2006; Barker et al., 2007, Ege et al., 2008). A number of preclinical and clinical intervention studies based on nutritional restrictions, high fat diets and functional ingredient supplementation (e.g. polyunsaturated fatty acids and probiotics) during pregnancy have revealed a close relationship between mother’s diet and infant’s health (Herrera et al., 2002; Church et al., 2009) supporting the fetal programming hypothesis proposed by Barker et al. (2007). Therefore, the interplay between both heredity and environmental factors (diet, microbes and xenobiotics) seems to affect every stage of development from conception to early postnatal period with potential long-term effects in child and adult health. Thus, it can be anticipated that further research on the convergence of both theories “hygienic” and “fetal programming” will better explain the current disease trends. The intestinal microbiota develops an array of physiological roles within the human body, shaping its metabolic abilities and immune responses. The intestinal colonization

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process of the newborn intestine seems to be a particularly relevant process to the infant’s health (Sanz et al., 2008a). The microbiota provides novel metabolic capacities and also regulates host gene expression to favor both host and microbe survival. In addition, microbial exposure through colonization of the newborn intestine is essential to fully boost the immune system and to regulate permeability, with long-term consequences on health. Microbiota acquisition and succession in the newborn’s intestine has been shown to determine susceptibility to infections and sensitization to dietary antigens. An adequate colonization process or the presence or absence of specific bacteria may favor the prevalence of the beneficial microbiota and a healthy status (Kalliomaki et al.2007). This may be achieved by dietary intervention based on probiotic and prebiotic administration. However, while probiotic administration only seems to provide temporary changes in a fully developed gut ecosystem, a practically sterile newborn intestine seems to be more amenable to intentional manipulations by dietary strategies. Evidence on the transmission of bacteria from the mother to the neonate has also encouraged the administration of probiotic-based products during the perinatal period and lactation to favor infants gut colonization with healthy strains. This practice also seems to provide additional benefits by influencing, for instance, breast-milk composition and immune functions on the mother-fetal interface. Therefore, the incorporation of probiotic based products into both maternal and infant nutrition is being investigated as a novel dietary approach to reduce disease risk in the light of both the hygienic and the fetal programming hypothesis. The current knowledge of the effectiveness and mechanisms by which probiotics could positively act at early stages of development favoring latter heath is review and discussed in the following sections.

2. Microbiota Acquisition and Succession in the Newborn Intestine Infants are born with a practically sterile intestine that is rapidly colonized after birth. The colonization during the first 12-24 hours of life is characterized by the presence of higher levels of facultative anaerobes (e.g. Enterobacteriaceae, Enterococcus, and Streptococcus) than strict anaerobic bacteria (e.g. Bifidobacterium, Bacteroides and Clostridium), but these proportions are reversed within a week after birth, when an anaerobic environment is established (Mackie et al., 1999). The infant microbiota, which is characterized by low diversity and instability, evolves into a typical adult microbiota over the first 24 months of life. Then, it becomes more stable and diverse, and seems to be unique for each individual (Zoetendal et al., 1998). The acquisition and succession of the gut microbiota depend on diverse external and internal host factors. External factors include the gestational age, intake of medicines, microbial load from the environment, mode of delivery, mother’s microbiota, number of siblings and type of feeding and solid diet. Internal factors include the gastrointestinal physiology that influences peristalsis, bile acid concentrations and gastrointestinal pH, as well as other genotypic determinants of gut colonization, whose knowledge is more limited (Björkstén, 2006; Sanz et al., 2008a). Gestational age has been suggested to influence the gut microbiota, particularly when comparing preterm and full-term infants due to differences in exposure to environmental

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microbes and antibiotics. In general, colonization of the intestine with beneficial bacteria has been shown to be delayed in preterm infants and the number of potentially pathogenic bacteria was higher than in healthy full-term infants. Comparisons of microbial succession patterns during the first 4 weeks of life revealed that E. coli, Enterococcus, and Klebsiella pneumoniae were commonly present and Bifidobacterium species were not in the microbiota of hospitalized preterm infants, in contrast to full-term infants (Schwiertz et al., 2003). In addition, the microbial patterns of preterm infants became more similar to one another over time, supporting the hypothesis that environmental factors influence the initial colonization of the newborn intestine. Other studies have also associated hospitalization and prematurity with higher prevalence and counts of Clostridium difficile (Penders et al., 2006). The fact that many preterm infants receive prophylactic antibiotics at birth may affect the intestinal colonization (Westerbeek et al., 2006) and, in fact, reductions in the numbers of Bifidobacterium and Bacteroides have been associated with this clinical practice (Penders et al., 2006). The mother’s microbiota (vaginal, intestinal, oral and cutaneous microbiota) is also thought to influence infant’s gut colonization (Mackie et al., 1999). During birth, exposure to the mother’s vaginal and intestinal microbiota due to the proximity between the birth canal and the anus is likely to influence the initial colonizers of the newborn intestine. After birth, cutaneous and oral mother’s microbiota will also be easily transferred to the newborn by kissing and sucking (Lindberg et al., 2004). In particular, a relation between the infant’s microbiota and type of delivery has been inferred in diverse studies. This has partly been explained by the lack of initial contact of infants delivered by caesarean section with the mother’s vaginal microbiota. Microbes from the vaginal canal and anus area seem to be able to enter the mouth and stomach of vaginally delivered infants within few minutes after birth. However, the vaginal microbiota seems to settle-down in the newborn intestine less easily than the maternal intestinal microbiota (Mackie et al., 1999). In general, caesarean-delivered infants have been shown to experience some delay in bacterial colonization, and their microbiota have presented aberrancies for up to 1 year (Adlerberth et al., 2006). Some studies showed that the fecal microbiota of caesarean-delivered infants harbors significantly fewer Bacteroides species compared with that of vaginally delivered infants (Grönlund et al., 2000; Adlerberth et al., 2006), which was suggested to be associated with maturation of humoral immunity (Grönlund et al., 2000). Bifidobacteria colonization was also shown to be delayed and reached lower levels in the caesarean delivered infants than in the vaginally delivered infants (Chen et al., 2007). Another study also revealed that the microbiota of the caesarean delivered group was less diverse, in terms of bacteria species, than the microbiota of the vaginally delivered group after 3 days of life (Biasucci et al., 2008). Moreover, Bifidobacterium species were absent in the microbiota of caesarean delivered infants, while B. longum and B. catenulatum groups were predominant in that of vaginally delivered neonates (Biasucci et al., 2008). In addition, mode of delivery seems to have significant effects on immunological functions of the infant, probably via its influence on gut microbiota colonization (Huurre et al. 2008a). At 1 month of age, the total gut bacterial counts in feces were higher in vaginally delivered infants than in caesarean section delivered infants, mainly due to higher bifidobacterial counts. In contrast, the total number of IgA-, IgG- and IgMsecreting cells was lower in infants born by vaginal delivery than in those born by caesarean

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section during the first year of life, possibly reflecting excessive antigen exposure across the vulnerable gut barrier in the last group (Huurre et al., 2008a). A large study on the contribution of external influences to the gut microbiota composition in early infancy, which included fecal samples from 1032 infants at 1 month of age, showed that infants born through caesarean section had lower numbers of Bifidobacterium and Bacteroides, whereas they were more often colonized with Clostridium difficile, compared with vaginally born infants (Penders et al., 2006). The number of siblings has also been related to the newborn microbiota since it implies an increase of microbial exposure by direct contact with the newborn. In some studies, infants with older siblings had slightly higher numbers of bifidobacteria compared with infants without siblings (Penders et al., 2006) but this was not demonstrated in all studies (Ahrné et al., 2005). Other studies showed transmission of intestinal bacteria from parents to their babies by comparison of fecal bacteria DNA patters, which were probably influenced by both genetic and environmental factors (Favier et al., 2003). The type of milk-feeding is regarded as one of the major environmental factors influencing gut colonization in early life. In general, Bifidobacterium are dominant in breastfed infants (up to 90% of the total fecal bacteria), while a more-diverse adult-like microbiota is found in formula-fed infants (Salminen & Isolauri, 2006). A large-scale population study has showed that exclusively formula-fed infants were more often colonized with E. coli, C. difficile, Bacteroides, and Lactobacillus, compared with breastfed infants (Penders et al., 2006). A delay in Bifidobacterium species colonization in formula-fed compared with breastfed babies has also been shown by other authors (Favier et al., 2003). In addition, differences in Bifidobacterium species composition as a function of the type of milk-feeding have been reported. Some authors showed that B. breve was predominant in breast-fed infants, whereas B. longum and B. adolescentis were found more often in formula-fed infants (Mitsuoka and Kaneuchi, 1977; Mevissen-Verhag et al., 1987). More recently, another comparative molecular analysis of the infant’s microbiota has showed that breast-fed infants had significantly higher levels of total Bifidobacterium and B. breve, and lower levels of B. adolescentis and B. catenulatum than formula-fed infants, resembling the bifidobacteria populations of the adult microbiota (Haarman & Knol, 2005). However, other authors have not found significant differences in Bifidobacterium species composition a function of the type of feeding (Favier et al., 2003). Breast milk seems to be a source of commensal bacteria to the infant gut, including Staphylococcus, Lactobacillus and Bifidobacterium (Martín et al., 2008). B. breve, B. adolescentis and B. bifidum have been isolated from milk in culture medium and, therefore, at least these species could be transferred alive to the newborn via breast-feeding. Breast-milk bifidobacteria composition was also analyzed by real-time PCR showing that B. longum was the most widely found species followed by B. animalis, B. bifidum and B. catenulatum although the detected prevalence of species was based on DNA detention but not on viability (Gueimonde et al., 2007). A recent study also showed that allergic mothers had significantly lower amounts of bifidobacteria in breast-milk compared with non-allergic mothers and their infants had concurrently lower counts of bifidobacteria in feces. Therefore, the maternal health status could derange the counts of bifidobacteria in breast-milk and influence the infants' fecal Bifidobacterium levels (Grönlund et al., 2007). Breast-milk also contains prebiotic substances (oligosaccharides), which stimulate the growth

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of Bifidobacterium and are one of the major bifidogenic factors (González et al., 2008). In fact, Bifidobacterium longum subsp. infantis ATCC 15697, an isolate from the infant gut, was shown to preferentially consume small mass oligosaccharides, which represent 63.9% of the total human milk oligosaccharides available (LoCascio et al., 2007). Accordingly, the complete genome sequence of this strain reflects a competitive ability to utilize milk-borne molecules, which lack a nutritive value to the neonate (Sela et al., 2008). Breast-milk contains other bioactive factors, such as secretory IgA, lactoferrin, and a range of cytokines, which have a remarkable effect on the development of the neonatal microbiota and mucosal immunity. In particular, IgA supplied by maternal milk has been shown to sequester commensal bacteria in the mice neonatal intestine, thereby delaying active development of IgA production (Kramer & Cebra, 1995), while the decline in maternal IgA supply increases intestinal bacterial colonization in pups (Inoue et al., 2005). In formula-fed infants no detectable levels of fecal IgA were found during the first 10 days of life, which might partially explain the development of a more-diverse microbiota in these infants at an early age (Bakker-Zierikzee et al., 2006). A few studies have demonstrated a relationship between the genotype and the gut microbial colonization process so far. Similarity between fecal bacteria DNA profiles of monozygotic twins was shown to be significantly greater than that between profiles of unrelated individuals (Zoetendal et al., 2001). Furthermore, the DNA profiles of the fecal microbiota of monozygotic twins were also significantly more similar than those of dizygotic twins (Stewart et al., 2005). It can be anticipated, that the host genotype may influence for instance the repertory of mucins, acting as bacterial adhesion sites in the intestinal mucosa, and the immune response, which altogether will restrict or allow the colonization of certain micro-organisms (Björkstén, 2006; Sanz et al., 2008a). However, some studies suggest that the shared environment influences the gut microbiota to a higher extent than the genotype (Palmer et al., 2007). In particular, studies on the evolution of mammals and their gut microbes by metagenomic approaches pointed out that the acquisition of a new diet is a fundamental driver for changes in gut bacterial diversity (Ley et al., 2008). This scientific evidence supports the hypothesis that dietary intervention strategies may play a critical role in health and disease prevention, via modulation of the gut microbiota and its functions. Consequently, probiotic and prebiotic based nutritional strategies could play a primary role in this field, which will become a reality as we improve our understanding of the roles of intestinal bacteria in the human body.

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3. Influence of the Intestinal Microbiota in host Physiology and Immunity during the Early Postnatal Period 3.1. Influence of the Intestinal Microbiota in Host Physiology and Metabolism The anatomy and physiology of the intestine depend on the interactions between the microbiota and the diet with the host epithelium, mucosal immune system and microvasculature (Hooper et al., 2002). After birth, the gastrointestinal tract undergoes vast structural and functional adaptations to be able to initially digest mother's milk and later solid food, during the weaning period. During the early suckling period, the morphological and functional gut development is reflected in a rapid intestinal growth and increased expression of digestive enzymes such as lactase. At weaning period, crypt hyperplasia and an increased expression of maltase, sucrase, and pancreatic trypsin also denote vast changes of the gastrointestinal tract to digest a more complex diet. It is known that the gut microbiota plays an important role in these gastrointestinal maturation processes from gnotobiology studies (Fåk et al., 2008). In germ-free animals the intestinal weight and surface area were decreased, the intestinal villi were thinner, and the shape of enterocytes was abnormal compared with conventionally raised mice (Berg, 1996). In contrast, the cecum was much larger in germ-free animals due to mucus and fiber accumulation and subsequent water retention when compared with conventionally raised animals (McCracken & Lorenz, 2001). In preterm formula-fed animal, the administration of specific bacteria immediately after birth limited the formulainduced mucosal atrophy, dysfunction, and pathogen load in preterm neonates. This strategy also increased the intestinal weight, mucosa proportion, villus height, RNA integrity, and brush border aminopeptidase A and N activities, reflecting the role of bacteria in intestinal physiology (Siggers et al., 2008). It is known that intestinal microbiota develops an important biochemical activity within the human body by both providing additional metabolic capacities encoded by the microbiome to the host (Gill et al., 2006) and by regulating diverse aspects of cellular differentiation and gene expression via host-microbe interactions (Hooper et al., 2002). The intestinal microbiota provides additional metabolic capacities, involved in the utilization of no digestible carbohydrates and host-derived glycoconjugates (e.g. chondroitin sulphate, mucin, hyaluronate and heparin), deconjugation and dehydroxylation of bile acids, cholesterol reduction and biosynthesis of vitamins (K and B group), isoprenoids and amino acids (e.g. lysine and threonine) (Hooper et al., 2002, Gill et al., 2006). In particular, the ability of the commensal microbiota to utilize complex dietary polysaccharides, that would otherwise be inaccessible to humans, may contribute to the ability of the host to harvest energy from the diet, which may represent 10% of the daily energy supply (Sanz et al., 2008b). In addition, B. thetaiotaomicron, a prominent commensal bacteria, has been shown to have ability to both hydrolyze host-derived glycans and influence the nature of those produced by the host by increasing expression of fucosylated ones to favor its own colonization (Hooper et al., 2001).

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The commensal microbiota has also been shown to induce expression of genes involved in the processing and absorption of dietary carbohydrates and complex lipids in the host (Hooper et al., 2001; Bäckhed et al., 2004). For instance, ileal expression of a monosaccharide transporter (Na+/glucose co-transporter) was induced in B. thetaiotaomicron mono-colonized mice, which would lead to increasing the absorption of dietary monosaccharides and short-chain fatty acids and, thereby, promote the novo synthesis of lipids in the liver (Hooper et al., 2001). The colonization of germ-free mice by conventional microbiota also increased liver expression of key enzymes (acetyl-CoA carboxylase and fatty acid synthase) involved in the de novo fatty acid biosynthetic pathways, and transcriptional factors (ChREBP and SREBP-1) involved in hepatocyte lipogenic responses to insulin and glucose (Bäckhed et al., 2004). In addition, the colonization reduced the levels of circulating fasting-induced adipose factor (Fiaf) and the skeletal muscle and liver levels of phosphorylated AMP-activated protein kinase, which altogether contribute to fat storage (Bäckhed, et al., 2007). The colonization of germ-free mice by B. thetaiotaomicron also increased the expression of other components involved in the host’s lipid absorption machinery, including a pancreatic-lipase related protein that hydrolyzes triacylglycerols, a cytosolic fatty acid binding protein (L-FABP) involved in intracellular trafficking of fatty acids, and the apolipoprotein A-IV that mediates export of triacylglycerols re-synthesized in the enterocyte (Hooper et al., 2001). The expression of genes involved in absorption of dietary metal ions was also modified by the microbiota, which for instance induced the expression of a high affinity copper transporter in the epithelium (Hooper et al, 2001). Furthermore, the colonization of the intestinal mucosa was also implicated in the regulation of the under laying microvasculature and angiogenesis (Stappenbeck, et al., 2002).

3.2. Influence of the Intestinal Microbiota in Host Immunity An adequate colonization process of the newborn intestine by commensal bacteria provides substantial stimuli for the maturation of the gut barrier function and immunity during the postnatal period, whereas alterations in this process may precede the development of immune-mediated diseases (Sanz et al., 2008a). After birth, the gastrointestinal system is immature and relatively permeable to macromolecules, but this high permeability declines with age until a more mature gut barrier is established at weaning. Commensal bacteria have been shown to regulate intestinal permeability for instance protecting against leakage of tight-junctions and alterations in cytoskeleton integrity associated with infections, stress and inflammatory conditions, contributing to the physical barrier that prevents the entry of harmful agents (Lutgendorff et al., 2008). The gut microbiota also influences the secretion of various protective substances by epithelial cells such as mucins and antimicrobial peptides. Commensal bacteria have been shown to regulate mucin gene expression by goblet cells modifying the glycosylation pattern and the expression of MUC-2 and MUC-3 genes, which may influence bacterial adhesion and colonization (Mack et al., 2003; Freitas et al., 2005; Caballero-Franco et al., 2007). The secretion of antimicrobial peptides (defensins, C-type lectins and angiogenins) by intestinal Paneth cells has been demonstrate to be stimulated by Gram-negative and Gram-positive (Ayabe et al., 2000; Hooper et al., 2003; Cash et al., 2006;

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Vaishnava et al., 2008), constituting a mechanism whereby commensal bacteria may influence gut ecology and regulate non-specific intestinal defenses during the postnatal period. Microbial colonization of germ-free animals has also an important impact on postnatal development of mucosal and systemic immunity. The gut-associated lymphoid tissue (GALT) is immature in germ-free mice, showing a reduced content of the lamina propria CD4+ T cells, IgA producing B cells and intraepithelial T cells. Systemic immunity is also affected by the absence of the microbiota and germ-free mice have decreased serum immunoglobulin levels and their mesenteric lymph nodes and spleens are smaller (Hrncir et al., 2008). The interaction of the gut microbiota with the GALT contributes to the production of secretoryIgA, modulation of cytokine and chemokine release, and development of balanced T helper (h) 1/Th2 responses and oral tolerance to innocuous antigens (Sanz et al., 2008a). The immune system should be regulated to such a way that can mount an effective innate and acquired mucosal and humoral response to eliminate the invader organism, but avoid collateral tissue damage and overreactions to harmless microbes and antigens. One of the key events in the regulation of appropriate immune responses is the differentiation between pathogenic and commensal bacteria. This is achieved by pattern-recognition receptors (Tolllike receptors [TLRs] and Nod-like receptors [NLRs]) expressed in epithelial and antigen presenting cells (macrophages and dendritic cells [DC]), which act as sentinels sensing environment and activating defenses in face to danger. In particular TLR-signaling commonly leads to the activation of nuclear factor kappa B (NF-κB) transcription pathway, with up regulation of major histocompatibility complexes and co stimulatory proteins, production of pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, and IL-8), and recruitment of other immune cells. Signaling through TLR also stimulates the maturation of DCs with enhanced ability to present antigens and activate T cells, T-cell co-stimulatory molecules (CD80 and CD86) and other activation markers. T-cell differentiation into Th 1, Th2 or regulatory T cells (Tregs) is also thought to depend on the type of TLRs involved (Winkler et al., 2007). Th1 responses are usually associated with inflammatory reactions and clearance of pathogenic bacteria and virus, Th2 responses with allergic responses and parasite clearance, and regulatory T cells (Th3 and Thr1) are essential in preventing overreactions (Sanz et al., 2007a). Some of the mechanism by which commensal bacteria, unlikely pathogens, have been shown to down-regulate pro-inflammatory responses include: (i) the inhibition of the NF-κB, either by regulating the nuclear export of NF-κB subunit relA (Kelly et al., 2004) or by inhibiting the IκB ubiquitination (Neish et al., 2000); (ii) the inhibition of TLR2-driven activation of NF-κB signaling via NOD signalling (Watanabe et al., 2008); (iii) the regulation of TLR expression and up-regulation of the negative regulator Tollip protein (Otte et al., 2004; Solano-Aguilar et al., 2008), (iv) the down-regulation of the expression of pro-inflammatory type I IFN related genes (Munakata et al., 2008), and (v) the induction of immunoregulatory cytokine production (IL-10 and TGF-β1) and Tregs (Hrncir et al., 2008).

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4. Influence of the Mother’s Diet and Environmental Exposures in Fetal Programming and Infant’s Health Scientific evidence indicates that the diet influences the fetal-maternal relationships with consequences on development and long-term health. The quality of the maternal diet during pregnancy has been related to offspring's risk of developing allergies, type 1 and type 2 diabetes, arteriosclerosis, cardiovascular disease and obesity (Bertino et al., 2006; Lamb et al., 2008; Yates et al., 2008). Pregnancy is seen as a period of maximum sensitivity to dietary influences since diverse organs and systems are in development in the fetus. In particular, immune and metabolic functions of the fetus are dependent on the mother and probably the refinement of their functions is already initiated inside the uterus. Successful pregnancy requires immunologic changes characterized by a dominance of Th2 and regulatory T cell effector responses in both mother and fetus, which help to maintain pregnancy and avoid the rejection of the immunologically incompatible fetus; however the persistence of this immune polarization favors the development of a long-lasting atopic phenotype latter in life (Calder et al., 2006; Kalliomaki et al., 2007). At least during the third trimester of human pregnancy, fetal T cells are able to mount antigen-specific responses to environmental and food-derived antigens and antigen-specific T cells are detectable in cord blood, indicating fetus sensitization in uterus (Calder et al., 2006). For example, the fetus mounted an immune response to rubella antigens whose mothers were infected with rubella during pregnancy. In infected fetuses, total IgM and IgA concentrations rose significantly, and rubella virus-specific IgM and IgA antibodies were detected at week 22 of pregnancy (Grangeot-Keros et al., 1988). A recent investigation has determined the relationships of cord blood immunoglobulin E (IgE) with maternal health conditions before and during pregnancy, showing inverse associations of cord blood IgE to seasonal allergens with positive maternal records for Toxoplasma gondii and rubella virus infections. Therefore, maternal immunity to certain pathogenic antigens may influence atopic sensitization in the fetus (Ege et al., 2008). Studies of the incidence of human pathogenic bacteria and virus (cytomegalovirus and herpes simplex virus type 1 and 2) in biopsy samples from the placenta and decidua of women with healthy pregnancies by PCR, showed that 38% of placental samples were positive for the selected microorganisms. Moreover, analyses of IgG isolated from the placenta support the hypothesis that immune responses suppress cytomegalovirus reactivation in the presence of pathogenic bacteria at the maternal-fetal interface, suggesting that at least microbial DNA at the placenta may drive immune responses (McDonagh et al., 2004). More recently, Bifidobacterium spp. and L. rhamnosus have also been identified in human placenta by quantitative real-time PCR recently (Satokari et al., 2009). Although cultivable bacteria were not detected, the possibility that fetus is exposed to bacterial components (e.g. DNA) via the mother’s placenta during pregnancy can not be disregarded. In particular, the unmethylated CpG oligodeoxynucleotide motifs of bacterial DNA are known to activate Toll-like receptor 9 and subsequently trigger Th-1-type immune responses, and this could be one of the mechanisms that contribute to program the infant's immune development during fetal life (Lee et al., 2006). A recent study has also found a close relationship between maternal and

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cord-blood specific IgE patterns to cow milk suggesting that the immune system can be stimulated by food allergens before birth (Bertino et al., 2006). Another study analyzed whether asthma prevention could start even earlier, before conception, by transfer of immunologic tolerance from the mother to the offspring in animal models (Polte et al., 2008). The offspring of naïve mothers had an asthma-like phenotype, while the offspring of tolerized mice with oral ovalbumin before conception were completely protected. The levels of allergen-specific IgG were increased in the sera of the mother, fetus, pup and breast milk, indication their involvement in tolerance transfer from the mother to the offspring (Polte et al., 2008). In addition, environmental exposure of the pregnant woman to microbes has been related to the allergic predisposition of the neonate. Therefore, the fetal immune system during the perinatal period can be responsive to environmental challenges (microbes or dietary compounds), which may not be longer effective in adulthood to prevent disease. Dietary supply of nutrients during conception and pregnancy may also influence the development of the fetus and offspring, and the risk of suffering metabolic diseases. Maternal obesity from exogenous origin and both maternal hyperglycemia and hyperlipidemia are thought to contribute to the development of metabolic disorders in the offspring. Studies on the effect of chronic high-fat diets on the development of the fetal metabolic systems in nonhuman primates showed that a developing fetus is highly vulnerable to excess of dietary lipids, independent of maternal diabetes and/or obesity, and that exposure to this diet may increase the risk of pediatric non-alcoholic fatty liver disease (McCurdy et al., 2009). Scientific evidence has also indicated that prenatal nutrition may affect dietary preferences and may contribute to more atherogenic lipid profiles later in life. Studies in animal models have suggested that fetal undernutrition can predispose to hypercholesterolemia and metabolic disorders directly by programming cholesterol metabolism and indirectly influencing lifestyle choices. Pregnant women exposed to famine in early gestation were more likely to consume a high-fat diet and they also seem to be less physically active (Lussana et al., 2008).

5. Probiotic and Prebiotic Concepts and Applications The colonization process of the newborn intestine is considered critical to the health status and risk of developing disease in early and later stages of life. Therefore, improving the characteristics of the gut microbiota particularly in the perinatal and postnatal periods is foreseen as a strategy to prevent the onset of disease (Sanz et al., 2008a). This could be achieved by the administration of probiotics, prebiotics or their combination (synbiotics). Probiotics are defined as live microbes that when administered in adequate amounts confer a health benefit to the host [FAO/WHO, 2002]. Prebiotics are non-digestible dietary ingredients that allow changes, both in the composition and/or activity of the gastrointestinal microbiota that confers benefits on the host’s health (Roberfroid, 2007). The genus Bifidobacterium is the predominant in the intestinal tract of infants; it represents about 3% of the total microbiota in the intestine of healthy adults and is associated with beneficial effects on health (Sanz et al., 2007a). The genus Lactobacillus also inhabits the gastrointestinal tract

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and some species are widely used in diverse food fermentations. These features have made these two bacterial genera the most attractive as probiotics for human consumption (Sanz et al., 2007b). Currently, these are incorporated into functional foods in the form of fermented milks, infant formula, cheese, ice cream and juices, and in pharmaceutical preparations. Milk fermented products constitute the best vehicle for probiotic bacteria since this matrix allows maintenance of viability and metabolic activity (Sanz et al., 2007b). Galacto-oligosaccharides and inulin-derivatives are the prebiotics most commonly commercialized in Europe (Haarman & Knol, 2005; Kelly, 2008). They are mainly added to infant formula to promote the prevalence of a microbiota composition similar to that resulting from breast-feeding during both milk-feeding and the weaning period. The administration of these prebiotic infant formulas have demonstrated to increase the total amount of fecal bifidobacteria and to modify the Bifidobacterium species composition, resembling that of breast-fed infants, when compared with infants receiving conventional formula (Haarman & Knol, 2005; Scholtens et al., 2006). The immaturity of the immune system and the increased epithelial permeability of the newborn intestine under lack of appropriate microbial stimulation seem to partly explain the increased prevalence of immune-mediated diseases in developed countries, such as allergies (the hygienic hypothesis). Initial attempts to control the development of allergies and food intolerance were based on the avoidance of the allergen in the diets, but the results were not satisfactory regarding long-term prevention. In recent times, novel methods including probiotic supplementation have been evaluated to counteracting the immunological and gut mucosal barrier dysfunction associated with these disorders, and to strengthening endogenous defense mechanisms. Moreover, while the administration of probiotics to adults with a fully developed intestinal microbiota produces only temporary colonization of the bacteria, the newborn intestine is more amenable to dietary and probiotic manipulation in the early postnatal period. Therefore, the use of probiotics early in life has been evaluated for preventing allergies, repeated infections and necrotizing enterocolitis, and provided some promising results in preclinical trials in animals (Siggers et al., 2008) and in clinical trials in humans (Lodinova-Zadnikova et al., 2003). A step beyond this concept has been the administration of probiotics, together or not with prebiotics, to pregnant women and their infants to influence both the intrauterine and postnatal development of the newborn, relaying on the “fetal programming theory”. These strategies are currently under investigation to prevent both immune and metabolic disorders with the hope that they will influence more effectively the infant’s health from conception onwards.

6. Influence of Maternal and Offspring Probiotic Intake in Animals Several studies have showed that exposure of pregnant mothers to specific probiotic bacteria prior to parturition and during lactation influences diverse physiological functions in the intestine of the offspring. The administration of the probiotic Lactobacillus plantarum 299v in the drinking water to pregnant and lactating rat dams until their pups had reached an age of 14 days resulted in the colonization of both mothers and pups gut by the bacterial

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strain and influenced gut growth and function of pups (Fåk et al., 2008). The small intestine, pancreas, spleen and liver weighed more in the probiotic colonized pups than in the control pups. The probiotic colonized pups also showed decreased gut permeability as compared to the control pups (Fåk et al., 2008). Nevertheless, the effects derived from maternal probiotic intake and their transmission to the offspring as such could be questioned since the administration of the probiotic through the drinking water could have facilitated direct contact and acquisition of the probiotic strain by the pups. The effects of L. rhamnosus GG supplementation to female mice intragastrically every day before conception, during pregnancy and lactation (perinatal supplementation group) or only during pregnancy (prenatal supplementation group) on the development of experimental allergic asthma in the offspring have also been investigated recently (Blümer et al., 2007). Intestinal colonization with L. rhamnosus GG was observed in mother mice, but not in the offspring. In spite of that, a reduced expression of TNF-α, IFN-γ, IL-5 and IL-10 was observed in splenic mononuclear cells of mice derived from mothers perinatally supplemented with L. rhamnosus GG. Moreover, allergic airway and peribronchial inflammation and goblet cell hyperplasia were significantly reduced in offspring of prenatally or perinatally mothers supplemented with L. rhamnosus GG as compared to control mice. Exposure to L. rhamnosus GG during pregnancy shifted the placental cytokine expression pattern with a markedly increased TNF-α level. Thus, the results suggest that this probiotic strain may exert beneficial effects on the development of experimental allergic asthma, when applied in a very early phase of life and that the effects are partly mediated via the placenta by induction of pro-inflammatory cell signals (Blümer et al., 2007). In fact, DNA from Bifidobacterium spp. and L. rhamnosus has also been identified in human placenta by quantitative real-time PCR and its influence on transmission of immune signals to the fetus through this route cannot be disregarded (Satokari et al., 2009). The oral supplementation of Bifidobacterium animalis subsp lactis Bb12 (Bb12) to sows during gestation and to their piglets for 91 days since birth led to the establishment of increased numbers of the probiotic in the piglets’ proximal colon compared to placebotreated piglets born to placebo-treated sows, Bb12-treated sows, or piglets born to placebo sows but treated with Bb12 immediately after birth. This effect was associated with a significant up-regulation of TLR-9 expression in proximal colon, but neither of TLR2 nor TLR4. This result suggests that exposure of the mother to Bb12 influenced both Bb12 load in the piglet in the presence of continuous daily feeding and significantly affected the host innate immune system development (Solano-Aguilar et al., 2008). The protective mechanisms of action of L. rhamnosus GG against atopic dermatitis development have also been studied in a mice model (NC/Nga mice) of human atopic dermatitis (Sawada et al., 2007). Maternal and infant mice were fed with food containing or not containing heat-treated L. rhamnosus GG during pregnancy and breastfeeding, and after weaning. While control NC/Nga mice raised under an air-uncontrolled condition spontaneously manifested typical skin lesions, probiotic feeding inhibited the onset and development of atopic skin lesions and reduced the numbers of mast cells and eosinophils in the affected skin sites. The probiotic-fed mice also showed a significant increase in plasma IL-10 levels and in the IL-10 mRNA expression in both Peyer's patches and mesenteric lymph nodes compared with control mice. However, there was no significant difference in

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the proportion of splenic CD4(+)CD25(+) regulatory T cells between the probiotic-fed mice and the control mice. Therefore, the ability of the probiotic L. rhamnosus GG to delay the onset and suppress the development of atopic dermatitis was mediated via a strong induction of IL-10 in intestinal lymphoid organs and at systemic level. Although the number of preclinical studies is limited, the accumulated scientific evidence suggests that maternal probiotic intake, particularly when occurring prior and after birth, influences the development of the offspring. However, these effects are not always correlated with the ability of the probiotic to colonize the offspring intestine. Moreover, animal studies also support a role for maternal probiotic intake in disease prevention and particularly in atopic dermatitis.

7. Influence of Maternal and Infant Probiotic Intake in Humans 7.1. Influence of Maternal Probiotic Intake in the Intestinal Microbiota of the Infants The clinical trials carried out to investigate the effects of the oral administration of probiotic bacteria only to the pregnant women or to both pregnant woman and their infants on children physiology and health are summarized in Table 1. A pilot study including six women, who were taking L. rhamnosus GG during late pregnancy but discontinued its consumption at the time of delivery, was carried out to evaluate the influence of probiotic intake in their children, who did not received the probiotic after birth (Schultz et al., 2004). The results showed that temporary colonization of the infant’s gut with L. rhamnosus GG was possible by colonizing the pregnant mother before delivery and that this colonization was stable for as long as 6 months, and could persist for up to 24 months (Schultz et al., 2004). Further studies showed that the administration of L. rhamnosus GG to mothers, 4 weeks before and 3 weeks after delivery, induced specific changes in the transfer and initial establishment of bifidobacteria in the neonates (Gueimonde et al, 2006). Infants whose mothers received L. rhamnosus GG showed a significantly higher presence of B. breve and lower of B. adolescentis than those from the placebo group at 5 days of age. B. adolescentis prevalence in the mother before delivery was correlated with its presence in the infant samples at 5 and 1 month and similar effects were detected for B. catenulatum and B. longum at 1 month; although these effects were only significant in the placebo group. Altogether these results suggest the transfer of bacteria from mother to the newborn. However, L. rhamnosus GG consumption also increased the bifidobacterial diversity in infants at 3 weeks and reduced the similarity of Bifidobacterium microbiota between mother and infant (Gueimonde et al., 2006), which partly contradicts the evidences on the transference of the fecal microbiota from the mother to the newborn.

