Milk Intelligence Mining Milk For Bioactive Substances Associated With Human Health - 2011 - International Dairy Journal PDF

International Dairy Journal 21 (2011) 377e401
Contents lists available at ScienceDirect
International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj
Review
Milk intelligence: Mining milk for bioactive substances associated

with human health
S. Mills a, R.P. Ross a, b, c, C. Hill a, c, d, G.F. Fitzgerald a, c, d, C. Stanton a, b, c, *
a
Food for Health Ireland, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland
Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland
c
Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
d
Department of Microbiology, University College Cork, Ireland
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2010
Received in revised form
16 December 2010
Accepted 20 December 2010
Milk has evolved as a complete food for the mammalian nourishment of its young. However, research is
unveiling an ever-accumulating range of bioactivities associated with milk substituents, emphasizing
a role in programming human health. One good example is the increased complexity of carbohydrates in
colostrum that may have a controlling inuence on the selection of gut microbiota in infants at a very early
stage of life. Milk can also affect processes outside the human gut e a proven example is the hypotensive
effect of milk bioactive peptides through angiotensin-I-converting enzyme (ACE) inhibition. However,
even more intriguing is the potential of milk constituents to inuence immune and neural networks
thereby affecting infection rates or mood, respectively. With the advent of bovine and human sequencing
omic technologies, scientists are set to unlock many of the mysteries/mechanisms of how milk is good for
you in ways that up to now were impossible to comprehend.
2011 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .378
Milk proteins and peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .378
2.1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
2.2.
Antihypertensive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
2.3.
Antithrombotic peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
2.4.
Opioid peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
2.5.
Casein phosphopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
2.6.
Immunomodulatory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
2.7.
Antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
2.8.
Cytomodulatory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
2.9.
Cysteine protease inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
2.10.
Mammary-associated serum amyloid A3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
3.1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
3.2.
Prebiotic effect of human milk oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
3.3.
Milk oligosaccharides and infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
3.4.
Milk oligosaccharides and the inflammatory immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
3.5.
Sialic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Milk lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
4.1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
4.2.
Milk fat globule membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
4.3.
Phospholipids and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
* Corresponding author. Teagasc, Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland. Tel.: 353 25 42606; fax: 353 25 42340.
E-mail address: Catherine.stanton@teagasc.ie (C. Stanton).
0958-6946/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2010.12.011
378
5.
6.
7.
S. Mills et al. / International Dairy Journal 21 (2011) 377e401
4.4.
Phospholipids and the immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
4.5.
Phospholipids and the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
4.6.
Polyunsaturated fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
4.7.
Conjugated linoleic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
4.8.
Increasing the conjugated linoleic acid content of milk and dairy foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
4.9.
Increasing eicosapentaenoic acid and docosahexaenoic acid content of milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
4.10.
Butyrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
4.11.
Medium chain fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
4.12.
Antimicrobial lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
Dairy calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .391
Microbial diversity inscribed in human milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
Conclusion and outlook for the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
1. Introduction
The relationship between diet and health is now well known to
be one of the keys to preventing disease and promoting wellbeing.
Indeed, it is on this basis that there has been major growth in the
market for functional foods. These are foods that exert a positive
inuence on human health over and above their nutritive value.
Dairy products hold a major share in this market, owed partly to the
fact that the manufacturing process of fermented dairy products
involves the addition of lactic acid bacteria (LAB), many of which
are now known to possess probiotic properties (Fuller, 1989) or
which themselves produce secondary metabolites with associated
health-promoting effects (Stanton, Ross, Fitzgerald, & Van Sindern,
2005). In addition, it is well known that milk itself is a rich source of
bioactive components that positively inuence host health. This is
probably not surprising given that milk can be considered the
ultimate food, being a complete food for newborns and the sole
food during the early stages of development.
Bovine milk is composed of approximately 5% lactose, 3.2%
protein, 4% lipid and 0.7% mineral salts (Sverin & Wenshui, 2005)
although the exact composition varies in response to a number of
factors including animal nutrition and stage of lactation. While milk
has always been known to have a pivotal role in bone and dental
health (Bonjour, Chevalley, Ferrari, & Rizzoli, 2005; Huncharek,
Muscat, & Kupelnick, 2008; Merritt, Qi, & Shi, 2006; Moore,
Bradlee, Gao, & Singer, 2008), more recent evidence has revealed
that it contains a plethora of components that encode functional
health benets far beyond that expected based on the nutritional
content alone. These encode specic lipids, complex carbohydrates
and peptide sequences encrypted within milk proteins that exert
activities that affect blood pressure or mood.
Milk provides the ultimate model for functional food development being endowed with nutritional, immunological and
biologically active components (Wong, de Souza, Kendall, Emam, &
Jenkins, 2006). Indeed, many milk components are now being
exploited as health-promoting ingredients in other food systems.
An extreme example is the production of recombinant human
milk proteins in plants with a view to developing food products
with enhanced nutrition for formula-fed infants as well as older
children and adults (Arakawa, Chong, Slattery, & Langridge, 1999).
Milk oligosaccharides have been shown to modulate both the
intestinal microbiota and the immune response, thus playing key
roles in defence against infection and allergy development in
formula-fed infants. In addition, milk lipids also possess therapeutic properties and efforts have been made to enhance the
presence of certain fatty acids in bovine milk, in particular the
bioactive lipid conjugated linoleic acid (CLA). Another particularly
exciting development is the Milk Genome Consortium that is
devoted to the understanding of how the biomolecules of milk are
manufactured and regulated and how they achieve their specic

functions (Ward & German, 2004).
This review aims to discuss the specic components of bovine
and human milk that have been associated with human health
through mechanisms that exceed basic nutrition. In particular, we
have focused on some specic examples derived from the protein,
namely bioactive peptides, the polysaccharide and lipid constituents of milk as well as dairy calcium. Finally, this review looks at
some exciting data highlighting the microbial diversity inscribed in
human milk for the benet of the infant.
2. Milk proteins and peptides
2.1. Introduction
The major protein fractions in bovine milk consist of caseins
and whey proteins including immunoglobulins, a-lactalbumin,
b-lactoglobulin, bovine serum albumin, immunoglobulin, lactoferrin
as well as proteoseepeptone fractions and transferrin (Table 1).
Lower amounts of other minor proteins and peptides also exist that
have for example, hormonal or other physiological activities, e.g.,
hormone releasing factor and the endogenous antibacterial system
(Sverin & Wenshui, 2005). In the last couple of decades, much
Table 1
Concentration and biological activity of major bovine milk proteins.a
Protein
Concentration
(g L1)
Function
Total caseins
26.0
a-casein
b-casein
k-casein
b-Lactoglobulin
13.0
9.3
3.3
6.3
3.2
a-Lactalbumin
1.2
Immunoglobulins
(A, M, and G)
Serum albumin
Lactoferrin
0.7
Ion carrier (Ca, PO4, Fe, Zn, Cu),

precursor of bioactive peptides
Precursor of bioactive peptides
Anticarcinogenic, weight management
Retinol carrier, binding fatty acids,
possible antioxidant
Lactose synthesis in mammary gland,
Ca carrier, immunomodulation
Immune protection
0.4
0.1
Lactoperoxidase
Lysozyme
0.003
0.0004
Miscellaneous
Proteoseepeptone
0.8
1.2
Glycomacropeptide
1.2
Total whey protein
Antimicrobial, antioxidative,
immunomodulation, iron absorption
Antimicrobial
Antimicrobial, synergistic effect
with immunoglobulins and lactoferrin
Function unknown but precursor
of bioactive protein and peptide in vitro
Antiviral, bidogenic
Adapted from Sverin and Wenshui (2005); see Walstra and Jenness (1984),
Yamauchi (1992) and Korhonen, Pihlanto-Leppala, Rantamaki, and Tupasela
(1998) for more details.
research has concentrated on the therapeutic potential of milk

