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MILK CONSUMPTION AND HEALTH
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FOOD AND BEVERAGE
CONSUMPTION AND HEALTH SERIES
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2009. ISBN: 978-1-60741-045-4
Marketing Food to Children and Adolescents

Nicoletta A. Wilks
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Red Wine and Health

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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
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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 vii
Chapter I Plant Sterols and Plant Stanols in Milk Products Used as Functional
Foods: Effects on Cardiovascular Risk Diseases Prevention 1
Fernando Ramos and David Saraiva
Chapter II Kefir and Health: A Perception 43
Zaheer Ahmed and Yanping Wang
Chapter III Fouling Reduction during Milk Processing Using Equipment
Surface Modification 71
Sundar Balasubramanian and Virendra M. Puri
Chapter IV Milk Fat/Sunflower Oil Blends as Trans Fat Replacers 87
Roberto J. Candal and María L. Herrera
Chapter V Probiotic Bacteria Isolated from Breast Milk for the Development
of New Functional Foods 115
G. Vinderola, A. Binetti and J. Reinheimer
Chapter VI Probiotics in Maternal and Early Infant Nutrition 125
Yolanda Sanz
Chapter VII Epilactose: Potential for Use as a Prebiotic 153
Susumu Ito, Jun Watanabe, Megumi Nishimukai, Hidenori Taguchi,
Hirokazu Matsui, Shigeki Hamada and Shigeaki Ito
Chapter VIII Lactoferrin as an Added-value Whey Component and a Healthy
Additive in Nutraceutical Drinks 163
Palmiro Poltronieri, Carla Vetrugno, Antonella Muscella, Santo
Marsigliante
vi Contents
Chapter IX Conjugated Linoleic Acid: An Anticancer Fatty Acid Found

in Milk and Meat 175
T. R. Dhiman, A. L. Ure and J. L. Walters
Chapter X Beneficial effects of Human Milk and Prebiotic-Like Fermented
Infant Formulas on the Intestinal Microflora and Immune system 215
Catherine J. Mullié, Daniel Izard and Marie-Bénédicte Romond
Index 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-
viii Ebbe Lange and Felix Vogel
carbonated beverage, possesses nutritional attributes due to its content of vitamins, protein
and minerals and therapeutic attributes contributed by its antibacterial spectrum, gastro-
intestinal proliferation, hypocholesterolemic effect, anti carcinogenic effect, lactic acid
content, b-galactosidase activity and bacterial colonization, improves immune system and is
also remedy for Helicobacter pylori infection which is only the property of kefir. Moreover,
on one side kefir is good dietetic beverage, and of particular interest of athletes, and on other
side the whole kefir is good for feeding premature infants because of good tolerance, and
adequate weight gain. Lots of work has been done on kefir from a health point of view, this
chapter summarizes all the data that has been completed to date. By reviewing the literature
the chemical, microbiological, nutritional and therapeutic characteristics of kefir have been
highlighted to justify its consumption as a healthy milk food.
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, Ni-
P-PTFE and LectrofluorTM-641) were used. Chemical analyses of the foulants indicate that
the coating integrity did not appear to be compromised for the LectrofluorTM-641 coatings.
However, there were trace amounts of fluorine present in the foulants adhering to the other
two coating types (AMC148-18 and Ni-P-PTFE). A preliminary cost estimate on the thermal
energy savings when using the coated surfaces indicate that there is substantial savings in
energy, further justifying the 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
x 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-density-
lipoprotein cholesterols were lowered, and 4) conversion of primary bile acids to secondary
bile acids was suppressed. Therefore, the conversion of lactose to epilactose may increase the
nutritional value of milk and whey.
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 ISBN: 978-1-60741-459-9
Editors: E. Lange and F. Vogel © 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: fjramos@ci.uc.pt
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 4-
desmethyl 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).
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 C7-
C8) 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 (24α-
methylcolest-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).
4-monomethylsterol
4,4-Dimethylsterol
HO HO
Gramisterol Cycloartanol
21
20 22
18 24
12 25
11 17 23 26
19 13 16
1 9
27
2 10 14 15
8
HO 3 7 HO
5
4 6
Cholesterol Sitosterol
Δ5 desmethylsterols
HO HO
Campesterol Stigmasterol
4-desmethylsterols
HO HO
Sitostanol Campestanol
Δ7 desmethylsterols
Δ5,7 desmethylsterols
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).
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 Total phytosterols

Barley * 59-83
*
Corn 178
*
Oats 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)
Table II. Phytosterols in fruit, mg/100 g edible portion (Adapted from Normén et al., 1999).
Fruit Total phytosterols

Apple 13
Banana 14
Clementine 16
Fig 22
Grapefruit 18
Honeydew melon 1.8
Kiwi 9.1
Lemon 18
Orange 24
Passion-fruit 44
Peach 15
Pear 12
Pineapple 17
Watermelon 1.3
Table III. Phytosterols in vegetables, mg/100 g edible portion (Adapted from Normén et al., 1999).
Vegetables Total phytosterols

Broccoli 39
Brussels sprouts 43
Carrot 16
Cauliflower 40
Celeriac 20
Celery 17
Chinese cabbage 8.5
Fennel 9.8
Kale 8.8
Leek 8.1
Mushrooms 18
Olives. green 35
Olives. black 50
Onion 8.4
Parsnip 27
Pepper. green 7.2
Potato 3.8
Radish 9.0
Sauerkraut 15
Swedish turnip 17
Tomato 4.7
White cabbage 13
Table IV. Phytosterols in vegetable oils, mg/100 g edible portion (Adapted from
Piironen et al., 2000a and Normén et al., 2007).
Vegetable oils Total phytosterols

Corn oil Crude 809-1557
Corn oil Refined 715-952
Cottonseed crude 431-539
Cottonseed refined 327-397
Olive Extra virgin 144-154
Olive Pomace 261-282
Palm Crude 71-117
Palm refined 39-61
Rapeseed Crude 513-979
Rapeseed refined 250-773
Rice bran Crude 3225
Rice bran Refined 1055
Soybean Crude 229-459
Soybean Refined 221-328
Sunflower Crude 374-725
Wheat germ 967
Sesame seed 472
Linseed 471
Oat 534
Peanut 251-315
Walnut 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 143-208
Brazil nuts 131
Cashew nuts 151-158
Coconut rasps 68
Hazelnuts 138
Linseeds 213
Peanuts 116-220
Pistachio nuts 297
Pumpkin seeds 94
Sesame seed 404
Sunflower seeds 322
Walnuts 128
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%
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;
Melńikov 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 Melńikov 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)
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
cholesterol-lowering efficacy of plant sterols diminished over time (comparing plasma

cholesterol levels after 1 and 2 months) while plant stanols maintained their cholesterol-
lowering 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 LDL-
cholesterol (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 co-
workerrs (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 8.5-
10.8%) 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; Waalkens-
Berendsen 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.
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 co-
authors (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.
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.
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 Percentage (%)

ß-sitosterol < 80
ß-sitostanol < 15
Campesterol < 40
Campestanol <5
Stigmasterol < 30
Brassicasterol <3
Other sterols/stanols <3
Total sterols/stanols 99
Still novel foods or ingredients could follow a simplified procedure, only requiring a
simple notification by the respective company. For this, they must be considered, in at least
one member state as "substantially equivalent" (composition, nutritional value, metabolism,
fixed use and level of undesirable substances) to pre-existent foods or ingredients (EFSA,
2003; 2005). In 2004, for the specific case of phytosterol and phytosterol esters enriched
dairy products European Commission adopted the 608/2004/EC Decision (EC, 2004f).
Accordingly, several enriched phytosterols dairy products can be introduced on the European
market, namely middle- and low-fat dairy products (with or without added fruits and/or
cereals) and fermented dairy products, like yogurts and cheese type products, with fat content
< 12g per 100g) and in which milk fat or proteins have been partial or totally substituted by
fat and/or plant proteins.
Additional EU regulations 2004/333/EC, 2004/334/EC, 2004/335/EC, 2004/336/EC and

2004/845/EC (EC 2004 a,b,c,d and e) specify the plant sterols and stanol profiles addition on
milk products, and are briefly presented on the Table VI. Furthermore, these regulations
define that plant sterols and stanols extracted from sources other than vegetable oil proper for
foods, should be free of contaminants, with purity higher than 99% of phytosterols (EC, 2004
a,b,c and d).
In the cases of Australia and New Zealand, the regulation of phytosterols enriched foods
is under the responsibility of a common organisation, Food Standards Australia New Zealand
(FSANZ). This organisation rules the incorporation of the referred compounds in foods
according its origin. Hence vegetable oils derived phytosterol esters can be incorporated in
milk if the following conditions are observed (FSANZ, 2006):
• milk present no more than 1.5g of total fat per 100g;

• milk is commercialised in a maximum of 1L packing volume;
• phytosterols are present in a concentration between 3.2 and 4.0g/L of milk .
They can also be incorporate in yogurt if the following conditions are observed
(FSANZ, 2006):
• with a concentration that do not exceed 1.5g/100g;
• the commercialization packing does not surpass a 200g capacity;
• the total amount of added phytosterol ester is in the range of 1.3-1.6g.
3.2. Market of Phytosterols Enriched Foods
3.2.1. Authorized Foods

Since the launching of a phytostanol esters enriched margarine in 1965, in Finland, the
global market of phytosterols enriched foods has been quickly expanded and diversified (Salo
et al., 2005). In fact, food type matrices are gradually developing. It is observed a gradual
substitution of high fat content foods for healthier alternatives, with a special attention to
foods that could be easier incorporated in the daily diet. Spreads and fermented milk type
products, like yoghurt and yoghurt drinks, were most commonly available (EFSA, 2008a).
Phytosterol enriched products that are or could be marketed outside the EU include juices, ice
creams, snack bars, white or whole-grain breads and buns, cereals, confectionery products
and cooking oils. In addition, GRAS status was recently given to phytosterol esters for use as
an ingredient in ground roasted coffee (FDA, 2005), to phytosterols and phytosterol esters for
use in pasta, noodles, soups, and puddings (FDA, 2006a), and to phytosterols for use in
different egg products (FDA, 2006b).
Tables VII and VIII, displays a list of phytosterol enriched foods and ingredients that
were authorized for commercialization in Europe and in the USA.
Currently, dairy products are the area that presents the higher growth in this kind food
industry/market. The first enriched dairy food, a yogurt enriched with phytostanol esters, was
introduced in United Kingdom, in 1999 (Salo et al., 2002). Dairy products with low fat
content are a suitable way to obtain phytosterols recommended daily dose, because they can
be considered as healthy foods and are easily included in the diet. The possibility to produce
a single dose that contains the optimal phytosterol daily amounts is another advantage of this
type of foods, since it facilitates the calculation of the estimated daily intake estimation (Salo
et al., 2002 and 2005).
Table VII. Phytosterol-enriched novel foods available on EU marketup to 2008

(Adapted from EC, 2008b).
Food type Status

Yellow fat spreads EC, 2000
Milk-based fruit drinks EC, 2004d
Milk-type products EC, 2004a,b and c
Fermented milk-type products EC, 2004a
Cheese-type products EC, 2004a and d
Yoghurt-type products EC, 2004b,c and d
Spicy sauces (including ketchup, mustard, marinades, dips, etc.) EC, 2004b
Salad dressings (including mayonnaise) EC, 2004a
Soya drinks EC, 2004a
Milk-based beverages EC, 2004e
Bakery products (rye bread) EC, 2006a and b
Oil EC, 2007
Rice drinks EC, 2008a
3.2.2. Market Characterization

In these days, the functional foods market is very small. In 2005, the European
phytosterols market was evaluated in 145million euros and could rise up to 311million euros
in 2012 ($US 184.6 million in 2005 to $US 395.2 million by 2012) (Frost & Sullivan, 2006b;
Jones & Jew, 2007). According to EFSA, it was commercialized in Europe, during 2007,
approximately 9,500 tons of phytosterols, what corresponds to 160 million euros. Of these,
80% were used in food products (EFSA, 2008a). Margarines are the most common products
in the market, corresponding to 75% of phytosterol enriched foods used in 2005 (EFSA,
2008a). Although plant stanols enriched products do not require previous approval as novel
food, they just correspond to 1/3 of the market, while plant sterols enriched products
represent the remaining 2/3 (EFSA, 2008a).
In the United Kingdom, consumer inquiries demonstrated that phytosterols enriched
products constitute a maximum of 12% of the total market of products to spread on, 15% of
the market of yogurts and yogurt drinks, and 6% of the milk total market (EFSA, 2008a).
American current market of phytosterols enriched foods is, also, slightly developed ($US
103.9 million or 81.9 million euros) in 2005 and estimates to rise to $US 196.7 million (155
million euros) in 2012. This fact could be justified by the American consumer's typical
behaviour that associates the maintenance of a healthy life style more to the consumption of
dietary supplements rather than of functional foods (Frost & Sullivan, 2006a)
Table VIII. Phytosterol-enriched foods and GRAS status in U.S.A up to 2008

(Adapted from http://www.cfsan.fda.gov/~rdb/opa-gras.html).
Food type Status

Vegetable oil spread FDA, 2000a
Vegetable oil spreads, dressings for salads, bars, and yogurt. FDA, 2000b
Vegetable oil, baking, frying, and salad dressings FDA, 2000c
Vegetable oil spreads, dressings for salad, health drinks, health bars, and
FDA, 2001
yogurt-type products
Margarine and vegetable-based spreads (margarine-like); yogurt and yogurt-
like products; milk-based juice beverages; ice cream and non-standardized
ice cream products; cream cheese and cream cheese-like products; snack bars
FDA, 2003
(health bars); salad dressing, mayonnaise, French dressing, and dressings for
salads; and white breads, white rolls and buns, and comparable non-
standardized white bread products
Ground roasted coffee FDA, 2005
Margarines and vegetable oil spreads, dressings for salads, beverages, snack
bars, dairy analogs (including soy milk, ice cream and cream substitutes),
cheese and cream, baked foods, ready-to-eat breakfast cereals, mayonnaise,
FDA, 2006a
pasta and noodles, sauces, salty snacks, processed soups, puddings, yogurt,
confections, vegetarian meat analogs, fruit/vegetable juices, edible vegetable
oils, including diacylglycerol oil.
Egg products, including egg whites and egg substitutes FDA, 2006b
Baked goods and baking mixes; fats and oils; frozen dairy desserts and
mixes; gelatins, puddings, and fillings; grain products and pastas; gravies and
FDA, 2006c
sauces; hard candy; milk; milk products; soft candy; soups and soup mixes;
and snack foods
3.3. Labelling
According to EFSA Scientific Committee on Food (SCF) and many independent studies,
it is recommended an intake of not more than 3g/d, mainly based on the fact that with intakes
higher than 2.5-3.0g/d, little additional benefit in LDL-C lowering is observed. (Ling &
Jones, 1995; Katan et al 2003; Berger et al., 2004; Kritchevsky, 2004; Gylling & Miettinen,
2005; Kritchevsky & Chen, 2005; Plat & Mensink, 2005; Salo et al. 2005; Trautwein &
Demonty, 2007).
Following the SCF recommendations, the European Commission adopted specific

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

portions/day) or a maximum of 3g of phytosterols (1 portion/day);
• drink packages should not contain more than 3g of phytosterols;
• the obligation of packing certain foods in single portions.
3.4. Intake Recommendations
Milk and dairy products daily consumption and the respective phytosterols suggested
intakes by manufacturers are shown on Table IX. It is clear that manufacturers follow the
SCF recommended phytosterols daily intake of 1.5-3.0g for an individual adult of medium
characteristics (SCF, 2002), as well as NCEP ATPIII (2001) and IAS (2003) which both
recommend 2 g/day to lower elevated LDL-C concentrations. For some products as milk
(1000ml) and cheeses, each portion supplies about a third of the phytosterols recommended
daily intake. So, the manufacturers suggest that this specific product should be consumed 3
times a day or, alternatively, that other phytosterols enriched foods should be additionally
consumed.
Fermented milk derived products, as yogurts, are usually the most available enriched
dairy food, as displayed by Table X, with a list of the available European market dairy
products at the end of 2007.
Table IX. Phytosterol concentrations in some dairy products and the recommended
daily consumption level (Adapted from EFSA, 2008a).
Product category Common packing Recommended Phytosterol Daily intake

size consumption concentration
Milk type products 1000ml 2-3x250ml 0.3 g/100ml 1.5-2.4g
Yoghurt type products 65-125ml 1-2x65-125ml 0.6-3.1 g/100ml 1.5-2.0g
Cheese type products 125g 3x30g 2.2 g/100g 2.1g
Cream cheese 200g 40-60g 5.0 g/100g 2-3g
Milk-based soft drink 1000ml 350ml 0.5 g/100ml 1.8g
Table X. Phytosterol-enriched dairy products available in EU at the end of 2007

(Adapted from EFSA, 2008a).
Member State Milk products

Austria Yoghurt drink
Belgium Yoghurt, yoghurt drink, milk, cheese spread
Bulgaria Not known
Cyprus Milk
Czech Republic Yoghurt, yoghurt drink, milk
Denmark Yoghurt, yoghurt drink, milk
Estonia Not known
Finland Milk, buttermilk, yoghurt, yoghurt drink
France Yoghurt, yoghurt drink, milk
Germany Yoghurt drink, milk, cheese
Greece Yoghurt, cheese spread
Hungary Not known
Ireland Yoghurt, yoghurt drink, cheese spread
Italy Yoghurt drink, milk, cheese spread
Latvia Not known
Lithuania Yoghurt drink
Luxembourg Cheese spread, yoghurt, yoghurt drink
Malta Yoghurt drink
Netherlands Yoghurt, yoghurt drink
Poland Yoghurt drink
Portugal Yoghurt, yoghurt drink, milk
Romania Not known
Slovakia Not known
Slovenia Yoghurt drink
Spain Yoghurt, yoghurt drink, milk
Sweden Milk
United Kingdom Yoghurt, yoghurt drink, milk, cheese
3.5. Technological Aspects
Initially, phytosterols were administered in the form of powder. However, great amounts
were needed to obtain an effective reduction in cholesterol blood levels. This was due to
phytosterols solubility, namely their water insolubility and weak solubility in fats and oils
that hinder its administration. These physical properties limited its applicability in foods and
in several pharmaceutical oral preparations. They were therefore considered, for a long time,
to have little practical health interest (Wester, 1999; Salo & Wester, 2005)
The incorporation of phytosterols in food to become easily available for the consumer
turned out to be an attention-grabbing challenge. This objective involves the production of
phytosterols with higher solubility in the intestinal tract, thus allowing a more efficient
cholesterol blood reduction (Rozner & Garti, 2006).
Until now, commercialised plant sterols and stanols have been obtained from by-products
of vegetable oil refinement and wood industrial processing (SCF, 2000; 2003a and b; Salo et
al., 2002; Moreau, 2004).
Vegetable oil phytosterols are usually isolated from vegetable oils, such as soy, corn, and
rapeseed oils, as well as from palm, cottonseed and peanut oils, during refinement
deodorising step (SCF, 2000; 2003a and b; Salo et al., 2002; Moreau, 2004), that results in a
distillate with circa 15–30% of phytosterols (Moreau, 2004). The sterol fraction is then
purified through solvents by a crystallization process (Salo et al., 2002; SCF, 2003a).
In wood processing industry, phytosterols are extracted from conifer trees. Wood extracts
are placed in an alkaline aqueous solution and phytosterols are then recovered as saponifiable
fraction, esterified phytosterols by fat and resinic acids and as many other neutral substances.
Phytosterols are then separated by liquid extraction in the case of acid esters or
crystallization, in the case of neutral substances. Saponifiable fraction is, subsequently
processed by a series of distillations. Phytosterols can also be recovered from the tall oil pitch
(tall oil sterols), which is the initial distillation residue. Plant sterols extracted from this oil
pitch and are, afterwards, crystallized (Salo et al., 2002; SCF, 2003a and b). Phytosterols
concentration in tall oil soap is 3-4% and in tall oil pitches about 10% (Salo et al., 2002). The
mixture composition between alimentary oils derived or wood derived plant sterols and
stanols are different. For instance, the relative concentration of sitosterol, campesterol,
stigmasterol and sitostanol is approximately 72%, 8.2%, 0.3%, and 15.3% in derived wood
processing products, and 45%, 26.8%, 19.3% and 2.1% in derived vegetable oils products,
respectively (Salo et al., 2002). Purified phytosterols make stable crystals and hold limited
water and oil solubility. At room temperature, free form phytosterols solubility has been
described as 0.01% in water (Engel & Knorr, 2004) and 3.5-4.0% (w/w) in oil (Christiansen
et al., 2002). At the same temperature, phytostanols solubility in oils and fats is inferior to
1% (w/w) (Wester, 2000). Due to this low solubility, phytosterols free form can take several
days to dissolve in biliary salt solutions (Armstrong & Carey, 1987; Ostlund et al., 1999).
Thus, an option is to promote a formulation that grants them a higher solubility in the
emulsified lipidic part of the alimentary digestion, as a way to improve an effective
cholesterol blood reduction.
3.5.1. Phytosterols Formulations
3.5.1.1. Esterified Phytosterols Formulations

The first alimentary products successfully used were fatty acids plant stanols esters, in a
strategy only later applied to plant sterols (Wester, 2000, Moreau et al., 2002). Phytosterols
esterification by fatty acids increases its oil solubility in about 10 times -oil solubility of 20-
30% (Jandacek et al., 1977; Mattson et al., 1982). Consequently, phytosterols incorporation
in alimentary oils or fats (i.e. margarine, butter, and fat spreads function the phytosterol esters
carriers) is the actual formulations state of the art (Wester, 2000; Trautwein et al., 2003).
The required dose of crystalline phytosterol to attain a 10% cholesterol reduction is 10-
20g/day, while for esterified phytosterols a similar effect could be attained with only 2-3
g/day. Besides, as esterified phytosterols are fat soluble, they could be evenly distributed in
the lipidic phase of the food mass (Salo et al., 2002). Esterification is carried out through
alimentary fatty acids esterification of the phytosterols free forms. Chemical reaction is made
at high temperatures under moderate vacuum, using a catalytic agent (SCF, 2003c; Salo et al.,
2005). When this reaction is complete, a wash procedure is performed to inactivate the
catalytic agent by lixiviation and deodorization procedures as the final steps, just as in the
case of alimentary oils (Salo et al., 2005). Also noteworthy, is the possibility of adjustment of
the phytosterols esters physical properties according to fatty acids composition. Depending
on the alimentary matrix to be enriched, esters can be adjusted for liquid or solid state at
room temperature, and, besides, they can, also, be modified in order to achieve more
nutritionally beneficial effects than the fat that they substitute (Wester, 2000; Salo et al.,
2005).
Recently, a new approach on the fatty acids esters involves the use of esterified plant
sterols with oil fish enriched with long chain poly-unsaturated fatty acids (n-3) (Ewart et al.,
2001) and have been shown to be effective in humans (Demonty et al., 2006).
Phytosterols fatty acid esters composition can vary without decreasing its cholesterol
blood reduction capacity. This fact was instantly observed in the earliest studies, when
esterified phytosterols with small, medium or long chain fatty acids have presented similar
effectiveness (Mattson et al., 1977).
3.5.1.2. Free Phytosterols Formulations

Until now, and besides esterification, several formulations have been developed to
reduce technological limitations of free phytosterols and to increase the possibility of
incorporation in food. To achieve the best possible formulation has been a permanent
challenge, especially since the observation that phytosterols effectiveness was dependent on
its physical properties, on its alimentary matrix solubility, and on the food fat content in
which they are incorporated.
A new formulation approach to increase the efficiency of phytosterols involves the use of
a microcrystalline suspension, which is prepared by heating a mixture of oils and phytosterols
at 100-110ºC, followed by a cooling at 90ºC. At this temperature, it is added water and the
resulting suspension is then agitated until it reaches room temperature. Using this
crystallization method, more than 30% of phytosterols can be added to fats and oils without
any food additive, because it acts as an emulsification agent. In the final suspension,
phytosterols appear in both dissolved and crystalline forms (Christiansen et al., 2001a; 2002).
Other formulations include, for instance, combinations of phytosterols and mineral salts,
by which an air jet micronizer forms with less than 20μm size (SCF, 2003c).
Phytosterols free form bioavailability can also be increased through its incorporation in
liposomes (Engel & Knorr, 2004).
The use of mesophase-stabilized compositions for free phytosterols incorporation in food
products, as well as phytosterol-protein complex, were patented by Kraft Foods, Inc. and
Monsanto Co., respectively (Corliss et al., 2000; Akashe & Miller, 2001). In the phytosterol-
protein complex, free phytosterols were first dissolved in vegetable oil and, only after,
proteins were added to act as transporters. The resulting complex is ready for use or could be
dehydrated (Corliss et al., 2000).
Another promising formulation seems to be phytosterols emulsification by lecithin.
Lecithin disperses in water forming micelles that include phytosterols, thus rendering them
water soluble. This formulation is compatible with foods without fat, just requiring small
amounts of phospholipids, and it is believed that it is more efficient in the solubility of
phytosterols than of triacilglycerols (Ostlund et al., 1999; Spilburg et al., 2003).
Phytosterols can also be dissolved in diacilglycerol. In fact, its solubility in
diacilglycerols is higher than in triacilglycerols, i.e. 6.0% and 1.3%, respectively.
Diacilglycerols are less abundant natural components in alimentary oils and fats, and are
currently used as emulsifier agents in foods. Up to now, diacilglycerol oil enriched in
phytosterols is commercialized in Japan (Salo et al., 2002), USA (FDA, 2006a) and EU (EC,
2007).
3.6. Phytosterols Alimentary Matrices
Margarines were the first commercial applications of phytosterols enriched foods.

Mattson and collaborators (1977; 1982), had earlier considered alimentary fat as a good
matrix for phytosterols, since cholesterol was also vehiculated by this kind of alimentary
matrix. Given this, vegetable oil spreads were used in most studies of phytosterols enriched
foods (Berger et al., 2004). But on the other hand, the incorporation of phytosterols in high
fat foods is not compliant with current dietary health recommendations (St-Onge & Jones,
2003). Furthermore, some alimentary cultures do not include margarines in daily diets
(Quilez et al., 2003; Winter, 2004). As a result, margarines are not the ideal alimentary
matrices for phytosterols recommended daily intake, due to their high fat content as well as
their uncommon use in some daily diet cultures (Salo et al., 2002).
Several new types of foods, including alternatives with low fat content or, even, without
fat are under research for market introduction as phytosterols enriched foods. However, some
limitations, in terms of effectiveness, have been reported, namely with no fat liquid
alimentary matrix. A key point in phytosterols action is its solubility level in alimentary
lipidic emulsified portion that is, is necessary to present a good distribution along the whole
lipidic portion in order to be efficiently incorporate in mixed micelles (Salo & Wester, 2005).
In the case of fat soluble phytosterols esters, solubility is not a problem; instead, the
decisive step is the ester’s hydrolysis by the non specific enzyme, cholesterol pancreatic
esterase. This enzyme secretion is stimulated by dietary emulsions that enter in the intestinal
lumen. In other words, low fat foods should be consumed together with the meal to reach a
greater effectiveness (Salo et al., 2005; Doornbos et al., 2006). In the case of free
phytosterols, solubility step represents a problem. Therefore, free sterols should be
incorporated in fat foods, unless they are activated by an appropriate procedure, like lecithin
emulsification (Ostlund, 2004).
Contrarily to its esterified form, free phytosterols efficiency, appears to be more
dependent on the alimentary matrix. Doornbos et al. (2006) investigated single dose (2.8-
3.2g) effectiveness of phytosterol esters enriched liquid yogurt with a total fat content of
2.2% or 3.3%, and consumed in different occasions (with or without meal). Their data
indicated that the effect of those products in cholesterol reduction was not dependent of the
fat content used. However, intake occasion was important. LDL-cholesterol reduction was
6.0% without meal and 9.4% with meal.
Noakes et al. (2005) evaluated low fat (0.54%) yogurts enriched with phytosterol esters
(1.7-1.8g/day) consumed as part of a normal diet. A LDL-cholesterol reduction of 5-6% was
observed. This reduction was considerably lower than data reported by Mensink et al. (2002).
Noakes et al. (2005) concluded that, in spite of existing differences between phytosterols
doses in both studies, the phytosterols intake along with meal in Mensink et al. (2002) study
was the reason for the improved effectiveness reported.
Low fat milk has also been studied as matrix for free and esterified phytosterols.
Thomsen et al. (2004) observed that daily intake of phytosterols (1.2-1.6g) enriched milk
containing 1.2% of fat, reduced blood LDL-cholesterol in 7-10% when consumed as part of a
typical Danish diet.
Clifton et al. (2004) investigated phytosterols (1.6g/day) esters enriched milk containing
2% of fat and consumed in at least two meals. They observed a LDL-cholesterol reduction of
16%. On this study, observed cholesterol reduction was superior to other matrices, such as
yogurt or cereals. Authors suggest that phytosterols are probably incorporated in the
membrane of milk globules, and thus becoming more easily available to be transferred to
micelle membranes.
In other low fat foods, phytosterols can be kept in small lipidic drops core, remaining
unavailable until fat digestion (Clifton et al., 2004).
The most controversial data in respect to phytosterol effects in the reduction of LDL-
cholesterol have been reported in liquid matrices without fat. Jones et al. (2003) observed
that free phytosterols (1.8g/day) enriched drinks with (up to 1 %) or without fat, consumed
along with meals did not resulted in superior reductions in LDL-cholesterol, comparatively to
a control diet.
However, in a latter study by Devaraj et al.(2004), the consumption of free phytosterols
enriched orange juice without fat (2g/day), with breakfast and dinner, a reduction of 12.4% in
LDL-cholesterol was observed. The same authors considered that in respect to the alimentary
matrix, the food vehicle used should be fatter to allow sufficient solubilization of phytosterols
and better micellar absorption. Furthermore, the authors concluded that solid alimentary
matrices would be more effective, owing to the increase of the contact time period between
the phytosterols and the gastrointestinal content, which favours the access to mixed micelles
(Jones et al., 2003).
When analysing the same results, Ostlund (2004) concluded that the lack of phytosterols
effectiveness was probably due to the absence of an appropriate free phytosterols
formulation.
3.7. Phytosterols Analytical Methodologies
Phytosterols reduction cholesterol properties contributed to a growing interest in its

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

One of the most critical steps in phytosterols analysis in different matrices is sample
preparation. Sterol oxidation prevention must be carried out by oxygen elimination. This
oxygen removal is usually done by using a nitrogen atmosphere (Lagarda et al., 2006).
3.7.1.1. Solvent Extraction

Applied extraction solvent depends of the matrix nature, of the physical state (liquid or
solid), and also of the free, esterified or glicosylated form in which phytosterols are present in
the matrix.
Phytosterols presented in plant tissues or in seed oils could be extracted by chloroform-
methanol, chloroform-methanol-water, hexane, methylene chloride or acetone.
Solid phase extraction (SPE) and supercritical fluid extraction (SFE) methodologies
could also be used to extract free and esterified phytosterols from oils or from fats (Lagarda
et al., 2006).
3.7.1.2. Saponification
As it was already referred, phytosterols are usually determined as free sterols
(Aitzetmuller et al., 1998; Piironen et al., 2000a). Lipidic extracts cannot include conjugated
polar phytosterols like esteril glycosids, because they are not soluble in apolar lipidic phases.
In this case, an alkaline hydrolyse is mandatory. After this saponification step, that release
sterols/stanols from their esters, the respective free forms could be extracted as
unsaponifiable fraction. According Piironen and collaborators (2000a), with this method, it is
possible to determine the great majority of the phytosterols.
Alkaline hydrolyse by ethanolic potassium hydroxide could be made at room or warm
temperatures. Nevertheless, hot saponification, with internal standard addition, is adequate
for the majority of enriched phytosterols matrices like, for instance, milk and yogurts
(Laakso, 2005; Ahmida et al., 2006; Santos et al., 2007). However, phytosterols glycoside
conjugates could not be hydrolysed by alkaline saponification. Consequently, this type of
phytosterols quantification through direct saponification methodology is not efficient. So, the
total phytosterols concentration determined is lower than the real value. Toivo and co-
workers (2000, 2001), suggested a previous step of acid hydrolyse (with HCl), before the
alkaline hydrolyse, as an alternative to release the phytosterols from the glycides. According
the same authors, combination of acid and alkaline hydrolyses gives better results than
alkaline hydrolyse, when used separately. Therefore, to obtain sterols totality (free, esterified
and glycosilated), in some natural sources, such as cereals, an acid hydrolyse must be applied
to the lipidic extract (Normén et al., 1999).
Notwithstanding, acid hydrolyse application is limited for countless factors. Firstly,
given the variety of alimentary products processed, not all the natural sources that have
glycoside phytosterols conjugated are part of common diet. Refined alimentary oils are an
example, since phytosterols are only presented in free and esterified forms. If glycoside
esterified forms are originally present, they are removed during refinement oil process (Toivo
et al., 2001). Besides, acid hydrolysis procedures are quite laborious and slowly for routine
analyses. On the other hand, its use in unstable sterols abundant matrices like, for instance 7-
phytosterols (7-avenasterol, 7-stigmasterol, etc.) and D5-avenasterol becomes quite

problematic (Lagarda et al., 2006).
3.7.1.3. Unsaponifiable Fraction Extraction

Usually, in alimentary matrices, as milk and yogurts, and in biological samples, as
plasma, unsapnifiable phytosterols fraction was extracted by apolar organic solvents, like n-
hexane (Ahmida et al., 2006; Santos et al., 2007). However, unsaponifiable phytosterols
fraction could also be extracted by other solvents, such as dichloromethane (Louter, 2004),
chloroform-methanol (Conchillo et al., 2005), chloroform-methanol-water (Piironen et al.,
2000a) or acetone (Claasen et al., 2000).
3.7.2. Determination
3.7.2.1. Gas Chromatography Analysis

Phytosterols GC separation is not possible without previous derivatization (Piironen et
al., 2000a; Lagarda et al., 2006). The phytosterols derivatization procedure usually used is a
transformation, by chemical reaction, on its silyl (or acetyl) derivatives which provide high
thermo-stability, low polarity and very well defined peaks with a more easily GC separation
and detection (Laakso, 2005). Derivatization reactives commonly used are N-methyl-N-
trimethylsylil-trifluoroacetamide (MSTFA) in pyridine and bis-(trimethyl silyl)-
trifluoroacetamide (BSTFA) containing 1% of trimethylchlorosilane (TMCS) which are
added to the dry sample residue (Lagarda et al., 2006).
As for chromatographic columns, in most laboratories, the packed ones have been
replaced by capillary columns. The latter columns offer short times of analysis, less peak
interferences; resolution improvement and high thermal stability, when compared with
packed ones (Abidi, 2001). GC methods with capillary column are also capable to separate
stanols from the correspondent sterols (Phillips et al., 1999). This excellent separation occurs
with common stationary phases, like 14% of cyano-propyl-phenyl-methyl-poli-siloxane (for
instance, DB-1701) and like 5% diphenyl-95% dimethyl-poli-siloxane (for instance, OV-5)
(Lagarda et al., 2006). As mobile phase, helium is usually used (Grandgirard et al., 2004).
Sterols/stanols quantification is, usually, done by internal standard (IS) methodology.
In this kind of methodology, IS should be added to the sample as soon as possible, to
compensate the losses that occur during extraction, transference, evaporation, and
derivatization steps, and thus correcting the analyte final quantification results.
The most common IS proposed in literature for sterols/stanols determinations include
betuline, cholestan, 5 β-cholestan-3 β-ol (coprostanol) and 5 α-cholestan-3 β-ol (cholestanol)
(Lagarda et al., 2006). An IS should be similar to the analyte, but IS right choice could be
problematic. Cholestan is a sterol hydrocarbon without the hydroxyl group in the position 3,
while betuline has two hydroxyl groups and its structure and chemical properties still are
more different than sterol ones.
Cholestanol (5 α-colestan-3 β-ol) should not be used for stanol analyses because samples
can contain cholestanol small amounts derived from cholesterol hydrogenation.
Coprostanol (5 β-cholestan-3 β- ol) seems to be the appropriate IS for most of the
matrices. It structure is similar to phytosterols, it is absent in alimentary samples and it FID
detector response is identical as for the determined analytes. So, no correction factors are
necessary for quantification (Laakso, 2005).
When IS method is used, detector response factors and linearity, for each sterol/stanol,
should be stabilized. Usually sterol/stanol amounts are calculated between peak area
comparison of analysed sterol/stanol and of IS. Then, as IS concentration is known, these
sterol (stanol)/IS ratios are used in calibration curve in order to easily quantify sample
phytosterol concentration (Conchillo et al., 2005; Johnsson & Dutta, 2006).
As referred previously, GC-FID or GC-MS are the selected methods for phytosterols
determination. However, GC-MS present some advantages relatively to GC-FID in sample
complex mixtures quantification (for instance, foods and biological samples), since only GC-
MS can confirm phytosterols identification as well as eluted peak purity evaluation (Piironen
et al., 2000a).
3.7.2.2. Liquid Chromatography Analysis

High performance liquid chromatography (HPLC) by ultraviolet (UV) detection can also
used to determine the main phytosterols in foods. HPLC offer, in relation to GC method, an
analyte no destructive alternative and no derivatization phytosterols are needed. However,
HPLC methods have low selectivity to separate phytosterols from corresponding phytostanols
(Piironen et al., 2000a).
Nonetheless, and while an effective HPLC application is not developed, namely by mass
detection, GC will continue to be the selected determination methodology for this type of
compounds.
4. Conclusion
Consumption of milk and dairy products enriched with phytosterols is a simple and safe
strategy to reduce blood cholesterol and, consequently, to avoid premature development of
cardiovascular diseases.
Recent studies confirm that phytosterols daily intake of 2g reduces blood LDL-
cholesterol levels by 10% (Katan et al 2003, EFSA, 2008a and b; AbuMweis et al., 2008;
Mannarino et al., 2009). However, no additional effect could be observed with an intake
higher than 3g. So, this dose should be established as the maximum daily intake.
Accordingly, dairy products seem to be a good matrix to obtain these phytosterols daily
doses (1-3g). Additionally, phytosterols enriched milk and yogurts low fat content, are
considered healthy foods with a high nutritional value and are easier to incorporate in any
type of diet.
5. Future Perspectives
Phytosterol enriched food can be considered an "open book", because many aspects and
health benefits are still under research (Trautwein & Demonty, 2007). In addition to their
well-established cholesterol-lowering effect that was described above, other potential health
benefits of phytosterols have been mentioned, namely immune stimulating (Calpe-Berdiel et
al., 2007) and anti-inflammatory activities (Navarro et al., 2001; Bouic, 2002,), as well as
beneficial effects against the development of different types of cancers, like breast, lung,
stomach and colon (Bradford & Awad, 2007) oesophagus (de Stefani et al., 2000), ovarian
(Mccann, et al., 2003) and prostate (Wilt et al., 1999; Berges et al., 2000) cancers,
respectively. However, evidence for such promising effects is still at a rudimentary stage and
more research is clearly needed to draw firm conclusions.
Given that, milk and yoghurts, while privileged phytosterols enriched foods, could be
considered as elected functional foods. Their nutritional allegations, today clearly proven,
could be substituted, in an early future, for no less interesting health allegations in the most
relevant subjects mentioned above.
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Chapter II
Kefir and Health: A Perception
Zaheer Ahmed2 and Yanping Wang*1

*1
School of Food Engineering and Biotechonolgy,
Tianjin University of Science & Technology, Tianjin 300457 China
2
Faculty of Sciences. Department of Home and Health Sciences.
Allama Iqbal Open University. H-8, Islamabad Pakistan
Abstract
Kefir is a fermented milk drink produced by the actions of bacteria and yeasts
contained in kefir grains, and is reported to have a unique taste and properties. Kefir, the
self-carbonated beverage, possesses nutritional attributes due to its content of vitamins,
protein and minerals and therapeutic attributes contributed by its antibacterial spectrum,
gastro-intestinal proliferation, hypocholesterolemic effect, anti carcinogenic effect, lactic
acid content, b-galactosidase activity and bacterial colonization, improves immune
system and is also remedy for Helicobacter pylori infection which is only the property of
kefir. Moreover, on one side kefir is good dietetic beverage, and of particular interest of
athletes, and on other side the whole kefir is good for feeding premature infants because
of good tolerance, and adequate weight gain. Lots of work has been done on kefir from a
health point of view, this chapter summarizes all the data that has been completed to date.
By reviewing the literature the chemical, microbiological, nutritional and therapeutic
characteristics of kefir have been highlighted to justify its consumption as a healthy milk
food.
*
ypwang40@yahoo.com
44 Zaheer Ahmed and Yanping Wang
Introduction
The benefits of probiotics have been recognized and explored for over a century.
Research in probiotics concentrates on modes of action, host-microbe interactions and such
specific questions as the influences on the human or animal immune system. Recently,
consumers’ growing preference for food with enhanced nutritional and therapeutic properties
has led to the inclusion of various cultured milk products as part of their diet based upon their
dietetic features. The increasing cost of health care, the steady increase in life expectancy and
the desire of the elderly for improved quality of their lives are driving factors for research and
development in the area of functional foods. Among a number of functional compounds
recognized so far, bioactive components from fermented foods and probiotics certainly take
the center stage due to their long tradition of safe use, and established and postulated
beneficial effects. Probiotics occur naturally in fermented food products such as yoghurt,
kefir, sauerkraut, cabbage kimchee, and soybean- based miso and natto (Sarkar 2008).
Among them kefir which is defined as the yogurt of the 21st century (Gorski 1994), has got
considerable attention of food scientists because of its unique and complex probiotic
properties.
Kefir is self-carbonated refreshing fermented milk with slight acidic taste, made from
kefir grains, a complex and specific mixture of bacteria and yeast held together by a
polysaccharide matrix. Kefir means “feel good” in Turkish and falls under the category of a
mixed lactic acid and ethanol fermented beverage (Kurmann, 1984). Kefir is also known as
Kefyr, Kephir, Kefer, Kiaphur, Knapon, Kepi and Kippi. Kefir distinguishes itself from the
traditional fermented milks (yogurt) and other fermented dairy products in that it is the
product of fermentation of milk in the presence of a mixed group of microflora confined to a
matrix of discrete ‘kefir grains’, which are recovered subsequent to fermentation (Malbaša et
al. 2009, Marshall & Cole, 1985). Kefir grains resemble small cauliflower florets or cooked
rice: measure 1–3 cm in length, are lobed, irregularly shaped, white to yellow-white in
colour, and have a slimy but firm texture (Plessas et al. 2007, Loretan et al. 2003). These
grains contain lactic acid bacteria (lactobacilli, lactococci, leuconostocs), acetic acid bacteria
and yeast mixture coupled together with casein and complex sugars by a matrix of
polyssacharide. Various lactic acid bacteria and yeasts have been identified as being present
in kefir grains, including L. brevis, L.helveticus, L. kefir, Leuconostoc mesenteroides,
Kluyveromyces lactis, K. marxianus and Pichia fermentans (Angulo et al. 1993, Lin et al.
1999). The micro-organisms contained within the kefir grains typically produce lactic acid,
antibiotics and several kinds of bactericide, such products inhibiting the proliferation of both
degrading and pathogenic microorganisms in kefir milk (Angulo et al. 1993).
Kefir occupies an important place in the human diet in many parts of the world including
Southwest Asia, Eastern and Northern Europe, North America, Japan (Otles and Cagindi,
2003), the Middle East, North Africa and Russia (Koroleva, 1982, IDF, 1988) due to its
nutritional and therapeutic significance. Especially in Soviet countries, kefir has, anecdotally,
been recommended for consumption by healthy people in order to lower the risk of chronic
diseases, and has also been provided to certain patients for the clinical treatment of a number
of gastrointestinal and metabolic diseases, hypertension, IHD and allergy (St-Onge et al.
2002; Farnworth & Mainville, 2003). Kefir has also been recommended for infants over the
Kefir and Health: A Perception 45
age of 6 months as mixed or artificial diet (Ivanova et al. 1980) and bifidokefir, containing
physiologically active cells of Bifidobacterium bifidum proved to be more efficacious than
ordinary kefir in eliminating intestinal infection in children (Murashova et al. 1997).
Production of Kefir
Kefir was first made from goat milk with kefir grains (Figure 1) in goat skin bag by
hanging in the house during winter and outside during summer (Ozer and Ozer, 1999).
Traditionally, kefir was produced by inoculating milk with kefir grains (Pijanowski, 1980) or
by the widely adopted European method, which involved use of bulk milk culture obtained
by kefir grains for milk inoculation (Puhan and Vogt, 1985). Kurmann (1984) classified kefir
under the class of mixed lactic acid and ethanol fermented milk and can be further sub-
classified as kefir obtained using kefir grains and artificial kefir obtained without kefir grains.
The traditional and industrial process of making kefir is presented in Figure 2a and Figure 2b
respectively. Kefir produced from pure cultures did not receive high sensory evaluation
scores in Canada unless it was sweetened (Duitschaever et al. 1987, 1991); Duitschaever et
al. (1987) also showed that only about 40% of people tasting natural kefir for the first time
gave it a positive taste rating. Addition of peach flavour, or modification of the fermentation
process (e.g. addition of lactococci, lactobacilli or yeasts) increased the acceptability of kefir,
compared to traditionally made kefir (Duitschaever et al. 1991; Muir et al. 1999).
To overcome these problems some biotechnological innovations in kefir production has
bee made (Sarkar 2008). Tratnik et al. (2006) recommended supplementation of goat milk
with whey protein concentrate at a level of 60.0-60.5 g/100 g proteins for enhancement in
ethanol production (0.35 ml/100 ml). To comply with the consumers demand for more
healthy foods, soya milk could be a suitable substitute for kefir production owing to its low
saturated fat and cholesterol (Berry, 2000), higher polyunsaturated fatty acids, lecithin,
linolenic acid, magnesium, iron, folic acid and vitamin E (Hermann, 1991).
At the present refreshing beverahge of kefir can be made by with fruit juice, molasses,
sugar, and any kind of milk such as cow, goat, sheep, camel, buffalo, or soy milk (Santos et
al. 2003, Harta et al. 2004). Fermentation of milk by traditional methods employing kefir
grains resulted in disparity in product quality due to diverse microflora and uncontrolled
fermentation. Two methods have been suggested to overcome the drawbacks of traditional
methods of kefir production, kefir can be produced either by simultaneous (Tamai et al. 1996)
or consecutive lactic acid and yeast fermentation (Beshkova et al., 2002). Probiotic kefir
capable of exhibiting antimicrobial properties could be obtained employing L. acidophilus,
Lactobacillus kefiranofuctens and Lactobacillus kefiranofaciens (Santos et al. 2003) or
Bifidobacterium bifidum (Murashova et al. 1997). Introduction of Candida kefyr during kefir
production may be advantageous as it did not disappear completely at pH 2.0, retains 97.2 per
cent viability in presence of 1per cent bile salts and not inhibited by most antibiotics
including tetracycline (You et al. 2006). In order to meet the consumer’s demand for
healthful foods in the current era of self-care and complementary medicine kefir with
enhanced dietetic properties could be obtained by co-inoculation of soya milk with yeasts
(Sacch. cerevesiae, Candida kefir), lactic acid bacteria (Streptococcus thermophilus,
Lactobacillus bulgaricus) and probiotic cultures (Lactobacillus acidophilus, Bifidobacterium

bifidum) (Sarkar 2008).
Figure 1 Kefir grains.
Chemical Composition of Kefir
The composition of kefir is variable and not well defined (Zubillaga et al. 2001). It
depends on the source and the fat content of milk, the composition of the grains or cultures
and the technological process of kefir (Kneifel and Mayer 1991, Bottazzi et al. 1994). Liut
Kevicius and Sarkinas (2004) reported kefir grains to contain 86.3 percent moisture, 4.5
percent protein, 1.2 percent ash and 0.03 percent fat. A typical kefir contains 89-90 percent
moisture, 0.2 percent lipids, 3.0 percent protein, 6.0 percent sugar, 0.7 percent ash (Ozer and
Ozer, 1999) and 1.0 percent each of lactic acid and alcohol (Webb et al., 1987). Kefir has
been reported to contain 1.98 g/l carbon dioxide and 0.48 percent alcohol (Beshkova et al.,
2002) and the content of carbon dioxide increased (201.7-277.0 ml/l) with increased
concentration (10-100 g/l) of kefir grains (Garrote et al., 1998). The major products formed
during fermentation are lactic acid, CO2 and alcohol. Diacetyl and acetaldehyde ( aromatic
compounds) (Zourari and Anifantakis, 1988, Beshkova et al. 2003), pyruvic acid, hoppuric
acid,acetic acid, propionic acid and butyric acid (Guzel-Seydim et al. 2000) are also present
in kefir. Diacetyl is produced by Str. Lactis subsp. diacetylactis and Leuconostoc sp.
(Libudzisz and Piatkiewicz, 1990). Commercial kefir contains half as much ortic acid, twice
as much pyruvic acid, nine time as much acetic acid, and about equal amount of uric acid as
does in commercial yoghurt (Dousset and Caillet 1993).
Storage of kefir at 48 °C for 21 days induced abatement in concentrations of ethanol to
0.08 percent, acetaldehyde to 11mg/g and a decline in acetoin to 16 ppm, however diacetyl
was not detected during either fermentation or storage (Guzel-Seydim et al. 2000). Kefir also
contains vitamins, macro and micro elements (Liut Kevicius and Sarkinas, 2004). Klyavinya
(1980) suggested a composition of 3.2-3.4 percent fat, 8.0 percentsolids-not-fats and 5.0
percent sucrose for kefir intended for infant feeding. The chemical composition of kefir is
given in Table 1.
Boiling of raw milk

↓
Cooling at 20-25 °C
↓
Incubation at 20-25 °C ← Kefir grain
↓
Fermentation at 20-25 °C, 18-24h
↓
Separation → Kefir grain
↓
Maturation and cooling at 4 °C
↓
Stored at 4 °C
Figure 2a. The traditional process of kefir. (Otles and Cagindi, 2003)
Raw milk
↓
Homogenization
↓
Pasteurization at 90-95 °C 5-10 min
↓
Cooling at 18-24 °C
↓
Incubation at 18-24 °C ← Kefir culture 2-8 %
↓
Fermentation 18-24 °C, 18-24 h
↓
Separation the coagulum
↓
Distribution in bottles
↓
Maturation 12-14/ 3-10 °C, 24 h
↓
Stored 4 °C
Figure 2b. The industrial process of kefir. (Otles and Cagindi, 2003)
Table 1. The chemical composition and nutritional values of kefir
Components 100 g Components 100 g

Energy 65 kcal Mineral content (g)
Fat (%) 3.5 Calcium 0.12
Protein (%) 3.3 Phosphorus 0.10
Lactose (%) 4.0 Magnesium 12
Water (%) 87.5 Potassium 0.15
Sodium 0.05
Milk acid (g) 0.8 Chloride 0.10
Ethyl alcohol (g) 0.9
Lactic acid (g) 1 Trace elements
Cholestrol (mg) 13 Iron (mg) 0.05
Phosphatateds (mg) 40 Copper (μg) 12
Molybdenum (μg) 5.5
Essential amino acids (g) Manganese (μg) 5
Tryptophan 0.05 Zinc (mg) 0.36
Phenylalanin+tyrosine 0.35
Leucine 0.34 Aromatic-compounds
Isoleucine 0.21 Acetaldehyde
Threonine 0.17 Diacetyl
Methionine+cystine 0.12 Acetoin
Lysine 0.27
Valine 0.22
Vitamins (mg)
A 0.06 B12 0.5
Carotene 0.02 Niacin 0.09
B1 0.04 C 1
B2 0.17 D 0.08
Source: Sarkar (2008), Liut Kevicius and Sarkinas (2004).
Microbiological Characteristics
Microflora of kefir grain and its approximate count in presented in Table 2. Microfora of
kefir grain is complex and not always constant, consisting of undefined species of bacteria
and yeasts (IDF, 1991). The microbial population found in kefir grains has been used as an
example of a symbiotic community (Margulis 1995); this symbiotic nature has made
identification and study of the constituent microorganisms within kefir grains difficult.
Abraham and Antoni (1999) reported that approximately 0.9 percent of the total weight of
wet kefir is represented by its microflora. Contents of bacteria in kefir varied from 6.4 ×104-
8.5 × 108 cfu/g, and yeasts from 1.5 × 105-3.7 × 108 cfu/g (Witthuhm et al. 2004). Irigoyen et
al. (2005) reported that besides a viable population of 108 cfu/ml of lactobacilli and
lactococci and 105 cfu/ml yeasts, kefir also had 106 cfu/ml acetic acid bacteria after 24 h of
fermentation.
Table 2. Microflora of kefir grains, kefir starter and kefir beverage
Microbial constituents Kefir grains Kefir starter Kefir beverage

(cfu/g) (cfu/ml) (cfu/ml)
Thermophilic lactobacilli 108 105 10-108
Mesophiilic lactobacilli 106-109 102-103 -
Lactococci 106 108-109 109
Leuconostoc 106 107-108 107-108
Acetic acid bacteria 108 105-106 104-105
Yeasts 106-108 105-106 104-105
Source: Bottazzi et al. (1994); Rea et al. (1996).
Bacterial cultures identified in kefir were Lactobacilli, Leuconostoc and Lactococcus