Table 1. Effects of maternal and infant probiotic intake in humans Probiotic/prebiotic/Diet L. rhamnosus GG L. rhamnosus GG Fermented milk and yogurt bacteria Lactobacillus casei DN11401

L. rhamnosus GG and LC705 B. breve Bb99 P. freudenreichii subsp. shermanii and galacto-oligosaccharides L. rhamnosus GG

Administration pattern Intake by healthy women at late pregnancy but not after delivery Intake by healthy women 4 weeks before and 3 weeks after delivery Oral intake by women at 34 weeks of pregnancy or vaginal application from first trimester onwards Intake by healthy women 6 weeks before delivery and during 6 weeks of lactation

Probiotic intake by women carrying fetus at allergy risk during the last month of pregnancy and by their infants until age of 6 months plus a prebiotic

Intake by mothers at family-risk of atopic eczema for 4 weeks before delivery and postnatally for 6 months

Trial outcome Probiotic colonization of the infant’s gut

Reference Schultz et al., 2004

Changes in bifidobacteria transfer and establishment in the neonates Reduction of genital infection risk

Gueimonde et al, 2006

Natural killer cell increase in mother’s peripheral blood and TNF-α decrease in breast-milk Decrease of gastrointestinal episodes in infants Increased resistance to respiratory infections in children during 2 years Trend to reduce IgE-associated diseases and prevented atopic eczema at 2 years Increase of fecal lactobacilli and bifidobacteria Risk reduction of atopic eczema for up to 7 y Increasing of TGF-β2 in mother’s milk

Ortiz-Andrellucchiet al., 2008

Othman et al., 2007

Kukkonen et al., 2007, 2008

Kalliomaki et al., 2007 Rautava et al., 2002

Table 1. Continued Probiotic/prebiotic/Diet L. rhamnosus GG and B. lactis Bb2

L. rhamnosus GG L. rhamnosus GG

L. rhamnosus GG and B. lactis Bb12 plus dietary counseling Dietary recommendations and L. rhamnosus GG and B. lactis L. rhamnosus GG and B. lactis Bb12 plus dietary counseling

Administration pattern Intake by pregnant women carrying fetus at allergy risk from the first trimester of pregnancy till the end of exclusive breastfeeding Intake by pregnant women carrying fetus at allergy risk for 36 weeks before delivery Intake by women at risk of atopic diseases from 4 to 6 weeks before delivery and postnatally for 6 months Intake by women from early till end of pregnancy Intake by women from first trimester of pregnancy onwards. Intake by healthy women from first trimester of pregnancy onwards

Trial outcome Modest increase of TGF-β2 in colostrums Reduced allergen sensitization in infants

Reference Huurre et al., 2008

No effect on fetal antigen-specific immune responses evaluated in cord blood cells No effect on incidence of atopic dermatitis

Boyle et al., 2008

Increased placental concentrations of linoleic and dihomo-gamma-linolenic acids. Highest and lowest intakes of specific nutrients associated with higher blood pressure in children at 6 months Reduced blood glucose concentrations and increased glucose tolerance during pregnancy and over the 12 months postpartum

Kaplas et al., 2007

Kopp et al., 2008

Aaltonen et al., 2008

Laitinen et al., 2008

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7.2. Influence of Maternal and Infant Probiotic Intake in Child Health The use of probiotics during pregnancy is under consideration due to the positive effects of some strains on diverse clinical situations such as infections (vaginal, respiratory and gastrointestinal) and allergy, as well as on the physiological development of immune and metabolic functions. The number of human clinical trials intended to validate such probiotic applications are rapidly increasing, some of which are showing moderate effectiveness in improving child’s health. The effectiveness of probiotics for preventing preterm labor and birth has been the objective of recent studies since the risk of this outcome in the presence of maternal infection is dramatically increased, reaching values of 30% to 50% (Othman et al., 2007). It has been suggested that specific probiotics could exert beneficial effects on such applications because of their ability to displace and inhibit pathogens and modulate immune responses by interfering with the inflammatory cascade that leads to preterm labor and delivery. So far, the two randomized controlled trials assessing the prevention of preterm birth in pregnant women and women planning pregnancy with the use of probiotics registered in 2006 have been reviewed (Othman et al., 2007). One study enrolled women after 34 weeks of pregnancy using oral fermented milk as probiotic, while the other study enrolled women diagnosed with bacterial vaginosis in early pregnancy that utilized commercially available yogurt vaginally. The results showed 81% reduction in the risk of genital infection with the use of probiotics. However, this was the only pre-specified clinical data available and there are, currently, insufficient data to assess impact on preterm birth and its complications. The prevention of AIDS and other infections in women and children by dietary intervention based on the probiotic concept has also been considered a possibility but still a distant reality (Reid and Devillard, 2004). The use of probiotic bacteria during pregnancy could be a mean to modulate immune development in fetus to reduce the risk of immune aberrancies and improve the host’s defenses. In this context, the effects of the consumption of milk fermented with the strain Lactobacillus casei DN11401 by pregnant women during 6 weeks before delivery and during 6 weeks of lactation were determined (Ortiz-Andrellucchi et al., 2008). Mothers supplemented with the probiotic showed a significant increase in natural killer cells in peripheral blood samples and a non-significant increase in T and B lymphocytes. Maternal milk also showed a decrease in the pro-inflammatory cytokine TNF-α and breast-fed child of the mothers who consumed L. casei showed fewer gastrointestinal episodes. The safety and effects of a mixture of 4 probiotic bacterial strains (L. rhamnosus GG and LC705, Bifidobacterium breve Bb99, and Propionibacterium freudenreichii subsp. shermanii) along with a prebiotic galacto-oligosaccharide has also been evaluated in pregnant women carrying high-risk children of allergic diseases and their infants. Pregnant women consumed a probiotic preparation or a placebo for 2 to 4 weeks before delivery and their infants received the same probiotics plus galacto-oligosaccharides for 6 months. No differences in growth, infant colic, morbidity or other adverse health effects between the two groups of children were found. Moreover, a slightly higher percentage of children had been prescribed antibiotics in the placebo group (28%) than in the probiotic group (23 %) during the

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intervention. Respiratory infections also occurred less frequently in the synbiotic group (3.7 versus 4.2 mean infections) throughout the follow-up period (Kukkonen et al., 2008). The administration of the probiotic L. rhamnosus GG to both pregnant mother and their babies demonstrated to reduce the incidence of atopic eczema for up to 7 years in the Finish population (Kalliomaki et al., 2007). L. rhamnosus GG was given prenatally to mothers, who had at least one first-degree relative with atopic eczema, allergic rhinitis, or asthma, for 4 weeks before expected delivery and postnatally for 6 months to their children. L. rhamnosus GG was effective in prevention of early atopic disease in children at high risk as determined by considering chronic recurring atopic eczema as the primary endpoint. The administration of probiotics to the pregnant and lactating mother was shown to increase the amount of antiinflammatory cytokine TGF-β2 in the mother’s milk, thereby increasing its immunoprotective potential (Rautava et al., 2002). The infants most likely to benefit from maternal probiotic supplementation were those with an elevated cord blood IgE concentration (Rautava et al., 2002). In addition, Huurre et al. (2008b) evaluated the effects of dietary counseling and probiotic supplementation (L. rhamnosus GG and B. lactis Bb2) to pregnant women, at risk of developing atopy, on their children. Children of atopic mothers, specifically when breastfed exclusively over 2.5 months or 6 months, had a higher risk of sensitization at the age of 12 months but this risk could be reduced by the use of probiotics during pregnancy and lactation. The preventive effects were considered to be the result of a beneficial change in breast milk composition characterized by a modest increase in TGF-β2 concentration (Huurre et al., 2008b); however, this increase did not reach statistical significance and it was only detected in the colostrums while disappeared after 1 month. To progress on the knowledge of the mechanisms of action beyond the effects of L. rhamnosus GG intake on atopic eczema prevention, Boyle et al., (2008) investigated whether this probiotic influenced fetal immune responses when administered to pregnant women for 36 week before delivery. The effects of stimulation of cord blood mononuclear cells from women who received the probiotic or placebo with heat-killed L. rhamnosus GG and ovalbumin were evaluated, without showing effects of the treatment on CD4(+) T cell proliferation, forkhead box P3 expression, dendritic cell phenotype or cytokine secretion. Therefore, L. rhamnosus GG supplementation to pregnant women failed to influence fetal antigen-specific immune responses and in particular transplacental immune effects were excluded as the mechanisms of probiotic action (Boyle et al., 2008). The effects of a mixture of 4 probiotic bacterial strains (L. rhamnosus GG and LC705, B. breve Bb99, and P. freudenreichii subsp. shermanii along with a prebiotic galacto-oligosaccharide on allergic disease prevention were also evaluated in pregnant women carrying high-risk children. Pregnant women consumed a probiotic preparation or a placebo for 2 to 4 weeks before delivery and their infants received the same probiotics plus galacto-oligosaccharides or placebo for 6 months. Probiotic treatment compared with placebo showed no effect on the cumulative incidence of allergic diseases, but tended to reduce IgE-associated (atopic) diseases and prevented atopic eczema at 2 years. In addition, lactobacilli and bifidobacteria more frequently colonized the intestine of supplemented infants, suggesting an inverse association between atopic diseases and gut colonization by probiotics (Kukkonen et al., 2007).

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In spite of the aforementioned evidence, the value of probiotics for primary prevention of atopic diseases is still controversial. An additional clinical double-blind, placebo-controlled trial has been carried out to study the preventive effect of the same probiotic, L. rhamnosus GG, on the development of atopic dermatitis in Germany (Kopp et al., 2008). Pregnant women from families with 1 or more members with an atopic disease received either the probiotic or placebo from 4 to 6 weeks before expected delivery, followed by a postnatal period of 6 months. In this case, supplementation with L. rhamnosus GG during pregnancy and early infancy neither reduced the incidence of atopic dermatitis nor altered the severity of atopic dermatitis in affected children, but it was associated with an increased rate of recurrent episodes of wheezing bronchitis at the age of 2 years. Therefore, the authors conclude that L. rhamnosus GG could not be generally recommended for primary prevention (Kopp et al., 2008). The metabolic effects of probiotic supplementation during the perinatal period also seem to be relevant to fetal programming and infant’s development and metabolism. A pilot intervention program in which participants received either a combination of dietary counseling and probiotics (L.rhamnosus GG and B. lactis Bb12), dietary counseling with placebo, or placebo alone from early pregnancy onwards, showed significant effects of the intervention on placental lipids. The major differences in placental fatty acids were attributable to a higher concentration of n-3 polyunsaturated fatty acids in both intervention arms than in controls. Further, dietary counseling with probiotics resulted in higher concentrations of linoleic (18:2n-6) and dihomo-gamma-linolenic acids (20:3n-6) comparing with either dietary counseling with placebo or controls (Kaplas et al., 2007). The impact of maternal nutrition with probiotic supplementation during pregnancy on infant blood pressure has also been evaluated (Aaltonen et al., 2008). Pregnant women were randomized into 3 groups, the first submitted to a modified dietary intake according to current recommendations and probiotics (diet/probiotics), the second followed dietary recommendations and received placebo (diet/placebo), and the third received placebo (control/placebo). Although these results were not completely conclusive, the highest and lowest intakes of specific nutrients, such as carbohydrates and monounsaturated fatty acids compared with the middle ones were associated with higher blood pressure in children at the age 6 months, suggesting that dietary counseling can program blood pressure. The effects of probiotic supplementation together with dietary counseling on glucose metabolism in pregnant women were evaluated further leading to more conclusive results (Laitinen et al., 2008). The study included three subgroups of pregnant woman at the first trimester of pregnancy, the first group received nutrition counseling to modify dietary intake according to current recommendations (diet/placebo), the second group received nutrition counseling and probiotics (L. rhamnosus GG and B. lactis Bb12; diet/probiotics) and the third group received placebo without nutritional counseling (control/placebo). Blood glucose concentrations were the lowest in the diet/probiotics group during pregnancy and over the 12 months’ postpartum period. Glucose tolerance was also better in the diet/probiotics group compared with the control/placebo group during the last trimester of pregnancy and over the 12-months’ postpartum period. Therefore, the study suggests that dietary counseling with probiotics can improved blood glucose control in a normoglycaemic population and thus may provide potential novel means for the prophylactic and therapeutic management of glucose disorders (Laitinen et al., 2008).

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Conclusions and Further Perspectives In the light of current scientific evidence, the potential of dietary interventions to improve the health of the mother and the infant is significant from conception onwards. Given the role of the intestinal microbiota in host immunity and metabolism, particularly at early developmental stages, dietary interventions based on probiotic and prebiotic administration could aid in health programming and disease prevention. To make it reality, further studies are needed to define the mechanisms by which intestinal bacterial may influence mothers physiology and the transmission routs of such effects to the offspring. The development of a larger number of clinical trials with different probiotic strains, in diverse physiologic and at risk conditions and during well-established intervention time-frames could be of great help to extend the probiotic concept applications and contribute to reduce the burden of disease within the modern lifestyle context.

Acknowledgments This work was supported by grants AGL2007-66126-C03-01/ALI and Consolider FunC-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation.

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In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter VII

Epilactose: Potential for Use as a Prebiotic Susumu Ito1, Jun Watanabe1, Megumi Nishimukai1, Hidenori Taguchi1, Hirokazu Matsui2, Shigeki Hamada2, and Shigeaki Ito2 1 2

Creative Research Initiative “Sousei”, Hokkaido University, Sapporo 001-0021, Japan Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Abstract Prebiotics are nondigestible food components that affect the host by stimulating the growth and/or activity of health-promoting bacteria in the colon and thus contribute to host health and well-being. Epilactose is the C2-epimer of lactose that is found in heatand alkali-treated milk. We found that a cellobiose 2-epimerase of Ruminococcus albus isolated from cow rumen efficiently converts lactose in milk and whey to epilactose. The enzymatic synthesis of epilactose has the advantage over chemical synthetic protocols reported to date of producing byproducts. A dietary intervention study showed that epilactose has potential for use as a prebiotic or prebiotic foodstuff. In the colon of rats fed epilactose, 1) growth of health-promoting lactobacilli and bifidobacteria was enhanced, 2) rates of mineral absorption were increased, 3) levels of plasma total cholesterol and non-high-density-lipoprotein cholesterols were lowered, and 4) conversion of primary bile acids to secondary bile acids was suppressed. Therefore, the conversion of lactose to epilactose may increase the nutritional value of milk and whey.

Introduction A probiotic is “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance” [1]. Lactobacillus and Bifidobacterium spp., which have been described as living microorganisms that exert health benefits,

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exemplify the concept of probiotics [2]. They have been reported to prevent and treat rotavirus infections and postantibiotic diarrhea [3], allergic diseases [4], and inflammatory bowel disease [5]. A prebiotic is usually defined as “a nondigestible food component that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health” [6]. Nondigestible saccharides such as dietary fibers and oligosaccharides are known to exhibit various healthpromoting biological activities [7, 8]. The growth of beneficial intestinal bifidobacteria is stimulated by dietary supplementation with prebiotics [9–11]. Through fortification with beneficial microflora, prebiotics may enhance the defense mechanisms of host animals, increase resistance to various health challenges, and accelerate recovery from gastrointestinal tract disturbances [12]. Some nondigestible saccharides promote mineral absorption, and the large intestine is involved in this beneficial effect [13, 14]. They are fermented by intestinal microorganisms, generating short-chain fatty acids (SCFA). Formation of SCFA in the large intestine has been proposed to be partly responsible for an increase in calcium absorption [15, 16]. In addition, nondigestible saccharides have been reported to improve lipid metabolism [17–20]. Lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid) [21–24] and lactulose (4-Oβ-D-galactopyranosyl-D-fructose) [25–29], which can be synthesized from lactose chemically or enzymatically, have been shown to promote intestinal adsorption of minerals and/or stimulate selective growth of bifidobacteria. Lactulose has been added to commercial infant feeding products. Tyler and Leatherwood [30] were the first to find that the ruminal Ruminococcus albus 7 (ATCC27210T) possessed an epimerization activity that catalyzed a hydroxyl stereoisomerism at the C-2 position of the glucose moiety of cellobiose and generated 4-O-β-D-glucopyranosyl-D-mannose. Recently, we reported the purification of cellobiose epimerases (CEs) from R. albus NE1 and Eubacterium cellulosolvens NE13 to homogeneity and the cloning and sequencing of their complete genes [31, 32]. Further, we showed that the enzymes convert lactose to epilactose (4-O-β-D-glactopyranosyl-Dmannose) [32, 33]. While considerable amounts of epilactose is known to be formed by heat and alkali treatments of cow milk [34, 35], CE has the advantage that it can produce epilactose directly from lactose without byproducts [36, 37]. However, the biological activities of this unusual sugar have not yet been examined. Here, we demonstrate that elilactose has potential for use as a prebiotic foodstuff and that the conversion of lactose to epilactose by CE may increase the nutritional value of milk and whey.

Biological Activities of Epilactose The purification of CE from R. albus NE1 and preparation of epilactose from lactose by the enzyme were conducted essentially by the method of Ito et al. (33). For the dietary intervention study, male Wistar-ST rats (4-week-old) were housed in individual stainlesssteel cages in a room with controlled temperature (22 ± 2°C) and allowed free access to a semipurified stock diet (sucrose-based, AIN-93G formula) and tap water for 5 days. The rats were then divided into two groups (n = 6), and each group was fed the control or 4.5%

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epilactose diet with freely available deionized water for 15 days. Body weight (BW) gains over 15 days were the same between rats fed control diet (99.8 ± 4.8 g) and those fed epilactose diet (104.0 ± 2.7 g). Further, liver and kidney weights relative to BW did not differ between the groups. Epilactose had no toxic effect at least up to 4.76 g/kg BW/day under our experimental conditions. Detailed analytical methods used in this study have been described in the literature [38, 39].

1. In vitro Digestion Stability, Bifodogenetic Activity, Tight Junction Permeability Epilactose was resistant to in vitro digestion by a crude preparation of rat intestinal enzyme(s). The growths of the human bifidobacteria Bifidobacterium bifidum, B. longum, B. breve, B. adolescentis, and B. catenalatum [40] were significantly increased in a medium containing 1.0% epilactose [33]. These results suggest that epilactose reaches the lower gastrointestinal tract without being digested, where it is utilized by bifidobacteria and lactobacilli. Epilactose increased net calcium absorption by increasing the paracellular transport through the control of tight junction of human Caco-2 cells, where it dosedependently decreased transepithelial electrical resistance across the cell monolayer [33].

2. Calcium Absorption in Small Intestine Calcium absorption rates were measured using everted sacs of jejunum and ileum isolated from rats fed control and epilactose diets [39]. The presence of epilactose was found to enhance calcium absorption in both jejunal and ileal sacs, regardless whether rats had been fed epilactose or control diet. The calcium absorption rates by jejunal and ileal sacs isolated from the epilactose-fed rats were similar to those by the sacs of control rats. These results indicate that the coexistence of intact epilactose with calcium in the small intestinal lumen enhances calcium absorption in a non-prebiotic way and that no adaptive change in the absorptive activity for calcium occurs in the small intestine due to feeding epilactose. Two distinct pathways are involved in intestinal calcium absorption, a transcellular pathway and a paracellular pathway [41]. Transcellular absorption is a saturable, carriermediated active transport pathway, whereas paracellular absorption through tight junctions is nonsaturable and diffusive and requires a gradient of calcium concentrations between luminal and basolateral sides. Passive absorption in the jejunum and ileum is the major absorptive pathway when calcium intake is adequate or high. Under our experimental conditions, rats were fed adequate calcium (5 g Ca/kg of diet), and therefore the absorption of calcium by epilactose may proceed mainly via the passive transport machinery [39].

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3. Population of Cecal Bacteria The number of total cecal anaerobes in epilactose-fed rats was higher than that in control rats when examined by a culture-based method. When measured using the real-time PCR with genus-specific primers, the logarithmic cecal lactobacilli number of epilactose-fed rats (9.46 ± 0.12 copies/g content) was significantly higher than that in control rats (8.73 ± 0.16 copies/g content). In addition, the logarithmic cecal bifidobacteria number of rats fed the epilactose diet reached 7.80 ± 0.28 copies/g content, being significantly higher than that of rats fed the control diet (5.63 ± 0.34 copies/g content). For enumeration of intestinal microbiota, 16S rRNA gene libraries were constructed from cecal DNA of rats using universal 16S rRNA-targeted primers. The populations of clones of harmful -Proteobacteria and Clostridia tended to be smaller in epilactose-fed rats than in control rats [38]. These results indicate that epilactose changes the intestinal microflora in a beneficial way. It is well known that bifidobacteria and lactobacilli ferment various kinds of sugars to produce organic acids [42]. Fructooligosaccharide is known to increase cecal wall weight by increasing organic acid concentrations in the cecum [43]. Indeed, the weights of the cecal wall and contents (g/100 g BW) in epilactose-fed rats were 0.38 ± 0.22 and 2.24 ± 0.09, respectively, while those in control rats were 0.23 ± 0.01 and 0.83 ± 0.08, respectively.

4. Organic acid Generation and Mineral Absorption The amount of total short-chain fatty acids (SCFA) (acetic, propionic, and butyric acids) in the whole cecal contents was found to be higher in epilactose-fed rats (447 ± 31 μmoles) than in control rats (138 ± 11 μmoles). Specifically, the amounts of acetic, propionic, and butyric acids in the epilactose-fed rats were 275 ± 19, 127 ± 8, and 46.2 ± 7.7 μmoles, respectively, whereas those in the control rats were 88.3 ± 6.9, 32.3 ± 3.2, and 17.1 ± 1.3 μmoles, respectively. The amount of total organic acids (SCFA, succinic acid, and lactic acid) in the whole cecal contents of epilactose-fed rats (677 ± 49 μmoles) was 5 times that of control rats (140 ± 11 μmoles). In particular, the amounts of succinic acid and lactic acid in the whole cecal contents of the epilactose-fed rats were 185 ± 27 and 44.9 ± 30.6 μmoles, respectively, values of which were very much higher than those of the control rats (1.52 ± 0.43 and 0.742 ± 0.167 μmoles, respectively). Consequently, the pH of the cecal contents of epilactose-fed rats was lowered by the organic acids to 6.62 ± 0.18, while that in control rats was 7.60 ± 0.04 [39]. Organic acids produced from nondigestible saccharides are thought to enlarge the large intestine and increase calcium solubility by lowering the pH in the luminal contents; as a result, calcium absorption is increased in the cecum [43, 44]. In fact, calcium solubility in the cecal contents was higher in epilactose-fed rats (32.6 ± 1.2%) than in control rats (13.9 ± 1.1%). Magnesium solubility in the cecum was also higher in rats fed epilactose (79.2 ± 3.4%) than in rats fed the control diet (59.9 ± 2.7%). These results suggest that epilactose intake increases mineral absorption in the large intestine by increasing absorptive area and mineral solubility, and the higher solubility may induce higher absorption in the epilactosefed rats.

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5. Levels of Cholesterols, Triglycerides, and Phospholipids Hara et al. demonstrated that SCFA inhibited hepatic cholesterol synthesis and lowered plasma cholesterol level in rats [19, 45]. It has been reported that acetic acid contributes to the serum cholesterol-lowering effect of oat bran in humans [46] and that propionic acid reduces cholesterol synthesis in humans and rats [47]. Also, a previous report has shown that sugar-beet fiber lowers cholesterol synthesis with a coordinated increase in generation of SCFA and that it increases fecal excretion of steroids [19]. We found that epilactose intake tended to decrease the plasma cholesterol level and elevated the level of SCFA in cecal contents of rats. Notably, the level of SCFA in the cecal contents was found to be negatively correlated with those of plasma total cholesterol and low- and very low-density lipoprotein cholesterols [39]. These results show that the SCFA generated by intestinal fermentation of epilactose contribute to the suppression of cholesterol synthesis and lowering of the plasma cholesterol level in rats. Feeding of epilactose did not have any significant effect on the levels of plasma triglycerides and phospholipids in rats under our experimental conditions.

6. Levels of Primary and Secondary Bile Acids The major primary bile acids in rats are chenodeoxycholic acid, α-muricholic acid (αMCA), and β-MCA [48]. These primary bile acids are transformed to secondary bile acids by intestinal bacteria [49]. The secondary bile acids, such as hyodeoxycholic acid (HDCA), are cytotoxic to colon cells and have been implicated as tumor promoters [50]. Ingestion of nondigestible oligosaccharides decrease the conversion of primary bile acids to secondary bile acids and reduced 1,2-dimethylhydrazine-induced precancerous colon lesions in rats [51]. We then examined the effects of epilactose on the levels of primary and secondary bile acids in the cecal contents of rats [39]. The total concentration of primary bile acids in dried feces was higher in rats fed the epilactose diet (63.99 ± 14.21 μmol/g) than in those fed the control diet (23.5 ± 4.04 μmol/g). The levels of secondary bile acids were almost the same in the control and epilactose-fed rats (25.9 ± 14.08 μmol/g and 19.55 ± 1.88 μmol/g, respectively). However, the level of HDCA was decreased to below the detection limit in the epilactose-fed rats from 16.60 ± 3.46 μmol/g in the control rats, while significantly higher concentrations of β-MCA were detected in the epilactose-fed rats (35.78 ± 6.9 μmol/g) than in the control rats (7.43 ± 1.67 μmol/g). The ratios of secondary bile acids to total primary bile acids were 1.21 ± 0.08 for the control rats and 0.38 ± 0.05 for the epilactose-fed rats. HDCA is known to be generated from β-MCA by an unidentified bacterium HDCA-1, and the introduction of this strain into germ-free rats facilitates the conversion of β-MCA to HDCA [52]. Our clone library analysis of 16S rRNA revealed that harmful bacteria tended to be fewer in the epilactose-fed rats than in the control rats. In rats fed the epilactose diet, the cecal content pH was around 6.6 [39]. At this pH, the proliferation of strain HDCA-1 is completely abolished [52]. These results suggest that epilactose reduces the population of some cecal bacteria that possess the ability to convert β-MCA to HDCA. Therefore, it is

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possible that the change in intestinal microbiota due to the ingestion of epilactose suppresses colon cancer by inhibiting the formation of secondary bile acids.

Conclusion We synthesized epilactose from lactose by the action of a CE from R. albus. Epilactose is nondigestible and has potential for use as a prebiotic that possesses several beneficial activities: stimulation of bifidobacteria growth, facilitation of mineral absorption, lowering of plasma total cholesterol and non-high-density-lipoprotein cholesterols, and suppression of the generation of secondary bile acids. CE can increase the value of milk and whey by preparing novel milk products that have prebiotic properties.

Ackknowledgments Purified epilactose was kindly provided by Drs. T. Yamamoto, M. Takada, and M. Yamamoto of Nihon Shokuhin Kako Co. Ltd. This study was supported by Special Coordination Funds for Promoting Science and Technology and by a National Project “Knowledge Cluster Initiative” (2nd stage, “Sapporo Biocluster Bio-S”) from The Ministry of Education, Science, Sports and Culture of Japan.

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In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter VIII

Lactoferrin as an Added-value Whey Component and a Healthy Additive in Nutraceutical Drinks Palmiro Poltronieri1,*, Carla Vetrugno2, Antonella Muscella2, Santo Marsigliante2 1

CNR-ISPA, Institute of Sciences of Food Productions, National Research Council, Lecce, Italy 2 Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, Lecce, Italy

Abstract Lactoferrin (Lf) is a whey protein with potential food applications to sustain human health. Lf is already added to infant formula milk powder so that, like breastmilk, it contains Lf to help build resistance to disease. One yogurt is added with Lf and produced by the Morinaga factory in The Nederlands. Lf binds iron, and can deliver it to increase iron availability. This ability seems to affect also microbes and fungi, although ironbound lactoferricin peptide seems to be as effective as the full protein. In this work it is shown the effect of Lf on MCF-7 cultured cells, i.e. the induction of apoptosis in the presence of sustained cell cycling driven by angiotensin-II growth factor. We thus show that Lf may have antiproliferative activity on selected cell types. Further work is needed to individuate the proteins interacting with Lf, and the downstream signalling that end in the shutting off of cell cycle effectors. We found that Lf-based emulsions storage with good stability up to 12 months. A milk or soy-milk beverage may be a convenient vehicle for delivery of Lf-based nutraceuticals.

*

Corresponding Author. E-mail: [email protected]

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Introduction Although there is no official definition of functional foods, it is generally considered that they are a group of foods which provide physiological benefits beyond those traditionally expected from food. Milk proteins have a great potential use as functional foods: healthy foods, nutraceuticals and food for specified human use, are one of the fields in constant growth in the food industry, as well as an emerging field of medical interest. Therefore, Health and Wellness has become an important internationally recognized, basic research program in many Research Departments of Food companies and Universities. Research in functional foods includes: a) utilization of biological models, including proteomics, to assess the molecular mechanisms and physiological effects of bioactive food components: b) development of bioprocessing techniques to isolate bioactive compounds or produce novel health promoting foods that maintain desirable quality and safety over an extended shelf-life; c) characterization of the molecular properties of novel food ingredients with health promoting activities. The milk / whey proteins show to possess important biological activities in terms of human health (Gill &Cross 2002; Clare et al. 2003; Smolenski et al. 2007). Milk proteins referenced in scientific publications are: High Mobility Group proteins (Yamamura et al. 1999), milk basic protein (Matsuoka et al. 2002), milk fat globule membrane proteins (Cavaletto et al. 2008) as xanthine oxidase, lactadherin, butyrophilin associated with phospholipids, whey acidic protein (Nukumi et al. 2007), lactoferrin (Lf) (Weinberg 2003; Wakabayashi et al. 2006); ceruloplasmin (Sokolov et al. 2006), lactoperoxidase (Severin and Wenshui 2005), alpha-lactalbumin (Teschemacher 2003), and beta-lactoglobulin (HernandezLedesma et al. 2008). New functional foods, enriched in bioactive milk proteins, may provide a healthy effect for the action of each constituent, in prevention of chronic pathologies and as co-adjuvant in maintaining physiological fitness. Whey proteins are usually denatured by heating during post-cheese processing. On one side, the economical costs of production and costs of whey discharge influence the manufacture process. Milk and dairy products containing whey proteins in their native state may constitute an interesting product innovation in milk industry. Lf is a natural defence protein with iron-binding ability, present in exocrine secretions and in body fluids that are commonly exposed to normal flora, acting as microbial barriers: milk, tears, nasal exudate, saliva, bronchial mucus, and gut surfaces. Additionally, Lf is produced in polymorphonuclear leukocytes and is deposited by these cells in septic sites. A principal function of Lf is that of scavenging non-protein-bound iron in body fluids and inflamed areas. Iron binding is a key property of Lf that accounts for many of its biological roles in host defence such as bacteriostasis and protection against oxygen radicals catalyzed by free iron (Crouch et al. 1992; Kruzel et al. 2006; Raghuveer et al. 2002). The Lf ingested with foods is processed by digestive proteases into peptides. One of these peptides is lactoferricin (Tomita et al. 2002), that includes the N-terminal lobe containing the first Fe2+ chelating domain, while another antimicrobial peptide is the 11residue lactoferrampin (Haney et al. 2007), known to possess antimicrobial activity. Recently, a conference on Lf bioactivity was held in the USA and proceedings were published as miniseries (Kruzel et al. 2007). Lf affects gut cell proliferation (Buccigrossi et

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al. 2007), bone cell activity (Cornish et al. 2004) and the synthesis of nitric oxide (Choe et al. 1999). Lf promotes antimicrobial activity by activating the immune system during transit in the gut. Anti-inflammatory effects of Lf have been shown by the inhibition of proinflammatory cytokine production (Kruzel et al. 2002) and the up regulation of antiinflammatory cytokines (Togawa et al. 2002). On the other hand, Lf may enhance directly or indirectly the immune response (in vitro and in vivo) by regulating the differentiation and activation of both T and B cells (Zimecki et al. 1991, Zimecki et al. 1995). Orally supplemented lactoferrin from bovine milk and Lf of other origin is purported to have beneficial effects on gut health of animals, as mouse/rats (Togawa et al. 2002), and dogs with the aim to improve the gut microflora (Pope et al. 2006). Furthermore, oral human lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy (Varadhachary et al. 2004) through the activation of non-specific immune system.

Results At first, we studied the colony forming potential of three cancer cell lines, MCF-7 breast cancer cells, HeLa cells, and SH-SY5Y neuroblastoma cells, in presence of three milk/whey proteins, i.e. Lactoferrin (Lf), β-lactoglobulin (β-Lg) and ά-lactalbumin (ά-La), at concentration of 10, 50 and 100 µg/ml. An anti-proliferative action of Lf on the MCF-7 cells was found at 50 µg/ml, evidenced as a decrease in the colony formation ability (Figure 1). It was decided to challenge MCF-7 cells using Lf at doses of 100 µg/ml and to investigate the intracellular mechanisms and the signalling pathways involved.

Figure 1.

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Figure 2.

In order to study the cell cycle state of cells, we counted the % of cells in cell cycle phases other than G0 by measuring the total DNA stained with propidium iodide in a FACSort cytofluorimeter (Becton Dickinson) (Figure 2). The effect of Lf administration on MCF-7 cells in the presence of 10% FBS was comparable to the effect of FBS withdrawal (Figure 3). The number of cells in S and G2/M phases decreased drastically, from 36% cells stimulated to proliferate, to just 6% of cells entering in mitosis. In order to understand the mechanisms of proliferation inhibition, two assays were designed. In the first, we analysed the up-regulation of the growth arrest protein p27kip, and the lowering of cell cycle related proteins CDK2 and cyclin E (Figure 4). In the presence of Lf, p27kip levels increased since the 12th hour, whereas CDK2 and cyclin E levels drastically lowered between the 24 and the 48 hour points. The mechanism by which Lf activates growth arrest was individuated in the activation of Protein Kinase C delta (PKC-δ) isoform, known to be involved in cell differentiation (figure 5). The western blot performed on cell lysates

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showed a four fold reduction in staining of unprocessed PKC-δ with appearance of the activated PKC-δ fragment (t-PKC-δ).

Figure 3.

Figure 4.

Figure 5.

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Figure 6.

On the other side, we found a second, pronounced effect of Lf on MCF-7 cells in the activation of apoptotic pathways. A well known marker of apoptosis is the cleavage of PARP protein: after the 9th hour of Lf treatment, we found the disappearance of the 116 kDa full length PARP protein, and the appearance of the 85 kDa cleaved product (figure 6). In addition, MCF-7 cells showed activation of caspase 9 between 6th and 9th hour after Lf addition, and activation of caspase 7 later on, at the 24th hour time point of experiment. Lf also induced the phosphorylation of p38 MAPK (figure 7a); when cells were incubated for 1 hour with 1 and 10 μM SB203580, an inhibitor of p38 MAPK, and then 100 µg/ml Lf was added for 24 hours, a reduction in caspase 7 activation was observed (figure 7b), suggesting an involvement of p38 MAPK in this process. We found that the p38 MAPK activity was also responsible for the phosphorylation of bcl-2 (figure 8a). In fact, Lf presence caused an increase in the phosphorylated bcl-2 form, and the pre-treatment with SB203580 drastically blocked this signal activation (figure 8b). It is proposed that Lf effect is mediated through p38 MAPK, triggering the release of bax protein from the complex with bcl-2, and the subsequent activation of the cell death caspases.

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Figure 7.

Figure 8.

In Jurkat cells, it was already shown that LF induces apoptosis through bcl-2 phosphorylation and the JNK-Bcl-2 signaling cascade (Lee et al. 2008). By treatment of Jurkat cells with the specific JNK inhibitor SP600125, but not the p38 MAPK inhibitor SB203580, is was shown that it is required for Lf-induced apoptosis. When JNK activation was abolished by SP600125, no Bcl-2 phosphorylation was detected, and the Lf-treated Jurkat cells did not undergo cell death. We also observed a low-metastatic phenotype in MCF-7 cells treated with Lf. Matrix metalloproteases are membrane-bound proteins involved in cell migration by actively degrading the extracellular matrices. MMP-2 levels were found three-fold decreased by western blot. Also the proteolytic activity in cell lysates was partially decreased, measured as gelatin proteolysis in an in-gel assay (figure 9).

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Figure 9.

In conclusion, it is possible to prospect a series of different beneficial effects on certain cancer cell types in the presence of Lf, i.e. growth arrest, reduction of metastatic potential, and cancer cell death. It is still not known which mechanism operates in the cell uptake of Lf, but other authors described the presence of Lf receptors (Suzuki et al. 2005, Mazurier and Hovanessian 2004) and on the internalisation of Lf and Lf-δ isoform (Legrand et al., 2004, Breton et al., 2004; Mariller et al. 2007).

Strategies for Delivery of Lf-Active Ingredient and to Increase LfConsumption in Foods A process of mild pasteurisation of Lf and Lf-containing food products has been described (Tomita et al., 2002). Adequate sources of bovine Lf and pasteurization processes are today available for the development of commercial food applications (Uzzan et al. 2007). Recently Lf was used, at doses between 250 mg to 500 mg, in infant milk powder and in yogurt, aiming to increase daily intake to the recommended amount of 0.5-1 gr (Soejima et al. 2007). Lactoferrin may be delivered through incorporation in food, as charge interaction with other food components. Methods of processing bioactive Lf include encapsulation, protection and delivering systems for functional food components using biopolymers, association colloids, emulsions, matrices. The various functional components need to be encapsulated; exploiting association colloids (micelles, vesicles, etc); emulsions (conventional, multilayer, multiple); biopolymer systems (matrices, gels, capsules, powders). We studied the possibility

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to include Lf in multilayered emulsions, using surface charged polysaccharides at optimal pH to form Lf-enriched oil-in-water emulsions (De Lorenzis et al., 2008). The Lf fraction used was devoid of bacterial contamination, since it was extracted and chromatographycally purified using a carboxymethylsepharose resin-filled column. From our results, Lf-based emulsions are stable for 12 months, with no disruption of physical-chemical property of the emulsion (figure 10). Emulsions may be used as food ingredient in different product type and form and contain only food-grade ingredients, as olive oil, may be enriched with ω-3 fatty acids, and designed to respond to specific needs of consumers and diets.

Figure 10.

Concluding Remarks It is possible to envisage different Lf applications on the basis of delivery systems and objectives. Considering the potential of ingested Lf to modulate the immune responses and the iron homeostasis (Paesano et al. 2008), an increase of daily consumption would provide benefits to the general health status. The production of Lf enriched foods and other nutraceutical supplements could be feasible considering the high stability of the Lf emulsions. Further studies are needed to prove absence of adverse effects during Lf local delivery, as in the case of management of local infection during transplants (van der Velden et al. 2008). Lf-containing liposomes, targeting diseased cells or organs, could be tested in

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clinical trials. Also in cases when other therapies alone show no effect or very low rate of success, the anticancer activity of Lf could be exploited in support to the therapeutic drugs.