proteins (Madureira, Pereira, Gomes, Pintado, & Malcata, 2007;
Teschemacher, Koch, & Brantl, 1997; Zimecki & Kruzel, 2007). For
example, it has been demonstrated that whey protein is superior to
other dietary proteins for suppression of tumor development, due to
components such as lactoferrin, b-lactoglobulin, a-lactalbumin and
serum albumin (Parodi, 2007). Indeed, under acidic conditions in the
presence of oleic acid, a-lactalbumin and oleic acid form a complex,
termed HAMLET (human a-lactalbumin made lethal to tumor cells)
which has been shown to inhibit a wide array of tumors via an
apoptosis-like event (Svanborg, Agerstam, Aronson, Bjerkvig, &
Duringer, 2003). The bovine counterpart of HAMLET, termed BAMLET, was recently shown to display potent cytotoxic activity against
eight cancer cell lines tested via a mechanism involving lysosomal
membrane permeabilisation (Rammer et al., 2010). A specialized
whey fraction (high in leucine, bioactive peptides and milk calcium)
with the commercial name Prolibra has been shown to promote
loss of body fat mass and greater preservation of lean muscle in
a randomized human clinical trial lasting 12 weeks (Frestedt, Zenk,
Kuskowski, Ward, & Bastian, 2008). Indeed, the utilisation of whey
as a functional food ingredient for weight management is attracting
much interest (Luhovyy, Akhavan, & Anderson, 2007). The special
membrane structure surrounding milk micro-lipid droplets,
composed of a lipid bilayer and proteins, termed the milk fat globule
membrane (MFGM), has also shown potential as a therapeutic agent
against many pathological conditions (Spitsberg, 2005). One of the
proteins isolated from MFGM, termed fatty acid binding protein
(FABP) has been shown to inhibit some breast cancer cell lines
(Spitsberg & Gorewit, 1997a, 2002; Spitsberg, Matitashvili, &
Gorewit, 1995). In addition, the onco-suppressor protein BRCA1
has also been found in both bovine and human MFGM (Spitsberg &
Gorewit, 1997b). Wang, Hirmo, Millen, and Wadstrom (2001c) also
demonstrated that glycoproteins of MFGM were capable of inhibiting the infection of Helicobacter pylori in a BALB/cA mouse model,
and the hemagglutination and adhesion of H. pylori in HeLa S3
379
monolayers. A 19 kDa protein derived from the degradation of proteoseepeptone component 3 following fermentation of fat-free milk
by a number of LAB enhanced monoclonal antibody production of
human hybridoma cells and stimulated immunoglobulin production
of human peripheral blood cells (Sugahara et al., 2005). Recently, the
basic protein fraction of milk termed milk basic protein (MBP) has
also been shown to have a direct effect on the strengthening of bones
in healthy human volunteers (Toba et al., 2001; Uenishi et al., 2007).
Indeed, various proteomic approaches are now being applied to
decipher the subcellular organization of the MFGM as discussed in
a review by Cavaletto, Giuffrida, and Conti (2008) and new studies
are revealing that differences in protein expression even occur in
MFGM between colostrum and day 7 milk (Reinhardt & Lippolis,
2008).
Bioactive peptides which exert numerous physiological
responses can also be generated from milk proteins in the gastrointestinal tract. Such bioactive peptides are latent or encrypted
within native protein precursors, thus proteolysis is required for
their release (Gobbetti, Minervini, & Rizzello, 2004). Indeed,
bioactive peptides have been generated from most of the major
proteins in both bovine and human milk (Sverin & Wenshui,
2005). Bioactive milk peptides were rst described in 1950, when
Mellander (1950) reported that ingestion of casein-derived phosphorylated peptides led to enhanced vitamin D-independent
calcication in rachitic infants. While bioactive peptides can be
generated from a variety of foods, milk proteins are generally
regarded as a very rich source and as a result have become
fundamental constituents of several commercially available functional food products and ingredients (Table 2). Bioactive peptides
from milk can be divided into the following categories based on
their physiological effect on the body or the protein from which
they have been derived; antihypertensive, antithrombotic, opioid,
casein phosphopeptides (CPPs), antimicrobial, cytomodulatory,
immunomodulatory, and miscellaneous peptides (Hayes, Stanton,
Fitzgerald, & Ross, 2007a).
Table 2
Milk-derived bioactive peptides in commercially available functional foods and ingredients.a
Bioactive peptide
Health claim
Product type
Brand name
Manufacturer
VPP, IPP from b-casein and k-casein

VPP, IPP from b-casein and k-casein
Reduction of blood pressure

Calpis
Evolus
Calpis Co., Japan

Valio, Finland
Whey peptides
BioZate
Davisco, USA
Casein-derived dodecapeptide FFVAPFPEVFGK

Casein-derived dodecapeptide FFVAPFPEVFGK
Glycomacropeptide k-casein f (106-169)
C12 Peption
Casein DP Peptio Drink
BioPURE-GMP
DMV, The Netherlands

Kanebo, Japan
Davisco, USA
as1-casein f (91e100) YLGYLEQLLR

Anticariogenic, antimicrobial,
antithrombic
Reduce stress
Sour milk
Fermented milk,
calcium enriched
Hydrolysed whey
protein isolate
Ingredient
Soft drink
Whey protein isolate
CSPHP ProDiet F200
Ingredia, France
Casein phosphopeptide
Glutamine-rich peptides
Helps mineral absorption

Immunomodulatory
Capolac
Tekkotsu Inryou
Kotsu Kotsu Calcium
CE90CPP
Glutamine Peptide
Arla Foods, Denmark

Suntory, Japan
Asahi, Japan
as1-casein f (1e6) RPKHPI, f (1e7)
Milk drink,
confectionary
Ingredient
Soft drink
Soft drink
Ingredient
Dry milk protein
hydrolysate
Fermented low
fat hard cheese
Ingredient
Ingredient
Festivo
Cysteine Peptide
PeptoPro
MTT Agrifood
Research, Finland
DSM, The Netherlands
Ingredient
Vivinal Alpha
Chewing gum
Margarine
Recaldent
Evolus Double
Effect Spread
RPKHPIK, f (1e9) RPKHPIKHQ,

Milk-derived peptide
Casein-derived peptide
Whey-derived peptide
Boost energy, improve sleep quality

Improves athletic performance
and muscle recovery
Aids relaxation and sleep
Casein phosphopeptides
Milk-derived peptides
Anticariogenic
Adapted and updated from Korhonen (2009), Korhonen and Pihlanto (2006), Hartmann and Meisel (2007); f denotes peptide fragment.
Borcula Domo Ingredients

(BDI), The Netherlands
Cadbury Enterprises
Valio, Finland
380
2.2. Antihypertensive peptides

Antihypertensive peptides inhibit ACE, a key enzyme involved in
the regulation of blood pressure (BP). These ACE inhibitors are
thought to be competitive inhibitors of ACE by preventing ACE from
synthesising the potent vasoconstrictor, angiotensin-II. In addition,
ACE also hydrolyses bradykinin, a vasodilator (Seppo, Jauhiainen,
Poussa, & Korpela, 2003). Milk fermentations using LAB or their
proteinases have been described as a strategy to release ACEinhibitory peptides from milk proteins, especially caseins (Hayes,
Ross, Fitzgerald, Hill, & Stanton, 2006; Hayes et al., 2007a, 2007b).
ACE inhibitory and antihypertensive peptides from dairy origin
contain up to 10 amino acids (Korhonen & Pihlanto, 2003; Meisel &
Bockelmann, 1999; Pihlanto, 2001; Saito, 2008). These peptides
have already been marketed (Table 2). For example, the ACEinhibitory peptides Val-Pro-Pro (Clare, Catignani, & Swaisgood,
2003; Hamel, Kielwein, & Teschemacher, 1985; Juillard et al.,
1995) and Ile-Pro-Pro (Chabance et al., 1995; Drouet et al., 1990)
derived from casein following fermentation with the strains
Lactobacillus helveticus and Saccharomyces cerevisiae, respectively,
are found in the commercial sour milk product, Calpis (Calpis, Co.
Ltd., Tokyo). The antihypertensive effect of Calpis was shown to
yield a systolic BP decrease of 17.7 mm Hg following administration
of 5 mL kg1 body weight of Calpis sour milk drink over an 8-h
period in spontaneously hypertensive rats (Yamamoto, Akino, &
Takano, 1994). Likewise, a signicant reduction in BP was
obtained in mildly hypertensive patients following oral consumption of 95 mL Calpis over an 8-week period (Hata et al., 1996).
More recently, casein hydrolysate containing Val-Pro-Pro and IlePro-Pro has been shown to improve vascular endothelial dysfunction in subjects with mild hypertension (Hirota et al., 2007).
Various traditional Swiss cheese varieties have also recently been
shown to contain, on average, similar concentrations of Val-Pro-Pro
and Ile-Pro-Pro to the recently developed fermented milk products
with BP-lowering properties (Table 2) (Butikofer, Meyer, Sieber,
Walther, & Wechsler, 2008). Moreover, bioavailability studies by
Foltz et al. (2007) demonstrated that the tri-peptide, Ile-Pro-Pro,
selectively escapes from intestinal degradation and reaches the
circulation undegraded. It was also demonstrated that Val-Pro-Pro
and Ile-Pro-Pro have the potential to inhibit ACE in a very similar
fashion to the current synthetic ACE inhibitors Captopril, Enalaprilat and Lisinopril, by hydrogen-bonding with similar residues in
the ACE catalytic site (Pina & Roque, 2008). Interestingly, antihypertensive peptides have now been produced using recombinant
DNA technologies, whereby recombinant fusion proteins have been
expressed in Escherichia coli, which are then puried and cleaved by
proteinase from a selected strain of L. helveticus (Losacco, Gallerani,
Gobbetti, Minervini, & De Leo, 2007). This technology will enable
scientists to design new peptides with even stronger inhibitory
activity and new therapeutic properties. Additionally, ACE inhibitors derived from casein or casokinins have been identied within
the sequences of human b- and k-casein (Kohmura, Nio, & Ariyoshi,
1990; Kohmura et al., 1989) and have also been generated by tryptic
digestion of a- and b-casein (Maruyama & Suzuki, 1982). Interestingly, as suggested by Hayes et al. (2007a) delivery of these
bioactive peptides to target organs may be enhanced by using
larger peptide sequences, which upon in vivo digestion with
proteinase and peptidase activities should release the antihypertensive peptide in the gastrointestinal tract.
2.3. Antithrombotic peptides
Antithrombotic peptides that interfere with the formation of
thrombi have also been identied in milk (Fiat, Migliore, & Jolles,
1993; Jauhiainen & Korpela, 2007; Zimecki & Kruzel, 2007).
Indeed, enzymatic hydrolysis of k-casein has resulted in some of the

