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Food and Beverage Consumption and Health Series

MILK CONSUMPTION AND HEALTH

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, 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 herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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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Ebbe Lange and Felix Vogel

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]

2

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).

4


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


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).


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

7


8

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


9

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%

10


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)


11

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

12


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.


13

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.

14


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.


15

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 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.

78

Sundar Balasubramanian and Virendra M. Puri

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.

Fouling Reduction during Milk Processing Using Equipment Surface Modification

79

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).


81

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).

82


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


83

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:

Milk Fat/Sunflower Oil Blends as Trans-Fat Replacers

89

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,

90

Roberto J. Candal and María L. Herrera

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


91

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

92


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

20

25

30

35

40

Temperature (°C)

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


94

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.


95

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.


96

HMF

40% SFO

b

Intensity

Intensity

a

10

15

20 2θ (°)

25

30

10

15

20

25

30

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


97

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


98

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.


99

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

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