References Breton, M., Mariller, C., Benaïssa, M., Caillaux, K., Browaeys, E., Masson, M., Vilain, J.P., Mazurier, J., Pierce, A. (2004) Expression of delta-lactoferrin induces cell cycle arrest. Biometals 17, 325-329. Buccigrossi, V., de Marco, G., Bruzzese, E., Ombrato, L., Bracale, I., Polito, G., Guarino, A. (2007) Lactoferrin Induces Concentration-Dependent Functional Modulation of Intestinal Proliferation and Differentiation. Pediatric Research 61, 410-414. Cavaletto, M., Giuffrida, M.G., Conti, A. (2008) Milk fat globule membrane components - a proteomic approach. Adv Exp Med Biol. 606, 129-41. Choe, Y.H. & Lee, S.W. (1999) Effect of lactoferrin on the production of tumor necrosis factor-alpha and nitric oxide. J. Cell Biochem. 76, 30–36. Clare, D.A., Catignani, G.L., Swaisgood, H.E. (2003) Biodefense properties of milk: the role of antimicrobial proteins and peptides. Curr Pharm Des. 9, 1239-1255. Cornish, J., Callon, K.E., Naot, D., Palmano, K.P., Banovic, T., Bava, U., Watson, M., Lin, J.-M., Tong, P.C., Chen, Q., Chan, V.A., Reid, H.E., Fazzalari, N., Baker, H.M., Baker, E.N., Haggarty, N.W., Grey, A.B., Reid, I.R. (2004) Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo. Endocrinology 145, 4366-4374. Crouch, S.P., Slater K.J., Fletcher, J. (1992) Regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin. Blood, 80, 235–240. De Lorenzis, E., Semeraro, C., De Blasi, M.D., Mita, G., Poltronieri, P. (2008) Emulsions based on the interactions between lactoferrin and chitosans. Food Biophysics, 3, 169173. Gill, H.S; & Cross, M.L. (2000) Anticancer properties of bovine milk. British J Nutrition, 84, 161-166. Haney, E.F., Lau, F., Vogel, H.J. (2007) Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin. Biochim. Biophys. Acta 1768, 355-2364. Hernández-Ledesma B, Recio I, Amigo L. (2008) Beta-lactoglobulin as source of bioactive peptides. Amino Acids 35, 57-65. Lee, S.-H., Park, S.W., Pyo, C.-W., Yoo, N.-K., Kim, J., Choi, S.-Y. (2008) Requirement of the JNK-associated Bcl-2 pathway for human lactoferrin-induced apoptosis in the Jurkat leukemia T cell line. Biochimie, 91, 102-108. Legrand, D., Vigié, K., Said, E.A., Elass, E., Masson, M., Slomianny, M.C., Carpentier, M., Briand, J.P., Mazurier, J., Hovanessian, A.G. (2004) Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells. Eur. J. Biochem. 271, 303317. Kruzel, M.L., Harari, Y., Mailman, D., Actor. J.K., Zimecki, M. (2002) Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice, Clin. Ex. Immunol. 130, 25–31.

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Kruzel, M.L., Bacsi, A., Choudhury, B., Sur, S., Boldogh, I. (2006) Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthma. Immunol. 119, 159-166. Kruzel, M.L., Actor, J.K., Boldogh, I., Zimecki, M. (2007) Lactoferrin in health and disease. Postepy Hig. Med. Dosw. (Online) 61, 261-267. Mariller, C., Benaissa, M., Hardiville, S., Breton, M., Pradelle, G., Mazurier, J., Pierce, A. (2007) Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression. FEBS J. 274, 2038-2053. Matsuoka, Y., Serizawa, A., Yoshioka, T., Yamamura, J., Morita, Y., Kawakami, H., Toba, Y., Takada, Y., Kumegawa, M. (2002) Cystatin C in milk basic protein (MBP) and its inhibitory effect on bone resorption in vitro. Biosci. Biotechnol. Biochem. 66, 2531-2536. Nukumi, N., Iwamori, T., Kano, K., Naito, K., Tojo, H. (2007) Reduction of tumorigenesis and invasion of human breast cancer cells by whey acidic protein (WAP). Cancer Lett. 252, 65-74. Paesano, R., Pietropaoli, M., Gessani, S., Valenti, P. (2008) The influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia. Biochimie, Advanced online, doi:10.1016/ j.biochi.2008.06.004. Pope, L., Flickinger, E., Karr-Lilienthal, L., Spears, J., Krammer, S., Fahey, G. (2006) Effects of lactoferrin supplementation on ileal and total tract nutrient digestibility, gastrointestinal microbial populations, and immune characteristics of ileal cannulated, healthy, adult dogs. Archives Animal Nutrition 60, 10-22. Raghuveer, T.S., McGuire, E.M., Martin, S.M., Wagner, B.A., Rebouché, C.J., Buettner, G.R., Widness, J.A. (2002) Lactoferrin in the preterm infants' diet attenuates ironinduced oxidation products. Pediatr Res. 52, 964-972. Séverin, S., Wenshui, X. (2005) Milk biologically active components as nutraceuticals: review. Crit Rev. Food Sci. Nutr. 45, 645-56. Smolenski, G., Haines, S., Kwan, F.Y., Bond, J., Farr, V., Davis, S.R., Stelwagen, K., Wheeler, T.T. (2007) Characterisation of host defence proteins in milk using a proteomic approach. J Proteome Res. Jan;6(1):207-15. Soejima T, Yamauchi K, Yamamoto T, Ohara Y, Nagao E, Kanbara K, Fujisawa M, Okuda Y, Namba S. (2007). Determination of bovine lactoferrin in lactoferrin-supplemented dairy products and raw milk by an automated latex assay. J. Dairy Sci. 74, 100-105. Sokolov, A.V., Pulina, M.O., Zakharova, E.T., Susorova, A.S., Runova, O.L., Kolodkin, N.I., Vasilyev, V.B. (2006) Identification and isolation from breast milk of ceruloplasminlactoferrin complex. Biochemistry (Moscow) 71, 160-166. Suzuki, Y.A., Lopez, V., Lönnerdal, B. (2005) Mammalian lactoferrin receptors: structure and function. Cell Mol. Life Sci. 62, 2560-2575. Teschemacher H. (2003) Opioid receptor ligands derived from food proteins. Curr. Pharm. Des. 9, 1331-144. Togawa, J., Nagase, H., Tanaka, K., Inamori, M., Nakajima A., Ueno, N., Saito, T., Sekihara, T. (2002) Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance. J. Gastroenterol. Hepatol. 17, 1291–1298.

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Tomita, M., Wakabayashi, H., Yamauchi, K., Teraguchi, S., Hayasawa, H. (2002) Bovine lactoferrin and lactoferricin derived from milk: production and applications. Biochem Cell Biol. 80, 109-112. Uzzan, M., Nechrebeki, J., Labuza, T.P. (2007) Thermal and storage stability of nutraceuticals in a milk beverage dietary supplement. J.Food Sci. 72, E109-14. van der Velden, W.J.F.M., Blijlevens, N.M.A., Donnelly J.P. (2008) The potential role of lactoferrin and derivatives in the management of infectious and inflammatory complications of hematology patients receiving a hematopoietic stem cell transplantation. Transplant Infectious Disease 10, 80-89. Varadhachary, A., Wolf, J.S., Petrak, K., O'Malley, B.W., Spadaro, M., Curcio, C., Forni, G., Pericle, F. (2004) Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy. Int. J. Cancer 111, 398-403. Wakabayashi, H., Yamauchi, K., Takase, M. (2006) Lactoferrin research, technology and applications. Int. Dairy J. 16, 1241- 1251. Yamamura, J., Takada, Y., Goto, M., Kumegawa, M., Aoe, S. (1999) High mobility grouplike protein in bovine milk stimulates the proliferation of osteoblastic MC3T3-E1 cells. Biochem. Biophys. Res. Commun. 1261, 113-117. Weinberg, E.D. (2003) The therapeutic potential of lactoferrin. Expert Opin Investig Drugs, 12, 841-851. Zimecki, M., Mazurier, J., Machnicki, M., Wieczorek, Z., Montreuil J., Spik, G. (1991) Immunostimulatory activity of lactotransferrin and maturation of CD4–CD8–murine thymocytes. Immunol. Lett. 30, 119–123. Zimecki, M., Mazurier, J., Spik G., Kapp, J.A. (1995) Human lactoferrin induces phenotypic and functional changes in murine splenic B cells. Immunology 86, 122–127.

In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter IX

Conjugated Linoleic Acid: An Anticancer Fatty Acid Found in Milk and Meat T. R. Dhiman*, A. L. Ure and J. L. Walters Department of Animal, Dairy and Veterinary Sciences Utah State University, Logan, Utah 84322-4815, USA

Abstract Conjugated linoleic acid (CLA) has been intensively studied recently, mainly because of its potential in protecting against cancer, atherogenesis, and diabetes. Conjugated linoleic acid is a collective term for a series of conjugated dienoic positional and geometrical isomers of linoleic acid, which among common human foods are found naturally in relative abundance in the milk and meat fat of ruminants. The cis-9, trans-11 isomer is the principle dietary form of CLA found in ruminant products, and is produced by partial ruminal biohydrogenation of linoleic acid or by endogenous synthesis in the tissues themselves. The CLA content in milk and meat is affected by several factors, such as an animal’s breed, age, diet, and management factors related to feed supplements affecting the diet. Conjugated linoleic acid in milk or meat has been shown to be a stable compound under normal cooking and storage conditions. Total CLA content in milk or dairy products ranges from 0.34 to 1.07% of total fat. Total CLA content in raw or processed beef ranges from 0.12 to 0.68% of total fat. It is currently estimated that the intake of the average adult consuming western diets is only one-third to one-half of the amount of CLA that has been shown to reduce cancer in animal studies. For this reason, *

Corresponding author: T. R. Dhiman, Phone: 435 797-2155; Fax: 435 797-2118; E-mail: [email protected] Approved as journal paper number 7679 of the Utah Agricultural Experiment Station, Utah State University, Logan, Utah 84322, USA.

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T. R. Dhiman, A. L. Ure and J. L. Walters increasing the CLA content of milk and meat has the potential to raise the nutritive and therapeutic values of dairy products and meat. Growing evidence suggests that consuming dairy products and meat enriched with CLA has beneficial effects on human health.

Introduction More than 2 decades ago, Michael Pariza and his colleagues at the University of Wisconsin, USA, observed that fat from grilled beef actually inhibited chemically induced cancer rather than promoting it in an animal model. These researchers identified the anticancer agent as a mixture of isomers of conjugated octadecadienoic acid [1]. The isomers were referred to collectively as conjugated linoleic acid (CLA). Since then an increasing number of studies using synthetic and natural CLA have shown that it can suppress cancer at a number of sites, including the mammary gland, skin, colon and forestomach [2-14]. In several human cancer studies, an inverse association was found between the level of CLA in the diet and the risk of developing cancer in breast adipose tissue [15-18]. Studies conducted with mice, chickens, and pigs have suggested a possible role of CLA (mainly the trans-10, cis-12 isomer) in decreasing body fat and increasing lean body mass [19-24]. A human study has suggested that CLA increases body mass without increasing body fat [25]. Several studies indicate that CLA may enhance immune function [26-31]. Conjugated linoleic acid has also been found to have antidiabetic and antiatherosclerotic properties in animal models [32-36]. Conjugated linoleic acid isomers are found naturally in foods, especially those of ruminant origin [37]. In ruminants, CLA is synthesized by ruminal bacteria using C18:2 or C18:3 as the precursor [38]. Conjugated linoleic acid isomers can also be synthesized in the laboratory from C18:2 or from sources high in C18:2, such as sunflower, safflower, soybean, or corn oils, by a reaction involving alkaline water isomerization [39] and isomerization in propylene glycol [40]. The objective of this chapter is to provide an overview of different CLA isomers, biosynthesis of CLA, CLA contents of milk and meat, factors affecting the level of CLA in foods, potential health benefits of CLA and CLA intakes in humans.

CLA Isomers The cis-9, trans-11 isomer is the principle dietary form of CLA exhibiting biological activity, and accounts for 73 to 94% of total CLA in milk, dairy products, meat, and processed meat products of ruminant origin [41-44]. In recent years, biological activities have been proposed for other CLA isomers, especially trans-10, cis-12 C18:2 (t10, c12 CLA), [20, 21]. Throughout the rest of the chapter, the cis double bond will be abbreviated as c and the trans double bond as t. The structures of c9, t11 CLA, t10, c12 CLA, and C18:2 are shown in Figure 1.

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A total of 17 natural CLA isomers have been found in milk, dairy products, beef, human milk, and human adipose tissue using silver ion-high performance liquid chromatography and gas chromatography-electron ionization mass spectrometry. Identified CLA isomers are t12, t14; t11, t13; t10, t12; t9, t11; t8, t10; t7, t9; t7, c9; t6, t8; c12, t14; t11, c13; c11, t13; c10, t12; c9, t11; c8, t10; c7, t9; c9, c11; and c11, c13. Bauman et al. [45] observed that butter contained c9, t11 (76.5%) and c7, t9 (6.7%) isomers. Sehat et al. [40] characterized the following distribution of CLA isomers in cheese fat: c9, t11 (78 to 84%); t7, c9 plus t8, c10 (8 to 13%); t11, c13 (1 to 2%); c12, t14 (<1%); and their total trans/trans isomers (5 to 9%). Recently, Fritsche et al. [46] identified the distribution of CLA isomers in beef samples and found that c9, t11 was the predominant isomer (72%), followed by the t7, c9 isomer (7.0%). Typical synthetic CLA isomer mixtures consist of c9, t11 (40.8 to 41.1%); t10, c12 (43.5 to 44.9%); and t9, t11/t10, t12 (4.6 to 10.0%) isomers [37, 40]. Christie et al. [39] and Fritsche [47] reported on a different synthetic CLA isomer mixture that contained c8, t10 (14%); c9, t11 (30%); t10, c12 (31%); and c11, t13 (24%). Most of the aforementioned CLA isomers are present in foods in very minute amounts and are of little known biological importance or have not been studied in detail. Therefore, the ensuing discussion in this chapter will focus on the two predominant forms of CLA, namely the c9, t11 and t10, c12 isomers.

Figure 1. Abbreviated chemical structures of ordinary C18:2 (linoleic acid) (A), and two major conjugated linoleic acids (CLA): c9, t11 isomer (B), and t10, c12 isomer (C).

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CLA Biosynthesis Conjugated linoleic acid originates from either ruminal biohydrogenation of C18:2 and C18:3 or from endogenous synthesis in tissues as shown in Figure 2. Ruminally, CLA is produced as an intermediate product during the biohydrogenation of dietary C18:2 or C18:3 to stearic acid (C18:0). Endogenously, CLA is synthesized from t-11, C18:1 vaccenic acid (TVA), another intermediate of ruminal biohydrogenation, via Δ9-desaturase [48]. The endogenous synthesis of CLA from TVA has been proposed as the major pathway of CLA synthesis in lactating cows, accounting for an estimated 78% of the CLA in milk fat [49, 50]. Ruminal biosynthesis

Endogenous synthesis (Mammary gland / adipose tissue)

Diets

α-C18:3

γ-C18:3

C18:2

I

α-C18:3, C18:2, γ-C18:3 I

I c9, t11, c15 C18:3

c6, c9, t11 C18:3 H

H

H t11, c15 C18:2

H c9, t 11 C18:2 (CLA)

H

H Trans-11 C18:1 (TVA)

c6, t11 C18:2 H

c9, t 11 C18:2 (CLA) Δ9-desaturase Trans-11 C18:1 (TVA)

H C18:0

C18:0

Figure 2. Proposed mechanism for CLA synthesis from ruminal biohydrogenation or endogenous synthesis. CLA, conjugated linoleic acid; TVA, trans vaccenic acid; I, isomerization reaction; and H, hydrogenation. Adapted and reproduced with permission. Reference: [56].

Ruminal Biohydrogenation Lipids in the ruminant diet are derived from forages, grains, and oil supplements. The lipid content in most ruminant diets ranges from 3-7% on a dietary dry matter (DM) basis. Most ruminant feeds of vegetable origin contain C18:2 and/or C18:3 as the predominant fatty acids. Among feeds, pasture grass diets fed consumed by ruminants are rich in C18:3, representing 48 to 56% of total fatty acids (FA). Corn or grass silages are rich in C18:2 (41% of FA) or C18:3 (46% of FA), respectively. Most plant seeds and oils are rich in C18:2, accounting for 53 to 69% of total FA. When consumed by ruminants, the lipid portion of these feeds undergoes two major processes in the rumen [51, 52]. In the first process, esterified plant lipids or triglycerides are

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quickly hydrolyzed to free FA by microbial lipases [53]. In the second process, the unsaturated free FA are rapidly hydrogenated by microorganisms in the rumen to produce more highly saturated end products. The c9, t11 isomer of CLA is the first intermediate product in the biohydrogenation of C18:2 by the enzyme linoleate isomerase (Figure 2), which is produced by the microorganism Butyrivibrio fibrisolvens [38] and other bacterial species. Part of the c9, t11 CLA is rapidly reduced to TVA or C18:0, [54-55], becoming available for absorption in the small intestine. Similar to the biohydrogenation of C18:2, the FA α and γ–C18:3, which are the predominant unsaturated FA in forages, also undergo isomerization and a series of reductions, ending with the formation of C18:0 in the case of complete biohydrogenation [56]. The c9, t11 CLA and TVA often escaping complete ruminal biohydrogenation are absorbed from the intestine and incorporated into milk fat [57, 58]. Studies with pure strains of ruminal bacteria have shown that most bacteria are capable of hydrogenating C18:2 to t-C18:1 and related isomers, but only a few have the ability to reduce C18:2 and C18:1 completely to C18:0 [59]. Interestingly, no single species of rumen bacteria catalyzes the complete biohydrogenation sequence [56, 60]. It has been suggested that the biohydrogenation pathways are affected by several factors related to the composition of the diet consumed by the animal, including the rumen environment and the bacterial population [58, 61-63].

Endogenous Synthesis It was originally assumed by the scientific community that the rumen was the primary site of origin of c9, t11 CLA in milk fat. Recently, however, it has been suggested that only a small portion of c9, t11 CLA escapes biohydrogenation in the rumen and that the major portion of c9, t11 CLA in milk comes from endogenous synthesis in the mammary gland via a pathway involving the desaturation of TVA by the Δ9-desaturase enzyme [49, 50, 64]. Several studies have been performed to confirm that the endogenous synthesis of CLA by Δ9-desaturase occurs in the mammary gland [50]. The actual estimated endogenous synthesis of c9, t11 CLA in milk fat was 64 [49], 78 [50], or 80% [65], of the total c9, t11 CLA with different correction factors used according to the extent of enzyme inhibition. There are reported species differences in the tissue distribution of Δ9-desaturase. Enzyme activity and mRNA abundance of Δ9-desaturase are highest in the liver of rodents; however, in growing sheep and cattle, adipose tissue is found to have the highest levels [6668]. In lactating ruminants, the highest activity of Δ9-desaturase is found in the mammary tissue [69]. There is very little research exploring the factors that influence and regulate Δ9desaturase activity in the tissues of ruminants.

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CLA Content in Milk and Meat Products In food, CLA is found in milk fat, the tissue fat of ruminant animals, and products derived from them. The CLA content of food products is influenced almost entirely by the CLA content of milk and meat fat and the processing methods used. Cow’s milk fat is the richest natural source of CLA. The total CLA content and the proportions of the c9, t11 CLA isomer in milk and dairy products from cows fed typical diets are in Table 1. These values can be used as guidelines to calculate the CLA intake by humans from dairy products available on the market. The average CLA content in milk and dairy products on the market was 0.53% of fat (range 0.34 to 1.07%). In fluid milk, CLA content ranged from 0.34 to 0.80% of fat with an average of 0.53%. The CLA in various types of cheeses was 0.34 to 1.07% of fat (average = 0.50%). The CLA in fermented dairy products including butter, ranged from 0.36 to 0.94% (average = 0.55%). The c9, t11 CLA isomer in all dairy products represented 79 to 94% of total CLA in milk fat. Table 1. Conjugated linoleic acid (CLA) contents in milk and dairy products Samples* Fluid milk products Whole milk [70, 78, 79, 86, 90, 94, 132, 137, 213, 224] Evaporated milk [213] UHT milk [214] Homogenized milk [37] Condensed milk [37], 214 Cultured buttermilk[132, 137] Cheeses Cheddar [40, 133, 137, 213, 215, 216] Feta [40] Cottage [37, 137, 213] Mozzarella [37, 40, 70, 133, 137, 213] Processed cheese [137, 213, 215, 217] Processed American [37, 40, 213] Processed Cheddar [40] Processed Parmesan [137] Fermented dairy products Plain yogurt [37, 133, 134, 137, 213, 214, 218] Lowfat yogurt [37] Butter [37, 133, 213, 214] Sour cream [37, 133, 137] Ice cream [37, 133]

Total CLA** (% of fat)

c9, t11CLA*** (%)

0.34-0.68 0.49 0.80 0.55 0.63-0.70 0.54-0.67

82-97 --92 82 89

0.40-0.53 0.49 0.45-0.59 0.34-0.50 0.41-1.07 0.36-0.50 0.50 0.53

78-82 81 83 78-95 75 79-93 84 --

0.38-0.88 0.44 0.47-0.94 0.46-0.75 0.36-0.50

83-84 86 78-88 78-90 76-86

* Numerical superscripts next to samples correspond to reference numbers cited in the reference section. ** Values are mean or minimum and maximum levels. *** Data are expressed as % of total CLA isomers.

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The CLA content and the proportions of c9, t11 CLA isomer in raw and processed meat products are summarized in Table 2. It is again prudent to emphasize that most of the research cited did not report total fat content of the products, and total actual CLA yields will depend upon fat percentage of the product. The average CLA content in meat products of ruminant origin available on the market was 0.46% of fat (range 0.12 to 1.20%) with the c9, t11 CLA isomer representing an average 73% of the total CLA. The CLA in meats of nonruminant origin averaged 0.16% of fat (range 0.06 to 0.25%) with the c9, t11 isomer representing 65% of the total CLA. The CLA content in turkey is almost twice the amount of that found in chicken, pork, or rabbit. In the case of non-ruminants, CLA may be produced by the conversion of TVA to CLA in the tissues or through the feeding of tallow, meat meal of ruminant origin, or synthetic CLA. Table 2. Conjugated linoleic acid (CLA) contents in meat and processed meat products Samples* Ruminants Beef Ground, [137, 46, 137, 219] Round [37, 136] Ribeye [136, 137] T-bone [136] Sirloin [136, 137] Frank [37] Smoked sausage [37] Veal [37] Lamb [37, 214, 220, 221, 222, 223] Non-ruminants Turkey [37, 214, 220] Turkey frank [37] Smoked turkey37 Pork [37, 220] Smoked bacon [37] Chicken [37, 214, 220] Rabbit [220]

Total CLA** (% of fat)

c9, t11CLA*** (%)

0.16-0.43 0.29-0.68 0.30-0.64 0.61 0.12-0.58 0.33 0.38 0.27 0.18-1.20

72-85 57-79 61 59 59 83 84 84 92

0.20-0.25 0.16 0.24 0.06-0.13 0.17 0.09-0.15 0.11

40-76 70 62 25-82 76 67-84 27

* Numerical superscripts next to samples correspond to reference numbers cited in the reference section. ** Values are mean or minimum and maximum levels. *** Data are expressed as % of total CLA isomers.

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Factors Affecting CLA Content in Milk Pasture, Conserved Forages, and Grain The CLA content in milk fat can be affected by a cow’s diet, breed, age, and the use of synthetic mixtures of CLA supplements. Among these factors, the diet is known to strongly influence the CLA content of milk, which is affected by feedstuffs such as pasture, conserved forages, plant seed oils, cereal grains, marine oils and marine feeds. A number of studies have shown the positive effects of pasture-based diets on the CLA content of milk fat. Dhiman et al. [70] reported that cows grazing pasture had 500% higher CLA content in milk fat (2.21% of total FA) as compared to cows fed a diet containing 50% conserved forage (hay and silages) and 50% grain (0.38% of total FA). Other researchers have also demonstrated that the CLA content of milk increased linearly as the proportion of fresh grass from pasture in the diet was increased [71-74]. Supplementing grain to cows grazing pasture decreases the CLA content of milk fat. Cows on pasture supplemented with 0, 6, or 12 kg/day of grain had 2.21, 1.43, and 0.89% CLA in milk fat, respectively [70]. Similarly, supplementing grain to cows receiving grass silage or replacing conserved grass in dairy cow diets with corn silage lowered the CLA content of milk [72, 75]. Corn silage contains 20 to 40% grain on a DM basis. The addition of grain to dairy diets decreases ruminal pH. A decrease in pH will change the microbial population and affect ruminal fermentation [62]. It has been suggested that the main ruminal biohydrogenating bacteria are cellulolytic [76, 77]. Reduction in ruminal pH decreases the population of cellulolytic bacteria and other microbes responsible for lipid biohydrogenation and the production of CLA and TVA [57]. Forage maturity and method of preservation also seem to be important factors influencing the CLA content of milk. Cows fed immature forages have higher levels of CLA in milk than cows fed mature forage. Cows fed grass silage cut at early heading, flowering, and second cutting had 1.14, 0.48, and 0.81% CLA in milk fat, respectively [75]. The high C18:3 content of immature grass and its low fiber content as compared to mature grass probably interact to increase the production of CLA and TVA.

Plant Oils and Seeds Feeding plant seed oils, such as sunflower, soybean, peanut, canola, and linseed oil, increased CLA content in milk [78-82]. These oils are rich in C18:2 and C18:3 FA. Studies have found that high levels of C18:2 and C18:3 (such as are found in most plant seed oils) result in increased production of CLA and TVA, with the TVA potentially being an additional substrate for the endogenous synthesis of c9, t11 CLA [56, 83, 84]. Besides directly increasing the yield of CLA and TVA, it is likely that C18:2 inhibits the final reduction of TVA, thus increasing its accumulation in the rumen [58] and subsequent availability to the animal. Feeding a diet containing soybean oil (4%) resulted in approximately a four-fold increase in CLA content of milk fat (2.08%) over the control diet without soybean oil (0.50% of milk

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fat), [85]. Supplementing peanut oil, sunflower oil, or linseed oil at 5.3% of dietary DM resulted in 1.33, 2.44, and 1.67% CLA in milk fat, respectively [78]. The specific FA that is most abundant in any given plant seed oil is very important in determining how much the oil will elevate the levels of CLA in milk fat. Oils rich in C18:2 are more effective at increasing CLA in milk fat as compared to oils rich in C18:3 or C18:1. Dhiman et al. [85] reported that linseed oil was not as efficient at increasing CLA content in milk fat as was soybean oil. Intact oil seeds are known to be less efficient than free oils at enhancing the CLA content of milk. Processing releases oil from the seeds, which then becomes available to the rumen microbes for biohydrogenation. Feeding raw seeds has little or no effect on the CLA content of milk fat because polyunsaturated fatty acids (PUFA) in the intact seeds are relatively unavailable to the rumen microbes for biohydrogenation [85]. However, if raw seeds are processed by grinding, roasting, micronizing, flaking, or extruding, those processed seeds are effective at increasing the CLA content in milk [71, 79, 86, 87-94]. Interestingly, cows grazing green grass that received 82% less C18:3 (received 102 g/day) had higher milk CLA content (2.21% of fat) [70] than cows fed diets containing conserved forages and grain supplemented with C18:3 (received 575 g/day) through linseed oil (1.67% CLA in milk fat) [78]. However, it should be pointed out that factors other than oil supply from pasture grass are also responsible for the higher CLA content observed in grazing cows. Cows grazed on pasture or fed forage alone will produce less milk, but with higher fat content, than cows fed conserved forage and grains. The milk yield of grazing cows is reduced by 50-60%, but the CLA content of milk is 300–400% higher than in milk from cows fed conserved forage and grain. Thus, the daily output of CLA from cows grazing on pasture will still be higher than from cows fed conserved forage and grain even with lower total milk yields. The situation differs when cows are fed plant oils, as plant oils enhance the CLA content of milk. Therefore, caution must be taken when comparing dietary influence on CLA content and daily CLA output.

Marine Oils and Feeds The feeding of fish oil to dairy cows has been shown to enhance the CLA and TVA contents of milk fat, but to reduce total fat content of milk [86, 91, 94, 95]. Feeding fish oil at 1.6% of the diet DM increased the CLA and TVA contents in milk fat from 0.16 and 1.03% in control group without fish oil to 1.55 and 7.50% in fish oil fed group, respectively [95]. Feeding diets containing 2% fish oil to dairy cows increased CLA and TVA contents in milk fat by 300 and 500%, respectively, as compared to milk fat from cows fed no fish oil [90, 96]. However, there was no additional increase in CLA and TVA contents when cows were fed 3% fish oil. The inclusion of marine feeds such as fish meal or sea algae in dairy cow diets has been shown to enhance the CLA content of milk [69, 85, 92, 97, 98]. The reduction of milk fat percentage is a common problem when feeding fish oil to lactating dairy cows and can influence the total CLA yield. Milk fat content was reduced by 20 to 25% when cows were fed diets containing 1.6 to 2.0% fish oil or 4% algae on a DM basis [90, 94, 95, 96, 98]. Researchers have also attempted to enhance CLA in milk fat by feeding combinations of fish and soybean oils or meals to dairy cows. In some studies, fish oil/fish meal was more

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effective at enhancing the CLA content of milk than adding similar amounts of soybean oil or combinations of fish oil and soybean oil through extruded soybeans or soybean meal [90, 91, 94, 97]. It is worth stating here that a component of fish oil may inhibit the growth of bacteria or production of bacterial enzymes responsible for the reduction of TVA to C18:0, creating conditions that are more favorable to the later tissue production of CLA from TVA. Therefore, C18:2 and C18:3 FA that were provided by the soybeans in the diet indirectly increased CLA synthesis. The origin of CLA in milk fat from cows fed fish oil is very possibly through the desaturation of TVA in the mammary gland by the Δ9-desaturase enzyme. The relationship between milk fat CLA and TVA is linear across a wide variety of feeding conditions [99].

Cow Management Systems Dairy cow management systems also influence the CLA content of milk. Jahreis et al. [72] collected milk samples over a period of one year from three farms with different management systems: 1) conventional farming with indoor feeding using preserved forages; 2) conventional farming with grazing during the summer season; and 3) ecological farming with no use of chemical fertilizers to produce forages and grazing during the summer season. The CLA content was 0.34, 0.61, and 0.80% of fat in milk from cows fed indoors, grazed during summer, and grazed in ecological farming conditions, respectively. Reasons for these results could be due, in part, to differences in vegetation or forage quality among the three systems. Depending on the season, CLA content in milk varied from 0.6 to 1.2% of milk fat, with content being higher in spring and summer than in winter [100-103]. These data suggest that the availability of fresh forages in spring and summer increases CLA content in milk fat as compared to mature forages in late summer or conserved forages in winter.

Cow Breed, Age, and Individual Variation Recent studies suggest that dairy cow breed can also influence the CLA content of milk. Montbeliard cows displayed a tendency to have higher CLA in milk fat (1.85%) as compared to Holstein-Friesian (1.66%) or Normande cows (1.64%) grazing on the same pasture [104]. Holstein-Friesian cows had higher CLA content in milk compared to Jerseys fed diets containing conserved forages and grains [105, 106, 107]. Conjugated linoleic acid content was also higher in milk fat from Holstein-Friesians (0.57%) than for Jersey cows (0.46%) when grazed on pasture [74]. Brown Swiss cows had higher CLA content in milk fat than Holstein-Friesian when fed similar diets [94, 106, 107]. The average difference in CLA content of milk fat among Brown Swiss, Holstein-Friesian, and Jersey breeds is 15 to 20% when fed similar diets. Brown Swiss cows have inherently higher CLA in milk fat, followed by the Holstein-Friesian and Jersey breeds.

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The CLA content in milk varies from cow to cow, even when the same diet is fed. Jiang et al. [57] and Stanton et al. [87] found substantial variation in the CLA content of milk (0.15% to 1.77% of fat) among individual cows fed the same diet. Kelly et al. [73, 78] observed a threefold variation in CLA content of milk among individual cows fed the same diet at a similar stage of lactation and producing milk with similar fat content.

Synthetic CLA Supplements Synthetic CLA isomers were fed in a ruminally-protected form to avoid ruminal biohydrogenation and enhance CLA in milk fat. The administration or feeding of CLA supplements to dairy cows caused a dramatic reduction in the fat content and total yield of milk fat, but resulted in a small increase in milk CLA content [82, 108, 109-115]. The highest transfer efficiencies for c9, t11 and t10, c12 isomers from supplement to milk were 11 and 4%, respectively [112-113].

Dietary Factors Affecting CLA in Meat Pastures and Conserved Forages As is the case with dairy cattle, grazing animals on pasture, feeding fresh forages or increasing the amount of forage in the diet will elevate the percentage of CLA as a proportion of total FA in meat from ruminants. Grazing beef steers on pasture or increasing the amount of silage in the diet increased the c9, t11 CLA content in meat fat by 29 to 45% compared to control [116, 117]. The increase in beef CLA content varies with the quality and quantity of forage in the animal’s diet. Beef from steers raised on green pasture had 200 to 500% more c9, t11 CLA as a proportion of fat as compared to steers fed an 87% corn grain-based feedlot diet [118, 119]. Rule et al. [120] observed that the percentage of c9, t11 isomer of CLA was higher in intramuscular fat of range cattle compared with that of steers fed a high-grain diet under feedlot conditions. The increase in c9, t11 CLA content in beef is not as dramatic as the increase seen in milk from cows grazed on pasture. This difference is probably due to differences in CLA production in the rumen or endogenous synthesis of CLA in intramuscular fat of beef cattle fed high-forage diets.

Plant Oils and Seeds Supplementing beef cattle diets with C18:2 or C18:3 rich plant oils has yielded variable results with regard to increasing the CLA content of beef. Feeding 4 to 6% of diet DM as soybean oil to beef cattle fed high grain diets either marginally increased or did not increase the c9, t11 CLA content of beef [121-124]. There was a small increase in the c9, t11 CLA content of fat from beef muscle when steers were fed 3 to 6% sunflower oil as compared to beef from cattle fed no oil (0.35 vs. 0.25% CLA in beef fat) [125, 126]. Feeding processed

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plant oil seeds has also resulted in marginal or no increase in CLA content of beef [117, 127129]. Feeding feed sources rich in C18:2 or C18:3 to dairy cows results in a 3 to 4 fold increase in the c9, t11 CLA content in milk fat [78, 85], but only marginal increases in beef fat. It is very possible that the mechanisms and routes of CLA synthesis (ruminal and endogenous) are different in the mammary gland and adipose tissue. Additional factors regulating the synthesis of CLA in the rumen, muscle, and mammary gland are poorly understood. Finishing beef cattle are typically fed diets containing 85 to 92% grain, whereas a typical diet for a high-producing dairy cow consists of only 50 to 60% grain. The lower proportion of c9, t11 CLA in beef fat as compared to milk fat in animals fed diets rich in C18:2 or C18:3 probably relates to the effects of the traditional high-grain, low-fiber diets fed to finishing cattle in the United States. It seems likely that the acidic ruminal pH often occurring in finishing beef cattle alters the microbial population involved in lipid biohydrogenation, and therefore influences the ruminal synthesis of CLA isomers. Grazing animals on pasture substantially increases CLA as a proportion of FA, but total fat content is reduced because of the lean beef from grassfed cattle. Therefore, increase in CLA content of beef should be evaluated based on total CLA available in the edible fat rather than concentrations in raw meat.

Animal Breed and Management Strategies Besides dietary factors, researchers have also studied the influence of beef cattle breed on CLA content in meat, and limited studies suggest that there is little breed effect. The CLA content in beef muscle was similar in European x British crossbreeds and 75% Wagyu cattle fed high-grain, barley-based diets [130]. Limousin cattle had only marginally higher CLA content in beef muscle as compared to Wagyu and Limousin x Wagyu cattle fed similar diets [131]. Existing literature indicates that the total CLA content (sum of c9, t11 and t10, c12 isomers) of beef varies from 0.17 to 1.35% of fat. The broad range of CLA content of beef is related to the wide variety of feeds offered, breed differences, and management strategies used to raise cattle.

Processing Effects on CLA Content of Milk and Meat The effects of processing conditions, dairy cultures, and storage conditions on CLA content of dairy products have been studied. Pasteurizing raw milk at 68.3°C for 30 min did not alter the CLA content of milk [1, 132]. Processing milk under normal conditions (up to 85˚C for 30 min) into dairy products such as yogurt, ice cream, sour cream, and cheese (Mozzarella, Gouda, and Cheddar) had no influence on the CLA content [79, 133, 134]. The CLA content of dairy products increases when milk is processed at higher temperatures.

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Clarification of ghee (butter oil) at 110 and 120°C increased the CLA content to 0.9 and 2.1% of fat, respectively, as compared to 0.6% in raw milk [135]. Available research suggests that CLA in milk fat is a stable compound under normal processing and storage conditions; however, processing dairy products at >80°C may slightly elevate the CLA content. There is not enough data available to form conclusions about the effects of different cultures and additives on the CLA content of dairy products. Influence of cooking temperatures and methods on the CLA content of beef have been studied by a few researchers. A majority of the results of such research [136, 137] suggest that the CLA content in meat products seems to be influenced by the raw material, and not by processing conditions such as cooking temperature. Different cooking methods (fried, baked, broiled, or microwave) and degree of doneness under normal conditions (60 to 80°C internal temperature in meat) did not alter the CLA content in beefsteaks (ribeye, round, T-bone, and sirloin) [136]. Storing cooked beef at 4°C for 7 days did not alter the CLA content [136]. Results from the above studies suggest that CLA in meat is a stable compound under normal cooking and storage conditions.

Health Benefits of CLA There is a growing interest in the levels of CLA in human diets because of accumulating evidence, largely based on animal studies, that suggests potential health benefits of CLA. Another reason is growing interest in natural foods, and that CLA occurs naturally in foods such as milk and meat derived from ruminants. Additionally, as discussed above animals grazing fresh green grass, “nature’s way of feeding ruminant animals”, results in 3 to 5-times more CLA in milk and meat than when animals are fed conserved forages and grains in confinement. Numerous physiological functions have been attributed to CLA, including cancer inhibition, antidiabetic effects, reduction of adipose tissue, alteration of body composition, reduction of atherosclerotic plaque formation, enhancement of immune response and help with bone formation (Table 3). Cancer Inhibition Conjugated linoleic acid has a variety of positive effects on cellular mechanisms in animal and human cancer cells that support its anticancer role. However, most of our understanding of the anticancer effects of CLA is based on animal models. Preliminary information from human studies shows positive health benefits. However, there is an urgent need to extend our understanding of anticancer effects of CLA in humans. The evidence concerning the promising role, and limitations, of CLA in the prevention of cancer is reviewed below.