most antithrombotic peptides to date from food sources. Thrombosis is a pathological condition that results in clots or thrombus
formation in arteries, veins or the chambers in the heart. From
a dairy point of view, it is interesting to note that comparisons can be
drawn between the mechanisms involved in milk clotting, dened
by the interaction of k-casein with chymosin and the mechanisms of
blood clotting, dened by the interaction of brinogen with
thrombin (Jolles, 1975; Jolles & Henschen, 1982; Rutherfurd & Gill,
2000). Having found structural similarities between bovine
k-casein and the human brinogen g-chain, Jolles, LoucheuxLefebvre, and Henschen (1978) hypothesised that both may have
evolved from a common ancestor during the past 450 million years.
Indeed, Jolles et al. (1978) demonstrated that several long k-casein
sections corresponding to 80% of the whole protein molecule have
counterparts in the brinogen g-chain, with 31e42% of the amino
acid residues occupying the same positions. In addition, several
common features were observed in the secondary structures of the
k-caseins and the brinogen g-chain (Jolles et al., 1978). Moreover,
the C-terminal sequence of the brinogen g-chain fragment (f)
(400e411) corresponding to the sequence HHLGGAKQAGDV
inhibits platelet aggregation and brinogen binding to ADP-activated platelets (Andrieux et al., 1989). The main antithrombotic
peptide isolated from bovine k-casein corresponding to f (106e116)
with the amino acid sequence MAIPPKKNQDK termed casoplatelin,
was also shown to inhibit ADP-induced platelet aggregation and
brinogen binding in a concentration-dependant manner (Jolles
et al., 1986) compounding the functional similarity between kcasein and brinogen.
Interestingly, it is thought that milk protein-derived antithrombotic peptides are absorbed into the bloodstream. For example,
two peptides from human and bovine k-caseinoglycopeptide have
been identied in the plasma of 5 day old newborns following
ingestion of a cow milk-based formula (Chabance et al., 1995). It is
also interesting to note that k-casein f (152e160) and f (155e160)
isolated as ACE inhibitors may also possess antithrombotic activity
(Gobbetti et al., 2004).
2.4. Opioid peptides
Opioid peptides are those peptides having pharmacological
similarities to opium. The caseins (as1-, as2,- b- and k-) and whey
proteins are potential sources of such opioid peptides. However, the
major opioid peptides are fragments of b-casein, called b-casomorphins (Clare & Swaisgood, 2000; Teschemacher et al., 1997). Similar
proteins have also been reported from human b-casein fractions
(Greenberg, Groves, & Dower, 1984; Yoshikawa, Yoshimura, & Chiba,
1984). On the other hand, all k-casein fragments, known as casoxins
behave as opioid antagonists (Sverin & Wenshui, 2005). Opioid
peptides have also been found encrypted within the primary
sequence of whey proteins such as lactoferrin, b-lactoglobulin, and
bovine serum albumin (Belem, Gibbs, & Lee, 1999; Rokka, Syvaoja,
Tuominen, & Korhonen, 1997). b-casomorphins are resistant to the
action of gastrointestinal enzymes (Read, Lord, Brantl, & Koch, 1990)
and have been associated with the following activities; antihypertensive, immunomodulatory, antidepressant, anti-secretory and
anti-diarrheal (Pihlanto, 2001). Opioid peptides are thought to be
biologically very potent; potentially, micromolar amounts may be
sufcient to exert physiological effects (Meisel, 1997; Meisel &
Fitzgerald, 2000). Systemic administration of a low dose
(1 mg kg1, i.p.) of bovine b-casomorphin-5 was shown to improve
the disturbance of learning and memory in mice (Sakaguchi, Koseki,
Wakamatsu, & Matsumura, 2006). In addition, b-casomorphin-7 was
shown to signicantly contribute to mucin production from both rat
and human intestinal mucin-producing cells using real time-PCR and
ELISA studies (Zoghbi et al., 2006). Since intestinal mucins play

a protective role in the gut, consumption of products containing bcasomorphin-7 could help to improve intestinal health by preventing the adherence of pathogens to the intestinal surface and thus
eliminate the onset of intestinal infections.
2.5. Casein phosphopeptides
CPPs refer to casein-derived phosphorylated peptides, which
contain single and multiple phosphoryl residues and are released
by enzymatic hydrolysis of as1-, as2-, b- and k-caseins both in vivo
and in vitro (Adamson & Reynolds, 1995, 1996; Clare & Swaisgood,
2000). These peptides efciently bind divalent cations such as Fe,
Mn, Cu and Se, due to the high content of negative charge and thus
may act as biocarriers. Several studies have investigated if CPPs in
the diet could increase calcium absorption. However, the results
have been conicting between human and animal studies (ScholzAhrens & Schrezenmeir, 2000). For example, 1.0 mg of CPP
administered extrinsically by gastric intubation to young male rats,
dramatically increased calcium absorption from calcium-fortied
milk compared with the control (Tsuchita, Suzuki, & Kuwata, 2001).
Also using an animal model, Erba, Ciappellano, and Testolin (2002)
demonstrated a similar positive effect but found that the ratios
between CPPs and calcium in the rat intestinal lumen was highly
benecial, with a ratio of 15 identied as the most efcient at
increasing mineral transport, where values above and below were
much less effective. Most recently, the addition of CPP was shown
to have a benecial effect on the absorption of calcium in growing
rats from CaCO3 added to both bovine and caprine milk (MoraGutierrez, Farrell, Attaie, McWhinney, & Wang, 2007). It was also
demonstrated that CPPs induce the inux of calcium into human
HT-29 cells, a rise which was dependant on CPP dose and did not
inuence ATP-induced release of calcium from intracellular stores
(Ferraretto, Signorile, Gravaghi, Fiorilli, & Tettamanti, 2001).
Human studies have been more controversial with regards the
positive effects of CPPs on calcium transport however. Indeed,
administration of 1 g of CPPs did not affect calcium metabolism
acutely in nine postmenopausal women following adminstration of
either CPP-enriched milk or CPP-enriched fermented milk (Narva,
Karkkainen, Poussa, Lamberg-Allardt, & Korpela, 2003). While the
effect of high doses of 2 well-dened CPP-enriched preparations on
15 volunteers consuming calcium lactate drinks did indicate
a positive effect of the CPPs on calcium absorption, it was concluded
that the differences in calcium absorption were unlikely to have any
biological signicance (Teucher et al., 2006). On the other hand,
CPPs have been shown to have anticariogenic properties and to
prevent enamel demineralisation (Aimutis, 2004; Grenby, Andrews,
Mistry, & Williams, 2001). Most recently, a highly diluted CPPamorphous calcium phosphate (CPP-ACP) preparation demonstrated potential as a tooth transport medium by preserving the
viability of an L929 broblastic cell line (Cehreli, Gurpinar, Onur, &
Dagli, 2008). CPPs are currently being used in commercial products including mineral drinks, nutritional supplements for children,
confectionary and products for dental care (Cross, Huq, & Reynolds,
2007; Luo & Wong, 2005) (Table 2).
2.6. Immunomodulatory peptides
Several immunomodulatory peptides have been found in bovine
and human milk (Gill, Doull, Rutherfurd, & Cross, 2000; Politis &
Chronopoulou, 2008). Indeed, casein-derived immunopeptides
have been shown to stimulate the phagocytic activities of both
human and murine macrophages and to protect against Klebsiella
pneumoniae infection in mice (Smacchi & Gobetti, 2000). Trypsinhydrolysed human milk was also found to contain
381
immunostimulating activity (Jolles et al., 1981). Immunomodulating peptides may play a key role in the proliferation and maturation
of T cells and natural killer cells in the newborn providing protection against a large number of bacteria, particularly enteric bacteria
(Clare et al., 2003; Clare & Swaisgood, 2000). Interestingly, the
peptide isracidin, f (1e23) of as1-casein obtained from the action of
chymosin, was shown to display antibacterial activity against
Staphylococcus aureus and Candida albicans, and also gave protection against mastitis following injection into the udder of sheep and
cow (Lahov & Regelson, 1996). Most recently, forced feeding
experiments on healthy animals with a malleable protein matrix
(MPM), composed of whey fermented by a LAB, capsular polysaccharides, vitamins, minerals and peptides generated during the
fermentation process was shown to stimulate the immune system
as seen by the increase in polymorphonuclear cell counts and
intracellular glutathione levels (Beaulieu, Dubuc, Beaudet, Dupont,
& Lemieux, 2007).
2.7. Antimicrobial peptides
Milk is a rich source of antimicrobial proteins and peptides,
capable of exerting antimicrobial activities comparable to antibiotics (Clare et al., 2003; Joerger, 2003; Lopez-Exposito & Recio,
2008; Sverin & Wenshui, 2005). This effect is due to the synergistic activity of naturally occurring peptides and defence proteins
besides immunoglobulins, such as lactoferrin, lactoperoxidase and
lysozyme and is greater than any individual contribution (Clare
et al., 2003; Sverin & Wenshui, 2005). The potent properties of
these agents can be reected by the example of bovine lactoferrin,
which has displayed strong antiviral activity against HIV and the
human cytomegalovirus, the latter of which is thought to act
synergistically in patients with acquired immunodeciency
syndrome (Floris, Recio, Berkhout, & Visser, 2003).
Most antimicrobial peptides reported to date have been released
from the parent milk protein following heat and/or alkali treatment
or enzymatic hydrolysis. For example, both bovine and human lactoferricin, corresponding to bovine lactoferrin f (17e41) and human
lactoferrin f (1e47) display antimicrobial activity against a broad
range of Gram-positive and Gram-negative bacteria, including the
food pathogen, Listeria monocytogenes (Clare et al., 2003; Floris et al.,
2003). Casein-derived antimicrobial peptides resulting after
fermentation by Lactobacillus acidophilus DPC6026 demonstrated
antibacterial activity against the pathogenic strains E. coli and
Enterobacter sakazakii, the latter of which can be problematic in milkbased infant formulas (Hayes et al., 2006). A synthetic 23-residue
peptide was generated from proteoseepeptone 3 f (113e135) termed
lactophoricin, which displayed antimicrobial activity against Grampositive and Gram-negative bacteria (Campagna, Mathot, Fleury,
Girardet, & Gaillard, 2004).
2.8. Cytomodulatory peptides
Cytomodulatory peptides which have been shown to inhibit cell
growth and stimulate the activity of immunocompetent and
neonatal intestinal cells, respectively, have been isolated from
a variety of fermented dairy products (Hayes et al., 2007a; Parodi,
2007). Several studies have displayed the cytomodulatory effects
of milk either in vivo or in vitro (Bif, Coradini, Larsen, Riva, & Di
Fronzo, 1997; Kampa et al., 1997; LeBlanc, Matar, Valdez, LeBlanc,
& Perdigon, 2002; de Moreno de LeBlanc, Matar, LeBlanc, &
Perdigon, 2005; Roy, Watanabe, & Tamai, 1999). Such peptides
can be classied as potential anti-carcinogens and have been
reported to act as specic signals that can inhibit the viability of
cancer cells (Meisel, 2005). Whey protein concentrate has been
attributed with anticancer activities, linked to its ability to donate
382
cysteine to the glutathione antioxidant system (GSH), one of the