(Witthuhm et al., 2004) and Wang et al. (2004) reported it to be consisted of Lactobacillus
delbrueckii subsp. delbrueckii (8), Lactobacillus delbrueckii subsp. bulgaricus (6),
Lactobacillus Kefir (2), Lactobacillus acidophilus (1), Enterococcus faecalis (7),
Enterococcus faecium (2), Streptococcus thermoplilus (1) and Lactococcus lactis subsp.
cremoris (3). Identified yeasts were Zygosaccharomyces, candida and Saccharomyces
(Witthuhm et al., 2004), Klyveromyces matxianus, Klyveromyces lactis and Saccharomyces
cerevesiae as the dominant flora and Saccharomyces unisporus, Zygosaccharomyces rouxii,
Torulaspora delbrus, Zygosaccharomyces rouxii, Torulaspora delbrueckii and
Debaryomyces hansenii (Loretan et al. 2003).
Nutritional Composition
Beyond its inherent high nutritional value as a source of proteins and calcium, kefir has a
long tradition of being regarded as good for health in countries where it is a staple in the diet
(Vinderola 2004). The nutritional attributes of kefir (Table 1) are due to its chemical
ingredients such as vitamins, protein and minerals and fermentation induced further
enhancement in its nutritional profiles. Limited information available regarding nutritional
characteristics of kefir are delineated underneath.
Vitamin Content
Kneifel and Mayer (1991) found that appreciable amounts of pyridoxine, vitamin B12,
folic acid and biotin were synthesized during kefir production, depending on the source of
kefir grains used, while thiamine and riboflavin levels were reduced. Several investigation
have, measured the quantity of kefir to determine whether fermentation changed levels of
vitamin compared to milk, but the results have not often been consistent. An early study of
the vitamin B12 content of kefir indicated that both during the fermentation and maturation
stages, the vitamin B12 content went down (Guzel-Seydim et al. 2000). Alm (1982) used
commercially available grains to produce kefir that had increased folic acid content compared
to starting milk. Kefir contained vitamin B1, vitamin B2, vitamin B5 (Liut Keviciusand and
Sarkinas, 2004), vitamin B1, Vitamin B12, folic acid, vitamin K (Otles & Cadingi, 2003) and
vitamin C (Khamnaeva et al. 2000). The vitamin content of kefir is influenced by the type of
milk as well as by supplementing flora. An abatement in vitamin B12 (34-37 percent) due to
the inclusion of Propionibacterium peterssoni and Propionibacterium pituitosum
(Roczniakova et al. 1974) and vitamin B12 (30 times) and folic acid (five times) with a slight
increase in pantothenic acid and vitamin B6 due to Propionibacterium freudenreichii subsp.
shermanii as supplementing organism (Cerna and Hrabova, 1982) were noted during the
manufacture of kefir.
Protein Content
Knowledge of grain proteins is limited (Abraham and Antoni 1999). A higher protein
content was found in kefir, when the grains were cultured in whey (Fil’ Chakova and
Koroleva, 1997) or in soy milk (Abraham and Antoni, 1999) than those cultured in milk.
Yuksekdag et al. (2004a) demonstrated the proteolytic activity of lactococci (13/21 strains)
isolated from kefir. Otles & Cadingi (2003) reported that during kefir fermentation process,
along with vitamin production, amion acids were also produced.Molokeev et al. (1998)
narrated that bifidofefir containing 2x107 cfu/ml bifidobacteria had higher contents of
glutamic acid and threonine than normal kefir. During fermentation of milk, there is a change
in amino acid profiles and higher amounts of threonine, serine, alanine, lysine and ammonia
are produced in kefir than in milk (Guzel-Seydim et al., 2003). Liut Kevicius and Sarkinas
(2004) reported amino acid profiles of kefir showing the presence of valine, isoleucine,
methionine, lysine, threonine, phenylalanine and tryptophan.
Sugar Contents
A typical kefir contains 6% sugar [Ozer & Ozer 1999], a major portion of the gelatinous
matrix containing kefir microflora. Sugar present in kefir is known as kefiran, a
heteropolysaccharide which is glucogalactan in nature. Kefiran improves viscosity and
viscoelastic properties of acid milk gels [Rimada et al. 2006] and itself forms gels at low
temperatures. Kefiran had been reported to form films isolated from LAB with low water
vapor permeability and extra ordinary flexibility, even higher than those corresponding to
low density polyethylene [Piermaria et al. 2009]. In addition, polysaccharides of kefir have
several health promoting properties such as inhibitory effects on rotavirus [Song et al. 2007],
immunomodulation or epithelium protection.
Mineral Content
Liut Kevicius and Sarkinas (2004) reported the presence of macro-elements such as
potassium, calcium, magnesium, phosphorus and micro-elements such as copper, zinc, iron,
manganese, cobalt, molybdenum in kefir.
Therapeutic Characteristics
Kefir exhibits varied therapeutic attributes due to the possession of different components
(Table 3), which are discussed below.
Anticarcinogenic Effect
Epidemiological studies have indicated a reduced risk of breast cancer in women who
consumed bovine fermented milk products (Redyy et al. 1983, Veer et al 1989). The
bioactive anticancer components present in fermented bovine milk include proteins and
peptides, which have been shown to have anti-tumorigenic activities in feeding trials both in
animal models of cancer and in cultured tumor cells (Svensson et al. 1999). The anti-
carcinogenic role of fermented milk products may be broadly classified into prevention of
cancer initiation and suppression of an initiated tumour by retarding the activity of enzymes
that convert procarcinogen to carcinogen or by activating the immune system (Kneating,
1985). Anticarcinogenic effect of kefir and kefir extract has been studied extensively.
Encouraging results regarding antitumor activities of kefir and isolated kefir extracts in
animal studies have been reported (Shiomi et al. 1982, Cevikbas et al. 1994, Furukawa et al
1990, Kubo et al 1992). Shiomi et al. (1982) showed that polysaccharides extracted from
kefir grains had antitumor activity in mice. Also, oral doses of 100 or 500 mg/kg of kefir fed
to mice with transplanted solid tumors of E-ascites carcinoma were shown to cause a
significant reduction in transplanted tumor size and activate the immunosuppressive activity
of the spleen (Kubo et al 1992). It has been shown that isolated strains of Streptococcus,
Lactobacillus and Leuconostoc (Hosono et al., 1990) and Streptococcus lactis subsp.
cremoris (Miyamoto et al., 1991) from kefir were able bind mutagens. Ingestion of kefir (2
g/Kg body weight) for nine days proved to be more effective for tumour inhibition than
yogurt (Furukawa et al., 1990), with better inhibition being noted (70.9 and 64.8 percent,
respectively) for soy milk Kefir than milk kefir (Liu et al., 2002). Kefiran, which is a water-
soluble glucogalactan, either isolated from kefir grain or produced by L.kefiranofaciens, a
strain isolated from kefir, (Wang et al. 2008) also have antitumor activity. However water
soluble polysaccharides containing kefir grain microflora are more efficacious than water
soluble polysaccharides for the suppression of tumors (Furukawa et al., 2000) and higher
dosages may be more effective if administered after the establishment of tumours (Murofushi
et al., 1983).
It has been concluded that antitumour properties of kefir may be either due to the
microorganisms or polysaccharides produced during fermentation (Liu et al. 2002), especially
lactobacillus (Santos et al. 2003). Guzel-Seydim et al. (2003) registered that based upon
previous research, milk protein and especially those with high concentration of sulphur
containing amino acids are important for anticarcinogenicity. De Moreno De Leblanc et al.
(2007) studied the involvment of immune cells in the antitumor effect of kefir in a murine
breast cancer model. He found that immune response in mammary gland played an important
role to avoid tumor growth. Chen et al (2007) studied the effect of kefir extract on
suppression of estrogen-dependent human breast cancer cells. He concluded that extracts
from early-stage kefir fermentation (kefir mother culture) and the final kefir product
demonstrated antiproliferative effects that were specific to breast cancer cells. Although the
bioactive component(s) and the mechanism of the antitumor activity were not clear, but his
study suggested the potential of kefir fermentation produced new peptides or other bioactive
compounds, which could have anticancer activities.
Figure 3 exhibits proposed sites and modes of action of Kefir throughout tumor cycle.
Antibacterial Spectrum
There are data to show that many lactobacilli are capable of producing a wide range of
antimicrobial compounds, including organic acids (lactic and acetic acids), carbon dioxide,
hydrogen peroxide, ethanol, diacetyl and peptides (bacteriocins) that may be beneficial not
only in the reduction of foodborne pathogens and spoilage bacteria during food production
and storage, but also in the treatment and prevention of gastrointestinal disorders and vaginal
infections (Zamfir et al. 1999 Zhou et al., 2008, Liu et al 2008, Simova et al. 2009 ).The
beneficial action of kefir can be partially attributed to the inhibition of pathogenic
microorganisms by metabolic products such as organic acids produced by kefir microflora
(Golowczyc et al 2008). The antibacterial activity of fermented milk products could be
attributed to metabolic end products present and/or antibacterial compounds produced by
starter cultures. Kefir has a higher bacteriostatic effect against Gram-negative organisms but
a better bactericidal effect against Gram positive organisms (Czamanski et al., 2004). The
antagonistic behaviour of kefir against Escherichia coli, Listeria monocytogenes, Yersinia
enterocolitica (Gulmez and Guven, 2003), Listeria innocua (Morgan et al., 2000),
Salmonella enteritidis (Czamanski et al., 2004) and Staphylococcus aureus (ATCC 29213),
Bacillus cereus (ATCC 11778), Salmonella enteritidis (ATCC 13076), Listeria
monocytogenes (ATCC 7644) and Escherichia coli (ATCC 8739) has been mentioned
(Ulusoy et al. 2007). Silva et al. (2008) studied the effect of antimicrobial activity of broth
(added with different sugars) with kefir grains and he found that kefir grains promoted the
hydrolysis of non-reducing sugars, which were converted into organic acids and substances
capable of producing inhibition halos in experiments with pathogenic microorganisms. The
inhibited species included: Candida albicans, Salmonella typhi, Shigella sonnei,
Staphylococcus aureus, and Escherichia coli.
Figure 3. Tumor formation in body and proposed sites of action of kefir.
In an in vitro study conducted by Rodrigues et al (2005a), kefir inhibited the growth of

Streptococcus pyogenes and Candida albicans. Santos et al. (2003) noted the antagonistic
behavior of isolated strains of lactobacilli from kefir grains against E. coli (43/58 strains), L.
monocytogenes (28/58 strains), Salmonella typhimurium (10/58 strains), S. enteritidis (22/58
strains), Shigella flexneri (36/58 strains) and Y. enterocolitica (47/58 strains). In another
study, strains of Lactococcus cremoris, Lc. lactis, Str. thermophilus and Str. durans, isolated
from kefir inhibited the growth of S. aureus (Yuksekdag et al 2004a). In the same study, two
strains of Lc. lactis and a strain of Lc. Cremoris inhibited the growth of E. coli and
Pseudomonas aeruginosa (Yuksekdag et al., 2004a). Witthuhn et al. (2004) also described a
strain of Str. thermophilus active against P. aeruginosa. The antibacterial activity of kefir
depends on the extent of fermentation. The populations of E. coli, L. monocytogenes and Y.
enterocolitica increase in kefir fermented for one day but only E. coli increased when
fermented for two days (Gulmez and Guven, 2003). The antibacterial activity of kefir against
various pathogens may be attributed to organic acids and specific antibodies produced by
acetic acid bacteria and yeasts (Koroleva, 1988), undissociated lactic and acetic acid
produced during fermentation (Garrote et al. 2000) or hydrogen peroxide produced by LAB
(Yuksekdag et al. 2004a, 2004b).
Effect on Immune System
Nutritional status has a major impact on the immune system (Vinderola et al. 2006a).
Numerous studies have demonstrated the beneficial effects of lactic acid bacteria (LAB) and
fermented dairy products in boosting specific or nonspecific immune responses (Gill, 1998;
Isolauri et al. 2004, Matar et al. 2001, Perdigon et al. 2001, Vinderola et al. 2004). Probiotic
microorganisms can exert their beneficial properties mainly through two mechanisms: direct
effects of the live microbial cells (probiotics) or indirect effects via metabolites of these cells
(biogenics). Biogenics are defined as food components derived from microbial activity which
provide health benefits without involving the intestinal microflora (Takano, 2002) while
irregularity in gut microflora functions could potentially contribute to a wide range of
diseases [Mai et al. 2009].
Vinderola et al (2005b) demonstrated the immunomodulating capacity of kefir in a
murine model, showing the importance of the dose and cell viability to obtain a Th2 or Th1
response. Kefir can increase the phagocytic activity of peritoneal and pulmonary
macrophages and can modulate the mucosal response at distant sites. In the latter study they
observed the effects of kefir microflora and its non-bacterial fraction on cytokine production
by cells of the innate immune system (peritoneal macrophages and adherent cells derived
from Peyer’s patches). Moreover they noticed a higher capacity of the products derived from
milk fermentation by kefir microflora (PMFKM), compared with kefir microflora itself, of
inducing the proliferation of IL-10 producing cells among adherent cells derived from
Peyer’s patches of mice(Vinderola et al. 2006a).In the most recent study, Vinderola et al.
(2006b) demonstrated the immunomodulating capacity of kefir in a murine model, aimed at
studying the immunomodulating capacity in vivo of the products derived from milk
fermentation by kefir microflora (PMFKM) on the gut. They observed that the PMFKM
induced a mucosal response and it was able to up and down regulate it for protective
immunity, maintaining the intestinal homeostasis, enhancing the IgA production at both the
small and large intestine level.
Table 3. Therapeutic attributes of kefir
Therapeutic attributes Therapeutic components References

Anti-carcinogenic effect Polysaccharide Furukawa et al. (2000)
Antimutagenicity Peptide Liu et al. (2005)
Antioxidant Liu et al. (2005)
Scavenging activity Peptide Liu et al. (2005)
Nagira et al.(1999a)
Mucositis Topuz et al. (2008)
Cholestrol lowering effect Polysaccharide Liu et al. (2006b)
Cholesterol degrading Vujicic et al. (1992)
enzyme
Immunomodulating capacity Kefir as whole Vinderola et al. (2005a)
Anti-allergenic properties kefir and soymilk kefir Liu et al. (2006)
Antibacterial spectrum Hydrogenperoxide Yuksekdag et al. (2004b)
Lactic acid Garrote et al. (2000)
Acetic acid Garrote et al. (2000)
Bacteriocin Santos et al. (2003)
Gastrointestinal proliferation Lactic acid bacteria Marquina et al. (2002)
b-galactosidase activity b-galactosidase enzyme De Vrese et al. (1992)
Bacterial colonization S-layer protein Garrote et al. (2004)
Antiflammatory Polysaccharide Moreira et al (2008)
Thoreux and Schmucker (2001) fed kefir produced from grains to young (6 months) and
old (26 months) rats and found an enhanced mucosal immune response in the young animals,
as shown by a higher anti-cholera toxin (CT) IgA response compared to controls. Both young
and old rats had significantly increased total non-specific IgGblood levels, and a decreased
systemic IgG response to CT. Taken together, Thoreux and Schmucker concluded that kefir,
like other probiotics, was exerting an adjuvant effect on the mucosal immune system, perhaps
produced by bacterial cell wall components.
Stimulation of the immune system may also occur due to the action of
exopolysaccharides found in kefir grains. Murofushi et al. (1983) used the method of La
Riviere et al. (1967) for the extraction of kefiran from kefir grains to produce a water-soluble
polysaccharide fraction that they fed to mice. The reduction in tumour growth that they
observed was linked to a cell-mediated response, and it appeared that the total dose of the
polysaccharide determined its effectiveness. Furukawa et al. (1992) have also shown that a
water-soluble fraction of kefir grains may act as a modulator of the immune response.
Vinderola et al (2006c) also observed the effects of kefir microflora and the non-bacterial
fraction on cytokine production by cells of the innate immunity-adherent populations of
Peyer’s patches and the peritoneal macrophages. Since kefir drink contains kefiran, it was of
interest to determine the effect of this exopolysaccharide on immune function. It was found
that the exopolysaccharide induced a gut mucosal response and it was able to up and down
regulate it for protective immunity, maintaining intestinal homeostasis, enhancing the IgA
production at both the small and large intestine level and influencing the systemic immunity
through the cytokines released to the circulating blood.
The effect of kefir exopolysaccharides on the immune system may be dependent on
whether the host is healthy or has developed any tumours. Furukawa et al. (1996) incubated
kefir grain polysaccharides with Peyer’s Patch (PP) cells from tumour-bearing mice and
found that the supernatant of this mixture enhanced proliferation of splenocytes from normal
mice and increased the mitogenic activities of lipopolysaccharides (LPS) and
phytohaemagglutinin- P (PHA-P) in splenocytes. They concluded that the polysaccharide
stimulated PP cells, causing them to secrete water-soluble factors that, in turn, enhanced the
mitogenic response of thymocytes and splenocytes in normal mice.
Anti-inflammatory
Rodrigues and coworkers (2005b) demonstrated the anti-inflammatory properties of kefir

and its polysaccharide extract. The results showed significant inhibition in the formation of
granuloma tissue for all the test groups, as compared to the blank group. Kefir suspensions in
molasses presented an inhibition of 41 ± 3% for the inflammatory process, fermented milk
prepared from kefir showed 44 ± 6% inhibition and kefiran extract 34 ± 15%. In order to
investigate the pharmacological effects of kefir, Lee and coworker (2007) used a mouse
asthma model, in which airway inflammation and airway remodeling was produced by
ovalbumin sensitization and challenge. BALB/c mice sensitized and challenged to
ovalbumin, were treated with kefir (50 mg/kg administered by intra-gastric mode) 1 h before
the ovalbumin challenge. Histological studies demonstrated that kefir substantially inhibited
ovalbumin-induced eosinophilia in lung tissue and mucus hypersecretion by goblet cells in
the airway. Kefir displayed anti-inflammatory and anti-allergic effects in a mouse asthma
model and may possess new therapeutic potential for the treatment of allergic bronchial
asthma. Allergic asthma is a complex chronic airway disorder that is characterized by airway
inflammation, lung eosinophilia, mucus hypersecretion by goblet cells, an elevated serum IgE
level, and airway hyperresponsiveness (AHR) (Elias et al. 2003). An orally administered L.
plantarum isolated from kefir, had also been reported to exert anti-inflammatory effects in a
mouse model (Lee et al. 2007).
Hypocholesterolemic Effect
Since the early studies of Mann & Spoerry (1974), there appears to have been an
increasing interest in the hypocholesterolaemic activity of fermented dairy products. A
number of studies have been performed with experimental animals, and also human subjects,
in order to elucidate the effect of fermented dairy products on serum cholesterol (Anderson &
Gilliland, 1999, St-Onge et al. 2000; Xiao et al. 2003). Although some contradictory results
have been obtained (Nakajima et al. 1992; de Roos et al. 1998), the majority of results from
these reports indicate that fermented dairy products, possibly including kefir, possess
hypocholesterolaemic properties.
Considerable attention has been given to the cholesterol level of foods due to its public
health significance as high levels are associated with greater risk of cardiovascular disease. A
higher population of LAB in kefir and their ability to bind up to 33.9 percent cholesterol led
to a direct reduction of cholesterol by cultures in the intestine (Hosono and Tanako, 1995).
Culturing of milk with kefir cultures at 248°C for 24 h induced an assimilation of cholesterol
by 28-65 percent and the corresponding figure reached to 41-84 percent after 48 h of
incubation (Vujicic et al. 1992). Application of invertase deficient yeasts such as
Saccharomyces cerevesiae (Tamai et al. 1996) was reported to exert a hypocholesterolemic
effect to consumers (Noh et al., 1997). Wojtowski et al. (2003) mentioned that sheep milk
kefir can be considered to be more advantageous for health effects than kefirs from cow or
goat milk due to the presence of higher levels of linoleic and/or linolenic acids.
Hydroxymethylglutaric and/or orotic acids presumably inhibit a rate-limiting enzyme in
cholesterol synthesis (Shahani and Chandan, 1979). Ozer and Ozer (1999) reported that the
hypocholesterolemic effect of kefir in humans may be attributed to a loss of orotic acids
during kefir fermentation, which are known to cause fat accumulation in the liver. Maeda et
al. (2004) reported that an exopolysaccharide produced by L. kefiranofaciens reduced serum
cholesterol level in rats when they consumed excessive dietary cholesterol. Liu et al (2006)
studied the effect of milk kefir and soya-milk kefir in cholesterol-fed hamsters. He also found
that the soyamilk, milk-kefir and soyamilk-kefir diets all tended towards a lowering of serum
triacylglycerol and total cholesterol concentrations, and a reduction of cholesterol
accumulation in the liver, the decrease in serum cholesterol concentration being mainly in the
non-HDL fraction. These findings demonstrate that kefir or its component may be considered
to be among the more promising food components in terms of preventing

hypocholesterolaemic action.
β-galactosidase Activity
Lactose maldigestion is the inability to completely digest lactose, the major carbohydrate
in virtually all mammalian milks. Lactose maldigestion affects approximately 75% of the
world’s adult population and occurs most often as the result of a genetically programmed
decrease in intestinal lactose activity after the age of 3 to 5 years. The use of fermented dairy
foods has long been employed as a strategy for overcoming lactose intolerance (Kolars et al
1984). This appears to be related to the presence of β-galactosidase in the yogurt starter
culture bacteria (Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii
subsp. bulgaricus) (Hertzler and Clancy 2003). Reduction in lactose content and the presence
of β-galactosidase activity in cultured milk products render it suitable for consumption by
persons classified as lactose-intolerant, attributed to better absorption of lactose than other
sources of sugar and autodigestion of lactose by its endogenous microorganisms. Kefir grains
possess β-galactosidase activity (De Vrese et al., 1992) and kefir may be equally effective as
yogurt for reducing breath hydrogen in lactose-intolerant adults (Hertzler and Clancy, 2003).
Kefir contains less lactose than milk (Dies, 2000) and have 60% higher β-galactosidase than
plain yoghurt and its ingestion improves lactose digestion and reduced the perceived severity
of flatulenceby 54-71 percent in contrast to milk (Hertzler and Clancy, 2003).
Gastrointestinal Proliferation
Gastrointestinal tract constitutes the largest interface between animals and their external
environment. As a barrier it prevents the penetration of harmful entities such as food antigens
(Umeda et al 2005). Fermented milk products are capable of restoring the normal lactic
intestinal flora by inhibiting undesirable flora and the antibacterial activity is influenced by
the culture activity, temperature and time of storage and the initial level of contamination.
Intake of kefir induced an abatement in LAB by ten-fold and a decline in levels of sulphite
reducing Clostridia by 100-fold in mice (Marquina et al., 2002), benefited protein digestion
and reduced glycemic index in female rats prevented (Urdaneta et al 2007), Campylobacter
jejuni colonization in the caecum of chicks (Zacconi et al., 2003), affected the intestinal
mucosal and systemic immune response in young rats (Troreux and Schmucker, 2001) and
induced mucosal resistance to gastrointestinal infection in mice (Liu et al., 2002). Kefir is
helpful in the treatment of post-operative patients or patients with gastrointestinal disorders
(Fil’ Chakova and Koroleva, 1997). Murashova et al. (1997) noted that infants with severe
intestinal infection receiving bifidokefir (kefir containing physiological cells of
Bifidobacterium bifidum) induced rapid inhibition of Salmonella and Shigella within 7-11
days of illness and the faecal flora reverted to normal after 4.8 ±0.8 days, whereas the
corresponding figures for the group of infants not receiving bifidokefir were 12-18 and 6.6 ±
0.9 days, respectively. Bifidokefir containing 5x107 Bifidobacteria/ml had a positive effect
on intestinal flora in humans and normalized intestinal Bifidobacteria: Lactobacilli balance
accompanied by a decline in the population of pathogens and abatement in lactic acid

bacteria within 7-14 days (Molokeev et al., 1998).
Bacterial Colonization
Lactic acid bacteria in food can transiently colonize the intestine and exert beneficial
effects (probiotic). Survival during intestinal transit or adhesion to epithelium or both seem to
be important for modifying the host’s immune reactivity. Because Successful seeding of
bacteria depends on their bile salt tolerance and ability to withstand the conditions prevailing
in the intestine (Schiffrin et al 1995). It has been observed that 85 percent of the isolated
species of Lactobacillus from kefir grains can resist oxgall and many of them are able adhere
to enterocyte-like cells (Santos et al., 2003). Similarly, yeasts like Kluyveromyces lactis and
Kluyveromyces ladderae are more acid tolerant and have the ability to adhere to human
intestine due to the proteinaceous factor (Kumura et al., 2004). Garrote et al. (2004) also
mentioned that the presence of S-layer protein in Lactobacillus kefir and Lactobacillus
parakefir was responsible for the adhesive properties to CaCo 2 cells, auto aggregation and
haemagglutination. Both specific attachment and multiplication on the surface of the
membrane of the gastro-intestinal epithelial cells are critical mechanisms in the establishment
of these organisms in the intestinal tract. Adhesion is not a necessary requirement for the
successful colonization of the intestine, but those organisms that do adhere may have more
effect on the physiological functioning of the intestinal tract (Cole and Fuller, 1984).
Anti-Diabetic Effect
Kwon and coworkers (2006) studied anti-diabetes functionality of Kefir culture-mediated

fermented soymilk supplemented with Rhodiola extracts. Their results indicated that Kefir
culture-mediated fermentation of soymilk supplemented with Rhodiola extracts resulted in
mobilization of total phenolics, which could be effectively designed as complimentary
therapies for postprandial hyperglycemia. Water-soluble or chloroform/ methanol-extracted
fractions from Kefram-Kefir were examined to evaluate the glucose uptake ability of L6
myotubes. As a result, the water-soluble fraction augmented the uptake of glucose in L6
myotubes both in the presence and absence of insulin stimulation. Estimation of intracellular
ROS level revealed that the water-soluble fraction of Kefram-Kefir reduced the intracellular
ROS level on both the undifferentiated and differentiated L6 cells linked to Type II diabetes
management. So it was suggested that the water-soluble fraction of Kefram-Kefir activates PI
3-kinase or other upstream molecules in the insulin signaling pathway, which resulted in the
augmentation of glucose uptake and its specific inhibition by wortmannin (Teruya et al
2002). Later on Maeda et al (2004) also found that kefiran supplementation demonstrated the
ability to significantly lower blood glucose in KKAy mice.
Antiallergic Properties
Food allergy is now recognized as a worldwide problem, and like other atopic disorders
its incidence appears to be increasing. Consumption of milk kefir and soymilk kefir
suppressed the IgE and IgG1 responses and altered the intestinal microflora in our
supplemented group, suggesting that milk kefir and soymilk kefir may be considered among
the more promising food components in terms of preventing food allergy and enhancement of
mucosal resistance to gastrointestinal pathogen infection (Liu et al 2006). Another
histological studies demonstrated that kefir substantially inhibited ovalbumin-induced
eosinophilia in lung tissue and mucus hypersecretion by goblet cells in the airway. Kefir
displayed anti-inflammatory and anti-allergic effects in a mouse asthma model and may
possess new therapeutic potential for the treatment of allergic bronchial asthma (Lee et al
2007).
Antioxidative Properties
Dietary constituents of antioxidative vitamins and other nutrients may play an important
role in protecting the body against oxidative damage. Guven et al (2003) carried out study to
investigate the protective effect of kefir against oxidative damage of CCl4 in mice, compared
with the well-known antioxidant vitamin E. Three-week-old Swiss Albino mice, weighing
22–26 g were used for the experiment. At the end of the microbiological analysis of kefir, the
averages of the total mesophilic aerobic colony counts, lactic acid bacteria, lactic
streptococci, enterococci, and yeasts were found to be 1.04 × 109, 9.87 × 108, 4.38 × 108,
7.80 × 104 and 1.26 ×105 CFU/ml, respectively. While both vitamin E and kefir were found
to have a protective effect aganist CCl4-induced damage, kefir was more protective (Guven et
al 2003).
Effect on Lipid and Blood Pressure Level
A study on the effects of kefiran (polysaccharide present in kefir) in animals

demonstrated that kefiran significantly suppressed increase of blood pressure and reduced the
serum cholesterol levels in SHRSP/Hos rats when subjects consumed excessive dietary
cholesterol. These results suggested that kefiran could be used as a functional food to prevent
some commonly occurring diseases (Maeda et al. 2004).
Protection against Apoptosis
UV irradiation produces reactive oxygen species from in skin cells and damage
melanocytes and other skin cells, causing liver spots, freckles, wrinkles and skin cancer. The
exposure of the intestine to ionizing radiation results apoptotic death of the stem cells. Nagira
and coworkers (1999a) studied the studied the scavenging effect of the Kefran-Kefir against
superoxide radicals and the protective effect of the Kefran–Kefir extract on UV damage of
human melanoma HMV-1 cells. When HMV-1 cells were treated with the Kefran-Kefir
extracts before, during or after UV irradiation, the alive cell number was remarkably
increased compared to the control values, proving that Kefran–Kefir can protect human
melanomas from UV damage. In another study they found that apoptosis of HMV-1 cells
caused by UV irradiation was also suppressed by the Kefir extract, suggesting that DNA
repair enhancing factors in the Kefir extract could decrease DNA damage by UV irradiation
and suppress apoptosis (Nagira et al 1999b). To evaluate the effect of fermented milk kefir on
X-ray-induced apoptosis in the colon of rats, Matsuu and colleague (2003) examined the
apoptotic index, the mean number of apoptotic cells detected by H&E staining per crypt in
the colon, in control rats and kefir-pretreated rats drinking kefir for 12 days before
irradiation. They demonstrated that kefir protected the colonic epithelial stem cell region, and
is extremely important for regeneration following radiation-induced apoptosis; this
antiapoptic effect of kefir was mediated through the inhibition of caspase-3 activation. Kefir
treatment may have possibilities to diminish side effects in the intestinal epithelium of
patients undergoing irradiation therapy for malignancy.
Conclusion
Recently, consumers’ growing preference for food with enhanced nutritional and
therapeutic properties has led to the inclusion of various cultured milk products as part of
their diet based upon their dietetic features. Among them kefir can attained a significantly
high ranking due to its long list of therapeutic properties. The self carbonated beverage, kefir
may be recommended as a dietary beverage owing to its nutritional attributes due to its
vitamin, protein and mineral content and therapeutic attributes, namely its antibacterial
spectrum, gastrointestinal proliferation, hypocholesterolemic effect, anticarcinogenic effect,
L(+) lactic acid content, β-galactosidase activity, protection against apoptosis, Effect on lipid
and blood pressure level antiallergic properties, antidibetic properties, anti-inflammatory and
bacterial colonization. Kefir is able to normalize intestinal microflora and is highly suitable
for consumption by normal and sick adults as well as infants.
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Editors: E. Lango and F. Vogel © 2009 Nova Science Publishers, Inc.
Chapter III
Fouling Reduction during Milk

Processing Using
Equipment Surface Modification
Sundar Balasubramanian1,* and Virendra M. Puri2

1
Department of Biological and Agricultural Engineering, Louisiana State University
AgCenter, 149 E.B. Doran Building, Baton Rouge, LA 70803, USA
2
Department of Agricultural and Biological Engineering, 229 Agricultural Engineering
Building, Pennsylvania State University, University Park, PA 16802, USA
Abstract
Fouling of equipment surfaces during milk processing is a phenomenon that needs to

be immediately addressed due to the increased energy utilization and production costs
encountered. Modifying the equipment surface is one method of reducing the incidence
of fouling. Research was carried out at the Pennsylvania State University using different
food-grade surface coatings to modify plate heat exchanger surface, and was tested for
their ability to reduce fouling during skim milk pasteurization. The results were
compared with traditional stainless steel 316 plate heat exchanger (PHE) surfaces
typically used in the food industry. Results after 6 h continuous testing using a pilot scale
PHE unit indicate that there was greater than 85% reduction in fouling when the three
coated surfaces (AMC 148-18, Ni-P-PTFE and LectrofluorTM-641) were used. Chemical
analyses of the foulants indicate that the coating integrity did not appear to be
compromised for the LectrofluorTM-641 coatings. However, there were trace amounts of
fluorine present in the foulants adhering to the other two coating types (AMC148-18 and
Ni-P-PTFE). A preliminary cost estimate on the thermal energy savings when using the
coated surfaces indicate that there is substantial savings in energy, further justifying the
*
Corresponding Author. E-mail: bsundar@lsu.edu; Tel: (225)-578-1072 (office).
72 Sundar Balasubramanian and Virendra M. Puri
use of these coated surfaces, and making them more attractive for possible
implementation in the food industry.
Introduction
Fouling is an undesirable side effect of thermal processing that results in an increase in

electrical and thermal energy usages due to the decrease in heat transfer coefficient and the
increase in pressure drop across the heat exchanger unit; thereby lowering the overall system
performance. Fouling occurs during thermal processing of foods rich in proteins; milk and
milk-based products. Unfolding of protein chains, commonly known as protein denaturation,
occurs when protein-rich foods are treated with heat. When these unfolded proteins
agglomerate and adhere to the food processing equipment surface, the phenomena is termed
as fouling. The resultant fouling layer is a low thermal conductivity layer and results in an
increase in the input thermal energy. Frequent cleaning of the fouling layer is required which
results in further increased energy and water usage. Further ramifications on the food product
quality and food safety aspects due to microbiological hazards, over heating or under heating
of foods cannot be ignored as a result of fouling.
Fouling of processing lines particularly plate heat exchangers has a profound economic
impact on the operating costs. A study estimates that about 80% of the total operating costs
involved in a typical dairy plant are attributed towards the effects of fouling (Bansal and
Chen, 2006). In dairy plants, the frequency of cleaning the fouled processing equipment is
higher than in other industries where fouling is prevalent. Cleaning the fouled equipment is a
time-consuming and energy-intensive process, consuming large quantities of water and
chemicals. For a typical dairy processing plant handling 283.91 m3 of milk per day an
average of approximately 416,395.3 m3 of water per year is required for cleaning (Rausch
and Powell, 1997). If the equipment is severely fouled or if the foulants adhere strongly with
the equipment surface, then the water and chemical requirement for cleaning the dairy
equipment further increases. On an average the CIP (cleaning-in-place) process accounts for
9.5% of the primary energy demand (energy consumption) in the Dutch dairy industry and
accounts for 0.14 to 0.30 MJ/cycle of the thermal energy requirement for milk pasteurization
(Ramirez et al., 2006). To add to this high energy requirement, the incidence of fouling
causes about 21% increase in the total energy consumption related to the operation and
cleaning of milk pasteurization units (Sandu and Singh, 1991). Thus, prolonging the
processing operation and minimizing the frequency of cleaning the foulants can be achieved
by minimizing or delaying the process of equipment fouling. This results in substantial
energy and cost savings.
Equipment surface properties like roughness, wettability, charge, and charge density
influence the protein adsorption (fouling). Hence, modification of the processing equipment
surface properties could reduce the rate of protein adsorption by minimizing the surface free
energy. This has led to an interest in investigating the role of modified surfaces on fouling
reduction. Primarily, two methods are used to modify the surface properties of equipment:
chemical methods and mechanical methods (Zettler et al., 2005). Machining, sand blasting,
and pulsed plasma combined with photolithographic techniques are some of the mechanical
Fouling Reduction during Milk Processing Using Equipment Surface Modification 73
means of altering surface roughness, thus altering the coefficient of friction and the frictional
forces acting on the food product flowing over the equipment surface (Pier Francesco et al.,
2006; Muller et al., 2006; Bretagnol et al., 2007).
In the chemical approach, use of anti-fouling coatings, ion implantation techniques, and
acid etching are some of the methods employed to modify the surface characteristics
(Rosmaninho and Melo, 2006; Ban et al., 2006; Braceras et al., 2007). There is significant
literature available on the use of non-stick anti-fouling coatings like Ni-P-PTFE (nickel
phosphorus polytetrafluroethylene) for reduction of fouling during processing of food
products (e.g., Zhao et al., 2005; Rosmaninho and Melo, 2006; Zhao and Liu, 2006).
However, there could be some concerns on the use of non-stick coatings in food processing
equipment owing to the possibility of coating degradation due to the processing conditions
and food components that come in touch of the coating material. This could lead to the
possibility of coating incursion into the processed food. Some studies have pointed out the
lower wear resistance and lower adhesion property of Ni-P-PTFE coatings on stainless steel
(Zhao and Liu, 2006; Wu et al., 2006). Hence, it is imperative to find a suitable commercially
available coating material that will have good binding properties with stainless steel as well
as be able to provide the necessary surface conditions that could reduce or prevent fouling.
A research effort was undertaken at the Department of Agricultural and Biological
Engineering, Pennsylvania State University to investigate the proof-of-concept of using
various food-grade coatings in reducing fouling during skim milk pasteurization. Successful
implementation of this technique will have a significant impact on reduction of energy
consumption in the food industry, improve food quality, reduce the extensive use of cleaning
chemicals, and reduce the use of valuable natural resources such as water. This chapter
summarizes the results obtained during the course of that study.
Materials and Methods
Plate Heat Exchanger Set-up
The plate heat exchanger (PHE) system for skim milk pasteurization has been described
by Balasubramanian and Puri (2008a) in detail. The PHE system was designed and installed
in-house at the Pilot Plant Facility of the Department of Agricultural and Biological
Engineering, the Pennsylvania State University. Briefly, the system consists of a PHE unit
(Model M6-MBase, Alfa Laval USA, Glen Allen, VA, USA) with heating and cooling
sections that were fed with hot water by heating and cooling sub-systems, and a data
acquisition unit. To accelerate the occurrence of fouling no re-generation section was used in
the study. This was in accordance to the findings of researchers who indicate that pre-heating
milk reduces the extent of fouling (de Jong 1997; Visser and Jeurnink 1997). The PHE unit
was operated in a single pass counter-current flow path.
The temperature of the fluids entering and leaving the PHE system was recorded using T-
type thermocouples (type T) installed at various locations in the process line including the
nine locations shown in Figure 1 to record the time-temperature history along the middle flow
channel. In addition, the thermocouple CT monitored the exiting fluid milk temperature,
which served to control the heater by turning it on or off. This control thermocouple ensured
that the temperature of the fluid milk leaving the heating channels to be 72±2oC. The time-
temperature data were collected every 2 s using LabView software (Version 6i, National
Instruments, Austin, TX, USA) via a data acquisition system.
Figure 1. Plate arrangement and milk flow direction in the heating section of the PHE system (top). The
bottom figure shows the plate with the thermocouples in 9 locations on the plate surface (denoted by dots)
and the control thermocouple in the flow channel (Recreated from Balasubramanian and Puri, 2008a).
Table 1. Physical properties and calculated overall heat transfer values for the control
(SS-316) and SS-316 coated surfaces in the PHE system
(Balasubramanian and Puri, 2009)
Surface type Thermal Continuous Coefficient of Calculated overall

conductivity usage friction1 heat transfer
1
temperature1, coefficient2, W/m2K
o
W/mk C
Control / SS-316 16.3 NA 0.15-0.25 244.3
Ni-P-PTFE 5.44 316 < 0.1 244.0
Lectrofluor-641TM 0.245 260 0.1-0.15 238.2
AMC148-18 1.4 229 0.2-0.3 243.2
1
Values obtained from the coating manufacturers.
2
Calculated using the equations available for determining the Nusselt, Prandtl, and Reynolds numbers.
The calculations were based on coating material of thickness of 13 microns (0.0005”) applied
uniformly onto the stainless steel plates of thickness 3 mm.
Food Grade Surface Coatings
After carefully studying the various properties (chemical resistance, thermal properties,
mechanical strength, coefficient of friction and cost of coating the material) of different food-
grade coating materials available in the market, three coating materials (Table 1) were
selected for this study. The various types of coatings applied are briefly discussed below.
Ni-P-PTFE Coatings
The final coating contained a blend of hard materials (like nickel and phosphorus) mixed
with finely suspended particles of a soft material, Teflon® (about 4 microns in size). The
coating was applied by an electroless nickel procedure. The strong carbon-fluorine bond in
Teflon® imparts chemical inertness and non-stick qualities to the surface. The phosphorus
increases the hardness of the surface and enhances its corrosion resistance property while
nickel helps the coated surface to maintain an appreciable thermal conductivity value.
Although the precise composition is not known due to the proprietary nature, Ni-P-PTFE
coatings usually contains about 15-25% Teflon,® 60-65% nickel and 10-12% phosphorus.
The TM-117P Ni-P-PTFE coating (Techmetals Inc., Dayton, OH, USA ) was applied
uniformly all over stainless steel-316 PHE plates (both faces) to a thickness of 20-25 µm,
yielding thermal conductivity of 5.44 Wm-1K-1 (Techmetals, 2008).
LectrofluorTM-641 Coatings
The polymer-based LectrofluorTM 641 coatings (General Magnaplate Corporation,
Linden, NJ, USA) have a lower thermal conductivity (0.25 Wm-1K-1) value than that of Ni-P-
PTFE coatings (General Magnaplate, 2007). The advantage of using polymer-based coatings
is that they are more resistant to the cleaning chemicals than Ni-P-PTFE coatings. Since these
coatings are already being used in the bio-medical industry (in surgical instruments and in
implants) they anticipated to have minimum interaction with the food products during
contact. These coatings are known to be deposited on the substrate either by standard
spraying method or electrostatic spraying method (the exact procedure is proprietary).
LectrofluorTM-641 – USDA, FDA, and AgriCanada code compliant – was applied uniformly
to one of the SS-316 PHE plates (both faces) to a thickness of 13-25 µm.
AMC148-18 Coatings
The AMC148-18 coatings (Advanced Materials Components Express, AMCX, Lemont,
PA, USA) was applied onto the cleaned SS-316 surface in a two-step process. In the first
step, the AMC-18 layer was reactively bonded to the SS-316 substrate through an
intermediate layer (complementary metal-oxide or nitride semiconductor) which was in turn
bonded to the SS-316 surface. Once the AMC-18 layer was formed, the AMC-148 coating
was applied to form a robust, low critical surface energy surface. The AMC-148 layer is FDA
approved to be GRAS (generally regarded as safe) and is approved for full contact. More
information regarding this coating is described elsewhere (Balasubramanian and Puri, 2008a).
The overall thickness of the coating was about 0.4 to 0.5 μm.
Fouling Experimentation
The fouling experiments were conducted using skim milk obtained from the Penn State
Creamery at two flow rates, namely 0.162 m3h-1channel-1 (typical flow rate in dairy industry)
and 0.144 m3h-1channel-1 (performance evaluation at reduced flow rate attributed to foulant
build-up). The system was optimized by a series of trial runs so that the temperatures of the
incoming skim milk and pasteurized skim milk entering the cooling section of the PHE were
maintained at 15 ± 3oC and 72 ± 2oC, respectively. A total of three experimental replications
were conducted at the nominal flow rate (0.162 m3h-1channel-1) for the control and the three
surface coatings forming a total of eighteen experimental trials. At the other flow rate, there
were three replications conducted for the control and two types of coated surfaces (Ni-P-
PTFE and LectrofluorTM 641). The same coated plate was used for all the replications and
flow rate condition tested.
At the end of each experimental run, the PHE unit was disassembled and the
experimental plate (control or coated plate) was removed and visually inspected for any
defects. The weight of the plate was recorded prior to and following the experiment (16-h
after plates were removed from the PHE unit and air dried). During the cleaning procedure, it
was ensured that the coated plates were not in the PHE unit. This is because of the
uncertainty of the coated plate’s reaction to strong acid and alkali solutions which are
typically used during cleaning. The PHE unit was again assembled with SS-316 plates alone
and the entire unit was cleaned thoroughly using alkali and acid based solutions as per the
cleaning protocols outlined by the Penn State Creamery (Balasubramanian and Puri, 2008b).
Statistical Analysis
One way analysis of variance (ANOVA) of the collected data was performed using the
PROC ANOVA procedure available in SAS (version 9.1, SAS Institute Inc., Cary, NC,
USA). The test was performed on the mean product pasteurization temperature values of the
control and coated plate experiment trials. One way ANOVA was also performed on the
weights of foulants collected from the different surfaces after the experiments (at the different
flow rates) to ascertain if there was any significant difference (at α=0.05) between the fouling
produced on the different surfaces.
To analyze the difference in the total thermal energy utilized by the pilot-scale system
containing the different coated surfaces and the control surface, Duncan multiple comparison
test was performed in one way ANOVA. Duncan multiple comparisons was also conducted
to determine if there was any difference between the average hot water temperatures entering
and leaving the PHE system due to the use of the different coated surfaces and the control
surface.
Analytical Characterization of Foulants
The foulants deposited on the plate surfaces were carefully removed and examined using
x-ray photo electron spectroscopy (XPS) to determine if the coating material entered the
processed food product. The procedure for analysis of the foulants by XPS is outlined by
Balasubramanian and Puri (2008a). The detection limit using this technique was in the range
of 0.1- 1.0 atom%.
Results
Visual Inspection of Fouled Plated Surface
Analysis of the average milk pasteurization temperatures showed no significant

differences (p>0.05) during the entire 6-h duration of testing with the coated and control
plates at the two different flow rates. This clearly indicates that similar pasteurization
temperatures were obtained during the course of the experiments using the different surfaces.
The fouling deposits on the test plates after the 6-h experiments under normal product
flow conditions are shown in Figure 2. There was a region on the top left corner of the plates
were similar fouling pattern was observed in all the plates types. This region of the plate was
where the milk flowing on the left side of the plate surface changes direction towards the exit,
and hence, encounters a reduction in velocity. Other than that region, the fouling patterns
observed in all the plate types were not similar. For SS-316 and graded Ni-P-PTFE (TM-
117P) 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.
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-P-
PTFE 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-P-
PTFE 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.
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.
Figure 3. Comparison of the XPS spectrum obtained from the skim milk foulants adhering to the different
surfaces (Balasubramanian and Puri, 2008a and 2008c).
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 Flow rate of 0.162 m3h-1channel-1 Flow rate of 0.144 m3h-1channel-1

Weight of Percent decrease Weight of Percent decrease
foulants the compared with foulants the compared with
next day (g) control next day next day (g) control next day
(%) (%)
Stainless steel 8.4 ± 0.51 0.0 13.3 ± 1.93 0.0
Ni-P-PTFE 1.0 ± 0.15 87.5 ± 0.03 1.9 ± 0.82 85.9 ± 0.04
(graded)
Lectrofluor-641TM 0.4 ± 0.23 94.7 ± 0.03 0.8 ± 0.06 94.2 ± 0.02
AMC-148-18 1.0 ± 0.31 87.6 ± 0.04 Not Applicable 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).
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 Surface type

SS-316 control Ni-P-PTFE LectrofluorTM AMC148-18
coated -641 coated coated
Mean hot water 78.7±0.5 81.9±2.6 80.6±0.6 76.8±0.2
temperature, oC (T1)
Ambient temperature, 20.5±0.9 21.4±0.2 23.5±0.4 19.7±0.9
o
C (T2)
Mean returning hot 59.9±0.5 66.4±3.4 65.1±0.4 59.4±0.3
water temperature, oC
(T3)
Heat required (Q1) for 37.28±0.88 38.79±1.84 36.56±0.09 36.54±0.47
heating water in the
tank from T2 to T1, MJ
1
Heat required (Q2) to 306.32±16.10 253.36±13.00 252.55±2.41 282.16±1.72
sustain T1 for 6 h, MJ
Total heat required (Q), 343.59±16.98 292.15±11.16 289.11±2.51 318.70±2.19
MJ
Total energy required, 95.44±4.72 81.15±3.10 80.30±0.70 88.52±0.61
kWh
2
Average percent change NA -14.97 -15.86 -7.25
in energy over the
control, %
1
Heat required to raise the temperature from T3 to T1.
2
Not applicable.
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
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.
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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
88 Roberto J. Candal and María L. Herrera
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.
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,
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 stearin-
rice 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 15-
19% 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. Reyes-
Hernández 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
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
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.
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%
C26 0.3 0.5 0.5 0.4 0.4
C28 0.0 0.5 0.5 0.4 0.4
C30 0.0 1.0 0.9 0.8 0.7
C32 0.0 2.1 2.0 1.8 1.4
C34 0.0 4.8 4.4 3.9 3.2
C36 0.3 8.6 7.9 7.2 6.3
C38 3.9 13.2 12.4 11.4 7.8
C40 0.0 8.0 7.3 6.6 7.2
C42 0.0 7.0 6.1 5.5 4.2
C44 0.0 7.5 6.5 5.8 4.8
C46 0.0 9.0 6.6 6.9 5.8
C48 0.1 11.0 10.0 8.3 6.6
C50 2.2 13.2 12.5 10.3 8.8
C52 20.1 9.2 10.0 9.9 13.1
C54 73.1 4.3 12.5 20.6 29.3
C54 (18:0) 0.8 0.3 1.4 0.3 0.5
C54 (18:1 trans) 0.0 1.0 0.8 0.5 0.5
C54 (18:1cis) 65.6 1.5 8.3 17.2 24.0
C54 (18:2) 6.7 1.5 2.0 2.6 4.3
MDP (°C) 40.2 40.4 38.8 37.4
80
70
60
50
SFC (%)
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).
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.
-1
-2
-3
dQ/dt
-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.
Table 2. Peak Temperature of Melting Endotherms and Total Melting Enthalpies

Corresponding to the Diagrams in Figure 2
Sample Shoulder First Endotherm Second Endotherm ΔH

(°Ca) (°Ca) (°Ca) (Jg-1b)
HMF 6.35 16.21 39.34 148.54
10% SFO in HMF 4.73 14.62 37.77 79.88
20% SFO in HMF -0.30 12.90 37.62 76.47
40% SFO in HMF -6.95 11.26 36.02 56.30
a
Standard deviation for all values were < ± 0.5°C.
b
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.
HMF a 40% SFO b
Intensity
Intensity
10 15 20 25 30 10 15 20 25 30
2θ (°) 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
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 Penetration Force (N)