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Function/Model used Carcinogenesis ▼Chemically induced mammary carcinogenesis in rats ▼Growth of transplantable breast cancer tumor cells in nude mice ▼Growth of transplantable prostate cancer tumor cells in nude mice ▼Stages of chemically induced skin tumorigenesis in mice ▼Chemically induced colon carcinogenesis in rats ▼Chemically induced forestomach carcinogenesis ►Carcinogenesis in Min mice Adipogenesis ▼Chicks, mice and rats ▼Human subjects ►Human (woman 20-41 years age) ►Weaned piglets fed high fat diet ►Fatty acid and glycerol metabolism in healthy weight-stable women Zambell et al. 2001) Atherosclerosis ▼Aortic plaque formation in hamster ▼Aortic atherosclerosis in rabbit Diabetic effects ▼Onset of diabetes in Zucker diabetic fatty male rats ▲Glucose tolerance and transport ▼Insulin sensitivity in mice Immune functions ▲Damage protection and lymphocyte proliferation in nursery pigs ►Young healthy women ▼Eicosanoid and histamine production ▲Onset of lupus in mouse model ▲Mitochondria protection from free radicals in rat liver Bone formation ▼Eicosanoid production in rats ▲Collagen synthesis in rats

Reference [2,4,5,12] [10,17] [7] [149,225] [151] [226] [227] [20,26,162,166] [184,179] [181] [228] [187]

[186] [32,157,229] [34] [230] [231] [171,199] [232] [31,200] [233] [234] [235] [203]

▲ = increased; ▼ = decreased; ► = no effect.

Ip and coworkers [2] were the first to show inhibition of cancer by the dietary administration of CLA. Female Sprague rats were fed a basal diet supplemented with 0, 0.5, 1.0 and 1.5% synthetically prepared CLA. Rats received their experimental diet 2 weeks before gavage treatment with the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). The experiment continued until study termination at 24 weeks after DMBA administration. Results showed a dose response reduction of 32 – 60% in the total number of mammary tumors. Later Ip et al. [3] showed that feeding as little as 0.05 to 0.5% of CLA in the diet resulted in dose responsive significant reduction in mammary tumors. The reduction amounted to 36-58% for CLA levels from 0.1 to 0.5% of the diet. It was also demonstrated

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that short-term, post initiation CLA feeding (4 or 8 weeks) was ineffective and reduction in tumor incidences was observed only in rats that received an uninterrupted CLAsupplemented diet for 20 weeks [4]. Researchers further demonstrated that CLA as a free fatty acid is as effective in reducing incidences of mammary tumor as the triglyceride form naturally found in foods. The magnitude of tumor inhibition was same irrespective of dietary fat level or saturated vs. unsaturated fat [5].

The above studies were conducted using synthetically prepared CLA. Feeding CLA-enriched butterfat to rats inhibited mammary tumor growth by 53% compared with rats fed butterfat with normal levels of CLA [138]. Another interesting observation in this study was that rats consuming the CLA-enriched butterfat accumulated more total CLA in the mammary gland and other tissues compared with those consuming synthetic CLA at the same dietary level of intake (Table 4). This study suggests that naturally occurring CLA may be metabolized and utilized differently than synthetic CLA. Hman consumption of TVA and its subsequent conversion to CLA in tissues should also be considered. Partially hydrogenated oils and meat and milk products contain significant amounts of TVA [139]. The distribution and activity of the Δ9-desaturase enzyme in humans is still debatable. Emken et al. [140] found no evidence of desaturation of TVA is plasma lipids of humans over a 48 hour period. However, Salminen conducted a study wherein adults consumed a diet high in either C18:0 or TVA for 5 weeks following the consumption of a high dairy product diet for 5 weeks [141]. Intakes of CLA for the dairy diet, C18:0, and TVA were 310, 90, and 40 mg/day, respectively. At the conclusion of the study, CLA concentration in serum lipids as a percent of total fat was 0.33, 0.17, and 0.43, respectively, suggesting that there was significant endogenous Δ9-desaturation of dietary TVA. Turpeinen et al [142] reported an average 19% conversion of TVA to CLA when human subjects consumed either 1.5, 3.0, or 4.5 g of TVA/day for 9 days. It is evident that the endogenous synthesis of CLA from TVA by Δ9-desaturase in humans requires further exploration and quantification. To determine if endogenously formed CLA from TVA is able to exert anticarcinogenic activity, rats were fed different levels of TVA (1-3% of diet) [143]. It was found that treatment with 2% TVA in the diet reduced the total number of premalignant lesions by 50%. It seems, therefore, that TVA also has anticarcinogenic effects, possibly through CLA. Table 4. Total CLA content in tissues of rats fed different sources of CLA Source of CLA1 Liver

Control butter fat CLA enriched butter Synthetic CLA2 1

2.6 15.7 10.2

Total CLA content Mammary Peritoneal fat fat μg/mg lipid 7.2 8.8 36.5 65.9 28.2 33.4

Plasma

5.5 23.3 12.5

CLA content of diets was 0.1, 0.8, and 0.8 g/100 g of dietary DM in control butter fat, CLA enriched butter fat and synthetic CLA diets, respectively. 2 Supplied by Nu-Chek-Prep, Elysian, MN. Reference: [138]

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Feeding of CLA affects different biological and biochemical parameters in the mammary gland, which induce changes that render the mammary gland less susceptible to cancer. The mechanisms whereby this occurs are not known, but some theories are that CLA reduces cell proliferation [144], alters various components of the cell cycle and induces apoptosis [145], or alters the expression of peroxisome proliferators-activated receptor-α (PPAR-α), [146, 147]. Another factor that is proposed to be involved in CLA anticarcinogenic activity is its metabolism as polyunsaturated fatty acids (PUFA). Feeding CLA resulted in decreased level of arachidonic acid, which is the substrate for eicosanoid biosynthesis [148]. It has also been shown that CLA is a chemoprotective fatty acid that inhibits phorbol ester-induced skin tumor promotion in mice [149] by reducing the formation of prostaglandin E2 [150]. Conjugated linoleic acid protected against 2-amino-3-methylimidzo[4,5f]quinoline-induced colon cancer in F344 rats [151, 152] by decreasing mucosal levels of prostaglandin E2 and arachidonic acid in a dose dependent manner. Yang et al. [153] studied antimutagenic efficacy of CLA in colon cancer using the (2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine) carcinogen that is present in cooked protein-rich food. Rats exposed to this carcinogen showed an 8 to 16-fold increase in cell mutation frequency in the colon. Supplementation of CLA in the diet reduced the mutation frequency in the distal colon by an average of 19%. There are very few research reports studying the anticarcinogenic effects of CLA in humans. A Finnish study reported decreased breast cancer risk in women consuming whole milk [154]. In another Finnish study Aro et al. [9] measured dietary and blood serum CLA in Finnish women patients (premenopausal and postmenopausal) with breast cancer from 1992 to 1995 and found that the CLA and TVA were lower in breast cancer postmenopausal patients than control women with no cancer. A study in an ethnically homogeneous region of France with 360 breast cancer patients revealed that the CLA content was higher in breast adipose tissues from control compared with cancer patients, suggesting higher the level of CLA in breast adipose tissue lower the incidences of breast cancer [155]. In summary, the proposed mechanisms of anticarcinogenic activity of CLA in vivo are through modulation of eicosanoids, increased apoptosis, reduced proliferation, and PPAR-α activation. Other mechanisms include reduction of murine mammary tumor metastasis by CLA that can affect later stages of disease, especially metastasis, which is crucial part in cancer prognosis. Details on the mechanism of anticarcinogenic activity of CLA can be found in recent reviews [156-159].

CLA and Body Energy Expenditure The energy expenditure effects of CLA are linked to its ability to reduce adipose tissue, alter body composition and reduce atherosclerotic plaque formation. The CLA isomer largely responsible for these effects is t10, c12 [160, 161]. The physiological effects of CLA related to body energy expenditure are discussed below.

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Body Composition Several studies have shown that CLA reduces body-fat accumulation and increases lean body mass in various animal models including mice ([23, 24, 162-165], rats [166-169], and pigs [170-176]. In these studies feeding 0.5 to 2.0 g of CLA /100 g of diet to growing animals resulted in less body fat and more lean mass. Adipose depot weights were 43-88% lower in mice fed the 0.5 to 1.0% CLA diet compared to control mice receiving no CLA [23, 162]. Adipose tissue reduction effects of CLA in these studies varied with adipose depot [167, 168] and strain/variety of the animal used [166]. Some researchers have related reduced adipose tissue weight in mice supplemented with CLA to increased energy expenditure and fat oxidation ([23, 177, 178]. Mice fed CLA had reduced lipase activity, enhanced lipolysis and increased muscle carnitine palmitoyltransferase activity [162, 163, 167], suggesting that CLA reduces adipose tissue by affecting some of the key enzymes involved in fat mobilization and storage. It has been suggested that the anabolic effect of the c9, t11 isomer of CLA precedes the reduction in fat by the t10, c12 isomer [160, 163]. Therefore, supplementing diets with both isomers would be more beneficial to health compared to a single isomer. There are few studies examining the effects of CLA on body composition in humans. Healthy Norwegian men and women (n=20) in a double-blind study received either control (5 men and 5 women) placebo capsules containing olive oil or capsules containing a synthetically prepared mixture of CLA (approximately equal proportions of t10, c12 and c9, t11 isomers) of 1.8 g/day for 12 weeks. Irrespective of gender, the group taking CLA had 4% reduction in body fat compared to control [179]. In another study by Blankson et al. [180] overweight subjects (n = 47) received either placebo (olive oil) or CLA at 1.7, 3.4, 5.1, 6.8 g /day for 12 weeks. Body fat decreased significantly only in subjects taking 3.4 or 6.8 g of CLA/day. These researchers concluded that a dose of 3.4 g of CLA/day was sufficient to reduce body fat in overweight adult people. In a short term (4-week) study conducted in the United States, healthy women (n = 17) received a placebo (sunflower oil) or 3.9 g of CLA/day. In the CLA fed group the fat mass was reduced by an average 0.2 kg (range -3.2 to +0.9 kg), with no change in body weight, whole body fat oxidation rate, lipolytic rate or fatty acid release from adipose tissue during exercise or rest periods [181, 182]. Feeding 2.7 g of CLA/day to obese human subjects (n = 80) for 6 months was ineffective in reducing body fat and weight [183]. In Sweden, men and women (n = 50) in a double blind study took 4.2 g CLA/day and 0.2 kg and 0.7 kg losses were observed in body fat mass in placebo and CLA groups, respectively [184]. Despite pronounced effects in animal models, the observed changes in body weight and fat mass due to CLA supplementation have been small and variable in human studies. The lack of response in human trials may be due to the short term nature of the studies, less control over the diet and lack of sensitive methods to detect small differences in body weight and fat mass.

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Atherosclerosis Considerably fewer studies have been published addressing the role of CLA in atherosclerosis compared to its other roles. However, the information available suggests that CLA reduces atherosclerotic plaque formation in rabbits and hamsters. Feeding 0.5 g of synthetically prepared CLA/day to rabbits on a hypercholesterolemic diet for 12 weeks reduced blood serum triglycerides and low density lipoprotein cholesterol levels, and resulted in less atherosclerotic plaque formation compared with control rabbits receiving no CLA [32]. Feeding CLA to hypercholesterolemic hamsters also reduced early aortic atherosclerotic plaque formation [185, 186]. However, in contrast to its protective role, CLA induced the formation of aortic fatty streaks in C557BI/6 mice fed an atherogenic diet [187]. While data in the above studies are conflicting, the majority of studies suggest that CLA can improve plasma lipoprotein metabolism and prevent atherosclerosis in animal models. The CLA isomer responsible for the effects on lipoprotein metabolism is t10, c12 [188]. There is a paucity of information on the effects of CLA on lipoprotein metabolism in humans. In a study by Noone et al. [189] human subjects were given 3 g/d of a blend of c9, t11 and t10, c12 CLA isomers (50:50 or 80:20) for 8 weeks. The 50:50 isomeric blend of CLA reduced plasma triacylglycerol concentration (-20%). Plasma triacylglycerol concentration has been identified as an independent risk factor for future chronic heart disease. The increased lipoprotein metabolism may be the mechanism for anti-atherogenic effects of CLA in humans, as has been shown in animal models.

CLA and Diabetes Impaired glucose tolerance and obesity are some of the risk factors for the development of type 2 diabetes. The t10, c12 CLA reduces body fat in animal models and it is this isomer that is implicated as an antidiabetic. It has been shown that CLA was equally effective as thiazolidinediones (a class of oral insulin sensitizing agents that improve glucose utilization without stimulating insulin release) in reducing fasting glucose in Zucker diabetic rats [190]. In a double blind study with human diabetics blood glucose and plasma leptin levels were decreased in CLA supplemented patients [191]. In type 2 diabetes feeding Zucker diabetic rats a synthetically prepared mix of CLA isomers reduced plasma insulin, triglycerides and leptin [34, 190]. In another study a synthetically prepared mix of CLA isomers improved impaired glucose tolerance, lowered adipose mass and enhanced glucose uptake into the muscle of type 2 diabetic rats [192]. Subsequently, Henrickson et al. [193] demonstrated that the improved glucose tolerance and insulin-stimulated glucose transport in the skeletal muscle of obese Zucker diabetic rats was due to the t10, c12 isomer with no effect due to the c9, t11 isomer of CLA. The, t10, c12 isomer of CLA promoted insulin resistance, increased serum glucose and insulin concentrations in ob/ob mice [194], and decreased insulin-stimulated glucose uptake in differentiating human preadipocytes [195]. It has been suggested that CLA delays the onset of diabetes in the Zucker diabetic rat model.

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The role of CLA in regulating body composition and obesity linked type 2 diabetes is not clearly understood. However, a significant correlation between body weight change and fasting glucose suggests that the mechanism by which of CLA reduces fasting blood glucose is related to its effect on lipogenesis. More long term studies are needed to understand the mechanisms.

CLA and Immunity The immune system is central to defense against diseases. Conjugated linoleic acid affects parameters related to immunity that may revolve around production of eicosanoids and immunoglobulins that are essential for immune functions. Eicosanoids are produced in the body from arachidonic acid by various types of immune cells to regulate cytokine synthesis and inflammation. Additionally, eicosanoids and cytokines affect a range of biological functions. Conjugated linoleic acid is a potent regulator of eicosanoid mediator release during immune response [196]. The physiological role of CLA in normal and immune-stimulated animals was studied. It was demonstrated that CLA protects against Escherichia coli-induced body weight loss in chicks and mice and reduces histamine-induced prostaglandin-E2 production in Guinea pig trachea [197, 198]. Recently, Cook et al. [199] showed that CLA not only enhances immune response, but also protects tissues from collateral damage by preventing weight loss, increasing feed intake, and prolonging life during the inflammatory process involving autoimmunity in mice and pigs. Rats fed a 1% CLA diet showed decreased levels of leukotriene-B4 and leukotriene-C4 in spleen and lungs [200]. In contrast, feeding 3.9 g of CLA/day to humans for 93 days resulted in no apparent alterations in eicosanoids (prostaglandin E2, leukotriene B-4) or cytokines [201]. Existing literature suggests that immune response of supplemental CLA may be through altered eicosanoid production, however, physiological effects of altered eicosanoid production are not clearly understood.

CLA and Bone Formation Dietary fat (CLA and omega-3 fatty acids) may influence bone formation and resorption by altering prostaglandin (PG) biosynthesis in bones [202, 203]. For example, PGE2 increased bone mass in rats [204], however, at high concentration it decreased bone growth [205]. Thus, PGE2 effects on bone formation may be dose related; that is stimulatory at low concentration and inhibitory at high concentration [203]. In a study by Watkins et al. [206] chicks fed butterfat showed reduced bone PGE2, elevated bone IGF-1 and increased bone formation rates of nearly 60% compared to those animals given diets containing corn oil. The authors concluded that bone effects were due to the presence of CLA and n-3 fatty acids in butter compared to higher n-3 fatty acids in corn oil. However, these beneficial effects of CLA on bone modeling have not been shown in any other studies. Dietary beef fat and a CLA supplement were able to maintain synthetic activity of osteoblastic cells and CLA was

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even able to rescue the reduced bone formation rate in rats given a diet high in omega-6 fatty acids [207]. Studies show an increase in percent of ash when CLA is fed to chicks [208]. This effect is presumed to be due to protection conferred by CLA against bone loss [20]. An increase in cytokines increases bone loss, and CLA appears to counter the effect of cytokines [206]. Cytokines are hormone-like mediators of immunity and inflammation that are produced by macrophages and other immune cells when they are stimulated. Osteoporosis is a major health problem in the U.S., and this effect of CLA could contribute to prevent osteoporosis. With limited information on effects of CLA on bone formation and resportion it is difficult to make firm conclusions. However, preliminary information from animal models is encouraging. To summarize overall health benefits of CLA, our understanding of health effects of CLA is primarily based on in vitro and animal studies along with a few studies in humans. Preliminary information shows that CLA may have a number of positive health benefits. However, the mechanisms of CLA action are not well understood. In addition to the need for understanding the mechanisms of CLA action, it is also imperative to determine if these beneficial physiological effects can be applied to human health. More long term human studies are needed because it is difficult to extrapolate information from animal studies to humans.

CLA Intake of Humans from Milk and Meat The question needs to be addressed of how much CLA humans are currently consuming and how much should be consumed in order to reap the health benefits and anticancer effects that have been observed from adding proper levels of CLA to the diet. Figure 3, panel A, shows the amounts of CLA provided in one normal serving of milk, cheese, lean beef, and poultry. Figure 3, panel B, shows the amounts of CLA provided by one serving of milk, cheese, beef, and poultry from CLA-enriched foods. One serving of milk is considered to be 227 ml and one serving of cheese is assumed to be 30 g. A serving of lean beef and poultry is assumed to be 100 g each. Fat intake (grams) from one serving of each food was calculated by multiplying the serving size by the fat content of the particular food. Conjugated linoleic acid intake (milligrams) from each particular food was then calculated by multiplying the CLA content by the calculated fat intake. Fat contents used were 3.5 [79], 32.0 [133], 6.4 [209], and 6.7% [210] for whole milk, cheese, beef, and poultry, respectively. The fat content of high-CLA beef was assumed to be 3.0% because pasture-grassfed beef contains considerably lower fat content than grain-fed beef. Average CLA values for low-CLA milk (0.53% of FA) and cheese (0.50% of FA) were taken from Table 1 and values for low-CLA beef (0.37% of FA) and poultry (0.19% of FA) were taken from Table 2. Conjugated linoleic acid contents used for high-CLA products were 2.21, 2.21, 1.35, and 0.19% of total FA for milk, cheese, beef, and poultry, respectively. The value used for poultry was the same in both panels because there is very little potential for CLA content to be increased in poultry products.

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Figure 3. Daily conjugated linoleic acid (CLA) intake by humans from one serving of low-CLA whole milk, cheese, beef, and poultry (A); and daily CLA intake from one serving of high-CLA whole milk, cheese, beef, and poultry (B).

Based on appropriate extrapolation of data from animals to humans, an adult human would require 0.72-0.80 g of CLA/d to inhibit tumor growth [211, 212]. A person eating one serving each of low-CLA whole milk, cheese, beef, and poultry per day would have a CLA intake of approximately 127 mg/day (Figure 3, panel A), which amounts to approximately 20% of the estimated daily requirement for humans shown to be an effective dose in animal models. However, a person consuming the high-CLA products would have a CLA intake of about 441 mg/day (Figure 3, panel B), which amounts to 60% of the estimated requirement for humans. It is apparent that the greatest potential for increasing the CLA intake of humans is to consume high-CLA containing dairy products (milk and cheese). Milk and dairy products from pasture-grassfed dairy cows are naturally enriched and have the highest contents of CLA. The consumption of grass-fed beef may also enhance CLA intake, though the increase would not be very substantial because total fat content is lower. Clearly, if the

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objective is to achieve health benefits from CLA, it is practical to do so through consumption of CLA-enriched milk and cheese. Human consumption of TVA and its subsequent conversion to CLA in tissues should also be considered. Partially hydrogenated oils and meat and milk products contain significant amounts of TVA [139], and a few studies have shown that there is some conversion of TVA to CLA in humans [142].

Conclusion Conjugated linoleic acid is unique because it occurs naturally in foods derived from ruminant animals. The ability to analyze and detect different isomers of CLA is improving, and this will be useful in future research investigating the biological role of individual isomers. Manipulating the diets of dairy and beef cattle and altering management practices on the farm can enhance the CLA contents of milk, dairy and beef products. The CLA contents of milk, dairy products, meat, and meat products vary widely. The amount of published research describing physiological and health benefits of CLA has expanded in recent years. However, little of this research has been done in humans. Conjugated linoleic acid intake by humans has the potential to be increased to a level that has been shown to be beneficial for health in animal models through the consumption of CLA-enriched dairy and beef products.

Acknowledgments The authors would like to acknowledge the Utah Agricultural Experiment Station, Utah State University for partial financial and technical support. Financial support for this research from Western Region Sustainable Agriculture, Cooperative Research Extension and Education, USDA is also acknowledged (Grant No. SW01-034).

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Adolf, R. (Eds.), Advances in Conjugated Linoleic Acid Research. Vol. 2, pp. 251-266. Champaign, IL: AOCS Press. [159] Banni, S., Heys, C. S. D., and Wahle, K. W. J. (2003). Conjugated linoleic acid as anticancer nutrients: Studies in vivo and cellular mechanisms. In: Sebedio, J., Christei, W. W., and Adolf, R. (Eds.), Advances in Conjugated Linoleic Acid Research. Vol. 2, pp. 267-281. Champaign, IL: AOCS Press. [160] Pariza, M. W., Park, Y., and Cook M. E. (2001). The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res., 40:283-298. [161] Granlund, L., Juvet, L. K., Pederson, J. I., and Nebb, H. I. (2003). Trans10, cis12conjugated linoleic acid prevents triacylglycerol accumulation in adipocytes by acting as a PPARγ modulator. J. Lipid Res., 44: 1441-1452. [162] Park, Y., Albright, K. J., Liu, W., Storkson, J. M., Cook, M. E., and Pariza, M. W. (1997). Effect of conjugated linoleic acid on body composition in mice. Lipids, 32 (8):853-858. [163] Park, Y., Albright, K. J., Storkson, J. M., Liu, W., Cook, M. E., and Pariza, M. W. (1999). Changes in body composition in mice during feeding and withrdrawal of conjugated linoleic acid. Lipids, 34:243-248. [164] Park, Y., Storkson, J. M., Albright, K. J., Liu, W., and Pariza, M. W. (1999). Evidence that the trans-10, cis-12 isomer of conjuaged linoleic acid induces body composition changes in mice. Lipids, 34:235-241. [165] Miner, J. L., Cederberg, C. A., Nielsen, M. K., Chen, X., and Baile, C. A. (2001). Conjugated linoleic acid (CLA), body fat, and apoptosis. Obes. Res., 9: 129-134. [166] Sisk, M., Hausman, D., Martin, R., and Azain, M. (2001). Dietary conjugated linoleic acid reduces adiposity in lean but not obese Zucker rats. J. Nutr., 131:1668-1674. [167] Rahman, S. M., Wang, Y., Yotsumoto, H., Cha, J., Han, S., Inoue, S., and Yanagita, T. (2001). Effects of conjugated linoleic acid on serum leptin concentrations, body-fat accumulation, and beta-oxidation of fatty acid in OLETF rats. Nutrition, 17:385-390. [168] Azain, M. J., Hausman, D. B., Sisk, M. B., Flatt, W. P., and Jewell, D. E. (2000). Dietary conjugated linoleic acid reduces rat adipose tissue cell size rather than cell number. J. Nutr., 130:1548-1554. [169] Stangl, G. I. (2000). Conjugated linoleic acids exhibit a strong fat-to-lean partitioning effect, reduce serum VLDL lipids and redistribute tissue lipids in food-restricted rats. J. Nutr., 130:1140-1146. [170] Stangl, G. I., Muller, H., and Kirchgessner, M. (1999). Conjugated linoleic acid effects on circulating hormones, metabolites and lipoproteins and its proportion in fasting serum and erythrocyte membranes of swine. Eur. J. Nutr., 38:271-277. [171] Bassaganaya-Riera, B., Hontecillas-Magarzo, R., Bregendahl, K., Wannenmuehler, M. J., and Zimmerman, D. R. (2001). Effects of dietary conjugated linoleic acid in nursery pigs of dirty and clean environments on growth, empty body composition, and immune competence. J. Anim. Sci., 79:714-721. [172] Dugan, M. E. R., Aalhus, J. L., Schaefer, A. L., and Kramer, J. K. G. (1997). The effect of conjugated linoleic acid on fat to lean repartitioning and feed conversions in pigs. Can. J. Anim., 77:723-725.

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In: Milk Consumption and Health Editors: E. Lango and F. Vogel

ISBN: 978-1-60741-459-9 © 2009 Nova Science Publishers, Inc.

Chapter X

Beneficial effects of Human Milk and Prebiotic-Like Fermented Infant Formulas on the Intestinal Microflora and Immune system Catherine J. Mullié1, Daniel Izard 2 and Marie-Bénédicte Romond 2 1

Faculté de Pharmacie, Université de Picardie Jules Verne 1 rue des Louvels, 80037 Amiens Cedex 1, France 2 Faculté des Sciences Pharmaceutiques et Biologiques, Université de Lille 2 – 3 rue du Professeur Laguesse – BP83, 59006 Lille Cedex, France

Abstract Mother’s milk remains the gold standard for the nutrition of human neonates. Thanks to its adaptable biochemical and immunological composition, mother’s milk allows for an optimal development of the intestinal microflora, especially by promoting the implantation and growth of some of the so-called health beneficial bacteria: bifidobacteria. When bifidobacteria are dominant in the intestinal flora, they are thought to help preventing gastrointestinal disorders, repress a potentially harmful proliferation of other intestinal bacteria and stimulate the priming of the neonate’s intestinal immune system. This is why, among other research trends, the latest infant formulas are attempting to reproduce this bifidogenic effect of mother’s milk through various ways such as the addition of exogenous bifidobacteria and/or of prebiotics (specific carbohydrate substrates promoting the growth of indigenous intestinal bifidobacteria). We will first review the beneficial effects of mother's milk and those putatively related to indigenous bacteria. The probiotic (feeding of live bifidobacteria) and prebiotic (feeding of specific carbohydrates) approaches to increase intestinal bifidobacteria will also be defined. Then, we will focus on prebiotics and on a novel approach to promote indigenous intestinal bifidobacteria: the use of an infant formula containing products of

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Catherine J. Mullié, Daniel Izard and Marie-Bénédicte Romond milk fermentation by Bifidobacterium breve strain C50. These fermentation products have previously been shown to have a bifidogenic effect on indigenous bifidobacteria, thus acting like prebiotics. We will compare the effect of this formula on the intestinal microflora establishment to the ones of mother’s milk and of a standard formula. We will also deal with the issue of specifically stimulating the growth of certain species of indigenous bifidobacteria, as some bacterial species belonging to this genus (e.g., Bifidobacterium adolescentis) have been shown to be linked with immunological conditions in neonates and young children such as atopic dermatitis.

Abbreviations AAD: Antibiotic-Associated Diarrhea; AD: Atopic Dermatitis; BbC50: Bifidobacterium breve C50; BT: Bacterial Translocation; CFU: Colony Forming Unit; FISH: Fluorescent In Situ Hybridization; FOS: Fructooligosaccharides; GOS: Galactooligossacharides; HMO: Human Milk Oligosaccharides; IL: Interleukin; MLN: Mesenteric Lymph Nodes; PCR: Polymerase Chain Reaction; TNF: Tumor Necrosis Factor; TLR: Toll-Like Receptor; VLBW: Very Low Birth Weight.

Taxonomic Warning Bifidobacterium lactis is now considered a subspecies of the Bifidobacterium animalis species (Masco et al., 2004). Therefore, trials reporting on either Bifidobacterium lactis Bb12 or Bifidobacterium animalis Bb12 use the same bacterial strain.

1. Introduction: Impact of Mother's Milk 1.1. Epidemiologic Data Breast-feeding has long been shown to induce protection against infectious diseases resulting in a decreased mortality/morbidity in breast-fed babies, especially for diarrheas (WHO, 2000). The lower incidence of conditions such as diarrhea, otitis media and

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respiratory infections are mainly reported for an exclusive breast-feeding of 3 months at least (Turck, 2005). The newborn immature immune system is replaced by specific and non-specific immune effectors in human milk such as immune cells, secretory Immunoglobulins A (sIgA) or lysozyme, for example. Human milk oligosaccharides (HMO) have also been proved to play a major part in reducing these infectious conditions (Kunz et al., 2000), mainly through acting as decoy receptor for pathogens and through their bifidogenic effect. Indeed, eversince the first observation of bacteria that were to become bifidobacteria in the stools of breast-fed infants by Tissier at the beginning of the 20th century, an infant intestinal flora dominated by bifidobacteria is thought to help preventing intestinal infectious diseases, such as viral gastroenteritis, but also extraintestinal infections. The effect of breast-feeding on the onset and prevalence of chronic diseases has also been investigated. Prevention of allergy, especially food allergy and atopic dermatitis, in atrisk infants (infants with at least one first-degree relative presenting with allergy) has been reported in numerous studies. Despite conflicting results, a 3-month exclusive breastfeeding seems to reduce the risk of atopic dermatitis, especially in allergy-prone infants (Gdalevich et al., 2001). Breast-fed children also have a decreased risk of childhood obesity, maybe thanks to an early regulation of energy intake at a low level and the prevention fat accumulation in tissues (Turck, 2005). Several epidemiologic studies report on this protection such as the works of Bergmann et al. (2003) or Grummer and Mei (2004). Cardiovascular disease prevention has also been the target of numerous observational studies searching for a protective effect of breastfeeding. A slight reduction in systolic blood pressure as well as a small decrease of adulthood cholesterolemia plead for such a protection, although results of observational studies are conflicting (Turck, 2005). This latter statement holds true for breastfeeding and type 1 diabetes, the most common autoimmune disease in childhood. Despite discordant results on the prevention of some chronic disorders, breastfeeding remains the gold standard for human infant nutrition. The only drawbacks of breastfeeding are mainly related to infectious diseases such as the transmission of Human Immunodeficience Virus (HIV) (4 to 22% risk) (Read, 2003) and Cytomegalovirus (CMV) (Hamprecht et al., 2001) to newborns by infected mothers. Therefore, breastfeeding is contraindicated in developed countries for mothers carrying HIV. For CMV, the contraindication is limited to women giving birth to preterm babies because of the potential seriousness of such a contamination.

1.2. On the Intestinal Microflora 1.2.1. In Term Infants The neonatal intestinal tract is sterile at birth. It is gradually colonized by bacteria belonging to the mother's genital tract and fecal flora in case of vaginal delivery while environmental bacteria also play a significant part in the colonization of babies delivered by caesarean section (Bezirtzoglou, 1997). Several studies have examined the bacterial flora of

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both breast-fed and bottle-fed infants using conventional plating and/or molecular techniques. Most of these works report a gradual colonization process of infants' gastrointestinal tract beginning with aerobic strains belonging to enterobacteria, streptococci and staphylococci. Maternal vaginal lactobacilli can also transiently colonize the infant but are subsequently replaced by lactobacilli from other sources. Once these first colonizers are implanted, they are thought to allow for a decrease in the redox potential of the intestine and then favor the implantation of anaerobic bacteria such as bifidobacteria. The latter bacteria usually become predominant within a week in breast-fed babies while dominance is reached later in bottle-fed infants (Rotimi et al., 1981 ; Yoshioka et al., 1983). Additionnally, using cultivation, formula-fed infants have been reported as displaying a more diverse and adult-like flora, (Bullen & Tearle, 1976; Benno et al., 1984; Balmer et al., 1989; Chierici et al., 1997). Towards the end of the 1990s, a debate on the accuracy of cultural approaches for the analysis of the intestinal microflora arose. At the same time, infant formulas were modified to try and improve the implantation of a gut flora similar to the one harbored by breast-fed babies. Subsequent works confirmed bifidobacteria as predominant inhabitants of both breast-fed and bottle-fed infants' gut. Additionally, in infants, up to 80% of intestinal bifidobacteria identified by molecular biology techniques could also be retrieved by culture of the same fecal samples (Saunier et al., 2003). Thus, culture-dependent techniques are still reliable as far as the exploration of the neonates' intestinal microbiota is concerned. The current state-of-the-art is that bifidobacteria are the major component of the infant flora. They represent 60 to 91% of the total flora in breast-fed infants vs. 28 to 75% in formula-fed infants between 12 and 20 days of life (Harmsen et al., 2000). In the Koala birth cohort study of the intestinal microbiota (in The Netherlands), the carriage prevalence (98.6%) as well as the counts in bifidobacteria were the highest at one month of age. Escherichia coli (87.7%), Bacteroides fragilis group (81.6%), lactobacilli (32.4%) and Clostridium difficile (25.0%) came next in terms of carriage prevalence (Penders et al., 2005; Penders et al., 2006). At one month of age, cesarean section still affected the bifidobacterial colonization while bottle-feeding only retained minor effect on bifidobacteria, possibly because of the wide use of prebiotic supplemented infant formulas in The Netherlands. However, we also demonstrated by using both cultural and molecular detection of human species belonging to the genus Bifidobacterium, that bifidobacteria were predominant in the gut of healthy bottle-fed neonates from 1 to 4 mo. of age (Mullié et al., 2006). An additional environmental factor that might impact the intestinal colonization with bifidobacteria is the presence of older siblings, as recently demonstrated by preliminary results obtained from our latest clinical study on the prevalence of bifidobacteria held on 30 breast-fed infants aged 2 to 5 mo. (Fig.1). However, this « sibling » effect did not last over time (Fig. 1).

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Figure 1. Intestinal colonisation with bifidobacteria in breast-fed infants from 2 to 5 months of age: influence of siblings (V1: 2 mo., V2: 3 mo., V3: 5 mo.) *: p<0.05 At the species level, Sakata et al. (2005), using cultural and terminal restriction fragment length polymorphism techniques, did not witness any difference in bifidobacterial species establishment between formula-fed, mixed-fed and breast-fed babies. However, along time, Bifidobacterium adolescentis counts were shown to decrease in breast-fed babies and in infants fed a prebiotic-supplemented formula while they were maintained in infants fed a standard formula (Haarman & Knol, 2005). Considering that species of the genus Bifidobacterium do not harbor the same enzymatic and antigenic potentials, it would therefore be of interest to further document their establishment in babies. If the carriage pevalence of bifidobacteria in bottle-fed infants nowadays tends to resemble the one of breast-fed neonates in countries using prebiotic-supplemented formulas, several differences in the microflora still remain. In the Koala birth cohort study, intestinal colonization with C. difficile, E. coli, B. fragilis group and lactobacilli was less frequent and their intestinal counts lower in breast-fed babies (Penders et al., 2005; Penders et al., 2006). As for intestinal colonization with enterobacteria, our latest preliminary results on 22 breastfed babies showed a constant overall level of colonization from 2 to 5 mo. of age: from 2.54 % of the total cultivable fecal flora at mo. 2 to 3.86 % at mo. 5 (median values, difference not statistically significant). However, at the species level, Escherichia coli carriage and colonization level significantly increased over time: - from 50% of carriers at mo. 2 to 82% at mo. 5 (p=0.048, Fisher’s exact test) ; - from 0.05% of the total cultivable flora at mo. 2 to 2.52% at mo. 5 (median values, p=0.034). As opposed to intestinal colonization with bifidobacteria, no significant influence of siblings on the intestinal colonization with enterobacteria was demonstrated in breast-fed infants.

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Other differences in the intestinal microflora balance were reported according to the feeding. Minor components of the fecal samples from breast-fed infants were mainly identified as lactobacilli and streptococci while samples from formula-fed infants contained more Bacteroides and clostridia. Altogether, enterococci, staphylococci, Bacteroides, and Veillonella could be isolated from both groups. But lactic acid bacteria were mainly isolated in breast-fed infants (Harmsen et al., 2000). Backing this observation, a higher carriage frequency was reported for total lactobacilli (p<0.01) and for L. rhamnosus (p<0.05) in breast-fed infants as compared to infants weaned at 6 mo. old (Ahrné et al., 2005). Higher fecal Bifidobacterium counts in breast-fed infants (exclusive breastfeeding for at least 3 mo.) were also recently reported by Rinne et al. (2005) as compared to formula-fed infants at 6 mo. (1.8×109 bacterial cells/g feces vs. 4.4×108, p<0.0004). Higher Lactobacillus/Enterococcus counts (2.9×108 vs. 1.8×108, p=0.01) were also described though differentiation between enterococci and lactobacilli was not undertaken in this clinical trial. These differences were no longer observed at 12 mo. of age. Apart from breast-fed infants, our latest work on the intestinal flora of neonates also included two additional groups randomly assigned at weaning (between age 2 and 3 mo., usually) to either a standard infant formula or Bifidobacterium breve C50 (BbC50) fermented formula (21 and 23 neonates, respectively). When a standard formula was instated at weaning, a significant increase in enterobacteria was noticed at month 5 (p=0.019) while bifidobacteria significantly decreased (p=0.006). Weaning and a standard infant formula thus modified the microbial balance but not straight away as counts in bifidobacteria and enterobacteria were not significantly different at month 3, as compared to month 2. Hence, differences in the dominant intestinal microflora isolated from breast-fed and bottle-fed infants tend to decrease with age, in terms of genera. However, further documentation is needed for both species ditribution in these dominant genera and the subdominant intestinal microbiota, as these species might be of importance in the regulation/maturation of the neonate's immune system or as a potential source of harmful bacteria (as for C. difficile and Clostridium perfringens, for example). 1.2.2. In Preterm and/or very Low Birth Weight (VLBW) Infants Studies on the implantation and development of preterm babies are scarcer than those held on term infants. Nevertheless, a colonization pattern distinct of that of term infants has been reported in several studies. Reports usually put forward a delayed colonization of the pre-term intestine by bifidobacteria (Westerbeek et al., 2006). Intestinal colonization with detectable levels of cultivable bifidobacteria usually takes up to one month. Coagulase negative staphylococci, enterococci and enterobacteria such as Klebsiella sp. and Enterobacter sp. are observed as the main components of the pre-term intestinal microflora (Gewolb et al., 1999; Schwierz et al., 2003). The use of antibiotics, the lack of full breastfeeding, the need for artificial respiration and other invasive interventions might account for such a situation. Indeed, Gewolb et al. (1999) report that pre-term infants fed human milk display a significantly more diverse intesinal flora at 20 and 30 days old than do formula-fed infants. Pre-term babies would therefore even more benefit from nutritional attempts to speed up and increase their intestinal colonization with bifidobacteria.