principal cellular protection mechanisms (Bounous, 2000).
Remarkably, the combination of a whey protein isolate, termed
Immunocal, with the anti-cancer drug baicalein enhanced the
cytotoxicity of baicalein by inducing more apoptosis in the human
hepatoma cell line Hep G2 (Tsai, Chang, Chen, & Lu, 2000). Most
recently, waste whey peptides from Mozzarella di Bufala Campana
cheese were shown to exert a signicant anti-proliferative effect on
a Caco-2 cell line (De Simone et al., 2009).
2.9. Cysteine protease inhibitors
The activity of some milk proteins as cysteine protease inhibitors
may also provide valuable therapeutic activities. Indeed, the
cysteine protease inhibitors of milk have been associated with
bactericidal activity and protection from bone resorption (Sano
et al., 2005). During bone resorption, osteoclasts secrete proteases
to digest bone matrix proteins such as collagen (Drake et al., 1996).
A 12 kDa cysteine protease inhibitor was puried from MBP and was
subsequently identied as bovine cystatin C (Matsuoka et al., 2002).
Moreover, both lactoferrin and b-casein have demonstrated
cysteine protease inhibition (Ohashi et al., 2003). Indeed, the 17mer at the C-terminus of lactoferrin showed 90% homology with the
sequences of a common active site of the cystatin family. The ability
to inhibit cysteine proteases has thus been postulated as an antimicrobial mechanism for both milk proteins (Ohashi et al., 2003).
factor (Gyorgy, Jeanloz, Von Nicholai, & Zilliken, 1974). Since then,
a large array of milk oligosaccharides was identied and emerging
techniques enable complete analysis of the oligosaccharide content
of human milk in particular (Ninoneuvo et al., 2006). Indeed,
oligosaccharides are a major component of human milk (Table 3). It
has been estimated that human milk contains 7e12 g L1 oligosaccharides (Boehm & Stahl, 2007), about 5e10% of the lactose
concentration. Comparison of the oligosaccharide content of
human milk with milk from other species indicates that human
milk is unique in terms of the complexity and content of its
oligosaccharides (Boehm & Stahl, 2007; Egge, 1993; Kunz, Rudloff,
Baier, Klein, & Strobel, 2000). Human milk oligosaccharides
(HMOs) achieve maximum concentration in the colostrum (above
20 g L1) after which they reach stability in the mature milk (about
12e14 g L1) (Coppa et al., 1993, 1999). Bovine milk on the other
hand harbours very low levels of oligosaccharides, in the region of
1 g L1 (Montreuil, 1960) (Table 3).
The monomers of milk oligosaccharides include D-glucose, Dgalactose, N-acetylglucosamine, L-fucose and sialic acid (N-acetylneuraminic acid), thus a large number of core structures can be
formed (Stahl, Thurl, Zeng, Karas, & Hillenkamp, 1994). However,
the variety is further increased due to a-glycosidic linkages of
fucose and/or sialic acid to the core molecules. The linkage of fucose
is genetically connected to the secretor/Lewis blood group status of
the individual mother (Boehm & Stahl, 2007; Kunz et al., 2000).
Indeed, the addition of fucose to an oligosaccharide by an a1,2
linkage is catalysed primarily by the secretor gene, Se (FUT2); the
2.10. Mammary-associated serum amyloid A3

Another example of a milk antimicrobial peptide is one which is
derived from mammary-associated serum amyloid A3 (M-SAA3),
which is highly abundant in colostrum of mammals but is present in
lower levels in mature milk (McDonald, Larson, Mack, & Weber,
2001). The proposed action of M-SAA3 is not through direct
killing, but via the induction of a host response affecting pathogen
binding to the gut surface. N-terminal sequencing data revealed
a conserved amino acid motif in the M-SAA3 proteins that has been
shown to enhance the mRNA expression of the specic mucin MUC3
by human intestinal cells (Larson, Wei, Weber, Mack, & McDonald,
2003), a phenomenon already discussed for the opioid peptide bcasomorphin-7. MUC3 prevents enteropathogen adherence to
epithelial cells. Indeed, peptides containing the species-conserved
amino acid motif have been shown to reduce the adherence of
enteropathogenic E. coli to human intestinal cells in culture by up to
80%. However, while the human 42-mer M-SAA3 protein and the
10-mer peptide (f (2e11) of the N-terminal region) were recently
shown to exhibit anti-infective activity in vitro, neither the protein
nor the peptide prevented enteric infection in the murine models
tested, which may be due to enzymatic degradation in the gastrointestinal tract or inactivity in the murine model (Gardiner et al.,
2009).
3. Oligosaccharides
3.1. Introduction
As early as 1905, the predominance of lactobacilli in the intestinal microbiota of breast-fed infants (in particular Lactobacillus
bidus now known as Bidobacterium bidum) was noted (Tissier,
1905). In contrast, E. coli was found to dominate in both precociously weaned infants and adults. It was hypothesised that breastfeeding afforded a protection against pathogen colonisation of the
gut, by promoting the growth and colonisation of L. bidus. Almost
70 years later, a glycan isolated from human milk was shown to
stimulate the growth of L. bidus, and was termed the bidus
Table 3
Compositions and concentrations of oligosaccharides in bovine milk, bovine colostrum and human milk.a
Oligosaccharide
Bovine milk Bovine colostrum