HMF 17 ± 3
10% SFO in HMF 7±2
20% SFO in HMF 4±1
40% SFO in HMF 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
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
2
Absorbance
1.5
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.
Induction Times of Crystallization
Supersaturation given by the variation in chemical potencial (Δμ) is defined as:
ΔHm
Δμ = ------- (Tm - Tc)
Tm (1)
melting enthalpy (ΔHm) divided by melting temperature (Tm) and multiplied by supercooling,
that is, the difference between melting and crystallization (Tc) temperatures. No kinetics
parameters are involved in this definition. However, the manner by which the thermodynamic
driving force for crystallization is achieved and the rate of development of this driving force
determine the rates of formation and growth of crystals. In particular, the rate of cooling can
substantially influence crystallization rate. Figure 5 shows the induction times obtained when
samples were crystallized at two cooling rates: 5.5°C/min and 0.1°C/min. Despite the small
differences in melting points (measured as Mettler Dropping Points, MDP) for different ratios
of SFO to HMF (Table 1), induction times were significantly different at p<0.05 between
samples. At slow cooling rates, there was a significant difference (p<0.05) in induction times
for HMF and 10% SFO blends and for 10% and 20% SFO blends at all Tc; and a significant
difference (p<0.01) for 20% and 40% SFO samples. Induction times were shorter at slow
cooling rate for the same supercooling, which is somewhat surprising. In general, when a fat
is crystallized at fast cooling rate (80 °C/min to a temperature below Tm of the α-polymorph),
the α-polymorph can be expected, whereas at the rates used in this study (i.e., 0.1°C/min), the
β’ or β polymorph is expected (Sato 1988). When palm oil was cooled at 0.1°C/min to
crystallization temperatures close to the melting point, the β’-form had shorter induction time
than the β-form. For the three main polymorphic forms of fats, induction times increase in the
order α, β’ and β (Chong 1993). A slower cooling rate might be expected to promote the
formation of more stable forms (β-polymorph) which have longer induction times. However,
the results obtained in this study were reversed, with faster cooling rate giving longer
induction times. The thermodynamic driving force was the same in both cases, but rapidly-
cooled samples (5.5°C/min) took longer at each temperature to crystallize. Similar results
were also found for hydrogenated sunflower seed oil, which has very different chemical
characteristics (rich in elaidic acid TAG, Herrera 1994). In nucleation, molecules need
sufficient time to organize and align with their neighbors to form stable nuclei. In the rapidly-
cooled sample, that organization took place primarily at crystallization temperature, whereas
the slower cooling process allowed reorganization to occur at warmer temperatures as the
sample was cooling to crystallization temperature. The end result was that the sample that
was cooled slowly needed less time at crystallization temperature to nucleate since much of
the molecular organization had already taken place by the time it reached crystallization
temperature.
140
120 HMFSC
Induction Times (min)
100 HMFFC
10%SFOSC
80 10%SFOFC
60 20%SFOSC
20%SFOFC
40 40%SFOSC
40%SFOFC
20
0
32 34 36 38 40
Temperature (°C)
Figure 5. Induction times of crystallization for slow cooling (solid symbols) and fast cooling (open symbols).
Actual Solid Fat Content
Figure 6 shows the increase in SFC with time for the 20% SFO in HMF blend
crystallized at slow rate (0.1°C/min, a) and fast rate (5.5°C/min, b). Zero time for these
graphs was the moment at which the samples reached crystallization temperature.
60
50
40
SFC (%)
30
20
10
0
0 10 20 30 40 50 60 70 80 90
Tim e (m in)
a)
60
50
40
SFC (%)
30
20
10
0
0 10 20 30 40 50 60 70 80 90
Tim e (m in)
b)
Figure 6. SFC with time for the 20% SFO in HMF blend crystallized at slow cooling rate (0.1°C/min, a) and
at fast cooling rate (5.5°C/min, b). Symbols: ■, □, ▲, Δ, ●, ○ ,♦ at 5, 10, 20, 25, 30, and 35°C, respectively.
The 20% blend crystallized slowly (a) at temperatures of 5, 10, 15 and 20°C had an
initial solid content of at least 90% of the final solid content (after 200 min), indicating that
most of the crystallization took place before the temperature reached the designated
crystallization temperature. At 30 and 35°C, a short induction period was necessary to start
crystallization. A 25°C, initial SFC was 50% of the final solid content. As expected, the final
SFC decreased as crystallization temperature increased, indicating the decrease in crystalline
phase volume as temperature increased. Crystallization with fast cooling (b) showed a
different behavior from that for slow cooling. Most of the crystallization process took place at
crystallization temperature. Below 25°C, there was no induction time of crystallization, and
curves showed a hyperbolic shape. However, a slight plateau in SFC was visible in all curves.
The SFC at the plateau decreased steadily both in rate and height as crystallization
temperature was increased. The second step of crystallization, above the plateau, was
sigmoidal in shape. For low supercoolings (crystallization temperatures above 25°C), curves
had sigmoidal shapes. At the beginning, there was an induction time when no fat crystallized,
which was followed by a period of rapid crystallization.
The thermodynamic driving force is one factor that influences the rate of crystallization.
However, other processing factors, such as heat and mass transfer during processing, also can
have significant effects on the rate of crystallization. For example, the manner by which the
thermodynamic driving force for crystallization is achieved and the rate of development of
this driving force determine the rate of formation and growth of crystals (Hartel 2001). In
particular, the rate of cooling can substantially influence crystallization rates since at slow
cooling rates, molecular organization takes place as the sample is slowly being cooled to
crystallization temperature. In contrast, rapid cooling forces the molecules to organized into
crystals under conditions farther from equilibrium. Faster cooling generally results in more
compound crystals formation (lower purity) and higher SFC compared with slower cooling.
Comparison of Figures 6 a and b, particularly at crystallization temperatures below 25°C,

shows that there is a higher SFC for fast than for slow cooling. At higher crystallization
temperatures (lower supercooling), the SFC is about the same for both cooling rates.
Crystallization properties of SFO/HMF blends remains compatible since crystallization
kinetics is fast and no lack of crystallization was found in any case. SFC values were
comparable to that obtained for hydrogenated sunflower seed oil (Herrera et al. 1999).
Microstructure
Many factors influence lipid crystallization, among them processing conditions and
composition of fat are especially relevant (Hartel 2001). Cooling rate, initial and final
temperatures, and agitation rate strongly modified crystallization kinetics. TAG organization,
fatty acid composition, minor lipids, and emulsifiers also determine crystallization rates and
induction times for crystallization. Crystal structure is mainly affected by all these factors. In
many applications of edible fats, the morphology and number of glyceride crystals determine
the suitability of the fat for a given purpose. For example, the morphology of TAG crystals is
related to the possibility of network formation to give a plastic fat. Rheological properties
such as hardness, spreadability, viscoelastic properties, flavor release and mouthfeel depend
in a complicated way on three main parameters: SFC, interaction between crystals, and
structure of the crystal network. These three parameters depend on crystallization process,
which is in turn affected by the conditions during crystallization. The rate of cooling, for
example, can substantially influence crystallization rates.
Light microscopy is a well-developed and increasingly used technique for studying the
microstructure of food systems in relation to their physical properties and processing
behavior. Figure 7 shows the effect of cooling rate on crystal morphology for the 10% SFO
sample crystallized without agitation on a microscope slide and kept isothermally for 30 min
at 30°C. At 30°C, the 10% SFO blend crystallized with high supercooling, as evidenced by
the small crystal size that appeared when samples were crystallized with the fast cooling rate
(a). These crystals were more transparent than the ones obtained with slow cooling (b) and
had lower amount of solids in each crystal. For the slow cooling rate, denser and larger
crystals, with a more regular boundary, were found. Typically, fewer crystals of higher purity
are obtained with slow cooling.
To better understand the processes of nucleation and growth under dynamic conditions,
we took samples from the glass cell periodically, beginning when the cell temperature
reached crystallization temperature and continuing for 3 h. As an example, Figure 8 shows
the morphology of crystals with time obtained when the mixture with 40% SFO in HMF was
crystallized isothermally at 35°C with fast (5.5°C/min, a) or slow rate (0.1°C/min), with an
agitation rate of 100 rpm. A few large crystals were formed under these processing
conditions. The photographs showed that they were not single crystals but grew by
aggregation. These crystals had a spherical shape consisting of small needle crystals.
Figure 7. Effect of cooling rate on crystal size and morphology for the 10% SFO in HMF blend cooled
isothermally without agitation at 30°C for 30 min. a) cooling rate 5.5°C/min, b) cooling rate 0.1°C/min.
a)
b)
Figure 8. Images of crystals corresponding to a 40% SFO in HMF blend crystallized isothermally at 35°C in
dynamic conditions. a) cooled at 5.5°C/min, 90 min, b) cooled at 0.1°C/min, 70 min. Scale as in Figure 7.
a)
b)
Figure 9. Images of crystals corresponding to a 10% SFO in HMF blend crystallized isothermally at fast
cooling rate without agitation: a) 25°C b) 35°C. Scale as in Figure 7.
Figure 9 shows the effect of temperature on crystal structure for the 10% SFO blend
crystallized at a fast cooling rate without agitation. Crystal size increased dramatically,
whereas crystal number decreased significantly with temperature. This effect was found in all
cases. At higher temperatures, fewer initial crystals were formed and their growth was
favored. These conditions favor crystal growth over nucleation (Hartel 2001). On the
contrary, when supercooling was higher (25°C), nucleation was favored and smaller crystals
appeared.
Effects of Emulsifiers
Emulsifiers are useful functional additives without which many food products would be
impossible to make. Emulsifiers typically act in multiphase systems in two main ways. The
first is as an emulsifying agent to enable two distinct phases to be combined in a stable quasi-
homogeneous state for an indefinite length of time. The second function of an emulsifier is
often to modify the behavior of the continuous phase of a food product so as to bring about a
specific effect or benefit, for example, the use of lecithin in chocolate to reduce the viscosity
of the product and improve the ease of handing and processability. In food, several
circumstances for controlling crystallization can be distinguished. In the first case, control of
size, distribution, shape and polymorphism of crystals is necessary to provide the desired
physical characteristics or quality. Here, the kinetics of nucleation and growth must be
controlled through formulation and processing conditions. In other products, controlling
crystallization means preventing crystallization from occurring even though the product is
thermodynamically prone to crystallize (Hartel 2001). Altering the physical properties of fats
and controlling the polymorphic transformation of TAG and their rheological behavior have
been important subjects for technologists and scientists. For example, the kinetics of β’ to β
transition is important from a technological point of view. It is clear that retarding
polymorphic transformation in solid fats can help, at least for some time, to delay loss in
quality. Emulsifiers, such as lecithin and monoglycerides, are used as both viscosity
controllers and as antibloom agents (Garti 1988). Some vegetable fats, such as partially
hydrogenated sunflower seed oil and low-erucic rapessed oil, have a strong tendency to form
β- crystals and cause sandiness in margarine. Several food emulsifiers, such as saturated and
unsaturated fatty acid monoglycerides, act as modifiers of crystal structure, thus helping to
prevent this unwanted phenomenon. The addition of 0.3% sorbitan tristearate inhibits the β’
to β transition in margarine (Madsen and Als !998). Probably the most important step for
controlling crystallization is nucleation. As a main strategy, the use of emulsifiers has drawn
attention for years. Emulsifiers alter the properties of the fat surface and the fat crystallization
process, resulting in an altered solid fat content and crystal size. In fat systems, some
emulsifiers act as heteronuclei (seeds or impurities that promote heterogeneous nucleation).
Nucleation of fat crystals is accelerated through the catalytic actions of such impurities. Other
emulsifiers affect the formation of critical nuclei and elongate induction times (Garti and
Yano 2001).
Sucrose esters can be used in food as emulsifiers because they are nontoxic, tasteless,
odorless, and are digested to sucrose and fatty acids in the stomach. Sucrose esters can also
be used in pharmaceuticals, cosmetics, foods, and in other products where a nonionic,
nontoxic, biodegradable emulsifier is required (Gupta et al. 1983). In addition to their major
function of producing and stabilizing emulsions, sucrose esters contribute to numerous other
functional roles as texturizer and film former. Typical applications are baked goods, fruit
coatings, and confectionary (Hasenhuettl 1997). Sucrose esters have a common feature that
makes them suitable as emulsifying agents: they are ambiphillic, possessing both lipophilic
and hydrophilic properties. The nature of this property is often expressed as the
hydrophilic/lipophilic balance (HLB) on a scale of 0 to 20, with low numbers indicative of
the oil-like tendency (Weyland 1997). The polyglycerol esters of fatty acids include a large
group of closely related compounds of complex composition. However, the individual
components are found as normal constituents of the human diet, i.e. glycerol, glycerol mono-,
di- and tri-fatty acid esters and individual fatty acids. Hydrolysis of the tri- and polyglycerol
esters in vitro with fresh pancreatic juice plus bile showed that 89 to 98% of oleate esters
were hydrolyzed. As a result of their multifunctional properties and harmless nature,
polyglycerol esters are widely used in many applications in the food and cosmetic industries.
They notably function as emulsifiers, dispersants, thickeners, solubilizers, spreading agents,

or emollients. More recently, new industrial applications based on polyglycerol esters have
been developed. This includes their utilization as antifogging and antistatic additives,
lubricants, or plasticizers.
The following data shows the effect of highly hydrophobic food emulsifiers with
different chemical structures on nucleation behavior. The selected emulsifiers were: a
palmitic sucrose ester with HLB = 1, a MDP of 58.0 and monoester content of 1 wt%, with
di-, tri-, and polyesters comprising 99 wt% and a polyglycerol stearic acid ester, DAS 7S,
with HLB = 3.7 and a melting point of 58.0°C. The sucrose head of P-170 has no chemical
similarity with the TAG structure, while polyglycerol alcohols are the result of glycerol
polymerization. The selected emulsifiers allow studying the effect of the hydrophilic heads.
Esters were added at concentrations of 0.1 wt% to HMF and the blends with SFO. The
selected concentration was within the concentrations usually employed in foods for these
esters.
Effect of Emulsifiers on Induction Times
Figure 10 shows values of induction times of nucleation vs. temperature for HMF and the
40% SFO blend, with and without addition of 0.1% of P-170 or DAS 7S, based on polarized
laser light turbidimetry. All samples showed similar behavior. A continuous curve can be
drawn, which means that when samples were crystallized at these temperatures, only one
polymorphic form was obtained (Ng 1990). A t-test at a confidence interval of 95% was
performed on the average induction times at every temperature for all systems. For HMF, no
significant differences were found at this level between the values of means below 313 K;
however, above this temperature, differences were significant, being notorious for low
supercooling (temperatures close to the melting point). For the blends with 40% SFO, the
same behavior was found, but differences were significant above 312 K. Sucrose ester P-170
elongated the induction times of all samples, and therefore delayed nucleation. On the
contrary, polyglycerol DAS 7S caused acceleration of nucleation. Shorter induction times
were found in all cases.
Effect of Emulsifiers on Polymorphism
Figure 11 shows X-ray diffraction patterns for HMF and the 40% SFO blend crystallized
with and without addition of emulsifiers at 35°C and a cooling rate of 5.5°C/min. HMF
showed the characteristics pattern of the β’ form with two strong signals at 4.3 and 3.9 Å.
Addition of emulsifiers did not modified the polymorphic behavior as may be noticed from
the patterns (a). As may be expected from the β tendency of SFO, HMF with addition of 40%
SFO had a shoulder at 4.6Å, indicating a trace amount of the β form (b). Addition of DAS 7S
increased the β’ tendency of the blend. This pattern did not show traces of the β form. Even
at the low concentration selected in this study (0.1%), the emulsifiers were able to modify the
polymorphic behavior of these systems. In agreement with these results, when polyglycerol
behenic acid esters were added to palm oil, they promoted the crystallization of the β’ form
from the melt, preventing the formation of granular crystals (Sakamoto 2003). These results
are important regarding technological applications, since different products require different
polymorphic forms to achieve the necessary macroscopic properties.
80
a
70
60
50
t (min)
40
30
20
10
0
310 312 314 316 318 320
T (°K)
80
b
70
60
50
t (min)
40
30
20
10
0
310 312 314 316 318 320
T (°K)
Figure 10. Induction times of crystallization vs. temperature for all samples: (a) HMF ( ♦ ) and with addition
of DAS 7S ( ○ ), and P-170 ( ■ ), (b) the 40% SFO blend ( ♦ ) and with addition of DAS 7S ( ○ ) and P-170 (
■ ). Data points are the average of three runs. Error bars are standard deviations.
Intensity
HMF
DAS 7S
P-170
10 15 20 25 30
2θ (°)
40% SFO
Intensity
DAS 7S
P-170
10 15 20 25 30
2θ (°)
Figure 11. Short spacings of (a) high melting fraction of milk fat (HMF) and with addition of emulsifiers, and
(b) the 40% sunflower oil (SFO) blend and with addition of emulsifiers.
a) b)
c)
Figure 12. Images of crystals corresponding to a 40% SFO in HMF blend crystallized isothermally at 35°C
for 60 min with a cooling rate of 5.5°C/min and an agitation rate of 100 rpm: a) 40% SFO blend, b) 40%
SFO blend with 0.1% of sucrose ester P-170, and c) 40% SFO blend with 0.1% of polyglycerol DAS-7S.
Effect of Emulsifiers on Microstructure
Figure 12 shows representative PLM images of the 40% SFO blend (a), with addition of
0.1% of P-170 (b), and with addition of 0.1% of DAS 7S (c), crystallized at 35°C, 100 rpm
and 5.5°C/min. The 40% SFO blend showed spherical agglomerated crystals that grew by
accumulation of small needle crystals separated by the liquid phase (a). Crystals were
brighter which indicated that they were form by more solid material. When P-170 was added
to HMF (b), crystals showed a different morphology. Spherical crystals with a less dense
crystal structure (related to the brightness of the crystals) were present. P-170 also diminished
crystal size, showing that P-170 affected not only nucleation but growth rate of the crystals as
well. These results were in agreement with the elongation of induction times found when P-
170 was added to the 40% SFO blend. It was reported that any factor that delays nucleation
produces this effect on crystal morphology (Herrera and Hartel 2000). Addition of DAS 7S to
the 40% SFO blend (c) accelerated crystallization. Images showed many small crystals
covering the field together with big spherical agglomerates. In agreement with our results,
Sakamoto et al. studying the isothermal behavior of palm oil by PLM found that polyglycerol
behenic acid esters accelerated nucleation. Palm oil crystals were smaller and their number
larger with the additives.
Effect of Emulsifiers on Rheology
Table 4 shows the effect of emulsifier addition on penetration force of HMF and its
blends with SFO crystallized at 35°C with agitation. As previously reported (Martini et al.
2002), primary crystals were formed under agitation at 35°C. After 90 min, samples were
cooled to 10°C, where crystallization continued without agitation. Confocal images of these
samples showed larger primary crystals formed at crystallization temperature, surrounded by
small and somewhat diffuse crystals in the background, which most likely were formed
during storage (Martini et al. 2002). Addition of sucrose esters significantly decreased
penetration force for all samples. After 24 h at 10°C, the SFC of all samples, both with and
without the addition of sucrose esters, was within the experimental error of the method (1%).
However, the average size of the primary crystals decreased significantly with addition of P-
170 and the microstructure of the final products were quite different (Martini and others
2002). DAS 7S promoted nucleation and therefore many small crystals were formed at
crystallization temperature. This polyglycerol ester increased penetration force. Although
emulsifiers did not affect the final SFC of the fats they had significant effect on the nature of
the crystalline microstructure. Therefore, they affected rheological properties. The effect of
emulsifiers on microstructure was related to their effects on crystallization kinetics.
Table 4. Effect of Emulsifier Addition on Penetration Force for HMF and its Blends
with SFO
Sample Penetration force (N)

Without emulsifiers With 0.1% P-170 With 0.1% DAS 7S
HMF 17 ± 3 9±2 20 ± 4
10% SFO in HMF 7±2 4±1 10 ± 2
20% SFO in HMF 4±1 2±1 7±3
40% SFO in HMF 2±1 1±1 4±2
Conclusion
Nucleation process was modified adding sucrose esters and polyglycerol esters. The
effect on induction times of nucleation (delay or acceleration) was related to the molecular
structure of emulsifiers and, more specifically, to the hydrophilic head. Both families of
emulsifiers seemed to follow a simple mechanism of incorporation into the crystal. However,
hydrophilic heads with no chemical similarity inhibited nucleation while heads with chemical
similarity promoted it. The emulsifiers selected in this study were also able to modify the
polymorphic behavior and crystalline microstructure of the low trans HMF/SFO blends. A
deep understanding of the role of sucrose and polyglycerols esters is a necessity to chocolate
producers, ice cream makers and food technologists.
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Chapter V
Probiotic Bacteria Isolated from Breast

Milk for the Development
of New Functional Foods
G. Vinderola, A. Binetti, and J. Reinheimer

Instituto de Lactología Industrial (INLAIN, UNL-CONICET),
Facultad de Ingeniería Química, Universidad Nacional del Litoral. 1º de Mayo 3250,
Santa Fe (3000), Argentina
Abstract
Baby’s intestine is (or was said to be) sterile at birth and gut microbiota development
is a gradual process after delivery. Quantitative and qualitative differences in
bifidobacterial and lactic acid bacteria levels and species composition have been shown
between breastfed and formula-fed infants, bifidobacteria being the most dominant
microorganisms in the former group. Establishment of the gut microbiota is a stepwise
process which provides the earliest and most massive source of microbial stimuli for the
normal maturation of the gut mucosal immune system, contributing to its development in
infancy and to the control of the gut-associated immunological homeostasis later in life.
Probiotic intervention in the neonatal period has attracted scientific interest after recent
demonstrations showing that specific strains reduce the symptoms and risk of allergic
and infectious diseases or improve feeding tolerance. However, no all early interventions
in children reported rendered positive results. The question of the right dose and the
specific pathologies that probiotic administration, to infants less than 6 month of age,
could be helpful for is still under a vigorous debate. Breast milk contains several factors,
including nutrients, antimicrobial agents, IgA antibodies and TGF-β, which contribute
beneficially to the immunologic maturation and well-being of the infant as well as factors
that promote the growth of bifidobacteria in the infant’s intestine. Additionally, healthy
breast milk contains significant numbers of bacteria. In 2003 it was reported the isolation
of lactobacilli from breast milk as potential probiotics. Breast milk seems to be a natural
116 G. Vinderola, A. Binetti and J. Reinheimer
source of probiotic bacteria for infants. In this context, supplementation of infant

formulas with these kinds of probiotics might beneficially alter the composition of the
microflora of formula-fed infants in such a way that it resembles that of breast-fed
infants. However, to date there is no available information concerning the technological
potential of these strains for their industrialization (growth in milk, resistance to lactic
acid, freezing or spray-drying, among others) if they are thought to be included in dairy
products or in formulas for infants.
Short Communication
Traditional Ideas Revised
For years, microbiological studies of breast milk have been restricted to the screening of
the presence of potential pathogenic bacteria, mainly related to clinical cases of mastitis.
Then, there is a limited quantity of reports about the isolation and analysis of commensal or
potential beneficial bacteria from breast milk of healthy women (Martín et al., 2003).
Moreover, the knowledge of the bacterial diversity in breast milk is very limited, and is
almost exclusively based on the use of culture media. The presence of non-cultivable
bacterial species might be overlooked if culture-independent molecular techniques are not
used (Martín et al., 2007a). Tissier (1900) introduced the idea that fetus is sterile in utero and
that bacterial colonization of the newborn intestinal tract begins during the transit through the
labour channel due to cross-contamination with vaginal and faecal bacteria of the maternal
microflora (Isolauri et al., 2001). Since then, these ideas have been widely accepted.
However, there is emerging and surprising evidence that unveil ways of mother-to-child
transmission of commensal microflora, during lactation and even before birth. Maybe one of
the first signs of the presence beneficial bacteria in breast milk was given in 1979 when West
et al. (1979) reported the isolation, among pathogenic species, of a Lactobacillus plantarum
strain from human milk. However, by that time, those finding were assumed to be a
contamination occurring during sample extraction (Gavin and Ostorv, 1977). Later on, RAPD
and PFGE analysis permitted to conclude that a strain of Lactobacillus salivarius, previously
isolated from feces of a one-month-old- breast-fed infant, could be detected in the breast milk
of his mother (Martín et al., 2006), suggesting that gut microbiota of breast-fed infants
reflects that of maternal breast milk. Recently, the presence of commensal bacteria was traced
back and they were found to occur in the umbilical cord blood and meconium of healthy
neonates and in murine amniotic fluid, suggesting that term fetuses are not completely sterile
and that a prenatal mother-to-child efflux of commensal bacteria may exist (Jiménez et al.,
2008a). This emerging evidence let us think that the traditional idea of a newborn with a
sterile intestine acquiring his gut intestine microflora exclusively from exogenous sources
should be put into test under the light of new molecular techniques indicating the presence of
commensal bacteria passing from the mother to the child via breast milk and even before
during the intrauterine life.
Probiotic Bacteria Isolated from Breast Milk for the Development … 117
Probiotics for Infants
Baby’s intestine, sterile at birth or having a low charge of mother’s commensal bacteria
(Jiménez et al., 2008a), is massively colonized by a gradual process and evolves quickly over
the first days of life, but quantity and species vary markedly over the first 2 years of life
(Kliger et al., 2007). The first colonizers are enterobacteria, streptococci and staphylococci,
followed by bifidobacteria (Rotimi and Duerden, 1981). Quantitative and qualitative
differences in bifidobacterial and lactic acid bacteria (LAB) concentrations and species
composition have been reported between breastfed and formula-fed infants, bifidobacteria
being the most dominant microorganisms in the former group (Harmsen et al., 2000). Breast-
fed infants are colonized predominantly by bifidobacteria and lactobacilli, whereas the
microflora of formula-fed infants is composed by a smaller proportion of bifidobacteria and
lactobacilli, dominating the anaerobic microorganisms, such as bacteroides and clostridia
(Harmsen et al., 2000; Kalliomaki et al., 2001; Penders et al., 2006). Establishment of the gut
microbiota is a stepwise process which provides the earliest and most massive source of
microbial stimuli (Rautava et al., 2004) for the normal maturation of the gut mucosal immune
system, contributing to its development in infancy and to control of the gut-associated
immunological homeostasis later in life (Rautava and Isolauri, 2002). It has been shown that
microbiota aberrancies precede the development of some atopic diseases (Kalliomaki et al.,
2001). Probiotic intervention in the neonatal period has attracted scientific interest after
recent demonstrations showing that specific strains reduce the symptoms and risk of allergic
and infectious diseases (Isolauri et al., 2001). Stratiki et al. (2007) demonstrated that the
administration of bovine milk supplemented with bifidobacteria for 7 days to preterm infants
decreased intestinal permeability and lead to increased head growth. Lee et al (2007) reported
that administration of probiotics to preterm infants led to Lactobacillus colonization in 27 out
of 73 patients and improved their feeding tolerance. Even if continuously administered during
the first 6 months after birth, it seems that no permanent establishment of a probiotic strain is
achieved (Gueimonde et al., 2006), but only a transient colonization of the gut leading to a
reduced prevalence of atopic eczema later in life. On the contrary, other studies showed no
effects on atopic dermatitis (Brouwer et al., 2006) or rectal bleeding (Szajewska et al., 2007)
after the administration of Lactobacillus GG to infant of less than 6 months of age. Even
undesired effects were observed followed the administration of L. casei Shirota during
lactation in an animal model (Ezendam and van Loveren, 2008). In other study where infants
were given daily 109 cells of L. acidophilus LAVRI-A1 during their 6 first months of life,
authors concluded that probiotic supplementation did not alter early innate immune responses
in this population at high risk of developing allergic disease (Taylor et al., 2006). Other
pathologies in which probiotic bacteria had been used are the prevention of antibiotic-
associated diarrhea, community-acquired diarrhea, necrotizing enterocolitis in premature
children, irritable bowel syndrome and constipation, infantile colic and atopic dermatitis
(Kliger et al., 2007). Although complementary feeding is not advised before the age of 6
months (Chouraqui et al., 2008), many results from clinical trials suggest that specific
probiotics might be useful in reducing the risk of certain pathologies but, which probiotics
should be used? Which doses shall be used? Should they be administered continuously or
cyclically (administration/withdrawal/administration)? Yet, a great number of issues need to
be resolved before general guidelines regarding the use of probiotics in the neonatal period
can be released (Rautava, 2007). The specific strain to be administered, its origin, its safety
and functional dose, the frequency of administration and the age of the individuals to which it
will be administered, seems to be crucial issues in order to warrant a safe and efficacious use
in newborn or infants.
Isolation and Characterization of Probiotic

Bacteria from Human Breast Milk
Breast milk contains several factors, including nutrients, antimicrobial agents, prebiotic
oligosaccharides, IgA antibodies, TGF-β, and the innate microbe receptor soluble (s)CD14,
which contribute beneficially to the immunologic maturation and well-being of the infant as
well as factors that promote the growth of bifidobacteria in the infant’s intestine (Rautava et
al., 2006). Additionally, healthy breast milk, for instance, contains significant numbers of
bacteria. These commensal bacteria include streptococci, lactobacilli, corynebacteria,
micrococci and propionibacteria (O’Sullivan et al., 2005). Bacterial species generally isolated
by agar plating breast milk samples include Staphylococcus epidermis, S. hominis, S. capitis,
S. aureus, Streptococcus salivarius, S. mitis, S. parasanguis, S. peores, Lactobacillus gasseri,
L. rhamnosus, L. acidophilus, L. plantarum, L. fermentum, L. salivarius, L. reuteri,
Enterococcus faecium and E. faecalis (Lara-Villoslada et al., 2007a). However, it seems that
breast milk microflora is much more diverse than suggested by culture-dependant methods if
molecular tools are used. For instance, Martín et al. (2007b) and Delgado et al. (2008)
reported isolates from breast milk belonging to genera as diverse as Weisella,
Propionibacterium, Serratia, Acinetobacter, Gemella, Pseudomonas, Veillonella, Kocuria,
Corynebacterium, Klebsiella, Rothia and Escherichia. In 2003, two European groups, one
from Finland (Heikkila and Saris, 2003) and another one from Spain (Martín et al., 2003)
simultaneously and independently reported the isolation of LAB from human breast milk.
Heikkila & Saris (2003) studied the bacterial diversity in human milk with focus on the
detection of bacteria with antimicrobial activity against Staphylococcus aureus, a known
causative agent of infectious mastitis in women. In that study, LAB such as L. rhamnosus, L.
crispatus, Lactococcus lactis and Leuconostoc mesenteroides were isolated from breast milk.
As L. crispatus is a predominant vaginal species, authors hypothesized that the infant may
have derived it from the vagina during delivery and then transmitted it to the maternal breast
skin during nursing or even to breast milk while suckling. However, in another research
(Martín et al., 2007b), the diversity of Lactobacillus in infant faeces, breast milk and vaginal
swabs from mothers whose neonates were born by vaginal delivery or caesarean section was
studied. Authors concluded that the bacterial composition of breast milk and infant faeces is
not related to the delivery method and that the origin of the LAB present in breast milk and in
the infant gut is still far from clear. None of the Lactobacillus species detected in vaginal
samples were present in breast milk provided by the women whose neonates were born by
vaginal delivery, a fact that suggests that transit through the vagina does not play a role in the
establishment of lactobacilli found in breast milk (Martín et al., 2007b).
Martín et al. (2003) isolated several L. gasseri strains from breast milk of 8 healthy
women on the fourth day postpartum. They found no RAPD correlation between strains
isolated from breast milk and strains isolated from breast skin or mammary areola, which
suggests that most breast milk LAB have an endogenous origin and that cross-contamination
with skin bacteria has, if any, a secondary role as LAB source for the infant gut (Martín et al.,
2003). A possible mechanism for commensal bacteria delivery from gut to breast milk has
been suggested. Dendritic cells can sample bacteria from the gut mucosa by penetration of
their dendrites into the gut lumen while preserving the integrity of the epithelial barrier
(Rescigno et al., 2001). Once inside dendritic cells, or any other antigen-presenting cell,
bacteria could spread to other locations by lymphatic circulation. It is known that, during the
lactation period, colonization of the mammary gland by cells of the immune system is a
selective process regulated by hormones. In this colonization, prokaryotic cells might be
involved as well. Some beneficial bacteria can have mechanisms to spread from the gut to
other organs in healthy hosts without leading to a pathogenic condition and at least some of
the LAB present in the maternal gut could reach the mammary gland through an endogenous
route (Martín et al., 2004).
The probiotic potential of strains isolated from breast milk was evaluated and authors
concluded that it was similar, at least, to that of the strains commonly used in commercial
probiotic products (Martín et al., 2005). Once evaluated and guaranteed the safety for oral
use of these news strains (Lara-Villoslada et al., 2007b), some functional properties of strains
of breast milk origin was determined. It was demonstrated their antimicrobial activity
(Olivares et al., 2006), the enhancement of the intestinal function in healthy adults (Olivares
et al., 2006), a differential capacity to modulate the gut immune response by inducing the
production of Th1 cytokines or IL-10 by two different strains (Díaz-Ropero et al., 2007) and
their potential to treat infectious mastitis if administered to mothers during lactation (Jiménez
et al., 2008b). Finally, Puleva Food S.L. (www.puleva.es) has recently industrialized one of
these lactobacilli strains and launched into the market Puleva Peques 2 Hereditum®, the first
dried milk containing probiotic bacteria isolated from breast milk and recommended for
infants from 6 moths onwards. However, no information about the specific strain used and
the level of viable cells present in the commercial product were available on the web site.
In this context, supplementation of infant formulas with these kinds of probiotics might
beneficially alter the composition of the microflora of formula-fed infants in such a way that
it resembles that of breast-fed infants (Ezendam and van Loveren, 2008). Cell viability of
probiotic bacteria in the food matrix is a prerequisite in order to guarantee their claimed
health effects (Galdeano and Perdigón, 2004; Ouwehand and Salminen, 1998). However,
even when a strain isolated from human milk has been successfully included in a commercial
dried milk, to date there is no available information concerning the technological
performance of strains from this ecological niche (growth in milk, resistance to lactic acid or
spray-drying), if they are thought to be included in dairy products or feed formulas for
infants.
Recently, 6 strains of Bifidobacterium lactis subsp. lactis have been isolated from breast
milk at our Institute (Instituto de Lactología Industrial, INLAIN UNL-CONICET, Santa Fe,
Argentina) (Zacarías et al., 2009). The samples were obtained from 16 different mothers
between days 1 and 12 after delivery, plated on agar media (MRS-cysteine and MRS-cysteine
acidified to pH 5.5 with acetic acid), and incubated anaerobically at 37ºC for 72h. In 5 of
those 16 mothers, two samples were obtained in two different days after delivery. Total
counts ranged from 3x101 to 1x105 CFU/ml in MRS-cysteine, and were predominantly cocci.
It is important to highlight that no lactobacilli were isolated from any of the samples,
contrarily to previous reports on the microbiological composition of breast milk (Lara-
Villoslada et al., 2007a; Martín et al., 2003; Martín et al., 2006). Bifidobacteria were detected
in 6 samples, mainly after the 1st day postpartum, at levels from 1x101 to 7.3x103 CFU/ml
(Figure 1). They were later identified by molecular tools. Isolates were preliminary
characterized according to different technological criteria such as storage stability as frozen (-
20ºC and -70ºC) cultures (in 20% skim milk) or as cultures suspended in 10% skim milk at
pH 6 or 4.5 for 4 weeks at 5ºC. Resistance to spray drying was evaluated as well. All the
isolated strains showed a high resistance to frozen storage for at least 6 months at -20ºC and -
70ºC. Strains also showed adequate stability during 4 weeks of refrigerated storage in milk at
5ºC, since cell viability losses were smaller than 0.5 log order CFU/ml at pH 6 or 4.5. Finally,
cell suspensions (108 CFU/ml) were spray-dried (Büchi mini spray dryer model B-290,
Flawil, Switzerland) and a very soluble white powder was obtained containing more than 109
CFU/ml and a moisture content less than 4% (w/w). Functional in vivo studies are presently
carried out in our laboratory to assess the probiotic potential of these bifidobacteria strains in
a murine model. This work represents the first report in Latinoamerica on the isolation and
preliminary technological characterization of bifidobacteria from breast milk, demonstrating
a satisfactory technological potential for their use in the elaboration of fermented milk
products or as adjunct in infant formulas if any probiotic property is observed for them.
4
Log (CFU/ml)
0
LM ( 5)
LM 5)
LM 0)
LM 7)
LM 0)
L M 2)
L M ( 6)
L M 5)
L M 7)
L M 1)
L M 1)
L M 1)
LM 1)
LM (1 )
LM (1 )
LM (1 )
LM (1 )
LM (1 )
LM (1 )
)
L M 2)
(1
1
2(
1
3(
4(
5(
5(
6(
7(
8(
9(
1
1
4
2(
3(
1(
10
11
12
13
14
15
16
LM
Sample
Figure 1. Total ( ) and bifidobacteria ( ) counts in MRS-cysteine agar (37ºC, 72h, anaerobiosis) of fresh
breast milk samples (LM) of 16 healthy women, between day 1 and 12 postpartum (the day of sampling is
indicated between brackets).
Concluding Remarks
Breast milk can be considered a natural symbiotic food containing diverse nutrients and
factors as well as functional bacteria (Lactobacillus and Bifidobacterium) able to satisfy the
nutritional and immunological needs of the neonate. Therefore, it is not surprising that the
bacterial composition of the infant faecal flora reflects the bacterial composition of breast
milk. The modulation of the intestinal microbiota by probiotic bacteria isolated from breast
milk has been shown to regulate the gut immune functions and to enhance defences against
intestinal pathogens and allergies. These facts open new perspectives for the isolation,
functional and technological characterization and addition of breast milk probiotics to infant
formulas, in order to get closer to the natural composition of breast milk, for children with
limited breast feeding or in cases where exclusive maternal care is not possible.
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Chapter VI
Probiotics in Maternal and

Early Infant Nutrition
Yolanda Sanz*
Microbial Ecophysiology and Nutrition Group. Institute of Agrochemistry and Food
Technology (IATA), Spanish National Research Council (CSIC),
PO Box 73, 46100 Burjassot, Valencia, Spain
Abstract
During pregnancy fetal development is entirely dependent on the mother.
Epidemiologic and clinical studies suggest that immunologic and metabolic profiles of
the pregnant uterus are responsive to mother’s diet. This evidence supports the
hypothesis that maternal nutrition may influence fetal programming and disease risk in
the offspring. After birth, the gastrointestinal tract undergoes vast structural and
functional adaptations under the stimulation of the microbiota and the diet that make
possible handle with antigens and digest milk and latter solid food. The intestinal
colonization process implies the activation of diverse metabolic functions either triggered
by host-microbe interactions or directly encoded by the genome of the microbiota
(microbiome). Moreover, microbial exposure through colonization process of the
newborn intestine is essential to regulate epithelial permeability and immune function,
with long-term consequences on host’s health. Bacterial composition and succession
during the intestinal colonization process have been shown to determine susceptibility to
infections and sensitization to dietary antigens. In this context, mammals seem to have a
developmental window within the perinatal and postnatal period, in which the host-gut
microbiota interactions are more influential in favoring later health. Probiotic and
prebiotic administration has been demonstrated to be a dietary strategy that at least
temporary modulates the microbiota composition and may favor a healthy status. These
*
Corresponding author. Tel.: 34 96 390 00 22; Fax: 34 96 363 63 01. E-mail: yolsanz@iata.csic.es
126 Yolanda Sanz
strategies have demonstrated moderate efficacy to reduce the risk of infections and
allergic diseases in early life. In recent years, the administration of probiotics to pregnant
and lactating mothers in addition to their newborns, along or not with prebiotics, has also
been evaluated to extend their applications and improve effectiveness by acting in these
critical developmental stages. This type of intervention has shown that specific probiotic
strains influence gut growth and immune function in the offspring of animal models.
Other studies have suggested that this dietary strategy may help to reduce the risk of
atopy, infections, and metabolic disorders in humans. The current knowledge of the
effectiveness and mechanisms by which the administration of probiotics to mothers and
infants could positively affect early stages of development, favoring latter heath is
review.
1. Introduction
The prevalence of metabolic diseases associated with obesity is increasing worldwide

presumably as a result of societal changes and environmental influences. In addition, whereas
hygienic conditions and medical advances have reduced the mortality from infectious
diseases in most countries, immune-mediated diseases, such as allergies and autoimmune
diseases, are becoming more prevalent worldwide. This has led to speculate that modern
lifestyle has altered the relationship between predisposing genes and environmental factors so
as to promote development of immune and metabolic disorders, which are becoming major
causes of concern and mortality in developed countries. The “hygienic hypothesis” proposed
by Strachan (1989) seems to explain reasonably well the increased prevalence of immune-
mediated diseases such as allergies. It is known that pregnancy favors Th2 lymphocyte
development in the mother and the fetus to prevent the fetus from rejection. However,
adequate exposure to microbes in early life is also essential to trigger Th1 lymphocyte
development that will prevent form persistence of the Th2 biased responses in the newborn
and thereby development of an allergic phenotype latter in life. Epidemiological and
intervention studies also suggest that diet and exposure to microbes influence the metabolic
and immunologic profiles of the pregnant uterus and the risk of immune and metabolic
diseases development in the offspring (Yajnik et al., 2006; Barker et al., 2007, Ege et al.,
2008). A number of preclinical and clinical intervention studies based on nutritional
restrictions, high fat diets and functional ingredient supplementation (e.g. polyunsaturated
fatty acids and probiotics) during pregnancy have revealed a close relationship between
mother’s diet and infant’s health (Herrera et al., 2002; Church et al., 2009) supporting the
fetal programming hypothesis proposed by Barker et al. (2007). Therefore, the interplay
between both heredity and environmental factors (diet, microbes and xenobiotics) seems to
affect every stage of development from conception to early postnatal period with potential
long-term effects in child and adult health. Thus, it can be anticipated that further research on
the convergence of both theories “hygienic” and “fetal programming” will better explain the
current disease trends.
The intestinal microbiota develops an array of physiological roles within the human
body, shaping its metabolic abilities and immune responses. The intestinal colonization
Probiotics in Maternal and Early Infant Nutrition 127
process of the newborn intestine seems to be a particularly relevant process to the infant’s
health (Sanz et al., 2008a). The microbiota provides novel metabolic capacities and also
regulates host gene expression to favor both host and microbe survival. In addition, microbial
exposure through colonization of the newborn intestine is essential to fully boost the immune
system and to regulate permeability, with long-term consequences on health. Microbiota
acquisition and succession in the newborn’s intestine has been shown to determine
susceptibility to infections and sensitization to dietary antigens. An adequate colonization
process or the presence or absence of specific bacteria may favor the prevalence of the
beneficial microbiota and a healthy status (Kalliomaki et al.2007). This may be achieved by
dietary intervention based on probiotic and prebiotic administration. However, while
probiotic administration only seems to provide temporary changes in a fully developed gut
ecosystem, a practically sterile newborn intestine seems to be more amenable to intentional
manipulations by dietary strategies. Evidence on the transmission of bacteria from the mother
to the neonate has also encouraged the administration of probiotic-based products during the
perinatal period and lactation to favor infants gut colonization with healthy strains. This
practice also seems to provide additional benefits by influencing, for instance, breast-milk
composition and immune functions on the mother-fetal interface. Therefore, the incorporation
of probiotic based products into both maternal and infant nutrition is being investigated as a
novel dietary approach to reduce disease risk in the light of both the hygienic and the fetal
programming hypothesis. The current knowledge of the effectiveness and mechanisms by
which probiotics could positively act at early stages of development favoring latter heath is
review and discussed in the following sections.
2. Microbiota Acquisition and Succession

in the Newborn Intestine
Infants are born with a practically sterile intestine that is rapidly colonized after birth.
The colonization during the first 12-24 hours of life is characterized by the presence of higher
levels of facultative anaerobes (e.g. Enterobacteriaceae, Enterococcus, and Streptococcus)
than strict anaerobic bacteria (e.g. Bifidobacterium, Bacteroides and Clostridium), but these
proportions are reversed within a week after birth, when an anaerobic environment is
established (Mackie et al., 1999). The infant microbiota, which is characterized by low
diversity and instability, evolves into a typical adult microbiota over the first 24 months of
life. Then, it becomes more stable and diverse, and seems to be unique for each individual
(Zoetendal et al., 1998). The acquisition and succession of the gut microbiota depend on
diverse external and internal host factors. External factors include the gestational age, intake
of medicines, microbial load from the environment, mode of delivery, mother’s microbiota,
number of siblings and type of feeding and solid diet. Internal factors include the
gastrointestinal physiology that influences peristalsis, bile acid concentrations and
gastrointestinal pH, as well as other genotypic determinants of gut colonization, whose
knowledge is more limited (Björkstén, 2006; Sanz et al., 2008a).
Gestational age has been suggested to influence the gut microbiota, particularly when
comparing preterm and full-term infants due to differences in exposure to environmental
128 Yolanda Sanz
microbes and antibiotics. In general, colonization of the intestine with beneficial bacteria has
been shown to be delayed in preterm infants and the number of potentially pathogenic
bacteria was higher than in healthy full-term infants. Comparisons of microbial succession
patterns during the first 4 weeks of life revealed that E. coli, Enterococcus, and Klebsiella
pneumoniae were commonly present and Bifidobacterium species were not in the microbiota
of hospitalized preterm infants, in contrast to full-term infants (Schwiertz et al., 2003). In
addition, the microbial patterns of preterm infants became more similar to one another over
time, supporting the hypothesis that environmental factors influence the initial colonization
of the newborn intestine. Other studies have also associated hospitalization and prematurity
with higher prevalence and counts of Clostridium difficile (Penders et al., 2006). The fact that
many preterm infants receive prophylactic antibiotics at birth may affect the intestinal
colonization (Westerbeek et al., 2006) and, in fact, reductions in the numbers of
Bifidobacterium and Bacteroides have been associated with this clinical practice (Penders et
al., 2006).
The mother’s microbiota (vaginal, intestinal, oral and cutaneous microbiota) is also
thought to influence infant’s gut colonization (Mackie et al., 1999). During birth, exposure to
the mother’s vaginal and intestinal microbiota due to the proximity between the birth canal
and the anus is likely to influence the initial colonizers of the newborn intestine. After birth,
cutaneous and oral mother’s microbiota will also be easily transferred to the newborn by
kissing and sucking (Lindberg et al., 2004). In particular, a relation between the infant’s
microbiota and type of delivery has been inferred in diverse studies. This has partly been
explained by the lack of initial contact of infants delivered by caesarean section with the
mother’s vaginal microbiota. Microbes from the vaginal canal and anus area seem to be able
to enter the mouth and stomach of vaginally delivered infants within few minutes after birth.
However, the vaginal microbiota seems to settle-down in the newborn intestine less easily
than the maternal intestinal microbiota (Mackie et al., 1999). In general, caesarean-delivered
infants have been shown to experience some delay in bacterial colonization, and their
microbiota have presented aberrancies for up to 1 year (Adlerberth et al., 2006). Some studies
showed that the fecal microbiota of caesarean-delivered infants harbors significantly fewer
Bacteroides species compared with that of vaginally delivered infants (Grönlund et al., 2000;
Adlerberth et al., 2006), which was suggested to be associated with maturation of humoral
immunity (Grönlund et al., 2000). Bifidobacteria colonization was also shown to be delayed
and reached lower levels in the caesarean delivered infants than in the vaginally delivered
infants (Chen et al., 2007). Another study also revealed that the microbiota of the caesarean
delivered group was less diverse, in terms of bacteria species, than the microbiota of the
vaginally delivered group after 3 days of life (Biasucci et al., 2008). Moreover,
Bifidobacterium species were absent in the microbiota of caesarean delivered infants, while
B. longum and B. catenulatum groups were predominant in that of vaginally delivered
neonates (Biasucci et al., 2008). In addition, mode of delivery seems to have significant
effects on immunological functions of the infant, probably via its influence on gut microbiota
colonization (Huurre et al. 2008a). At 1 month of age, the total gut bacterial counts in feces
were higher in vaginally delivered infants than in caesarean section delivered infants, mainly
due to higher bifidobacterial counts. In contrast, the total number of IgA-, IgG- and IgM-
secreting cells was lower in infants born by vaginal delivery than in those born by caesarean
section during the first year of life, possibly reflecting excessive antigen exposure across the
vulnerable gut barrier in the last group (Huurre et al., 2008a). A large study on the
contribution of external influences to the gut microbiota composition in early infancy, which
included fecal samples from 1032 infants at 1 month of age, showed that infants born through
caesarean section had lower numbers of Bifidobacterium and Bacteroides, whereas they were
more often colonized with Clostridium difficile, compared with vaginally born infants
(Penders et al., 2006).
The number of siblings has also been related to the newborn microbiota since it implies
an increase of microbial exposure by direct contact with the newborn. In some studies, infants
with older siblings had slightly higher numbers of bifidobacteria compared with infants
without siblings (Penders et al., 2006) but this was not demonstrated in all studies (Ahrné et
al., 2005). Other studies showed transmission of intestinal bacteria from parents to their
babies by comparison of fecal bacteria DNA patters, which were probably influenced by both
genetic and environmental factors (Favier et al., 2003).
The type of milk-feeding is regarded as one of the major environmental factors
influencing gut colonization in early life. In general, Bifidobacterium are dominant in breast-
fed infants (up to 90% of the total fecal bacteria), while a more-diverse adult-like microbiota
is found in formula-fed infants (Salminen & Isolauri, 2006). A large-scale population study
has showed that exclusively formula-fed infants were more often colonized with E. coli, C.
difficile, Bacteroides, and Lactobacillus, compared with breastfed infants (Penders et al.,
2006). A delay in Bifidobacterium species colonization in formula-fed compared with breast-
fed babies has also been shown by other authors (Favier et al., 2003). In addition, differences
in Bifidobacterium species composition as a function of the type of milk-feeding have been
reported. Some authors showed that B. breve was predominant in breast-fed infants, whereas
B. longum and B. adolescentis were found more often in formula-fed infants (Mitsuoka and
Kaneuchi, 1977; Mevissen-Verhag et al., 1987). More recently, another comparative
molecular analysis of the infant’s microbiota has showed that breast-fed infants had
significantly higher levels of total Bifidobacterium and B. breve, and lower levels of B.
adolescentis and B. catenulatum than formula-fed infants, resembling the bifidobacteria
populations of the adult microbiota (Haarman & Knol, 2005). However, other authors have
not found significant differences in Bifidobacterium species composition a function of the
type of feeding (Favier et al., 2003). Breast milk seems to be a source of commensal bacteria
to the infant gut, including Staphylococcus, Lactobacillus and Bifidobacterium (Martín et al.,
2008). B. breve, B. adolescentis and B. bifidum have been isolated from milk in culture
medium and, therefore, at least these species could be transferred alive to the newborn via
breast-feeding. Breast-milk bifidobacteria composition was also analyzed by real-time PCR
showing that B. longum was the most widely found species followed by B. animalis, B.
bifidum and B. catenulatum although the detected prevalence of species was based on DNA
detention but not on viability (Gueimonde et al., 2007). A recent study also showed that
allergic mothers had significantly lower amounts of bifidobacteria in breast-milk compared
with non-allergic mothers and their infants had concurrently lower counts of bifidobacteria in
feces. Therefore, the maternal health status could derange the counts of bifidobacteria in
breast-milk and influence the infants' fecal Bifidobacterium levels (Grönlund et al., 2007).
Breast-milk also contains prebiotic substances (oligosaccharides), which stimulate the growth
130 Yolanda Sanz
of Bifidobacterium and are one of the major bifidogenic factors (González et al., 2008). In
fact, Bifidobacterium longum subsp. infantis ATCC 15697, an isolate from the infant gut, was
shown to preferentially consume small mass oligosaccharides, which represent 63.9% of the
total human milk oligosaccharides available (LoCascio et al., 2007). Accordingly, the
complete genome sequence of this strain reflects a competitive ability to utilize milk-borne
molecules, which lack a nutritive value to the neonate (Sela et al., 2008). Breast-milk
contains other bioactive factors, such as secretory IgA, lactoferrin, and a range of cytokines,
which have a remarkable effect on the development of the neonatal microbiota and mucosal
immunity. In particular, IgA supplied by maternal milk has been shown to sequester
commensal bacteria in the mice neonatal intestine, thereby delaying active development of
IgA production (Kramer & Cebra, 1995), while the decline in maternal IgA supply increases
intestinal bacterial colonization in pups (Inoue et al., 2005). In formula-fed infants no
detectable levels of fecal IgA were found during the first 10 days of life, which might
partially explain the development of a more-diverse microbiota in these infants at an early
age (Bakker-Zierikzee et al., 2006).
A few studies have demonstrated a relationship between the genotype and the gut
microbial colonization process so far. Similarity between fecal bacteria DNA profiles of
monozygotic twins was shown to be significantly greater than that between profiles of
unrelated individuals (Zoetendal et al., 2001). Furthermore, the DNA profiles of the fecal
microbiota of monozygotic twins were also significantly more similar than those of dizygotic
twins (Stewart et al., 2005). It can be anticipated, that the host genotype may influence for
instance the repertory of mucins, acting as bacterial adhesion sites in the intestinal mucosa,
and the immune response, which altogether will restrict or allow the colonization of certain
micro-organisms (Björkstén, 2006; Sanz et al., 2008a). However, some studies suggest that
the shared environment influences the gut microbiota to a higher extent than the genotype
(Palmer et al., 2007). In particular, studies on the evolution of mammals and their gut
microbes by metagenomic approaches pointed out that the acquisition of a new diet is a
fundamental driver for changes in gut bacterial diversity (Ley et al., 2008). This scientific
evidence supports the hypothesis that dietary intervention strategies may play a critical role in
health and disease prevention, via modulation of the gut microbiota and its functions.
Consequently, probiotic and prebiotic based nutritional strategies could play a primary role in
this field, which will become a reality as we improve our understanding of the roles of
intestinal bacteria in the human body.
3. Influence of the Intestinal Microbiota

in host Physiology and Immunity during
the Early Postnatal Period
3.1. Influence of the Intestinal Microbiota in Host Physiology and

Metabolism
The anatomy and physiology of the intestine depend on the interactions between the
microbiota and the diet with the host epithelium, mucosal immune system and
microvasculature (Hooper et al., 2002). After birth, the gastrointestinal tract undergoes vast
structural and functional adaptations to be able to initially digest mother's milk and later solid
food, during the weaning period. During the early suckling period, the morphological and
functional gut development is reflected in a rapid intestinal growth and increased expression
of digestive enzymes such as lactase. At weaning period, crypt hyperplasia and an increased
expression of maltase, sucrase, and pancreatic trypsin also denote vast changes of the
gastrointestinal tract to digest a more complex diet. It is known that the gut microbiota plays
an important role in these gastrointestinal maturation processes from gnotobiology studies
(Fåk et al., 2008). In germ-free animals the intestinal weight and surface area were decreased,
the intestinal villi were thinner, and the shape of enterocytes was abnormal compared with
conventionally raised mice (Berg, 1996). In contrast, the cecum was much larger in germ-free
animals due to mucus and fiber accumulation and subsequent water retention when compared
with conventionally raised animals (McCracken & Lorenz, 2001). In preterm formula-fed
animal, the administration of specific bacteria immediately after birth limited the formula-
induced mucosal atrophy, dysfunction, and pathogen load in preterm neonates. This strategy
also increased the intestinal weight, mucosa proportion, villus height, RNA integrity, and
brush border aminopeptidase A and N activities, reflecting the role of bacteria in intestinal
physiology (Siggers et al., 2008).
It is known that intestinal microbiota develops an important biochemical activity within
the human body by both providing additional metabolic capacities encoded by the
microbiome to the host (Gill et al., 2006) and by regulating diverse aspects of cellular
differentiation and gene expression via host-microbe interactions (Hooper et al., 2002).
The intestinal microbiota provides additional metabolic capacities, involved in the
utilization of no digestible carbohydrates and host-derived glycoconjugates (e.g. chondroitin
sulphate, mucin, hyaluronate and heparin), deconjugation and dehydroxylation of bile acids,
cholesterol reduction and biosynthesis of vitamins (K and B group), isoprenoids and amino
acids (e.g. lysine and threonine) (Hooper et al., 2002, Gill et al., 2006). In particular, the
ability of the commensal microbiota to utilize complex dietary polysaccharides, that would
otherwise be inaccessible to humans, may contribute to the ability of the host to harvest
energy from the diet, which may represent 10% of the daily energy supply (Sanz et al.,
2008b). In addition, B. thetaiotaomicron, a prominent commensal bacteria, has been shown to
have ability to both hydrolyze host-derived glycans and influence the nature of those
produced by the host by increasing expression of fucosylated ones to favor its own
colonization (Hooper et al., 2001).
132 Yolanda Sanz
The commensal microbiota has also been shown to induce expression of genes involved
in the processing and absorption of dietary carbohydrates and complex lipids in the host
(Hooper et al., 2001; Bäckhed et al., 2004). For instance, ileal expression of a
monosaccharide transporter (Na+/glucose co-transporter) was induced in B. thetaiotaomicron
mono-colonized mice, which would lead to increasing the absorption of dietary
monosaccharides and short-chain fatty acids and, thereby, promote the novo synthesis of
lipids in the liver (Hooper et al., 2001). The colonization of germ-free mice by conventional
microbiota also increased liver expression of key enzymes (acetyl-CoA carboxylase and fatty
acid synthase) involved in the de novo fatty acid biosynthetic pathways, and transcriptional
factors (ChREBP and SREBP-1) involved in hepatocyte lipogenic responses to insulin and
glucose (Bäckhed et al., 2004). In addition, the colonization reduced the levels of circulating
fasting-induced adipose factor (Fiaf) and the skeletal muscle and liver levels of
phosphorylated AMP-activated protein kinase, which altogether contribute to fat storage
(Bäckhed, et al., 2007). The colonization of germ-free mice by B. thetaiotaomicron also
increased the expression of other components involved in the host’s lipid absorption
machinery, including a pancreatic-lipase related protein that hydrolyzes triacylglycerols, a
cytosolic fatty acid binding protein (L-FABP) involved in intracellular trafficking of fatty
acids, and the apolipoprotein A-IV that mediates export of triacylglycerols re-synthesized in
the enterocyte (Hooper et al., 2001). The expression of genes involved in absorption of
dietary metal ions was also modified by the microbiota, which for instance induced the
expression of a high affinity copper transporter in the epithelium (Hooper et al, 2001).
Furthermore, the colonization of the intestinal mucosa was also implicated in the regulation
of the under laying microvasculature and angiogenesis (Stappenbeck, et al., 2002).
3.2. Influence of the Intestinal Microbiota in Host Immunity
An adequate colonization process of the newborn intestine by commensal bacteria