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1.3. On the Maturation of the Intestinal Immune System The germ-free status of the intrauterine environment favors humoral immunity through T-helper 2 (Th2) type cytokine response over cellular immunity driven by T-helper 1 (Th1) response (Cumming and Thompson, 1997). During early infancy, the immature gut associated lymphoid tissue (GALT), is gradually confronted to increasing amounts of dietary and microbial antigens. These early contacts seem to be decisive for the later development of an adequate and appropriate immune response. The ability to discriminate between harmless and harmful antigens, termed tolerance, is believed to occur primarily in the gut and is facilitated by specialized B and T cells, production of sIgA, recognition of the commensal microflora by Toll-like receptors (TLR) and skewed Th2 response. Hence, failure to regulate tolerance and active immune responses could contribute to food-related allergy, autoimmunity, and inflammatory bowel disorders. At birth, the intestinal immune system is immature with very few IgA-producing B cells present in peripheral blood of newborns. Consequently, the presence of IgA is low and IgM is the major initial antibody type. After 1–2 months, IgA becomes the dominant antibody in the intestine and reaches adult levels at the end of the first year of life (Ouwehand et al., 2002). However, exclusive breast-feeding for 3 months had no significant effect on the total number of IgM-, IgA-secreting cells at 3 months of age whereas the number of IgG-secreting cells was increased (Rinne et al., 2005). Nevertheless, breast milk possesses other putative ways to optimize maturation of the immune system. One example of such mechanims is the presentation of already processed dietary antigens by the mother's gut mucosa (Strobel, 2001). Human milk also contains a wide variety of specific factors such as cytokines like transforming growth factor-β (TGF-β, the expression of which directly correlates with the mucosal IgA antibody response in infants) and the innate soluble microbe receptor sCD14 (Oddy et al., 1999 ; Rinne et al., 2005). Cytokines, especially interleukins (IL), are poorly expressed by the neonate. Breast-milk has been shown to contain both T helper1 (Th1), T helper 2 (Th2) and T helper 3 (Th3) cytokines (Hawkes et al., 2002). Th1 pathway leads to cellular immunity and involves cytokines such as IL-2, IL-12, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). IL-4, IL-5, IL-6 and IL-13 are implied in the Th2 pathway, generating humoral immunity through antibody secretion with IgE responses, in particular. Also termed Tregulatory1 (Treg1) pathway, Th3 response leads to oral tolerance and antiinflammatory regulation, involving IL-10 and TGF-β. This considerable intake of cytokines, likely produced in the mammary gland, has the potential to positively influence the development and proliferation of many immunoactive cell types belonging to the infant immune system. Among these cytokines, high levels of IL-2, a potent T lymphocyte regulator, can also be found (Bryan et al., 2006). In this study, milk IL-2 levels aditionnally correlated with those detected in paired serum samples. Moreover, immune cells harvested from milk samples were shown to be able to produce IL-2, especially when stimulated by concanavaline A (Bryan et al., 2006). Therefore, through providing additional IL-2 to the neonate, breast-milk could potentially aid the maturation of the immune system. Immune cells contained in human milk are composed of macrophages (55-60%), neutrophils (30-40%) and lymphocytes (5-10%). These latter are mainly activated T lymphocytes that compensate for the immature function of neonatal T cells and promote their

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maturation, with an influence not limited to the intestinal immune system thanks to their potential passage through the neonate's intestinal mucosa (Field, 2005). Modulation of the immune system by breast-milk can also be achieved through its content in various growth factors and hormones such as cortisol, epidermal growth factor, insulin-like growth factor and many others. Nucleotides and long chain polyinsaturated fatty acids (conjugated linoleic acid, arachidonic acid and docohexaenoic acid, mainly) are thought to be implicated in the immune system development and maturation (Field, 2005). Of more interest for this chapter, indirect stimulation of the GALT is also occurring via the specific intestinal microbiota developped by breast-fed babies, especially through HMO promoting the growth of bifidobacteria in infants' intestine (Beerens et al., 1980). Several mechanisms have been put forward to explain the stimulative effect of bacteria, especially bifidobacteria, on the immune system (MacPherson et al., 2004; Forchielli and Walker, 2005; Kelly and Conway, 2005): i. a stimulation of secretory IgA (sIgA) production and secretion; ii. an induction of T cell activation; iii. the regulation of Toll-like receptor (TLR) expression (TLR2 and TLR4, more specifically); iv. regulation of cytokine production; v. the production of a balanced T helper response (Th1=Th2=Th3) or the prevention of an imbalanced T helper response (Th1>Th2 or Th1
1.4. Limits to Human Milk Bifidogenic Effect? Though the mode of nutrition is of utmost importance, it is not the only factor influencing the implantation of the intestinal microbiota. Various factors such as gestational age, mode of delivery, local environment and antibiotic treatments have also been demonstrated as playing an important part in the development of the neonatal intestinal flora (Fanaro et al., 2003). Infant populations from various geographical origins have been shown to display heterogenous intestinal colonization with bifidobacteria, although breast-fed over several months (Sepp et al., 1997).

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Cesarean section has long been shown has favoring the implantation of environmental strains in the infant's gut (Bezirtzoglou & Romond, 1990 ; Bezirtzoglou, 1997). In a recent work, in comparison with vaginal delivery at home, cesarean section resulted in lower colonization rates and counts of bifidobacteria and B. fragilis-group species, whereas prevalence and counts of C. difficile and counts of E. coli were higher (Penders et al., 2006). Oral use of antibiotics (mainly amoxicillin) by the infant during the first month of life also resulted in decreased numbers of bifidobacteria and B. fragilis-group species (Penders et al., 2006).

2. Putative Beneficial Effects of Intestinal Bifidobacteria The first species of Bifidobacterium was isolated by Tissier (1900) who named it after its peculiar shape: Bacillus bifidus. In 1920, this strain was renamed Lactobacillus bifidus (Holland, 1920) and it was not until 1965 that bifidobacteria were taxonomicaly separated from lactobacilli (Sebald, 1965). Because a largely predominant bifidobacterial flora was observed in breast-fed infants, who besides show a greater resistance to infectious diseases than do bottle-fed infants, Tissier suggested that the "bifid" bacteria could be administered to patients with diarrhoea to help restore a healthy gut flora. Meanwhile, Elie Metchnikoff (1908) observed that: "The dependence of intestinal microbes on food makes it possible to adopt measures to modify the flora in our bodies and to replace harmful microbes by useful microbes". The idea of generating a predominant bifidobacterial flora through the consumption of live bifidobacteria then fell out of fashion until a renewal of interest for these bacteria occurred in the seventies, gradually made its way and reached its highest in the nineties of the former century. The inclusion of probiotic bifidobacteria in feeding formulas was then attempted. However, whether bifidobacteria actively protect or only bear witness to the infant’s good health is still a matter of debate. Numerous studies and trials have demonstrated the activity of probiotic bifidobacteria. Nevertheless, the mechanisms involved have still not been totally elucidated. Here, we will first review the putative beneficial effects of indigenous bifidobacteria on infants' health before assessing the probiotic and prebiotic approaches to reproduce human milk bifidogenic effects.

2.1. Prevention and/or Treatment of Gastrointestinal Disorders Mechanisms underlying the protective effect induce by the presence/absence of intestinal bifidobacteria such as barrier effect, regulation of bacterial translocation (BT) or adjuvant effect can be better understood using results obtained on gnotobiotic animal models (Mullié et al., 2005 ; Romond et al., 2007). We will mainly focus here on clinical evidence.

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2.1.1. Viral Acute Gastroenteritis Gastroenteritis of viral origin, especially rotavirus infection, is the main cause of acute diarrheas in infants and children all over the world. In developing countries, it is probably one of the main factors of infant mortality. Firstly, osmotic diarrhea occurs followed by a second phase corresponding to active viral replication. Oral rehydration solutions prevent dehydration but do not shorten the duration of diarrhea. Breast-feeding has long been associated with a decreased incidence of gastrointestinal infections (Bullen & Willis, 1971; Howie et al., 1990), possibly because of its bifidogenic effect creating an acidic intestinal environment inhospitable to infectious organisms (Bullen & Willis, 1971; Duffy et al., 1986). The protective effect of probiotic bifidobacteria has been shown by Saavedra et al. (1994) in a double-blind placebo-controlled trial. Feeding of Bifidobacterium bifidum associated with Streptococcus thermophilus in a standard milk formula significantly reduced the incidence of acute diarrhea and the rotavirus shedding in hospitalised infants. Diarrhea occurred in 7% of the infants receiving the probiotic and in 31% of the control subjects (p=0.035) and shedding rotavirus occurred in 10% of children versus 39% respectively (p=0.025). In another work, a formula supplemented with Bifidobacterium lactis Bb12 (106 cfu/g of powder) given to 3-8 mo. old infants did not significantly reduce the incidence of diarrhea compared to a control formula (28.3 vs. 38.7%, respectively) but the mean number of days with diarrhea per infant was reduced in the supplemented group (1.15 ± 2.5 vs. 2.3 ± 4.5 days in the control group, p=0.0002) (Chouraqui et al., 2004). Thus, bifidobacteria may provide some of the protective or alleviating effects of breastfeeding against acute gastroenteritis. 2.1.2. Post-Antibiotic Diarrhea Antibiotic administration is frequent in infants, especially in preterms. Antibiotherapy is known to induce a disruption of the intestinal microflora balance in both infants and adults (Sullivan et al., 2001; Fanaro et al., 2003). Lately, a probiotic formula containing 107 colony forming unit (CFU) Bifidobacterium lactis and 106 CFU Streptococcus thermophilus was shown to reduce the frequency of postantibiotic diarrhea in infants (Correa et al., 2005). Infants were aged 6 to 36 months and received either this or an unsupplemented commercial infant from the onset of antibiotherapy and for a duration of 15 days. Infants were clinically followed up for 15 additional days. A reduction in antibiotic-associated diarrhea (AAD) was witnessed in the probiotic group with a 16% incidence as compared with a 31% incidence in the non-supplemented group. 2.1.3. Other Gastrointestinal Disorders An infant formula supplemented with either a dose of 1.106 or 1.107 CFU/g Bifidobacterium lactis and Streptococcus thermophilus was recently assessed for tolerance in infants aged 3 to 24 months at enrollment (Saavedra et al., 2004). In addition to normal growth rates, this clinical trial reported a lower frequency of colic or irritability (p≈0.001) and a lower frequency of antibiotic use (p≈0.001) in infants receiving the supplemented formula as compared with infants fed the unsupplemented formula given as control.

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Infant formulas supplemented with a 1.107 CFU/g dose of either Bifidobacterium lactis Bb12 or Lactobacillus reuteri ATCC 55730 were tested for 12 weeks on 1 to 4 mo. old healthy infants (Weizman et al., 2005). Control babies had significantly more febrile episodes compared with those fed B. lactis or L. reuteri. Controls also had more diarrhea episodes and episodes of longer duration. Bifidobacterium longum BL999 was also lately assessed at the dose of 2.107 CFU/g along with 4 g of a prebiotic mixture (GOS/FOS ratio: 9/1) (Puccio et al., 2007). Infants were aged less than 14 days at the study onset and followed until their 112th day. A higher stool frequency was recorded in the experimental group as compared to control (2.2 ± 0.7 vs. 1.8 ± 0.9, respectively, p=0.018) along with a lower risk of constipation (p=0.03).

2.2. Immunomodulation and Prevention of Allergic Conditions (Food Allergy, Atopic Dermatitis, Celiac Disease) Human beings are exposed to numerous food antigens. The intestinal mucosa is efficient in assimilating antigens encountered along the enteric route. But high-level antigen exposure during the first few months of life may predispose individuals to allergic sensitisation. The immature gut barrier may lead to aberrant antigen transfer and immune responses and so explain vulnerability to oral tolerance breakdown at an early age. Enteric antigens are frequently derived from food and allergic reactions to foods are common. Gastrointestinal microflora promotes potentially anti-allergenic processes (i) T-helper 1 type immunity, (ii) generation of TGFβ which has an essential role in suppressing Th2induced allergic inflammation and inducing oral tolerance, and (iii) IgA production. Kalliomäki et al. (2001) showed the essential role of endogenous microflora by demonstrating that a reduced ratio of bifidobacteria to clostridia in early gut microflora precedes the development of atopy and atopic disease. He et al. (2001) investigated the differences between Bifidobacterium strains in the faeces of allergic and healthy infants. Healthy infants had typical infant bifidobacterial flora (B. bifidum, B. breve and B. infantis). Allergic infants were mainly colonised with B. adolescentis. Bifidobacteria from allergic patients were also found to adhere less to the human mucus than strains from the healthy infants. However, these results remain controversial. Indeed, a recent study by Adlerberth et al. (2007), held on infants born in Italy, Great Britain and Sweden, showed no significant association between the composition of the intestinal microflora and the development of food allergy and atopic dermatitis. Several recent studies report on differences in intestinal gut microbiota of infants with and without eczema (Murray et al., 2005; Mah et al., 2006; Adlerberth et al., 2007). While Adleberth et al. (2007) did not witness any difference in the fecal microbiota of infants with or without eczema; Murray et al. (2005) showed that fecal counts in bifidobacteria were lower in infants suffering from eczema. Both Bifidobacterium and Clostridium counts were also found to be lower in toddlers with eczema as compared to controls (p=0.003 and p=0.012, respectively) while lactic acid bacteria and enterococci were more numerous in toddlers suffering from eczema (Mah et al., 2006).

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However, not all Bifidobacterium species seem to be beneficial from an immunomodulatory point of view. Just like Bifidobacterium adolescentis was found more frequently in the microflora of allergic infants, the fecal carriage of Bifidobacterium pseudocatenulatum in 3 to 6 months-old infants has lately been associated with exclusive formula-feeding and an increased risk of developing atopic eczema (carriage prevalence : 26% vs. 4% in infants with and without eczema, respectively ; p=0.04) (Gore et al., 2007). In the same study, the carriage of B. bifidum was associated with breast-feeding (p=0.01). Additionnaly, recent data reported a higher prevalence of B. catenulatum in biopsies obtained from controls than in biopsies from patients suffering from either active or non-active celiac disease. B. dentium prevalence was higher in feces of non-active celiac disease patients than in controls. On another hand, the numbers of total bifidobacteria and B. longum species were lower in both patients with active and non-active celiac disease, as compared to controls (Collado et al., 2008). These results point out the importance of not only trying to speed up and increase the implantation of bifidobacteria in the intestine of bottle-fed neonates but also of favoring the so-called “infant” Bifidobacterium species such as B. bifidum, B. breve and B.longum-B.infantis. These species appear to be linked with a health-positive maturation and regulation while species more frequently found in adults (B. adolescentis, B. catenulatum-B. pseudocatenulatum, B. dentium) tend to be reported as linked with deleterious health conditions. Hence, studies focusing on increasing bifidobacteria in infants should not only focus on the overall bifidobacterial carriage prevalence and counts but also on identifying the species promoted, as experienced in one of our previous work (Mullié et al., 2004). Although human milk complex and dynamic composition cannot be mimicked by industrial products, manufacturers of infant formulas are attempting to reproduce its effects on the intestinal microbiota and immune system through adding components such as pro- and prebiotics to their products.

3. Probiotics to Reproduce Human Milk Effects Probiotics were originally defined as ‘live microbial food supplements which beneficially affect the host animal by improving its intestinal microbial balance’ (Fuller, 1989).

3.1. On the Intestinal Microbiota Balance 3.1.1. Pre-Term Infants Intestinal colonization of VLBW infants by Bifidobacterium breve YIT 4010 was observed following a daily oral inake of 0.5×109 cfu for 28 days by Kitajima et al. (1997). Colonization occurred within two weeks and lasted well after the oral administration was discontinued as most infants still exhibited high detectable levels of B. breve YIT 4010 at the end of the 8-week follow-up. Colonized infants also had a significantly higher weight gain between 4 and 8 weeks of life. Mohan et al. (2006) showed that the consumption of a Bifidobacterium lactis Bb12 supplemented infant formula (2.109 cells/g powder) for 21 days induced a rise in

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bifidobacterial counts in preterm infants (p=0.001). This rise was witnessed through culturedependent and -independent techniques. A simultaneous decrease in Enterobacteriaceae viable counts was also reported (p=0.015). 3.1.2. Term Infants Langhendries et al. (1995) fed an acidified infant formula containing 106 CFU live bifidobacteria/g of powder to healthy full-term newborns for the two first months of life. This resulted in a similar and significantly higher intestinal colonization frequency with bifidobacteria for the breast-fed and Bifidobacterium formula supplemented groups as compared to infants receiving the control formula. However, in infants colonized with bifidobacteria, counts were similar whatever the group. Remarkably, this paper was the only clinical trial reporting on the effect of a Bifidobacterium- enriched formula on the intestinal flora of term infants until recently. In a work by Bakker-Zierikzee et al. (2005) studying the effects of both pre- and probiotics, infants received a formula supplemented with 6.1010 CFU B. animalis (Bb12)/L vs. the same standard non-supplemented formula and a breast-fed group. Fecal samples were retrieved on day 5, 10, 28, 60, 120 and 160. Using Fluorescent in situ hybridization (FISH) enumeration technique, no significant difference was witnessed in the percentage on bifidobacteria in the total flora throughout the 160 days in this trial. The authors account for these discrepancies by stating that Langhendries et al. (1995) did not give any quantitative data on the stimulation of bifidobacteria but differences in enumeration techniques can also be put forward.

3.2. On the Maturation of the Intestinal Immune System and Prevention of Allergic Diseases Majamaa and Isolauri (1997) suggest that probiotic bacteria such as bifidobacteria may promote endogenous barrier mechanisms in the patients with atopic dermatitis and food allergy. The exact mechanisms by which probiotics may affect atopic disease remain speculative. However, there is increasing evidence that specific input from the faecal flora to the innate immune system is essential for the establishment and maintenance of oral tolerance. Isolauri et al. (1993) showed that probiotic use in primary prevention of atopic disease was based on its ability to reverse increased intestinal permeability. Adhesion of probiotics to the intestinal mucosa is also considered important to modulate the immune system (Schiffrin et al., 1997). Hattori et al. (2003) administered lyophilised bifidobacteria (B. breve M-16V) to children with atopic dermatitis who had a Bifidobacterium-deficient microflora. Changes in the fecal microflora were observed after one month (increase in the proportions of bifidobacteria and decrease in aerobic bacteria within the total flora) as well as a significant improvement in allergic symptoms (cutaneous symptom score and total allergic score). However, no correlation could be demonstrated between changes in fecal microflora and improvements in allergic symptoms. Oral introduction of probiotics could also help in the treatment of food allergies by alleviating intestinal inflammation as intestinal microorganisms have been suggested to

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down-regulate allergic inflammation by counter-balancing T-helper cell type 2 responses (Kirjavainen et al., 1999). Recently, mucosal immunologic maturation was assessed in term infants at 3, 7 and 12 months in artificially fed infants before 2 mo. of age (Rautava et al., 2006). They received an infant formula supplemented with 1.1010 cfu L. rhamnosus GG and 1.1010 Bifidobacterium lactis Bb12 daily until 12 mo. of age. Determination of the number of IgA secreting cells (total IgA and cow's milk-specific IgA : casein and β-lactoglobulin), seric TGF-β and sCD14 levels. No significant difference between total IgA-secreting cell numbers or TGF-β levels. Higher number of cow's milk specific IgA secreting cells in the probiotic group at 7 mo. of age (p=0.043) and higher levels of seric sCD14 (p=0.046) at 12 mo. of age. Lately, an Australian paper reporting on a 6 month-feeding of Lactobacillus acidophilus to allergy-prone infants did not reduce the risk of atopic dermatitis but moreover tended to increase the risk of allergen sensitization in such a predisposed population (Taylor et al., 2007).

3.3. Safety Considerations and Conclusions The latter statement, though made for a probiotic Lactobacillus strain, along with consideration on beneficial and deleterious Bifidobacterium strain, shows a careful clinical evaluation of each probiotic strain and its effects is needed before being widely administered to infants through a commercial product. Apart from this, most clinical studies aim at evaluating the tolerance and safety of probiotic supplemented formula with mainly growth criteria. Infectious adverse effects of live bifidobacteria such as bacteriemia or meningitis have however also been reported in high-risk populations (Sussman et al., 1986 ; Hata at al., 1988 ; Gasser, 1994 ; Nakazawa et al., 1996) but prospective controlled studies on the effects of feeding live active bacteria in infants for any extended period of time are scarce. Saavedra et al. (2004) reported on such a study held on 3 mo. old infants at entry and fed Bifidobacterium lactis Bb12 at a dose of either 106 or 107 cfu/g formula for as long as infants had a minimum daily intake of 240 mL of the supplemented formula (resulting in a mean participation length of about 7 months). Supplemented infants showed similar growth rates as controls and no peculiar adverse effects in supplemented groups, whatever the probiotic dose. Weizman and Alsheikh (2006) also recently reported on the safety and tolerance of a formula supplemented with B. lactis Bb12. Infants were less than 4 months old at entry and received the formula for 4 weeks only and reached similar conclusions. In the global threat of antibiotic-resistance, transfer of resistance from a probiotic strain to indigenous intestinal strain may raise concern. Bifidobacteria appear as relatively safe in this regard as they display low natural and acquired resistances to antibiotics (Moubareck et al., 2005). Nevertheless, B. lactis Bb12 was found to be resistant to vancomycin (Mohan et al., 2006) and could therefore be a potential source of vancomycin-resistance spread. Recently, recommandations from the EU-Prosafe project further emphasised the need for control of antibiotic-resistance in probiotic strains (Vankerckhoven et al., 2007).

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3.3.1. Pre-Term Infants Special care must be taken when preterm babies are considered. In this peculiar population, an increased permeability of the intestinal epithelial lining may lead to bacterial translocation and, subsequently, to sepsis. Therefore, the administration of high doses of live bacteria has been questioned in this population as it might trigger unexpected infectious/inflammatory conditions.

4. Prebiotics to Reproduce Human Milk Effects Prebiotics are defined as “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improve host health” (Gibson & Roberfroid, 1995). Most prebiotics used as food ingredients belong to fructooligosaccharides (FOS) and galactooligosaccharides (GOS). Breast-milk is a complex mixture of over 100 different branched oligosaccharides, monosaccharides and various sugar derivatives (Coppa et al., 1999). These oligosaccharides mainly consist of a lactose core substituted with N-acetyl glucosamine, galactose, fucose and sialic acid (Kunz & Rudolff, 1993 ; Kunz et al., 2000) and are poorly digested in the upper gastrointestinal tract (Engfer et al., 2000). One of the major explanation for Bifidobacterium dominance in breast-fed infants is that bifidobacteria are uniquely adapted to the use of these human milk oligosaccharides (HMO) (Schell et al., 2002). HMO therefore could act as a model of natural prebiotics (Edwards et al., 2002) and to adapt infant formulas, supplementation with these undigestible carbohydrates has been attempted.

4.1. On the Intestinal Microflora Balance Surprinsingly, there is substantially more literature on the effect of prebiotics than on the effect of probiotics on the infant intestinal microbiota composition. 4.1.1. Pre-Term Infants Boehm et al. (2002) assessed the effect of a formula enriched with 10g/L of a GOS/FOS mixture (9:1 ratio) on the intestinal microbiota of preterm (gestational age < 32 weeks) infants using cultural techniques. The GOS/FOS ratio was chosen to mimic the molecular size distribution of HMO. This supplemented formula was compared to the same formula without oligosaccharides (replaced by a similar amount of maltodextrines) and human milk effects. Samples were retrieved at 7, 14 and 28 days of formula feeding. Bifidobacteria were readily detected in the 7th day sample of all infants. Counts in bifidobacteria increased over time in all three groups (mother's milk, unsupplemented formula and supplemented formula). After the 28 day feeding period, counts in bifidobacteria were significantly higher in the supplemented group as compared to the standard formula (p=0.0008) and were similar to

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those witnessed in babies fed human milk. Lactobacilli also had increasing counts over time but no significant difference was observed at any time, whatever the feeding mode. More recently, another clinical trial assessed the sole FOS as oligosaccharide supplement in preterms (Kapiki et al., 2007). Babies with a maximum gestational age of 36 weeks were included and fed a formula supplemented with FOS (i.e. inulin) at 4g/L for 14 days. Stool samples were retrieved on the first and th seventh day. Rise in counts and colonization prevalence of bifidobacteria (p= 0.032 and p=0.030, respectively). Rise in Bacteroides counts (p=0.029) was also witnessed along with a decrease in counts of Escherichia coli and enterococci. (p =0.029 and p =0.025, respectively). 4.1.2. Term Infants Moro et al. (2002) tested two supplemented formulas at 4 or 8 g/L of the same GOS/FOS mixture (9:1 ratio) as the one studied by Boehm et al. (2002) on preterms. Higher counts in bifidobacteria and lactobacilli were observed after a 28-day feeding period with both supplemented formulas as compared to control (p<0.001). A dose-dependent effect was also reported, but for bifidobacteria only, as the group receiving 8g/L oligosaccharides had significantly more fecal bifidobacteria than the 4g/L one (p<0,01). No significant effect was witnessed on Bacteroides, Clostridium species, E. coli, Enterobacter, Citrobacter, Proteus, Klebsiella, and Candida. In a recent double-blind, prospective, randomized clinical trial, Costalos et al. (2007) report on the intestinal colonization of term infants by bifidobacteria and clostridia receiving a formula enriched with 4g/L mixture of GOS and long chain FOS. Babies aged less than 14 days were enrolled. Stool sampling took place at entry and 6 weeks later and bifidobacteria were enumerated by FISH (Fluorescent in situ hybridization). Counts in E. coli, bifidobacteria and clostridia were similar with and without prebiotic supplementation at 6 weeks. Bakker- Zierikzee et al. (2005) tested for 16 weeks a formula enriched with 6 g/L of a GOS/FOS (9:1) blend on healthy full-term infants. These authors also used FISH enumeration of bifidobacteria. No significant difference in bifidobacterial counts was observed, whatever the sampling time and the feeding group. Using quantitative real-time PCR, Haarman & Knol (2005) showed an increase of the bifidobacterial population (54.8 ± 9.8% to 73.4 ± 4.0% of the total flora, p=0.047) in healthy full-term infants receiving for weeks a 8.0 g/L FOS/GOS supplemented formula. A rise in Lactobacillus counts was also detected following supplementation (0.8 ± 0.3% versus 4.4 ± 1.4%, p=0.019) (Haarman & Knol, 2006). Additionally, Bifidobacterium species distribution in supplemented infants was more similar to that of breast-fed infants while nonsupplemented infants mainly harbored B. adolescentis and B. catenulatum group species. As mentioned earlier in this paper, these species are considered as belonging to a more adult-like microflora and as putatively pejorative for health (found in allergic-prone infants) (Haarman & Knol, 2005). In a similar trial using molecular detection technique, Penders et al. (2006) showed that infants fed exclusively with a formula supplemented with a mixture of GOS/FOS had higher counts of bifidobacteria and lactobacilli in their stools, compared with infants fed an unsupplemented formula. Molecular techniques are therefore backing up results using conventional cultural techniques.

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Euler et al. (2005) report on a clinical trial using only FOS as supplement at 1.5 or 3g/L on 2 to 6 weeks-old vaginally delivered term infants. Counts in clostridia and bifidobacteria were increased on the seventh day (p=0.0356 and p=0.045, respectively) only in infants receiving a 1.5g/L FOS supplementation. This increase did not last as counts in both bacterial groups were not statistically different between groups 7 days after FOS supplementation has ceased. When compared to human milk-fed controls, infants fed FOS supplemented formula also had higher Bacteroides and Enterococcus counts. Acidic oligosaccharides (2 g/L) have also been used to try and modify the neonate intestinal microflora in addition or not to a mixture of FOS/GOS at 6g/L (Fanaro et al., 2005). Only the combination of oligosaccharides increased the fecal counts in bifidobacteria and lactobacilli (p<0.01). No modification of the other components of the intestinal microflora was reported. The authors reach the conclusion that acid oligosaccharides alone cannot benefically modify the intestinal microflora, maybe because of the low dosage used to supplement the standard formula. As can be seen, over the last decade, there has been a florishing literature addressing the effects of prebiotics on the intestinal microflora composition. However, general conclusions are difficult to draw. Firstly because various oligosaccharides have been used though most trials have been held with FOS and GOS. Secondly because variations in feeding periods (7 days to 6 weeks), doses (1.5 to 10.0 g/L) and dose-effect responses are reported. Most of the times, these supplemented formula are proved as bifidogenic but the effect on lactobacilli is sometimes more contrasted. While most works report on an overwise unchanged microflora, some studies report acknowledge a similar rise in Bacteroides counts while opposite results are obtained with enterococci (Euler et al., 2005 ; Kapiki et al., 2007).

4.2. On the Maturation of the Intestinal Immune System and Prevention of Allergic Diseases Results of studies reporting on these issues are scarcer. The direct effect of prebiotics on the immune status was only found two papers assessing clinical symptoms described as atopic dermatitis (AD) and eczema. The immune mechanisms underlying was only investigated in a couple of studies. Roller et al. (2004) reported an up-regulation of IL-10 and IFN γ production along with a regulation of the cecal IgA response in rats following a 4-week ingestion of inulin enriched oligofructose. However, these results were hardly confirmed in human adults (Roller et al., 2007). Moro et al. (2006) studied the incidence of AD in 259 allergy-prone infants over the first 6 months of life. Term infants received either a hydrolysed protein formula supplemented with 8g/L of a GOS/FOS mixture (9:1) or with 8 g/L maltodextrines (placebo) for 6 months. Stool microflora was assessed in a subgroup of 98 infants. AD was diagnosed in the first 6 months of life in 9.8% of infants in the prebiotic group and in 23.1% of infants in the placebo group (p=0.014). At 6 months, higher counts in bifidobacteria were observed in the prebiotic subgroup of infants whose fecal microflora had been evaluated (10.28 log CFU/g vs. 8.65 log CFU/g, p<0.0001).

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Ziegler et al. (2007) tested another type of oligosaccharide blend (see below). A statistical difference with the control group was detected for eczema with the 4g/L supplemented group showing a higher incidence than in both the control group and the 8 g/L supplemented group (18% vs. 7%, p=0.046 and 18% vs. 4%, p=0.008, respectively). However, the paper does not explain how eczema was evaluated in these children. The first paper hypothesizes on an indirect effect of prebiotics on the development and maturation of the immune system through modifications of the intestinal microflora while the second mostly focus on tolerance. Therefore, up to this day, no direct effect of prebiotics on the immune system has been reported and additional studies could prove useful in unraveling the mechanisms behind the immunomodulating effects of prebiotics.

4.3. Safety Considerations and Conclusions As for probiotics, most studies on prebiotic supplementation of infant formulas focus on their gastrointestinal tolerance and growth rates. Even though several European expert panels acknowledge fructooligosaccharides and galactooligosaccharides to be safe at doses up to 8g/L for infants, in 2004, the European Society for Pediatric Gastroenterology, Hepatology and Nutrition's committee on nutrition concluded that oligosaccharides mixtures in infants formula have not demonstrated adverse effects and that further evaluation was needed before a general recommendation on the use of oligosaccharides supplementation in infancy as a prophylactic or therapeutic treatment could be made (Agostini et al., 2004). However, in a recent assay on infant formulas with 4 and 8 g/L of a prebiotic blend (polydextrose, galactooligosaccharides and lactulose), adverse effects were more frequent in both supplemented groups vs. control. Diarrhea (p=0.008), eczema (p=0.046) and irritability (p=0.027) were statistically more frequent with the supplemented formulas (Ziegler et al. 2007). Although similar growth rates were recorded throughout the 120 days of follow-up, infants receiving the highest prebiotic dose also had the highest dropout rate resulting from feeding intolerance. The potential risk of intolerance to prebiotics, especially with high dose supplementations, must therefore be carefully assessed versus the benefits of prebiotics. Additionally, another issue in fragile infants such as the premature is bacterial translocation (BT). BT might be favored by high doses of unfermented sugars reaching the neonate intestine. This is one of the reason why a new approach to mimic human milk effects on the intestinal microbiota was attempted. This approach relies on milk fermentation products by strain B. breve C50. The final product is deprived of live bifidobacteria. Hence, it cannot be termed a probitioc and does not bear the putative threat of inflammatory/infectious conditions triggered by probiotics in fragile populations.

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5. A New Approach to Mimic Human Milk Effects: Bifidobacterial Products Derived from Milk Fermentation In the late 1980s, protection against AAD was observed in adults consuming milk fermented with bifidobacteria and lactic acid bacteria but not in adults fed milk fermented with the sole lactic acid bacteria (Colombel et al., 1987). The analysis of the intestinal flora showed that protection was associated with a repressed clostridial flora. Thereafter, strains of bifidobacteria and the compounds they generated through milk fermentation were assessed in animal models and humans to see how they reproduced this repressive effect on intestinal clostridia (Romond et al., 1997 ; Mullié et al., 2002). Bifidobacterium breve C50 (BbC50) products exhibited the best repressive effect on clostridia. Hence, a heat-treated infant formula fermented with Bifidobacterium breve C50 (BbC50) and Streptococcus thermophilus 065 was developed and marketed (Danone, France). In this formula, S. thermophilus 065 is mainly playing the part of a fermentation starter and producer of β-galactosidase. The fermentation is started in the presence of 107 bacteria, a population reaching 109 colony-forming units/mL after a 7-hour incubation at 37°C. After milk fermentation by the two strains, the formula is ridden of its live bacteria through heating at 80°C for 15 sec. Therefore, from an infectious point of view, it can be considered as safe to administer, even to preterm infants. The formula is then supplemented with various nutritive elements, homogenized at 250 bars and spray-dried at 200°C. The final nutrient composition of this infant formula is (grams per 100 g): proteins, 12.4; lipids, 21; lactose, 46.6; maltodextrin, 14; vitamins and minerals. We will thereafter review the pieces of evidence backing the beneficial effects on the intestinal flora and immune system of this formula as well as the latest clinical trials on infants held with the commercial product.

5.1. Rationale for the Use of such Products (Mouse Models and In Vitro Assays) In addition to the repressive effect on clostridia, BbC50 products were shown to display an in vivo repressive effect on Bacteroides fragilis group as well as a bifidogenic activity associated with a decrease in fecal pH (Romond et al., 1997). A further trial held on healthy human volunteers showed that a 7-day intake of BbC50 whey reproduced the modifications in the intestinal microflora witnessed in mice. The effects of a 7-day oral intake of BbC50 milk fermentation products on the intestinal microflora and enzymatic activities of healthy adults were further compared to those of BbC50 and Streptococcus thermophilus 065 commercial product (Romond et al., 1998). Significantly lower fecal counts in Bacteroides fragilis, clostridial spores, and Clostridium perfringens were retrieved after consumption of either milk preparation. An increase in fecal bifidobacteria was also witnessed but only in the group receiving BbC50 alone milk fermentation products. Hence, the beneficial effects of these wheys on the intestinal microbiota balance can be put on BbC50 milk fermentation

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products and not on a synergistic effect with S. thermophilus 065 ones. An increase in fecal bifidobacteria was also witnessed but only in the group receiving the sole BbC50 milk fermentation products. The reduction in clostridia and Bacteroides fragilis was therefore not supported by the overgrowth of intestinal bifidobacteria. Further analysis did not provide evidence of an in vitro antibiotic-like effect against Bacteroides fragilis and Clostridium. Investigations carried out on animal models to elucidate the nature of bioactive compounds contained in BbC50 cell-free wheys indicate that lactosidase activity (Mullié et al., 2002) or glycoproteic compounds produced through milk fermentation are the best candidates. Other works report on the immunomodulatory properties of BbC50 milk fermentation products in animal models. A trial reporting on the effects of a 10-week oral treatment with B. breve C50 and S. thermophilus 065 secretion products observed an enhanced Th1 response and intestinal barrier function in an IL-10 deficient mouse model (Ménard et al., 2005). In this study, a higher number of CD4+ and CD8+ lymphocytes were found to produce interferon (IFN)-γ in mesenteric lymph nodes (MLN) when mice had orally ingested the culture medium of BbC50 and S. thermophilus 065, compared to control mice or mice ingesting both live bacteria. Additionally, the secretion of three pro-inflammatory cytokines: IFN-γ, Tumor Necrosis Factor (TNF)-α and IL-12 was found to be significantly higher in mice treated with BbC50 and S. thermophilus 065 culture medium in answer to LPS stimulation. Moreover, the direct oral administration of the commercial product to mice does not alter the development of oral tolerance to ovalbumine (Ménard et al., 2006). Mice were fed this formula for 1 week before and 5 weeks after oral tolerance to ovalbumine induction. Moreover, immunisation of mice with ovalbumine led to higher IgG titers in mice receiving the heat-treated formula (16.45 ± 1.24 and 15.46 ± 0.79, respectively; p=0.012) while antiovalbumine IgE titers did not rise, accounting for a specific stimulation of Th1 but not Th2 response by the formula (Ménard et al., 2006). Another important data provided by the same authors indicate that BbC50 fermentation products can cross an intestinal monolayer of intestinal cells and retain antiinflammatory properties. Therefore, a systemic immunomodulatory effect can be expected for these products (Ménard et al., 2004). Lately, Hoarau et al. (2006) investigated the immunomodulatory properties of BbC50 supernatants on dendritic cells (potent antigen-presenting cells). BbC50 supernatants were shown to increase CD83, CD86, and HLA-DR expression on dendritic cells as compared to control or LPS stimulation. They also prolonged dendritic cell survival with high IL-10 and low IL-12 productions, likely through an up-regulation of Bcl-xL and Phospho-Bad, compared to LPS-stimulated cells. BbC50 supernatants also induced activation of TLR-2 transfected cells.