(g L1)
(g L-1)
Lactose
40e50
40e50
Trace
e
e
e
e
e
e
e
e
e
NRb
NR
NR
NR
NR
NR
NR
0.03e0.06
0.019
0.095
Trace
Trace
Trace
e
e
e
0.047
Trace (3
0.028
Trace (3
Trace (1
Trace (2
NR
e
Neutral oligosaccharides
Lacto-N-tetraose
Lacto-N-fucopentaose I
Lacto-N-fucopentaose II
Lacto-N-fucopentaose III
Lacto-N-difucohexaose I
Lacto-N-novopentaose
N-acetylgalactosaminylglucose
N-acetylgalactosyl-lactose
a30 -galactosyl-lactose
b30 -galactosyl-lactose
60 -galactosyl-lactose
N-acetyl-lactoseamine
N-acetyl-galactosaminyl-lactose
Acidic oligosaccharides
NeuAc(a2e6)lactose
NeuAc(a2e3)lactose
N-glycoylneuraminyl-lactose
NeuAc-lacto-N-tetraose a
NeuAc-lacto-N-tetraose c
NeuAc2-lacto-N-tetraose
6-Sialyl-lactosamine
3-Sialyl-galactosyl-lactose
Disialyl-lactose
Sialyl-lactose-1-phosphate
Sialyl-lactose-6-phosphate
3-Glycolyl-neuraminyl-lactose
6-Glycolyl-neuraminyl-lactose
GlcNAcb(1e3)Galb(1e4)
GlcNAcb(1e3)Galb(1e4)Glc
Human milk
(g L-1)
55e70
0.5e1.5
1.2e1.7
0.3e1.0
0.01e0.2
0.1e0.2
0.3e0.5
0.1e0.3
0.03e0.2
0.1e0.6
0.2e0.6
mmol L1)
mmol L1)
mmol L1)
mmol L1)
e
a
Adapted from Mehra and Kelly (2006); for details see Kunz and Rudloff (2002),
Gopal and Gill (2000), Nakamura and Urashima (2004).
b
NR: oligosaccharides detected and structurally characterized, but concentration
not reported.
addition of fucose by an a1,3 or a1,4 linkage is catalysed by fucosyltransferases produced by the Lewis gene, Le (FUT3) or other a1,3
transferase genes (FUT4, 5, 6, 7, and 9) of this family (Oriol,
Mollicone, Cailleau, Balanzino, & Breton, 1999). Synthesis of all of
these molecules transpires in the breast starting with lactose at the
reducing end (Boehm & Stahl, 2003; Kunz et al., 2000). Interestingly, milk fucosyloligosaccharide expression has been shown to
change both qualitatively and quantitatively over the course of
lactation showing a relative decrease over the rst year (Chaturvedi
et al., 2001). Moreover, concentrations of certain HMOs in colostrum have recently been shown to uctuate even between day 1
and day 3 of lactation (Asakuma et al., 2007; 2008). HMOs, which
can be divided into neutral and acidic fractions, have been shown to
exert a variety of physiological functions (Coppa, Bruni, Morelli,
Soldi, & Gabrielli, 2004; Coppa, Zampini, Galeazzi, & Gabrielli,
2006; German, Freeman, Lebrilla, & Mills, 2008; Hickey, 2009;
Kunz & Rudloff, 2008; Kunz et al., 2000; Newburg, 2009;
Newburg, Ruiz-Palacois, & Morrow, 2005). Moreover, a recent
study has also demonstrated that HMOs have the potential to
inuence various stages in gastrointestinal development in vitro by
inducing growth inhibition or differentiation depending on the
oligosaccharide and cell line tested (Kuntz, Rudloff, & Kunz, 2008).
3.2. Prebiotic effect of human milk oligosaccharides
Interestingly, an analysis of the fecal microbial communities of
106 individual mammals indicated that host diet had a major
inuence on bacterial diversity (Ley et al., 2008). In keeping with
this notion, it has been suggested that a clue to the ability of the
host to select for different intestinal bacteria may come from
understanding the role of HMOs in the infant gut as prebiotics
(Ninonuevo et al., 2007). Indeed, selection pressures in the gut,
such as carbohydrate availability have shaped the intestinal metagenome such that the gut microbiota in adults is dominated by
members of just 2 divisions of bacteria- the Bacteroidetes and the
Firmicutes and one member of Archaea, Methanobrevibacter smithii
(Eckburg et al., 2005). Interestingly, a metagenomics approach
studying genomic and genetic diversity in our distal gut microbiome demonstrated the abundance of Bidobacterium phylotypes
(Gill et al., 2006). Moreover, a recently dened minimal human gut
metagenome, described as the bacterial functions involved in gut
homeostasis, includes functions involved in compex polysaccharide
degradation and synthesis of short chain fatty acids (SCFAs)
amongst others (Qin et al., 2010).
HMOs are one of the rst selective pressures enforced on the gut
microbiota and in doing so exert a prebiotic effect which has been
conrmed in many studies (Coppa et al., 2006). Remarkably, at
birth the human gastrointestinal tract is sterile however, various
factors affect microbial colonisation (Mackie, Sghir, & Gaskins,
1999; Mountzouris & Gibson, 2003; Orrhage & Nord, 1999)
including mode of delivery (Gronlund, Lehtonen, Eerola, & Kero,
1999), type of feeding (Orrhage & Nord, 1999) and antibiotic
therapy (Burman, Berglund, Huovinen, & Tullus, 1999; Kalenic,
Francetic, Polak, Zele-Starcevic, & Bencic, 1993). The intestinal
microbiota of the breast-fed infant is represented mainly by bidobacteria and lactobacilli largely due to the fact that bidobacteria
can metabolise the neutral oligosaccharide fraction of human milk
(Harmsen et al., 2000). In supporting this, a recent study has shown
that the strain Bidobacterium infantis can grow on puried HMOs
as a sole carbon source (Ward, Ninonuevo, Mills, Lebrilla, &
German, 2006). Of ve bidobacteria strains tested in a follow-up
study, Bidobacterium longum biovar infantis was shown to achieve
highest cell density suggesting that HMOs may even selectively
promote the growth of certain bidobacteria strains (Ward,
Ninonuevo, Mills, Lebrilla, & German, 2007). LoCascio et al.
383
(2007) demonstrated that the infant gut isolate B. longum biovar

infantis ATCC 15697 possessed fucosidase and sialidase activities,
not present in other tested strains and preferentially consumed
small mass oligosaccharides, representing 64% of the total HMOs
available. Indeed, the sequenced genome of this strain revealed the
presence of a milk-active gene suite reecting potential adaptation
to the infant host that included a 43 kilobase pair cluster dedicated
to the uptake and processing of HMOs (Sela et al., 2008). Several
bidobacterial strains have been shown to harbour a lacto-N-biosidase enzyme that liberates lacto-N-biose I from lacto-N-tetraose,
a major component of HMOs, which was not observed in other
bacteria such as clostridia, bacteroides and lactobacilli (Wada et al.,
2008). On the contrary, bidobacteria and lactobacilli represent
only 40e60% of the intestinal microbiota of bottle-fed infants, with
the remaining composed of Enterobacteriaceae and Bacteroides
(Harmsen et al., 2000; Ouwehand, Isolauri, & Salminen, 2002;
Rubaltelli, Biadaioli, Pecile, & Nicoletti, 1998).
The effects of an intestinal microbiota dominated by bidobacteria in breast-fed infants are extremely benecial and include
inhibition of pathogen colonisation (Knol et al., 2005a), modulation
of mucosal physiology and barrier function, and modulation of the
systemic immunologic and inammatory responses as emphasized
by the Committee on Nutrition of the European Society of
Pediatric Gastroenterology, Hepatology and Nutrition in 2004
(ESPGHAN Committee on Nutrition, 2004). Furthermore, it has
been shown that the composition of the gut metagenome can have
consequences for an individuals predisposition to obesity, a trend
which has been observed in both animal models and humans
(Backhed & Crawford, 2009; Backhed et al., 2004; Cani & Delzenne,
2009; DiBaise et al., 2008; Tsukumo, Carvalho, Carvalho-Filho, &
Saad, 2009). Indeed, an increase in the proportion of Firmicutes
relative to Bacteroidetes has been detected in both obese humans
and animals. Several mechanisms linking gut microbiota to energy
metabolism in the host have been proposed including energy
harvest from the diet, synthesis of gut peptides involved in energy
homeostasis, and regulation of fat storage (Cani & Delzenne, 2009).
Almost 40 oligosaccharides have been identied in bovine milk,
which is used in the manufacture of infant formula, and in
comparison to HMOs these oligosaccharides are composed of
shorter oligomeric chains and are signicantly more anionic with
approx. 70% being sialylated (Tao et al., 2008). Since analogues of
HMOs are not commercially available, oligosaccharides from other
sources have thus been assessed for their bidogenic effects in
humans and subsequent use in infant formula. The prebiotic
mixture fructo- and galacto-oligosaccharides (FOS and GOS,
respectively) have proven highly effective as supplements for
promoting the growth of bidobacteria and decreasing the numbers
of pathogenic microorganisms (Boehm et al., 2005; Bruzzese,
Volpicelli, Squaglia, Tartaglione, & Guarino, 2006; Fanaro et al.,
2005a; Miniello, Moro, & Armenio, 2003; Moro & Arslanoglu, 2005).
FOS are of vegetable origin and are found in onions, asparagus,
artichokes and tomatoes (Bornet, 1994; Crittenden & Playne, 1996),
whereas GOS are derived from bovine milk (Montserrat &
Santamaria-Orleans, 2001). Both FOS and GOS harbour the properties of soluble alimentary bres, entering the intestinal tract
intact where they are then fermented by the colonic microbiota
(Montserrat & Santamaria-Orleans, 2001). Analysis of the transcriptional response of B. longum following growth in human milk,
formula milk containing GOS and FOS and semi-synthetic medium
with glucose demonstrated substantial similarity in the transcriptomes for human milk and formula compared with that of the
semi-synthetic medium (Gonzalez, Klaassens, Malinen, de Vos, &
Vaughan, 2008). However, only a few genes implicated in carbohydrate metabolism were similarly upregulated during growth in
human milk and formula milk containing GOS and FOS, which, as
384
the authors suggest, may provide guidance for optimizing formula