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

influence gut ecology and regulate non-specific intestinal defenses during the postnatal
period.
Microbial colonization of germ-free animals has also an important impact on postnatal
development of mucosal and systemic immunity. The gut-associated lymphoid tissue (GALT)
is immature in germ-free mice, showing a reduced content of the lamina propria CD4+ T
cells, IgA producing B cells and intraepithelial T cells. Systemic immunity is also affected by
the absence of the microbiota and germ-free mice have decreased serum immunoglobulin
levels and their mesenteric lymph nodes and spleens are smaller (Hrncir et al., 2008). The
interaction of the gut microbiota with the GALT contributes to the production of secretory-
IgA, modulation of cytokine and chemokine release, and development of balanced T helper
(h) 1/Th2 responses and oral tolerance to innocuous antigens (Sanz et al., 2008a). The
immune system should be regulated to such a way that can mount an effective innate and
acquired mucosal and humoral response to eliminate the invader organism, but avoid
collateral tissue damage and overreactions to harmless microbes and antigens. One of the key
events in the regulation of appropriate immune responses is the differentiation between
pathogenic and commensal bacteria. This is achieved by pattern-recognition receptors (Toll-
like receptors [TLRs] and Nod-like receptors [NLRs]) expressed in epithelial and antigen
presenting cells (macrophages and dendritic cells [DC]), which act as sentinels sensing
environment and activating defenses in face to danger. In particular TLR-signaling
commonly leads to the activation of nuclear factor kappa B (NF-κB) transcription pathway,
with up regulation of major histocompatibility complexes and co stimulatory proteins,
production of pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, and IL-8), and
recruitment of other immune cells. Signaling through TLR also stimulates the maturation of
DCs with enhanced ability to present antigens and activate T cells, T-cell co-stimulatory
molecules (CD80 and CD86) and other activation markers. T-cell differentiation into Th 1,
Th2 or regulatory T cells (Tregs) is also thought to depend on the type of TLRs involved
(Winkler et al., 2007). Th1 responses are usually associated with inflammatory reactions and
clearance of pathogenic bacteria and virus, Th2 responses with allergic responses and parasite
clearance, and regulatory T cells (Th3 and Thr1) are essential in preventing overreactions
(Sanz et al., 2007a). Some of the mechanism by which commensal bacteria, unlikely
pathogens, have been shown to down-regulate pro-inflammatory responses include: (i) the
inhibition of the NF-κB, either by regulating the nuclear export of NF-κB subunit relA (Kelly
et al., 2004) or by inhibiting the IκB ubiquitination (Neish et al., 2000); (ii) the inhibition of
TLR2-driven activation of NF-κB signaling via NOD signalling (Watanabe et al., 2008); (iii)
the regulation of TLR expression and up-regulation of the negative regulator Tollip protein
(Otte et al., 2004; Solano-Aguilar et al., 2008), (iv) the down-regulation of the expression of
pro-inflammatory type I IFN related genes (Munakata et al., 2008), and (v) the induction of
immunoregulatory cytokine production (IL-10 and TGF-β1) and Tregs (Hrncir et al., 2008).
134 Yolanda Sanz
4. Influence of the Mother’s Diet

and Environmental Exposures
in Fetal Programming and Infant’s Health
Scientific evidence indicates that the diet influences the fetal-maternal relationships with
consequences on development and long-term health. The quality of the maternal diet during
pregnancy has been related to offspring's risk of developing allergies, type 1 and type 2
diabetes, arteriosclerosis, cardiovascular disease and obesity (Bertino et al., 2006; Lamb et
al., 2008; Yates et al., 2008). Pregnancy is seen as a period of maximum sensitivity to dietary
influences since diverse organs and systems are in development in the fetus. In particular,
immune and metabolic functions of the fetus are dependent on the mother and probably the
refinement of their functions is already initiated inside the uterus.
Successful pregnancy requires immunologic changes characterized by a dominance of
Th2 and regulatory T cell effector responses in both mother and fetus, which help to maintain
pregnancy and avoid the rejection of the immunologically incompatible fetus; however the
persistence of this immune polarization favors the development of a long-lasting atopic
phenotype latter in life (Calder et al., 2006; Kalliomaki et al., 2007). At least during the third
trimester of human pregnancy, fetal T cells are able to mount antigen-specific responses to
environmental and food-derived antigens and antigen-specific T cells are detectable in cord
blood, indicating fetus sensitization in uterus (Calder et al., 2006). For example, the fetus
mounted an immune response to rubella antigens whose mothers were infected with rubella
during pregnancy. In infected fetuses, total IgM and IgA concentrations rose significantly,
and rubella virus-specific IgM and IgA antibodies were detected at week 22 of pregnancy
(Grangeot-Keros et al., 1988). A recent investigation has determined the relationships of cord
blood immunoglobulin E (IgE) with maternal health conditions before and during pregnancy,
showing inverse associations of cord blood IgE to seasonal allergens with positive maternal
records for Toxoplasma gondii and rubella virus infections. Therefore, maternal immunity to
certain pathogenic antigens may influence atopic sensitization in the fetus (Ege et al., 2008).
Studies of the incidence of human pathogenic bacteria and virus (cytomegalovirus and herpes
simplex virus type 1 and 2) in biopsy samples from the placenta and decidua of women with
healthy pregnancies by PCR, showed that 38% of placental samples were positive for the
selected microorganisms. Moreover, analyses of IgG isolated from the placenta support the
hypothesis that immune responses suppress cytomegalovirus reactivation in the presence of
pathogenic bacteria at the maternal-fetal interface, suggesting that at least microbial DNA at
the placenta may drive immune responses (McDonagh et al., 2004). More recently,
Bifidobacterium spp. and L. rhamnosus have also been identified in human placenta by
quantitative real-time PCR recently (Satokari et al., 2009). Although cultivable bacteria were
not detected, the possibility that fetus is exposed to bacterial components (e.g. DNA) via the
mother’s placenta during pregnancy can not be disregarded. In particular, the unmethylated
CpG oligodeoxynucleotide motifs of bacterial DNA are known to activate Toll-like receptor
9 and subsequently trigger Th-1-type immune responses, and this could be one of the
mechanisms that contribute to program the infant's immune development during fetal life
(Lee et al., 2006). A recent study has also found a close relationship between maternal and
cord-blood specific IgE patterns to cow milk suggesting that the immune system can be
stimulated by food allergens before birth (Bertino et al., 2006). Another study analyzed
whether asthma prevention could start even earlier, before conception, by transfer of
immunologic tolerance from the mother to the offspring in animal models (Polte et al., 2008).
The offspring of naïve mothers had an asthma-like phenotype, while the offspring of tolerized
mice with oral ovalbumin before conception were completely protected. The levels of
allergen-specific IgG were increased in the sera of the mother, fetus, pup and breast milk,
indication their involvement in tolerance transfer from the mother to the offspring (Polte et
al., 2008). In addition, environmental exposure of the pregnant woman to microbes has been
related to the allergic predisposition of the neonate. Therefore, the fetal immune system
during the perinatal period can be responsive to environmental challenges (microbes or
dietary compounds), which may not be longer effective in adulthood to prevent disease.
Dietary supply of nutrients during conception and pregnancy may also influence the
development of the fetus and offspring, and the risk of suffering metabolic diseases. Maternal
obesity from exogenous origin and both maternal hyperglycemia and hyperlipidemia are
thought to contribute to the development of metabolic disorders in the offspring. Studies on
the effect of chronic high-fat diets on the development of the fetal metabolic systems in
nonhuman primates showed that a developing fetus is highly vulnerable to excess of dietary
lipids, independent of maternal diabetes and/or obesity, and that exposure to this diet may
increase the risk of pediatric non-alcoholic fatty liver disease (McCurdy et al., 2009).
Scientific evidence has also indicated that prenatal nutrition may affect dietary preferences
and may contribute to more atherogenic lipid profiles later in life. Studies in animal models
have suggested that fetal undernutrition can predispose to hypercholesterolemia and
metabolic disorders directly by programming cholesterol metabolism and indirectly
influencing lifestyle choices. Pregnant women exposed to famine in early gestation were
more likely to consume a high-fat diet and they also seem to be less physically active
(Lussana et al., 2008).
5. Probiotic and Prebiotic Concepts

and Applications
The colonization process of the newborn intestine is considered critical to the health
status and risk of developing disease in early and later stages of life. Therefore, improving the
characteristics of the gut microbiota particularly in the perinatal and postnatal periods is
foreseen as a strategy to prevent the onset of disease (Sanz et al., 2008a). This could be
achieved by the administration of probiotics, prebiotics or their combination (synbiotics).
Probiotics are defined as live microbes that when administered in adequate amounts confer a
health benefit to the host [FAO/WHO, 2002]. Prebiotics are non-digestible dietary
ingredients that allow changes, both in the composition and/or activity of the gastrointestinal
microbiota that confers benefits on the host’s health (Roberfroid, 2007). The genus
Bifidobacterium is the predominant in the intestinal tract of infants; it represents about 3% of
the total microbiota in the intestine of healthy adults and is associated with beneficial effects
on health (Sanz et al., 2007a). The genus Lactobacillus also inhabits the gastrointestinal tract
136 Yolanda Sanz
and some species are widely used in diverse food fermentations. These features have made
these two bacterial genera the most attractive as probiotics for human consumption (Sanz et
al., 2007b). Currently, these are incorporated into functional foods in the form of fermented
milks, infant formula, cheese, ice cream and juices, and in pharmaceutical preparations. Milk
fermented products constitute the best vehicle for probiotic bacteria since this matrix allows
maintenance of viability and metabolic activity (Sanz et al., 2007b). Galacto-oligosaccharides
and inulin-derivatives are the prebiotics most commonly commercialized in Europe (Haarman
& Knol, 2005; Kelly, 2008). They are mainly added to infant formula to promote the
prevalence of a microbiota composition similar to that resulting from breast-feeding during
both milk-feeding and the weaning period. The administration of these prebiotic infant
formulas have demonstrated to increase the total amount of fecal bifidobacteria and to modify
the Bifidobacterium species composition, resembling that of breast-fed infants, when
compared with infants receiving conventional formula (Haarman & Knol, 2005; Scholtens et
al., 2006).
The immaturity of the immune system and the increased epithelial permeability of the
newborn intestine under lack of appropriate microbial stimulation seem to partly explain the
increased prevalence of immune-mediated diseases in developed countries, such as allergies
(the hygienic hypothesis). Initial attempts to control the development of allergies and food
intolerance were based on the avoidance of the allergen in the diets, but the results were not
satisfactory regarding long-term prevention. In recent times, novel methods including
probiotic supplementation have been evaluated to counteracting the immunological and gut
mucosal barrier dysfunction associated with these disorders, and to strengthening endogenous
defense mechanisms. Moreover, while the administration of probiotics to adults with a fully
developed intestinal microbiota produces only temporary colonization of the bacteria, the
newborn intestine is more amenable to dietary and probiotic manipulation in the early
postnatal period. Therefore, the use of probiotics early in life has been evaluated for
preventing allergies, repeated infections and necrotizing enterocolitis, and provided some
promising results in preclinical trials in animals (Siggers et al., 2008) and in clinical trials in
humans (Lodinova-Zadnikova et al., 2003). A step beyond this concept has been the
administration of probiotics, together or not with prebiotics, to pregnant women and their
infants to influence both the intrauterine and postnatal development of the newborn, relaying
on the “fetal programming theory”. These strategies are currently under investigation to
prevent both immune and metabolic disorders with the hope that they will influence more
effectively the infant’s health from conception onwards.
6. Influence of Maternal and Offspring

Probiotic Intake in Animals
Several studies have showed that exposure of pregnant mothers to specific probiotic
bacteria prior to parturition and during lactation influences diverse physiological functions in
the intestine of the offspring. The administration of the probiotic Lactobacillus plantarum
299v in the drinking water to pregnant and lactating rat dams until their pups had reached an
age of 14 days resulted in the colonization of both mothers and pups gut by the bacterial
strain and influenced gut growth and function of pups (Fåk et al., 2008). The small intestine,
pancreas, spleen and liver weighed more in the probiotic colonized pups than in the control
pups. The probiotic colonized pups also showed decreased gut permeability as compared to
the control pups (Fåk et al., 2008). Nevertheless, the effects derived from maternal probiotic
intake and their transmission to the offspring as such could be questioned since the
administration of the probiotic through the drinking water could have facilitated direct
contact and acquisition of the probiotic strain by the pups. The effects of L. rhamnosus GG
supplementation to female mice intragastrically every day before conception, during
pregnancy and lactation (perinatal supplementation group) or only during pregnancy
(prenatal supplementation group) on the development of experimental allergic asthma in the
offspring have also been investigated recently (Blümer et al., 2007). Intestinal colonization
with L. rhamnosus GG was observed in mother mice, but not in the offspring. In spite of that,
a reduced expression of TNF-α, IFN-γ, IL-5 and IL-10 was observed in splenic mononuclear
cells of mice derived from mothers perinatally supplemented with L. rhamnosus GG.
Moreover, allergic airway and peribronchial inflammation and goblet cell hyperplasia were
significantly reduced in offspring of prenatally or perinatally mothers supplemented with L.
rhamnosus GG as compared to control mice. Exposure to L. rhamnosus GG during
pregnancy shifted the placental cytokine expression pattern with a markedly increased TNF-α
level. Thus, the results suggest that this probiotic strain may exert beneficial effects on the
development of experimental allergic asthma, when applied in a very early phase of life and
that the effects are partly mediated via the placenta by induction of pro-inflammatory cell
signals (Blümer et al., 2007). In fact, DNA from Bifidobacterium spp. and L. rhamnosus has
also been identified in human placenta by quantitative real-time PCR and its influence on
transmission of immune signals to the fetus through this route cannot be disregarded
(Satokari et al., 2009).
The oral supplementation of Bifidobacterium animalis subsp lactis Bb12 (Bb12) to sows
during gestation and to their piglets for 91 days since birth led to the establishment of
increased numbers of the probiotic in the piglets’ proximal colon compared to placebo-
treated piglets born to placebo-treated sows, Bb12-treated sows, or piglets born to placebo
sows but treated with Bb12 immediately after birth. This effect was associated with a
significant up-regulation of TLR-9 expression in proximal colon, but neither of TLR2 nor
TLR4. This result suggests that exposure of the mother to Bb12 influenced both Bb12 load in
the piglet in the presence of continuous daily feeding and significantly affected the host
innate immune system development (Solano-Aguilar et al., 2008).
The protective mechanisms of action of L. rhamnosus GG against atopic dermatitis
development have also been studied in a mice model (NC/Nga mice) of human atopic
dermatitis (Sawada et al., 2007). Maternal and infant mice were fed with food containing or
not containing heat-treated L. rhamnosus GG during pregnancy and breastfeeding, and after
weaning. While control NC/Nga mice raised under an air-uncontrolled condition
spontaneously manifested typical skin lesions, probiotic feeding inhibited the onset and
development of atopic skin lesions and reduced the numbers of mast cells and eosinophils in
the affected skin sites. The probiotic-fed mice also showed a significant increase in plasma
IL-10 levels and in the IL-10 mRNA expression in both Peyer's patches and mesenteric
lymph nodes compared with control mice. However, there was no significant difference in
138 Yolanda Sanz
the proportion of splenic CD4(+)CD25(+) regulatory T cells between the probiotic-fed mice
and the control mice. Therefore, the ability of the probiotic L. rhamnosus GG to delay the
onset and suppress the development of atopic dermatitis was mediated via a strong induction
of IL-10 in intestinal lymphoid organs and at systemic level.
Although the number of preclinical studies is limited, the accumulated scientific evidence
suggests that maternal probiotic intake, particularly when occurring prior and after birth,
influences the development of the offspring. However, these effects are not always correlated
with the ability of the probiotic to colonize the offspring intestine. Moreover, animal studies
also support a role for maternal probiotic intake in disease prevention and particularly in
atopic dermatitis.
7. Influence of Maternal and Infant Probiotic

Intake in Humans
7.1. Influence of Maternal Probiotic Intake in the Intestinal

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

L. rhamnosus GG Intake by healthy women at late Probiotic colonization of the infant’s gut Schultz et al., 2004
pregnancy but not after delivery
L. rhamnosus GG Intake by healthy women 4 weeks before Changes in bifidobacteria transfer and Gueimonde et al, 2006
and 3 weeks after delivery establishment in the neonates
Fermented milk Oral intake by women at 34 weeks of Reduction of genital infection risk Othman et al., 2007
and yogurt bacteria pregnancy or vaginal application from
first trimester onwards
Lactobacillus casei Intake by healthy women 6 weeks before Natural killer cell increase in mother’s Ortiz-Andrellucchiet al.,
DN11401 delivery and during 6 weeks of lactation peripheral blood and TNF-α decrease in 2008
breast-milk
Decrease of gastrointestinal episodes in
infants
L. rhamnosus GG and Probiotic intake by women carrying Increased resistance to respiratory infections Kukkonen et al., 2007,
LC705 fetus at allergy risk during the last in children during 2 years 2008
B. breve Bb99 month of pregnancy and by their infants Trend to reduce IgE-associated diseases and
P. freudenreichii subsp. until age of 6 months plus a prebiotic prevented atopic eczema at 2 years
shermanii and Increase of fecal lactobacilli and
galacto-oligosaccharides bifidobacteria
L. rhamnosus GG Intake by mothers at family-risk of Risk reduction of atopic eczema for up to 7 y Kalliomaki et al., 2007
atopic eczema for 4 weeks before Increasing of TGF-β2 in mother’s milk Rautava et al., 2002
delivery and postnatally for 6 months
Table 1. Continued
Probiotic/prebiotic/Diet Administration pattern Trial outcome Reference

L. rhamnosus GG and Intake by pregnant women carrying fetus Modest increase of TGF-β2 in colostrums Huurre et al., 2008
B. lactis Bb2 at allergy risk from the first trimester of Reduced allergen sensitization in infants
pregnancy till the end of exclusive breast-
feeding
L. rhamnosus GG Intake by pregnant women carrying fetus No effect on fetal antigen-specific immune Boyle et al., 2008
at allergy risk for 36 weeks before delivery responses evaluated in cord blood cells
L. rhamnosus GG Intake by women at risk of atopic diseases No effect on incidence of atopic dermatitis Kopp et al., 2008
from 4 to 6 weeks before delivery and
postnatally for 6 months
L. rhamnosus GG and B. Intake by women from early till end of Increased placental concentrations of Kaplas et al., 2007
lactis Bb12 plus dietary pregnancy linoleic and dihomo-gamma-linolenic
counseling acids.
Dietary recommendations Intake by women from first trimester of Highest and lowest intakes of specific Aaltonen et al., 2008
and L. rhamnosus GG and pregnancy onwards. nutrients associated with higher blood
B. lactis pressure in children at 6 months
L. rhamnosus GG and B. Intake by healthy women from first Reduced blood glucose concentrations and Laitinen et al., 2008
lactis Bb12 plus dietary trimester of pregnancy onwards increased glucose tolerance during
counseling pregnancy and over the 12 months
postpartum
7.2. Influence of Maternal and Infant Probiotic Intake

in Child Health
The use of probiotics during pregnancy is under consideration due to the positive effects
of some strains on diverse clinical situations such as infections (vaginal, respiratory and
gastrointestinal) and allergy, as well as on the physiological development of immune and
metabolic functions. The number of human clinical trials intended to validate such probiotic
applications are rapidly increasing, some of which are showing moderate effectiveness in
improving child’s health. The effectiveness of probiotics for preventing preterm labor and
birth has been the objective of recent studies since the risk of this outcome in the presence of
maternal infection is dramatically increased, reaching values of 30% to 50% (Othman et al.,
2007). It has been suggested that specific probiotics could exert beneficial effects on such
applications because of their ability to displace and inhibit pathogens and modulate immune
responses by interfering with the inflammatory cascade that leads to preterm labor and
delivery. So far, the two randomized controlled trials assessing the prevention of preterm
birth in pregnant women and women planning pregnancy with the use of probiotics registered
in 2006 have been reviewed (Othman et al., 2007). One study enrolled women after 34 weeks
of pregnancy using oral fermented milk as probiotic, while the other study enrolled women
diagnosed with bacterial vaginosis in early pregnancy that utilized commercially available
yogurt vaginally. The results showed 81% reduction in the risk of genital infection with the
use of probiotics. However, this was the only pre-specified clinical data available and there
are, currently, insufficient data to assess impact on preterm birth and its complications. The
prevention of AIDS and other infections in women and children by dietary intervention based
on the probiotic concept has also been considered a possibility but still a distant reality (Reid
and Devillard, 2004).
The use of probiotic bacteria during pregnancy could be a mean to modulate immune
development in fetus to reduce the risk of immune aberrancies and improve the host’s
defenses. In this context, the effects of the consumption of milk fermented with the strain
Lactobacillus casei DN11401 by pregnant women during 6 weeks before delivery and during
6 weeks of lactation were determined (Ortiz-Andrellucchi et al., 2008). Mothers
supplemented with the probiotic showed a significant increase in natural killer cells in
peripheral blood samples and a non-significant increase in T and B lymphocytes. Maternal
milk also showed a decrease in the pro-inflammatory cytokine TNF-α and breast-fed child of
the mothers who consumed L. casei showed fewer gastrointestinal episodes. The safety and
effects of a mixture of 4 probiotic bacterial strains (L. rhamnosus GG and LC705,
Bifidobacterium breve Bb99, and Propionibacterium freudenreichii subsp. shermanii) along
with a prebiotic galacto-oligosaccharide has also been evaluated in pregnant women carrying
high-risk children of allergic diseases and their infants. Pregnant women consumed a
probiotic preparation or a placebo for 2 to 4 weeks before delivery and their infants received
the same probiotics plus galacto-oligosaccharides for 6 months. No differences in growth,
infant colic, morbidity or other adverse health effects between the two groups of children
were found. Moreover, a slightly higher percentage of children had been prescribed
antibiotics in the placebo group (28%) than in the probiotic group (23 %) during the
142 Yolanda Sanz
intervention. Respiratory infections also occurred less frequently in the synbiotic group (3.7
versus 4.2 mean infections) throughout the follow-up period (Kukkonen et al., 2008).
The administration of the probiotic L. rhamnosus GG to both pregnant mother and their
babies demonstrated to reduce the incidence of atopic eczema for up to 7 years in the Finish
population (Kalliomaki et al., 2007). L. rhamnosus GG was given prenatally to mothers, who
had at least one first-degree relative with atopic eczema, allergic rhinitis, or asthma, for 4
weeks before expected delivery and postnatally for 6 months to their children. L. rhamnosus
GG was effective in prevention of early atopic disease in children at high risk as determined
by considering chronic recurring atopic eczema as the primary endpoint. The administration
of probiotics to the pregnant and lactating mother was shown to increase the amount of anti-
inflammatory cytokine TGF-β2 in the mother’s milk, thereby increasing its
immunoprotective potential (Rautava et al., 2002). The infants most likely to benefit from
maternal probiotic supplementation were those with an elevated cord blood IgE concentration
(Rautava et al., 2002). In addition, Huurre et al. (2008b) evaluated the effects of dietary
counseling and probiotic supplementation (L. rhamnosus GG and B. lactis Bb2) to pregnant
women, at risk of developing atopy, on their children. Children of atopic mothers,
specifically when breastfed exclusively over 2.5 months or 6 months, had a higher risk of
sensitization at the age of 12 months but this risk could be reduced by the use of probiotics
during pregnancy and lactation. The preventive effects were considered to be the result of a
beneficial change in breast milk composition characterized by a modest increase in TGF-β2
concentration (Huurre et al., 2008b); however, this increase did not reach statistical
significance and it was only detected in the colostrums while disappeared after 1 month. To
progress on the knowledge of the mechanisms of action beyond the effects of L. rhamnosus
GG intake on atopic eczema prevention, Boyle et al., (2008) investigated whether this
probiotic influenced fetal immune responses when administered to pregnant women for 36
week before delivery. The effects of stimulation of cord blood mononuclear cells from
women who received the probiotic or placebo with heat-killed L. rhamnosus GG and
ovalbumin were evaluated, without showing effects of the treatment on CD4(+) T cell
proliferation, forkhead box P3 expression, dendritic cell phenotype or cytokine secretion.
Therefore, L. rhamnosus GG supplementation to pregnant women failed to influence fetal
antigen-specific immune responses and in particular transplacental immune effects were
excluded as the mechanisms of probiotic action (Boyle et al., 2008). The effects of a mixture
of 4 probiotic bacterial strains (L. rhamnosus GG and LC705, B. breve Bb99, and P.
freudenreichii subsp. shermanii along with a prebiotic galacto-oligosaccharide on allergic
disease prevention were also evaluated in pregnant women carrying high-risk children.
Pregnant women consumed a probiotic preparation or a placebo for 2 to 4 weeks before
delivery and their infants received the same probiotics plus galacto-oligosaccharides or
placebo for 6 months. Probiotic treatment compared with placebo showed no effect on the
cumulative incidence of allergic diseases, but tended to reduce IgE-associated (atopic)
diseases and prevented atopic eczema at 2 years. In addition, lactobacilli and bifidobacteria
more frequently colonized the intestine of supplemented infants, suggesting an inverse
association between atopic diseases and gut colonization by probiotics (Kukkonen et al.,
2007).
In spite of the aforementioned evidence, the value of probiotics for primary prevention of
atopic diseases is still controversial. An additional clinical double-blind, placebo-controlled
trial has been carried out to study the preventive effect of the same probiotic, L. rhamnosus
GG, on the development of atopic dermatitis in Germany (Kopp et al., 2008). Pregnant
women from families with 1 or more members with an atopic disease received either the
probiotic or placebo from 4 to 6 weeks before expected delivery, followed by a postnatal
period of 6 months. In this case, supplementation with L. rhamnosus GG during pregnancy
and early infancy neither reduced the incidence of atopic dermatitis nor altered the severity of
atopic dermatitis in affected children, but it was associated with an increased rate of recurrent
episodes of wheezing bronchitis at the age of 2 years. Therefore, the authors conclude that L.
rhamnosus GG could not be generally recommended for primary prevention (Kopp et al.,
2008).
The metabolic effects of probiotic supplementation during the perinatal period also seem
to be relevant to fetal programming and infant’s development and metabolism. A pilot
intervention program in which participants received either a combination of dietary
counseling and probiotics (L.rhamnosus GG and B. lactis Bb12), dietary counseling with
placebo, or placebo alone from early pregnancy onwards, showed significant effects of the
intervention on placental lipids. The major differences in placental fatty acids were
attributable to a higher concentration of n-3 polyunsaturated fatty acids in both intervention
arms than in controls. Further, dietary counseling with probiotics resulted in higher
concentrations of linoleic (18:2n-6) and dihomo-gamma-linolenic acids (20:3n-6) comparing
with either dietary counseling with placebo or controls (Kaplas et al., 2007). The impact of
maternal nutrition with probiotic supplementation during pregnancy on infant blood pressure
has also been evaluated (Aaltonen et al., 2008). Pregnant women were randomized into 3
groups, the first submitted to a modified dietary intake according to current recommendations
and probiotics (diet/probiotics), the second followed dietary recommendations and received
placebo (diet/placebo), and the third received placebo (control/placebo). Although these
results were not completely conclusive, the highest and lowest intakes of specific nutrients,
such as carbohydrates and monounsaturated fatty acids compared with the middle ones were
associated with higher blood pressure in children at the age 6 months, suggesting that dietary
counseling can program blood pressure. The effects of probiotic supplementation together
with dietary counseling on glucose metabolism in pregnant women were evaluated further
leading to more conclusive results (Laitinen et al., 2008). The study included three subgroups
of pregnant woman at the first trimester of pregnancy, the first group received nutrition
counseling to modify dietary intake according to current recommendations (diet/placebo), the
second group received nutrition counseling and probiotics (L. rhamnosus GG and B. lactis
Bb12; diet/probiotics) and the third group received placebo without nutritional counseling
(control/placebo). Blood glucose concentrations were the lowest in the diet/probiotics group
during pregnancy and over the 12 months’ postpartum period. Glucose tolerance was also
better in the diet/probiotics group compared with the control/placebo group during the last
trimester of pregnancy and over the 12-months’ postpartum period. Therefore, the study
suggests that dietary counseling with probiotics can improved blood glucose control in a
normoglycaemic population and thus may provide potential novel means for the prophylactic
and therapeutic management of glucose disorders (Laitinen et al., 2008).
144 Yolanda Sanz
Conclusions and Further Perspectives
In the light of current scientific evidence, the potential of dietary interventions to

improve the health of the mother and the infant is significant from conception onwards.
Given the role of the intestinal microbiota in host immunity and metabolism, particularly at
early developmental stages, dietary interventions based on probiotic and prebiotic
administration could aid in health programming and disease prevention. To make it reality,
further studies are needed to define the mechanisms by which intestinal bacterial may
influence mothers physiology and the transmission routs of such effects to the offspring. The
development of a larger number of clinical trials with different probiotic strains, in diverse
physiologic and at risk conditions and during well-established intervention time-frames could
be of great help to extend the probiotic concept applications and contribute to reduce the
burden of disease within the modern lifestyle context.
Acknowledgments
This work was supported by grants AGL2007-66126-C03-01/ALI and Consolider Fun-

C-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation.
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Chapter VII
Epilactose: Potential
for Use as a Prebiotic
Susumu Ito1, Jun Watanabe1, Megumi Nishimukai1, Hidenori

Taguchi1, Hirokazu Matsui2, Shigeki Hamada2, and Shigeaki Ito2
1
Creative Research Initiative “Sousei”, Hokkaido University, Sapporo 001-0021, Japan
2
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Abstract
Prebiotics are nondigestible food components that affect the host by stimulating the
growth and/or activity of health-promoting bacteria in the colon and thus contribute to
host health and well-being. Epilactose is the C2-epimer of lactose that is found in 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-density-lipoprotein cholesterols were lowered, and 4)
conversion of primary bile acids to secondary bile acids was suppressed. Therefore, the
conversion of lactose to epilactose may increase the nutritional value of milk and whey.
Introduction
A probiotic is “a live microbial feed supplement which beneficially affects the host
animal by improving its intestinal microbial balance” [1]. Lactobacillus and Bifidobacterium
spp., which have been described as living microorganisms that exert health benefits,
154 Susumu Ito, Jun Watanabe, Megumi Nishimukai et al.
exemplify the concept of probiotics [2]. They have been reported to prevent and treat
rotavirus infections and postantibiotic diarrhea [3], allergic diseases [4], and inflammatory
bowel disease [5]. A prebiotic is usually defined as “a nondigestible food component that
beneficially affects the host by selectively stimulating the growth and/or activity of one or a
limited number of bacteria in the colon, and thus improves host health” [6]. Nondigestible
saccharides such as dietary fibers and oligosaccharides are known to exhibit various health-
promoting biological activities [7, 8]. The growth of beneficial intestinal bifidobacteria is
stimulated by dietary supplementation with prebiotics [9–11]. Through fortification with
beneficial microflora, prebiotics may enhance the defense mechanisms of host animals,
increase resistance to various health challenges, and accelerate recovery from gastrointestinal
tract disturbances [12]. Some nondigestible saccharides promote mineral absorption, and the
large intestine is involved in this beneficial effect [13, 14]. They are fermented by intestinal
microorganisms, generating short-chain fatty acids (SCFA). Formation of SCFA in the large
intestine has been proposed to be partly responsible for an increase in calcium absorption [15,
16]. In addition, nondigestible saccharides have been reported to improve lipid metabolism
[17–20].
Lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid) [21–24] and lactulose (4-O-
β-D-galactopyranosyl-D-fructose) [25–29], which can be synthesized from lactose
chemically or enzymatically, have been shown to promote intestinal adsorption of minerals
and/or stimulate selective growth of bifidobacteria. Lactulose has been added to commercial
infant feeding products. Tyler and Leatherwood [30] were the first to find that the ruminal
Ruminococcus albus 7 (ATCC27210T) possessed an epimerization activity that catalyzed a
hydroxyl stereoisomerism at the C-2 position of the glucose moiety of cellobiose and
generated 4-O-β-D-glucopyranosyl-D-mannose. Recently, we reported the purification of
cellobiose epimerases (CEs) from R. albus NE1 and Eubacterium cellulosolvens NE13 to
homogeneity and the cloning and sequencing of their complete genes [31, 32]. Further, we
showed that the enzymes convert lactose to epilactose (4-O-β-D-glactopyranosyl-D-
mannose) [32, 33]. While considerable amounts of epilactose is known to be formed by heat
and alkali treatments of cow milk [34, 35], CE has the advantage that it can produce
epilactose directly from lactose without byproducts [36, 37]. However, the biological
activities of this unusual sugar have not yet been examined.
Here, we demonstrate that elilactose has potential for use as a prebiotic foodstuff and that
the conversion of lactose to epilactose by CE may increase the nutritional value of milk and
whey.
Biological Activities of Epilactose
The purification of CE from R. albus NE1 and preparation of epilactose from lactose by
the enzyme were conducted essentially by the method of Ito et al. (33). For the dietary
intervention study, male Wistar-ST rats (4-week-old) were housed in individual stainless-
steel cages in a room with controlled temperature (22 ± 2°C) and allowed free access to a
semipurified stock diet (sucrose-based, AIN-93G formula) and tap water for 5 days. The rats
were then divided into two groups (n = 6), and each group was fed the control or 4.5%
Epilactose: Potential for Use as a Prebiotic 155
epilactose diet with freely available deionized water for 15 days. Body weight (BW) gains
over 15 days were the same between rats fed control diet (99.8 ± 4.8 g) and those fed
epilactose diet (104.0 ± 2.7 g). Further, liver and kidney weights relative to BW did not differ
between the groups. Epilactose had no toxic effect at least up to 4.76 g/kg BW/day under our
experimental conditions. Detailed analytical methods used in this study have been described
in the literature [38, 39].
1. In vitro Digestion Stability, Bifodogenetic Activity, Tight

Junction Permeability
Epilactose was resistant to in vitro digestion by a crude preparation of rat intestinal

enzyme(s). The growths of the human bifidobacteria Bifidobacterium bifidum, B. longum, B.
breve, B. adolescentis, and B. catenalatum [40] were significantly increased in a medium
containing 1.0% epilactose [33]. These results suggest that epilactose reaches the lower
gastrointestinal tract without being digested, where it is utilized by bifidobacteria and
lactobacilli. Epilactose increased net calcium absorption by increasing the paracellular
transport through the control of tight junction of human Caco-2 cells, where it dose-
dependently decreased transepithelial electrical resistance across the cell monolayer [33].
2. Calcium Absorption in Small Intestine
Calcium absorption rates were measured using everted sacs of jejunum and ileum
isolated from rats fed control and epilactose diets [39]. The presence of epilactose was found
to enhance calcium absorption in both jejunal and ileal sacs, regardless whether rats had been
fed epilactose or control diet. The calcium absorption rates by jejunal and ileal sacs isolated
from the epilactose-fed rats were similar to those by the sacs of control rats. These results
indicate that the coexistence of intact epilactose with calcium in the small intestinal lumen
enhances calcium absorption in a non-prebiotic way and that no adaptive change in the
absorptive activity for calcium occurs in the small intestine due to feeding epilactose.
Two distinct pathways are involved in intestinal calcium absorption, a transcellular
pathway and a paracellular pathway [41]. Transcellular absorption is a saturable, carrier-
mediated active transport pathway, whereas paracellular absorption through tight junctions is
nonsaturable and diffusive and requires a gradient of calcium concentrations between luminal
and basolateral sides. Passive absorption in the jejunum and ileum is the major absorptive
pathway when calcium intake is adequate or high. Under our experimental conditions, rats
were fed adequate calcium (5 g Ca/kg of diet), and therefore the absorption of calcium by
epilactose may proceed mainly via the passive transport machinery [39].
3. Population of Cecal Bacteria
The number of total cecal anaerobes in epilactose-fed rats was higher than that in control
rats when examined by a culture-based method. When measured using the real-time PCR
with genus-specific primers, the logarithmic cecal lactobacilli number of epilactose-fed rats
(9.46 ± 0.12 copies/g content) was significantly higher than that in control rats (8.73 ± 0.16
copies/g content). In addition, the logarithmic cecal bifidobacteria number of rats fed the
epilactose diet reached 7.80 ± 0.28 copies/g content, being significantly higher than that of
rats fed the control diet (5.63 ± 0.34 copies/g content). For enumeration of intestinal
microbiota, 16S rRNA gene libraries were constructed from cecal DNA of rats using
universal 16S rRNA-targeted primers. The populations of clones of harmful -Proteobacteria
and Clostridia tended to be smaller in epilactose-fed rats than in control rats [38]. These
results indicate that epilactose changes the intestinal microflora in a beneficial way.
It is well known that bifidobacteria and lactobacilli ferment various kinds of sugars to
produce organic acids [42]. Fructooligosaccharide is known to increase cecal wall weight by
increasing organic acid concentrations in the cecum [43]. Indeed, the weights of the cecal
wall and contents (g/100 g BW) in epilactose-fed rats were 0.38 ± 0.22 and 2.24 ± 0.09,
respectively, while those in control rats were 0.23 ± 0.01 and 0.83 ± 0.08, respectively.
4. Organic acid Generation and Mineral Absorption
The amount of total short-chain fatty acids (SCFA) (acetic, propionic, and butyric acids)
in the whole cecal contents was found to be higher in epilactose-fed rats (447 ± 31 μmoles)
than in control rats (138 ± 11 μmoles). Specifically, the amounts of acetic, propionic, and
butyric acids in the epilactose-fed rats were 275 ± 19, 127 ± 8, and 46.2 ± 7.7 μmoles,
respectively, whereas those in the control rats were 88.3 ± 6.9, 32.3 ± 3.2, and 17.1 ± 1.3
μmoles, respectively. The amount of total organic acids (SCFA, succinic acid, and lactic
acid) in the whole cecal contents of epilactose-fed rats (677 ± 49 μmoles) was 5 times that of
control rats (140 ± 11 μmoles). In particular, the amounts of succinic acid and lactic acid in
the whole cecal contents of the epilactose-fed rats were 185 ± 27 and 44.9 ± 30.6 μmoles,
respectively, values of which were very much higher than those of the control rats (1.52 ±
0.43 and 0.742 ± 0.167 μmoles, respectively). Consequently, the pH of the cecal contents of
epilactose-fed rats was lowered by the organic acids to 6.62 ± 0.18, while that in control rats
was 7.60 ± 0.04 [39].
Organic acids produced from nondigestible saccharides are thought to enlarge the large
intestine and increase calcium solubility by lowering the pH in the luminal contents; as a
result, calcium absorption is increased in the cecum [43, 44]. In fact, calcium solubility in the
cecal contents was higher in epilactose-fed rats (32.6 ± 1.2%) than in control rats (13.9 ±
1.1%). Magnesium solubility in the cecum was also higher in rats fed epilactose (79.2 ±
3.4%) than in rats fed the control diet (59.9 ± 2.7%). These results suggest that epilactose
intake increases mineral absorption in the large intestine by increasing absorptive area and
mineral solubility, and the higher solubility may induce higher absorption in the epilactose-
fed rats.
5. Levels of Cholesterols, Triglycerides, and Phospholipids
Hara et al. demonstrated that SCFA inhibited hepatic cholesterol synthesis and lowered
plasma cholesterol level in rats [19, 45]. It has been reported that acetic acid contributes to
the serum cholesterol-lowering effect of oat bran in humans [46] and that propionic acid
reduces cholesterol synthesis in humans and rats [47]. Also, a previous report has shown that
sugar-beet fiber lowers cholesterol synthesis with a coordinated increase in generation of
SCFA and that it increases fecal excretion of steroids [19]. We found that epilactose intake
tended to decrease the plasma cholesterol level and elevated the level of SCFA in cecal
contents of rats. Notably, the level of SCFA in the cecal contents was found to be negatively
correlated with those of plasma total cholesterol and low- and very low-density lipoprotein
cholesterols [39]. These results show that the SCFA generated by intestinal fermentation of
epilactose contribute to the suppression of cholesterol synthesis and lowering of the plasma
cholesterol level in rats. Feeding of epilactose did not have any significant effect on the levels
of plasma triglycerides and phospholipids in rats under our experimental conditions.
6. Levels of Primary and Secondary Bile Acids
The major primary bile acids in rats are chenodeoxycholic acid, α-muricholic acid (α-
MCA), and β-MCA [48]. These primary bile acids are transformed to secondary bile acids by
intestinal bacteria [49]. The secondary bile acids, such as hyodeoxycholic acid (HDCA), are
cytotoxic to colon cells and have been implicated as tumor promoters [50]. Ingestion of
nondigestible oligosaccharides decrease the conversion of primary bile acids to secondary
bile acids and reduced 1,2-dimethylhydrazine-induced precancerous colon lesions in rats
[51].
We then examined the effects of epilactose on the levels of primary and secondary bile
acids in the cecal contents of rats [39]. The total concentration of primary bile acids in dried
feces was higher in rats fed the epilactose diet (63.99 ± 14.21 μmol/g) than in those fed the
control diet (23.5 ± 4.04 μmol/g). The levels of secondary bile acids were almost the same in
the control and epilactose-fed rats (25.9 ± 14.08 μmol/g and 19.55 ± 1.88 μmol/g,
respectively). However, the level of HDCA was decreased to below the detection limit in the
epilactose-fed rats from 16.60 ± 3.46 μmol/g in the control rats, while significantly higher
concentrations of β-MCA were detected in the epilactose-fed rats (35.78 ± 6.9 μmol/g) than
in the control rats (7.43 ± 1.67 μmol/g). The ratios of secondary bile acids to total primary
bile acids were 1.21 ± 0.08 for the control rats and 0.38 ± 0.05 for the epilactose-fed rats.
HDCA is known to be generated from β-MCA by an unidentified bacterium HDCA-1, and
the introduction of this strain into germ-free rats facilitates the conversion of β-MCA to
HDCA [52]. Our clone library analysis of 16S rRNA revealed that harmful bacteria tended to
be fewer in the epilactose-fed rats than in the control rats. In rats fed the epilactose diet, the
cecal content pH was around 6.6 [39]. At this pH, the proliferation of strain HDCA-1 is
completely abolished [52]. These results suggest that epilactose reduces the population of
some cecal bacteria that possess the ability to convert β-MCA to HDCA. Therefore, it is
possible that the change in intestinal microbiota due to the ingestion of epilactose suppresses
colon cancer by inhibiting the formation of secondary bile acids.
Conclusion
We synthesized epilactose from lactose by the action of a CE from R. albus. Epilactose is

nondigestible and has potential for use as a prebiotic that possesses several beneficial
activities: stimulation of bifidobacteria growth, facilitation of mineral absorption, lowering of
plasma total cholesterol and non-high-density-lipoprotein cholesterols, and suppression of the
generation of secondary bile acids. CE can increase the value of milk and whey by preparing
novel milk products that have prebiotic properties.
Ackknowledgments
Purified epilactose was kindly provided by Drs. T. Yamamoto, M. Takada, and M.

Yamamoto of Nihon Shokuhin Kako Co. Ltd. This study was supported by Special
Coordination Funds for Promoting Science and Technology and by a National Project
“Knowledge Cluster Initiative” (2nd stage, “Sapporo Biocluster Bio-S”) from The Ministry
of Education, Science, Sports and Culture of Japan.
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Chapter VIII
Lactoferrin as an Added-value Whey

Component and a Healthy Additive in
Nutraceutical Drinks
Palmiro Poltronieri1,*, Carla Vetrugno2,

Antonella Muscella2, Santo Marsigliante2
1
CNR-ISPA, Institute of Sciences of Food Productions, National Research Council,
Lecce, Italy
2
Department of Biological and Environmental Sciences and Technologies
(Di.S.Te.B.A.), University of Salento, Lecce, Italy
Abstract
Lactoferrin (Lf) is a whey protein with potential food applications to sustain human
health. Lf is already added to infant formula milk powder so that, like breastmilk, it
contains Lf to help build resistance to disease. One yogurt is added with Lf and produced
by the Morinaga factory in The Nederlands. Lf binds iron, and can deliver it to increase
iron availability. This ability seems to affect also microbes and fungi, although 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.
We found that Lf-based emulsions storage with good stability up to 12 months. A
milk or soy-milk beverage may be a convenient vehicle for delivery of Lf-based
nutraceuticals.
*
Corresponding Author. E-mail: palmiro.poltronieri@ispa.cnr.it
164 P. Poltronieri, C. Vetrugno, A. Muscella et al.
Introduction
Although there is no official definition of functional foods, it is generally considered that

they are a group of foods which provide physiological benefits beyond those traditionally
expected from food. Milk proteins have a great potential use as functional foods: healthy
foods, nutraceuticals and food for specified human use, are one of the fields in constant
growth in the food industry, as well as an emerging field of medical interest. Therefore,
Health and Wellness has become an important internationally recognized, basic research
program in many Research Departments of Food companies and Universities. Research in
functional foods includes: a) utilization of biological models, including proteomics, to assess
the molecular mechanisms and physiological effects of bioactive food components: b)
development of bioprocessing techniques to isolate bioactive compounds or produce novel
health promoting foods that maintain desirable quality and safety over an extended shelf-life;
c) characterization of the molecular properties of novel food ingredients with health
promoting activities.
The milk / whey proteins show to possess important biological activities in terms of
human health (Gill &Cross 2002; Clare et al. 2003; Smolenski et al. 2007). Milk proteins
referenced in scientific publications are: High Mobility Group proteins (Yamamura et al.
1999), milk basic protein (Matsuoka et al. 2002), milk fat globule membrane proteins
(Cavaletto et al. 2008) as xanthine oxidase, lactadherin, butyrophilin associated with
phospholipids, whey acidic protein (Nukumi et al. 2007), lactoferrin (Lf) (Weinberg 2003;
Wakabayashi et al. 2006); ceruloplasmin (Sokolov et al. 2006), lactoperoxidase (Severin and
Wenshui 2005), alpha-lactalbumin (Teschemacher 2003), and beta-lactoglobulin (Hernandez-
Ledesma et al. 2008). New functional foods, enriched in bioactive milk proteins, may provide
a healthy effect for the action of each constituent, in prevention of chronic pathologies and as
co-adjuvant in maintaining physiological fitness. Whey proteins are usually denatured by
heating during post-cheese processing. On one side, the economical costs of production and
costs of whey discharge influence the manufacture process. Milk and dairy products
containing whey proteins in their native state may constitute an interesting product
innovation in milk industry.
Lf is a natural defence protein with iron-binding ability, present in exocrine secretions
and in body fluids that are commonly exposed to normal flora, acting as microbial barriers:
milk, tears, nasal exudate, saliva, bronchial mucus, and gut surfaces. Additionally, Lf is
produced in polymorphonuclear leukocytes and is deposited by these cells in septic sites. A
principal function of Lf is that of scavenging non-protein-bound iron in body fluids and
inflamed areas. Iron binding is a key property of Lf that accounts for many of its biological
roles in host defence such as bacteriostasis and protection against oxygen radicals catalyzed
by free iron (Crouch et al. 1992; Kruzel et al. 2006; Raghuveer et al. 2002).
The Lf ingested with foods is processed by digestive proteases into peptides. One of
these peptides is lactoferricin (Tomita et al. 2002), that includes the N-terminal lobe
containing the first Fe2+ chelating domain, while another antimicrobial peptide is the 11-
residue lactoferrampin (Haney et al. 2007), known to possess antimicrobial activity.
Recently, a conference on Lf bioactivity was held in the USA and proceedings were
published as miniseries (Kruzel et al. 2007). Lf affects gut cell proliferation (Buccigrossi et
Lactoferrin as an Added-value Whey Component and a Healthy Additive … 165
al. 2007), bone cell activity (Cornish et al. 2004) and the synthesis of nitric oxide (Choe et al.
1999). Lf promotes antimicrobial activity by activating the immune system during transit in
the gut. Anti-inflammatory effects of Lf have been shown by the inhibition of pro-
inflammatory cytokine production (Kruzel et al. 2002) and the up regulation of anti-
inflammatory cytokines (Togawa et al. 2002). On the other hand, Lf may enhance directly or
indirectly the immune response (in vitro and in vivo) by regulating the differentiation and
activation of both T and B cells (Zimecki et al. 1991, Zimecki et al. 1995). Orally
supplemented lactoferrin from bovine milk and Lf of other origin is purported to have
beneficial effects on gut health of animals, as mouse/rats (Togawa et al. 2002), and dogs with
the aim to improve the gut microflora (Pope et al. 2006). Furthermore, oral human lactoferrin
inhibits growth of established tumors and potentiates conventional chemotherapy
(Varadhachary et al. 2004) through the activation of non-specific immune system.
Results
At first, we studied the colony forming potential of three cancer cell lines, MCF-7 breast
cancer cells, HeLa cells, and SH-SY5Y neuroblastoma cells, in presence of three milk/whey
proteins, i.e. Lactoferrin (Lf), β-lactoglobulin (β-Lg) and ά-lactalbumin (ά-La), at
concentration of 10, 50 and 100 µg/ml. An anti-proliferative action of Lf on the MCF-7 cells
was found at 50 µg/ml, evidenced as a decrease in the colony formation ability (Figure 1). It
was decided to challenge MCF-7 cells using Lf at doses of 100 µg/ml and to investigate the
intracellular mechanisms and the signalling pathways involved.
Figure 1.
Figure 2.
In order to study the cell cycle state of cells, we counted the % of cells in cell cycle
phases other than G0 by measuring the total DNA stained with propidium iodide in a
FACSort cytofluorimeter (Becton Dickinson) (Figure 2).
The effect of Lf administration on MCF-7 cells in the presence of 10% FBS was
comparable to the effect of FBS withdrawal (Figure 3). The number of cells in S and G2/M
phases decreased drastically, from 36% cells stimulated to proliferate, to just 6% of cells
entering in mitosis.
In order to understand the mechanisms of proliferation inhibition, two assays were
designed. In the first, we analysed the up-regulation of the growth arrest protein p27kip, and
the lowering of cell cycle related proteins CDK2 and cyclin E (Figure 4). In the presence of
Lf, p27kip levels increased since the 12th hour, whereas CDK2 and cyclin E levels drastically
lowered between the 24 and the 48 hour points. The mechanism by which Lf activates growth
arrest was individuated in the activation of Protein Kinase C delta (PKC-δ) isoform, known to
be involved in cell differentiation (figure 5). The western blot performed on cell lysates
showed a four fold reduction in staining of unprocessed PKC-δ with appearance of the
activated PKC-δ fragment (t-PKC-δ).
Figure 3.
Figure 4.
Figure 5.
Figure 6.
On the other side, we found a second, pronounced effect of Lf on MCF-7 cells in the
activation of apoptotic pathways. A well known marker of apoptosis is the cleavage of PARP
protein: after the 9th hour of Lf treatment, we found the disappearance of the 116 kDa full
length PARP protein, and the appearance of the 85 kDa cleaved product (figure 6).
In addition, MCF-7 cells showed activation of caspase 9 between 6th and 9th hour after Lf
addition, and activation of caspase 7 later on, at the 24th hour time point of experiment. Lf
also induced the phosphorylation of p38 MAPK (figure 7a); when cells were incubated for 1
hour with 1 and 10 μM SB203580, an inhibitor of p38 MAPK, and then 100 µg/ml Lf was
added for 24 hours, a reduction in caspase 7 activation was observed (figure 7b), suggesting
an involvement of p38 MAPK in this process. We found that the p38 MAPK activity was
also responsible for the phosphorylation of bcl-2 (figure 8a). In fact, Lf presence caused an
increase in the phosphorylated bcl-2 form, and the pre-treatment with SB203580 drastically
blocked this signal activation (figure 8b). It is proposed that Lf effect is mediated through
p38 MAPK, triggering the release of bax protein from the complex with bcl-2, and the
subsequent activation of the cell death caspases.
Figure 7.
Figure 8.
In Jurkat cells, it was already shown that LF induces apoptosis through bcl-2
phosphorylation and the JNK-Bcl-2 signaling cascade (Lee et al. 2008). By treatment of
Jurkat cells with the specific JNK inhibitor SP600125, but not the p38 MAPK inhibitor
SB203580, is was shown that it is required for Lf-induced apoptosis. When JNK activation
was abolished by SP600125, no Bcl-2 phosphorylation was detected, and the Lf-treated
Jurkat cells did not undergo cell death.
We also observed a low-metastatic phenotype in MCF-7 cells treated with Lf. Matrix
metalloproteases are membrane-bound proteins involved in cell migration by actively
degrading the extracellular matrices. MMP-2 levels were found three-fold decreased by
western blot. Also the proteolytic activity in cell lysates was partially decreased, measured as
gelatin proteolysis in an in-gel assay (figure 9).
Figure 9.
In conclusion, it is possible to prospect a series of different beneficial effects on certain

cancer cell types in the presence of Lf, i.e. growth arrest, reduction of metastatic potential,
and cancer cell death. It is still not known which mechanism operates in the cell uptake of Lf,
but other authors described the presence of Lf receptors (Suzuki et al. 2005, Mazurier and
Hovanessian 2004) and on the internalisation of Lf and Lf-δ isoform (Legrand et al., 2004,
Breton et al., 2004; Mariller et al. 2007).
Strategies for Delivery of Lf-Active Ingredient and to Increase Lf-