5.2. Effect of B. Breve C50 Milk Fermentation Products on Acute Diarrhea A study by Thibault et al. (2004) reported on the use of the BbC50 and S. thermophilus 065 infant formula on acute diarrhea. At enrollment, infants were aged 4 to 6 mo. and had

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ceased breast-feeding for at least one month. During 5 months, infants received either a formula fermented with BbC50 and Streptococcus thermophilus 065 or a standard formula with the same basic nutritionnal composition. Both formulas were well accepted and tolerated, allowing for normal growth and good diet compliance. No significant differences were recorded in the incidence, the number or the duration of diarrhea episodes. A lower number of dehydration cases in the fermented formula group (p=0.01) was however reported as well as fewer oral rehydration salts prescriptions and formula switches (p=0.003 and p=0.0001, respectively). This indicates a reduced severity of diarrhea episodes in infants fed the fermented formula.

5.3. Effect of Bifidobacterium Breve C50 Milk Fermentation Products on the Intestinal Flora and Immune System of Healthy Full-Term Infants A global evaluation of the immune response to the BbC50 formula was performed in healthy infants (Indrio et al., 2007). T lymphocyte maturation is known to take place in the neonate's thymus. The thymus has previously been shown to grow from birth till eight months of age. Additionally, breastfed infants have bigger sized thymus than formula-fed ones. In a recent work, BbC50 fermented infant formula was shown to significantly increase thymus size in healthy term infants, compared to a standard infant formula (Indrio et al., 2007). Thymus size was assessed through ultrasound examinations and thymus index calculated in 90 term infants on the third day of life and on the first, second, third and fourth months thereafter. Thirty infants were breast-fed while 30 others received the BbC50 fermented formula and 30 the control formula. Infants receiving BbC50 formula had a significantly higher thymus index as compared to the standard formula (p<0.036) but still remained lower than that in breast-fed infants (p<0.042). In this study, breast-fed and BbC50 fermented infant formula-fed infants also had significantly lower stool pH values than babies fed the standard formula throughout the 4 mo. survey (p<0.001). Therefore, BbC50 fermented formula tends to favor thymus development as compared to a standard formula and hence T lymphocyte maturation in neonates. This immunomodulatory effect could partly be relayed by the intestinal flora. In a double-blind, placebo controlled, randomised clinical trial, BbC50 infant formula was shown to induce higher counts in bifidobacteria within 3 months (Mullié et al., 2004) (Table 1). In this study, thirty (seven boys and 23 girls) full-term infants born between August 1999 and January 2000 at the maternité Pavillon de la Sainte Famille (Clinique du Bois, Lille, France) were enrolled within the first days after birth. As their mothers had previously decided not to breastfeed, newborns received either the placebo or the BbC50 fermented infant formula (FIF) from inclusion until 4 months of age. The study protocol and consent procedures were approved by the local ethics committee (Comité Consultatif des Personnes se prêtant à la Recherche Biomédicale de Lille). Infant formulas were supplied as powder in numbered containers (Blédina SA, Steenvoorde, France). Their basal nutrient compositions were similar (grams per 100 mL: 1.45 g of protein, 8.3 g of carbohydrates, and 3.5 g of fat per 100 mL). According to the French vaccination program, they received an injection of Pentacoq®

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(vaccine against diphtheria and tetanus toxoids, poliomyelitis virus, Haemophilus influenzae, and Bordetella pertussis; Pasteur Mérieux Serums and Vaccines, Lyon, France) at 2, 3, and 4 months. Infants were followed up to the age of 5 months. Stools were collected for IgA quantification at 3 (before the second Pentacoq® injection), 3.5, and 4 months (after the second Pentacoq® injection). Total IgA and anti–poliomyelitis-specific IgA titers were measured by ELISA (Mullié et al., 2004). Fecal samples to enumerate cultivable bifidobacteria, lactobacilli, enterobacteria, clostridia, Bacteroides fragilis group and total cultivable fecal flora were collected at 1, 2, 3, and 4 months, on swabs that allow for the survival of anaerobic bacteria. All microbiological samples were processed within 48 hours from collection. Appropriate dilutions of the fecal samples were seeded onto the following culture media: 1. Horse blood agar (Columbia agar base; Oxoid, Dardilly, France) supplemented with glucose (0.5%) and cysteine · HCl (0.03%) to enumerate eubacteria, cocci, and clostridia; 2. Columbia agar base (Oxoid) supplemented with glucose (0.5%) and cysteine–HCl (0.03%) for clostridial spores (using fecal dilutions heated for 10 min at 70°C); 3. Beerens (Beerens, 1990) and MRS agar for bifidobacteria and lactobacilli; 4. Bile Bacteroides Esculin (BBE) agar for Bacteroides fragilis group; 5. Eosin Methylene Blue (EMB) agar for enterobacteria; 6. Bile Esculin Azide (BEA) agar for enterococci. Additionally, Clostridium perfringens vegetative forms were more specifically searched for using lactose-sulfite broth incubated at 46°C for 48 hours and the most probable number method (Beerens et al., 1982). Plates were incubated in an anaerobic chamber (La Calhène, Vélizy, France) for 5 days for the enumaration of Bacteroides fragilis group, bifidobacteria and clostridia (vegetative and sporulated forms). Aerobic incubation was carried out for 48 hours at 37°C. Each type of colony was subcultured on Rosenow broth, Gram-stained, and tested for aerobic susceptibility and catalase production. The sum of the various bacteria recovered gave the total cultivable flora expressed as CFU/mL. The counts in cultivable bifidobacteria, enterobacteria and clostridia were expressed as the percentage of the total cultivable flora. Bacteria are considered as belonging to the dominant flora when their proportion is ≥1% of the total cultivable flora (Holdeman et al., 1976). The detection limit of the culture method was 0.01%. Bifidobacteria were identified at the species level by a multiplex PCR technique using species-specific primers previously described (Mullié et al., 2003). The other bacterial groups were identified by using API systems (Biomérieux, Marcy l’Etoile, France). IgA titers and total cultivable bifidobacterial proportions were analyzed by nonparametric ANOVA for repeated measures (Conover’s method). Fisher exact test was used to evaluate the difference in colonization percentages by the various bifidobacterial species between the feeding groups. Mann-Whitney test was used to compare antipoliovirus IgA titers between infants who harbored B. longum-infantis at 4 mo and those who did not. Spearman rank correlation coefficient was used to correlate fecal bacterial counts with each other and with IgA concentration. Bacterial proportions were reported as mean ± SD.

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Twenty babies (9 controls and 11 BbC50) completed the study. As reported earlier (Mullié et al., 2004), counts in bifidobacteria rose from month 1 to month 4 only in the BbC50 group, corroborating the formerly described bifidogenic effect of this formula (Table 1). The population of enterobacteria (mainly identified as Escherichia coli) did not vary with the type of formula (Table 1). However, enterobacteria proportions significantly decreased with time in both group (p<0.05 at months 3 and 4, Table 1).

Table 1. Fecal colonization of infants receiving either the control or the BbC50 fermented formula Month 1

Month 2

Month 3

Month 4

Contro BbC50 l

Control BbC50 Control BbC50

Control

BbC50

28.1 ± 17.25

28.0 ± 25.06

32.4 ± 17.26

38.7 ± 30.52

34.2 ± 24.87

50.8 ± 25.91*

35.1 ± 20.20

52.0 ± 23.95*

Enterobacteriaa 10.2 ± 11.11

19.5 ± 22.74

15.2 ± 16.25

6.7 ± 8.16

3.9 ± 4.48**

3.7 ± 2.67**

4.2 ± 4.98**

4.0 ± 4.48**

C. perfringens vegetative formsb

-7.4 ± 0.99

-8.1 ± 0.74

-8.1 ± 1.15

-9.1 ± 0.42

-7.4 ± 0.99

-8.3 ± 1.44

-6.0 ± 0.10***

Bifidobacteriaa

-7.9 ± 0.79

a

expressed as percentage of the total cultivable flora (mean ± SD). expressed as log cfu/mL. * p<0.05. ** p<0.05, compared to values at month 1. *** Significantly different from values in the control group at month 4 (p<0.05). b

Negative correlations were found between counts in bifidobacteria and enterobacteria in both control and BbC50 infants at 4 month of age (rs=-0,543 and rs=-0,623; p<0.05, respectively). At an early stage (1 and 2 months), the negative correlation was only observed in the group fed BbC50 fermented formula (rs=-0.749, p<0.05 and rs=-0.718, p<0.02, respectively). As mentioned above (§ 1. 2. 1.), a similar negative correlation was witnessed in breast-fed infants at 2 months of age. An early and specific modification induced by human milk as well as BbC50 fermented formula on the equilibrium between enterobacteria (usually the first colonizers of the neonate's gut) and bifidobacteria therefore seems to take place. A “pH effect” could be put forward to explain the inverted relationship between counts in enterobacteria and bifidobacteria. Indeed, when the latter colonize the intestine at a high level, the end-products of their sugar metabolism (mainly acetic, lactic and propionic acids) generate a decrease in the intestinal pH which is less favorable to the iron intake of enterobacteria leading to their reduced growth. A decreased pH has already been reported for BbC50 fermented formula (Indrio et al., 2007). Nevertheless, this cannot be the only mechanism underlying the repression of enterobacteria in the BbC50 group as the decrease in

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their intestinal counts had begun before (at 2 months of age) the rise in bifidobacteria was significant (at 3 months of age, Table 1). An alternative (but not exclusive) explanation could be a modification of the intestinal flora in response to a change in intestinal mucins or substrates produced by infants along with the maturation process. At an early stage, the maturation process could be stimulated to a similar extent in BbC50 and breast-fed infants. As bifidobacteria have a large panel of glycosidasic activities (Schell et al., 2002), they may be more efficient in using these substrates than enterobacteria.

Table 2. Correlations between counts in enterobacteria and counts in Bifidobacterium species

B. breve (1 month) B. breve (4 months) B. bifidum (1 month) a

All infants rs=-0.682 (p=0.0256) rs=-0.440 (p=0.05) NS

Control NSa NS rs=0.659 (p=0.075)

BbC50 rs=-0.895 (p=0.00113) NS NS

Not significant.

This balance might be involved in the immune regulation. BbC50 was found to preferentially stimulate some of the Bifidobacterium species (i.e. B. breve and B. longuminfantis)(Mullié et al., 2004). And an increased poliovirus-specific intestinal IgA response coincided with the bifidobacterial promotion. Species-specific correlations with counts in enterobacteria indicate that a peculiar species cannot be singled out to account for the negative correlation witnessed between enterobacteria and bifidobacteria (Table 2). At month 1, B. breve was the sole species exhibiting a negative correlation with enterobacteria. This relationship disappears with time, possibly as B. breve prevalence decreases and the species is replaced by “adult species” such as B. adolescentis or B. catenulatum-pseudocatenulatum (Mullié et al., 2006). However, at month 4, the negative correlation between B. breve and enterobacteria counts becomes significant once again despite the decrease in B. breve carriage prevalence. But at that time, the relation was observed independently from the type of feeding. Even breast-fed babies show the same balance (personal data). But it seems that a comparable microbial balance affects the immune system in various ways according to the type of feeding. At 4 months of age, no correlation was observed between antipolovirus IgA titers and E. coli in BbC50 whereas a negative correlation was found in the control group (rs=-0.829, p=0.021). Therefore, a possible inhibitory effect on antipoliovirus IgA response induced by E. coli intestinal population could be suppressed by the intake of BbC50 infant formula. In addition, when Clostridium perfringens vegetative forms are considered, significantly lower counts were found in BbC50 fecal samples at month 4, as compared to control group values at the same sampling time (Table 1). Moreover, the number of babies who were never colonized with detectable levels of vegetative C. perfringens tended to be higher in the BbC50 group (Control=0/9 vs. BbC50=4/11, p=0.094). Such an effect warrants further

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investigations. Infants and children with short bowel syndrom display frequent septicemia with Gram negative bacteria when they are colonized by C. perfringens. A specific nutrition preventing the expression of C. perfringens toxin would therefore be helpful in this peculiar population. BbC50 fermented infant formula displays a positive influence on the full-term neonate intestinal flora as a bifidogenic effect is witnessed along with a repression of enterobacteria and vegetative forms of C. perfringens at month 4. Moreover, so-called infant species (i.e. B. breve and B. longum-infantis) are favored. This point is of great interest as B. adolescentis and B. catenulatum-pseudocatenulatum (“adult” bifidobacterial species) are more frequently isolated from allergy-prone or atopic infants. Additionally, a stimulation of the immune can also be acknowledged for this fermented infant formula. Further investigations should be undertaken to elucidate the precise mechanism(s) of this immune stimulation. In our latest work comparing the effects of a standard infant formula and BbC50 fermented infant formula instated after weaning with those of a continuous breast-feeding, preliminary results showed that while an increase in enterobacteria and decrease in bifidobacteria was witnessed when a standard infant formula was instated after weaning (see paragraph 1. 2. 1. above), significantly less modifications to the intestinal balance between enterobacteria and bifidobacteria were triggered by the BbC50 fermented infant formula. Indeed, bacterial counts and colonization levels were closer to those registered with human milk.

6. Conclusion Experimental data show that infant formulas have been improved so as to generate the implantation of an intestinal flora close to the one of breast-fed infants in terms of dominance of the genus Bifidobacterium. Whether this global bifidogenic effect is sufficient to prevent the development of chronic conditions such as allergy remains to be ascertained. A highly hygienic lifestyle and other environmental factors also impact the intestinal microflora and most probably bear some responsibility in triggering chronic diseases. The study of a beneficial intestinal balance should therefore not be limited to bifidobacteria as a whole but also take into account Bifidobacterium species, other components of the gut microbiota as well as their relationships. BbC50 milk fermentation products could represent an interesting alternative to classic pre-/probiotics in the modulation of the intestinal flora, especially in the development of an adapted nutrition in fragile populations such as premature babies and at-risk infants (atopy, allergy, type 1 diabetes, cardio-vascular diseases etc.). Nevertheless, their use should be validated in large preventive trials targeting such situations.

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Strobel, S. (2001) Immunity induced after a feed of antigen during early life: oral tolerance vs. sensitisation. Proc. Nutr. Soc., 60, 437-442. Sullivan, Å., Edlund, C. & Nord, C.E. (2001) Effect of antimicrobial agents on the ecological balance of human microflora. Lancet Infect. Dis., 1, 101-114. Sussman, J.I., Baron, E.J., Goldberg, S.M., Kaplan, M.H., & Pizzarello, R.A. (1986) Clinical manifestations and therapy of Lactobacillus endocarditis: report of a case and review of the literature. Rev. Infect. Dis., 8, 771-776. Thibault, H., Aubert-Jacquin, C. & Goulet, O. (2004) Effects of long-term consumption of a fermented infant formula (with Bifidobacterium breve C50 and Streptococcus thermophilus 065) on acute diarrhea in healthy infants. J. Pediatr. Gastroenterol. Nutr., 39, 147–152. Tissier, H. (1900) Recherche sur la flore intestinale normale et pathologique du nourrisson. Thesis. University of Paris. Vankerckhoven. V., Huys. G., Vancanneyt. M., Vael. C., Klare, I., Romond, M.B., Entenza, J.M., Moreillon, P., Wind, R.D., Knol, J., Wiertz, E., Pot, B., Vaughan, E.E., Kahlmeter, G., & Goossens H. (2007) Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE project. Trends Food Sci. Technol., doi:10.1016/j.tifs.2007.07.013. Weesterbek, E.A.M., van den Berg, A., Lafeber, H.N., Knol, J., Fetter, W.P.F., & van Elburg, R.M. (2006) The intestinal bacterial colonisation in preterm infants: a review of the literature. Clin. Nutr., 25, 361-368. Weizman, Z., Asli, G. & Alsheikh, A. (2005) Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents. Pediatrics, 115, 5-9. Weizman, Z. & Alsheikh, A. (2006) Safety and tolerance of a probiotic formula in early infancy comparing two probiotic agents: a pilot study. J. Am. Coll. Nutr., 25, 415-419. WHO collaborative study team on the role of breastfeeding on the prevention of mortality (2000) Effect of breast feeding on infant and child mortality due to infectious diseases in less developed countries: a pooled analysis. Lancet, 355, 451-455. Yoshioka, H., Iseki, K. & Fujita K. (1983) Development and differences of intestinal flora in the neonatal period in breast-fed and bottle-fed infants. Pediatrics, 72, 317-321. Ziegler, E., Vanderhoof, J.A., Petschow, B., Mitmesser, S.H., Stolz, S.I., Harris, C.L., & Berseth, C.L. (2007) Term infants fed formula supplemented with selected blends of prebiotics grow normally and have soft stools similar to those reported for breast-fed infants. J. Pediatr. Gastroenterol. Nutr., 44, 359-S64.

Index A abatement, 46, 50, 57 ABC, 10 absorption, x, 3, 10, 11, 13, 25, 30, 31, 35, 37, 38, 39, 40, 41, 42, 57, 132, 153, 154, 155, 156, 158, 159, 160, 161, 179 accounting, 178, 234 accumulation, 182, 191, 208 accuracy, 218 acetaldehyde, 46 acetate, 159, 161 acetic acid, 44, 46, 48, 52, 53, 120, 157 acetone, 26, 27 acidic, 44, 164, 173, 186, 224 Acinetobacter, 118 activated receptors, 207 activation, ix, 60, 98, 125, 133, 150, 165, 166, 168, 169, 190, 222, 234, 242 active transport, 155 acute, 30, 158, 224, 234, 247 Adams, 84, 145 adaptation, 149, 246 additives, 91, 104, 106, 110, 187 adenocarcinoma, 197 adenosine, 10 adenosine triphosphate, 10 adhesion, 58, 73, 78, 85, 130, 132, 243 adhesion strength, 78 adhesive properties, 58 adipocytes, 208 adipose, 132, 176, 177, 179, 186, 187, 190, 191, 192, 198, 201, 205, 208, 212, 213 adipose tissue, 176, 177, 179, 186, 187, 190, 191, 201, 205, 208, 212, 213

adiposity, 208, 213, 240 adjustment, 22 administration, ix, x, 2, 21, 62, 67, 68, 115, 117, 122, 123, 125, 127, 131, 135, 136, 138, 142, 144, 148, 150, 161, 166, 173, 185, 188, 209, 224, 226, 229, 234, 243 adsorption, 72, 154, 161 adult, xi, 19, 33, 57, 68, 126, 127, 129, 145, 160, 173, 175, 191, 195, 218, 221, 230, 238, 239 adult population, 57 adulteration, 36 adulthood, 135, 217 adults, 57, 60, 62, 119, 123, 135, 136, 189, 224, 226, 231, 233 aerobic, 59, 218, 227, 236 aerobic bacteria, 227 affect, 182, 190, 193, 202 Africa, 44 agar, 118, 119, 120, 236 age, ix, xi, 45, 57, 115, 117, 127, 128, 130, 132, 136, 138, 139, 142, 143, 175, 182, 188, 205, 218, 219, 220, 221, 222, 225, 228, 229, 230, 235, 237, 238, 244, 245 agent, 22, 23, 104, 118, 158, 176 agents, ix, 23, 29, 105, 115, 118, 123, 132, 192, 247 aggregates, 97 aggregation, 58, 102 aging, 95, 212 aid, 88, 144, 221 AIDS, 141, 245 air, 23, 76, 137 airway hyperresponsiveness, 56 airway inflammation, 55, 145, 173 alanine, 50 Albino, 59 alcohol, 4, 46, 48

250

Index

alcohols, 106 algae, 4, 183, 203 ALI, 144 alkali, x, 76, 153, 154 alkaline, 21, 25, 26, 161, 176 alkaline media, 161 alkylation, 3 allergens, 134, 145 allergic asthma, 137 allergic inflammation, 225, 228 allergic reaction, 225 allergic rhinitis, 142 allergic sensitisation, 225 allergy, 44, 59, 121, 139, 140, 141, 217, 221, 225, 227, 228, 231, 239, 243, 244 alpha, 67, 144, 149, 164, 172 ALT, 133, 221, 222 alternative, viii, 2, 26, 28, 87, 88, 89, 122, 238, 239 alternatives, viii, 16, 23, 87, 88, 112, 113, 114 alters, 33, 150, 186, 190, 206 AMF, 91, 92 amino, 48, 50, 52, 62, 131, 190, 207 amino acid, 48, 50, 52, 62, 131 amino acids, 48, 52, 131 aminopeptidase, 131 ammonia, 50 amniotic, 116 amniotic fluid, 116 amorphous, 85 anabolic, 191 anaemia, 173 anaerobe, 160, 246 anaerobes, 127, 156 anaerobic, 117, 127, 147, 218, 236 anaerobic bacteria, 127, 218, 236 analysis of variance, 76 anatomy, 131 anemia, 159 angiogenesis, 132, 150 animal models, x, 51, 126, 135, 159, 176, 187, 191, 192, 194, 195, 196, 200, 223, 233, 234 animal studies, xi, 51, 138, 175, 187, 194 animals, 4, 9, 55, 56, 57, 59, 131, 133, 136, 154, 158, 165, 180, 185, 186, 187, 191, 193, 195, 196, 198 Animals, 136 ANOVA, 76, 236 antagonistic, 52, 53 anthracene, 188 anti-atherogenic, 192

antibacterial, viii, 43, 52, 53, 57, 60, 61, 65, 68 antibiotic, 61, 117, 222, 224, 228, 234, 241 antibiotics, 44, 45, 128, 141, 220, 223, 228 antibody, 62, 146, 221, 244 anticancer, 30, 51, 52, 172, 176, 187, 194, 208 anticancer activity, 172 Anti-carcinogenic, 54 antidiabetic, 176, 187, 192, 210 antigen, 119, 129, 133, 134, 140, 142, 145, 173, 198, 210, 225, 234, 247 antigen presenting cells, 133 antigen-presenting cell, 119, 234 anti-inflammatory, 221 antimicrobial protein, 172 antioxidant, 37, 59, 64 Antioxidative, 59 antitumor, 51, 52, 63 anti-tumor, 51 anus, 128 APC, 212 API, 236 apolipoprotein A-I, 132 Apolipoprotein E, 37 apoptosis, x, 60, 65, 67, 163, 168, 169, 172, 190, 197, 207, 208, 213 apoptotic, 59, 168, 197 apoptotic cells, 60 apoptotic pathway, 168 application, 26, 28, 31, 33, 40, 64, 139, 145 aqueous solution, 21, 30 arachidonic acid, 190, 193, 222 Argentina, 87, 115, 119, 124 aromatic compounds, 46 arrest, 166, 170, 172 arteriosclerosis, 134 artery, 39, 41, 42 ascites, 51 aseptic, 30 ash, 46, 194 Asia, 44, 65, 84 assessment, 40, 121, 122, 148, 247 assimilation, 56 association, 176, 197 asthma, 55, 59, 61, 63, 135, 137, 142, 149, 173, 245 atherogenesis, xi, 175 atherosclerosis, 9, 151, 188, 192, 199, 209, 210, 213 atherosclerotic plaque, 9, 187, 190, 192 atherosclerotic vascular disease, 35 athletes, viii, 43 atmosphere, 26

Index atoms, 3 atopic dermatitis, xi, 117, 121, 137, 138, 140, 143, 150, 216, 217, 225, 227, 228, 231, 242, 243, 244, 246 atopic eczema, 117, 139, 142, 226, 240, 242 atopy, x, 122, 126, 142, 147, 225, 239, 243 ATP, 10, 37 atrophy, 131 attachment, 58 Australia, 16, 111, 112 Austria, 20 autoimmune disease, 126, 217 autoimmune diseases, 126 autoimmunity, 121, 147, 193, 221 autosomal recessive, 13 availability, x, 163, 182, 184 avoidance, 136 azoxymethane, 162

B B cells, 133, 165, 174, 221 B lymphocytes, 141 babies, 129, 142, 146, 216, 217, 218, 219, 220, 222, 225, 229, 230, 235, 237, 238, 239 Bacillus, 52, 223 bacteria, vii, ix, x, xi, 43, 44, 45, 48, 49, 52, 53, 54, 57, 58, 59, 62, 64, 65, 66, 67, 68, 115, 116, 117, 118, 119, 121, 122, 123, 127, 128, 129, 130, 131, 132, 133, 134, 136, 138, 139, 141, 144, 145, 147, 148, 150, 151, 153, 154, 157, 159, 161, 176, 179, 182, 184, 200, 201, 202, 206, 215, 217, 220, 222, 223, 225, 227, 228, 229, 233, 234, 236, 239, 242, 243, 244, 246 bacterial, viii, xi, 43, 54, 55, 60, 85, 116, 118, 121, 122, 123, 128, 130, 132, 134, 136, 141, 142, 144, 146, 148, 149, 150, 151, 171, 179, 184, 216, 217, 220, 223, 229, 231, 232, 236, 239, 242, 246, 247 bacterial cells, 220 bacterial contamination, 171 bacterial strains, 141, 142 bacteriocin, 64, 69 bacteriocins, 52, 66 bacteriostatic, 52 bacterium, 157 Baked goods, 18 baking, 18, 62 barley, 186, 206 barrier, 57, 98, 119, 129, 132, 223, 225, 227, 234, 244

251

barriers, 164 basic research, 164 Bcl-2, 169, 172 Bcl-xL, 234 BEA, 236 beef, xi, 175, 176, 177, 185, 186, 187, 193, 194, 195, 196, 200, 204, 205, 206, 212 behavior, ix, 53, 88, 91, 94, 95, 101, 102, 105, 106, 110, 111, 112 Belgium, 20, 63, 112 beneficial effect, xi, 9, 22, 29, 44, 53, 58, 64, 67, 135, 137, 141, 154, 165, 170, 176, 193, 215, 223, 233 benefits, vii, 14, 29, 36, 41, 44, 54, 90, 127, 135, 153, 164, 171, 176, 187, 194, 196, 232 benign, 30, 42 benign prostatic hyperplasia, 30, 42 benzo(a)pyrene, 212 Best Practice, 63 beverages, 17, 18, 32, 33, 35, 64 bile, x, 10, 12, 13, 29, 45, 58, 105, 127, 131, 153, 157, 158, 161 bile acids, x, 131, 153, 157, 158, 161 binding, 11, 35, 73, 81, 132, 150, 160, 164, 172, 211 binding energy, 81 bioactive compounds, 52, 164, 234 bioavailability, 23, 158 biochemical, xi, 215 biodegradable, 105 biodiversity, 145 biological activity, 176 biological models, 164 biomass, 64 Biometals, 172 biopolymer, 170 biopolymers, 170 biopsies, 226 biopsy, 134 biosafety, 244 biosynthesis, 3, 34, 39, 131, 176, 190, 193, 213 biosynthetic pathways, 132 biotechnological, 45 biotechnology, 33 biotin, 49 birth, ix, 115, 116, 117, 125, 127, 128, 131, 132, 135, 137, 138, 141, 148, 217, 218, 219, 221, 235, 240, 245 birthweight, 242 bison, 205 bleaching, 6

252

Index

bleeding, 117, 123 blends, ix, 36, 40, 88, 89, 92, 94, 95, 99, 102, 106, 110, 111, 210, 247 blood, viii, 2, 3, 9, 10, 11, 14, 19, 21, 22, 24, 28, 33, 35, 39, 55, 58, 59, 60, 61, 64, 66, 68, 87, 88, 116, 134, 139, 140, 141, 142, 143, 144, 145, 190, 192, 193, 202, 205, 211, 217, 221, 236, 243, 246 blood glucose, 58, 64, 140, 143, 192, 193 blood plasma, 202, 205 blood pressure, 59, 60, 64, 140, 143, 144, 217 blood stream, 10, 11 blot, 166, 169 body, 176, 187, 190, 191, 192, 193, 198, 208, 209 body composition, 187, 190, 191, 193, 198, 208, 209 body fat, 176, 191, 192, 198, 208, 209 body fluid, 164 body weight, 9, 13, 51, 191, 193 Boeing, 42 bone density, 198 bone growth, 193 bone loss, 194 bone mass, 193, 211 bone remodeling, 211 bone resorption, 173 borderline, 61 bounds, 4 bovine, 51, 60, 117, 123, 165, 170, 172, 173, 174, 200, 201, 202, 203, 204, 240 bowel, 64, 144, 159, 221, 239 boys, 235 Brazil, 8 breakdown, 225 breakfast, 18, 24, 34 breast, 216, 217, 218, 219, 220, 221, 222, 223, 226, 227, 229, 230, 235, 237, 238, 239, 240, 241, 242, 243, 245, 246 Breast, 216, 217, 221, 224, 229, 242 breast cancer, 51, 52, 61, 67, 165, 173, 188, 190, 197, 198, 207 breast feeding, 121, 241, 243, 245, 247 breast milk, ix, 115, 116, 118, 119, 120, 121, 122, 123, 135, 142, 148, 173, 221, 240 breastfeeding, 137, 217, 220, 224, 242, 245, 247 brevis, 44, 63 Britain, 225 bronchial asthma, 56, 59 bronchitis, 143 Brussels, 7, 63 Buenos Aires, 87 buffalo, 45

Bulgaria, 20 Burkholderia, 160 butyric, 46, 156 by-products, vii, 1, 6, 21

C cabbage, 7, 44 CAD, 13 caecum, 57, 159 caesarean section, 118, 128, 217 calcium, 49, 51, 78, 154, 155, 156, 159, 160, 161, 204 calibration, 28 Campylobacter jejuni, 57, 69 Canada, 45, 111 cancer, xi, 51, 52, 63, 165, 170, 175, 176, 187, 188, 190, 196, 197, 198, 206, 207, 245 cancer cells, 52, 61, 165, 173, 187, 197 candida, 49 Candida, 45, 52, 53, 68, 230 candidates, 234 capillary, 25, 27, 29, 39 carbohydrate, xi, 57, 62, 202, 215 carbohydrates, xi, 131, 132, 143, 158, 159, 215, 229, 235 carbon, 3, 46, 52, 62, 75, 92 Carbon, 93 carbon atoms, 3 carbon dioxide, 46, 52 carbon tetrachloride, 62 carcinogen, 51, 188, 190 carcinogenesis, 188, 196, 197, 207, 210 carcinogenic, viii, 43, 51 carcinoma, 31, 51, 61 cardiovascular disease, vii, 1, 2, 3, 9, 28, 35, 56, 134 Cardiovascular disease, 217 cardiovascular risk, 33 carotene, 33, 39, 40, 198 carotenoids, 19, 38 carrier, 12, 155 casein, 44, 228 caspase, 60, 168 CAT, 62 catabolic, 198 catalase, 236 cattle, 179, 185, 186, 196, 204, 205, 206 Caucasus, 63 CBS, 68 CD8+, 234 CDK2, 166

Index CEC, 123 cecum, 131, 156, 159, 161, 207 cell, x, 2, 29, 38, 54, 55, 60, 61, 68, 95, 102, 120, 133, 137, 139, 155, 162, 163, 164, 165, 166, 168, 169, 170, 172, 174, 190, 208, 211, 221, 222, 228, 234, 242, 244 cell culture, 61 cell cycle, x, 163, 166, 172, 190 cell death, 168, 169, 170 cell differentiation, 133, 166 cell growth, 29 cell line, 165, 172 cell lines, 165 cellular immunity, 221 cellulose, 161 cereals, 4, 5, 6, 15, 16, 18, 24, 26, 34 certificate, 14 ceruloplasmin, 164, 173 cesarean section, 218, 223, 240 CFA, 154, 156 channels, 74 charge density, 72 , 15, 18, 20, 21, 32, 78, 136, 164, 177, 180, 186, 194, 195, 199, 202, 212 chemical approach, 73 chemical composition, 47, 48, 89, 92, 94, 95 chemical properties, ix, 27, 40, 88, 90 chemical structures, vii, 1, 3, 106, 177 chemicals, 72, 73, 75, 78 chemokine, 133 chemokines, 133 chemotherapy, 165, 174 chicken, 181, 205 chickens, 9, 176 chicks, 39, 57, 193, 194, 211 child care centers, 247 child mortality, 247 childhood, 217, 240, 242, 245 children, ix, xi, 19, 45, 63, 65, 115, 117, 121, 122, 138, 139, 140, 141, 142, 143, 149, 150, 158, 216, 217, 224, 227, 232, 239, 245, 246 China, 43, 64, 68 chloride, 26 Chloride, 48 chloroform, 26, 27, 58 chocolate, 91, 95, 105, 111 cholera, 55 cholesterol, vii, viii, x, 1, 2, 3, 4, 9, 10, 11, 12, 13, 14, 19, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 56, 59, 60,

253

61, 62, 64, 65, 67, 68, 87, 88, 131, 135, 153, 157, 158, 159, 161, 162, 192, 205 cholesterol lowering agents, 29 chromatography, 25, 29, 31, 92, 177, 199 chronic diseases, 44, 217, 239 chronic disorders, 217 circulation, 119 cis, xi, 89, 175, 176, 198, 199, 200, 202, 203, 204, 205, 206, 208, 212 classification, 14, 246 cleaning, 72, 73, 75, 76, 78, 79 cleavage, 168 clinical, 220, 223, 224, 227, 228, 230, 231, 233, 235, 242, 243 clinical symptoms, 231, 243 clinical trial, 9, 117, 136, 138, 141, 144, 150, 172, 220, 224, 227, 230, 231, 233, 235 clinical trials, 9, 117, 136, 138, 141, 144, 172, 233 clinics, 122 clone, 157 cloning, 154 CMV, 217 Co, 10, 23, 61, 64, 158, 159 CO2, 46 coatings, viii, 71, 73, 75, 76, 78, 79, 81, 83, 84, 85, 90, 105 cobalt, 51 Cochrane, 149 coconut, 90 coconut oil, 90 coeliac disease, 149, 241 coenzyme, 201 coffee, 16, 18 cohort, 218, 219, 240, 245 colic, 117, 224 colitis, 147, 150, 173 collateral, 133, 193, 210 collateral damage, 193, 210 colloids, 170 colon, x, 29, 60, 137, 153, 154, 157, 158, 159, 176, 188, 190, 207, 229, 245 colon cancer, 158, 190, 245 colon carcinogenesis, 188, 207 colonisation, 146, 150, 219, 246, 247 colonization, viii, ix, 43, 54, 57, 58, 60, 116, 117, 119, 122, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 136, 138, 139, 142, 144, 149, 150, 217, 218, 219, 220, 222, 223, 226, 227, 230, 236, 237, 239 colonizers, 117, 128, 218, 237