milk to better simulate the bidogenic effects of human breast
milk. Interestingly, Bouhnik et al. (1996) demonstrated that ingestion of FOS at a level of 12.5 g day1 led to an increase in colonic
bidobacteria in healthy volunteers. Likewise, Boehm et al. (2002)
demonstrated that supplementation of preterm formula with
10 g L1 of GOS and FOS stimulated the growth of bidobacteria in
preterm infants resulting in stool samples with similar characteristics to those from preterm infants who had received human milk.
Indeed, several studies have reported similar effects of feeding
infants GOS/FOS formula, resulting in an intestinal microecology
with similarities to that of breast-fed infants (Bakkar-Zierikzee
et al., 2005; Knol et al., 2005b). Most recently, Costalos, Kapiki,
Apostolou, and Papathoma (2008) demonstrated that infants fed
a formula containing GOS/FOS produced softer stools with
a microbiota having a signicantly lower proportion of both clostridia and E. coli and a higher proportion of bidobacteria as
compared with infants given a standard infant formula. Interestingly, infant formula supplemented with acidic oligosaccharides
derived from pectin hydrolysate did not exert any signicant
inuence on the intestinal microecology of term infants (Fanaro
et al., 2005b). The subsequent health benets of consuming the
GOS/FOS mixture in infants have also been documented. Indeed,
consumption of a supplemented hypoallergenic formula containing
8 g L1 of GOS/FOS for 6 months was shown to reduce the incidence
of atopic dermatitis in a double-blind, randomized, placebocontrolled trial involving 259 infants at risk for atopy during the
rst six months of age (Moro et al., 2006) possibly due to their
bidogenic effects. In addition, consumption of the oligosaccharide
prebiotics by healthy term infants with a parental history of atopy
was shown to reduce the number of infectious episodes and the
incidence of recurring infections, particularly respiratory infections
during the rst 6 months of life in a double-blind, placebocontrolled trial (Arslanoglu, Moro, & Boehm, 2007). In a follow-up
study, it was demonstrated that infants in the GOS/FOS group had
fewer episodes of respiratory tract infections (P < 0.1), fewer fever
episodes (P < 0.00001), and fewer antibiotic prescriptions
(P < 0.05) during the rst two years of life, demonstrating that the
protective effects lasted beyond the 6 month intervention period
(Arslanoglu et al., 2008). In addition, consumption of supplemented
formula containing GOS/FOS for 6 months was shown to induce
a benecial antibody prole following vaccination at 3 months with
Hexavac against a.o. diphteria, tetanus, polio (DTP) by reducing the
total Ig response and modulating the immune response towards
cows milk protein, while leaving the response to vaccination intact
(van Hoffen et al., 2009).
The resulting by-products from the fermentation of prebiotic
substances by the gut microbiota, in particular SCFAs are capable of
exerting numerous physiological effects and are thus essential
biological components in maintaining health and evading disease
(Bruzzese et al., 2006; Roy, Kien, Bouthillier, & Levy, 2006). Moreover, a reduction of SCFA concentrations in the gut has also been
observed in patients with inammatory bowel disease (IBD)
(Treem, Ahsan, Shoup, & Hyams, 1994). Butyrate is perhaps the
most inuential SCFA in terms of its physiological effects, serving as
the principal energy source for colonic epithelial cells and as
regulator to a number of genes associated with cell differentiation,
proliferation and apoptosis. Thus, butyrate has a profound inuence on the aetiology of various colonic diseases including cancer,
IBD and the regulation of lipid metabolism (Scheppach & Weiler,
2004). Interestingly, a recent study demonstrated that the
eukaryotic G-protein-coupled receptor, GPR43 is the relevant
receptor for SCFA effects on immune cells (Maslowski et al., 2009).
Indeed, using murine models, stimulation of GPR43 by SCFAs was
necessary for the normal resolution of certain inammatory
responses whereas GPR43-decient mice (Gpr43/) demonstrated

exacerbated inammation in models of colitis, asthma and arthritis
in the presence of SCFAs (Maslowski et al., 2009). Since butyrate is
also produced in bovine milk, the physiological effects of this fatty
acid are discussed in more detail in the Lipids section.
3.3. Milk oligosaccharides and infection
It has long been recognised that breast-fed infants suffer from
lower rates of enteric infections such as infectious diarrhoea,
a factor that has been primarily attributed to both the secretory
antibodies and prebiotic factors in human milk. However, while the
prebiotic effect exerts a competitive inhibition on enteric pathogenic bacteria and undoubtedly ne-tunes the immune response
against infection, the inhibition of pathogen binding to host cell
ligands afforded by oligosaccharides is believed to be the primary
means of protection for breast-fed infants against enteric
pathogens.
During pathogenesis, the majority of enteric pathogens use host
cell-surface glycans to bind to their target cells. In many cases, this
adhesion is mediated by lectins present on the surface of the
pathogenic microorganism. It is now known that soluble oligosaccharides from milk competitively inhibit the ability of pathogens
to bind to receptors in the gut by binding bacterial lectins (Newburg
et al., 2005). The bacteria are then swept away by ciliary action,
leaving the host mucosa untouched.
Several in vitro studies have yielded results demonstrating the
protective capabilities of HMOs. For example, the a1,2-linked
fucosylated glyconjugates expressed in human milk have been
shown to inhibit binding of Campylobacter to intestinal cells in vivo,
a bacterium known to be a signicant cause of diarrhoea worldwide
(Ruiz-Palacois, Cervantes, Ramos, Chavez-Munguia, & Newburg,
2003). Fucosylated oligosaccharides have also demonstrated an
ability to bind to members of the calicivirus family of enteric
pathogens which have been recognised as a major cause of diarrhoea in humans, especially infants (Marionneau et al., 2002;
Ruvoen-Clouet, Ganiere, Andre-Fontaine, Blanchard, & Le Pendu,
2000). Rotavirus, on the other hand, was shown to be inhibited
by a mucin-associated 46 kDa milk glycoprotein called lactadherin
(Newburg et al., 2005; Yolken et al., 1992). Removal of sialic acid
from lactadherin renders the glycoprotein ineffectual against
inhibiting rotavirus, suggesting that the glycan portion of the
molecule may be responsible for inhibition. Similarly, HMOs were
shown to strongly inhibit hemagglutination mediated by enterotoxigenic E. coli (ETEC) and uropathogenic E. coli (UPEC) strains,
which decreased when the oligosaccharides were desialylated
(Martin-Sosa, Martin, & Hueso, 2002).
Interestingly, although bovine milk oligosaccharides were less
efcient at inhibiting the hemagglutination of the ETEC strains,
they were considered good inhibitors of the UPEC strains (MartinSosa et al., 2002). HMOs have also demonstrated an ability to
inhibit the activities of bacterial toxins. Research by Otnaess,
Laegreid, and Ertresvag (1983) demonstrated that the activities of
the labile toxin of E. coli and cholera toxin were inhibited by the
ganglioside fraction of human milk. Various treatments of the
inhibitory component indicated that it was indeed an oligosaccharide or glyconjugate (glycan). Later, it was demonstrated that
the oligosaccharides of human milk containing a1,2-linked fucose
structures were also capable of binding the stable toxin of E. coli
(Newberg, Pickering, McCleur, & Cleary, 1990). Interestingly, studies
using T84 cells derived from human enterocytes indicated that the
oligosaccharide inhibits stable toxin by binding its cellular
receptor; the extracellular domain of guanylate cyclase, an enzyme
responsible for producing guanosine monophosphate (GMP).
Elevated levels of GMP as a result of stable toxin binding result in
loss of chloride and bicarbonate transport, culminating in the efux

of uid and electrolytes on a cellular level, which transpires as
secretory diarrhoea in the host. However, binding of the extracellular domain of guanylate cyclase by the oligosaccharide competitively inhibits binding by stable toxin of E. coli (Crane, Azar, Stam, &
Newburg, 1994). Newburg, Chaturvedi, Crane, Cleary, and Pickering
(1995) estimated that the inhibitory oligosaccharide is active at
a concentration of approximately 30 parts per billion, the concentration at which it occurs in human milk.
Studies in human populations have demonstrated that the
protective capabilities of HMOs observed in vitro are also active in
vivo. Morrow et al. (2004) collected data and samples from 93
breast-feeding mother-infant pairs from birth to 2 years with infant
feeding and diarrhoea data collected weekly. Milk samples
obtained 1e5 weeks postpartum were analysed for oligosaccharide
content. Moderate to severe diarrhoea of all causes occurred less
often in infants whose milk contained high levels of 2-linked
fucosyloligosaccharides as a percent of milk oligosaccharide. Other
studies have also demonstrated the signicance of 2-linked fucosyloligosaccharides for inhibition of pathogen binding during
breast feeding against infectious diarrhoea (Morrow, Ruiz-Palacois,
Jiang, & Newburg, 2005; Newburg et al., 2003). Most recently, it was
demonstrated that early consumption of HMOs through breast
feeding was also associated with less reported respiratory and
gastrointestinal illness in infants (Stepans et al., 2006).
The prevention of bacterial adhesion as observed by human milk
glycans is an attractive option for the development of new therapeutic agents targeting infections of mucosal surfaces in particular,
e.g., infections of the intestine, urinary tract, nasopharyngeal tract,
etc. The concept that the infectious agent is neutralised through
binding of the oligosaccharide is appealing, as the infectious agent
is still available to provoke an immune response. However,
manufacturing and delivery challenges must yet be addressed, but
based on the information encoded in human milk glycans, carbohydrate-based therapies offer great potential for future antimicrobial drugs as discussed by Sharon and Ofek (2000) and
Bavington and Page (2005).
3.4. Milk oligosaccharides and the inammatory immune response
Interestingly, milk oligosaccharides have been shown to play
a signicant role in the activities of the inammatory immune
response. Inammation is a complex multi-step process providing
a non-specic defence mechanism against tissue injury. During the
inammatory process, injured tissue cells release chemical signals
called inammatory mediators that activate the endothelium of
nearby capillaries. This activation results in the release of cellsurface adhesion molecules referred to as selectins, which are
glycoprotein in nature, and essential for the formation of plateletneutrophil complexes (PNC) (Jungi, Spycher, Nydegger, & Barandun,
1986; Kannagi, 2002; Ley, 2003; Rhee et al., 2003; Varki, 1994).
These heterogenous cell aggregates represent a large subpopulation of neutrophils with a greater capacity for phagocytosis and
increased production of reactive oxygen species (ROS) (Peters et al.,
1999). Selectins are also essential components in the cell adhesion
cascade leading to the extravasation of blood cells into the tissue
during inammatory processes (McEver, 1997). PNC formation
requires P-selectin on activated platelets and P-selectin glycoprotein ligand 1 (PSGL-1) on neutrophils (Larson et al., 1989). This
interaction induces signalling pathways leading to an increased
expression of adhesion molecules, including b 2 integrin CD11b/
CD18 (Piccardoni et al., 2001). However, the excessive recruitment
of such cell aggregrates can have deleterious consequences, leading
to the destruction of healthy tissue as observed in many diseases
(Carden & Granger, 2000; Laroux & Grisham, 2001; Schon et al.,
385
2002). Milk oligosaccharides have been shown to harbour