Consumption in Foods
A process of mild pasteurisation of Lf and Lf-containing food products has been

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

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

objectives. Considering the potential of ingested Lf to modulate the immune responses and
the iron homeostasis (Paesano et al. 2008), an increase of daily consumption would provide
benefits to the general health status. The production of Lf enriched foods and other
nutraceutical supplements could be feasible considering the high stability of the Lf
emulsions. Further studies are needed to prove absence of adverse effects during Lf local
delivery, as in the case of management of local infection during transplants (van der Velden
et al. 2008). Lf-containing liposomes, targeting diseased cells or organs, could be tested in
clinical trials. Also in cases when other therapies alone show no effect or very low rate of
success, the anticancer activity of Lf could be exploited in support to the therapeutic drugs.
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Chapter IX
Conjugated Linoleic Acid:

An Anticancer Fatty Acid Found
in Milk and Meat
T. R. Dhiman*, A. L. Ure and J. L. Walters

Department of Animal, Dairy and Veterinary Sciences
Utah State University, Logan, Utah 84322-4815, USA
Abstract
Conjugated linoleic acid (CLA) has been intensively studied recently, mainly
because of its potential in protecting against cancer, atherogenesis, and diabetes.
Conjugated linoleic acid is a collective term for a series of conjugated dienoic positional
and geometrical isomers of linoleic acid, which among common human foods are found
naturally in relative abundance in the milk and meat fat of ruminants. The cis-9, trans-11
isomer is the principle dietary form of CLA found in ruminant products, and is produced
by partial ruminal biohydrogenation of linoleic acid or by endogenous synthesis in the
tissues themselves. The CLA content in milk and meat is affected by several factors, such
as an animal’s breed, age, diet, and management factors related to feed supplements
affecting the diet. Conjugated linoleic acid in milk or meat has been shown to be a stable
compound under normal cooking and storage conditions. Total CLA content in milk or
dairy products ranges from 0.34 to 1.07% of total fat. Total CLA content in raw or
processed beef ranges from 0.12 to 0.68% of total fat. It is currently estimated that the
intake of the average adult consuming western diets is only one-third to one-half of the
amount of CLA that has been shown to reduce cancer in animal studies. For this reason,
*
Corresponding author: T. R. Dhiman, Phone: 435 797-2155; Fax: 435 797-2118; E-mail:
trdhiman@cc.usu.edu
Approved as journal paper number 7679 of the Utah Agricultural Experiment Station, Utah State
University, Logan, Utah 84322, USA.
176 T. R. Dhiman, A. L. Ure and J. L. Walters
increasing the CLA content of milk and meat has the potential to raise the nutritive and
therapeutic values of dairy products and meat. Growing evidence suggests that
consuming dairy products and meat enriched with CLA has beneficial effects on human
health.
Introduction
More than 2 decades ago, Michael Pariza and his colleagues at the University of
Wisconsin, USA, observed that fat from grilled beef actually inhibited chemically induced
cancer rather than promoting it in an animal model. These researchers identified the
anticancer agent as a mixture of isomers of conjugated octadecadienoic acid [1]. The isomers
were referred to collectively as conjugated linoleic acid (CLA). Since then an increasing
number of studies using synthetic and natural CLA have shown that it can suppress cancer at
a number of sites, including the mammary gland, skin, colon and forestomach [2-14]. In
several human cancer studies, an inverse association was found between the level of CLA in
the diet and the risk of developing cancer in breast adipose tissue [15-18].
Studies conducted with mice, chickens, and pigs have suggested a possible role of CLA
(mainly the trans-10, cis-12 isomer) in decreasing body fat and increasing lean body mass
[19-24]. A human study has suggested that CLA increases body mass without increasing
body fat [25]. Several studies indicate that CLA may enhance immune function [26-31].
Conjugated linoleic acid has also been found to have antidiabetic and antiatherosclerotic
properties in animal models [32-36].
Conjugated linoleic acid isomers are found naturally in foods, especially those of
ruminant origin [37]. In ruminants, CLA is synthesized by ruminal bacteria using C18:2 or
C18:3 as the precursor [38]. Conjugated linoleic acid isomers can also be synthesized in the
laboratory from C18:2 or from sources high in C18:2, such as sunflower, safflower, soybean, or
corn oils, by a reaction involving alkaline water isomerization [39] and isomerization in
propylene glycol [40].
The objective of this chapter is to provide an overview of different CLA isomers,
biosynthesis of CLA, CLA contents of milk and meat, factors affecting the level of CLA in
foods, potential health benefits of CLA and CLA intakes in humans.
CLA Isomers
The cis-9, trans-11 isomer is the principle dietary form of CLA exhibiting biological
activity, and accounts for 73 to 94% of total CLA in milk, dairy products, meat, and
processed meat products of ruminant origin [41-44]. In recent years, biological activities have
been proposed for other CLA isomers, especially trans-10, cis-12 C18:2 (t10, c12 CLA), [20,
21]. Throughout the rest of the chapter, the cis double bond will be abbreviated as c and the
trans double bond as t. The structures of c9, t11 CLA, t10, c12 CLA, and C18:2 are shown in
Figure 1.
Conjugated Linoleic Acid: An Anticancer Fatty Acid Found in Milk and Meat 177
A total of 17 natural CLA isomers have been found in milk, dairy products, beef, human
milk, and human adipose tissue using silver ion-high performance liquid chromatography and
gas chromatography-electron ionization mass spectrometry. Identified CLA isomers are t12,
t14; t11, t13; t10, t12; t9, t11; t8, t10; t7, t9; t7, c9; t6, t8; c12, t14; t11, c13; c11, t13; c10,
t12; c9, t11; c8, t10; c7, t9; c9, c11; and c11, c13. Bauman et al. [45] observed that butter
contained c9, t11 (76.5%) and c7, t9 (6.7%) isomers. Sehat et al. [40] characterized the
following distribution of CLA isomers in cheese fat: c9, t11 (78 to 84%); t7, c9 plus t8, c10
(8 to 13%); t11, c13 (1 to 2%); c12, t14 (<1%); and their total trans/trans isomers (5 to 9%).
Recently, Fritsche et al. [46] identified the distribution of CLA isomers in beef samples and
found that c9, t11 was the predominant isomer (72%), followed by the t7, c9 isomer (7.0%).
Typical synthetic CLA isomer mixtures consist of c9, t11 (40.8 to 41.1%); t10, c12 (43.5
to 44.9%); and t9, t11/t10, t12 (4.6 to 10.0%) isomers [37, 40]. Christie et al. [39] and
Fritsche [47] reported on a different synthetic CLA isomer mixture that contained c8, t10
(14%); c9, t11 (30%); t10, c12 (31%); and c11, t13 (24%).
Most of the aforementioned CLA isomers are present in foods in very minute amounts
and are of little known biological importance or have not been studied in detail. Therefore,
the ensuing discussion in this chapter will focus on the two predominant forms of CLA,
namely the c9, t11 and t10, c12 isomers.
Figure 1. Abbreviated chemical structures of ordinary C18:2 (linoleic acid) (A), and two major
conjugated linoleic acids (CLA): c9, t11 isomer (B), and t10, c12 isomer (C).
CLA Biosynthesis
Conjugated linoleic acid originates from either ruminal biohydrogenation of C18:2 and
C18:3 or from endogenous synthesis in tissues as shown in Figure 2. Ruminally, CLA is
produced as an intermediate product during the biohydrogenation of dietary C18:2 or C18:3 to
stearic acid (C18:0). Endogenously, CLA is synthesized from t-11, C18:1 vaccenic acid (TVA),
another intermediate of ruminal biohydrogenation, via Δ9-desaturase [48]. The endogenous
synthesis of CLA from TVA has been proposed as the major pathway of CLA synthesis in
lactating cows, accounting for an estimated 78% of the CLA in milk fat [49, 50].
Ruminal biosynthesis Endogenous synthesis
(Mammary gland / adipose tissue)
Diets
α-C18:3 C18:2 γ-C18:3 α-C18:3, C18:2, γ-C18:3
I I
I
c9, t11, c15 C18:3 c6, c9, t11 C18:3
H H
H H
t11, c15 C18:2 c9, t 11 C18:2 (CLA) c6, t11 C18:2 c9, t 11 C18:2 (CLA)
Δ9-desaturase
H H H
Trans-11 C18:1 (TVA) Trans-11 C18:1 (TVA)
C18:0 C18:0
Figure 2. Proposed mechanism for CLA synthesis from ruminal biohydrogenation or endogenous
synthesis. CLA, conjugated linoleic acid; TVA, trans vaccenic acid; I, isomerization reaction; and H,
hydrogenation. Adapted and reproduced with permission. Reference: [56].
Ruminal Biohydrogenation
Lipids in the ruminant diet are derived from forages, grains, and oil supplements. The
lipid content in most ruminant diets ranges from 3-7% on a dietary dry matter (DM) basis.
Most ruminant feeds of vegetable origin contain C18:2 and/or C18:3 as the predominant fatty
acids. Among feeds, pasture grass diets fed consumed by ruminants are rich in C18:3,
representing 48 to 56% of total fatty acids (FA). Corn or grass silages are rich in C18:2 (41%
of FA) or C18:3 (46% of FA), respectively. Most plant seeds and oils are rich in C18:2,
accounting for 53 to 69% of total FA.
When consumed by ruminants, the lipid portion of these feeds undergoes two major
processes in the rumen [51, 52]. In the first process, esterified plant lipids or triglycerides are
quickly hydrolyzed to free FA by microbial lipases [53]. In the second process, the
unsaturated free FA are rapidly hydrogenated by microorganisms in the rumen to produce
more highly saturated end products.
The c9, t11 isomer of CLA is the first intermediate product in the biohydrogenation of
C18:2 by the enzyme linoleate isomerase (Figure 2), which is produced by the microorganism
Butyrivibrio fibrisolvens [38] and other bacterial species. Part of the c9, t11 CLA is rapidly
reduced to TVA or C18:0, [54-55], becoming available for absorption in the small intestine.
Similar to the biohydrogenation of C18:2, the FA α and γ–C18:3, which are the predominant
unsaturated FA in forages, also undergo isomerization and a series of reductions, ending with
the formation of C18:0 in the case of complete biohydrogenation [56]. The c9, t11 CLA and
TVA often escaping complete ruminal biohydrogenation are absorbed from the intestine and
incorporated into milk fat [57, 58].
Studies with pure strains of ruminal bacteria have shown that most bacteria are capable
of hydrogenating C18:2 to t-C18:1 and related isomers, but only a few have the ability to reduce
C18:2 and C18:1 completely to C18:0 [59]. Interestingly, no single species of rumen bacteria
catalyzes the complete biohydrogenation sequence [56, 60].
It has been suggested that the biohydrogenation pathways are affected by several factors
related to the composition of the diet consumed by the animal, including the rumen
environment and the bacterial population [58, 61-63].
Endogenous Synthesis
It was originally assumed by the scientific community that the rumen was the primary
site of origin of c9, t11 CLA in milk fat. Recently, however, it has been suggested that only a
small portion of c9, t11 CLA escapes biohydrogenation in the rumen and that the major
portion of c9, t11 CLA in milk comes from endogenous synthesis in the mammary gland via
a pathway involving the desaturation of TVA by the Δ9-desaturase enzyme [49, 50, 64].
Several studies have been performed to confirm that the endogenous synthesis of CLA
by Δ9-desaturase occurs in the mammary gland [50]. The actual estimated endogenous
synthesis of c9, t11 CLA in milk fat was 64 [49], 78 [50], or 80% [65], of the total c9, t11
CLA with different correction factors used according to the extent of enzyme inhibition.
There are reported species differences in the tissue distribution of Δ9-desaturase.
Enzyme activity and mRNA abundance of Δ9-desaturase are highest in the liver of rodents;
however, in growing sheep and cattle, adipose tissue is found to have the highest levels [66-
68]. In lactating ruminants, the highest activity of Δ9-desaturase is found in the mammary
tissue [69]. There is very little research exploring the factors that influence and regulate Δ9-
desaturase activity in the tissues of ruminants.
CLA Content in Milk and Meat Products
In food, CLA is found in milk fat, the tissue fat of ruminant animals, and products
derived from them. The CLA content of food products is influenced almost entirely by the
CLA content of milk and meat fat and the processing methods used. Cow’s milk fat is the
richest natural source of CLA. The total CLA content and the proportions of the c9, t11 CLA
isomer in milk and dairy products from cows fed typical diets are in Table 1. These values
can be used as guidelines to calculate the CLA intake by humans from dairy products
available on the market.
The average CLA content in milk and dairy products on the market was 0.53% of fat
(range 0.34 to 1.07%). In fluid milk, CLA content ranged from 0.34 to 0.80% of fat with an
average of 0.53%. The CLA in various types of cheeses was 0.34 to 1.07% of fat (average =
0.50%). The CLA in fermented dairy products including butter, ranged from 0.36 to 0.94%
(average = 0.55%). The c9, t11 CLA isomer in all dairy products represented 79 to 94% of
total CLA in milk fat.
Table 1. Conjugated linoleic acid (CLA) contents in milk and dairy products
Samples* Total CLA** c9, t11CLA***

(% of fat) (%)
Fluid milk products
Whole milk [70, 78, 79, 86, 90, 94, 132, 137, 213, 224] 0.34-0.68 82-97
Evaporated milk [213] 0.49 --
UHT milk [214] 0.80 --
Homogenized milk [37] 0.55 92
Condensed milk [37], 214 0.63-0.70 82
Cultured buttermilk[132, 137] 0.54-0.67 89
Cheeses
Cheddar [40, 133, 137, 213, 215, 216] 0.40-0.53 78-82
Feta [40] 0.49 81
Cottage [37, 137, 213] 0.45-0.59 83
Mozzarella [37, 40, 70, 133, 137, 213] 0.34-0.50 78-95
Processed cheese [137, 213, 215, 217] 0.41-1.07 75
Processed American [37, 40, 213] 0.36-0.50 79-93
Processed Cheddar [40] 0.50 84
Processed Parmesan [137] 0.53 --
Fermented dairy products
Plain yogurt [37, 133, 134, 137, 213, 214, 218] 0.38-0.88 83-84
Lowfat yogurt [37] 0.44 86
Butter [37, 133, 213, 214] 0.47-0.94 78-88
Sour cream [37, 133, 137] 0.46-0.75 78-90
Ice cream [37, 133] 0.36-0.50 76-86
* Numerical superscripts next to samples correspond to reference numbers cited in the reference
section.
** Values are mean or minimum and maximum levels.
*** Data are expressed as % of total CLA isomers.
The CLA content and the proportions of c9, t11 CLA isomer in raw and processed meat
products are summarized in Table 2. It is again prudent to emphasize that most of the
research cited did not report total fat content of the products, and total actual CLA yields will
depend upon fat percentage of the product. The average CLA content in meat products of
ruminant origin available on the market was 0.46% of fat (range 0.12 to 1.20%) with the c9,
t11 CLA isomer representing an average 73% of the total CLA. The CLA in meats of non-
ruminant origin averaged 0.16% of fat (range 0.06 to 0.25%) with the c9, t11 isomer
representing 65% of the total CLA. The CLA content in turkey is almost twice the amount of
that found in chicken, pork, or rabbit. In the case of non-ruminants, CLA may be produced by
the conversion of TVA to CLA in the tissues or through the feeding of tallow, meat meal of
ruminant origin, or synthetic CLA.
Table 2. Conjugated linoleic acid (CLA) contents in meat and processed meat products
Samples* Total CLA** c9, t11CLA***

(% of fat) (%)
Ruminants
Beef
Ground, [137, 46, 137, 219] 0.16-0.43 72-85
Round [37, 136] 0.29-0.68 57-79
Ribeye [136, 137] 0.30-0.64 61
T-bone [136] 0.61 59
Sirloin [136, 137] 0.12-0.58 59
Frank [37] 0.33 83
Smoked sausage [37] 0.38 84
Veal [37] 0.27 84
Lamb [37, 214, 220, 221, 222, 223] 0.18-1.20 92
Non-ruminants
Turkey [37, 214, 220] 0.20-0.25 40-76
Turkey frank [37] 0.16 70
Smoked turkey37 0.24 62
Pork [37, 220] 0.06-0.13 25-82
Smoked bacon [37] 0.17 76
Chicken [37, 214, 220] 0.09-0.15 67-84
Rabbit [220] 0.11 27
* Numerical superscripts next to samples correspond to reference numbers cited in the reference
section.
** Values are mean or minimum and maximum levels.
*** Data are expressed as % of total CLA isomers.
Factors Affecting CLA Content in Milk
Pasture, Conserved Forages, and Grain
The CLA content in milk fat can be affected by a cow’s diet, breed, age, and the use of
synthetic mixtures of CLA supplements. Among these factors, the diet is known to strongly
influence the CLA content of milk, which is affected by feedstuffs such as pasture, conserved
forages, plant seed oils, cereal grains, marine oils and marine feeds.
A number of studies have shown the positive effects of pasture-based diets on the CLA
content of milk fat. Dhiman et al. [70] reported that cows grazing pasture had 500% higher
CLA content in milk fat (2.21% of total FA) as compared to cows fed a diet containing 50%
conserved forage (hay and silages) and 50% grain (0.38% of total FA). Other researchers
have also demonstrated that the CLA content of milk increased linearly as the proportion of
fresh grass from pasture in the diet was increased [71-74].
Supplementing grain to cows grazing pasture decreases the CLA content of milk fat.
Cows on pasture supplemented with 0, 6, or 12 kg/day of grain had 2.21, 1.43, and 0.89%
CLA in milk fat, respectively [70]. Similarly, supplementing grain to cows receiving grass
silage or replacing conserved grass in dairy cow diets with corn silage lowered the CLA
content of milk [72, 75]. Corn silage contains 20 to 40% grain on a DM basis. The addition
of grain to dairy diets decreases ruminal pH. A decrease in pH will change the microbial
population and affect ruminal fermentation [62]. It has been suggested that the main ruminal
biohydrogenating bacteria are cellulolytic [76, 77]. Reduction in ruminal pH decreases the
population of cellulolytic bacteria and other microbes responsible for lipid biohydrogenation
and the production of CLA and TVA [57].
Forage maturity and method of preservation also seem to be important factors
influencing the CLA content of milk. Cows fed immature forages have higher levels of CLA
in milk than cows fed mature forage. Cows fed grass silage cut at early heading, flowering,
and second cutting had 1.14, 0.48, and 0.81% CLA in milk fat, respectively [75]. The high
C18:3 content of immature grass and its low fiber content as compared to mature grass
probably interact to increase the production of CLA and TVA.
Plant Oils and Seeds
Feeding plant seed oils, such as sunflower, soybean, peanut, canola, and linseed oil,
increased CLA content in milk [78-82]. These oils are rich in C18:2 and C18:3 FA. Studies have
found that high levels of C18:2 and C18:3 (such as are found in most plant seed oils) result in
increased production of CLA and TVA, with the TVA potentially being an additional
substrate for the endogenous synthesis of c9, t11 CLA [56, 83, 84]. Besides directly
increasing the yield of CLA and TVA, it is likely that C18:2 inhibits the final reduction of
TVA, thus increasing its accumulation in the rumen [58] and subsequent availability to the
animal.
Feeding a diet containing soybean oil (4%) resulted in approximately a four-fold increase
in CLA content of milk fat (2.08%) over the control diet without soybean oil (0.50% of milk
fat), [85]. Supplementing peanut oil, sunflower oil, or linseed oil at 5.3% of dietary DM
resulted in 1.33, 2.44, and 1.67% CLA in milk fat, respectively [78]. The specific FA that is
most abundant in any given plant seed oil is very important in determining how much the oil
will elevate the levels of CLA in milk fat. Oils rich in C18:2 are more effective at increasing
CLA in milk fat as compared to oils rich in C18:3 or C18:1. Dhiman et al. [85] reported that
linseed oil was not as efficient at increasing CLA content in milk fat as was soybean oil.
Intact oil seeds are known to be less efficient than free oils at enhancing the CLA content
of milk. Processing releases oil from the seeds, which then becomes available to the rumen
microbes for biohydrogenation. Feeding raw seeds has little or no effect on the CLA content
of milk fat because polyunsaturated fatty acids (PUFA) in the intact seeds are relatively
unavailable to the rumen microbes for biohydrogenation [85]. However, if raw seeds are
processed by grinding, roasting, micronizing, flaking, or extruding, those processed seeds are
effective at increasing the CLA content in milk [71, 79, 86, 87-94].
Interestingly, cows grazing green grass that received 82% less C18:3 (received 102 g/day)
had higher milk CLA content (2.21% of fat) [70] than cows fed diets containing conserved
forages and grain supplemented with C18:3 (received 575 g/day) through linseed oil (1.67%
CLA in milk fat) [78]. However, it should be pointed out that factors other than oil supply
from pasture grass are also responsible for the higher CLA content observed in grazing cows.
Cows grazed on pasture or fed forage alone will produce less milk, but with higher fat
content, than cows fed conserved forage and grains. The milk yield of grazing cows is
reduced by 50-60%, but the CLA content of milk is 300–400% higher than in milk from cows
fed conserved forage and grain. Thus, the daily output of CLA from cows grazing on pasture
will still be higher than from cows fed conserved forage and grain even with lower total milk
yields. The situation differs when cows are fed plant oils, as plant oils enhance the CLA
content of milk. Therefore, caution must be taken when comparing dietary influence on CLA
content and daily CLA output.
Marine Oils and Feeds
The feeding of fish oil to dairy cows has been shown to enhance the CLA and TVA
contents of milk fat, but to reduce total fat content of milk [86, 91, 94, 95]. Feeding fish oil at
1.6% of the diet DM increased the CLA and TVA contents in milk fat from 0.16 and 1.03%
in control group without fish oil to 1.55 and 7.50% in fish oil fed group, respectively [95].
Feeding diets containing 2% fish oil to dairy cows increased CLA and TVA contents in milk
fat by 300 and 500%, respectively, as compared to milk fat from cows fed no fish oil [90, 96].
However, there was no additional increase in CLA and TVA contents when cows were fed
3% fish oil. The inclusion of marine feeds such as fish meal or sea algae in dairy cow diets
has been shown to enhance the CLA content of milk [69, 85, 92, 97, 98]. The reduction of
milk fat percentage is a common problem when feeding fish oil to lactating dairy cows and
can influence the total CLA yield. Milk fat content was reduced by 20 to 25% when cows
were fed diets containing 1.6 to 2.0% fish oil or 4% algae on a DM basis [90, 94, 95, 96, 98].
Researchers have also attempted to enhance CLA in milk fat by feeding combinations of
fish and soybean oils or meals to dairy cows. In some studies, fish oil/fish meal was more
effective at enhancing the CLA content of milk than adding similar amounts of soybean oil or
combinations of fish oil and soybean oil through extruded soybeans or soybean meal [90, 91,
94, 97].
It is worth stating here that a component of fish oil may inhibit the growth of bacteria or
production of bacterial enzymes responsible for the reduction of TVA to C18:0, creating
conditions that are more favorable to the later tissue production of CLA from TVA.
Therefore, C18:2 and C18:3 FA that were provided by the soybeans in the diet indirectly
increased CLA synthesis. The origin of CLA in milk fat from cows fed fish oil is very
possibly through the desaturation of TVA in the mammary gland by the Δ9-desaturase
enzyme. The relationship between milk fat CLA and TVA is linear across a wide variety of
feeding conditions [99].
Cow Management Systems
Dairy cow management systems also influence the CLA content of milk. Jahreis et al.
[72] collected milk samples over a period of one year from three farms with different
management systems: 1) conventional farming with indoor feeding using preserved forages;
2) conventional farming with grazing during the summer season; and 3) ecological farming
with no use of chemical fertilizers to produce forages and grazing during the summer season.
The CLA content was 0.34, 0.61, and 0.80% of fat in milk from cows fed indoors, grazed
during summer, and grazed in ecological farming conditions, respectively. Reasons for these
results could be due, in part, to differences in vegetation or forage quality among the three
systems.
Depending on the season, CLA content in milk varied from 0.6 to 1.2% of milk fat, with
content being higher in spring and summer than in winter [100-103]. These data suggest that
the availability of fresh forages in spring and summer increases CLA content in milk fat as
compared to mature forages in late summer or conserved forages in winter.
Cow Breed, Age, and Individual Variation
Recent studies suggest that dairy cow breed can also influence the CLA content of milk.
Montbeliard cows displayed a tendency to have higher CLA in milk fat (1.85%) as compared
to Holstein-Friesian (1.66%) or Normande cows (1.64%) grazing on the same pasture [104].
Holstein-Friesian cows had higher CLA content in milk compared to Jerseys fed diets
containing conserved forages and grains [105, 106, 107]. Conjugated linoleic acid content
was also higher in milk fat from Holstein-Friesians (0.57%) than for Jersey cows (0.46%)
when grazed on pasture [74]. Brown Swiss cows had higher CLA content in milk fat than
Holstein-Friesian when fed similar diets [94, 106, 107]. The average difference in CLA
content of milk fat among Brown Swiss, Holstein-Friesian, and Jersey breeds is 15 to 20%
when fed similar diets. Brown Swiss cows have inherently higher CLA in milk fat, followed
by the Holstein-Friesian and Jersey breeds.
The CLA content in milk varies from cow to cow, even when the same diet is fed. Jiang
et al. [57] and Stanton et al. [87] found substantial variation in the CLA content of milk
(0.15% to 1.77% of fat) among individual cows fed the same diet. Kelly et al. [73, 78]
observed a threefold variation in CLA content of milk among individual cows fed the same
diet at a similar stage of lactation and producing milk with similar fat content.
Synthetic CLA Supplements
Synthetic CLA isomers were fed in a ruminally-protected form to avoid ruminal

biohydrogenation and enhance CLA in milk fat. The administration or feeding of CLA
supplements to dairy cows caused a dramatic reduction in the fat content and total yield of
milk fat, but resulted in a small increase in milk CLA content [82, 108, 109-115]. The highest
transfer efficiencies for c9, t11 and t10, c12 isomers from supplement to milk were 11 and
4%, respectively [112-113].
Dietary Factors Affecting CLA in Meat
Pastures and Conserved Forages
As is the case with dairy cattle, grazing animals on pasture, feeding fresh forages or
increasing the amount of forage in the diet will elevate the percentage of CLA as a proportion
of total FA in meat from ruminants. Grazing beef steers on pasture or increasing the amount
of silage in the diet increased the c9, t11 CLA content in meat fat by 29 to 45% compared to
control [116, 117]. The increase in beef CLA content varies with the quality and quantity of
forage in the animal’s diet. Beef from steers raised on green pasture had 200 to 500% more
c9, t11 CLA as a proportion of fat as compared to steers fed an 87% corn grain-based feedlot
diet [118, 119]. Rule et al. [120] observed that the percentage of c9, t11 isomer of CLA was
higher in intramuscular fat of range cattle compared with that of steers fed a high-grain diet
under feedlot conditions. The increase in c9, t11 CLA content in beef is not as dramatic as
the increase seen in milk from cows grazed on pasture. This difference is probably due to
differences in CLA production in the rumen or endogenous synthesis of CLA in
intramuscular fat of beef cattle fed high-forage diets.
Plant Oils and Seeds
Supplementing beef cattle diets with C18:2 or C18:3 rich plant oils has yielded variable
results with regard to increasing the CLA content of beef. Feeding 4 to 6% of diet DM as
soybean oil to beef cattle fed high grain diets either marginally increased or did not increase
the c9, t11 CLA content of beef [121-124]. There was a small increase in the c9, t11 CLA
content of fat from beef muscle when steers were fed 3 to 6% sunflower oil as compared to
beef from cattle fed no oil (0.35 vs. 0.25% CLA in beef fat) [125, 126]. Feeding processed
plant oil seeds has also resulted in marginal or no increase in CLA content of beef [117, 127-
129].
Feeding feed sources rich in C18:2 or C18:3 to dairy cows results in a 3 to 4 fold increase in
the c9, t11 CLA content in milk fat [78, 85], but only marginal increases in beef fat. It is very
possible that the mechanisms and routes of CLA synthesis (ruminal and endogenous) are
different in the mammary gland and adipose tissue. Additional factors regulating the
synthesis of CLA in the rumen, muscle, and mammary gland are poorly understood.
Finishing beef cattle are typically fed diets containing 85 to 92% grain, whereas a typical
diet for a high-producing dairy cow consists of only 50 to 60% grain. The lower proportion of
c9, t11 CLA in beef fat as compared to milk fat in animals fed diets rich in C18:2 or C18:3
probably relates to the effects of the traditional high-grain, low-fiber diets fed to finishing
cattle in the United States. It seems likely that the acidic ruminal pH often occurring in
finishing beef cattle alters the microbial population involved in lipid biohydrogenation, and
therefore influences the ruminal synthesis of CLA isomers. Grazing animals on pasture
substantially increases CLA as a proportion of FA, but total fat content is reduced because of
the lean beef from grassfed cattle. Therefore, increase in CLA content of beef should be
evaluated based on total CLA available in the edible fat rather than concentrations in raw
meat.
Animal Breed and Management Strategies
Besides dietary factors, researchers have also studied the influence of beef cattle breed
on CLA content in meat, and limited studies suggest that there is little breed effect. The CLA
content in beef muscle was similar in European x British crossbreeds and 75% Wagyu cattle
fed high-grain, barley-based diets [130]. Limousin cattle had only marginally higher CLA
content in beef muscle as compared to Wagyu and Limousin x Wagyu cattle fed similar diets
[131].
Existing literature indicates that the total CLA content (sum of c9, t11 and t10, c12
isomers) of beef varies from 0.17 to 1.35% of fat. The broad range of CLA content of beef is
related to the wide variety of feeds offered, breed differences, and management strategies
used to raise cattle.
Processing Effects on CLA Content

of Milk and Meat
The effects of processing conditions, dairy cultures, and storage conditions on CLA
content of dairy products have been studied. Pasteurizing raw milk at 68.3°C for 30 min did
not alter the CLA content of milk [1, 132]. Processing milk under normal conditions (up to
85˚C for 30 min) into dairy products such as yogurt, ice cream, sour cream, and cheese
(Mozzarella, Gouda, and Cheddar) had no influence on the CLA content [79, 133, 134]. The
CLA content of dairy products increases when milk is processed at higher temperatures.
Clarification of ghee (butter oil) at 110 and 120°C increased the CLA content to 0.9 and
2.1% of fat, respectively, as compared to 0.6% in raw milk [135].
Available research suggests that CLA in milk fat is a stable compound under normal
processing and storage conditions; however, processing dairy products at >80°C may slightly
elevate the CLA content. There is not enough data available to form conclusions about the
effects of different cultures and additives on the CLA content of dairy products.
Influence of cooking temperatures and methods on the CLA content of beef have been
studied by a few researchers. A majority of the results of such research [136, 137] suggest
that the CLA content in meat products seems to be influenced by the raw material, and not by
processing conditions such as cooking temperature. Different cooking methods (fried, baked,
broiled, or microwave) and degree of doneness under normal conditions (60 to 80°C internal
temperature in meat) did not alter the CLA content in beefsteaks (ribeye, round, T-bone, and
sirloin) [136]. Storing cooked beef at 4°C for 7 days did not alter the CLA content [136].
Results from the above studies suggest that CLA in meat is a stable compound under normal
cooking and storage conditions.
Health Benefits of CLA
There is a growing interest in the levels of CLA in human diets because of

accumulating evidence, largely based on animal studies, that suggests potential
health benefits of CLA. Another reason is growing interest in natural foods,
and that CLA occurs naturally in foods such as milk and meat derived from
ruminants. Additionally, as discussed above animals grazing fresh green grass,
“nature’s way of feeding ruminant animals”, results in 3 to 5-times more CLA
in milk and meat than when animals are fed conserved forages and grains in
confinement. Numerous physiological functions have been attributed to CLA,
including cancer inhibition, antidiabetic effects, reduction of adipose tissue,
alteration of body composition, reduction of atherosclerotic plaque formation,
enhancement of immune response and help with bone formation (Table 3).
Cancer Inhibition
Conjugated linoleic acid has a variety of positive effects on cellular mechanisms in

animal and human cancer cells that support its anticancer role. However, most of our
understanding of the anticancer effects of CLA is based on animal models. Preliminary
information from human studies shows positive health benefits. However, there is an urgent
need to extend our understanding of anticancer effects of CLA in humans. The evidence
concerning the promising role, and limitations, of CLA in the prevention of cancer is
reviewed below.
Table 3. Physiological functions of conjugated linoleic acid (CLA)
Function/Model used Reference

Carcinogenesis
▼Chemically induced mammary carcinogenesis in rats [2,4,5,12]
▼Growth of transplantable breast cancer tumor cells in nude mice [10,17]
▼Growth of transplantable prostate cancer tumor cells in nude mice [7]
▼Stages of chemically induced skin tumorigenesis in mice [149,225]
▼Chemically induced colon carcinogenesis in rats [151]
▼Chemically induced forestomach carcinogenesis [226]
►Carcinogenesis in Min mice [227]
Adipogenesis
▼Chicks, mice and rats [20,26,162,166]
▼Human subjects [184,179]
►Human (woman 20-41 years age) [181]
►Weaned piglets fed high fat diet [228]
►Fatty acid and glycerol metabolism in healthy weight-stable women [187]
Zambell et al. 2001)
Atherosclerosis
▼Aortic plaque formation in hamster [186]
▼Aortic atherosclerosis in rabbit [32,157,229]
Diabetic effects
▼Onset of diabetes in Zucker diabetic fatty male rats [34]
▲Glucose tolerance and transport [230]
▼Insulin sensitivity in mice [231]
Immune functions
▲Damage protection and lymphocyte proliferation in nursery pigs [171,199]
►Young healthy women [232]
▼Eicosanoid and histamine production [31,200]
▲Onset of lupus in mouse model [233]
▲Mitochondria protection from free radicals in rat liver [234]
Bone formation
▼Eicosanoid production in rats [235]
▲Collagen synthesis in rats [203]
▲ = increased; ▼ = decreased; ► = no effect.
Ip and coworkers [2] were the first to show inhibition of cancer by the dietary
administration of CLA. Female Sprague rats were fed a basal diet supplemented with 0, 0.5,
1.0 and 1.5% synthetically prepared CLA. Rats received their experimental diet 2 weeks
before gavage treatment with the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). The
experiment continued until study termination at 24 weeks after DMBA administration.
Results showed a dose response reduction of 32 – 60% in the total number of mammary
tumors. Later Ip et al. [3] showed that feeding as little as 0.05 to 0.5% of CLA in the diet
resulted in dose responsive significant reduction in mammary tumors. The reduction
amounted to 36-58% for CLA levels from 0.1 to 0.5% of the diet. It was also demonstrated
that short-term, post initiation CLA feeding (4 or 8 weeks) was ineffective and reduction in
tumor incidences was observed only in rats that received an uninterrupted CLA-
supplemented diet for 20 weeks [4]. Researchers further demonstrated that CLA as a free
fatty acid is as effective in reducing incidences of mammary tumor as the triglyceride form
naturally found in foods. The magnitude of tumor inhibition was same irrespective of dietary
fat level or saturated vs. unsaturated fat [5].
The above studies were conducted using synthetically prepared CLA. Feeding
CLA-enriched butterfat to rats inhibited mammary tumor growth by 53%
compared with rats fed butterfat with normal levels of CLA [138]. Another
interesting observation in this study was that rats consuming the CLA-enriched
butterfat accumulated more total CLA in the mammary gland and other tissues
compared with those consuming synthetic CLA at the same dietary level of
intake (Table 4). This study suggests that naturally occurring CLA may be
metabolized and utilized differently than synthetic CLA.
Hman consumption of TVA and its subsequent conversion to CLA in tissues should also
be considered. Partially hydrogenated oils and meat and milk products contain significant
amounts of TVA [139]. The distribution and activity of the Δ9-desaturase enzyme in humans
is still debatable. Emken et al. [140] found no evidence of desaturation of TVA is plasma
lipids of humans over a 48 hour period. However, Salminen conducted a study wherein adults
consumed a diet high in either C18:0 or TVA for 5 weeks following the consumption of a high
dairy product diet for 5 weeks [141]. Intakes of CLA for the dairy diet, C18:0, and TVA were
310, 90, and 40 mg/day, respectively. At the conclusion of the study, CLA concentration in
serum lipids as a percent of total fat was 0.33, 0.17, and 0.43, respectively, suggesting that
there was significant endogenous Δ9-desaturation of dietary TVA. Turpeinen et al [142]
reported an average 19% conversion of TVA to CLA when human subjects consumed either
1.5, 3.0, or 4.5 g of TVA/day for 9 days. It is evident that the endogenous synthesis of CLA
from TVA by Δ9-desaturase in humans requires further exploration and quantification.
To determine if endogenously formed CLA from TVA is able to exert anticarcinogenic
activity, rats were fed different levels of TVA (1-3% of diet) [143]. It was found that
treatment with 2% TVA in the diet reduced the total number of premalignant lesions by 50%.
It seems, therefore, that TVA also has anticarcinogenic effects, possibly through CLA.
Table 4. Total CLA content in tissues of rats fed different sources of CLA
Source of CLA1 Total CLA content

Liver Mammary Peritoneal Plasma
fat fat
μg/mg lipid
Control butter fat 2.6 7.2 8.8 5.5
CLA enriched butter 15.7 36.5 65.9 23.3
Synthetic CLA2 10.2 28.2 33.4 12.5
1
CLA content of diets was 0.1, 0.8, and 0.8 g/100 g of dietary DM in control butter fat, CLA enriched
butter fat and synthetic CLA diets, respectively.
2
Supplied by Nu-Chek-Prep, Elysian, MN.
Reference: [138]
Feeding of CLA affects different biological and biochemical parameters in the mammary
gland, which induce changes that render the mammary gland less susceptible to cancer. The
mechanisms whereby this occurs are not known, but some theories are that CLA reduces cell
proliferation [144], alters various components of the cell cycle and induces apoptosis [145],
or alters the expression of peroxisome proliferators-activated receptor-α (PPAR-α), [146,
147]. Another factor that is proposed to be involved in CLA anticarcinogenic activity is its
metabolism as polyunsaturated fatty acids (PUFA). Feeding CLA resulted in decreased level
of arachidonic acid, which is the substrate for eicosanoid biosynthesis [148].
It has also been shown that CLA is a chemoprotective fatty acid that inhibits phorbol
ester-induced skin tumor promotion in mice [149] by reducing the formation of prostaglandin
E2 [150]. Conjugated linoleic acid protected against 2-amino-3-methylimidzo[4,5-
f]quinoline-induced colon cancer in F344 rats [151, 152] by decreasing mucosal levels of
prostaglandin E2 and arachidonic acid in a dose dependent manner. Yang et al. [153] studied
antimutagenic efficacy of CLA in colon cancer using the (2-amino-1-methyl-6
phenylimidazo[4,5-b]pyridine) carcinogen that is present in cooked protein-rich food. Rats
exposed to this carcinogen showed an 8 to 16-fold increase in cell mutation frequency in the
colon. Supplementation of CLA in the diet reduced the mutation frequency in the distal colon
by an average of 19%.
There are very few research reports studying the anticarcinogenic effects of CLA in
humans. A Finnish study reported decreased breast cancer risk in women consuming whole
milk [154]. In another Finnish study Aro et al. [9] measured dietary and blood serum CLA in
Finnish women patients (premenopausal and postmenopausal) with breast cancer from 1992
to 1995 and found that the CLA and TVA were lower in breast cancer postmenopausal
patients than control women with no cancer.
A study in an ethnically homogeneous region of France with 360 breast cancer patients
revealed that the CLA content was higher in breast adipose tissues from control compared
with cancer patients, suggesting higher the level of CLA in breast adipose tissue lower the
incidences of breast cancer [155].
In summary, the proposed mechanisms of anticarcinogenic activity of CLA in vivo are
through modulation of eicosanoids, increased apoptosis, reduced proliferation, and PPAR-α
activation. Other mechanisms include reduction of murine mammary tumor metastasis by
CLA that can affect later stages of disease, especially metastasis, which is crucial part in
cancer prognosis. Details on the mechanism of anticarcinogenic activity of CLA can be found
in recent reviews [156-159].
CLA and Body Energy Expenditure
The energy expenditure effects of CLA are linked to its ability to reduce adipose tissue,
alter body composition and reduce atherosclerotic plaque formation. The CLA isomer largely
responsible for these effects is t10, c12 [160, 161]. The physiological effects of CLA related
to body energy expenditure are discussed below.
Body Composition
Several studies have shown that CLA reduces body-fat accumulation and increases lean
body mass in various animal models including mice ([23, 24, 162-165], rats [166-169], and
pigs [170-176]. In these studies feeding 0.5 to 2.0 g of CLA /100 g of diet to growing animals
resulted in less body fat and more lean mass. Adipose depot weights were 43-88% lower in
mice fed the 0.5 to 1.0% CLA diet compared to control mice receiving no CLA [23, 162].
Adipose tissue reduction effects of CLA in these studies varied with adipose depot [167, 168]
and strain/variety of the animal used [166].
Some researchers have related reduced adipose tissue weight in mice supplemented with
CLA to increased energy expenditure and fat oxidation ([23, 177, 178]. Mice fed CLA had
reduced lipase activity, enhanced lipolysis and increased muscle carnitine
palmitoyltransferase activity [162, 163, 167], suggesting that CLA reduces adipose tissue by
affecting some of the key enzymes involved in fat mobilization and storage. It has been
suggested that the anabolic effect of the c9, t11 isomer of CLA precedes the reduction in fat
by the t10, c12 isomer [160, 163]. Therefore, supplementing diets with both isomers would
be more beneficial to health compared to a single isomer.
There are few studies examining the effects of CLA on body composition in humans.
Healthy Norwegian men and women (n=20) in a double-blind study received either control (5
men and 5 women) placebo capsules containing olive oil or capsules containing a
synthetically prepared mixture of CLA (approximately equal proportions of t10, c12 and c9,
t11 isomers) of 1.8 g/day for 12 weeks. Irrespective of gender, the group taking CLA had 4%
reduction in body fat compared to control [179]. In another study by Blankson et al. [180]
overweight subjects (n = 47) received either placebo (olive oil) or CLA at 1.7, 3.4, 5.1, 6.8 g
/day for 12 weeks. Body fat decreased significantly only in subjects taking 3.4 or 6.8 g of
CLA/day. These researchers concluded that a dose of 3.4 g of CLA/day was sufficient to
reduce body fat in overweight adult people.
In a short term (4-week) study conducted in the United States, healthy women (n = 17)
received a placebo (sunflower oil) or 3.9 g of CLA/day. In the CLA fed group the fat mass
was reduced by an average 0.2 kg (range -3.2 to +0.9 kg), with no change in body weight,
whole body fat oxidation rate, lipolytic rate or fatty acid release from adipose tissue during
exercise or rest periods [181, 182]. Feeding 2.7 g of CLA/day to obese human subjects (n =
80) for 6 months was ineffective in reducing body fat and weight [183]. In Sweden, men and
women (n = 50) in a double blind study took 4.2 g CLA/day and 0.2 kg and 0.7 kg losses
were observed in body fat mass in placebo and CLA groups, respectively [184].
Despite pronounced effects in animal models, the observed changes in body weight and
fat mass due to CLA supplementation have been small and variable in human studies. The
lack of response in human trials may be due to the short term nature of the studies, less
control over the diet and lack of sensitive methods to detect small differences in body weight
and fat mass.
Atherosclerosis
Considerably fewer studies have been published addressing the role of CLA in
atherosclerosis compared to its other roles. However, the information available suggests that
CLA reduces atherosclerotic plaque formation in rabbits and hamsters. Feeding 0.5 g of
synthetically prepared CLA/day to rabbits on a hypercholesterolemic diet for 12 weeks
reduced blood serum triglycerides and low density lipoprotein cholesterol levels, and resulted
in less atherosclerotic plaque formation compared with control rabbits receiving no CLA
[32]. Feeding CLA to hypercholesterolemic hamsters also reduced early aortic atherosclerotic
plaque formation [185, 186]. However, in contrast to its protective role, CLA induced the
formation of aortic fatty streaks in C557BI/6 mice fed an atherogenic diet [187]. While data
in the above studies are conflicting, the majority of studies suggest that CLA can improve
plasma lipoprotein metabolism and prevent atherosclerosis in animal models. The CLA
isomer responsible for the effects on lipoprotein metabolism is t10, c12 [188].
There is a paucity of information on the effects of CLA on lipoprotein metabolism in
humans. In a study by Noone et al. [189] human subjects were given 3 g/d of a blend of c9,
t11 and t10, c12 CLA isomers (50:50 or 80:20) for 8 weeks. The 50:50 isomeric blend of
CLA reduced plasma triacylglycerol concentration (-20%). Plasma triacylglycerol
concentration has been identified as an independent risk factor for future chronic heart
disease. The increased lipoprotein metabolism may be the mechanism for anti-atherogenic
effects of CLA in humans, as has been shown in animal models.
CLA and Diabetes
Impaired glucose tolerance and obesity are some of the risk factors for the development
of type 2 diabetes. The t10, c12 CLA reduces body fat in animal models and it is this isomer
that is implicated as an antidiabetic. It has been shown that CLA was equally effective as
thiazolidinediones (a class of oral insulin sensitizing agents that improve glucose utilization
without stimulating insulin release) in reducing fasting glucose in Zucker diabetic rats [190].
In a double blind study with human diabetics blood glucose and plasma leptin levels were
decreased in CLA supplemented patients [191].
In type 2 diabetes feeding Zucker diabetic rats a synthetically prepared mix of CLA
isomers reduced plasma insulin, triglycerides and leptin [34, 190]. In another study a
synthetically prepared mix of CLA isomers improved impaired glucose tolerance, lowered
adipose mass and enhanced glucose uptake into the muscle of type 2 diabetic rats [192].
Subsequently, Henrickson et al. [193] demonstrated that the improved glucose tolerance and
insulin-stimulated glucose transport in the skeletal muscle of obese Zucker diabetic rats was
due to the t10, c12 isomer with no effect due to the c9, t11 isomer of CLA. The, t10, c12
isomer of CLA promoted insulin resistance, increased serum glucose and insulin
concentrations in ob/ob mice [194], and decreased insulin-stimulated glucose uptake in
differentiating human preadipocytes [195]. It has been suggested that CLA delays the onset
of diabetes in the Zucker diabetic rat model.
The role of CLA in regulating body composition and obesity linked type 2 diabetes is not
clearly understood. However, a significant correlation between body weight change and
fasting glucose suggests that the mechanism by which of CLA reduces fasting blood glucose
is related to its effect on lipogenesis. More long term studies are needed to understand the
mechanisms.
CLA and Immunity
The immune system is central to defense against diseases. Conjugated linoleic acid
affects parameters related to immunity that may revolve around production of eicosanoids
and immunoglobulins that are essential for immune functions. Eicosanoids are produced in
the body from arachidonic acid by various types of immune cells to regulate cytokine
synthesis and inflammation. Additionally, eicosanoids and cytokines affect a range of
biological functions. Conjugated linoleic acid is a potent regulator of eicosanoid mediator
release during immune response [196].
The physiological role of CLA in normal and immune-stimulated animals was studied. It
was demonstrated that CLA protects against Escherichia coli-induced body weight loss in
chicks and mice and reduces histamine-induced prostaglandin-E2 production in Guinea pig
trachea [197, 198]. Recently, Cook et al. [199] showed that CLA not only enhances immune
response, but also protects tissues from collateral damage by preventing weight loss,
increasing feed intake, and prolonging life during the inflammatory process involving
autoimmunity in mice and pigs. Rats fed a 1% CLA diet showed decreased levels of
leukotriene-B4 and leukotriene-C4 in spleen and lungs [200]. In contrast, feeding 3.9 g of
CLA/day to humans for 93 days resulted in no apparent alterations in eicosanoids
(prostaglandin E2, leukotriene B-4) or cytokines [201]. Existing literature suggests that
immune response of supplemental CLA may be through altered eicosanoid production,
however, physiological effects of altered eicosanoid production are not clearly understood.
CLA and Bone Formation
Dietary fat (CLA and omega-3 fatty acids) may influence bone formation and resorption
by altering prostaglandin (PG) biosynthesis in bones [202, 203]. For example, PGE2
increased bone mass in rats [204], however, at high concentration it decreased bone growth
[205]. Thus, PGE2 effects on bone formation may be dose related; that is stimulatory at low
concentration and inhibitory at high concentration [203]. In a study by Watkins et al. [206]
chicks fed butterfat showed reduced bone PGE2, elevated bone IGF-1 and increased bone
formation rates of nearly 60% compared to those animals given diets containing corn oil. The
authors concluded that bone effects were due to the presence of CLA and n-3 fatty acids in
butter compared to higher n-3 fatty acids in corn oil. However, these beneficial effects of
CLA on bone modeling have not been shown in any other studies. Dietary beef fat and a
CLA supplement were able to maintain synthetic activity of osteoblastic cells and CLA was
even able to rescue the reduced bone formation rate in rats given a diet high in omega-6 fatty
acids [207].
Studies show an increase in percent of ash when CLA is fed to chicks [208]. This effect
is presumed to be due to protection conferred by CLA against bone loss [20]. An increase in
cytokines increases bone loss, and CLA appears to counter the effect of cytokines [206].
Cytokines are hormone-like mediators of immunity and inflammation that are produced by
macrophages and other immune cells when they are stimulated. Osteoporosis is a major
health problem in the U.S., and this effect of CLA could contribute to prevent osteoporosis.
With limited information on effects of CLA on bone formation and resportion it is difficult to
make firm conclusions. However, preliminary information from animal models is
encouraging.
To summarize overall health benefits of CLA, our understanding of health effects of
CLA is primarily based on in vitro and animal studies along with a few studies in humans.
Preliminary information shows that CLA may have a number of positive health benefits.
However, the mechanisms of CLA action are not well understood. In addition to the need for
understanding the mechanisms of CLA action, it is also imperative to determine if these
beneficial physiological effects can be applied to human health. More long term human
studies are needed because it is difficult to extrapolate information from animal studies to
humans.
CLA Intake of Humans from Milk and Meat
The question needs to be addressed of how much CLA humans are currently consuming
and how much should be consumed in order to reap the health benefits and anticancer effects
that have been observed from adding proper levels of CLA to the diet. Figure 3, panel A,
shows the amounts of CLA provided in one normal serving of milk, cheese, lean beef, and
poultry. Figure 3, panel B, shows the amounts of CLA provided by one serving of milk,
cheese, beef, and poultry from CLA-enriched foods. One serving of milk is considered to be
227 ml and one serving of cheese is assumed to be 30 g. A serving of lean beef and poultry is
assumed to be 100 g each. Fat intake (grams) from one serving of each food was calculated
by multiplying the serving size by the fat content of the particular food. Conjugated linoleic
acid intake (milligrams) from each particular food was then calculated by multiplying the
CLA content by the calculated fat intake. Fat contents used were 3.5 [79], 32.0 [133], 6.4
[209], and 6.7% [210] for whole milk, cheese, beef, and poultry, respectively. The fat content
of high-CLA beef was assumed to be 3.0% because pasture-grassfed beef contains
considerably lower fat content than grain-fed beef. Average CLA values for low-CLA milk
(0.53% of FA) and cheese (0.50% of FA) were taken from Table 1 and values for low-CLA
beef (0.37% of FA) and poultry (0.19% of FA) were taken from Table 2. Conjugated linoleic
acid contents used for high-CLA products were 2.21, 2.21, 1.35, and 0.19% of total FA for
milk, cheese, beef, and poultry, respectively. The value used for poultry was the same in both
panels because there is very little potential for CLA content to be increased in poultry
products.
Figure 3. Daily conjugated linoleic acid (CLA) intake by humans from one serving of low-CLA whole
milk, cheese, beef, and poultry (A); and daily CLA intake from one serving of high-CLA whole milk,
cheese, beef, and poultry (B).
Based on appropriate extrapolation of data from animals to humans, an adult human

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

of CLA-enriched milk and cheese.
Human consumption of TVA and its subsequent conversion to CLA in tissues should
also be considered. Partially hydrogenated oils and meat and milk products contain
significant amounts of TVA [139], and a few studies have shown that there is some
conversion of TVA to CLA in humans [142].
Conclusion
Conjugated linoleic acid is unique because it occurs naturally in foods derived from
ruminant animals. The ability to analyze and detect different isomers of CLA is improving,
and this will be useful in future research investigating the biological role of individual
isomers. Manipulating the diets of dairy and beef cattle and altering management practices on
the farm can enhance the CLA contents of milk, dairy and beef products. The CLA contents
of milk, dairy products, meat, and meat products vary widely. The amount of published
research describing physiological and health benefits of CLA has expanded in recent years.
However, little of this research has been done in humans. Conjugated linoleic acid intake by
humans has the potential to be increased to a level that has been shown to be beneficial for
health in animal models through the consumption of CLA-enriched dairy and beef products.
Acknowledgments
The authors would like to acknowledge the Utah Agricultural Experiment Station, Utah
State University for partial financial and technical support. Financial support for this research
from Western Region Sustainable Agriculture, Cooperative Research Extension and
Education, USDA is also acknowledged (Grant No. SW01-034).
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Chapter X
Beneficial effects of Human Milk