254

Index

colorectal cancer, 67 Columbia, 236 commensals, 150 commercial, 224, 228, 233, 234 commercialization, 14, 15, 16 communication, 204 communities, 146, 151 community, 3, 48, 117, 151, 179 competence, 208 competition, 10 compliance, 235 complications, 141, 174 components, vii, x, 4, 23, 35, 36, 44, 51, 54, 55, 57, 59, 61, 62, 66, 73, 85, 91, 98, 105, 132, 134, 153, 164, 170, 172, 173, 190, 211, 220, 222, 226, 231, 239, 242 composites, 31 composition, ix, x, xi, 15, 21, 22, 29, 36, 39, 46, 47, 48, 61, 64, 65, 66, 75, 89, 91, 92, 94, 95, 97, 102, 105, 115, 117, 118, 119, 120, 121, 123, 125, 127, 129, 135, 142, 149, 150, 179, 187, 190, 191, 193, 198, 201, 203, 204, 205, 206, 208, 209, 211, 212, 213, 215, 225, 226, 229, 231, 233, 235, 240, 241, 242, 243, 245, 246 compositions, 235 compounds, 2, 3, 4, 12, 16, 25, 26, 28, 29, 30, 41, 44, 48, 52, 105, 135, 164, 222, 233, 234, 244 concentrates, 36, 44 concentration, 2, 6, 11, 16, 20, 21, 26, 28, 36, 38, 46, 52, 56, 106, 142, 143, 157, 159, 162, 165, 189, 192, 193, 202, 204, 211, 212, 236 conception, 126, 135, 136, 137, 144, 149 conductivity, 72, 75, 77, 79, 83 confidence, 106 confidence interval, 106 configuration, 88 confinement, 187 Congress, 61, 111, 113, 124 conifer, 21 conjugated dienes, 199 consent, 235 constipation, 64, 117, 225 consumers, 44, 45, 56, 60, 88, 171 consumption, vii, viii, 1, 3, 4, 9, 12, 13, 14, 17, 18, 19, 20, 24, 31, 39, 43, 44, 57, 60, 67, 72, 73, 136, 138, 141, 145, 146, 148, 171, 189, 195, 196, 223, 226, 233, 246, 247 contact time, 25 contaminant, 81 contaminants, 16

contamination, 57, 116, 119, 171, 217 control, ix, 24, 25, 31, 39, 42, 60, 67, 74, 76, 77, 79, 81, 82, 83, 84, 105, 115, 117, 136, 137, 143, 150, 154, 155, 156, 157, 182, 183, 185, 189, 190, 191, 192, 198, 203, 204, 210, 224, 225, 227, 228, 230, 232, 234, 235, 237, 238, 242, 245 control group, 183, 224, 232, 237, 238 controlled studies, 228 controlled trials, 29, 141 convergence, 126 conversion, x, 90, 153, 154, 157, 181, 189, 196 conversion degrees, 90 Cookies, 114 cooking, xi, 16, 84, 90, 175, 187 cooling, 23, 73, 76, 79, 90, 95, 97, 99, 100, 101, 102, 103, 104, 106, 109 cooling process, 99 copper, 51, 132, 204, 205 corn, 4, 21, 176, 182, 185, 193, 204 coronary artery disease, 39, 41, 42 coronary heart disease, viii, 2, 34, 42, 87, 88 correction factors, 28, 179 correlation, 119, 193, 227, 236, 237, 238 correlation coefficient, 236 correlations, 237, 238 corrosion, 75 cortisol, 222 Corynebacterium, 118 cosmetics, 105 cost saving, 72 costs, viii, 4, 71, 72, 164 costs of production, 164 counseling, 140, 142, 143, 147 covering, 110 cow milk, 68, 135, 154, 212, 243 cows, 178, 180, 182, 183, 184, 185, 186, 195, 200, 201, 202, 203, 204, 206, 212 CRC, 61, 161 crystal growth, 104, 111 crystal structure, 90, 104, 105, 110 crystalline, 22, 23, 85, 92, 101, 110, 111 crystallization, ix, 10, 21, 23, 88, 90, 91, 92, 95, 97, 98, 99, 100, 101, 102, 105, 107, 110, 112, 113, 114 crystallization kinetics, 102, 110, 112, 114 crystals, 10, 21, 91, 95, 97, 99, 101, 102, 103, 104, 105, 107, 109, 110 cultivation, 218 culture, 29, 33, 45, 52, 57, 58, 61, 63, 66, 116, 118, 129, 156, 161, 200, 218, 227, 234, 236

Index culture media, 116, 236 CVD, vii, 1, 9, 13 cycling, x, 163 Cyprus, 20 Cystatin, 173 Cystatin C, 173 cysteine, 119, 120, 236 cytokine, 54, 55, 133, 137, 141, 142, 165, 172, 173, 193, 221, 222 cytokine response, 221 cytokines, 55, 67, 119, 130, 133, 165, 193, 194, 221, 234 cytomegalovirus, 134 cytometry, 161 cytoskeleton, 132 cytosolic, 132 cytotoxic, 157 cytotoxicity, 33 Czech Republic, 20

D dairy, vii, ix, xi, 1, 3, 14, 15, 17, 18, 19, 20, 28, 36, 44, 53, 56, 57, 61, 63, 65, 66, 67, 72, 76, 84, 85, 111, 116, 119, 164, 173, 175, 176, 177, 180, 182, 183, 184, 185, 186, 187, 189, 195, 196, 200, 201, 202, 203, 204, 206, 207, 211 dairy industry, 72, 76, 84, 85 dairy products, vii, ix, xi, 1, 3, 14, 15, 17, 19, 20, 28, 44, 53, 56, 61, 63, 66, 67, 116, 119, 164, 173, 175, 176, 177, 180, 186, 187, 195, 196, 206, 207, 211 danger, 133 Darcy, 197 database, 38 de novo, 132, 204 death, vii, 1, 2, 59, 64 deaths, 2 decisions, 15 defects, 76 defense, 136, 154, 159, 193, 242 defense mechanisms, 136, 154 defenses, 133, 141 deficiency, 173 definition, vii, 13, 19, 88, 99, 164 deformation, 96 degradation, 73, 161 degrading, 44, 54, 169 degree, 217 dehydration, 224, 235

255

delivery, ix, xi, 29, 115, 118, 119, 127, 128, 138, 139, 140, 141, 142, 143, 145, 147, 163, 171, 217, 222, 223 denaturation, 72 dendrites, 119 dendritic cell, 119, 133, 142, 234, 242, 243 Dendritic cells, 119, 123 Denmark, 20 density, x, 35, 153, 158, 159, 192, 198 deposition, 77, 85, 209 deposits, 77, 78 depression, 198, 201, 210 derivatives, 25, 27, 34, 136, 174, 198, 199, 212, 229 dermatitis, xi, 117, 137, 143, 216, 217, 225, 227, 228, 231, 242, 243, 244, 246 detection, 25, 27, 28, 65, 77, 84, 118, 121, 157, 160, 218, 230, 236, 242 detention, 129 developed countries, 2, 126, 136, 217, 247 developmental origins, 145 deviation, 95 diabetes, xi, 58, 63, 134, 135, 175, 188, 192, 193, 210, 217, 239 diacylglycerol, 18 diarrhea, 117, 154, 158, 216, 224, 225, 234, 241, 247 diarrhoea, 223, 246 dienes, 199 diet, vii, ix, xi, 2, 4, 10, 12, 13, 14, 16, 17, 19, 23, 24, 25, 26, 28, 30, 34, 35, 36, 39, 44, 49, 60, 67, 91, 105, 125, 126, 127, 130, 131, 134, 135, 143, 144, 146, 147, 148, 150, 154, 155, 156, 157, 159, 173, 175, 176, 178, 179, 182, 183, 184, 185, 186, 188, 189, 190, 191, 192, 193, 194, 197, 205, 206, 235 dietary, viii, x, xi, 10, 17, 18, 23, 24, 31, 36, 37, 38, 39, 40, 41, 56, 59, 60, 87, 88, 125, 127, 130, 131, 132, 134, 135, 136, 140, 141, 142, 143, 144, 146, 147, 153, 154, 159, 162, 174, 175, 176, 178, 183, 186, 188, 189, 190, 197, 198, 200, 201, 202, 203, 204, 205, 207, 208, 210, 211, 212, 221 dietary fat, 146, 189, 203, 204, 212 dietary fiber, 154 dietary intake, 37, 143, 200 dietary supplementation, 154, 202, 204, 210, 212 diets, 23, 36, 56, 126, 135, 136, 155, 171, 178, 180, 182, 183, 184, 185, 186, 187, 189, 191, 193, 196, 201, 202, 205, 206, 212 differential scanning calorimetry, 90 differentiation, 131, 133, 165, 166, 220 diffraction, 95

Index

256

digestibility, 173 digestion, 22, 24, 57, 62, 155 digestive enzymes, 131 diphtheria, 236 diseases, vii, x, 1, 2, 54, 59, 117, 126, 132, 136, 139, 140, 141, 142, 143, 154, 216, 217, 223, 239 disorder, 13, 56 distillation, 6, 21 distribution, 24, 105, 111, 177, 179, 189, 206, 212, 229, 230 diversity, 37, 116, 118, 121, 122, 127, 130, 138 dizygotic, 130 dizygotic twins, 130 DNA, 60, 129, 130, 134, 137, 149, 156, 166 DNA damage, 60 DNA repair, 60 dogs, 165, 173 dominance, 134, 218, 229, 239 doped, 85 dosage, 231 double blind study, 191, 192 double bonds, 88, 89 download, 35 down-regulation, 133 dressings, 17, 18, 32, 95 drinking, 60, 136 drinking water, 136 drugs, 2, 149, 172 dry matter, 178 drying, ix, 116, 119, 120 DSC, 90, 94 duration, 68, 77, 121, 197, 224, 225, 235 dyslipidemia, 30

E E. coli, 53, 128, 129, 219, 223, 230, 238 Eastern Europe, 65 eating, vii, 1, 195 ecological, 119, 184, 247 ecology, 133, 148 ecosystem, 127, 145, 148 eczema, 142, 147, 225, 226, 231, 232, 240, 242, 243 Education, 37, 196 egg, 16, 18, 89 eicosanoid, 190, 193, 210 eicosanoids, 190, 193 elaboration, 120 elderly, 44 election, 246

electrical resistance, 155 electrodes, 84 electron, 77 electrophoresis, 151 ELISA, 236 elk, 205 elongation, 110 embryo, 98 embryos, 98 emotional, 242 emulsification, 23, 24 emulsifier, 23, 104, 105, 110 emulsifying agents, 105 emulsions, xi, 24, 105, 163, 170, 171 encapsulated, 170 encapsulation, 170 endocarditis, 247 endocytosis, 172 endogenous, 225, 227 endotherms, 94 energy, viii, 11, 71, 72, 73, 79, 82, 83, 84, 87, 88, 98, 131, 190, 191, 198, 209, 217 energy consumption, 72, 73 energy efficiency, 84 energy supply, 131 enrollment, 224, 234 enteric, 225 enteritis, 241 enterococci, 59, 220, 225, 230, 231, 236 enterocolitis, 117, 136, 150 environment, 34, 127, 130, 133, 146, 179, 221, 222, 224 environmental, 217, 223, 239 environmental factors, 126, 128, 129, 239 environmental influences, 126 enzymatic, x, 25, 90, 153, 161, 219, 233, 241 enzyme secretion, 24 enzymes, 13, 51, 131, 132, 154, 184, 191 eosinophilia, 56, 59 eosinophils, 137 epidemiologic studies, 217 epidermal growth factor, 222 epidermis, 118, 207 epithelial cell, 58, 132, 145, 148, 149 epithelial cells, 58, 132, 145, 148, 149 epithelial stem cell, 60 epithelium, 50, 58, 60, 131, 132, 148, 159, 160, 197, 207 equilibrium, 101, 237 equol, 159

Index erythrocyte, 208 erythrocyte membranes, 208 Escherichia coli, 52, 62, 148, 158, 193, 218, 219, 230, 237, 245 esophagus, 31 essential fatty acids, 89 ester, 11, 16, 24, 30, 31, 34, 35, 36, 38, 40, 106, 109, 110, 190, 207 esterases, 11, 24 esterification, 2, 11, 22 esters, 11, 13, 15, 16, 17, 19, 21, 22, 24, 26, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 105, 106, 107, 110, 111, 199 Estonia, 20 estrogen, 52, 61, 197 etching, 73, 84 ethanol, 44, 45, 46, 52 ethics, 235 eukaryotes, 2 Euler, 231, 241 Europe, vii, 1, 14, 16, 17, 44, 136 European, 232, 240 European Commission, 15, 19 European Parliament, 31, 32, 33 European Union, 15, 25, 31, 32, 33 evaporation, 27 evidence, xi, 176, 187, 189, 207, 223, 227, 233, 234 evolution, 36, 130, 147, 148 ewe, 203 examinations, 235 exclusion, 69, 83 excretion, 157 exercise, 191 exocrine, 164 exogenous, xi, 215 exopolysaccharides, 55 experimental condition, 155, 157 expert, 232 exposure, x, 59, 125, 126, 127, 128, 129, 135, 136, 137, 148, 225 Exposure, 137 expression, 190, 197 external environment, 57 external influences, 129 extraction, 21, 25, 26, 27, 55, 116 extrapolation, 195 exudate, 164

257

F fabrication, 113 faecal, 30, 41, 57, 116, 121, 150, 160, 227, 240, 241, 245 faecal bacteria, 30, 116 failure, 221 family, 36, 139 famine, 135, 148 FAO, viii, 87, 88, 111, 135, 145 farming, 184 farms, 184 fasting, 132, 192, 193, 208 fasting glucose, 192, 193 fat, viii, xi, 2, 3, 10, 15, 16, 17, 21, 22, 23, 24, 25, 28, 32, 34, 36, 37, 38, 40, 41, 46, 47, 56, 87, 88, 89, 91, 92, 93, 94, 95, 96, 97, 99, 101, 102, 105, 108, 112, 113, 114, 132, 135, 144, 148, 159, 164, 172, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 208, 209, 211, 212, 217, 235 fat soluble, 22, 24, 37 fats, ix, 18, 21, 22, 23, 26, 29, 40, 47, 88, 89, 91, 92, 94, 95, 96, 97, 99, 102, 105, 110, 111, 112, 113, 114, 201 FDA, viii, 14, 15, 16, 18, 23, 33, 34, 75, 87, 88, 111, 114 fecal, 217, 218, 220, 225, 226, 227, 230, 231, 233, 236, 238, 240, 242, 243, 246 feces, 116, 122, 128, 129, 146, 157, 220, 226, 240 Federal Register, 111 feeding, viii, ix, xi, 43, 47, 51, 115, 117, 121, 122, 127, 129, 136, 137, 140, 149, 150, 154, 155, 181, 183, 184, 185, 187, 188, 191, 192, 193, 198, 205, 206, 207, 208, 215, 217, 218, 220, 223, 226, 228, 229, 230, 231, 232, 236, 238, 239, 241, 243, 245, 247 females, 2 femoral bone, 159 fermentation, xi, 44, 45, 46, 48, 49, 50, 51, 53, 54, 56, 58, 60, 61, 66, 68, 157, 182, 216, 232, 233, 234, 239 fertilizers, 184 fetal, ix, 125, 126, 127, 134, 135, 136, 140, 142, 143, 145, 146, 148, 150 fetal growth, 150 fetus, 116, 126, 134, 135, 137, 139, 140, 141 fetuses, 116, 134 fever, 150

258

Index

fiber, 4, 131, 157, 159, 182, 186 fiber content, 182 FID, 25, 28, 92 Fife, 202 film, 105 films, 50 financial support, 83 Finland, 14, 16, 20, 34, 41, 42, 118 fish, 22, 31, 145, 183, 184, 203, 206 FISH, 216, 227, 230 fish meal, 183, 203 fish oil, 145, 183, 184, 203, 206 Fisher exact test, 236 fitness, 164 flame, 25, 92 flame ionization detector (FID), 92 flavor, 91, 92, 102, 203 flexibility, 50, 92 flora, xi, 49, 50, 57, 64, 121, 122, 123, 148, 164, 215, 217, 218, 219, 220, 222, 223, 225, 227, 230, 233, 236, 237, 238, 239, 240, 241, 242, 243, 246, 247 flow, 73, 74, 76, 77, 78, 79, 82, 83, 92 fluid, 26, 73, 84, 91, 116, 180 fluid extract, 26, 91 fluorescence, 161 fluorescence in situ hybridization, 161 fluorine, viii, 71, 75, 81, 83 focusing, 226 folding, 67 folic acid, 45, 49 food, vii, viii, ix, x, 1, 2, 6, 9, 12, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25, 29, 30, 31, 32, 33, 34, 35, 36, 37, 41, 42, 43, 44, 52, 54, 57, 58, 59, 60, 64, 65, 68, 71, 72, 73, 75, 77, 79, 83, 84, 87, 88, 89, 91, 95, 102, 104, 105, 106, 111, 119, 121, 122, 125, 131, 134, 136, 137, 145, 153, 154, 163, 164, 170, 173, 180, 190, 194, 199, 200, 205, 208, 211, 217, 221, 223, 225, 226, 227, 229, 240, 244 food allergy, 59, 217, 225, 227, 244 Food and Drug Administration, 14, 15, 88 Food and Drug Administration (FDA), 15, 88 food industry, vii, viii, 1, 14, 17, 71, 73, 164 food production, 52 , 17, 23, 29, 35, 44, 73, 75, 89, 95, 104, 170, 180 food safety, 72 fortification, 154 fouling, viii, 71, 72, 73, 76, 77, 78, 79, 81, 83, 84, 85 Fourier, 90 fractionation, 89, 91, 92

France, 20, 73, 113, 190, 198, 215, 233, 235, 236, 246 free energy, 72, 85, 98 free radicals, 188 freeze-dried, 67 freezing, ix, 116 friction, 73, 75, 85 frost, 34 fructooligosaccharides, 159, 161, 162, 229, 232, 240, 244 fructose, 154 fruit juice, 45 fruits, vii, 1, 4, 6, 15, 19, 31, 38, 89 frying, 18 FTIR, 90 functional changes, 174 fungi, x, 163 fusiform, 161

G gas, 25, 26, 29, 39, 41, 42, 92, 177, 199 gas chromatograph, 25, 26, 29, 41, 92, 177, 199 gastrectomy, 159 gastric, 55 gastroenteritis, 217, 224, 241 gastrointestinal, ix, xi, 10, 25, 44, 52, 57, 59, 60, 125, 127, 131, 132, 135, 139, 141, 145, 148, 149, 151, 154, 155, 173, 215, 218, 224, 229, 232, 241, 246 gastrointestinal tract, ix, 10, 125, 131, 135, 148, 151, 154, 155, 218, 229, 241, 246 GC, 25, 27, 28, 29, 40, 92, 246 gel, 151, 169 gelatin, 169 gels, 50, 66, 170 gender, 191 gene, 41, 127, 131, 132, 145, 156, 160, 173, 209, 244 gene expression, 127, 131, 132, 145, 173, 209 generation, 33, 42, 73, 157, 158, 225 genes, 126, 132, 133, 154, 197 genetics, 150, 206 Geneva, 42, 111 genome, x, 125, 130, 150, 246 genotype, 130, 151, 209 Germany, 20, 143 gestation, 135, 137 gestational age, 127, 222, 229, 230 Gibbs, 98

Index Gibbs free energy, 98 girls, 235 gland, 52, 119, 176, 179, 184, 186, 189, 190, 196, 197, 206, 207, 221 glass, 102 glucose, 58, 64, 67, 132, 140, 143, 147, 154, 159, 192, 193, 199, 206, 210, 236 glucose metabolism, 143 glucose regulation, 147 glucose tolerance, 140, 192, 199 glutamic acid, 50 glycans, 131, 148 glycemic index, 57 glycerol, 105, 106, 188, 209 glycoconjugates, 131 glycol, 176 glycoside, 26 glycosides, 4 glycosylation, 132, 146 goals, 222 goat milk, 45, 56 goblet cells, 56, 59, 132 gold, xi, 215, 217 gold standard, xi, 215, 217 Gore, 226, 242 grain, 16, 18, 48, 50, 51, 55, 61, 62, 63, 65, 66, 182, 183, 185, 186, 194, 204, 205 grains, vii, 1, 4, 6, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 55, 57, 58, 60, 61, 62, 63, 64, 66, 68, 97, 178, 182, 183, 184, 187 Gram-negative, 52, 132 Gram-positive, 132 grants, 22, 144 graph, 98 grass, 113, 178, 182, 183, 187, 195, 200, 204, 205 grazing, 182, 183, 184, 185, 187 Great Britain, 225 Greece, 20 groups, 27, 37, 55, 88, 118, 128, 141, 143, 154, 191, 220, 227, 228, 229, 231, 232, 236 growth, vii, ix, x, xi, 3, 17, 29, 52, 53, 55, 62, 64, 65, 97, 99, 101, 102, 104, 105, 110, 111, 115, 117, 118, 119, 126, 129, 131, 137, 141, 145, 150, 153, 154, 158, 163, 164, 165, 166, 170, 174, 184, 189, 193, 195, 197, 198, 208, 209, 210, 211, 215, 221, 222, 224, 228, 229, 232, 235, 237, 241 growth factor, x, 163, 211, 221, 222 growth rate, 110, 224, 228, 232 GST, 62 guidelines, 9, 14, 35, 36, 118, 180

259

Guinea, 193, 210 gut, ix, x, 13, 54, 55, 64, 68, 115, 116, 117, 118, 119, 121, 122, 123, 125, 127, 128, 129, 130, 131, 132, 133, 135, 136, 138, 139, 142, 144, 145, 146, 147, 148, 149, 150, 164, 218, 221, 222, 223, 225, 237, 239, 243, 245

H halos, 52 handling, 72 hanging, 36, 45 hardness, 75, 102, 112 harvest, 6, 131 hazards, 72 HDL, 56 healing, 66 health, vii, viii, x, xi, 1, 2, 3, 9, 14, 15, 18, 21, 23, 29, 33, 36, 37, 40, 41, 42, 43, 44, 49, 50, 54, 56, 64, 90, 111, 119, 123, 125, 126, 127, 129, 130, 134, 135, 136, 138, 141, 144, 153, 163, 164, 165, 171, 173, 176, 187, 191, 194, 196, 213, 215, 223, 226, 229, 230, 244 health care, 44 health effects, 14, 56, 119, 123, 141, 194 health status, 129, 135, 171 heart, viii, 2, 33, 34, 36, 42, 87, 88, 192 Heart, 30, 35, 38, 42 heart disease, 33, 36, 88, 192 heat, viii, x, 71, 72, 73, 74, 79, 82, 83, 84, 85, 90, 101, 137, 142, 150, 153, 154, 160, 233, 234, 244 heat transfer, 72, 74, 79 heating, 23, 72, 73, 74, 77, 79, 82, 92, 94, 164, 233 heating rate, 92 height, 96, 101, 131 Helicobacter pylori, viii, 43 helium, 27 hematological, 198 hematology, 174 hematopoietic, 174 hematopoietic stem cell, 174 hepatocyte, 132 hepatocytes, 11, 161 heredity, 126 herpes simplex virus type 1, 134 heterogeneous, 105 heterozygote, 13 heterozygotes, 13, 36 hexane, 26, 27 high fat, 2, 16, 23, 126, 188

260

Index

high pressure, 161 high risk, 88, 117, 142 high temperature, 22 high-fat, 135, 148 high-level, 225 high-risk, 141, 142, 228, 246 high-risk populations, 228 histamine, 188, 193, 198, 210 histological, 59 HIV, 217 HLA, 234 Hm, 99 Holland, 223, 243 homeostasis, ix, 54, 55, 115, 117, 150, 171, 173 homogeneity, 154 homogenized, 233 hormones, 30, 119, 205, 208, 222 hospital, 246 hospitalization, 128 hospitalized, 128, 150, 246 host, ix, x, 44, 55, 58, 61, 125, 127, 130, 131, 132, 135, 137, 141, 144, 146, 150, 151, 153, 164, 173, 226, 229 hot water, 73, 76, 77, 82 household, 64, 150 HPLC, 25, 28, 90 HR, 56 human, vii, x, xi, 2, 4, 9, 12, 35, 37, 38, 39, 41, 44, 52, 56, 58, 59, 61, 64, 65, 105, 116, 118, 119, 121, 122, 124, 126, 130, 131, 134, 136, 137, 141, 146, 148, 149, 151, 155, 158, 159, 161, 163, 164, 165, 172, 173, 175, 176, 177, 187, 189, 191, 192, 194, 195, 197, 206, 210, 211, 212, 215, 217, 218, 220, 221, 222, 223, 225, 226, 229, 231, 232, 233, 237, 239, 240, 241, 242, 243, 244, 245, 246, 247 human immunodeficiency virus, 245 human milk, 116, 118, 119, 121, 122, 124, 130, 146, 148, 177, 212, 217, 220, 221, 222, 223, 226, 229, 231, 232, 237, 239, 240, 241, 242, 243 human subjects, 35, 56, 189, 191, 192, 210 humans, x, 9, 13, 22, 38, 39, 56, 57, 60, 66, 126, 131, 136, 139, 157, 160, 176, 180, 187, 189, 190, 191, 192, 193, 194, 195, 196, 206, 209, 233, 246 humoral immunity, 128, 146, 147, 221 Hungary, 20 hybridization, 227, 230 hydro, 105, 106, 111 hydrocarbon, 27 hydrodynamic, 79 hydrogen, 52, 53, 57

hydrogen peroxide, 52, 53 hydrogenation, ix, 27, 88, 89, 178, 200 hydrolysis, 11, 13, 24, 27, 52, 61, 200, 241 hydrolyzed, 105, 179 hydrophilic, 105, 106, 111 hydrophobic, 2, 10, 106 hydrophobicity, 68 hydroxide, 26 hydroxyl, 3, 4, 27, 154 hydroxyl groups, 27 hygiene, 123, 150 hygienic, 126, 127, 136, 144, 239 hyperbolic, 101 hypercholesterolemia, 34, 135 hyperglycemia, 58, 135 hyperlipidemia, 135 hyperplasia, 30, 42, 131, 137, 207 hyperproliferation, 9 hypersensitivity, 65 hypertension, 44 hypertensive, 67 hypocholesterolemic, viii, 2, 9, 11, 43, 56, 60 hypothesis, ix, 123, 125, 126, 127, 128, 130, 134, 136

I ice, 16, 18, 111, 136, 186, 191, 202, 234 id, viii, 24, 71, 213, 228, 237 identification, 25, 28, 34, 48, 63, 68, 121, 242, 243, 244 IFN, 133, 137, 221, 231, 234 IgE, 56, 59, 134, 139, 142, 145, 221, 234 IGF, 193, 211 IGF-1, 193, 211 IgG, 55, 128, 134, 221, 234 IHD, 44 IL-1, 54, 119, 133, 137, 221, 231, 234 IL-10, 54, 119, 133, 137, 221, 231, 234 IL-13, 221 IL-2, 221 IL-4, 221 IL-6, 221 IL-8, 133 ileum, 155 images, 109, 110 immune cells, 52, 133, 193, 194, 217, 221, 222 immune function, x, 55, 121, 125, 127, 176, 193, 245 immune regulation, 238

Index immune response, 30, 52, 53, 55, 57, 68, 117, 119, 123, 126, 130, 133, 134, 140, 141, 142, 145, 165, 171, 187, 193, 198, 210, 221, 225, 235, 241, 244, 245 immune system, viii, ix, xi, 43, 44, 51, 53, 54, 55, 62, 65, 115, 117, 119, 127, 131, 133, 135, 136, 137, 165, 173, 193, 215, 217, 220, 221, 222, 226, 227, 232, 233, 238, 245 immunity, 54, 55, 67, 68, 122, 130, 132, 133, 134, 144, 145, 148, 193, 194, 221, 225, 243, 244 immunodeficiency, 245 immunoglobulin, 133, 134, 193, 211 immunological, ix, xi, 115, 117, 121, 128, 136, 148, 215, 242 immunomodulation, 50 immunomodulatory, 149, 226, 234, 235 immunosuppressive, 51 impaired glucose tolerance, 192, 199 implants, 75 implementation, viii, 14, 72, 73 impurities, 97, 105 in situ, 227, 230 in situ hybridization, 161, 227, 230 in utero, 116 in vitro, 9, 10, 30, 37, 53, 61, 68, 105, 147, 148, 155, 165, 173, 194, 201, 202, 234 in vivo, 10, 30, 54, 120, 146, 147, 165, 172, 190, 208, 233 incidence, viii, 59, 71, 72, 79, 134, 140, 142, 143, 216, 224, 231, 232, 235, 244 inclusion, vii, 44, 50, 60, 183, 223, 235 incubation, 56, 233, 236 India, 89, 114, 245 Indian, 89, 206 indication, 19, 135 indigenous, xi, 145, 150, 215, 223, 228 Indigenous, 146, 149 indirect effect, 53, 232 induction, x, 97, 98, 99, 101, 102, 105, 106, 110, 111, 133, 137, 138, 163, 222, 234 induction period, 98, 101 induction time, 97, 98, 99, 101, 102, 105, 106, 110, 111 industrial, 21, 40, 45, 47, 106, 226 industrial application, 106 industrial processing, 21 industrialization, ix, 116 industry, vii, viii, 1, 14, 17, 21, 72, 73, 75, 76, 82, 84, 85, 164 inertness, 75

261

infancy, ix, 115, 117, 121, 123, 129, 143, 146, 149, 221, 232, 241, 245, 247 infant colic, 141 infant formulas, ix, xi, 116, 119, 120, 121, 136, 215, 218, 226, 229, 232, 239, 246 infant mortality, 224 infants, ix, x, 44, 57, 60, 115, 116, 117, 119, 121, 122, 123, 126, 127, 128, 129, 135, 136, 138, 139, 140, 141, 142, 144, 145, 146, 148, 150, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 Infants, 117, 127, 138, 217, 220, 224, 225, 226, 227, 228, 229, 230, 235, 236, 239 infection, viii, 43, 45, 57, 59, 139, 141, 147, 171, 224, 243 infections, x, 52, 65, 125, 127, 132, 134, 136, 139, 141, 142, 148, 149, 154, 217, 222, 224, 242, 247 infectious, ix, 115, 117, 118, 119, 121, 122, 126, 174, 216, 217, 223, 224, 229, 232, 233, 247 infectious diseases, ix, 115, 117, 126, 216, 217, 223, 247 inflammation, 30, 56, 121, 137, 145, 147, 173, 193, 194, 225, 227 inflammatory, 29, 30, 55, 59, 60, 63, 66, 132, 133, 137, 141, 142, 154, 165, 172, 174, 193, 221, 229, 232, 234 inflammatory bowel disease, 154 inflammatory responses, 133, 172 influence, 179, 182, 183, 184, 186, 193, 204 infrared, 90, 211 infrared spectroscopy, 90 ingestion, vii, 1, 3, 5, 57, 158, 159, 160, 231 inhibition, 11, 13, 35, 51, 52, 55, 57, 58, 60, 91, 133, 149, 161, 165, 166, 179, 187, 188 inhibitor, 35, 168, 169 inhibitory, 50, 173, 193, 197, 207, 238 inhibitory effect, 50, 173, 197, 238 inhospitable, 224 initiation, 51, 189 injection, 198, 235 innate immunity, 55, 68, 146, 243 innovation, 2, 164 Innovation, 144 inoculation, 45 Inspection, 77, 78 instability, 127 instruments, 75 insulin, 58, 67, 132, 158, 192, 211, 222 insulin signaling, 58

262

Index

insulin-like growth factor, 211, 222 integrity, viii, 71, 78, 81, 119, 131, 132 interaction, 68, 75, 97, 102, 133, 147, 170 interactions, ix, 44, 97, 125, 131, 146, 150, 172 interest, 187 interface, 57, 98, 127, 134, 148, 150 interference, 2, 33 interferon, 147, 148, 221, 234 interferon (IFN), 234 interleukin-1, 147 interleukins, 221 interval, 97 intervention, ix, x, 115, 117, 121, 126, 127, 130, 141, 142, 143, 144, 153, 154 intervention strategies, 130 intestinal flora, xi, 57, 121, 215, 217, 220, 222, 227, 233, 235, 238, 239, 242, 246, 247 intestinal tract, 21, 58, 116, 135, 217, 241 intestinal villi, 131 intestine, ix, x, 56, 58, 59, 115, 116, 117, 118, 125, 127, 128, 130, 131, 132, 135, 136, 138, 142, 146, 147, 150, 154, 156, 161, 179, 218, 220, 221, 222, 226, 232, 237, 240, 242 intragastrically, 137 intramuscular, 185, 205, 206 intravenous, 206 inulin, 136, 230, 231, 245 invasive, 220 Investigations, 150, 234 ion implantation, 73, 84 ionization, 25, 92, 177 ionizing radiation, 59 ions, 132 Ireland, 20 iron, x, 45, 51, 148, 159, 163, 164, 171, 172, 173, 237 iron deficiency, 173 irradiation, 60 irritability, 224, 232 irritable bowel syndrome, 117 isolation, ix, 34, 115, 116, 118, 120, 121, 173, 240 isoleucine, 50 isomerization, 161, 176, 178, 179 isomers, xi, 175, 176, 177, 179, 180, 181, 185, 186, 191, 192, 196, 197, 199, 200, 202, 204, 206, 208, 210, 212 isoprenoid, 3 isothermal, 110 isotopes, 160 ISPA, 163

Italy, 20, 163, 225

J Japan, 23, 44, 61, 62, 153, 158 Japanese, 62, 65, 66, 159 jejunum, 155 JNK, 169, 172 Jun, v, 77, 84, 153

K kappa, 133 kappa B, 133 kernel, 89 kidney, 155 kinase, 58, 132, 166, 173 kinetics, 35, 39, 66, 99, 102, 105, 110, 112, 114, 209 King, 147 knowledge, 211 Korean, 67

L LAB, 50, 53, 56, 57, 117, 118, 119 labeling, 32, 88, 111 labor, 141 labour, 116, 149 lactase, 131 lactating, x, 126, 136, 142, 149, 178, 179, 183, 200, 201, 202, 203 lactation, 116, 117, 119, 121, 122, 127, 136, 139, 141, 142, 145, 148, 185, 204, 241 lactic acid, viii, ix, 43, 44, 45, 46, 53, 58, 59, 60, 62, 64, 65, 66, 67, 68, 115, 117, 119, 122, 156, 220, 225, 233, 242, 246 lactic acid bacteria, ix, 44, 45, 53, 58, 59, 62, 64, 65, 66, 67, 68, 115, 117, 122, 220, 225, 233, 242, 246 lactobacillus, 52 Lactobacillus, 45, 49, 51, 57, 58, 60, 61, 63, 64, 65, 66, 68, 69, 116, 117, 118, 121, 122, 123, 129, 135, 136, 139, 141, 145, 146, 147, 148, 149, 150, 153, 158, 220, 223, 225, 228, 230, 242, 243, 245, 247 lactoferricin, x, 163, 164, 174 lactoferrin, 130, 164, 165, 172, 173, 174 lactoglobulin, 164, 165, 172, 228 lactoperoxidase, 164

Index lactose, x, 57, 61, 62, 63, 153, 154, 158, 160, 161, 229, 233, 236 lactose intolerance, 57 lamina, 133 large intestine, 54, 55, 148, 154, 156 large-scale, 129 laser, 106 latex, 173 Latvia, 20 LDL, 2, 9, 12, 13, 14, 18, 19, 24, 25, 28, 39, 41, 42, 208 lead, 225, 229 leakage, 132 lean body mass, 176, 191 lecithin, 23, 24, 38, 41, 45, 105 lectin, 145, 146 left ventricle, 33 legislation, 15 leptin, 192, 208 lesions, 137, 157, 189, 206, 207 Leuconostoc, 44, 46, 49, 51, 118 leukemia, 172 leukocytes, 66, 164, 246 life expectancy, 2, 44 life quality, 2 lifestyle, 2, 126, 135, 144, 239 ligands, 149, 173 light beam, 98 limitations, 2, 22, 24, 187 linear, 184 linoleic acid, xi, 88, 175, 176, 177, 178, 180, 181, 184, 187, 188, 190, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 222 linolenic acid, 45, 56, 140, 143, 202 lipase, 89, 132, 191 lipases, 11, 179, 200 lipid, 22, 24, 25, 26, 31, 33, 34, 35, 38, 41, 60, 67, 91, 97, 102, 114, 132, 135, 148, 154, 159, 161, 178, 182, 186, 189, 202, 205, 210, 213 Lipid, 26, 29, 35, 36, 37, 39, 40, 41, 42, 59, 111, 112, 113, 114, 200, 202, 208, 210, 212 lipid metabolism, 34, 38, 41, 154, 159, 161, 205, 210 lipid peroxidation, 213 lipid profile, 31, 33, 41, 135, 148 lipids, 34, 36, 37, 41, 42, 46, 61, 64, 91, 102, 114, 132, 135, 143, 159, 178, 189, 200, 205, 206, 208, 211, 233 lipodystrophy, 213 lipolysis, 10, 191

263

lipophilic, 105 lipopolysaccharide, 55, 146 lipoprotein, 31, 35, 157, 192, 206 lipoproteins, 37, 199, 208 liposomes, 23, 171 liquid chromatography, 25, 28, 29, 39, 42, 90, 177, 199 liquid phase, 109 liquids, 90 Listeria monocytogenes, 52, 62 literature, 229, 231 Lithuania, 20 liver, 39, 56, 59, 132, 135, 137, 155, 161, 179, 188, 213 liver disease, 135 liver spots, 59 Livestock, 205 London, 65, 240, 245 losses, 27, 120, 191 Louisiana, 71 Louisiana State University, 71 low birthweight, 242 low density polyethylene, 50 low temperatures, 50 low-density, 2, 35, 157 low-density lipoprotein, 2, 35, 157 LPO, 62 LPS, 55, 172, 234 lubricants, 106 lumen, 11, 24, 119, 155 luminal, 147, 155, 156. 193 lupus, 188, 213 lupus erythematosus, 213 Luxembourg, 20 lymph, 11, 133, 137, 234 lymph node, 133, 137, 234 lymphatic, 119 lymphocyte, 126, 188, 198, 221, 235, 240 lymphocytes, 141, 221, 234 lymphoid, 133, 138, 221 lymphoid organs, 138 lymphoid tissue, 133, 221 lysine, 50, 131 lysozyme, 217