epitopes similar to selectin-binding ligands. Furthermore, since it
has been conrmed that lactose-derived oligosaccharides in infants
survive the gastrointestinal tract and are excreted in the urine
(Rudloff, Pohlentz, Diekmann, Egge, & Kunz, 1996), such oligosaccharides may have a role to play in anti-inammatory processes.
Interestingly, sialyl-Lewis ligands on milk oligosaccharides were
shown to have the greatest capacity for binding selectins in vitro
(Rudloff, Stefan, Pohlentz, & Kunz, 2002).
In an attempt to decipher the exact mechanisms involved Bode
Rudloff, Kunz, Strobel, and Klein (2004b) demonstrated that the
acidic fraction of HMOs competes with PSGL-1 for P-selectin binding
thus reducing PNC formation. Indeed, the acidic fraction reduced
PNC formation up to 20%, which was similar to the effect seen with
sialyl-Lewis X, a physiological binding determinant for selectins. A
dose-dependent decrease in b 2 integrin expression was also
observed in associated neutrophils. Moreover, several active
components within the acidic component of HMOs were identied
as determinants for selectin binding, such as 30 -sialyl-lactose and 30 sialyl-3-fucosyl-lactose (Bode et al., 2004a). Interestingly, a study by
Schumacher, Bendas, Stahl, and Beermann (2006) identied the
acidic fraction of HMOs as effective agents in interfering with
P-selectin ligand binding, suggesting that molecular charge is
important for binding interactions. However, total HMOs were not as
effective as the acidic fraction alone, suggesting that HMOs modulate
rather than block the function of P-selectin (Schumacher et al., 2006).
SCFAs resulting from the degradation of HMOs (particularly
acetate and propionate) are now known to bind GPR43, which is
expressed by cell types involved in innate immunity, and in doing
so regulate immune and inammatory responses (Maslowski et al.,
2009). Indeed, re-colonisation of germ-free mice, suffering from
induced colitis, with gut microbiota showed a marked reduction in
inammation. Likewise, treatment of these germ-free mice with
acetate markedly improved disease indices. Therefore, SCFA-GPR43
interactions provide another mechanism whereby HMOs can
regulate the immune and inammatory responses.
Several modern-day diseases have resulted from an overshooting immune response. The lower incidence of infectious and
inammatory diseases in breast-fed versus formula-fed infants
suggests that HMOs may play a signicant role in anti-inammatory processes. For example, necrotising enterocolitis (NEC),
a disease primarily observed in premature infants results from
pathogen colonisation and adhesion to the intestinal surface
resulting in tissue necrosis. However, excessive leukocyte inltration, followed by the production of ROS have also been shown to
play a major role in its progression (Bode et al., 2004a; Musemeche,
Caplan, Hsueh, Sun, & Kelly, 1991). The lower incidence of NEC in
breast-fed infants suggests that HMOs might inuence the onset of
the disease by preventing an amplied immune response.
3.5. Sialic acid
Another milk oligosaccharide that has been the recipient of
considerable attention in recent years is N-acetylneuraminic acid,
also known as sialic acid. Several oligosaccharides found in milk
tend to be sialylated, consisting of a core structure with a lactose
unit at the reducing end and at least one N-acetylneuraminic acid
unit at the non-reducing end (Wang & Brand-Miller, 2003). The
most abundant sialyloligosaccharides of human milk found at
several stages of lactation were 30 -sialyl-3-fucosyl-lactose and sialyllacto-N-tetraoses (a, b and c), while 60 -sialyllactosamine and 30 sialyllactose were the most abundant sialyloligosaccharides in
bovine milk (Martin-Sosa, Martin, Garcia-Pardo, & Hueso, 2003).
Interestingly, sialic acid is also found in high concentrations in
the mammalian nervous system, with the majority being present in
386
gangliosides (65%) followed by glycoproteins (32%) and the

remaining 3% as free sialic acid (Brunngraber, Witting, Haberland, &
Brown, 1972). As suggested by Wang and Brand-Miller (2003), the
sialic acid moieties of gangliosides and glycoproteins in the brain
frontal cortex play both a structural and functional role and probably participate in a variety of cellular events, such as cell recognition, cell-to-cell contact, receptor binding and modulation,
immunological properties and biosignal transduction. Gangliosides
are complex glycosphingolipids, which make up 10% of the total
lipid mass in the brain, containing different numbers of negatively
charged sialic acid moieties (Wang & Brand-Miller, 2003). While
the exact functions of gangliosides still remain relatively poorly
understood, it has been hypothesised that they are involved in the
formation of memory (Wang & Brand-Miller, 2003). Brain glycoproteins are also related to brain function in the formation of
memory and learning (Bogoch, 1977; Schmidt, 1989). The repulsive
effects of the negatively charged sialic acid may serve to prevent
cell aggregation (Schauer & Kamerling, 1997). On the other hand,
cell adhesion may be facilitated through positively charged
substances or calcium (Wang & Brand-Miller, 2003). Indeed,
binding of calcium to ganglioside sialic acid is of great importance
in the function of nervous tissues (Rosenberg, 1995; Schauer &
Kamerling, 1997). Thus, it has been postulated that sialic acid is
the actual receptor for neurotransmitters in the central nervous
system (CNS) (von Itzstein & Thomson, 1997).
Several studies have demonstrated that children who have been
breast-fed as babies display higher intelligence skills than children
who were formula-fed (Fergusson, Beautrais, & Silva, 1982; Lucas,
Morley, & Cole, 1998; Lucas, Morley, Cole, Lister, & Leeson-Payne,
1992; Rodgers, 1978; Smith, Durkin, Hinton, Bellinger, & Kuhn,
2003). In an intriguing study, Wang, McVeagh, Petocz, and BrandMiller (2003) examined the brain frontal cortex gray matter of 25
infants who died of sudden infant death syndrome and whose
feeding pattern had been previously recorded in 21 cases. It was
found that ganglioside-bound and protein-bound sialic acid
concentrations were 32% and 22% higher, respectively, in the frontal
cortex gray matter of breast-fed infants. Moreover, it was demonstrated that the concentration of sialic acid in saliva of breast-fed
infants was twice the level of sialic acid found in the saliva of
formula-fed infants (Wang et al., 2001b).
In a more recent study, it was shown that feeding piglets
protein-bound sialic acid during early development enhanced
learning and memory and increased the expression of 2 genes
associated with learning in developing piglets, namely a2,8-sialyltransferase II and IV (Wang et al., 2007b). Furthermore, within
2 min of injection of 3-day old male domestic piglets with N-acetylneuraminic acid-6-14C, 80% of the activity was removed from
the blood. At 120 min, the brain contained signicantly more
radioactivity than the liver, pancreas, heart and spleen, but less
than the kidneys, proving that an exogenous source of sialic acid is
capable of crossing the blood-brain barrier (Wang, Downing,
Petocz, Brand-Miller, & Bryden, 2007a).
The potential signicance of sialic acid for early brain development has led to an assessment of the sialic acid contents of both
bovine milk and infant formulas. Interestingly, Asakuma et al.
(2007) demonstrated that the concentration of 30 -sialyllactoses in
human colostrum was signicantly higher on day 1 of the rst three
days of lactation than on day 2 and 3, but the levels of 60 - sialyllactoses and sialyllactose-N-tetraose were higher on day 3 than
on day 1, indicating that during the rst 3 days of lactation the
concentration of sialyloligosaccharides changes in accordance with
the physiological demands of the newborn infant. However, infant
formulas were shown to contain less than 25% sialic acid than that
found in mature human milk (Wang, Brand-Miller, McVeagh, &
Petocz, 2001a). Thus, further studies are required to assess the
effectiveness and safety of sialic acid supplemented formulas.