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

1
Faculté de Pharmacie, Université de Picardie Jules Verne 1
rue des Louvels, 80037 Amiens Cedex 1, France
2
Faculté des Sciences Pharmaceutiques et Biologiques, Université de Lille 2 – 3
rue du Professeur Laguesse – BP83, 59006 Lille Cedex, France
Abstract
Mother’s milk remains the gold standard for the nutrition of human neonates.
Thanks to its adaptable biochemical and immunological composition, mother’s milk
allows for an optimal development of the intestinal microflora, especially by promoting
the implantation and growth of some of the so-called health beneficial bacteria:
bifidobacteria. When bifidobacteria are dominant in the intestinal flora, they are thought
to help preventing gastrointestinal disorders, repress a potentially harmful proliferation of
other intestinal bacteria and stimulate the priming of the neonate’s intestinal immune
system. This is why, among other research trends, the latest infant formulas are
attempting to reproduce this bifidogenic effect of mother’s milk through various ways
such as the addition of exogenous bifidobacteria and/or of prebiotics (specific
carbohydrate substrates promoting the growth of indigenous intestinal bifidobacteria).
We will first review the beneficial effects of mother's milk and those putatively related to
indigenous bacteria. The probiotic (feeding of live bifidobacteria) and prebiotic (feeding
of specific carbohydrates) approaches to increase intestinal bifidobacteria will also be
defined. Then, we will focus on prebiotics and on a novel approach to promote
indigenous intestinal bifidobacteria: the use of an infant formula containing products of
216 Catherine J. Mullié, Daniel Izard and Marie-Bénédicte Romond
milk fermentation by Bifidobacterium breve strain C50. These fermentation products

have previously been shown to have a bifidogenic effect on indigenous bifidobacteria,
thus acting like prebiotics. We will compare the effect of this formula on the intestinal
microflora establishment to the ones of mother’s milk and of a standard formula. We will
also deal with the issue of specifically stimulating the growth of certain species of
indigenous bifidobacteria, as some bacterial species belonging to this genus (e.g.,
Bifidobacterium adolescentis) have been shown to be linked with immunological
conditions in neonates and young children such as atopic dermatitis.
Abbreviations
AAD: Antibiotic-Associated Diarrhea;

AD: Atopic Dermatitis;
BbC50: Bifidobacterium breve C50;
BT: Bacterial Translocation;
CFU: Colony Forming Unit;
FISH: Fluorescent In Situ Hybridization;
FOS: Fructooligosaccharides;
GOS: Galactooligossacharides;
HMO: Human Milk Oligosaccharides;
IL: Interleukin;
MLN: Mesenteric Lymph Nodes;
PCR: Polymerase Chain Reaction;
TNF: Tumor Necrosis Factor;
TLR: Toll-Like Receptor;
VLBW: Very Low Birth Weight.
Taxonomic Warning
Bifidobacterium lactis is now considered a subspecies of the Bifidobacterium animalis

species (Masco et al., 2004). Therefore, trials reporting on either Bifidobacterium lactis Bb12
or Bifidobacterium animalis Bb12 use the same bacterial strain.
1. Introduction: Impact of Mother's Milk
1.1. Epidemiologic Data
Breast-feeding has long been shown to induce protection against infectious diseases
resulting in a decreased mortality/morbidity in breast-fed babies, especially for diarrheas
(WHO, 2000). The lower incidence of conditions such as diarrhea, otitis media and
Human Milk and New Prebiotic-Like Fermented Infant Formulas 217
respiratory infections are mainly reported for an exclusive breast-feeding of 3 months at least
(Turck, 2005).
The newborn immature immune system is replaced by specific and non-specific immune
effectors in human milk such as immune cells, secretory Immunoglobulins A (sIgA) or
lysozyme, for example. Human milk oligosaccharides (HMO) have also been proved to play
a major part in reducing these infectious conditions (Kunz et al., 2000), mainly through
acting as decoy receptor for pathogens and through their bifidogenic effect. Indeed, eversince
the first observation of bacteria that were to become bifidobacteria in the stools of breast-fed
infants by Tissier at the beginning of the 20th century, an infant intestinal flora dominated by
bifidobacteria is thought to help preventing intestinal infectious diseases, such as viral
gastroenteritis, but also extraintestinal infections.
The effect of breast-feeding on the onset and prevalence of chronic diseases has also
been investigated. Prevention of allergy, especially food allergy and atopic dermatitis, in at-
risk infants (infants with at least one first-degree relative presenting with allergy) has been
reported in numerous studies. Despite conflicting results, a 3-month exclusive breastfeeding
seems to reduce the risk of atopic dermatitis, especially in allergy-prone infants (Gdalevich et
al., 2001).
Breast-fed children also have a decreased risk of childhood obesity, maybe thanks to an
early regulation of energy intake at a low level and the prevention fat accumulation in tissues
(Turck, 2005). Several epidemiologic studies report on this protection such as the works of
Bergmann et al. (2003) or Grummer and Mei (2004).
Cardiovascular disease prevention has also been the target of numerous observational
studies searching for a protective effect of breastfeeding. A slight reduction in systolic blood
pressure as well as a small decrease of adulthood cholesterolemia plead for such a protection,
although results of observational studies are conflicting (Turck, 2005). This latter statement
holds true for breastfeeding and type 1 diabetes, the most common autoimmune disease in
childhood.
Despite discordant results on the prevention of some chronic disorders, breastfeeding
remains the gold standard for human infant nutrition. The only drawbacks of breastfeeding
are mainly related to infectious diseases such as the transmission of Human
Immunodeficience Virus (HIV) (4 to 22% risk) (Read, 2003) and Cytomegalovirus (CMV)
(Hamprecht et al., 2001) to newborns by infected mothers. Therefore, breastfeeding is
contraindicated in developed countries for mothers carrying HIV. For CMV, the
contraindication is limited to women giving birth to preterm babies because of the potential
seriousness of such a contamination.
1.2. On the Intestinal Microflora
1.2.1. In Term Infants

The neonatal intestinal tract is sterile at birth. It is gradually colonized by bacteria
belonging to the mother's genital tract and fecal flora in case of vaginal delivery while
environmental bacteria also play a significant part in the colonization of babies delivered by
caesarean section (Bezirtzoglou, 1997). Several studies have examined the bacterial flora of
both breast-fed and bottle-fed infants using conventional plating and/or molecular techniques.
Most of these works report a gradual colonization process of infants' gastrointestinal tract
beginning with aerobic strains belonging to enterobacteria, streptococci and staphylococci.
Maternal vaginal lactobacilli can also transiently colonize the infant but are subsequently
replaced by lactobacilli from other sources. Once these first colonizers are implanted, they
are thought to allow for a decrease in the redox potential of the intestine and then favor the
implantation of anaerobic bacteria such as bifidobacteria. The latter bacteria usually become
predominant within a week in breast-fed babies while dominance is reached later in bottle-fed
infants (Rotimi et al., 1981 ; Yoshioka et al., 1983). Additionnally, using cultivation,
formula-fed infants have been reported as displaying a more diverse and adult-like flora,
(Bullen & Tearle, 1976; Benno et al., 1984; Balmer et al., 1989; Chierici et al., 1997).
Towards the end of the 1990s, a debate on the accuracy of cultural approaches for the
analysis of the intestinal microflora arose. At the same time, infant formulas were modified to
try and improve the implantation of a gut flora similar to the one harbored by breast-fed
babies. Subsequent works confirmed bifidobacteria as predominant inhabitants of both
breast-fed and bottle-fed infants' gut. Additionally, in infants, up to 80% of intestinal
bifidobacteria identified by molecular biology techniques could also be retrieved by culture
of the same fecal samples (Saunier et al., 2003). Thus, culture-dependent techniques are still
reliable as far as the exploration of the neonates' intestinal microbiota is concerned.
The current state-of-the-art is that bifidobacteria are the major component of the infant
flora. They represent 60 to 91% of the total flora in breast-fed infants vs. 28 to 75% in
formula-fed infants between 12 and 20 days of life (Harmsen et al., 2000). In the Koala birth
cohort study of the intestinal microbiota (in The Netherlands), the carriage prevalence
(98.6%) as well as the counts in bifidobacteria were the highest at one month of age.
Escherichia coli (87.7%), Bacteroides fragilis group (81.6%), lactobacilli (32.4%) and
Clostridium difficile (25.0%) came next in terms of carriage prevalence (Penders et al., 2005;
Penders et al., 2006). At one month of age, cesarean section still affected the bifidobacterial
colonization while bottle-feeding only retained minor effect on bifidobacteria, possibly
because of the wide use of prebiotic supplemented infant formulas in The Netherlands.
However, we also demonstrated by using both cultural and molecular detection of human
species belonging to the genus Bifidobacterium, that bifidobacteria were predominant in the
gut of healthy bottle-fed neonates from 1 to 4 mo. of age (Mullié et al., 2006). An additional
environmental factor that might impact the intestinal colonization with bifidobacteria is the
presence of older siblings, as recently demonstrated by preliminary results obtained from our
latest clinical study on the prevalence of bifidobacteria held on 30 breast-fed infants aged 2 to
5 mo. (Fig.1). However, this « sibling » effect did not last over time (Fig. 1).
90,00
80,00 *
70,00
bifidobacteria (% total cultivable bateria)
60,00
50,00
0 sibling
> 1 sibling
40,00
30,00
20,00
10,00
0,00
V1 V2 V3
Figure 1. Intestinal colonisation with bifidobacteria in breast-fed infants from 2 to 5

months of age: influence of siblings
(V1: 2 mo., V2: 3 mo., V3: 5 mo.)
*: p<0.05
At the species level, Sakata et al. (2005), using cultural and terminal restriction fragment
length polymorphism techniques, did not witness any difference in bifidobacterial species
establishment between formula-fed, mixed-fed and breast-fed babies. However, along time,
Bifidobacterium adolescentis counts were shown to decrease in breast-fed babies and in
infants fed a prebiotic-supplemented formula while they were maintained in infants fed a
standard formula (Haarman & Knol, 2005). Considering that species of the genus
Bifidobacterium do not harbor the same enzymatic and antigenic potentials, it would
therefore be of interest to further document their establishment in babies.
If the carriage pevalence of bifidobacteria in bottle-fed infants nowadays tends to
resemble the one of breast-fed neonates in countries using prebiotic-supplemented formulas,
several differences in the microflora still remain. In the Koala birth cohort study, intestinal
colonization with C. difficile, E. coli, B. fragilis group and lactobacilli was less frequent and
their intestinal counts lower in breast-fed babies (Penders et al., 2005; Penders et al., 2006).
As for intestinal colonization with enterobacteria, our latest preliminary results on 22 breast-
fed babies showed a constant overall level of colonization from 2 to 5 mo. of age: from 2.54
% of the total cultivable fecal flora at mo. 2 to 3.86 % at mo. 5 (median values, difference not
statistically significant). However, at the species level, Escherichia coli carriage and
colonization level significantly increased over time:
- from 50% of carriers at mo. 2 to 82% at mo. 5 (p=0.048, Fisher’s exact test) ;
- from 0.05% of the total cultivable flora at mo. 2 to 2.52% at mo. 5 (median values,
p=0.034).
As opposed to intestinal colonization with bifidobacteria, no significant influence of siblings
on the intestinal colonization with enterobacteria was demonstrated in breast-fed infants.
Other differences in the intestinal microflora balance were reported according to the
feeding. Minor components of the fecal samples from breast-fed infants were mainly
identified as lactobacilli and streptococci while samples from formula-fed infants contained
more Bacteroides and clostridia. Altogether, enterococci, staphylococci, Bacteroides, and
Veillonella could be isolated from both groups. But lactic acid bacteria were mainly isolated
in breast-fed infants (Harmsen et al., 2000). Backing this observation, a higher carriage
frequency was reported for total lactobacilli (p<0.01) and for L. rhamnosus (p<0.05) in
breast-fed infants as compared to infants weaned at 6 mo. old (Ahrné et al., 2005).
Higher fecal Bifidobacterium counts in breast-fed infants (exclusive breastfeeding for at
least 3 mo.) were also recently reported by Rinne et al. (2005) as compared to formula-fed
infants at 6 mo. (1.8×109 bacterial cells/g feces vs. 4.4×108, p<0.0004). Higher
Lactobacillus/Enterococcus counts (2.9×108 vs. 1.8×108, p=0.01) were also described though
differentiation between enterococci and lactobacilli was not undertaken in this clinical trial.
These differences were no longer observed at 12 mo. of age.
Apart from breast-fed infants, our latest work on the intestinal flora of neonates also
included two additional groups randomly assigned at weaning (between age 2 and 3 mo.,
usually) to either a standard infant formula or Bifidobacterium breve C50 (BbC50) fermented
formula (21 and 23 neonates, respectively). When a standard formula was instated at
weaning, a significant increase in enterobacteria was noticed at month 5 (p=0.019) while
bifidobacteria significantly decreased (p=0.006). Weaning and a standard infant formula thus
modified the microbial balance but not straight away as counts in bifidobacteria and
enterobacteria were not significantly different at month 3, as compared to month 2.
Hence, differences in the dominant intestinal microflora isolated from breast-fed and
bottle-fed infants tend to decrease with age, in terms of genera. However, further
documentation is needed for both species ditribution in these dominant genera and the sub-
dominant intestinal microbiota, as these species might be of importance in the
regulation/maturation of the neonate's immune system or as a potential source of harmful
bacteria (as for C. difficile and Clostridium perfringens, for example).
1.2.2. In Preterm and/or very Low Birth Weight (VLBW) Infants

Studies on the implantation and development of preterm babies are scarcer than those
held on term infants. Nevertheless, a colonization pattern distinct of that of term infants has
been reported in several studies. Reports usually put forward a delayed colonization of the
pre-term intestine by bifidobacteria (Westerbeek et al., 2006). Intestinal colonization with
detectable levels of cultivable bifidobacteria usually takes up to one month. Coagulase
negative staphylococci, enterococci and enterobacteria such as Klebsiella sp. and
Enterobacter sp. are observed as the main components of the pre-term intestinal microflora
(Gewolb et al., 1999; Schwierz et al., 2003). The use of antibiotics, the lack of full breast-
feeding, the need for artificial respiration and other invasive interventions might account for
such a situation. Indeed, Gewolb et al. (1999) report that pre-term infants fed human milk
display a significantly more diverse intesinal flora at 20 and 30 days old than do formula-fed
infants. Pre-term babies would therefore even more benefit from nutritional attempts to speed
up and increase their intestinal colonization with bifidobacteria.
1.3. On the Maturation of the Intestinal Immune System
The germ-free status of the intrauterine environment favors humoral immunity through
T-helper 2 (Th2) type cytokine response over cellular immunity driven by T-helper 1 (Th1)
response (Cumming and Thompson, 1997). During early infancy, the immature gut
associated lymphoid tissue (GALT), is gradually confronted to increasing amounts of dietary
and microbial antigens. These early contacts seem to be decisive for the later development of
an adequate and appropriate immune response. The ability to discriminate between harmless
and harmful antigens, termed tolerance, is believed to occur primarily in the gut and is
facilitated by specialized B and T cells, production of sIgA, recognition of the commensal
microflora by Toll-like receptors (TLR) and skewed Th2 response. Hence, failure to regulate
tolerance and active immune responses could contribute to food-related allergy,
autoimmunity, and inflammatory bowel disorders.
At birth, the intestinal immune system is immature with very few IgA-producing B cells
present in peripheral blood of newborns. Consequently, the presence of IgA is low and IgM is
the major initial antibody type. After 1–2 months, IgA becomes the dominant antibody in the
intestine and reaches adult levels at the end of the first year of life (Ouwehand et al., 2002).
However, exclusive breast-feeding for 3 months had no significant effect on the total number
of IgM-, IgA-secreting cells at 3 months of age whereas the number of IgG-secreting cells
was increased (Rinne et al., 2005). Nevertheless, breast milk possesses other putative ways to
optimize maturation of the immune system. One example of such mechanims is the
presentation of already processed dietary antigens by the mother's gut mucosa (Strobel,
2001). Human milk also contains a wide variety of specific factors such as cytokines like
transforming growth factor-β (TGF-β, the expression of which directly correlates with the
mucosal IgA antibody response in infants) and the innate soluble microbe receptor sCD14
(Oddy et al., 1999 ; Rinne et al., 2005). Cytokines, especially interleukins (IL), are poorly
expressed by the neonate. Breast-milk has been shown to contain both T helper1 (Th1), T
helper 2 (Th2) and T helper 3 (Th3) cytokines (Hawkes et al., 2002). Th1 pathway leads to
cellular immunity and involves cytokines such as IL-2, IL-12, interferon-γ (IFN-γ) and tumor
necrosis factor-α (TNF-α). IL-4, IL-5, IL-6 and IL-13 are implied in the Th2 pathway,
generating humoral immunity through antibody secretion with IgE responses, in particular.
Also termed Tregulatory1 (Treg1) pathway, Th3 response leads to oral tolerance and anti-
inflammatory regulation, involving IL-10 and TGF-β. This considerable intake of cytokines,
likely produced in the mammary gland, has the potential to positively influence the
development and proliferation of many immunoactive cell types belonging to the infant
immune system. Among these cytokines, high levels of IL-2, a potent T lymphocyte
regulator, can also be found (Bryan et al., 2006). In this study, milk IL-2 levels aditionnally
correlated with those detected in paired serum samples. Moreover, immune cells harvested
from milk samples were shown to be able to produce IL-2, especially when stimulated by
concanavaline A (Bryan et al., 2006). Therefore, through providing additional IL-2 to the
neonate, breast-milk could potentially aid the maturation of the immune system.
Immune cells contained in human milk are composed of macrophages (55-60%),
neutrophils (30-40%) and lymphocytes (5-10%). These latter are mainly activated T
lymphocytes that compensate for the immature function of neonatal T cells and promote their
maturation, with an influence not limited to the intestinal immune system thanks to their
potential passage through the neonate's intestinal mucosa (Field, 2005).
Modulation of the immune system by breast-milk can also be achieved through its
content in various growth factors and hormones such as cortisol, epidermal growth factor,
insulin-like growth factor and many others. Nucleotides and long chain polyinsaturated fatty
acids (conjugated linoleic acid, arachidonic acid and docohexaenoic acid, mainly) are thought
to be implicated in the immune system development and maturation (Field, 2005).
Of more interest for this chapter, indirect stimulation of the GALT is also occurring via
the specific intestinal microbiota developped by breast-fed babies, especially through HMO
promoting the growth of bifidobacteria in infants' intestine (Beerens et al., 1980). Several
mechanisms have been put forward to explain the stimulative effect of bacteria, especially
bifidobacteria, on the immune system (MacPherson et al., 2004; Forchielli and Walker, 2005;
Kelly and Conway, 2005):
i. a stimulation of secretory IgA (sIgA) production and secretion;

ii. an induction of T cell activation;
iii. the regulation of Toll-like receptor (TLR) expression (TLR2 and TLR4, more
specifically);
iv. regulation of cytokine production;
v. the production of a balanced T helper response (Th1=Th2=Th3) or the prevention of
an imbalanced T helper response (Th1>Th2 or Th1<Th2)
Human milk therefore achieves two important goals (i) protecting the neonate from
infections through its many defensive constituents and (ii) optimizing the maturation of the
(gut) immune system through various direct or indirect mechanisms involving both molecules
and/or cells.
Infant formulas do not contain immune cells or hormonal components but many of the
other constituents of human milk identified as immunostimulative have been added to
standard formulas to reproduce breast-fed infants GALT maturation. We will thereafter focus
on compounds added in attempt to manipulate the intestinal microbiota and induce a
beneficial increase in bifidobacteria, thus eliciting the neonatal GALT.
1.4. Limits to Human Milk Bifidogenic Effect?
Though the mode of nutrition is of utmost importance, it is not the only factor
influencing the implantation of the intestinal microbiota. Various factors such as gestational
age, mode of delivery, local environment and antibiotic treatments have also been
demonstrated as playing an important part in the development of the neonatal intestinal flora
(Fanaro et al., 2003).
Infant populations from various geographical origins have been shown to display
heterogenous intestinal colonization with bifidobacteria, although breast-fed over several
months (Sepp et al., 1997).
Cesarean section has long been shown has favoring the implantation of environmental
strains in the infant's gut (Bezirtzoglou & Romond, 1990 ; Bezirtzoglou, 1997). In a recent
work, in comparison with vaginal delivery at home, cesarean section resulted in lower
colonization rates and counts of bifidobacteria and B. fragilis-group species, whereas
prevalence and counts of C. difficile and counts of E. coli were higher (Penders et al., 2006).
Oral use of antibiotics (mainly amoxicillin) by the infant during the first month of life
also resulted in decreased numbers of bifidobacteria and B. fragilis-group species (Penders et
al., 2006).
2. Putative Beneficial Effects

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

bifidobacteria such as barrier effect, regulation of bacterial translocation (BT) or adjuvant
effect can be better understood using results obtained on gnotobiotic animal models (Mullié
et al., 2005 ; Romond et al., 2007). We will mainly focus here on clinical evidence.
2.1.1. Viral Acute Gastroenteritis

Gastroenteritis of viral origin, especially rotavirus infection, is the main cause of acute
diarrheas in infants and children all over the world. In developing countries, it is probably
one of the main factors of infant mortality. Firstly, osmotic diarrhea occurs followed by a
second phase corresponding to active viral replication. Oral rehydration solutions prevent
dehydration but do not shorten the duration of diarrhea.
Breast-feeding has long been associated with a decreased incidence of gastrointestinal
infections (Bullen & Willis, 1971; Howie et al., 1990), possibly because of its bifidogenic
effect creating an acidic intestinal environment inhospitable to infectious organisms (Bullen
& Willis, 1971; Duffy et al., 1986).
The protective effect of probiotic bifidobacteria has been shown by Saavedra et al.
(1994) in a double-blind placebo-controlled trial. Feeding of Bifidobacterium bifidum
associated with Streptococcus thermophilus in a standard milk formula significantly reduced
the incidence of acute diarrhea and the rotavirus shedding in hospitalised infants. Diarrhea
occurred in 7% of the infants receiving the probiotic and in 31% of the control subjects
(p=0.035) and shedding rotavirus occurred in 10% of children versus 39% respectively
(p=0.025). In another work, a formula supplemented with Bifidobacterium lactis Bb12 (106
cfu/g of powder) given to 3-8 mo. old infants did not significantly reduce the incidence of
diarrhea compared to a control formula (28.3 vs. 38.7%, respectively) but the mean number
of days with diarrhea per infant was reduced in the supplemented group (1.15 ± 2.5 vs. 2.3 ±
4.5 days in the control group, p=0.0002) (Chouraqui et al., 2004).
Thus, bifidobacteria may provide some of the protective or alleviating effects of
breastfeeding against acute gastroenteritis.
2.1.2. Post-Antibiotic Diarrhea

Antibiotic administration is frequent in infants, especially in preterms. Antibiotherapy is
known to induce a disruption of the intestinal microflora balance in both infants and adults
(Sullivan et al., 2001; Fanaro et al., 2003).
Lately, a probiotic formula containing 107 colony forming unit (CFU) Bifidobacterium
lactis and 106 CFU Streptococcus thermophilus was shown to reduce the frequency of post-
antibiotic diarrhea in infants (Correa et al., 2005). Infants were aged 6 to 36 months and
received either this or an unsupplemented commercial infant from the onset of antibiotherapy
and for a duration of 15 days. Infants were clinically followed up for 15 additional days. A
reduction in antibiotic-associated diarrhea (AAD) was witnessed in the probiotic group with a
16% incidence as compared with a 31% incidence in the non-supplemented group.
2.1.3. Other Gastrointestinal Disorders

An infant formula supplemented with either a dose of 1.106 or 1.107 CFU/g
Bifidobacterium lactis and Streptococcus thermophilus was recently assessed for tolerance in
infants aged 3 to 24 months at enrollment (Saavedra et al., 2004). In addition to normal
growth rates, this clinical trial reported a lower frequency of colic or irritability (p≈0.001)
and a lower frequency of antibiotic use (p≈0.001) in infants receiving the supplemented
formula as compared with infants fed the unsupplemented formula given as control.
Infant formulas supplemented with a 1.107 CFU/g dose of either Bifidobacterium lactis
Bb12 or Lactobacillus reuteri ATCC 55730 were tested for 12 weeks on 1 to 4 mo. old
healthy infants (Weizman et al., 2005). Control babies had significantly more febrile episodes
compared with those fed B. lactis or L. reuteri. Controls also had more diarrhea episodes and
episodes of longer duration.
Bifidobacterium longum BL999 was also lately assessed at the dose of 2.107 CFU/g
along with 4 g of a prebiotic mixture (GOS/FOS ratio: 9/1) (Puccio et al., 2007). Infants were
aged less than 14 days at the study onset and followed until their 112th day. A higher stool
frequency was recorded in the experimental group as compared to control (2.2 ± 0.7 vs. 1.8 ±
0.9, respectively, p=0.018) along with a lower risk of constipation (p=0.03).
2.2. Immunomodulation and Prevention of Allergic Conditions

(Food Allergy, Atopic Dermatitis, Celiac Disease)
Human beings are exposed to numerous food antigens. The intestinal mucosa is efficient
in assimilating antigens encountered along the enteric route. But high-level antigen exposure
during the first few months of life may predispose individuals to allergic sensitisation. The
immature gut barrier may lead to aberrant antigen transfer and immune responses and so
explain vulnerability to oral tolerance breakdown at an early age. Enteric antigens are
frequently derived from food and allergic reactions to foods are common.
Gastrointestinal microflora promotes potentially anti-allergenic processes (i) T-helper 1
type immunity, (ii) generation of TGFβ which has an essential role in suppressing Th2-
induced allergic inflammation and inducing oral tolerance, and (iii) IgA production.
Kalliomäki et al. (2001) showed the essential role of endogenous microflora by
demonstrating that a reduced ratio of bifidobacteria to clostridia in early gut microflora
precedes the development of atopy and atopic disease. He et al. (2001) investigated the
differences between Bifidobacterium strains in the faeces of allergic and healthy infants.
Healthy infants had typical infant bifidobacterial flora (B. bifidum, B. breve and B. infantis).
Allergic infants were mainly colonised with B. adolescentis. Bifidobacteria from allergic
patients were also found to adhere less to the human mucus than strains from the healthy
infants. However, these results remain controversial. Indeed, a recent study by Adlerberth et
al. (2007), held on infants born in Italy, Great Britain and Sweden, showed no significant
association between the composition of the intestinal microflora and the development of food
allergy and atopic dermatitis.
Several recent studies report on differences in intestinal gut microbiota of infants with
and without eczema (Murray et al., 2005; Mah et al., 2006; Adlerberth et al., 2007). While
Adleberth et al. (2007) did not witness any difference in the fecal microbiota of infants with
or without eczema; Murray et al. (2005) showed that fecal counts in bifidobacteria were
lower in infants suffering from eczema. Both Bifidobacterium and Clostridium counts were
also found to be lower in toddlers with eczema as compared to controls (p=0.003 and
p=0.012, respectively) while lactic acid bacteria and enterococci were more numerous in
toddlers suffering from eczema (Mah et al., 2006).
However, not all Bifidobacterium species seem to be beneficial from an

immunomodulatory point of view. Just like Bifidobacterium adolescentis was found more
frequently in the microflora of allergic infants, the fecal carriage of Bifidobacterium
pseudocatenulatum in 3 to 6 months-old infants has lately been associated with exclusive
formula-feeding and an increased risk of developing atopic eczema (carriage prevalence :
26% vs. 4% in infants with and without eczema, respectively ; p=0.04) (Gore et al., 2007). In
the same study, the carriage of B. bifidum was associated with breast-feeding (p=0.01).
Additionnaly, recent data reported a higher prevalence of B. catenulatum in biopsies obtained
from controls than in biopsies from patients suffering from either active or non-active celiac
disease. B. dentium prevalence was higher in feces of non-active celiac disease patients than
in controls. On another hand, the numbers of total bifidobacteria and B. longum species were
lower in both patients with active and non-active celiac disease, as compared to controls
(Collado et al., 2008). These results point out the importance of not only trying to speed up
and increase the implantation of bifidobacteria in the intestine of bottle-fed neonates but also
of favoring the so-called “infant” Bifidobacterium species such as B. bifidum, B. breve and
B.longum-B.infantis. These species appear to be linked with a health-positive maturation and
regulation while species more frequently found in adults (B. adolescentis, B. catenulatum-B.
pseudocatenulatum, B. dentium) tend to be reported as linked with deleterious health
conditions. Hence, studies focusing on increasing bifidobacteria in infants should not only
focus on the overall bifidobacterial carriage prevalence and counts but also on identifying the
species promoted, as experienced in one of our previous work (Mullié et al., 2004).
Although human milk complex and dynamic composition cannot be mimicked by
industrial products, manufacturers of infant formulas are attempting to reproduce its effects
on the intestinal microbiota and immune system through adding components such as pro- and
prebiotics to their products.
3. Probiotics to Reproduce Human Milk Effects
Probiotics were originally defined as ‘live microbial food supplements which beneficially
affect the host animal by improving its intestinal microbial balance’ (Fuller, 1989).
3.1. On the Intestinal Microbiota Balance
3.1.1. Pre-Term Infants

Intestinal colonization of VLBW infants by Bifidobacterium breve YIT 4010 was
observed following a daily oral inake of 0.5×109 cfu for 28 days by Kitajima et al. (1997).
Colonization occurred within two weeks and lasted well after the oral administration was
discontinued as most infants still exhibited high detectable levels of B. breve YIT 4010 at the
end of the 8-week follow-up. Colonized infants also had a significantly higher weight gain
between 4 and 8 weeks of life.
Mohan et al. (2006) showed that the consumption of a Bifidobacterium lactis Bb12
supplemented infant formula (2.109 cells/g powder) for 21 days induced a rise in
bifidobacterial counts in preterm infants (p=0.001). This rise was witnessed through culture-
dependent and -independent techniques. A simultaneous decrease in Enterobacteriaceae
viable counts was also reported (p=0.015).
3.1.2. Term Infants

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

Prevention of Allergic Diseases
Majamaa and Isolauri (1997) suggest that probiotic bacteria such as bifidobacteria may
promote endogenous barrier mechanisms in the patients with atopic dermatitis and food
allergy. The exact mechanisms by which probiotics may affect atopic disease remain
speculative. However, there is increasing evidence that specific input from the faecal flora to
the innate immune system is essential for the establishment and maintenance of oral
tolerance. Isolauri et al. (1993) showed that probiotic use in primary prevention of atopic
disease was based on its ability to reverse increased intestinal permeability. Adhesion of
probiotics to the intestinal mucosa is also considered important to modulate the immune
system (Schiffrin et al., 1997). Hattori et al. (2003) administered lyophilised bifidobacteria
(B. breve M-16V) to children with atopic dermatitis who had a Bifidobacterium-deficient
microflora. Changes in the fecal microflora were observed after one month (increase in the
proportions of bifidobacteria and decrease in aerobic bacteria within the total flora) as well as
a significant improvement in allergic symptoms (cutaneous symptom score and total allergic
score). However, no correlation could be demonstrated between changes in fecal microflora
and improvements in allergic symptoms.
Oral introduction of probiotics could also help in the treatment of food allergies by
alleviating intestinal inflammation as intestinal microorganisms have been suggested to
down-regulate allergic inflammation by counter-balancing T-helper cell type 2 responses

(Kirjavainen et al., 1999).
Recently, mucosal immunologic maturation was assessed in term infants at 3, 7 and 12
months in artificially fed infants before 2 mo. of age (Rautava et al., 2006). They received an
infant formula supplemented with 1.1010 cfu L. rhamnosus GG and 1.1010 Bifidobacterium
lactis Bb12 daily until 12 mo. of age. Determination of the number of IgA secreting cells
(total IgA and cow's milk-specific IgA : casein and β-lactoglobulin), seric TGF-β and sCD14
levels. No significant difference between total IgA-secreting cell numbers or TGF-β levels.
Higher number of cow's milk specific IgA secreting cells in the probiotic group at 7 mo. of
age (p=0.043) and higher levels of seric sCD14 (p=0.046) at 12 mo. of age.
Lately, an Australian paper reporting on a 6 month-feeding of Lactobacillus acidophilus
to allergy-prone infants did not reduce the risk of atopic dermatitis but moreover tended to
increase the risk of allergen sensitization in such a predisposed population (Taylor et al.,
2007).
3.3. Safety Considerations and Conclusions
The latter statement, though made for a probiotic Lactobacillus strain, along with
consideration on beneficial and deleterious Bifidobacterium strain, shows a careful clinical
evaluation of each probiotic strain and its effects is needed before being widely administered
to infants through a commercial product.
Apart from this, most clinical studies aim at evaluating the tolerance and safety of
probiotic supplemented formula with mainly growth criteria. Infectious adverse effects of live
bifidobacteria such as bacteriemia or meningitis have however also been reported in high-risk
populations (Sussman et al., 1986 ; Hata at al., 1988 ; Gasser, 1994 ; Nakazawa et al., 1996)
but prospective controlled studies on the effects of feeding live active bacteria in infants for
any extended period of time are scarce. Saavedra et al. (2004) reported on such a study held
on 3 mo. old infants at entry and fed Bifidobacterium lactis Bb12 at a dose of either 106 or
107 cfu/g formula for as long as infants had a minimum daily intake of 240 mL of the
supplemented formula (resulting in a mean participation length of about 7 months).
Supplemented infants showed similar growth rates as controls and no peculiar adverse effects
in supplemented groups, whatever the probiotic dose. Weizman and Alsheikh (2006) also
recently reported on the safety and tolerance of a formula supplemented with B. lactis Bb12.
Infants were less than 4 months old at entry and received the formula for 4 weeks only and
reached similar conclusions.
In the global threat of antibiotic-resistance, transfer of resistance from a probiotic strain
to indigenous intestinal strain may raise concern. Bifidobacteria appear as relatively safe in
this regard as they display low natural and acquired resistances to antibiotics (Moubareck et
al., 2005). Nevertheless, B. lactis Bb12 was found to be resistant to vancomycin (Mohan et
al., 2006) and could therefore be a potential source of vancomycin-resistance spread.
Recently, recommandations from the EU-Prosafe project further emphasised the need for
control of antibiotic-resistance in probiotic strains (Vankerckhoven et al., 2007).

Special care must be taken when preterm babies are considered. In this peculiar
population, an increased permeability of the intestinal epithelial lining may lead to bacterial
translocation and, subsequently, to sepsis. Therefore, the administration of high doses of live
bacteria has been questioned in this population as it might trigger unexpected
infectious/inflammatory conditions.
4. Prebiotics to Reproduce Human Milk Effects
Prebiotics are defined as “non-digestible food ingredients that beneficially affect the host
by selectively stimulating the growth and/or activity of one or a limited number of bacteria in
the colon, and thus improve host health” (Gibson & Roberfroid, 1995). Most prebiotics used
as food ingredients belong to fructooligosaccharides (FOS) and galactooligosaccharides
(GOS).
Breast-milk is a complex mixture of over 100 different branched oligosaccharides,
monosaccharides and various sugar derivatives (Coppa et al., 1999). These oligosaccharides
mainly consist of a lactose core substituted with N-acetyl glucosamine, galactose, fucose and
sialic acid (Kunz & Rudolff, 1993 ; Kunz et al., 2000) and are poorly digested in the upper
gastrointestinal tract (Engfer et al., 2000). One of the major explanation for Bifidobacterium
dominance in breast-fed infants is that bifidobacteria are uniquely adapted to the use of these
human milk oligosaccharides (HMO) (Schell et al., 2002). HMO therefore could act as a
model of natural prebiotics (Edwards et al., 2002) and to adapt infant formulas,
supplementation with these undigestible carbohydrates has been attempted.
4.1. On the Intestinal Microflora Balance
Surprinsingly, there is substantially more literature on the effect of prebiotics than on the
effect of probiotics on the infant intestinal microbiota composition.

Boehm et al. (2002) assessed the effect of a formula enriched with 10g/L of a GOS/FOS
mixture (9:1 ratio) on the intestinal microbiota of preterm (gestational age < 32 weeks)
infants using cultural techniques. The GOS/FOS ratio was chosen to mimic the molecular
size distribution of HMO. This supplemented formula was compared to the same formula
without oligosaccharides (replaced by a similar amount of maltodextrines) and human milk
effects. Samples were retrieved at 7, 14 and 28 days of formula feeding. Bifidobacteria were
readily detected in the 7th day sample of all infants. Counts in bifidobacteria increased over
time in all three groups (mother's milk, unsupplemented formula and supplemented formula).
After the 28 day feeding period, counts in bifidobacteria were significantly higher in the
supplemented group as compared to the standard formula (p=0.0008) and were similar to
those witnessed in babies fed human milk. Lactobacilli also had increasing counts over time
but no significant difference was observed at any time, whatever the feeding mode.
More recently, another clinical trial assessed the sole FOS as oligosaccharide supplement
in preterms (Kapiki et al., 2007). Babies with a maximum gestational age of 36 weeks were
included and fed a formula supplemented with FOS (i.e. inulin) at 4g/L for 14 days. Stool
samples were retrieved on the first and th seventh day. Rise in counts and colonization
prevalence of bifidobacteria (p= 0.032 and p=0.030, respectively). Rise in Bacteroides counts
(p=0.029) was also witnessed along with a decrease in counts of Escherichia coli and
enterococci. (p =0.029 and p =0.025, respectively).
4.1.2. Term Infants

Moro et al. (2002) tested two supplemented formulas at 4 or 8 g/L of the same GOS/FOS
mixture (9:1 ratio) as the one studied by Boehm et al. (2002) on preterms. Higher counts in
bifidobacteria and lactobacilli were observed after a 28-day feeding period with both
supplemented formulas as compared to control (p<0.001). A dose-dependent effect was also
reported, but for bifidobacteria only, as the group receiving 8g/L oligosaccharides had
significantly more fecal bifidobacteria than the 4g/L one (p<0,01). No significant effect was
witnessed on Bacteroides, Clostridium species, E. coli, Enterobacter, Citrobacter, Proteus,
Klebsiella, and Candida.
In a recent double-blind, prospective, randomized clinical trial, Costalos et al. (2007)
report on the intestinal colonization of term infants by bifidobacteria and clostridia receiving
a formula enriched with 4g/L mixture of GOS and long chain FOS. Babies aged less than 14
days were enrolled. Stool sampling took place at entry and 6 weeks later and bifidobacteria
were enumerated by FISH (Fluorescent in situ hybridization). Counts in E. coli,
bifidobacteria and clostridia were similar with and without prebiotic supplementation at 6
weeks.
Bakker- Zierikzee et al. (2005) tested for 16 weeks a formula enriched with 6 g/L of a
GOS/FOS (9:1) blend on healthy full-term infants. These authors also used FISH
enumeration of bifidobacteria. No significant difference in bifidobacterial counts was
observed, whatever the sampling time and the feeding group.
Using quantitative real-time PCR, Haarman & Knol (2005) showed an increase of the
bifidobacterial population (54.8 ± 9.8% to 73.4 ± 4.0% of the total flora, p=0.047) in healthy
full-term infants receiving for weeks a 8.0 g/L FOS/GOS supplemented formula. A rise in
Lactobacillus counts was also detected following supplementation (0.8 ± 0.3% versus 4.4 ±
1.4%, p=0.019) (Haarman & Knol, 2006). Additionally, Bifidobacterium species distribution
in supplemented infants was more similar to that of breast-fed infants while non-
supplemented infants mainly harbored B. adolescentis and B. catenulatum group species. As
mentioned earlier in this paper, these species are considered as belonging to a more adult-like
microflora and as putatively pejorative for health (found in allergic-prone infants) (Haarman
& Knol, 2005). In a similar trial using molecular detection technique, Penders et al. (2006)
showed that infants fed exclusively with a formula supplemented with a mixture of GOS/FOS
had higher counts of bifidobacteria and lactobacilli in their stools, compared with infants fed
an unsupplemented formula. Molecular techniques are therefore backing up results using
conventional cultural techniques.
Euler et al. (2005) report on a clinical trial using only FOS as supplement at 1.5 or 3g/L
on 2 to 6 weeks-old vaginally delivered term infants. Counts in clostridia and bifidobacteria
were increased on the seventh day (p=0.0356 and p=0.045, respectively) only in infants
receiving a 1.5g/L FOS supplementation. This increase did not last as counts in both bacterial
groups were not statistically different between groups 7 days after FOS supplementation has
ceased. When compared to human milk-fed controls, infants fed FOS supplemented formula
also had higher Bacteroides and Enterococcus counts.
Acidic oligosaccharides (2 g/L) have also been used to try and modify the neonate
intestinal microflora in addition or not to a mixture of FOS/GOS at 6g/L (Fanaro et al., 2005).
Only the combination of oligosaccharides increased the fecal counts in bifidobacteria and
lactobacilli (p<0.01). No modification of the other components of the intestinal microflora
was reported. The authors reach the conclusion that acid oligosaccharides alone cannot
benefically modify the intestinal microflora, maybe because of the low dosage used to
supplement the standard formula.
As can be seen, over the last decade, there has been a florishing literature addressing the
effects of prebiotics on the intestinal microflora composition. However, general conclusions
are difficult to draw. Firstly because various oligosaccharides have been used though most
trials have been held with FOS and GOS. Secondly because variations in feeding periods (7
days to 6 weeks), doses (1.5 to 10.0 g/L) and dose-effect responses are reported. Most of the
times, these supplemented formula are proved as bifidogenic but the effect on lactobacilli is
sometimes more contrasted. While most works report on an overwise unchanged microflora,
some studies report acknowledge a similar rise in Bacteroides counts while opposite results
are obtained with enterococci (Euler et al., 2005 ; Kapiki et al., 2007).
4.2. On the Maturation of the Intestinal Immune System and

Prevention of Allergic Diseases
Results of studies reporting on these issues are scarcer. The direct effect of prebiotics on
the immune status was only found two papers assessing clinical symptoms described as
atopic dermatitis (AD) and eczema. The immune mechanisms underlying was only
investigated in a couple of studies. Roller et al. (2004) reported an up-regulation of IL-10 and
IFN γ production along with a regulation of the cecal IgA response in rats following a 4-week
ingestion of inulin enriched oligofructose. However, these results were hardly confirmed in
human adults (Roller et al., 2007).
Moro et al. (2006) studied the incidence of AD in 259 allergy-prone infants over the first
6 months of life. Term infants received either a hydrolysed protein formula supplemented
with 8g/L of a GOS/FOS mixture (9:1) or with 8 g/L maltodextrines (placebo) for 6 months.
Stool microflora was assessed in a subgroup of 98 infants. AD was diagnosed in the first 6
months of life in 9.8% of infants in the prebiotic group and in 23.1% of infants in the placebo
group (p=0.014). At 6 months, higher counts in bifidobacteria were observed in the prebiotic
subgroup of infants whose fecal microflora had been evaluated (10.28 log CFU/g vs. 8.65 log
CFU/g, p<0.0001).
Ziegler et al. (2007) tested another type of oligosaccharide blend (see below). A
statistical difference with the control group was detected for eczema with the 4g/L
supplemented group showing a higher incidence than in both the control group and the 8 g/L
supplemented group (18% vs. 7%, p=0.046 and 18% vs. 4%, p=0.008, respectively).
However, the paper does not explain how eczema was evaluated in these children.
The first paper hypothesizes on an indirect effect of prebiotics on the development and
maturation of the immune system through modifications of the intestinal microflora while the
second mostly focus on tolerance. Therefore, up to this day, no direct effect of prebiotics on
the immune system has been reported and additional studies could prove useful in unraveling
the mechanisms behind the immunomodulating effects of prebiotics.
4.3. Safety Considerations and Conclusions
As for probiotics, most studies on prebiotic supplementation of infant formulas focus on

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

Bifidobacterial Products Derived from
Milk Fermentation
In the late 1980s, protection against AAD was observed in adults consuming milk
fermented with bifidobacteria and lactic acid bacteria but not in adults fed milk fermented
with the sole lactic acid bacteria (Colombel et al., 1987). The analysis of the intestinal flora
showed that protection was associated with a repressed clostridial flora. Thereafter, strains of
bifidobacteria and the compounds they generated through milk fermentation were assessed in
animal models and humans to see how they reproduced this repressive effect on intestinal
clostridia (Romond et al., 1997 ; Mullié et al., 2002). Bifidobacterium breve C50 (BbC50)
products exhibited the best repressive effect on clostridia.
Hence, a heat-treated infant formula fermented with Bifidobacterium breve C50 (BbC50)
and Streptococcus thermophilus 065 was developed and marketed (Danone, France). In this
formula, S. thermophilus 065 is mainly playing the part of a fermentation starter and producer
of β-galactosidase. The fermentation is started in the presence of 107 bacteria, a population
reaching 109 colony-forming units/mL after a 7-hour incubation at 37°C. After milk
fermentation by the two strains, the formula is ridden of its live bacteria through heating at
80°C for 15 sec. Therefore, from an infectious point of view, it can be considered as safe to
administer, even to preterm infants. The formula is then supplemented with various nutritive
elements, homogenized at 250 bars and spray-dried at 200°C. The final nutrient composition
of this infant formula is (grams per 100 g): proteins, 12.4; lipids, 21; lactose, 46.6;
maltodextrin, 14; vitamins and minerals.
We will thereafter review the pieces of evidence backing the beneficial effects on the
intestinal flora and immune system of this formula as well as the latest clinical trials on
infants held with the commercial product.
5.1. Rationale for the Use of such Products (Mouse Models and
In Vitro Assays)
In addition to the repressive effect on clostridia, BbC50 products were shown to display
an in vivo repressive effect on Bacteroides fragilis group as well as a bifidogenic activity
associated with a decrease in fecal pH (Romond et al., 1997). A further trial held on healthy
human volunteers showed that a 7-day intake of BbC50 whey reproduced the modifications
in the intestinal microflora witnessed in mice. The effects of a 7-day oral intake of BbC50
milk fermentation products on the intestinal microflora and enzymatic activities of healthy
adults were further compared to those of BbC50 and Streptococcus thermophilus 065
commercial product (Romond et al., 1998). Significantly lower fecal counts in Bacteroides
fragilis, clostridial spores, and Clostridium perfringens were retrieved after consumption of
either milk preparation. An increase in fecal bifidobacteria was also witnessed but only in the
group receiving BbC50 alone milk fermentation products. Hence, the beneficial effects of
these wheys on the intestinal microbiota balance can be put on BbC50 milk fermentation
products and not on a synergistic effect with S. thermophilus 065 ones. An increase in fecal
bifidobacteria was also witnessed but only in the group receiving the sole BbC50 milk
fermentation products. The reduction in clostridia and Bacteroides fragilis was therefore not
supported by the overgrowth of intestinal bifidobacteria. Further analysis did not provide
evidence of an in vitro antibiotic-like effect against Bacteroides fragilis and Clostridium.
Investigations carried out on animal models to elucidate the nature of bioactive compounds
contained in BbC50 cell-free wheys indicate that lactosidase activity (Mullié et al., 2002) or
glycoproteic compounds produced through milk fermentation are the best candidates.
Other works report on the immunomodulatory properties of BbC50 milk fermentation
products in animal models. A trial reporting on the effects of a 10-week oral treatment with
B. breve C50 and S. thermophilus 065 secretion products observed an enhanced Th1 response
and intestinal barrier function in an IL-10 deficient mouse model (Ménard et al., 2005). In
this study, a higher number of CD4+ and CD8+ lymphocytes were found to produce
interferon (IFN)-γ in mesenteric lymph nodes (MLN) when mice had orally ingested the
culture medium of BbC50 and S. thermophilus 065, compared to control mice or mice
ingesting both live bacteria. Additionally, the secretion of three pro-inflammatory cytokines:
IFN-γ, Tumor Necrosis Factor (TNF)-α and IL-12 was found to be significantly higher in
mice treated with BbC50 and S. thermophilus 065 culture medium in answer to LPS
stimulation.
Moreover, the direct oral administration of the commercial product to mice does not alter
the development of oral tolerance to ovalbumine (Ménard et al., 2006). Mice were fed this
formula for 1 week before and 5 weeks after oral tolerance to ovalbumine induction.
Moreover, immunisation of mice with ovalbumine led to higher IgG titers in mice receiving
the heat-treated formula (16.45 ± 1.24 and 15.46 ± 0.79, respectively; p=0.012) while anti-
ovalbumine IgE titers did not rise, accounting for a specific stimulation of Th1 but not Th2
response by the formula (Ménard et al., 2006).
Another important data provided by the same authors indicate that BbC50 fermentation
products can cross an intestinal monolayer of intestinal cells and retain antiinflammatory
properties. Therefore, a systemic immunomodulatory effect can be expected for these
products (Ménard et al., 2004).
Lately, Hoarau et al. (2006) investigated the immunomodulatory properties of BbC50
supernatants on dendritic cells (potent antigen-presenting cells). BbC50 supernatants were
shown to increase CD83, CD86, and HLA-DR expression on dendritic cells as compared to
control or LPS stimulation. They also prolonged dendritic cell survival with high IL-10 and
low IL-12 productions, likely through an up-regulation of Bcl-xL and Phospho-Bad,
compared to LPS-stimulated cells. BbC50 supernatants also induced activation of TLR-2
transfected cells.
5.2. Effect of B. Breve C50 Milk Fermentation Products on Acute

Diarrhea
A study by Thibault et al. (2004) reported on the use of the BbC50 and S. thermophilus
065 infant formula on acute diarrhea. At enrollment, infants were aged 4 to 6 mo. and had
ceased breast-feeding for at least one month. During 5 months, infants received either a
formula fermented with BbC50 and Streptococcus thermophilus 065 or a standard formula
with the same basic nutritionnal composition. Both formulas were well accepted and
tolerated, allowing for normal growth and good diet compliance. No significant differences
were recorded in the incidence, the number or the duration of diarrhea episodes. A lower
number of dehydration cases in the fermented formula group (p=0.01) was however reported
as well as fewer oral rehydration salts prescriptions and formula switches (p=0.003 and
p=0.0001, respectively). This indicates a reduced severity of diarrhea episodes in infants fed
the fermented formula.
5.3. Effect of Bifidobacterium Breve C50 Milk Fermentation

Products on the Intestinal Flora and Immune System of Healthy
Full-Term Infants
A global evaluation of the immune response to the BbC50 formula was performed in
healthy infants (Indrio et al., 2007). T lymphocyte maturation is known to take place in the
neonate's thymus. The thymus has previously been shown to grow from birth till eight
months of age. Additionally, breastfed infants have bigger sized thymus than formula-fed
ones. In a recent work, BbC50 fermented infant formula was shown to significantly increase
thymus size in healthy term infants, compared to a standard infant formula (Indrio et al.,
2007). Thymus size was assessed through ultrasound examinations and thymus index
calculated in 90 term infants on the third day of life and on the first, second, third and fourth
months thereafter. Thirty infants were breast-fed while 30 others received the BbC50
fermented formula and 30 the control formula. Infants receiving BbC50 formula had a
significantly higher thymus index as compared to the standard formula (p<0.036) but still
remained lower than that in breast-fed infants (p<0.042). In this study, breast-fed and BbC50
fermented infant formula-fed infants also had significantly lower stool pH values than babies
fed the standard formula throughout the 4 mo. survey (p<0.001). Therefore, BbC50
fermented formula tends to favor thymus development as compared to a standard formula and
hence T lymphocyte maturation in neonates.
This immunomodulatory effect could partly be relayed by the intestinal flora. In a
double-blind, placebo controlled, randomised clinical trial, BbC50 infant formula was shown
to induce higher counts in bifidobacteria within 3 months (Mullié et al., 2004) (Table 1). In
this study, thirty (seven boys and 23 girls) full-term infants born between August 1999 and
January 2000 at the maternité Pavillon de la Sainte Famille (Clinique du Bois, Lille, France)
were enrolled within the first days after birth. As their mothers had previously decided not to
breastfeed, newborns received either the placebo or the BbC50 fermented infant formula
(FIF) from inclusion until 4 months of age. The study protocol and consent procedures were
approved by the local ethics committee (Comité Consultatif des Personnes se prêtant à la
Recherche Biomédicale de Lille). Infant formulas were supplied as powder in numbered
containers (Blédina SA, Steenvoorde, France). Their basal nutrient compositions were similar
(grams per 100 mL: 1.45 g of protein, 8.3 g of carbohydrates, and 3.5 g of fat per 100 mL).
According to the French vaccination program, they received an injection of Pentacoq®
(vaccine against diphtheria and tetanus toxoids, poliomyelitis virus, Haemophilus influenzae,
and Bordetella pertussis; Pasteur Mérieux Serums and Vaccines, Lyon, France) at 2, 3, and 4
months. Infants were followed up to the age of 5 months. Stools were collected for IgA
quantification at 3 (before the second Pentacoq® injection), 3.5, and 4 months (after the
second Pentacoq® injection). Total IgA and anti–poliomyelitis-specific IgA titers were
measured by ELISA (Mullié et al., 2004). Fecal samples to enumerate cultivable
bifidobacteria, lactobacilli, enterobacteria, clostridia, Bacteroides fragilis group and total
cultivable fecal flora were collected at 1, 2, 3, and 4 months, on swabs that allow for the
survival of anaerobic bacteria. All microbiological samples were processed within 48 hours
from collection. Appropriate dilutions of the fecal samples were seeded onto the following
culture media:
1. Horse blood agar (Columbia agar base; Oxoid, Dardilly, France) supplemented with
glucose (0.5%) and cysteine · HCl (0.03%) to enumerate eubacteria, cocci, and
clostridia;
2. Columbia agar base (Oxoid) supplemented with glucose (0.5%) and cysteine–HCl
(0.03%) for clostridial spores (using fecal dilutions heated for 10 min at 70°C);
3. Beerens (Beerens, 1990) and MRS agar for bifidobacteria and lactobacilli;
4. Bile Bacteroides Esculin (BBE) agar for Bacteroides fragilis group;
5. Eosin Methylene Blue (EMB) agar for enterobacteria;
6. Bile Esculin Azide (BEA) agar for enterococci.
Additionally, Clostridium perfringens vegetative forms were more specifically searched

for using lactose-sulfite broth incubated at 46°C for 48 hours and the most probable number
method (Beerens et al., 1982).
Plates were incubated in an anaerobic chamber (La Calhène, Vélizy, France) for 5 days
for the enumaration of Bacteroides fragilis group, bifidobacteria and clostridia (vegetative
and sporulated forms). Aerobic incubation was carried out for 48 hours at 37°C.
Each type of colony was subcultured on Rosenow broth, Gram-stained, and tested for
aerobic susceptibility and catalase production. The sum of the various bacteria recovered
gave the total cultivable flora expressed as CFU/mL. The counts in cultivable bifidobacteria,
enterobacteria and clostridia were expressed as the percentage of the total cultivable flora.
Bacteria are considered as belonging to the dominant flora when their proportion is ≥1% of
the total cultivable flora (Holdeman et al., 1976). The detection limit of the culture method
was 0.01%. Bifidobacteria were identified at the species level by a multiplex PCR technique
using species-specific primers previously described (Mullié et al., 2003). The other bacterial
groups were identified by using API systems (Biomérieux, Marcy l’Etoile, France).
IgA titers and total cultivable bifidobacterial proportions were analyzed by
nonparametric ANOVA for repeated measures (Conover’s method). Fisher exact test was
used to evaluate the difference in colonization percentages by the various bifidobacterial
species between the feeding groups. Mann-Whitney test was used to compare antipoliovirus
IgA titers between infants who harbored B. longum-infantis at 4 mo and those who did not.
Spearman rank correlation coefficient was used to correlate fecal bacterial counts with each
other and with IgA concentration. Bacterial proportions were reported as mean ± SD.
Twenty babies (9 controls and 11 BbC50) completed the study. As reported earlier
(Mullié et al., 2004), counts in bifidobacteria rose from month 1 to month 4 only in the
BbC50 group, corroborating the formerly described bifidogenic effect of this formula (Table
1).
The population of enterobacteria (mainly identified as Escherichia coli) did not vary with
the type of formula (Table 1). However, enterobacteria proportions significantly decreased
with time in both group (p<0.05 at months 3 and 4, Table 1).
Table 1. Fecal colonization of infants receiving either the control or the BbC50
fermented formula
Month 1 Month 2 Month 3 Month 4