M M.O., 173 machinery, 132, 155 macromolecules, 132

264

Index

macrophage, 198 macrophages, 54, 55, 133, 194, 221 Madison, 113, 114 magnesium, 45, 51, 160 magnetic, 92 mainstream, vii maintenance, 2, 17, 18, 136, 227 major histocompatibility complex, 133 malabsorption, 159 males, 2 malignancy, 60 Malta, 20 maltodextrin, 233 Mammalian, 173 mammals, x, 2, 125, 130 management, xi, 35, 36, 58, 143, 171, 174, 175, 184, 186, 196, 244 management practices, 196 manganese, 51 Manganese, 48 mango, 89 Manhattan, 84 manipulation, 136, 201 manufacturer, 92 manufacturing, 4 MAPK, 168, 169 margarine, 16, 18, 22, 30, 38, 41, 90, 92, 94, 95, 105, 112 market, 14, 15, 16, 17, 18, 20, 23, 32, 33, 75, 89, 119, 180, 181 markets, 3 Mars, 163 mass, 176, 177, 191, 192, 199, 209 mass spectrometry, 25, 29, 177 mass transfer, 101 mast cells, 137 mastitis, 116, 118, 119, 121, 122 maternal, ix, 116, 118, 119, 121, 125, 127, 128, 129, 134, 135, 137, 138, 139, 141, 142, 143, 144, 145, 146, 147 maternal care, 121 maternofetal, 149 Matrices, 23 matrix, 22, 23, 24, 25, 26, 28, 44, 50, 85, 119, 136 maturation, ix, 49, 115, 117, 118, 123, 128, 131, 132, 133, 146, 174, 220, 221, 222, 226, 228, 232, 235, 238, 242, 245 maturation process, 131, 238 MBP, 173 MCA, 157

meals, 24, 183 measurement, 96, 97, 112 measures, 96, 223, 236 meat, xi, 18, 64, 175, 176, 180, 181, 185, 186, 187, 189, 196, 200, 205, 209, 211, 212 mechanical properties, 96 meconium, 116, 122 media, 116, 119, 161, 216, 236 median, 219 mediators, 194, 211 medication, 19 medicine, 2, 45 melanoma, 59, 61, 65 melon, 7 melt, 107 melting, ix, 88, 89, 91, 92, 93, 94, 95, 96, 98, 99, 106, 108 melting temperature, 90, 92, 98, 99 membranes, 3, 24, 208 men, 31, 35, 39, 40, 61, 67, 160, 161, 191 meningitis, 228, 245 meta-analysis, 12, 14, 29, 242 metabolic, ix, 44, 52, 125, 126, 131, 134, 135, 136, 141, 143, 205, 209, 210, 243 metabolic disorder, x, 126, 135, 136 metabolic systems, 135 metabolism, 13, 15, 31, 34, 36, 37, 38, 39, 41, 66, 67, 135, 143, 144, 154, 159, 160, 161, 188, 190, 192, 198, 200, 202, 205, 209, 210, 211, 237 metabolites, 3, 39, 53, 197, 207, 208, 244 metal ions, 132 metastasis, 190, 197 metastatic, 61, 169, 170 methanol, 10, 26, 27, 58 methionine, 50 methylation, 199 methylene, 26 methylene chloride, 26 mice, 39, 42, 51, 54, 55, 57, 58, 59, 61, 62, 64, 65, 66, 67, 121, 130, 131, 132, 133, 135, 137, 144, 146, 147, 150, 159, 172, 176, 188, 190, 191, 192, 193, 197, 198, 208, 209, 212, 213, 233, 234, 244, 246 micelles, 10, 23, 24, 25, 29, 37, 38, 170 microarray, 148 microbes, x, 126, 128, 130, 133, 135, 146, 149, 150, 163, 182, 183, 223 Microbes, 128, 144, 240

Index microbial, ix, x, 48, 53, 61, 64, 66, 68, 115, 117, 125, 127, 128, 129, 130, 134, 136, 148, 153, 164, 173, 179, 182, 186, 200, 220, 221, 226, 238 Microbial, 49, 64, 125, 133, 200, 202 microbiota, ix, 64, 115, 116, 117, 121, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 136, 138, 144, 145, 146, 147, 148, 149, 156, 158, 162, 218, 220, 222, 225, 226, 229, 232, 233, 239, 240, 242, 243, 244, 245, 246 Microbiota, 127, 131, 132, 138, 147, 149, 226 microflora, ix, xi, 30, 44, 45, 48, 50, 51, 52, 54, 55, 59, 60, 61, 64, 66, 68, 116, 117, 118, 119, 122, 145, 150, 154, 156, 159, 162, 165, 215, 218, 219, 220, 221, 224, 225, 226, 227, 230, 231, 232, 233, 239, 240, 241, 242, 243, 244, 245, 246, 247 Microflora, vi, 48, 49, 60, 215, 217, 229 microorganism, 179 microorganisms, ix, 44, 48, 51, 52, 53, 57, 63, 115, 117, 134, 153, 161, 179, 200, 227 micro-organisms, 130 microscopy, 102 microsomes, 201, 213 microstructure, ix, 88, 91, 95, 102, 110, 111, 113 microvasculature, 131, 132 microwave, 187 Middle East, 44 middle-aged, 31 migration, 81, 169 milk fermentation, xi, 54, 68, 216, 232, 233, 234, 239 milligrams, 194 minerals, viii, 43, 49, 154, 233 Ministry of Education, 158 mitochondria, 213 mitogenic, 55, 61 mitosis, 166 mixing, 89, 92 MMP-2, 169 mobility, 174 model system, 98, 112 modeling, 193, 211 models, x, 9, 41, 51, 126, 135, 159, 164, 176, 187, 191, 192, 194, 195, 196, 200, 223, 233, 234 modulation, 30, 66, 121, 130, 133, 158, 173, 190, 198, 207, 239, 242, 243, 246 modulus, 96 moisture, 46, 120 moisture content, 120 molasses, 45, 55 molecular biology, 218

265

molecular mechanisms, 164 molecular structure, 111 molecules, 2, 10, 58, 98, 99, 101, 130, 133, 222 molybdenum, 51 momentum, 77 monolayer, 155, 234 monolayers, 123 mononuclear cells, 137, 142, 147, 172, 211, 243 monosaccharides, 132, 160, 229 monounsaturated fat, 143 monounsaturated fatty acids, 143 monozygotic twins, 130 morbidity, 141, 216 morphogenesis, 206 morphological, 131 morphology, 95, 102, 103, 110 mortality, 2, 126, 216, 224, 247 Moscow, 61, 63, 173 mothers, x, 118, 119, 126, 129, 134, 136, 138, 139, 141, 142, 144, 147, 149, 217, 235, 243 moths, 119 mouse, 30, 55, 59, 63, 64, 145, 151, 165, 188, 198, 207, 210, 213, 234 mouse model, 30, 56, 188, 234 mouth, 128 mRNA, 137, 179, 201 MRS, 119, 120, 236 mucin, 131, 132, 145, 148 mucosa, 119, 130, 131, 132, 159, 197, 207, 221, 222, 225, 227 mucosal barrier, 136 mucus, 56, 59, 131, 164, 225 mucus hypersecretion, 56, 59 multiplication, 58 murine model, 54, 120, 173 muscle, 2, 9, 67, 132, 185, 186, 191, 192, 204, 205, 206, 210, 212, 213 muscle cells, 9, 67 muscle weakness, 2 mutagenic, 42, 62 mutation, 36, 190, 207 mutations, 13

N Na+, 132 N-acety, 229 National Research Council, 125, 163

266

Index

natural, vii, ix, 1, 3, 4, 12, 14, 23, 26, 41, 45, 68, 73, 91, 95, 115, 121, 141, 147, 164, 176, 177, 180, 187, 196, 228, 229 natural food, 12, 187 natural killer cell, 141 natural resources, 73 necrosis, 172, 221 needs, 194 neonatal, ix, 61, 115, 117, 122, 130, 148, 217, 221, 222, 243, 247 neonate, xi, 121, 123, 127, 130, 135, 215, 220, 221, 222, 231, 232, 235, 237, 239 neonates, xi, 116, 118, 121, 123, 128, 131, 138, 139, 145, 146, 147, 215, 218, 219, 220, 226, 235, 246 neoplasia, 212 Netherlands, 20, 38, 65, 67, 113, 218 network, 96, 97, 102 neuroblastoma, 165 neutralization, 6 neutrophils, 221 New England, 63 New Jersey, 84 New York, 36, 37, 38, 39, 40, 63, 64, 111, 112, 113, 114, 200, 202, 244 New Zealand, 16, 203 Newton, 97 Ni, viii, 71, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 85 Niacin, 48 nickel, 73, 75, 78, 84 Nielsen, 208 nitric acid, 78 nitric oxide, 165, 172 nitride, 75 nitrogen, 26 NMR, 92, 93 nodes, 234 non-alcoholic fatty liver, 135 non-enzymatic, 213 nonionic, 105 nonparametric, 236 nontoxic, 105 Norfolk, 39 normal, ix, xi, 2, 6, 13, 24, 50, 55, 57, 60, 61, 77, 105, 115, 117, 123, 159, 164, 175, 186, 187, 189, 193, 194, 224, 235, 246 normal conditions, 186, 187 North Africa, 44 North America, 44, 122 N-terminal, 164 nuclear, 92, 133, 147

nuclear magnetic resonance, 92 nucleation, 97, 99, 102, 104, 105, 106, 110, 111 nuclei, 97, 99, 105, 111 nucleus, 3, 97 nursing, 118 Nusselt, 74 nutraceutical, 171 nutraceuticals, vii, xi, 163, 164, 173, 174 nutrient, vii, 88, 111, 146, 173, 198, 210, 233, 235 nutrients, viii, ix, 37, 59, 87, 88, 115, 118, 121, 135, 140, 143, 208, 242 nutrition, vii, viii, ix, xi, 2, 14, 34, 38, 39, 62, 87, 88, 111, 125, 127, 135, 143, 144, 145, 215, 217, 222, 232, 239, 240, 242 nuts, 4, 6, 8, 14, 39

O oat, 68, 157 obese, 191, 192, 208, 209, 210 obesity, 126, 134, 135, 144, 149, 192, 193, 209, 217 obligate, 13, 36 obligation, 19 Ohio, 85 oil, ix, 4, 6, 8, 10, 16, 18, 21, 22, 23, 26, 30, 31, 32, 33, 35, 36, 42, 88, 89, 92, 93, 94, 95, 96, 99, 102, 105, 107, 108, 110, 112, 145, 171, 178, 182, 183, 184, 185, 187, 191, 193, 202, 203, 204, 205, 206, 209, 212 oils, vii, ix, 1, 4, 5, 6, 8, 16, 18, 21, 22, 23, 25, 26, 29, 34, 88, 89, 92, 176, 178, 182, 183, 185, 189, 196 oligofructose, 158, 159, 231, 245 oligomerization, 150 oligosaccharide, 141, 142, 230, 232, 240, 241, 243 oligosaccharides, 118, 129, 136, 139, 141, 142, 147, 148, 150, 154, 157, 159, 160, 217, 229, 230, 231, 232, 240, 241, 243, 244 Oligosaccharides, 216, 241, 243 olive, 4, 171, 191 Oman, 201 omega-3, 145, 193 omega-6, 194 online, 33, 34, 173 Opioid, 173 optimal health, vii oral, 21, 35, 51, 62, 67, 68, 119, 128, 133, 135, 137, 138, 141, 150, 165, 192, 209, 221, 225, 226, 227, 233, 234, 235, 244, 247 orange juice, 24, 31

Index organic, 27, 52, 53, 62, 156 organic solvents, 27 organism, 50, 133, 202 osmotic, 224 osteoblastic cells, 193 osteoporosis, vii, 194 otitis media, 216 output, 183 ovarian cancer, 37 ovariectomized, 159 overweight, 191, 209, 240, 242 oxidation, 9, 26, 36, 173, 191, 208 oxidation products, 36, 173 oxidation rate, 191 oxidative, 59 oxidative damage, 59 oxide, 75, 165, 172 oxides, 31, 35 oxygen, 26, 164

P p38, 168, 169 PA, 71, 75 Pacific, 84 Pakistan, 43, 65 palm oil, 4, 89, 95, 99, 107, 110 pancreas, 137 pancreatic, 24, 105, 131, 132 pantothenic acid, 50 paper, 227, 228, 230, 232 parameter, 97 parasite, 133 parents, 129 Paris, 113, 246, 247 PARP, 168 particles, 75, 78, 85 passive, 155 pasta, 16, 18 pasteurization, viii, 71, 72, 73, 76, 77, 79, 81, 82, 83, 84, 170 pasture, 178, 182, 183, 184, 185, 186, 194, 195, 201, 204 pathogenesis, 61 pathogenic, 44, 52, 116, 119, 128, 133, 134, 158 pathogens, 52, 53, 58, 68, 121, 133, 141, 148, 217 pathology, 13 pathways, 132, 155, 165, 168, 179 patients, 13, 19, 34, 37, 44, 57, 60, 67, 117, 159, 174, 190, 192, 223, 225, 226, 227, 245

267

PCR, 121, 129, 134, 137, 146, 148, 156, 216, 230, 236, 242, 244, 245 pectin, 241 pediatric, 135, 242, 245 Pennsylvania, viii, 71, 73 peptide, x, 163, 164, 172 Peptide, 54 peptides, 51, 52, 67, 132, 164, 172 perinatal, x, 125, 127, 135, 137, 143 peripheral blood, 139, 141, 211, 221, 243 peripheral blood mononuclear cell, 211, 243 peristalsis, 127 peritoneal, 54, 55 permeability, x, 2, 3, 50, 117, 123, 125, 127, 132, 136, 137, 227, 229, 243 permeation, 67 peroxidation, 213 Peroxisome, 207 personal, 238 pertussis, 236 PGE, 198, 210 pH, 45, 120, 127, 156, 157, 171, 182, 186, 233, 235, 237, 242, 243 pH values, 235 phagocytic, 54 pharmaceutical, 21, 91, 136 pharmaceuticals, 105 pharmacological, 2, 55 phenotype, 126, 134, 142, 169 phenotypes, 203 phenotypic, 174 phenylalanine, 50 phorbol, 190, 207 phosphate, 78, 85 phospholipids, 10, 23, 91, 157, 164 phosphorus, 51, 73, 75 phosphorylation, 168, 169 photographs, 102 physical chemistry, 31 physical properties, 21, 22, 89, 95, 102, 105 physicochemical, ix, 63, 88, 89 physicochemical properties, ix, 88, 89 physiological, vii, 57, 58, 62, 126, 136, 141, 164, 187, 190, 193, 194, 196, 200, 212, 241 physiology, 127, 131, 138, 144 phytochemicals, 37 phytohaemagglutinin, 55 phytosterols, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42

268

Index

pig, 193, 199, 210 pigs, 33, 150, 176, 188, 191, 193, 208, 209, 210, 213 pilot study, 123, 138, 159, 247 pitch, 21 placebo, 35, 121, 137, 138, 141, 142, 143, 147, 158, 191, 224, 231, 235, 244 placenta, 134, 137, 149 placental, 134, 137, 140, 143, 146, 147 planning, 141 plant sterols, vii, 1, 2, 3, 4, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 21, 22, 29, 30, 31, 36, 37, 38, 39, 40, 41, 42 plants, 3, 4, 29, 31, 72 plaque, 187, 188, 190, 192 plasma, x, 12, 13, 27, 34, 35, 38, 39, 41, 42, 67, 72, 84, 137, 153, 157, 158, 159, 189, 192, 199, 201, 202, 205, 206, 209 plastic, 89, 102 plasticity, 90, 203 play, 9, 59, 118, 130, 217 Poland, 20, 63 polarity, 27 polarization, 134 poliovirus, 238, 244 pollen, 173 polyesters, 106 polyethylene, 50 polymer, 75, 78 polymer-based, 75, 78 polymerization, 106 polymorphism, ix, 88, 91, 94, 95, 105, 111, 112, 114, 219 polymorphonuclear, 164 polysaccharide, 44, 55, 59, 63, 65, 66 polysaccharides, 50, 51, 55, 131, 171 polyunsaturated fat, 45, 89, 126, 143, 183, 190, 203, 212 polyunsaturated fatty acid, 45, 89, 126, 143, 183, 190, 203, 212 polyunsaturated fatty acids, 45, 89, 126, 143, 183, 190, 203 population, 9, 13, 39, 48, 56, 57, 58, 61, 68, 117, 129, 142, 143, 148, 157, 179, 182, 186, 228, 229, 230, 233, 237, 238, 239 pork, 181 Portugal, 1, 20 positive correlation, 13 postmenopausal, 190, 197 postmenopausal women, 197 postpartum, 119, 120, 140, 143

postpartum period, 143 potassium, 26, 51 poultry, 194, 195, 211 powder, x, 21, 41, 84, 120, 163, 170, 224, 226, 227, 235 power, 62 PPARγ, 208, 210 Prandtl, 74 prebiotics, x, xi, 126, 135, 136, 147, 154, 158, 159, 160, 215, 226, 229, 231, 232, 242, 245, 247 precipitation, 30 preclinical, 126, 136, 138 preference, 44, 60, 148 pregnancy, ix, 125, 126, 134, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149 pregnant, ix, 19, 125, 126, 135, 136, 138, 140, 141, 142, 143, 150, 173 pregnant women, 136, 138, 140, 141, 142, 143, 150, 173 premature babies, 239 premature infant, viii, 43 prematurity, 128 premenopausal, 190 preparation, 233 pressure, 59, 60, 64, 72, 92, 140, 143, 144, 161, 217 preterm infants, 117, 122, 123, 128, 150, 173, 227, 233, 240, 243, 244, 246, 247 prevention, vii, 1, 3, 9, 14, 26, 35, 51, 52, 63, 117, 121, 123, 130, 135, 136, 138, 141, 142, 143, 144, 147, 149, 158, 164, 187, 196, 197, 211, 217, 222, 227, 241, 242, 246, 247 preventive, 142, 143, 239 primates, 135, 148 priming, xi, 215 principle, xi, 175, 176 probe, 96 probiotic, ix, x, xi, 44, 46, 58, 62, 63, 65, 68, 115, 117, 119, 120, 121, 122, 123, 126, 127, 130, 136, 137, 138, 139, 141, 142, 143, 144, 145, 147, 148, 149, 150, 153, 206, 215, 223, 224, 227, 228, 246, 247 Probiotics, v, 44, 63, 117, 122, 123, 125, 135, 145, 147, 149, 158, 226, 244 procedures, 235 producers, 111 production, viii, 21, 45, 49, 50, 54, 55, 61, 62, 63, 66, 69, 71, 90, 119, 130, 133, 146, 159, 165, 171, 172, 174, 182, 184, 185, 188, 193, 200, 201, 202, 207, 211, 221, 222, 225, 231, 236, 243 production costs, viii, 71

Index prognosis, 190 program, 83, 134, 143, 164, 235 programming, ix, 125, 126, 127, 135, 136, 143, 144 proinflammatory, 67 pro-inflammatory, 133, 137, 141, 165, 234 pro-inflammatory response, 133 prokaryotic, 119 prokaryotic cell, 119 proliferation, viii, xi, 43, 44, 54, 55, 60, 61, 142, 157, 162, 164, 166, 174, 188, 190, 197, 207, 215, 221 promote, xi, 215, 221, 227 property, viii, 43, 73, 75, 105, 120, 164, 171 prophylactic, 65, 128, 143, 172, 232 propionic acid, 46, 66, 157, 237 propylene, 176 prostaglandin, 190, 193, 211, 213 prostate, 29, 188 prostate cancer, 188 proteases, 164 protection, 9, 50, 60, 149, 164, 170, 188, 194, 216, 217, 233 protective mechanisms, 137 protective role, 192 protein, viii, x, 23, 31, 35, 43, 45, 46, 49, 50, 52, 54, 57, 58, 60, 68, 72, 84, 132, 133, 163, 164, 166, 168, 172, 173, 174, 190, 203, 209, 211, 231, 235, 241 protein denaturation, 72 proteins, vii, x, 11, 15, 16, 23, 35, 45, 49, 50, 51, 62, 72, 123, 133, 146, 163, 164, 165, 166, 169, 172, 173, 233, 241 Proteobacteria, 156 proteolysis, 169 proteomics, 164 protocol, 235 protocols, x, 76, 153 Pseudomonas, 53, 118 Pseudomonas aeruginosa, 53 PTFE, viii, 71, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 85 puberty, 205 public health, 56 PUFA, ix, 88, 89, 183, 190 purification, 154 pyrene, 212 pyridoxine, 49 pyruvic, 46

269

Q qualitative differences, ix, 115, 117 quality of life, 14 Quebec, 111

R radiation, 60, 65 Radiation, 65 radio, 92 radius, 98 rain, 185 random, 111 range, 13, 14, 16, 52, 54, 77, 82, 90, 91, 95, 130, 180, 181, 185, 186, 191, 193 RAPD, 116, 119 rat, 29, 61, 136, 145, 155, 159, 160, 161, 162, 188, 192, 196, 197, 199, 206, 207, 208, 210, 213 rats, x, 31, 35, 41, 42, 55, 56, 57, 59, 60, 65, 67, 68, 121, 153, 154, 155, 156, 157, 159, 161, 162, 165, 173, 188, 189, 190, 191, 192, 193, 197, 206, 207, 208, 211, 213, 231, 243, 245 raw material, 6, 187 reactive oxygen, 59 reactive oxygen species, 59 reactivity, 58 reality, 35, 130, 141, 144 real-time, 230, 242, 245 receptors, 133, 170, 173, 207, 221 recognition, 133, 221 recovery, 41, 154 redox, 218 reducing sugars, 52 reduction, 182, 183, 184, 185, 187, 188, 190, 191, 197, 207, 217, 224, 234 regeneration, 60 regression, 9 regular, 102 regulation, 3, 11, 15, 16, 132, 133, 137, 147, 149, 150, 165, 166, 210, 217, 220, 221, 222, 223, 226, 231, 234, 238 regulations, vii, 1, 3, 16, 19 rehydration, 224, 235 reinforcement, 14 rejection, 126, 134 relationship, 13, 40, 41, 126, 130, 134, 184, 237, 238 relationships, 134, 146, 239 relatives, 13

Index

270

relevance, 97 remodeling, 55 repair, 60 replication, 224 repression, 237, 239 reproduction, 42 research, xi, 215, 244 research and development, 44 residential, 241 resin, 171 resistance, ix, x, 57, 59, 73, 75, 84, 116, 119, 120, 139, 144, 154, 155, 163, 192, 198, 223, 228 resolution, 25, 27 resources, 73, 79 respiration, 220 respiratory, 139, 141, 217 responsiveness, 42 restriction fragment length polymorphis, 219 retail, 211 retardation, 92 retention, 131 Reynolds, 74, 79 Reynolds number, 74, 79 rheological properties, 91, 95, 97, 110 riboflavin, 49 rice, 4, 33, 44, 90 risk, viii, ix, 2, 9, 13, 30, 31, 33, 36, 39, 44, 51, 56, 87, 88, 115, 117, 125, 126, 127, 134, 135, 139, 140, 141, 142, 144, 146, 147, 176, 190, 192, 197, 198, 206, 207, 217, 225, 226, 228, 232, 239, 246 risk factors, 192 RNA, 131 roasted coffee, 16, 18 rodents, 179 Romania, 20 room temperature, 21, 22, 23 ROS, 58 rotavirus, 50, 154, 224, 241, 246 roughness, 72 rubella, 134, 145, 146 rubella virus, 134, 145, 146 ruminant, xi, 175, 176, 178, 180, 181, 187, 196 Russia, 44 rye, 4, 17, 32

S S. thermophilus, 233, 234 SA, 235

safety, 13, 14, 15, 30, 35, 39, 40, 41, 118, 119, 141, 147, 164, 228, 246 salad dressings, 18, 32, 95 Salen, 13, 40 saliva, 164 Salmonella, 52, 53, 57 salt, 22, 29, 58 salts, 10, 23, 45, 204, 235 sample, 25, 26, 27, 28, 41, 96, 98, 99, 101, 102, 116, 119, 123, 229 sampling, 120, 230, 238 sand, 72 saponins, 3 SAS, 76 saturated fat, viii, 45, 87, 88, 89 saturated fatty acids, 88 savings, viii, 71, 79, 82, 84 scanning calorimetry, 94 scientific community, 3, 179 scores, 45 SD, 236, 237 search, 34 searching, 217 secrete, 55, 244 secretin, 221 secretion, 11, 24, 132, 142, 145, 148, 204, 221, 222, 234, 244 seed, 8, 26, 36, 92, 99, 102, 105, 182, 183, 201, 205 seeding, 58 seeds, 4, 6, 8, 14, 39, 89, 105, 178, 183, 186, 202, 212 selecting, 149 selectivity, 28 self-care, 45 semiconductor, 75 semimembranosus, 204 sensing, 133 sensitivity, 97, 134, 188 sensitization, x, 55, 67, 125, 127, 134, 140, 142, 147, 228, 246 separation, 27, 34, 199 sepsis, 229 septicemia, 239 sequencing, 154, 160 series, xi, 175, 179 serine, 50 serum, 30, 31, 33, 34, 35, 36, 37, 38, 39, 41, 56, 59, 60, 61, 67, 133, 157, 161, 189, 190, 192, 197, 206, 208, 211, 221 sesame, 14

Index severity, 41, 42, 57, 143, 150, 235 sex, 30 sex hormones, 30 shape, 101, 102, 105, 131, 146, 223 shaping, 126 shear, 78, 79, 96 sheep, 45, 56, 68, 179, 212 Shigella, 52, 53, 57 short-term, 38, 189, 205 shoulder, 95, 106 sialic acid, 229 sibling, 218 siblings, 127, 129, 218, 219 SIC, 125 side effects, 2, 36, 60 signaling, 58, 133, 147, 169, 210 signalling, x, 133, 163, 165 signals, 95, 106, 137 signs, 78, 81, 116 silica, 84 siloxane, 27 silver, 177, 199 similarity, 106, 111, 138 single crystals, 102 sites, 10, 52, 53, 54, 68, 130, 137, 164, 176 skeletal muscle, 132, 192 skin, 45, 59, 118, 119, 137, 148, 176, 188, 190, 207 skin cancer, 59 Slovakia, 20, 205 Slovenia, 21 small intestine, 137, 155, 179 smooth muscle cells, 9 software, 74 solid solutions, 94 solid state, 22 solid tumors, 51 solubility, 2, 10, 21, 22, 23, 24, 35, 156 solvent, 26, 89, 91, 97 solvents, 21, 27 South Africa, 64, 68 soy, xi, 18, 21, 39, 41, 45, 50, 51, 163, 212 soybean, 39, 44, 89, 176, 182, 183, 185, 202, 203, 205 soybeans, 184, 202, 203, 204, 205 Spain, 21, 60, 118, 125 Spearman rank correlation coefficient, 236 species, ix, xi, 48, 52, 58, 59, 63, 65, 115, 116, 117, 118, 128, 129, 136, 146, 161, 179, 203, 216, 218, 219, 220, 223, 226, 230, 236, 238, 239, 241, 242, 243, 245, 246

271

specificity, 25 spectrophotometric, 25 spectrophotometric method, 25 spectroscopy, 77 spectrum, viii, 43, 54, 60, 66, 80 speculation, 207 speed, 220, 226 S-phase, 173 spleen, 51, 137, 193 Sprague-Dawley rats, 31 squamous cell, 31 squamous cell carcinoma, 31 stability, viii, xi, 27, 41, 85, 87, 88, 91, 120, 163, 171, 174 stages, x, 49, 98, 126, 127, 135, 144, 190 stainless steel, viii, 71, 73, 74, 75, 85, 96 standard deviation, 107 Standards, 16 staphylococcal, 144 staphylococci, 117, 218, 220 Staphylococcus, 52, 118, 129, 148 Staphylococcus aureus, 52, 118, 148 statin, 31 statins, 2, 30 steel, viii, 71, 73, 74, 75, 78, 81, 85, 96, 154 stem cells, 59 sterile, ix, 115, 116, 117, 122, 127, 217 steroid, 2, 3, 161, 162 steroid hormone, 2 steroid hormones, 2 steroids, 157 sterols, vii, 1, 3, 4, 9, 10, 12, 13, 15, 16, 21, 24, 25, 26, 27, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 41, 91, 161 stock, 154 stomach, 29, 63, 105, 128 storage, xi, 46, 52, 57, 62, 63, 95, 97, 110, 120, 124, 132, 144, 163, 174, 175, 186, 187, 191, 206 strain, xi, 51, 53, 66, 96, 116, 117, 119, 122, 130, 137, 141, 148, 157, 160, 191, 216, 223, 228, 232, 243, 246 strains, ix, x, 50, 51, 53, 63, 66, 68, 115, 117, 119, 121, 122, 123, 126, 127, 141, 142, 144, 148, 149, 179, 201, 218, 223, 225, 228, 233 strategies, x, 2, 126, 127, 130, 136, 186, 205 strength, 75, 78, 198 streptococci, 59, 117, 118, 218, 220 Streptomyces, 41 stress, 96, 132, 148, 209, 242 subgroups, 143

272

Index

substitutes, 18 substitution, 16 substrates, xi, 3, 62, 215, 238 sucrose, 47, 105, 106, 109, 110, 111, 113, 154 suffering, 135, 173, 225, 226 sugar, 6, 45, 46, 50, 57, 154, 157, 159, 229, 237 sugars, 44, 52, 156, 232 sulfuric acid, 84 sulphate, 131 sulphur, 52 summer, 45, 184 Sun, 41 sunflower, ix, 31, 88, 89, 92, 93, 94, 95, 96, 99, 102, 105, 108, 176, 182, 183, 185, 191, 201, 205, 206 supercooling, 97, 99, 102, 104, 106 supercritical, 26, 91 supernatant, 55, 61 superoxide, 59 supplemental, 193 supplements, viii, xi, 17, 18, 87, 88, 171, 175, 178, 182, 185, 203, 226 suppliers, 4 supply, viii, 87, 88, 130, 135, 183 suppression, 51, 52, 157, 158 surface area, 131 surface energy, 75, 98 surface modification, 84 surface properties, 72, 85 surface roughness, 73 surfactant, 64 surgical, 75 surveillance, 14, 19, 242 survival, 127, 234, 236, 242 susceptibility, x, 125, 127, 236, 244 suspensions, 55, 120 sustainability, 41 Sweden, 21, 38, 191, 225 Switzerland, 39, 42, 111, 120 symbiotic, 48, 121 symbols, 100 symptom, 227 symptoms, ix, 115, 117, 227, 231, 243 synbiotics, 135 synergistic, 234 synergistic effect, 234 synthesis, x, xi, 3, 12, 13, 41, 56, 132, 153, 157, 160, 161, 165, 175, 178, 179, 182, 184, 185, 186, 188, 189, 193, 201, 202, 204 systemic immune response, 57 systems, 184, 236

systolic blood pressure, 217

T T cell, 133, 134, 138, 142, 146, 172, 221, 222 T lymphocyte, 221, 235 Taiwan, 64 taste, vii, 2, 43, 44, 45 taxonomic, 244 taxonomy, 244 T-cell, 133 Teflon, 75, 78 temperature, 21, 23, 57, 73, 76, 77, 82, 90, 91, 92, 94, 95, 98, 99, 100, 101, 102, 104, 106, 107, 110, 154, 187 temperature gradient, 77 tetanus, 236 tetracycline, 45 TGF, ix, 115, 118, 133, 139, 140, 142, 221, 225, 228 T-helper cell, 228 therapeutic, 232 therapy, 60, 123, 247 thermal energy, viii, 71, 72, 76, 79, 82, 83 thermal expansion, 78 thermal properties, 75, 94, 95 thermal stability, 27, 85 thermodynamic, 97, 99, 101 Thermophilic, 49 thiazolidinediones, 192 threat, 228, 232 threonine, 50, 131 thromboxane, 33 thymocytes, 55, 174 thymus, 235, 243 Tibet, 68 tight junction, 123, 155, 160 time, 218, 219, 228, 229, 230, 237, 238 time-frame, 144 timing, 197 tissue, 55, 59, 133, 176, 177, 179, 180, 184, 186, 187, 190, 191, 198, 201, 205, 206, 208, 209, 211, 212, 213, 221 titanium, 84 Titanium, 84 TLR, 133, 137, 216, 221, 222, 234 TLR2, 133, 137, 222 TLR4, 137, 222 TLR9, 147 TNF, 133, 137, 139, 141, 216, 221, 234 TNF-α, 133

Index Tocopherol, 40, 90 toddlers, 225, 243 Tokyo, 65 tolerance, viii, ix, 43, 58, 62, 115, 117, 122, 123, 133, 135, 140, 143, 149, 188, 192, 221, 224, 225, 227, 228, 232, 234, 244, 246, 247 Toll-like, 133, 134, 149, 221, 222, 242 tomato, 77, 84 total cholesterol, x, 2, 56, 153, 157, 158 total energy, 72, 82, 209 toxic, 155 toxic effect, 155 toxicity, 2, 35 toxin, 55, 239 trachea, 193, 210 tradition, 44, 49 training, 198 trans, viii, xi, 87, 88, 89, 93, 111, 112, 113, 114, 175, 176, 177, 178, 198, 199, 200, 202, 203, 204, 205, 206, 208, 212 transcription, 133, 173 transcription factor, 173 transcriptional, 132, 146 transesterification, 35 transfer, 35, 72, 74, 79, 135, 138, 139, 146, 148, 149, 185, 225, 228 transference, 27, 138 transformation, 27, 105 transforming growth factor, 221 transgenic, 4, 42 transgenic mice, 42 transgenic plants, 4 transition, 95, 105 transitions, 95 translocation, 223, 229, 232, 246 transmission, 116, 127, 129, 137, 144, 211, 217, 242, 245 transparent, 102 transplantation, 174 transport, 11, 155, 159, 188, 192, 210, 244 trees, 21, 89 Tregs, 133 triacylglycerols, 114, 132 trial, 35, 67, 76, 81, 121, 123, 143, 147, 150, 158, 220, 224, 227, 230, 231, 233, 234, 235, 241, 243, 244, 246 triggers, 148 triglyceride, 189, 201 triglycerides, 157, 178, 192 Triglycerides, 157

273

trypsin, 131 tryptophan, 50 Tryptophan, 48 tumor, 51, 52, 62, 65, 67, 157, 172, 188, 189, 190, 195, 197, 207, 221 tumor cells, 51, 67, 188, 207 tumor growth, 52, 62, 65, 189, 195 tumor metastasis, 190, 197 tumor necrosis factor, 172, 221 tumorigenesis, 173, 188 tumors, 51, 165, 174, 188, 198 tumour, 51, 55, 64 tumour growth, 55 tumours, 51, 55 Turkey, 181 TVA, 178, 179, 181, 182, 183, 184, 189, 190, 196 twins, 130 type 1 diabetes, 217, 239 type 2 diabetes, 134, 192, 193 tyrosine, 48

U ulcerative colitis, 158 ultrasound, 235 ultraviolet, 28 umbilical cord blood, 116 uncertainty, 76 undernutrition, 135, 151 unfolded, 72 United Kingdom, 17, 21, 240 United Nations, viii, 87, 88 United States, 14, 29, 31, 39, 186, 191, 245 uric acid, 46 Uruguay, 31 USDA, 75, 196 Utah, 175, 196 uterus, ix, 125, 126, 134 UV, 28, 59, 65

V vaccination, 235 vaccine, 123, 236 vacuum, 22 vagina, 118, 122 Valdez, 64 Valencia, 125 validity, vii

Index

274 valine, 50 values, xi, 41, 48, 60, 74, 76, 94, 95, 96, 102, 106, 141, 156, 176, 180, 194, 219, 235, 237, 238 vancomycin, 228 vapor, 50 variable, 185, 191 variables, 210 variance, 76 variation, 99, 185, 201, 203, 242 vascular, 239 vascular disease, 35, 39, 239 vascular diseases, 239 vegetable oil, ix, 4, 6, 8, 16, 18, 21, 23, 25, 29, 34, 40, 42, 88, 89 vegetables, 4, 5, 6, 7, 38 vegetarians, 9 vegetation, 184 velocity, 77, 96 villus, 131 viral, 217, 224 viral gastroenteritis, 217 virus, 133, 134, 145, 146, 236, 245 virus infection, 134 viscoelastic properties, 50, 102 viscosity, 50, 96, 97, 105 visible, 19, 101 vision, 19 vitamin A, 213 vitamin B1, 49 vitamin B12, 49 vitamin B2, 50 vitamin B6, 50 vitamin C, 50 Vitamin C, 49 vitamin D, 2 vitamin E, 45, 59, 205 vitamin K, 50 vitamins, viii, 43, 46, 49, 59, 61, 131, 233 VLBW, 216, 220, 226 VLDL, 208 vulnerability, 225

W warrants, 238 water, 10, 21, 23, 26, 27, 33, 50, 51, 55, 58, 62, 72, 73, 82, 84, 131, 136, 154, 176 water vapor, 50 water-soluble, 51, 55, 58 weakness, 2

wear, 73, 79, 85 web, 119 weight gain, viii, 43, 226 weight loss, 193 Weinberg, 164, 174 well-being, ix, x, 115, 118, 153 wellness, 14 western blot, 166, 169 western diet, xi, 175 wettability, 72 wheat, 4, 41, 66, 159 wheat germ, 4 wheezing, 143 whey, x, 45, 50, 153, 154, 158, 163, 164, 165, 173, 233, 244, 246 WHO, 2, 42, 88, 111, 135, 145, 216, 247 winter, 45, 184 Wisconsin, 113, 114, 176 Wistar rats, 161 withdrawal, 36, 117, 166, 198 witness, 219, 223, 225 women, 19, 39, 40, 42, 51, 61, 116, 118, 119, 120, 121, 122, 134, 135, 136, 138, 139, 140, 141, 142, 143, 150, 173, 188, 190, 191, 197, 213, 217 wood, 21, 42 workers, 2, 9, 11, 12, 13, 25, 26 World Health Organization (WHO), viii, 87, 88 writing, 3

X xenobiotics, 126 XPS, 77, 80, 81 X-ray diffraction (XRD), 90, 95, 106

Y yeast, 44, 45, 63, 66, 67 Yeasts, 49 Yemen, 89 yield, 96, 182, 183, 185, 204 yogurt, x, 16, 17, 18, 24, 44, 51, 57, 60, 62, 66, 139, 141, 163, 170, 180, 186, 206, 212

Z zinc, 48, 51, 160