Contro BbC50 Control BbC50 Control BbC50 Control BbC50
l
Bifidobacteriaa 28.1 ± 28.0 ± 32.4 ± 38.7 ± 34.2 ± 50.8 ± 35.1 ± 52.0 ±
17.25 25.06 17.26 30.52 24.87 25.91* 20.20 23.95*
Enterobacteriaa 10.2 ± 19.5 ± 15.2 ± 6.7 ± 3.9 ± 3.7 ± 4.2 ± 4.0 ±
11.11 22.74 16.25 8.16 4.48** 2.67** 4.98** 4.48**
C. perfringens -7.9 ± -7.4 ± -8.1 ± -8.1 ± -9.1 ± -7.4 ± -8.3 ± -6.0 ±
vegetative 0.79 0.99 0.74 1.15 0.42 0.99 1.44 0.10***
formsb
a
expressed as percentage of the total cultivable flora (mean ± SD).
b
expressed as log cfu/mL.
*
p<0.05.
**
p<0.05, compared to values at month 1.
***
Significantly different from values in the control group at month 4 (p<0.05).
Negative correlations were found between counts in bifidobacteria and enterobacteria in

both control and BbC50 infants at 4 month of age (rs=-0,543 and rs=-0,623; p<0.05,
respectively). At an early stage (1 and 2 months), the negative correlation was only observed
in the group fed BbC50 fermented formula (rs=-0.749, p<0.05 and rs=-0.718, p<0.02,
respectively). As mentioned above (§ 1. 2. 1.), a similar negative correlation was witnessed in
breast-fed infants at 2 months of age. An early and specific modification induced by human
milk as well as BbC50 fermented formula on the equilibrium between enterobacteria (usually
the first colonizers of the neonate's gut) and bifidobacteria therefore seems to take place.
A “pH effect” could be put forward to explain the inverted relationship between counts
in enterobacteria and bifidobacteria. Indeed, when the latter colonize the intestine at a high
level, the end-products of their sugar metabolism (mainly acetic, lactic and propionic acids)
generate a decrease in the intestinal pH which is less favorable to the iron intake of
enterobacteria leading to their reduced growth. A decreased pH has already been reported for
BbC50 fermented formula (Indrio et al., 2007). Nevertheless, this cannot be the only
mechanism underlying the repression of enterobacteria in the BbC50 group as the decrease in
their intestinal counts had begun before (at 2 months of age) the rise in bifidobacteria was
significant (at 3 months of age, Table 1). An alternative (but not exclusive) explanation could
be a modification of the intestinal flora in response to a change in intestinal mucins or
substrates produced by infants along with the maturation process. At an early stage, the
maturation process could be stimulated to a similar extent in BbC50 and breast-fed infants.
As bifidobacteria have a large panel of glycosidasic activities (Schell et al., 2002), they may
be more efficient in using these substrates than enterobacteria.
Table 2. Correlations between counts in enterobacteria and counts in

Bifidobacterium species
All infants Control BbC50

B. breve (1 month) rs=-0.682 NSa rs=-0.895
(p=0.0256) (p=0.00113)
B. breve (4 months) rs=-0.440 NS NS
(p=0.05)
B. bifidum (1 month) NS rs=0.659 NS
(p=0.075)
a
Not significant.
This balance might be involved in the immune regulation. BbC50 was found to
preferentially stimulate some of the Bifidobacterium species (i.e. B. breve and B. longum-
infantis)(Mullié et al., 2004). And an increased poliovirus-specific intestinal IgA response
coincided with the bifidobacterial promotion. Species-specific correlations with counts in
enterobacteria indicate that a peculiar species cannot be singled out to account for the
negative correlation witnessed between enterobacteria and bifidobacteria (Table 2). At month
1, B. breve was the sole species exhibiting a negative correlation with enterobacteria. This
relationship disappears with time, possibly as B. breve prevalence decreases and the species
is replaced by “adult species” such as B. adolescentis or B. catenulatum-pseudocatenulatum
(Mullié et al., 2006). However, at month 4, the negative correlation between B. breve and
enterobacteria counts becomes significant once again despite the decrease in B. breve
carriage prevalence. But at that time, the relation was observed independently from the type
of feeding. Even breast-fed babies show the same balance (personal data). But it seems that a
comparable microbial balance affects the immune system in various ways according to the
type of feeding. At 4 months of age, no correlation was observed between antipolovirus IgA
titers and E. coli in BbC50 whereas a negative correlation was found in the control group
(rs=-0.829, p=0.021). Therefore, a possible inhibitory effect on antipoliovirus IgA response
induced by E. coli intestinal population could be suppressed by the intake of BbC50 infant
formula.
In addition, when Clostridium perfringens vegetative forms are considered, significantly
lower counts were found in BbC50 fecal samples at month 4, as compared to control group
values at the same sampling time (Table 1). Moreover, the number of babies who were never
colonized with detectable levels of vegetative C. perfringens tended to be higher in the
BbC50 group (Control=0/9 vs. BbC50=4/11, p=0.094). Such an effect warrants further
investigations. Infants and children with short bowel syndrom display frequent septicemia
with Gram negative bacteria when they are colonized by C. perfringens. A specific nutrition
preventing the expression of C. perfringens toxin would therefore be helpful in this peculiar
population.
BbC50 fermented infant formula displays a positive influence on the full-term neonate
intestinal flora as a bifidogenic effect is witnessed along with a repression of enterobacteria
and vegetative forms of C. perfringens at month 4. Moreover, so-called infant species (i.e. B.
breve and B. longum-infantis) are favored. This point is of great interest as B. adolescentis
and B. catenulatum-pseudocatenulatum (“adult” bifidobacterial species) are more frequently
isolated from allergy-prone or atopic infants. Additionally, a stimulation of the immune can
also be acknowledged for this fermented infant formula. Further investigations should be
undertaken to elucidate the precise mechanism(s) of this immune stimulation.
In our latest work comparing the effects of a standard infant formula and BbC50
fermented infant formula instated after weaning with those of a continuous breast-feeding,
preliminary results showed that while an increase in enterobacteria and decrease in
bifidobacteria was witnessed when a standard infant formula was instated after weaning (see
paragraph 1. 2. 1. above), significantly less modifications to the intestinal balance between
enterobacteria and bifidobacteria were triggered by the BbC50 fermented infant formula.
Indeed, bacterial counts and colonization levels were closer to those registered with human
milk.
6. Conclusion
Experimental data show that infant formulas have been improved so as to generate the
implantation of an intestinal flora close to the one of breast-fed infants in terms of dominance
of the genus Bifidobacterium. Whether this global bifidogenic effect is sufficient to prevent
the development of chronic conditions such as allergy remains to be ascertained. A highly
hygienic lifestyle and other environmental factors also impact the intestinal microflora and
most probably bear some responsibility in triggering chronic diseases. The study of a
beneficial intestinal balance should therefore not be limited to bifidobacteria as a whole but
also take into account Bifidobacterium species, other components of the gut microbiota as
well as their relationships.
BbC50 milk fermentation products could represent an interesting alternative to classic
pre-/probiotics in the modulation of the intestinal flora, especially in the development of an
adapted nutrition in fragile populations such as premature babies and at-risk infants (atopy,
allergy, type 1 diabetes, cardio-vascular diseases etc.). Nevertheless, their use should be
validated in large preventive trials targeting such situations.
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prebiotics grow normally and have soft stools similar to those reported for breast-fed
infants. J. Pediatr. Gastroenterol. Nutr., 44, 359-S64.
Index
adiposity, 208, 213, 240

A
adjustment, 22
abatement, 46, 50, 57 administration, ix, x, 2, 21, 62, 67, 68, 115, 117, 122,
ABC, 10 123, 125, 127, 131, 135, 136, 138, 142, 144, 148,
absorption, x, 3, 10, 11, 13, 25, 30, 31, 35, 37, 38, 150, 161, 166, 173, 185, 188, 209, 224, 226, 229,
39, 40, 41, 42, 57, 132, 153, 154, 155, 156, 158, 234, 243
159, 160, 161, 179 adsorption, 72, 154, 161
accounting, 178, 234 adult, xi, 19, 33, 57, 68, 126, 127, 129, 145, 160,
accumulation, 182, 191, 208 173, 175, 191, 195, 218, 221, 230, 238, 239
accuracy, 218 adult population, 57
acetaldehyde, 46 adulteration, 36
acetate, 159, 161 adulthood, 135, 217
acetic acid, 44, 46, 48, 52, 53, 120, 157 adults, 57, 60, 62, 119, 123, 135, 136, 189, 224, 226,
acetone, 26, 27 231, 233
acidic, 44, 164, 173, 186, 224 aerobic, 59, 218, 227, 236
Acinetobacter, 118 aerobic bacteria, 227
activated receptors, 207 affect, 182, 190, 193, 202
activation, ix, 60, 98, 125, 133, 150, 165, 166, 168, Africa, 44
169, 190, 222, 234, 242 agar, 118, 119, 120, 236
active transport, 155 age, ix, xi, 45, 57, 115, 117, 127, 128, 130, 132, 136,
acute, 30, 158, 224, 234, 247 138, 139, 142, 143, 175, 182, 188, 205, 218, 219,
Adams, 84, 145 220, 221, 222, 225, 228, 229, 230, 235, 237, 238,
adaptation, 149, 246 244, 245
additives, 91, 104, 106, 110, 187 agent, 22, 23, 104, 118, 158, 176
adenocarcinoma, 197 agents, ix, 23, 29, 105, 115, 118, 123, 132, 192, 247
adenosine, 10 aggregates, 97
adenosine triphosphate, 10 aggregation, 58, 102
adhesion, 58, 73, 78, 85, 130, 132, 243 aging, 95, 212
adhesion strength, 78 aid, 88, 144, 221
adhesive properties, 58 AIDS, 141, 245
adipocytes, 208 air, 23, 76, 137
adipose, 132, 176, 177, 179, 186, 187, 190, 191, 192, airway hyperresponsiveness, 56
198, 201, 205, 208, 212, 213 airway inflammation, 55, 145, 173
adipose tissue, 176, 177, 179, 186, 187, 190, 191, alanine, 50
201, 205, 208, 212, 213 Albino, 59
alcohol, 4, 46, 48
250 Index
alcohols, 106 antibacterial, viii, 43, 52, 53, 57, 60, 61, 65, 68
algae, 4, 183, 203 antibiotic, 61, 117, 222, 224, 228, 234, 241
ALI, 144 antibiotics, 44, 45, 128, 141, 220, 223, 228
alkali, x, 76, 153, 154 antibody, 62, 146, 221, 244
alkaline, 21, 25, 26, 161, 176 anticancer, 30, 51, 52, 172, 176, 187, 194, 208
alkaline media, 161 anticancer activity, 172
alkylation, 3 Anti-carcinogenic, 54
allergens, 134, 145 antidiabetic, 176, 187, 192, 210
allergic asthma, 137 antigen, 119, 129, 133, 134, 140, 142, 145, 173, 198,
allergic inflammation, 225, 228 210, 225, 234, 247
allergic reaction, 225 antigen presenting cells, 133
allergic rhinitis, 142 antigen-presenting cell, 119, 234
allergic sensitisation, 225 anti-inflammatory, 221
allergy, 44, 59, 121, 139, 140, 141, 217, 221, 225, antimicrobial protein, 172
227, 228, 231, 239, 243, 244 antioxidant, 37, 59, 64
alpha, 67, 144, 149, 164, 172 Antioxidative, 59
ALT, 133, 221, 222 antitumor, 51, 52, 63
alternative, viii, 2, 26, 28, 87, 88, 89, 122, 238, 239 anti-tumor, 51
alternatives, viii, 16, 23, 87, 88, 112, 113, 114 anus, 128
alters, 33, 150, 186, 190, 206 APC, 212
AMF, 91, 92 API, 236
amino, 48, 50, 52, 62, 131, 190, 207 apolipoprotein A-I, 132
amino acid, 48, 50, 52, 62, 131 Apolipoprotein E, 37
amino acids, 48, 52, 131 apoptosis, x, 60, 65, 67, 163, 168, 169, 172, 190,
aminopeptidase, 131 197, 207, 208, 213
ammonia, 50 apoptotic, 59, 168, 197
amniotic, 116 apoptotic cells, 60
amniotic fluid, 116 apoptotic pathway, 168
amorphous, 85 application, 26, 28, 31, 33, 40, 64, 139, 145
anabolic, 191 aqueous solution, 21, 30
anaemia, 173 arachidonic acid, 190, 193, 222
anaerobe, 160, 246 Argentina, 87, 115, 119, 124
anaerobes, 127, 156 aromatic compounds, 46
anaerobic, 117, 127, 147, 218, 236 arrest, 166, 170, 172
anaerobic bacteria, 127, 218, 236 arteriosclerosis, 134
analysis of variance, 76 artery, 39, 41, 42
anatomy, 131 ascites, 51
anemia, 159 aseptic, 30
angiogenesis, 132, 150 ash, 46, 194
animal models, x, 51, 126, 135, 159, 176, 187, 191, Asia, 44, 65, 84
192, 194, 195, 196, 200, 223, 233, 234 assessment, 40, 121, 122, 148, 247
animal studies, xi, 51, 138, 175, 187, 194 assimilation, 56
animals, 4, 9, 55, 56, 57, 59, 131, 133, 136, 154, association, 176, 197
158, 165, 180, 185, 186, 187, 191, 193, 195, 196, asthma, 55, 59, 61, 63, 135, 137, 142, 149, 173, 245
198 atherogenesis, xi, 175
Animals, 136 atherosclerosis, 9, 151, 188, 192, 199, 209, 210, 213
ANOVA, 76, 236 atherosclerotic plaque, 9, 187, 190, 192
antagonistic, 52, 53 atherosclerotic vascular disease, 35
anthracene, 188 athletes, viii, 43
anti-atherogenic, 192 atmosphere, 26
Index 251
atoms, 3 barriers, 164

atopic dermatitis, xi, 117, 121, 137, 138, 140, 143, basic research, 164
150, 216, 217, 225, 227, 228, 231, 242, 243, 244, Bcl-2, 169, 172
246 Bcl-xL, 234
atopic eczema, 117, 139, 142, 226, 240, 242 BEA, 236
atopy, x, 122, 126, 142, 147, 225, 239, 243 beef, xi, 175, 176, 177, 185, 186, 187, 193, 194, 195,
ATP, 10, 37 196, 200, 204, 205, 206, 212
atrophy, 131 behavior, ix, 53, 88, 91, 94, 95, 101, 102, 105, 106,
attachment, 58 110, 111, 112
Australia, 16, 111, 112 Belgium, 20, 63, 112
Austria, 20 beneficial effect, xi, 9, 22, 29, 44, 53, 58, 64, 67,
autoimmune disease, 126, 217 135, 137, 141, 154, 165, 170, 176, 193, 215, 223,
autoimmune diseases, 126 233
autoimmunity, 121, 147, 193, 221 benefits, vii, 14, 29, 36, 41, 44, 54, 90, 127, 135,
autosomal recessive, 13 153, 164, 171, 176, 187, 194, 196, 232
availability, x, 163, 182, 184 benign, 30, 42
avoidance, 136 benign prostatic hyperplasia, 30, 42
azoxymethane, 162 benzo(a)pyrene, 212
Best Practice, 63
beverages, 17, 18, 32, 33, 35, 64
B
bile, x, 10, 12, 13, 29, 45, 58, 105, 127, 131, 153,
157, 158, 161
B cells, 133, 165, 174, 221
bile acids, x, 131, 153, 157, 158, 161
B lymphocytes, 141
binding, 11, 35, 73, 81, 132, 150, 160, 164, 172, 211
babies, 129, 142, 146, 216, 217, 218, 219, 220, 222,
binding energy, 81
225, 229, 230, 235, 237, 238, 239
bioactive compounds, 52, 164, 234
Bacillus, 52, 223
bioavailability, 23, 158
bacteria, vii, ix, x, xi, 43, 44, 45, 48, 49, 52, 53, 54,
biochemical, xi, 215
57, 58, 59, 62, 64, 65, 66, 67, 68, 115, 116, 117,
biodegradable, 105
118, 119, 121, 122, 123, 127, 128, 129, 130, 131,
biodiversity, 145
132, 133, 134, 136, 138, 139, 141, 144, 145, 147,
biological activity, 176
148, 150, 151, 153, 154, 157, 159, 161, 176, 179,
biological models, 164
182, 184, 200, 201, 202, 206, 215, 217, 220, 222,
biomass, 64
223, 225, 227, 228, 229, 233, 234, 236, 239, 242,
Biometals, 172
243, 244, 246
biopolymer, 170
bacterial, viii, xi, 43, 54, 55, 60, 85, 116, 118, 121,
biopolymers, 170
122, 123, 128, 130, 132, 134, 136, 141, 142, 144,
biopsies, 226
146, 148, 149, 150, 151, 171, 179, 184, 216, 217,
biopsy, 134
220, 223, 229, 231, 232, 236, 239, 242, 246, 247
biosafety, 244
bacterial cells, 220
biosynthesis, 3, 34, 39, 131, 176, 190, 193, 213
bacterial contamination, 171
biosynthetic pathways, 132
bacterial strains, 141, 142
biotechnological, 45
bacteriocin, 64, 69
biotechnology, 33
bacteriocins, 52, 66
biotin, 49
bacteriostatic, 52
birth, ix, 115, 116, 117, 125, 127, 128, 131, 132,
bacterium, 157
135, 137, 138, 141, 148, 217, 218, 219, 221, 235,
Baked goods, 18
240, 245
baking, 18, 62
birthweight, 242
barley, 186, 206
bison, 205
barrier, 57, 98, 119, 129, 132, 223, 225, 227, 234,
bleaching, 6
244
252 Index
bleeding, 117, 123 Bulgaria, 20

blends, ix, 36, 40, 88, 89, 92, 94, 95, 99, 102, 106, Burkholderia, 160
110, 111, 210, 247 butyric, 46, 156
blood, viii, 2, 3, 9, 10, 11, 14, 19, 21, 22, 24, 28, 33, by-products, vii, 1, 6, 21
35, 39, 55, 58, 59, 60, 61, 64, 66, 68, 87, 88, 116,
C
134, 139, 140, 141, 142, 143, 144, 145, 190, 192,
193, 202, 205, 211, 217, 221, 236, 243, 246 cabbage, 7, 44
blood glucose, 58, 64, 140, 143, 192, 193 CAD, 13
blood plasma, 202, 205 caecum, 57, 159
blood pressure, 59, 60, 64, 140, 143, 144, 217 caesarean section, 118, 128, 217
blood stream, 10, 11 calcium, 49, 51, 78, 154, 155, 156, 159, 160, 161,
blot, 166, 169 204
body, 176, 187, 190, 191, 192, 193, 198, 208, 209 calibration, 28
body composition, 187, 190, 191, 193, 198, 208, 209 Campylobacter jejuni, 57, 69
body fat, 176, 191, 192, 198, 208, 209 Canada, 45, 111
body fluid, 164 cancer, xi, 51, 52, 63, 165, 170, 175, 176, 187, 188,
body weight, 9, 13, 51, 191, 193 190, 196, 197, 198, 206, 207, 245
Boeing, 42 cancer cells, 52, 61, 165, 173, 187, 197
bone density, 198 candida, 49
bone growth, 193 Candida, 45, 52, 53, 68, 230
bone loss, 194 candidates, 234
bone mass, 193, 211 capillary, 25, 27, 29, 39
bone remodeling, 211 carbohydrate, xi, 57, 62, 202, 215
bone resorption, 173 carbohydrates, xi, 131, 132, 143, 158, 159, 215, 229,
borderline, 61 235
bounds, 4 carbon, 3, 46, 52, 62, 75, 92
bovine, 51, 60, 117, 123, 165, 170, 172, 173, 174, Carbon, 93
200, 201, 202, 203, 204, 240 carbon atoms, 3
bowel, 64, 144, 159, 221, 239 carbon dioxide, 46, 52
boys, 235 carbon tetrachloride, 62
Brazil, 8 carcinogen, 51, 188, 190
breakdown, 225 carcinogenesis, 188, 196, 197, 207, 210
breakfast, 18, 24, 34 carcinogenic, viii, 43, 51
breast, 216, 217, 218, 219, 220, 221, 222, 223, 226, carcinoma, 31, 51, 61
227, 229, 230, 235, 237, 238, 239, 240, 241, 242, cardiovascular disease, vii, 1, 2, 3, 9, 28, 35, 56, 134
243, 245, 246 Cardiovascular disease, 217
Breast, 216, 217, 221, 224, 229, 242 cardiovascular risk, 33
breast cancer, 51, 52, 61, 67, 165, 173, 188, 190, carotene, 33, 39, 40, 198
197, 198, 207 carotenoids, 19, 38
breast feeding, 121, 241, 243, 245, 247 carrier, 12, 155
breast milk, ix, 115, 116, 118, 119, 120, 121, 122, casein, 44, 228
123, 135, 142, 148, 173, 221, 240 caspase, 60, 168
breastfeeding, 137, 217, 220, 224, 242, 245, 247 CAT, 62
brevis, 44, 63 catabolic, 198
Britain, 225 catalase, 236
bronchial asthma, 56, 59 cattle, 179, 185, 186, 196, 204, 205, 206
bronchitis, 143 Caucasus, 63
Brussels, 7, 63 CBS, 68
Buenos Aires, 87 CD8+, 234
buffalo, 45 CDK2, 166
Index 253
CEC, 123 61, 62, 64, 65, 67, 68, 87, 88, 131, 135, 153, 157,
cecum, 131, 156, 159, 161, 207 158, 159, 161, 162, 192, 205
cell, x, 2, 29, 38, 54, 55, 60, 61, 68, 95, 102, 120, cholesterol lowering agents, 29
133, 137, 139, 155, 162, 163, 164, 165, 166, 168, chromatography, 25, 29, 31, 92, 177, 199
169, 170, 172, 174, 190, 208, 211, 221, 222, 228, chronic diseases, 44, 217, 239
234, 242, 244 chronic disorders, 217
cell culture, 61 circulation, 119
cell cycle, x, 163, 166, 172, 190 cis, xi, 89, 175, 176, 198, 199, 200, 202, 203, 204,
cell death, 168, 169, 170 205, 206, 208, 212
cell differentiation, 133, 166 classification, 14, 246
cell growth, 29 cleaning, 72, 73, 75, 76, 78, 79
cell line, 165, 172 cleavage, 168
cell lines, 165 clinical, 220, 223, 224, 227, 228, 230, 231, 233, 235,
cellular immunity, 221 242, 243
cellulose, 161 clinical symptoms, 231, 243
cereals, 4, 5, 6, 15, 16, 18, 24, 26, 34 clinical trial, 9, 117, 136, 138, 141, 144, 150, 172,
certificate, 14 220, 224, 227, 230, 231, 233, 235
ceruloplasmin, 164, 173 clinical trials, 9, 117, 136, 138, 141, 144, 172, 233
cesarean section, 218, 223, 240 clinics, 122
CFA, 154, 156 clone, 157
channels, 74 cloning, 154
charge density, 72 CMV, 217
, 15, 18, 20, 21, 32, 78, 136, 164, 177, 180, 186, 194, Co, 10, 23, 61, 64, 158, 159
195, 199, 202, 212 CO2, 46
chemical approach, 73 coatings, viii, 71, 73, 75, 76, 78, 79, 81, 83, 84, 85,
chemical composition, 47, 48, 89, 92, 94, 95 90, 105
chemical properties, ix, 27, 40, 88, 90 cobalt, 51
chemical structures, vii, 1, 3, 106, 177 Cochrane, 149
chemicals, 72, 73, 75, 78 coconut, 90
chemokine, 133 coconut oil, 90
chemokines, 133 coeliac disease, 149, 241
chemotherapy, 165, 174 coenzyme, 201
chicken, 181, 205 coffee, 16, 18
chickens, 9, 176 cohort, 218, 219, 240, 245
chicks, 39, 57, 193, 194, 211 colic, 117, 224
child care centers, 247 colitis, 147, 150, 173
child mortality, 247 collateral, 133, 193, 210
childhood, 217, 240, 242, 245 collateral damage, 193, 210
children, ix, xi, 19, 45, 63, 65, 115, 117, 121, 122, colloids, 170
138, 139, 140, 141, 142, 143, 149, 150, 158, 216, colon, x, 29, 60, 137, 153, 154, 157, 158, 159, 176,
217, 224, 227, 232, 239, 245, 246 188, 190, 207, 229, 245
China, 43, 64, 68 colon cancer, 158, 190, 245
chloride, 26 colon carcinogenesis, 188, 207
Chloride, 48 colonisation, 146, 150, 219, 246, 247
chloroform, 26, 27, 58 colonization, viii, ix, 43, 54, 57, 58, 60, 116, 117,
chocolate, 91, 95, 105, 111 119, 122, 125, 126, 127, 128, 129, 130, 131, 132,
cholera, 55 133, 135, 136, 138, 139, 142, 144, 149, 150, 217,
cholesterol, vii, viii, x, 1, 2, 3, 4, 9, 10, 11, 12, 13, 218, 219, 220, 222, 223, 226, 227, 230, 236, 237,
14, 19, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 33, 239
34, 35, 36, 37, 38, 39, 40, 41, 42, 45, 56, 59, 60, colonizers, 117, 128, 218, 237
254 Index
colorectal cancer, 67 contamination, 57, 116, 119, 171, 217

Columbia, 236 control, ix, 24, 25, 31, 39, 42, 60, 67, 74, 76, 77, 79,
commensals, 150 81, 82, 83, 84, 105, 115, 117, 136, 137, 143, 150,
commercial, 224, 228, 233, 234 154, 155, 156, 157, 182, 183, 185, 189, 190, 191,
commercialization, 14, 15, 16 192, 198, 203, 204, 210, 224, 225, 227, 228, 230,
communication, 204 232, 234, 235, 237, 238, 242, 245
communities, 146, 151 control group, 183, 224, 232, 237, 238
community, 3, 48, 117, 151, 179 controlled studies, 228
competence, 208 controlled trials, 29, 141
competition, 10 convergence, 126
compliance, 235 conversion, x, 90, 153, 154, 157, 181, 189, 196
complications, 141, 174 conversion degrees, 90
components, vii, x, 4, 23, 35, 36, 44, 51, 54, 55, 57, Cookies, 114
59, 61, 62, 66, 73, 85, 91, 98, 105, 132, 134, 153, cooking, xi, 16, 84, 90, 175, 187
164, 170, 172, 173, 190, 211, 220, 222, 226, 231, cooling, 23, 73, 76, 79, 90, 95, 97, 99, 100, 101, 102,
239, 242 103, 104, 106, 109
composites, 31 cooling process, 99
composition, ix, x, xi, 15, 21, 22, 29, 36, 39, 46, 47, copper, 51, 132, 204, 205
48, 61, 64, 65, 66, 75, 89, 91, 92, 94, 95, 97, 102, corn, 4, 21, 176, 182, 185, 193, 204
105, 115, 117, 118, 119, 120, 121, 123, 125, 127, coronary artery disease, 39, 41, 42
129, 135, 142, 149, 150, 179, 187, 190, 191, 193, coronary heart disease, viii, 2, 34, 42, 87, 88
198, 201, 203, 204, 205, 206, 208, 209, 211, 212, correction factors, 28, 179
213, 215, 225, 226, 229, 231, 233, 235, 240, 241, correlation, 119, 193, 227, 236, 237, 238
242, 243, 245, 246 correlation coefficient, 236
compositions, 235 correlations, 237, 238
compounds, 2, 3, 4, 12, 16, 25, 26, 28, 29, 30, 41, corrosion, 75
44, 48, 52, 105, 135, 164, 222, 233, 234, 244 cortisol, 222
concentrates, 36, 44 Corynebacterium, 118
concentration, 2, 6, 11, 16, 20, 21, 26, 28, 36, 38, 46, cosmetics, 105
52, 56, 106, 142, 143, 157, 159, 162, 165, 189, cost saving, 72
192, 193, 202, 204, 211, 212, 236 costs, viii, 4, 71, 72, 164
conception, 126, 135, 136, 137, 144, 149 costs of production, 164
conductivity, 72, 75, 77, 79, 83 counseling, 140, 142, 143, 147
confidence, 106 covering, 110
confidence interval, 106 cow milk, 68, 135, 154, 212, 243
configuration, 88 cows, 178, 180, 182, 183, 184, 185, 186, 195, 200,
confinement, 187 201, 202, 203, 204, 206, 212
Congress, 61, 111, 113, 124 CRC, 61, 161
conifer, 21 crystal growth, 104, 111
conjugated dienes, 199 crystal structure, 90, 104, 105, 110
consent, 235 crystalline, 22, 23, 85, 92, 101, 110, 111
constipation, 64, 117, 225 crystallization, ix, 10, 21, 23, 88, 90, 91, 92, 95, 97,
consumers, 44, 45, 56, 60, 88, 171 98, 99, 100, 101, 102, 105, 107, 110, 112, 113,
consumption, vii, viii, 1, 3, 4, 9, 12, 13, 14, 17, 18, 114
19, 20, 24, 31, 39, 43, 44, 57, 60, 67, 72, 73, 136, crystallization kinetics, 102, 110, 112, 114
138, 141, 145, 146, 148, 171, 189, 195, 196, 223, crystals, 10, 21, 91, 95, 97, 99, 101, 102, 103, 104,
226, 233, 246, 247 105, 107, 109, 110
contact time, 25 cultivation, 218
contaminant, 81 culture, 29, 33, 45, 52, 57, 58, 61, 63, 66, 116, 118,
contaminants, 16 129, 156, 161, 200, 218, 227, 234, 236
Index 255
culture media, 116, 236 delivery, ix, xi, 29, 115, 118, 119, 127, 128, 138,
CVD, vii, 1, 9, 13 139, 140, 141, 142, 143, 145, 147, 163, 171, 217,
cycling, x, 163 222, 223
Cyprus, 20 denaturation, 72
Cystatin, 173 dendrites, 119
Cystatin C, 173 dendritic cell, 119, 133, 142, 234, 242, 243
cysteine, 119, 120, 236 Dendritic cells, 119, 123
cytokine, 54, 55, 133, 137, 141, 142, 165, 172, 173, Denmark, 20
193, 221, 222 density, x, 35, 153, 158, 159, 192, 198
cytokine response, 221 deposition, 77, 85, 209
cytokines, 55, 67, 119, 130, 133, 165, 193, 194, 221, deposits, 77, 78
234 depression, 198, 201, 210
cytomegalovirus, 134 derivatives, 25, 27, 34, 136, 174, 198, 199, 212, 229
cytometry, 161 dermatitis, xi, 117, 137, 143, 216, 217, 225, 227,
cytoskeleton, 132 228, 231, 242, 243, 244, 246
cytosolic, 132 detection, 25, 27, 28, 65, 77, 84, 118, 121, 157, 160,
cytotoxic, 157 218, 230, 236, 242
cytotoxicity, 33 detention, 129
Czech Republic, 20 developed countries, 2, 126, 136, 217, 247
developmental origins, 145
deviation, 95
D
diabetes, xi, 58, 63, 134, 135, 175, 188, 192, 193,
210, 217, 239
dairy, vii, ix, xi, 1, 3, 14, 15, 17, 18, 19, 20, 28, 36,
diacylglycerol, 18
44, 53, 56, 57, 61, 63, 65, 66, 67, 72, 76, 84, 85,
diarrhea, 117, 154, 158, 216, 224, 225, 234, 241, 247
111, 116, 119, 164, 173, 175, 176, 177, 180, 182,
diarrhoea, 223, 246
183, 184, 185, 186, 187, 189, 195, 196, 200, 201,
dienes, 199
202, 203, 204, 206, 207, 211
diet, vii, ix, xi, 2, 4, 10, 12, 13, 14, 16, 17, 19, 23,
dairy industry, 72, 76, 84, 85
24, 25, 26, 28, 30, 34, 35, 36, 39, 44, 49, 60, 67,
dairy products, vii, ix, xi, 1, 3, 14, 15, 17, 19, 20, 28,
91, 105, 125, 126, 127, 130, 131, 134, 135, 143,
44, 53, 56, 61, 63, 66, 67, 116, 119, 164, 173,
144, 146, 147, 148, 150, 154, 155, 156, 157, 159,
175, 176, 177, 180, 186, 187, 195, 196, 206, 207,
173, 175, 176, 178, 179, 182, 183, 184, 185, 186,
211
188, 189, 190, 191, 192, 193, 194, 197, 205, 206,
danger, 133
235
Darcy, 197
dietary, viii, x, xi, 10, 17, 18, 23, 24, 31, 36, 37, 38,
database, 38
39, 40, 41, 56, 59, 60, 87, 88, 125, 127, 130, 131,
de novo, 132, 204
132, 134, 135, 136, 140, 141, 142, 143, 144, 146,
death, vii, 1, 2, 59, 64
147, 153, 154, 159, 162, 174, 175, 176, 178, 183,
deaths, 2
186, 188, 189, 190, 197, 198, 200, 201, 202, 203,
decisions, 15
204, 205, 207, 208, 210, 211, 212, 221
defects, 76
dietary fat, 146, 189, 203, 204, 212
defense, 136, 154, 159, 193, 242
dietary fiber, 154
defense mechanisms, 136, 154
dietary intake, 37, 143, 200
defenses, 133, 141
dietary supplementation, 154, 202, 204, 210, 212
deficiency, 173
diets, 23, 36, 56, 126, 135, 136, 155, 171, 178, 180,
definition, vii, 13, 19, 88, 99, 164
182, 183, 184, 185, 186, 187, 189, 191, 193, 196,
deformation, 96
201, 202, 205, 206, 212
degradation, 73, 161
differential scanning calorimetry, 90
degrading, 44, 54, 169
differentiation, 131, 133, 165, 166, 220
degree, 217
diffraction, 95
dehydration, 224, 235
256 Index
digestibility, 173 electrical resistance, 155

digestion, 22, 24, 57, 62, 155 electrodes, 84
digestive enzymes, 131 electron, 77
diphtheria, 236 electrophoresis, 151
diseases, vii, x, 1, 2, 54, 59, 117, 126, 132, 136, 139, ELISA, 236
140, 141, 142, 143, 154, 216, 217, 223, 239 elk, 205
disorder, 13, 56 elongation, 110
distillation, 6, 21 embryo, 98
distribution, 24, 105, 111, 177, 179, 189, 206, 212, embryos, 98
229, 230 emotional, 242
diversity, 37, 116, 118, 121, 122, 127, 130, 138 emulsification, 23, 24
dizygotic, 130 emulsifier, 23, 104, 105, 110
dizygotic twins, 130 emulsifying agents, 105
DNA, 60, 129, 130, 134, 137, 149, 156, 166 emulsions, xi, 24, 105, 163, 170, 171
DNA damage, 60 encapsulated, 170
DNA repair, 60 encapsulation, 170
dogs, 165, 173 endocarditis, 247
dominance, 134, 218, 229, 239 endocytosis, 172
doped, 85 endogenous, 225, 227
dosage, 231 endotherms, 94
double blind study, 191, 192 energy, viii, 11, 71, 72, 73, 79, 82, 83, 84, 87, 88,
double bonds, 88, 89 98, 131, 190, 191, 198, 209, 217
download, 35 energy consumption, 72, 73
down-regulation, 133 energy efficiency, 84
dressings, 17, 18, 32, 95 energy supply, 131
drinking, 60, 136 enrollment, 224, 234
drinking water, 136 enteric, 225
drugs, 2, 149, 172 enteritis, 241
dry matter, 178 enterococci, 59, 220, 225, 230, 231, 236
drying, ix, 116, 119, 120 enterocolitis, 117, 136, 150
DSC, 90, 94 environment, 34, 127, 130, 133, 146, 179, 221, 222,
duration, 68, 77, 121, 197, 224, 225, 235 224
dyslipidemia, 30 environmental, 217, 223, 239
environmental factors, 126, 128, 129, 239
environmental influences, 126
E
enzymatic, x, 25, 90, 153, 161, 219, 233, 241
enzyme secretion, 24
E. coli, 53, 128, 129, 219, 223, 230, 238
enzymes, 13, 51, 131, 132, 154, 184, 191
Eastern Europe, 65
eosinophilia, 56, 59
eating, vii, 1, 195
eosinophils, 137
ecological, 119, 184, 247
epidemiologic studies, 217
ecology, 133, 148
epidermal growth factor, 222
ecosystem, 127, 145, 148
epidermis, 118, 207
eczema, 142, 147, 225, 226, 231, 232, 240, 242, 243
epithelial cell, 58, 132, 145, 148, 149
Education, 37, 196
epithelial cells, 58, 132, 145, 148, 149
egg, 16, 18, 89
epithelial stem cell, 60
eicosanoid, 190, 193, 210
epithelium, 50, 58, 60, 131, 132, 148, 159, 160, 197,
eicosanoids, 190, 193
207
elaboration, 120
equilibrium, 101, 237
elderly, 44
equol, 159
election, 246
Index 257
erythrocyte, 208
F
erythrocyte membranes, 208
Escherichia coli, 52, 62, 148, 158, 193, 218, 219,
fabrication, 113
230, 237, 245
faecal, 30, 41, 57, 116, 121, 150, 160, 227, 240, 241,
esophagus, 31
245
essential fatty acids, 89
faecal bacteria, 30, 116
ester, 11, 16, 24, 30, 31, 34, 35, 36, 38, 40, 106, 109,
failure, 221
110, 190, 207
family, 36, 139
esterases, 11, 24
famine, 135, 148
esterification, 2, 11, 22
FAO, viii, 87, 88, 111, 135, 145
esters, 11, 13, 15, 16, 17, 19, 21, 22, 24, 26, 30, 31,
farming, 184
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 105,
farms, 184
106, 107, 110, 111, 199
fasting, 132, 192, 193, 208
Estonia, 20
fasting glucose, 192, 193
estrogen, 52, 61, 197
fat, viii, xi, 2, 3, 10, 15, 16, 17, 21, 22, 23, 24, 25,
etching, 73, 84
28, 32, 34, 36, 37, 38, 40, 41, 46, 47, 56, 87, 88,
ethanol, 44, 45, 46, 52
89, 91, 92, 93, 94, 95, 96, 97, 99, 101, 102, 105,
ethics, 235
108, 112, 113, 114, 132, 135, 144, 148, 159, 164,
eukaryotes, 2
172, 175, 176, 177, 178, 179, 180, 181, 182, 183,
Euler, 231, 241
184, 185, 186, 187, 188, 189, 191, 192, 193, 194,
Europe, vii, 1, 14, 16, 17, 44, 136
195, 197, 198, 199, 200, 201, 202, 203, 204, 205,
European, 232, 240
206, 208, 209, 211, 212, 217, 235
European Commission, 15, 19
fat soluble, 22, 24, 37
European Parliament, 31, 32, 33
fats, ix, 18, 21, 22, 23, 26, 29, 40, 47, 88, 89, 91, 92,
European Union, 15, 25, 31, 32, 33
94, 95, 96, 97, 99, 102, 105, 110, 111, 112, 113,
evaporation, 27
114, 201
evidence, xi, 176, 187, 189, 207, 223, 227, 233, 234
FDA, viii, 14, 15, 16, 18, 23, 33, 34, 75, 87, 88, 111,
evolution, 36, 130, 147, 148
114
ewe, 203
fecal, 217, 218, 220, 225, 226, 227, 230, 231, 233,
examinations, 235
236, 238, 240, 242, 243, 246
exclusion, 69, 83
feces, 116, 122, 128, 129, 146, 157, 220, 226, 240
excretion, 157
Federal Register, 111
exercise, 191
feeding, viii, ix, xi, 43, 47, 51, 115, 117, 121, 122,
exocrine, 164
127, 129, 136, 137, 140, 149, 150, 154, 155, 181,
exogenous, xi, 215
183, 184, 185, 187, 188, 191, 192, 193, 198, 205,
exopolysaccharides, 55
206, 207, 208, 215, 217, 218, 220, 223, 226, 228,
experimental condition, 155, 157
229, 230, 231, 232, 236, 238, 239, 241, 243, 245,
expert, 232
247
exposure, x, 59, 125, 126, 127, 128, 129, 135, 136,
females, 2
137, 148, 225
femoral bone, 159
Exposure, 137
fermentation, xi, 44, 45, 46, 48, 49, 50, 51, 53, 54,
expression, 190, 197
56, 58, 60, 61, 66, 68, 157, 182, 216, 232, 233,
external environment, 57
234, 239
external influences, 129
fertilizers, 184
extraction, 21, 25, 26, 27, 55, 116
fetal, ix, 125, 126, 127, 134, 135, 136, 140, 142, 143,
extrapolation, 195
145, 146, 148, 150
exudate, 164
fetal growth, 150
fetus, 116, 126, 134, 135, 137, 139, 140, 141
fetuses, 116, 134
fever, 150
258 Index
fiber, 4, 131, 157, 159, 182, 186 France, 20, 73, 113, 190, 198, 215, 233, 235, 236,
fiber content, 182 246
FID, 25, 28, 92 free energy, 72, 85, 98
Fife, 202 free radicals, 188
film, 105 freeze-dried, 67
films, 50 freezing, ix, 116
financial support, 83 friction, 73, 75, 85
Finland, 14, 16, 20, 34, 41, 42, 118 frost, 34
fish, 22, 31, 145, 183, 184, 203, 206 fructooligosaccharides, 159, 161, 162, 229, 232, 240,
FISH, 216, 227, 230 244
fish meal, 183, 203 fructose, 154
fish oil, 145, 183, 184, 203, 206 fruit juice, 45
Fisher exact test, 236 fruits, vii, 1, 4, 6, 15, 19, 31, 38, 89
fitness, 164 frying, 18
flame, 25, 92 FTIR, 90
flame ionization detector (FID), 92 functional changes, 174
flavor, 91, 92, 102, 203 fungi, x, 163
flexibility, 50, 92 fusiform, 161
flora, xi, 49, 50, 57, 64, 121, 122, 123, 148, 164,
215, 217, 218, 219, 220, 222, 223, 225, 227, 230,
G
233, 236, 237, 238, 239, 240, 241, 242, 243, 246,
247
gas, 25, 26, 29, 39, 41, 42, 92, 177, 199
flow, 73, 74, 76, 77, 78, 79, 82, 83, 92
gas chromatograph, 25, 26, 29, 41, 92, 177, 199
fluid, 26, 73, 84, 91, 116, 180
gastrectomy, 159
fluid extract, 26, 91
gastric, 55
fluorescence, 161
gastroenteritis, 217, 224, 241
fluorescence in situ hybridization, 161
gastrointestinal, ix, xi, 10, 25, 44, 52, 57, 59, 60,
fluorine, viii, 71, 75, 81, 83
125, 127, 131, 132, 135, 139, 141, 145, 148, 149,
focusing, 226
151, 154, 155, 173, 215, 218, 224, 229, 232, 241,
folding, 67
246
folic acid, 45, 49
gastrointestinal tract, ix, 10, 125, 131, 135, 148, 151,
food, vii, viii, ix, x, 1, 2, 6, 9, 12, 14, 15, 16, 17, 19,
154, 155, 218, 229, 241, 246
20, 21, 22, 23, 25, 29, 30, 31, 32, 33, 34, 35, 36,
GC, 25, 27, 28, 29, 40, 92, 246
37, 41, 42, 43, 44, 52, 54, 57, 58, 59, 60, 64, 65,
gel, 151, 169
68, 71, 72, 73, 75, 77, 79, 83, 84, 87, 88, 89, 91,
gelatin, 169
95, 102, 104, 105, 106, 111, 119, 121, 122, 125,
gels, 50, 66, 170
131, 134, 136, 137, 145, 153, 154, 163, 164, 170,
gender, 191
173, 180, 190, 194, 199, 200, 205, 208, 211, 217,
gene, 41, 127, 131, 132, 145, 156, 160, 173, 209,
221, 223, 225, 226, 227, 229, 240, 244
244
food allergy, 59, 217, 225, 227, 244
gene expression, 127, 131, 132, 145, 173, 209
Food and Drug Administration, 14, 15, 88
generation, 33, 42, 73, 157, 158, 225
Food and Drug Administration (FDA), 15, 88
genes, 126, 132, 133, 154, 197
food industry, vii, viii, 1, 14, 17, 71, 73, 164
genetics, 150, 206
food production, 52
Geneva, 42, 111
, 17, 23, 29, 35, 44, 73, 75, 89, 95, 104, 170, 180
genome, x, 125, 130, 150, 246
food safety, 72
genotype, 130, 151, 209
fortification, 154
Germany, 20, 143
fouling, viii, 71, 72, 73, 76, 77, 78, 79, 81, 83, 84, 85
gestation, 135, 137
Fourier, 90
gestational age, 127, 222, 229, 230
fractionation, 89, 91, 92
Gibbs, 98
Index 259
Gibbs free energy, 98 Guinea, 193, 210

girls, 235 gut, ix, x, 13, 54, 55, 64, 68, 115, 116, 117, 118, 119,
gland, 52, 119, 176, 179, 184, 186, 189, 190, 196, 121, 122, 123, 125, 127, 128, 129, 130, 131, 132,
197, 206, 207, 221 133, 135, 136, 138, 139, 142, 144, 145, 146, 147,
glass, 102 148, 149, 150, 164, 218, 221, 222, 223, 225, 237,
glucose, 58, 64, 67, 132, 140, 143, 147, 154, 159, 239, 243, 245
192, 193, 199, 206, 210, 236
glucose metabolism, 143
H
glucose regulation, 147
glucose tolerance, 140, 192, 199
halos, 52
glutamic acid, 50
handling, 72
glycans, 131, 148
hanging, 36, 45
glycemic index, 57
hardness, 75, 102, 112
glycerol, 105, 106, 188, 209
harvest, 6, 131
glycoconjugates, 131
hazards, 72
glycol, 176
HDL, 56
glycoside, 26
healing, 66
glycosides, 4
health, vii, viii, x, xi, 1, 2, 3, 9, 14, 15, 18, 21, 23,
glycosylation, 132, 146
29, 33, 36, 37, 40, 41, 42, 43, 44, 49, 50, 54, 56,
goals, 222
64, 90, 111, 119, 123, 125, 126, 127, 129, 130,
goat milk, 45, 56
134, 135, 136, 138, 141, 144, 153, 163, 164, 165,
goblet cells, 56, 59, 132
171, 173, 176, 187, 191, 194, 196, 213, 215, 223,
gold, xi, 215, 217
226, 229, 230, 244
gold standard, xi, 215, 217
health care, 44
Gore, 226, 242
health effects, 14, 56, 119, 123, 141, 194
grain, 16, 18, 48, 50, 51, 55, 61, 62, 63, 65, 66, 182,
health status, 129, 135, 171
183, 185, 186, 194, 204, 205
heart, viii, 2, 33, 34, 36, 42, 87, 88, 192
grains, vii, 1, 4, 6, 43, 44, 45, 46, 48, 49, 50, 51, 52,
Heart, 30, 35, 38, 42
53, 55, 57, 58, 60, 61, 62, 63, 64, 66, 68, 97, 178,
heart disease, 33, 36, 88, 192
182, 183, 184, 187
heat, viii, x, 71, 72, 73, 74, 79, 82, 83, 84, 85, 90,
Gram-negative, 52, 132
101, 137, 142, 150, 153, 154, 160, 233, 234, 244
Gram-positive, 132
heat transfer, 72, 74, 79
grants, 22, 144
heating, 23, 72, 73, 74, 77, 79, 82, 92, 94, 164, 233
graph, 98
heating rate, 92
grass, 113, 178, 182, 183, 187, 195, 200, 204, 205
height, 96, 101, 131
grazing, 182, 183, 184, 185, 187
Helicobacter pylori, viii, 43
Great Britain, 225
helium, 27
Greece, 20
hematological, 198
groups, 27, 37, 55, 88, 118, 128, 141, 143, 154, 191,
hematology, 174
220, 227, 228, 229, 231, 232, 236
hematopoietic, 174
growth, vii, ix, x, xi, 3, 17, 29, 52, 53, 55, 62, 64, 65,
hematopoietic stem cell, 174
97, 99, 101, 102, 104, 105, 110, 111, 115, 117,
hepatocyte, 132
118, 119, 126, 129, 131, 137, 141, 145, 150, 153,
hepatocytes, 11, 161
154, 158, 163, 164, 165, 166, 170, 174, 184, 189,
heredity, 126
193, 195, 197, 198, 208, 209, 210, 211, 215, 221,
herpes simplex virus type 1, 134
222, 224, 228, 229, 232, 235, 237, 241
heterogeneous, 105
growth factor, x, 163, 211, 221, 222
heterozygote, 13
growth rate, 110, 224, 228, 232
heterozygotes, 13, 36
GST, 62
hexane, 26, 27
guidelines, 9, 14, 35, 36, 118, 180
high fat, 2, 16, 23, 126, 188
260 Index
high pressure, 161 hydrogen peroxide, 52, 53

high risk, 88, 117, 142 hydrogenation, ix, 27, 88, 89, 178, 200
high temperature, 22 hydrolysis, 11, 13, 24, 27, 52, 61, 200, 241
high-fat, 135, 148 hydrolyzed, 105, 179
high-level, 225 hydrophilic, 105, 106, 111
high-risk, 141, 142, 228, 246 hydrophobic, 2, 10, 106
high-risk populations, 228 hydrophobicity, 68
histamine, 188, 193, 198, 210 hydroxide, 26
histological, 59 hydroxyl, 3, 4, 27, 154
HIV, 217 hydroxyl groups, 27
HLA, 234 hygiene, 123, 150
Hm, 99 hygienic, 126, 127, 136, 144, 239
Holland, 223, 243 hyperbolic, 101
homeostasis, ix, 54, 55, 115, 117, 150, 171, 173 hypercholesterolemia, 34, 135
homogeneity, 154 hyperglycemia, 58, 135
homogenized, 233 hyperlipidemia, 135
hormones, 30, 119, 205, 208, 222 hyperplasia, 30, 42, 131, 137, 207
hospital, 246 hyperproliferation, 9
hospitalization, 128 hypersensitivity, 65
hospitalized, 128, 150, 246 hypertension, 44
host, ix, x, 44, 55, 58, 61, 125, 127, 130, 131, 132, hypertensive, 67
135, 137, 141, 144, 146, 150, 151, 153, 164, 173, hypocholesterolemic, viii, 2, 9, 11, 43, 56, 60
226, 229 hypothesis, ix, 123, 125, 126, 127, 128, 130, 134,
hot water, 73, 76, 77, 82 136
household, 64, 150
HPLC, 25, 28, 90
I
HR, 56
human, vii, x, xi, 2, 4, 9, 12, 35, 37, 38, 39, 41, 44,
ice, 16, 18, 111, 136, 186, 191, 202, 234
52, 56, 58, 59, 61, 64, 65, 105, 116, 118, 119,
id, viii, 24, 71, 213, 228, 237
121, 122, 124, 126, 130, 131, 134, 136, 137, 141,
identification, 25, 28, 34, 48, 63, 68, 121, 242, 243,
146, 148, 149, 151, 155, 158, 159, 161, 163, 164,
244
165, 172, 173, 175, 176, 177, 187, 189, 191, 192,
IFN, 133, 137, 221, 231, 234
194, 195, 197, 206, 210, 211, 212, 215, 217, 218,
IgE, 56, 59, 134, 139, 142, 145, 221, 234
220, 221, 222, 223, 225, 226, 229, 231, 232, 233,
IGF, 193, 211
237, 239, 240, 241, 242, 243, 244, 245, 246, 247
IGF-1, 193, 211
human immunodeficiency virus, 245
IgG, 55, 128, 134, 221, 234
human milk, 116, 118, 119, 121, 122, 124, 130, 146,
IHD, 44
148, 177, 212, 217, 220, 221, 222, 223, 226, 229,
IL-1, 54, 119, 133, 137, 221, 231, 234
231, 232, 237, 239, 240, 241, 242, 243
IL-10, 54, 119, 133, 137, 221, 231, 234
human subjects, 35, 56, 189, 191, 192, 210
IL-13, 221
humans, x, 9, 13, 22, 38, 39, 56, 57, 60, 66, 126,
IL-2, 221
131, 136, 139, 157, 160, 176, 180, 187, 189, 190,
IL-4, 221
191, 192, 193, 194, 195, 196, 206, 209, 233, 246
IL-6, 221
humoral immunity, 128, 146, 147, 221
IL-8, 133
Hungary, 20
ileum, 155
hybridization, 227, 230
images, 109, 110
hydro, 105, 106, 111
immune cells, 52, 133, 193, 194, 217, 221, 222
hydrocarbon, 27
immune function, x, 55, 121, 125, 127, 176, 193,
hydrodynamic, 79
245
hydrogen, 52, 53, 57
immune regulation, 238
Index 261
immune response, 30, 52, 53, 55, 57, 68, 117, 119, infancy, ix, 115, 117, 121, 123, 129, 143, 146, 149,
123, 126, 130, 133, 134, 140, 141, 142, 145, 165, 221, 232, 241, 245, 247
171, 187, 193, 198, 210, 221, 225, 235, 241, 244, infant colic, 141
245 infant formulas, ix, xi, 116, 119, 120, 121, 136, 215,
immune system, viii, ix, xi, 43, 44, 51, 53, 54, 55, 218, 226, 229, 232, 239, 246
62, 65, 115, 117, 119, 127, 131, 133, 135, 136, infant mortality, 224
137, 165, 173, 193, 215, 217, 220, 221, 222, 226, infants, ix, x, 44, 57, 60, 115, 116, 117, 119, 121,
227, 232, 233, 238, 245 122, 123, 126, 127, 128, 129, 135, 136, 138, 139,
immunity, 54, 55, 67, 68, 122, 130, 132, 133, 134, 140, 141, 142, 144, 145, 146, 148, 150, 217, 218,
144, 145, 148, 193, 194, 221, 225, 243, 244 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,
immunodeficiency, 245 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,
immunoglobulin, 133, 134, 193, 211 239, 240, 241, 242, 243, 244, 245, 246, 247
immunological, ix, xi, 115, 117, 121, 128, 136, 148, Infants, 117, 127, 138, 217, 220, 224, 225, 226, 227,
215, 242 228, 229, 230, 235, 236, 239
immunomodulation, 50 infection, viii, 43, 45, 57, 59, 139, 141, 147, 171,
immunomodulatory, 149, 226, 234, 235 224, 243
immunosuppressive, 51 infections, x, 52, 65, 125, 127, 132, 134, 136, 139,
impaired glucose tolerance, 192, 199 141, 142, 148, 149, 154, 217, 222, 224, 242, 247
implants, 75 infectious, ix, 115, 117, 118, 119, 121, 122, 126,
implementation, viii, 14, 72, 73 174, 216, 217, 223, 224, 229, 232, 233, 247
impurities, 97, 105 infectious diseases, ix, 115, 117, 126, 216, 217, 223,
in situ, 227, 230 247
in situ hybridization, 161, 227, 230 inflammation, 30, 56, 121, 137, 145, 147, 173, 193,
in utero, 116 194, 225, 227
in vitro, 9, 10, 30, 37, 53, 61, 68, 105, 147, 148, 155, inflammatory, 29, 30, 55, 59, 60, 63, 66, 132, 133,
165, 173, 194, 201, 202, 234 137, 141, 142, 154, 165, 172, 174, 193, 221, 229,
in vivo, 10, 30, 54, 120, 146, 147, 165, 172, 190, 232, 234
208, 233 inflammatory bowel disease, 154
incidence, viii, 59, 71, 72, 79, 134, 140, 142, 143, inflammatory responses, 133, 172
216, 224, 231, 232, 235, 244 influence, 179, 182, 183, 184, 186, 193, 204
inclusion, vii, 44, 50, 60, 183, 223, 235 infrared, 90, 211
incubation, 56, 233, 236 infrared spectroscopy, 90
India, 89, 114, 245 ingestion, vii, 1, 3, 5, 57, 158, 159, 160, 231
Indian, 89, 206 inhibition, 11, 13, 35, 51, 52, 55, 57, 58, 60, 91, 133,
indication, 19, 135 149, 161, 165, 166, 179, 187, 188
indigenous, xi, 145, 150, 215, 223, 228 inhibitor, 35, 168, 169
Indigenous, 146, 149 inhibitory, 50, 173, 193, 197, 207, 238
indirect effect, 53, 232 inhibitory effect, 50, 173, 197, 238
induction, x, 97, 98, 99, 101, 102, 105, 106, 110, inhospitable, 224
111, 133, 137, 138, 163, 222, 234 initiation, 51, 189
induction period, 98, 101 injection, 198, 235
induction time, 97, 98, 99, 101, 102, 105, 106, 110, innate immunity, 55, 68, 146, 243
111 innovation, 2, 164
industrial, 21, 40, 45, 47, 106, 226 Innovation, 144
industrial application, 106 inoculation, 45
industrial processing, 21 Inspection, 77, 78
industrialization, ix, 116 instability, 127
industry, vii, viii, 1, 14, 17, 21, 72, 73, 75, 76, 82, instruments, 75
84, 85, 164 insulin, 58, 67, 132, 158, 192, 211, 222
inertness, 75 insulin signaling, 58
262 Index
insulin-like growth factor, 211, 222 Italy, 20, 163, 225

integrity, viii, 71, 78, 81, 119, 131, 132
interaction, 68, 75, 97, 102, 133, 147, 170
J
interactions, ix, 44, 97, 125, 131, 146, 150, 172
interest, 187
Japan, 23, 44, 61, 62, 153, 158
interface, 57, 98, 127, 134, 148, 150
Japanese, 62, 65, 66, 159
interference, 2, 33
jejunum, 155
interferon, 147, 148, 221, 234
JNK, 169, 172
interferon (IFN), 234
Jun, v, 77, 84, 153
interleukin-1, 147
interleukins, 221
interval, 97 K
intervention, ix, x, 115, 117, 121, 126, 127, 130,
141, 142, 143, 144, 153, 154 kappa, 133
intervention strategies, 130 kappa B, 133
intestinal flora, xi, 57, 121, 215, 217, 220, 222, 227, kernel, 89
233, 235, 238, 239, 242, 246, 247 kidney, 155
intestinal tract, 21, 58, 116, 135, 217, 241 kinase, 58, 132, 166, 173
intestinal villi, 131 kinetics, 35, 39, 66, 99, 102, 105, 110, 112, 114, 209
intestine, ix, x, 56, 58, 59, 115, 116, 117, 118, 125, King, 147
127, 128, 130, 131, 132, 135, 136, 138, 142, 146, knowledge, 211
147, 150, 154, 156, 161, 179, 218, 220, 221, 222, Korean, 67
226, 232, 237, 240, 242
intragastrically, 137 L
intramuscular, 185, 205, 206
intravenous, 206 LAB, 50, 53, 56, 57, 117, 118, 119
inulin, 136, 230, 231, 245 labeling, 32, 88, 111
invasive, 220 labor, 141
Investigations, 150, 234 labour, 116, 149
ion implantation, 73, 84 lactase, 131
ionization, 25, 92, 177 lactating, x, 126, 136, 142, 149, 178, 179, 183, 200,
ionizing radiation, 59 201, 202, 203
ions, 132 lactation, 116, 117, 119, 121, 122, 127, 136, 139,
Ireland, 20 141, 142, 145, 148, 185, 204, 241
iron, x, 45, 51, 148, 159, 163, 164, 171, 172, 173, lactic acid, viii, ix, 43, 44, 45, 46, 53, 58, 59, 60, 62,
237 64, 65, 66, 67, 68, 115, 117, 119, 122, 156, 220,
iron deficiency, 173 225, 233, 242, 246
irradiation, 60 lactic acid bacteria, ix, 44, 45, 53, 58, 59, 62, 64, 65,
irritability, 224, 232 66, 67, 68, 115, 117, 122, 220, 225, 233, 242, 246
irritable bowel syndrome, 117 lactobacillus, 52
isolation, ix, 34, 115, 116, 118, 120, 121, 173, 240 Lactobacillus, 45, 49, 51, 57, 58, 60, 61, 63, 64, 65,
isoleucine, 50 66, 68, 69, 116, 117, 118, 121, 122, 123, 129,
isomerization, 161, 176, 178, 179 135, 136, 139, 141, 145, 146, 147, 148, 149, 150,
isomers, xi, 175, 176, 177, 179, 180, 181, 185, 186, 153, 158, 220, 223, 225, 228, 230, 242, 243, 245,
191, 192, 196, 197, 199, 200, 202, 204, 206, 208, 247
210, 212 lactoferricin, x, 163, 164, 174
isoprenoid, 3 lactoferrin, 130, 164, 165, 172, 173, 174
isothermal, 110 lactoglobulin, 164, 165, 172, 228
isotopes, 160 lactoperoxidase, 164
ISPA, 163
Index 263
lactose, x, 57, 61, 62, 63, 153, 154, 158, 160, 161, lipophilic, 105
229, 233, 236 lipopolysaccharide, 55, 146
lactose intolerance, 57 lipoprotein, 31, 35, 157, 192, 206
lamina, 133 lipoproteins, 37, 199, 208
large intestine, 54, 55, 148, 154, 156 liposomes, 23, 171
large-scale, 129 liquid chromatography, 25, 28, 29, 39, 42, 90, 177,
laser, 106 199
latex, 173 liquid phase, 109
Latvia, 20 liquids, 90
LDL, 2, 9, 12, 13, 14, 18, 19, 24, 25, 28, 39, 41, 42, Listeria monocytogenes, 52, 62
208 literature, 229, 231
lead, 225, 229 Lithuania, 20
leakage, 132 liver, 39, 56, 59, 132, 135, 137, 155, 161, 179, 188,
lean body mass, 176, 191 213
lecithin, 23, 24, 38, 41, 45, 105 liver disease, 135
lectin, 145, 146 liver spots, 59
left ventricle, 33 Livestock, 205
legislation, 15 London, 65, 240, 245
leptin, 192, 208 losses, 27, 120, 191
lesions, 137, 157, 189, 206, 207 Louisiana, 71
Leuconostoc, 44, 46, 49, 51, 118 Louisiana State University, 71
leukemia, 172 low birthweight, 242
leukocytes, 66, 164, 246 low density polyethylene, 50
life expectancy, 2, 44 low temperatures, 50
life quality, 2 low-density, 2, 35, 157
lifestyle, 2, 126, 135, 144, 239 low-density lipoprotein, 2, 35, 157
ligands, 149, 173 LPO, 62
light beam, 98 LPS, 55, 172, 234
limitations, 2, 22, 24, 187 lubricants, 106
linear, 184 lumen, 11, 24, 119, 155
linoleic acid, xi, 88, 175, 176, 177, 178, 180, 181, luminal, 147, 155, 156. 193
184, 187, 188, 190, 193, 194, 195, 196, 197, 198, lupus, 188, 213
199, 200, 201, 202, 203, 204, 205, 206, 207, 208, lupus erythematosus, 213
209, 210, 211, 212, 213, 222 Luxembourg, 20
linolenic acid, 45, 56, 140, 143, 202 lymph, 11, 133, 137, 234
lipase, 89, 132, 191 lymph node, 133, 137, 234
lipases, 11, 179, 200 lymphatic, 119
lipid, 22, 24, 25, 26, 31, 33, 34, 35, 38, 41, 60, 67, lymphocyte, 126, 188, 198, 221, 235, 240
91, 97, 102, 114, 132, 135, 148, 154, 159, 161, lymphocytes, 141, 221, 234
178, 182, 186, 189, 202, 205, 210, 213 lymphoid, 133, 138, 221
Lipid, 26, 29, 35, 36, 37, 39, 40, 41, 42, 59, 111, lymphoid organs, 138
112, 113, 114, 200, 202, 208, 210, 212 lymphoid tissue, 133, 221
lipid metabolism, 34, 38, 41, 154, 159, 161, 205, 210 lysine, 50, 131
lipid peroxidation, 213 lysozyme, 217
lipid profile, 31, 33, 41, 135, 148
lipids, 34, 36, 37, 41, 42, 46, 61, 64, 91, 102, 114,
M
132, 135, 143, 159, 178, 189, 200, 205, 206, 208,
211, 233
M.O., 173
lipodystrophy, 213
machinery, 132, 155
lipolysis, 10, 191
macromolecules, 132
264 Index
macrophage, 198 meals, 24, 183

macrophages, 54, 55, 133, 194, 221 measurement, 96, 97, 112
Madison, 113, 114 measures, 96, 223, 236
magnesium, 45, 51, 160 meat, xi, 18, 64, 175, 176, 180, 181, 185, 186, 187,
magnetic, 92 189, 196, 200, 205, 209, 211, 212
mainstream, vii mechanical properties, 96
maintenance, 2, 17, 18, 136, 227 meconium, 116, 122
major histocompatibility complex, 133 media, 116, 119, 161, 216, 236
malabsorption, 159 median, 219
males, 2 mediators, 194, 211
malignancy, 60 medication, 19
Malta, 20 medicine, 2, 45
maltodextrin, 233 melanoma, 59, 61, 65
Mammalian, 173 melon, 7
mammals, x, 2, 125, 130 melt, 107
management, xi, 35, 36, 58, 143, 171, 174, 175, 184, melting, ix, 88, 89, 91, 92, 93, 94, 95, 96, 98, 99,
186, 196, 244 106, 108
management practices, 196 melting temperature, 90, 92, 98, 99
manganese, 51 membranes, 3, 24, 208
Manganese, 48 men, 31, 35, 39, 40, 61, 67, 160, 161, 191
mango, 89 meningitis, 228, 245
Manhattan, 84 meta-analysis, 12, 14, 29, 242
manipulation, 136, 201 metabolic, ix, 44, 52, 125, 126, 131, 134, 135, 136,
manufacturer, 92 141, 143, 205, 209, 210, 243
manufacturing, 4 metabolic disorder, x, 126, 135, 136
MAPK, 168, 169 metabolic systems, 135
margarine, 16, 18, 22, 30, 38, 41, 90, 92, 94, 95, 105, metabolism, 13, 15, 31, 34, 36, 37, 38, 39, 41, 66,
112 67, 135, 143, 144, 154, 159, 160, 161, 188, 190,
market, 14, 15, 16, 17, 18, 20, 23, 32, 33, 75, 89, 192, 198, 200, 202, 205, 209, 210, 211, 237
119, 180, 181 metabolites, 3, 39, 53, 197, 207, 208, 244
markets, 3 metal ions, 132
Mars, 163 metastasis, 190, 197
mass, 176, 177, 191, 192, 199, 209 metastatic, 61, 169, 170
mass spectrometry, 25, 29, 177 methanol, 10, 26, 27, 58
mass transfer, 101 methionine, 50
mast cells, 137 methylation, 199
mastitis, 116, 118, 119, 121, 122 methylene, 26
maternal, ix, 116, 118, 119, 121, 125, 127, 128, 129, methylene chloride, 26
134, 135, 137, 138, 139, 141, 142, 143, 144, 145, mice, 39, 42, 51, 54, 55, 57, 58, 59, 61, 62, 64, 65,
146, 147 66, 67, 121, 130, 131, 132, 133, 135, 137, 144,
maternal care, 121 146, 147, 150, 159, 172, 176, 188, 190, 191, 192,
maternofetal, 149 193, 197, 198, 208, 209, 212, 213, 233, 234, 244,
Matrices, 23 246
matrix, 22, 23, 24, 25, 26, 28, 44, 50, 85, 119, 136 micelles, 10, 23, 24, 25, 29, 37, 38, 170
maturation, ix, 49, 115, 117, 118, 123, 128, 131, microarray, 148
132, 133, 146, 174, 220, 221, 222, 226, 228, 232, microbes, x, 126, 128, 130, 133, 135, 146, 149, 150,
235, 238, 242, 245 163, 182, 183, 223
maturation process, 131, 238 Microbes, 128, 144, 240
MBP, 173
MCA, 157
Index 265
microbial, ix, x, 48, 53, 61, 64, 66, 68, 115, 117, molecular mechanisms, 164
125, 127, 128, 129, 130, 134, 136, 148, 153, 164, molecular structure, 111
173, 179, 182, 186, 200, 220, 221, 226, 238 molecules, 2, 10, 58, 98, 99, 101, 130, 133, 222
Microbial, 49, 64, 125, 133, 200, 202 molybdenum, 51
microbiota, ix, 64, 115, 116, 117, 121, 123, 125, momentum, 77
126, 127, 128, 129, 130, 131, 132, 133, 135, 136, monolayer, 155, 234
138, 144, 145, 146, 147, 148, 149, 156, 158, 162, monolayers, 123
218, 220, 222, 225, 226, 229, 232, 233, 239, 240, mononuclear cells, 137, 142, 147, 172, 211, 243
242, 243, 244, 245, 246 monosaccharides, 132, 160, 229
Microbiota, 127, 131, 132, 138, 147, 149, 226 monounsaturated fat, 143
microflora, ix, xi, 30, 44, 45, 48, 50, 51, 52, 54, 55, monounsaturated fatty acids, 143
59, 60, 61, 64, 66, 68, 116, 117, 118, 119, 122, monozygotic twins, 130
145, 150, 154, 156, 159, 162, 165, 215, 218, 219, morbidity, 141, 216
220, 221, 224, 225, 226, 227, 230, 231, 232, 233, morphogenesis, 206
239, 240, 241, 242, 243, 244, 245, 246, 247 morphological, 131
Microflora, vi, 48, 49, 60, 215, 217, 229 morphology, 95, 102, 103, 110
microorganism, 179 mortality, 2, 126, 216, 224, 247
microorganisms, ix, 44, 48, 51, 52, 53, 57, 63, 115, Moscow, 61, 63, 173
117, 134, 153, 161, 179, 200, 227 mothers, x, 118, 119, 126, 129, 134, 136, 138, 139,
micro-organisms, 130 141, 142, 144, 147, 149, 217, 235, 243
microscopy, 102 moths, 119
microsomes, 201, 213 mouse, 30, 55, 59, 63, 64, 145, 151, 165, 188, 198,
microstructure, ix, 88, 91, 95, 102, 110, 111, 113 207, 210, 213, 234
microvasculature, 131, 132 mouse model, 30, 56, 188, 234
microwave, 187 mouth, 128
Middle East, 44 mRNA, 137, 179, 201
middle-aged, 31 MRS, 119, 120, 236
migration, 81, 169 mucin, 131, 132, 145, 148
milk fermentation, xi, 54, 68, 216, 232, 233, 234, mucosa, 119, 130, 131, 132, 159, 197, 207, 221, 222,
239 225, 227
milligrams, 194 mucosal barrier, 136
minerals, viii, 43, 49, 154, 233 mucus, 56, 59, 131, 164, 225
Ministry of Education, 158 mucus hypersecretion, 56, 59
mitochondria, 213 multiplication, 58
mitogenic, 55, 61 murine model, 54, 120, 173
mitosis, 166 muscle, 2, 9, 67, 132, 185, 186, 191, 192, 204, 205,
mixing, 89, 92 206, 210, 212, 213
MMP-2, 169 muscle cells, 9, 67
mobility, 174 muscle weakness, 2
model system, 98, 112 mutagenic, 42, 62
modeling, 193, 211 mutation, 36, 190, 207
models, x, 9, 41, 51, 126, 135, 159, 164, 176, 187, mutations, 13
191, 192, 194, 195, 196, 200, 223, 233, 234
modulation, 30, 66, 121, 130, 133, 158, 173, 190,
N
198, 207, 239, 242, 243, 246
modulus, 96
Na+, 132
moisture, 46, 120
N-acety, 229
moisture content, 120
National Research Council, 125, 163
molasses, 45, 55
molecular biology, 218
266 Index
natural, vii, ix, 1, 3, 4, 12, 14, 23, 26, 41, 45, 68, 73, nuclear magnetic resonance, 92
91, 95, 115, 121, 141, 147, 164, 176, 177, 180, nucleation, 97, 99, 102, 104, 105, 106, 110, 111
187, 196, 228, 229 nuclei, 97, 99, 105, 111
natural food, 12, 187 nucleus, 3, 97
natural killer cell, 141 nursing, 118
natural resources, 73 Nusselt, 74
necrosis, 172, 221 nutraceutical, 171
needs, 194 nutraceuticals, vii, xi, 163, 164, 173, 174
neonatal, ix, 61, 115, 117, 122, 130, 148, 217, 221, nutrient, vii, 88, 111, 146, 173, 198, 210, 233, 235
222, 243, 247 nutrients, viii, ix, 37, 59, 87, 88, 115, 118, 121, 135,
neonate, xi, 121, 123, 127, 130, 135, 215, 220, 221, 140, 143, 208, 242
222, 231, 232, 235, 237, 239 nutrition, vii, viii, ix, xi, 2, 14, 34, 38, 39, 62, 87, 88,
neonates, xi, 116, 118, 121, 123, 128, 131, 138, 139, 111, 125, 127, 135, 143, 144, 145, 215, 217, 222,
145, 146, 147, 215, 218, 219, 220, 226, 235, 246 232, 239, 240, 242
neoplasia, 212 nuts, 4, 6, 8, 14, 39
Netherlands, 20, 38, 65, 67, 113, 218
network, 96, 97, 102
O
neuroblastoma, 165
neutralization, 6
oat, 68, 157
neutrophils, 221
obese, 191, 192, 208, 209, 210
New England, 63
obesity, 126, 134, 135, 144, 149, 192, 193, 209, 217
New Jersey, 84
obligate, 13, 36
New York, 36, 37, 38, 39, 40, 63, 64, 111, 112, 113,
obligation, 19
114, 200, 202, 244
Ohio, 85
New Zealand, 16, 203
oil, ix, 4, 6, 8, 10, 16, 18, 21, 22, 23, 26, 30, 31, 32,
Newton, 97
33, 35, 36, 42, 88, 89, 92, 93, 94, 95, 96, 99, 102,
Ni, viii, 71, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 85
105, 107, 108, 110, 112, 145, 171, 178, 182, 183,
Niacin, 48
184, 185, 187, 191, 193, 202, 203, 204, 205, 206,
nickel, 73, 75, 78, 84
209, 212
Nielsen, 208
oils, vii, ix, 1, 4, 5, 6, 8, 16, 18, 21, 22, 23, 25, 26,
nitric acid, 78
29, 34, 88, 89, 92, 176, 178, 182, 183, 185, 189,
nitric oxide, 165, 172
196
nitride, 75
oligofructose, 158, 159, 231, 245
nitrogen, 26
oligomerization, 150
NMR, 92, 93
oligosaccharide, 141, 142, 230, 232, 240, 241, 243
nodes, 234
oligosaccharides, 118, 129, 136, 139, 141, 142, 147,
non-alcoholic fatty liver, 135
148, 150, 154, 157, 159, 160, 217, 229, 230, 231,
non-enzymatic, 213
232, 240, 241, 243, 244
nonionic, 105
Oligosaccharides, 216, 241, 243
nonparametric, 236
olive, 4, 171, 191
nontoxic, 105
Oman, 201
Norfolk, 39
omega-3, 145, 193
normal, ix, xi, 2, 6, 13, 24, 50, 55, 57, 60, 61, 77,
omega-6, 194
105, 115, 117, 123, 159, 164, 175, 186, 187, 189,
online, 33, 34, 173
193, 194, 224, 235, 246
Opioid, 173
normal conditions, 186, 187
optimal health, vii
North Africa, 44
oral, 21, 35, 51, 62, 67, 68, 119, 128, 133, 135, 137,
North America, 44, 122
138, 141, 150, 165, 192, 209, 221, 225, 226, 227,
N-terminal, 164
233, 234, 235, 244, 247
nuclear, 92, 133, 147
orange juice, 24, 31
Index 267
organic, 27, 52, 53, 62, 156 PCR, 121, 129, 134, 137, 146, 148, 156, 216, 230,
organic solvents, 27 236, 242, 244, 245
organism, 50, 133, 202 pectin, 241
osmotic, 224 pediatric, 135, 242, 245
osteoblastic cells, 193 Pennsylvania, viii, 71, 73
osteoporosis, vii, 194 peptide, x, 163, 164, 172
otitis media, 216 Peptide, 54
output, 183 peptides, 51, 52, 67, 132, 164, 172
ovarian cancer, 37 perinatal, x, 125, 127, 135, 137, 143
ovariectomized, 159 peripheral blood, 139, 141, 211, 221, 243
overweight, 191, 209, 240, 242 peripheral blood mononuclear cell, 211, 243
oxidation, 9, 26, 36, 173, 191, 208 peristalsis, 127
oxidation products, 36, 173 peritoneal, 54, 55
oxidation rate, 191 permeability, x, 2, 3, 50, 117, 123, 125, 127, 132,
oxidative, 59 136, 137, 227, 229, 243
oxidative damage, 59 permeation, 67
oxide, 75, 165, 172 peroxidation, 213
oxides, 31, 35 Peroxisome, 207
oxygen, 26, 164 personal, 238
pertussis, 236
PGE, 198, 210
P
pH, 45, 120, 127, 156, 157, 171, 182, 186, 233, 235,
237, 242, 243
p38, 168, 169
pH values, 235
PA, 71, 75
phagocytic, 54
Pacific, 84
pharmaceutical, 21, 91, 136
Pakistan, 43, 65
pharmaceuticals, 105
palm oil, 4, 89, 95, 99, 107, 110
pharmacological, 2, 55
pancreas, 137
phenotype, 126, 134, 142, 169
pancreatic, 24, 105, 131, 132
phenotypes, 203
pantothenic acid, 50
phenotypic, 174
paper, 227, 228, 230, 232
phenylalanine, 50
parameter, 97
phorbol, 190, 207
parasite, 133
phosphate, 78, 85
parents, 129
phospholipids, 10, 23, 91, 157, 164
Paris, 113, 246, 247
phosphorus, 51, 73, 75
PARP, 168
phosphorylation, 168, 169
particles, 75, 78, 85
photographs, 102
passive, 155
physical chemistry, 31
pasta, 16, 18
physical properties, 21, 22, 89, 95, 102, 105
pasteurization, viii, 71, 72, 73, 76, 77, 79, 81, 82, 83,
physicochemical, ix, 63, 88, 89
84, 170
physicochemical properties, ix, 88, 89
pasture, 178, 182, 183, 184, 185, 186, 194, 195, 201,
physiological, vii, 57, 58, 62, 126, 136, 141, 164,
204
187, 190, 193, 194, 196, 200, 212, 241
pathogenesis, 61
physiology, 127, 131, 138, 144
pathogenic, 44, 52, 116, 119, 128, 133, 134, 158
phytochemicals, 37
pathogens, 52, 53, 58, 68, 121, 133, 141, 148, 217
phytohaemagglutinin, 55
pathology, 13
phytosterols, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
pathways, 132, 155, 165, 168, 179
15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28,
patients, 13, 19, 34, 37, 44, 57, 60, 67, 117, 159,
29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42
174, 190, 192, 223, 225, 226, 227, 245
268 Index
pig, 193, 199, 210 postpartum period, 143

pigs, 33, 150, 176, 188, 191, 193, 208, 209, 210, 213 potassium, 26, 51
pilot study, 123, 138, 159, 247 poultry, 194, 195, 211
pitch, 21 powder, x, 21, 41, 84, 120, 163, 170, 224, 226, 227,
placebo, 35, 121, 137, 138, 141, 142, 143, 147, 158, 235
191, 224, 231, 235, 244 power, 62
placenta, 134, 137, 149 PPARγ, 208, 210
placental, 134, 137, 140, 143, 146, 147 Prandtl, 74
planning, 141 prebiotics, x, xi, 126, 135, 136, 147, 154, 158, 159,
plant sterols, vii, 1, 2, 3, 4, 5, 9, 10, 11, 12, 13, 14, 160, 215, 226, 229, 231, 232, 242, 245, 247
15, 16, 17, 19, 21, 22, 29, 30, 31, 36, 37, 38, 39, precipitation, 30
40, 41, 42 preclinical, 126, 136, 138
plants, 3, 4, 29, 31, 72 preference, 44, 60, 148
plaque, 187, 188, 190, 192 pregnancy, ix, 125, 126, 134, 135, 137, 138, 139,
plasma, x, 12, 13, 27, 34, 35, 38, 39, 41, 42, 67, 72, 140, 141, 142, 143, 144, 145, 146, 147, 149
84, 137, 153, 157, 158, 159, 189, 192, 199, 201, pregnant, ix, 19, 125, 126, 135, 136, 138, 140, 141,
202, 205, 206, 209 142, 143, 150, 173
plastic, 89, 102 pregnant women, 136, 138, 140, 141, 142, 143, 150,
plasticity, 90, 203 173
play, 9, 59, 118, 130, 217 premature babies, 239
Poland, 20, 63 premature infant, viii, 43
polarity, 27 prematurity, 128
polarization, 134 premenopausal, 190
poliovirus, 238, 244 preparation, 233
pollen, 173 pressure, 59, 60, 64, 72, 92, 140, 143, 144, 161, 217
polyesters, 106 preterm infants, 117, 122, 123, 128, 150, 173, 227,
polyethylene, 50 233, 240, 243, 244, 246, 247
polymer, 75, 78 prevention, vii, 1, 3, 9, 14, 26, 35, 51, 52, 63, 117,
polymer-based, 75, 78 121, 123, 130, 135, 136, 138, 141, 142, 143, 144,
polymerization, 106 147, 149, 158, 164, 187, 196, 197, 211, 217, 222,
polymorphism, ix, 88, 91, 94, 95, 105, 111, 112, 227, 241, 242, 246, 247
114, 219 preventive, 142, 143, 239
polymorphonuclear, 164 primates, 135, 148
polysaccharide, 44, 55, 59, 63, 65, 66 priming, xi, 215
polysaccharides, 50, 51, 55, 131, 171 principle, xi, 175, 176
polyunsaturated fat, 45, 89, 126, 143, 183, 190, 203, probe, 96
212 probiotic, ix, x, xi, 44, 46, 58, 62, 63, 65, 68, 115,
polyunsaturated fatty acid, 45, 89, 126, 143, 183, 117, 119, 120, 121, 122, 123, 126, 127, 130, 136,
190, 203, 212 137, 138, 139, 141, 142, 143, 144, 145, 147, 148,
polyunsaturated fatty acids, 45, 89, 126, 143, 183, 149, 150, 153, 206, 215, 223, 224, 227, 228, 246,
190, 203 247
population, 9, 13, 39, 48, 56, 57, 58, 61, 68, 117, Probiotics, v, 44, 63, 117, 122, 123, 125, 135, 145,
129, 142, 143, 148, 157, 179, 182, 186, 228, 229, 147, 149, 158, 226, 244
230, 233, 237, 238, 239 procedures, 235
pork, 181 producers, 111
Portugal, 1, 20 production, viii, 21, 45, 49, 50, 54, 55, 61, 62, 63,
positive correlation, 13 66, 69, 71, 90, 119, 130, 133, 146, 159, 165, 171,
postmenopausal, 190, 197 172, 174, 182, 184, 185, 188, 193, 200, 201, 202,
postmenopausal women, 197 207, 211, 221, 222, 225, 231, 236, 243
postpartum, 119, 120, 140, 143 production costs, viii, 71
Index 269
prognosis, 190
Q
program, 83, 134, 143, 164, 235
programming, ix, 125, 126, 127, 135, 136, 143, 144
qualitative differences, ix, 115, 117
proinflammatory, 67
quality of life, 14
pro-inflammatory, 133, 137, 141, 165, 234
Quebec, 111
pro-inflammatory response, 133
prokaryotic, 119
prokaryotic cell, 119 R
proliferation, viii, xi, 43, 44, 54, 55, 60, 61, 142,
157, 162, 164, 166, 174, 188, 190, 197, 207, 215, radiation, 60, 65
221 Radiation, 65
promote, xi, 215, 221, 227 radio, 92
property, viii, 43, 73, 75, 105, 120, 164, 171 radius, 98
prophylactic, 65, 128, 143, 172, 232 rain, 185
propionic acid, 46, 66, 157, 237 random, 111
propylene, 176 range, 13, 14, 16, 52, 54, 77, 82, 90, 91, 95, 130,
prostaglandin, 190, 193, 211, 213 180, 181, 185, 186, 191, 193
prostate, 29, 188 RAPD, 116, 119
prostate cancer, 188 rat, 29, 61, 136, 145, 155, 159, 160, 161, 162, 188,
proteases, 164 192, 196, 197, 199, 206, 207, 208, 210, 213
protection, 9, 50, 60, 149, 164, 170, 188, 194, 216, rats, x, 31, 35, 41, 42, 55, 56, 57, 59, 60, 65, 67, 68,
217, 233 121, 153, 154, 155, 156, 157, 159, 161, 162, 165,
protective mechanisms, 137 173, 188, 189, 190, 191, 192, 193, 197, 206, 207,
protective role, 192 208, 211, 213, 231, 243, 245
protein, viii, x, 23, 31, 35, 43, 45, 46, 49, 50, 52, 54, raw material, 6, 187
57, 58, 60, 68, 72, 84, 132, 133, 163, 164, 166, reactive oxygen, 59
168, 172, 173, 174, 190, 203, 209, 211, 231, 235, reactive oxygen species, 59
241 reactivity, 58
protein denaturation, 72 reality, 35, 130, 141, 144
proteins, vii, x, 11, 15, 16, 23, 35, 45, 49, 50, 51, 62, real-time, 230, 242, 245
72, 123, 133, 146, 163, 164, 165, 166, 169, 172, receptors, 133, 170, 173, 207, 221
173, 233, 241 recognition, 133, 221
Proteobacteria, 156 recovery, 41, 154
proteolysis, 169 redox, 218
proteomics, 164 reducing sugars, 52
protocol, 235 reduction, 182, 183, 184, 185, 187, 188, 190, 191,
protocols, x, 76, 153 197, 207, 217, 224, 234
Pseudomonas, 53, 118 regeneration, 60
Pseudomonas aeruginosa, 53 regression, 9
PTFE, viii, 71, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, regular, 102
85 regulation, 3, 11, 15, 16, 132, 133, 137, 147, 149,
puberty, 205 150, 165, 166, 210, 217, 220, 221, 222, 223, 226,
public health, 56 231, 234, 238
PUFA, ix, 88, 89, 183, 190 regulations, vii, 1, 3, 16, 19
purification, 154 rehydration, 224, 235
pyrene, 212 reinforcement, 14
pyridoxine, 49 rejection, 126, 134
pyruvic, 46 relationship, 13, 40, 41, 126, 130, 134, 184, 237, 238
relationships, 134, 146, 239
relatives, 13
270 Index
relevance, 97 safety, 13, 14, 15, 30, 35, 39, 40, 41, 118, 119, 141,
remodeling, 55 147, 164, 228, 246
repair, 60 salad dressings, 18, 32, 95
replication, 224 Salen, 13, 40
repression, 237, 239 saliva, 164
reproduction, 42 Salmonella, 52, 53, 57
research, xi, 215, 244 salt, 22, 29, 58
research and development, 44 salts, 10, 23, 45, 204, 235
residential, 241 sample, 25, 26, 27, 28, 41, 96, 98, 99, 101, 102, 116,
resin, 171 119, 123, 229
resistance, ix, x, 57, 59, 73, 75, 84, 116, 119, 120, sampling, 120, 230, 238
139, 144, 154, 155, 163, 192, 198, 223, 228 sand, 72
resolution, 25, 27 saponins, 3
resources, 73, 79 SAS, 76
respiration, 220 saturated fat, viii, 45, 87, 88, 89
respiratory, 139, 141, 217 saturated fatty acids, 88
responsiveness, 42 savings, viii, 71, 79, 82, 84
restriction fragment length polymorphis, 219 scanning calorimetry, 94
retail, 211 scientific community, 3, 179
retardation, 92 scores, 45
retention, 131 SD, 236, 237
Reynolds, 74, 79 search, 34
Reynolds number, 74, 79 searching, 217
rheological properties, 91, 95, 97, 110 secrete, 55, 244
riboflavin, 49 secretin, 221
rice, 4, 33, 44, 90 secretion, 11, 24, 132, 142, 145, 148, 204, 221, 222,
risk, viii, ix, 2, 9, 13, 30, 31, 33, 36, 39, 44, 51, 56, 234, 244
87, 88, 115, 117, 125, 126, 127, 134, 135, 139, seed, 8, 26, 36, 92, 99, 102, 105, 182, 183, 201, 205
140, 141, 142, 144, 146, 147, 176, 190, 192, 197, seeding, 58
198, 206, 207, 217, 225, 226, 228, 232, 239, 246 seeds, 4, 6, 8, 14, 39, 89, 105, 178, 183, 186, 202,
risk factors, 192 212
RNA, 131 selecting, 149
roasted coffee, 16, 18 selectivity, 28
rodents, 179 self-care, 45
Romania, 20 semiconductor, 75
room temperature, 21, 22, 23 semimembranosus, 204
ROS, 58 sensing, 133
rotavirus, 50, 154, 224, 241, 246 sensitivity, 97, 134, 188
roughness, 72 sensitization, x, 55, 67, 125, 127, 134, 140, 142, 147,
rubella, 134, 145, 146 228, 246
rubella virus, 134, 145, 146 separation, 27, 34, 199
ruminant, xi, 175, 176, 178, 180, 181, 187, 196 sepsis, 229
Russia, 44 septicemia, 239
rye, 4, 17, 32 sequencing, 154, 160
series, xi, 175, 179
serine, 50
S
serum, 30, 31, 33, 34, 35, 36, 37, 38, 39, 41, 56, 59,
60, 61, 67, 133, 157, 161, 189, 190, 192, 197,
S. thermophilus, 233, 234
206, 208, 211, 221
SA, 235
sesame, 14
Index 271
severity, 41, 42, 57, 143, 150, 235 specificity, 25

sex, 30 spectrophotometric, 25
sex hormones, 30 spectrophotometric method, 25
shape, 101, 102, 105, 131, 146, 223 spectroscopy, 77
shaping, 126 spectrum, viii, 43, 54, 60, 66, 80
shear, 78, 79, 96 speculation, 207
sheep, 45, 56, 68, 179, 212 speed, 220, 226
Shigella, 52, 53, 57 S-phase, 173
short-term, 38, 189, 205 spleen, 51, 137, 193
shoulder, 95, 106 Sprague-Dawley rats, 31
sialic acid, 229 squamous cell, 31
sibling, 218 squamous cell carcinoma, 31
siblings, 127, 129, 218, 219 stability, viii, xi, 27, 41, 85, 87, 88, 91, 120, 163,
SIC, 125 171, 174
side effects, 2, 36, 60 stages, x, 49, 98, 126, 127, 135, 144, 190
signaling, 58, 133, 147, 169, 210 stainless steel, viii, 71, 73, 74, 75, 85, 96
signalling, x, 133, 163, 165 standard deviation, 107
signals, 95, 106, 137 Standards, 16
signs, 78, 81, 116 staphylococcal, 144
silica, 84 staphylococci, 117, 218, 220
siloxane, 27 Staphylococcus, 52, 118, 129, 148
silver, 177, 199 Staphylococcus aureus, 52, 118, 148
similarity, 106, 111, 138 statin, 31
single crystals, 102 statins, 2, 30
sites, 10, 52, 53, 54, 68, 130, 137, 164, 176 steel, viii, 71, 73, 74, 75, 78, 81, 85, 96, 154
skeletal muscle, 132, 192 stem cells, 59
skin, 45, 59, 118, 119, 137, 148, 176, 188, 190, 207 sterile, ix, 115, 116, 117, 122, 127, 217
skin cancer, 59 steroid, 2, 3, 161, 162
Slovakia, 20, 205 steroid hormone, 2
Slovenia, 21 steroid hormones, 2
small intestine, 137, 155, 179 steroids, 157
smooth muscle cells, 9 sterols, vii, 1, 3, 4, 9, 10, 12, 13, 15, 16, 21, 24, 25,
software, 74 26, 27, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 41,
solid solutions, 94 91, 161
solid state, 22 stock, 154
solid tumors, 51 stomach, 29, 63, 105, 128
solubility, 2, 10, 21, 22, 23, 24, 35, 156 storage, xi, 46, 52, 57, 62, 63, 95, 97, 110, 120, 124,
solvent, 26, 89, 91, 97 132, 144, 163, 174, 175, 186, 187, 191, 206
solvents, 21, 27 strain, xi, 51, 53, 66, 96, 116, 117, 119, 122, 130,
South Africa, 64, 68 137, 141, 148, 157, 160, 191, 216, 223, 228, 232,
soy, xi, 18, 21, 39, 41, 45, 50, 51, 163, 212 243, 246
soybean, 39, 44, 89, 176, 182, 183, 185, 202, 203, strains, ix, x, 50, 51, 53, 63, 66, 68, 115, 117, 119,
205 121, 122, 123, 126, 127, 141, 142, 144, 148, 149,
soybeans, 184, 202, 203, 204, 205 179, 201, 218, 223, 225, 228, 233
Spain, 21, 60, 118, 125 strategies, x, 2, 126, 127, 130, 136, 186, 205
Spearman rank correlation coefficient, 236 strength, 75, 78, 198
species, ix, xi, 48, 52, 58, 59, 63, 65, 115, 116, 117, streptococci, 59, 117, 118, 218, 220
118, 128, 129, 136, 146, 161, 179, 203, 216, 218, Streptomyces, 41
219, 220, 223, 226, 230, 236, 238, 239, 241, 242, stress, 96, 132, 148, 209, 242
243, 245, 246 subgroups, 143
272 Index
substitutes, 18 systolic blood pressure, 217

substitution, 16
substrates, xi, 3, 62, 215, 238
T
sucrose, 47, 105, 106, 109, 110, 111, 113, 154
suffering, 135, 173, 225, 226
T cell, 133, 134, 138, 142, 146, 172, 221, 222
sugar, 6, 45, 46, 50, 57, 154, 157, 159, 229, 237
T lymphocyte, 221, 235
sugars, 44, 52, 156, 232
Taiwan, 64
sulfuric acid, 84
taste, vii, 2, 43, 44, 45
sulphate, 131
taxonomic, 244
sulphur, 52
taxonomy, 244
summer, 45, 184
T-cell, 133
Sun, 41
Teflon, 75, 78
sunflower, ix, 31, 88, 89, 92, 93, 94, 95, 96, 99, 102,
temperature, 21, 23, 57, 73, 76, 77, 82, 90, 91, 92,
105, 108, 176, 182, 183, 185, 191, 201, 205, 206
94, 95, 98, 99, 100, 101, 102, 104, 106, 107, 110,
supercooling, 97, 99, 102, 104, 106
154, 187
supercritical, 26, 91
temperature gradient, 77
supernatant, 55, 61
tetanus, 236
superoxide, 59
tetracycline, 45
supplemental, 193
TGF, ix, 115, 118, 133, 139, 140, 142, 221, 225, 228
supplements, viii, xi, 17, 18, 87, 88, 171, 175, 178,
T-helper cell, 228
182, 185, 203, 226
therapeutic, 232
suppliers, 4
therapy, 60, 123, 247
supply, viii, 87, 88, 130, 135, 183
thermal energy, viii, 71, 72, 76, 79, 82, 83
suppression, 51, 52, 157, 158
thermal expansion, 78
surface area, 131
thermal properties, 75, 94, 95
surface energy, 75, 98
thermal stability, 27, 85
surface modification, 84
thermodynamic, 97, 99, 101
surface properties, 72, 85
Thermophilic, 49
surface roughness, 73
thiazolidinediones, 192
surfactant, 64
threat, 228, 232
surgical, 75
threonine, 50, 131
surveillance, 14, 19, 242
thromboxane, 33
survival, 127, 234, 236, 242
thymocytes, 55, 174
susceptibility, x, 125, 127, 236, 244
thymus, 235, 243
suspensions, 55, 120
Tibet, 68
sustainability, 41
tight junction, 123, 155, 160
Sweden, 21, 38, 191, 225
time, 218, 219, 228, 229, 230, 237, 238
Switzerland, 39, 42, 111, 120
time-frame, 144
symbiotic, 48, 121
timing, 197
symbols, 100
tissue, 55, 59, 133, 176, 177, 179, 180, 184, 186,
symptom, 227
187, 190, 191, 198, 201, 205, 206, 208, 209, 211,
symptoms, ix, 115, 117, 227, 231, 243
212, 213, 221
synbiotics, 135
titanium, 84
synergistic, 234
Titanium, 84
synergistic effect, 234
TLR, 133, 137, 216, 221, 222, 234
synthesis, x, xi, 3, 12, 13, 41, 56, 132, 153, 157, 160,
TLR2, 133, 137, 222
161, 165, 175, 178, 179, 182, 184, 185, 186, 188,
TLR4, 137, 222
189, 193, 201, 202, 204
TLR9, 147
systemic immune response, 57
TNF, 133, 137, 139, 141, 216, 221, 234
systems, 184, 236
TNF-α, 133
Index 273
Tocopherol, 40, 90 trypsin, 131

toddlers, 225, 243 tryptophan, 50
Tokyo, 65 Tryptophan, 48
tolerance, viii, ix, 43, 58, 62, 115, 117, 122, 123, tumor, 51, 52, 62, 65, 67, 157, 172, 188, 189, 190,
133, 135, 140, 143, 149, 188, 192, 221, 224, 225, 195, 197, 207, 221
227, 228, 232, 234, 244, 246, 247 tumor cells, 51, 67, 188, 207
Toll-like, 133, 134, 149, 221, 222, 242 tumor growth, 52, 62, 65, 189, 195
tomato, 77, 84 tumor metastasis, 190, 197
total cholesterol, x, 2, 56, 153, 157, 158 tumor necrosis factor, 172, 221
total energy, 72, 82, 209 tumorigenesis, 173, 188
toxic, 155 tumors, 51, 165, 174, 188, 198
toxic effect, 155 tumour, 51, 55, 64
toxicity, 2, 35 tumour growth, 55
toxin, 55, 239 tumours, 51, 55
trachea, 193, 210 Turkey, 181
tradition, 44, 49 TVA, 178, 179, 181, 182, 183, 184, 189, 190, 196
training, 198 twins, 130
trans, viii, xi, 87, 88, 89, 93, 111, 112, 113, 114, type 1 diabetes, 217, 239
175, 176, 177, 178, 198, 199, 200, 202, 203, 204, type 2 diabetes, 134, 192, 193
205, 206, 208, 212 tyrosine, 48
transcription, 133, 173
transcription factor, 173
U
transcriptional, 132, 146
transesterification, 35
ulcerative colitis, 158
transfer, 35, 72, 74, 79, 135, 138, 139, 146, 148, 149,
ultrasound, 235
185, 225, 228
ultraviolet, 28
transference, 27, 138
umbilical cord blood, 116
transformation, 27, 105
uncertainty, 76
transforming growth factor, 221
undernutrition, 135, 151
transgenic, 4, 42
unfolded, 72
transgenic mice, 42
United Kingdom, 17, 21, 240
transgenic plants, 4
United Nations, viii, 87, 88
transition, 95, 105
United States, 14, 29, 31, 39, 186, 191, 245
transitions, 95
uric acid, 46
translocation, 223, 229, 232, 246
Uruguay, 31
transmission, 116, 127, 129, 137, 144, 211, 217, 242,
USDA, 75, 196
245
Utah, 175, 196
transparent, 102
uterus, ix, 125, 126, 134
transplantation, 174
UV, 28, 59, 65
transport, 11, 155, 159, 188, 192, 210, 244
trees, 21, 89
Tregs, 133 V
triacylglycerols, 114, 132
trial, 35, 67, 76, 81, 121, 123, 143, 147, 150, 158, vaccination, 235
220, 224, 227, 230, 231, 233, 234, 235, 241, 243, vaccine, 123, 236
244, 246 vacuum, 22
triggers, 148 vagina, 118, 122
triglyceride, 189, 201 Valdez, 64
triglycerides, 157, 178, 192 Valencia, 125
Triglycerides, 157 validity, vii
274 Index
valine, 50 wear, 73, 79, 85

values, xi, 41, 48, 60, 74, 76, 94, 95, 96, 102, 106, web, 119
141, 156, 176, 180, 194, 219, 235, 237, 238 weight gain, viii, 43, 226
vancomycin, 228 weight loss, 193
vapor, 50 Weinberg, 164, 174
variable, 185, 191 well-being, ix, x, 115, 118, 153
variables, 210 wellness, 14
variance, 76 western blot, 166, 169
variation, 99, 185, 201, 203, 242 western diet, xi, 175
vascular, 239 wettability, 72
vascular disease, 35, 39, 239 wheat, 4, 41, 66, 159
vascular diseases, 239 wheat germ, 4
vegetable oil, ix, 4, 6, 8, 16, 18, 21, 23, 25, 29, 34, wheezing, 143
40, 42, 88, 89 whey, x, 45, 50, 153, 154, 158, 163, 164, 165, 173,
vegetables, 4, 5, 6, 7, 38 233, 244, 246
vegetarians, 9 WHO, 2, 42, 88, 111, 135, 145, 216, 247
vegetation, 184 winter, 45, 184
velocity, 77, 96 Wisconsin, 113, 114, 176
villus, 131 Wistar rats, 161
viral, 217, 224 withdrawal, 36, 117, 166, 198
viral gastroenteritis, 217 witness, 219, 223, 225
virus, 133, 134, 145, 146, 236, 245 women, 19, 39, 40, 42, 51, 61, 116, 118, 119, 120,
virus infection, 134 121, 122, 134, 135, 136, 138, 139, 140, 141, 142,
viscoelastic properties, 50, 102 143, 150, 173, 188, 190, 191, 197, 213, 217
viscosity, 50, 96, 97, 105 wood, 21, 42
visible, 19, 101 workers, 2, 9, 11, 12, 13, 25, 26
vision, 19 World Health Organization (WHO), viii, 87, 88
vitamin A, 213 writing, 3
vitamin B1, 49
vitamin B12, 49
X
vitamin B2, 50
vitamin B6, 50
xenobiotics, 126
vitamin C, 50
XPS, 77, 80, 81
Vitamin C, 49
X-ray diffraction (XRD), 90, 95, 106
vitamin D, 2
vitamin E, 45, 59, 205
vitamin K, 50 Y
vitamins, viii, 43, 46, 49, 59, 61, 131, 233
VLBW, 216, 220, 226 yeast, 44, 45, 63, 66, 67
VLDL, 208 Yeasts, 49
vulnerability, 225 Yemen, 89
yield, 96, 182, 183, 185, 204
yogurt, x, 16, 17, 18, 24, 44, 51, 57, 60, 62, 66, 139,
W 141, 163, 170, 180, 186, 206, 212
warrants, 238
water, 10, 21, 23, 26, 27, 33, 50, 51, 55, 58, 62, 72, Z
73, 82, 84, 131, 136, 154, 176
water vapor, 50 zinc, 48, 51, 160
water-soluble, 51, 55, 58
weakness, 2

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NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Chapter IX Conjugated Linoleic Acid: An Anticancer Fatty Acid Found

by the genome of the microbiota (microbiome). Moreover, microbial exposure through

Plant Sterols and Plant Stanols in Milk

Fernando Ramos* and David Saraiva

2. Plant Sterols and Plant Stanols

2.1. Nomenclature, Chemical Structures and Properties

2.2. Natural Sources of Phytosterols

Figure 1. Representative figure of structures of 4,4-dimethylsterols, 4 –methylsterols and 4-desmethylsterols

Table I. Phytosterols in cereals and derived products, mg/100 g edible portion

Cereal grains Total phytosterols

Fruit Total phytosterols

Vegetables Total phytosterols

Vegetable oils Total phytosterols

Nuts and seeds Total phytosterols

2.3. Estimated Average Intakes of Phytosterols

2.4. Prevention of Cardiovascular Diseases

2.4.1. Mechanisms of Cholesterolemia Reduction

2.4.1.1. Competition between Cholesterol and Phytosterols for Mixed

2.4.1.2. Phytosterols and Cholesterol Co-crystallization

2.4.1.3. Reducing Cholesterol Absorption via Competition with Cholesterol

2.4.1.4. Inhibition of Enzymes Involved in Phytosterols Absorption Process

2.5. Hypocholesterolemic Comparison between Plant Sterols

Whether plant sterols or stanols have a larger hypocholesterolemic effect is subject of

cholesterol-lowering efficacy of plant sterols diminished over time (comparing plasma

2.6. Phytosterols Safety Use

Finally, supplementary phytosterols safety information collected during, at least, 5 years

3. Milk and other Dairy Products Enriched

Beneficial effects on blood cholesterol reduction by phytosterols enriched foods have

Phytosterols Percentage (%)

Additional EU regulations 2004/333/EC, 2004/334/EC, 2004/335/EC, 2004/336/EC and

• milk present no more than 1.5g of total fat per 100g;

3.2. Market of Phytosterols Enriched Foods

3.2.1. Authorized Foods

Table VII. Phytosterol-enriched novel foods available on EU marketup to 2008

Food type Status

3.2.2. Market Characterization

Table VIII. Phytosterol-enriched foods and GRAS status in U.S.A up to 2008

Food type Status

Following the SCF recommendations, the European Commission adopted specific

• the easiness to be divided in portions (each one containing a maximum of 1g (3

3.4. Intake Recommendations

Product category Common packing Recommended Phytosterol Daily intake

Table X. Phytosterol-enriched dairy products available in EU at the end of 2007

Member State Milk products

3.5. Technological Aspects

3.5.1. Phytosterols Formulations

3.5.1.1. Esterified Phytosterols Formulations

3.5.1.2. Free Phytosterols Formulations

3.6. Phytosterols Alimentary Matrices

Margarines were the first commercial applications of phytosterols enriched foods.

3.7. Phytosterols Analytical Methodologies