Interestingly, an assessment of bovine colostrum and milk during
prepartum and early lactation suggested that bovine colostrum,
especially that collected after parturition may be a suitable source
of 30 sialyllactoses as an additive by the food or pharmaceutical
industries (Nakamura et al., 2003).
Isomeric differences exist amongst mammalian sialic acids,
which may have consequences for human health and disease.
Indeed, while humans produce N-acetylneuraminic acid other
mammals synthesise N-glycolylneuraminic acid (Neu5Gc), a mutation estimated to have occurred w2e3 million years ago, just
before the emergence of the genus Homo (Wood & Collard, 1999). A
Neu5Gc-binding pathogen such as Non-Human Hominid (NHH)
malaria, combined with genetic drift caused by ancestral demography, has been proposed as a possible selection mechanism, suggesting that sialic acid-related genes are a hot-spot for genetic and
physiological changes in human evolution (Varki, 2010). However,
humans can acquire Neu5Gc from dietary sources including red
meats and, to a lesser extent, milk products (Tangvoranuntakul
et al., 2003); this is detected as foreign by the immune system
and may be recognised by Neu5Gc-binding pathogens. Thus, it has
been suggested that Neu5Gc antibodies contribute to red meat
aggravation of diseases by stimulating chronic inammation, and
that Neu5Gc-containing epithelial cells may play a role in red meatrelated food poisoning (Varki, 2010). Moreover, human carcinomas
have been shown to accumulate dietary Neu5Gc (Yin et al., 2006)
and anti-Neu5Gc antibodies have been shown to enhance Neu5Gcpositive tumors in mice (Hedlund, Padler-Karavani, Varki, & Varki,
2008). However, it has been suggested that high levels of these
antibodies may kill tumor cells and even protect certain individuals
with very high levels from some cancers (Varki, 2010).
While the concepts proposed have been deduced from sound
scientic evidence many of these are speculative and require
further investigation. But as with all biological systems, and milk is
no exception, one must take into account the interplay between all
substituents and the host. Thus, while consumption of Neu5Gc
from bovine milk may contribute to human disease phenotypes,
milk itself is composed of a multitude of health-promoting factors,
which one could speculate, could also negate, or at least minimize
such deleterious effects.
4. Milk lipids
4.1. Introduction
Both bovine and human milk fat are rich sources of bioactive
substances offering a plethora of ingredients for the development
of functional food products (German & Dillard, 2006; Haug et al.,
2007). Structurally, milk fat occurs as micro droplets surrounded
by the MFGM, containing mainly triacylglycerols, esters and retinol
esters in the core, and primarily phospholipids and cholesterol in
the membrane (Argov, Lemay, & German, 2008; Jensen, 1999;
Mather, 2000). In the case of human milk, 40e55% of the total
energy intake of the offspring is obtained from milk fat (German &
Dillard, 2006), while it also supplies essential nutrients such as
essential fatty acids and the fat soluble vitamins, A, D, E and K.
Triacylglycerols represent 97e98% of the total lipids in the milk of
most species, while the level of cholesterol in milk is low when
compared with other food sources (Fox & McSweeney, 1998).
4.2. Milk fat globule membrane
In a review by Spitsberg (2005), the associated health benets of
bovine MFGM were discussed in detail. Interestingly, components
of the MFGM have exhibited anticancer, anticholesterolemic and
antimicrobial effects, with some of these effects attributable to the

protein fraction. Recently, it was demonstrated that milk fat globules, MFGM and the glycosphingolipids lactosylceramide, and the
gangliosides GM3 and GD3 were capable of binding ETEC strains
and inhibiting bacterial haemagglutination (Sanchez-Juanes,
Alonso, Zancada, & Hueso, 2009). Moreover, feeding cows a diet
rich in polyunsaturated fatty acids (PUFAs) has been shown to affect
the composition of the MFGM, reecting changes in fatty acid
synthesis in the mammary gland resulting in a higher amount of
phospholipids (18%), which was related to a smaller size of milk fat
globules, an increase in the concentration of sphingomyelin (30%),
and a higher content of stearic acid (1.7-fold), unsaturated fatty
acids (1.36-fold) and C18:1 trans fatty acids (3.7-fold) (Lopez et al.,
2008).
4.3. Phospholipids and cancer
While much of the health benets of MFGM have been associated
with proteins, the phospholipid content has also received signicant
attention for its associated health effects. Indeed, Spitsberg (2005)
has proposed that the consumption of MFGM as a neutraceutical
or dairy food, or the consumption of food products enriched with
MFGM conveys health benets due to the presence of phospholipids.
The three main phospholipids of MFGM include sphingomyelin,
phosphatidyl choline and phosphatidyl ethanolamine (Spitsberg,
2005). Several cellular processes are affected by phospholipids,
from growth and development to memory, stress responses, and
myelination in the CNS (McDaniel, Maier, & Einstein, 2003; Oshida
et al., 2003b). Furthermore, the sphingomyelin component of the
MFGM has exhibited anticancer effects (Berra, Colombo,
Sottocornola, & Giacosa, 2002; Hertervig, Nilsson, Cheng, & Duan,
2003; Lemonnier et al., 2003; Parodi, 2001) and has been shown to
be an effective inhibitor of intestinal absorption of cholesterol in rats
(Noh & Koo, 2004).
Sphingomyelin belongs to one of the most complex classes of
lipids in nature, namely the sphingolipids. Such lipids contain an
amide-linked fatty acid and a long chain (sphingoid) base. They are
located in cellular membranes, lipoproteins (especially low density
lipoproteins, LDL) and other lipid-rich structures such as skin
(Akalin, Gonc, & Unal, 2006). It is now known that these complex
sphingolipids and their breakdown products (ceramides and
sphingosines) are highly bioactive compounds that play essential
roles in cell growth, survival and death (Akalin et al., 2006; Cuvillier
& Levade, 2003; Vesper et al., 1999). Interestingly, orally administered sphingolipids have been shown to prevent colon cancer
development in different rodent models (Schmelz, 2004). These
dietary sphingolipids are digested throughout the small intestine
and colon by intestinal or bacterial enzymes (Schmelz, Crall,
Larocque, Dillehay, & Merrill, 1994).
Earlier studies have also shown that feeding sphingomyelin to
mice in amounts equivalent to those found in foods such as dairy
products (0.025e0.1%) (Vesper et al., 1999) inhibits the early stages
of colon carcinogenesis and decreases the ratio of malignant
adenocarcinomas to benign adenomas (Dillehay, Webb, Schmelz, &
Merrill, 1994; Schmelz et al., 1996). In addition to sphingomyelin,
the glycosphingolipids of milk, namely glucosylceramide, lactosylceramide, and ganglioside GD3 have been shown to inhibit the
early stages of chemically induced colon cancer in female CF1 mice
(Schmelz, Sullards, Dillehay, & Merrill, 2000). The genetic mutations
associated with colon tumors are often the result of defects in the
adenomatous polyposis coli (APC)/b-catenin regulatory system.
Feeding C57B1/6JMin/ mice (harbouring a truncated APC gene
product) diets supplemented with ceramide, sphingomyelin, glucosylceramide, lactosylceramide, and ganglioside GD3 reduced the
number of tumors in all regions of the intestine (Schmelz et al.,
387
2001). It was concluded that dietary sphingolipids, via their digestion products, bypass or correct defect(s) in the (APC)/b-catenin
regulatory pathway (Schmelz et al., 2001).
Similar effects were also observed in human colon cancer cell
lines. Studies using HT-29 and HCT-116 human colon cancer cells
demonstrated that both sphingosine and ceramide inhibited
growth and caused death of the colon cancer cells in time and
concentration-dependent manners, where the 4,5-trans double
bond of ceramide was found to be essential for its inhibitory effects
(Ahn & Schroeder, 2002). Apoptosis has been suggested as the
mechanism of action since both the sphingoid bases and ceramide
were shown to cause chromatin and nuclear condensation, and
fragmentation of DNA. Moreover, the sphingoid bases were shown
to arrest the cell cycle at the G2/M phase and cause accumulation in
the S phase.
Sphingolipid metabolites have shown potential as chemotherapeutic and chemopreventive agents. The sphingoid bases sphingosine and sphinganine were shown to preferentially inhibit breast
cancer cells and eliminate stem cells from which most breast cancer
cells arise (Ahn, Chang, & Schroeder, 2006). In contrast, sphingosine-1-phosphate has been shown to stimulate cell growth and
suppress apoptosis by acting through G-protein coupled receptors
present on mammalian cells, thus stimulating cell proliferation,
angiogenesis and inhibiting apoptosis (Oskouian & Saba, 2007;
Spiegel & Milstien, 2003). The enzyme responsible for sphingosine-1-phosphate synthesis, namely sphingosine kinase 1, behaves
as an oncogene in experimental systems (Oskouian & Saba, 2007).
Moreover, sphingosine-1-phosphate-specic antibodies slow
tumor progression and angiogenesis in mice murine xenograft and
allograft models (Oskouian & Saba, 2007). As suggested by
Oskouian and Saba (2007), knowledge of this nature may have
implications regarding colon cancer screening, dietary chemoprevention and therapeutics.
4.4. Phospholipids and the immune system
Sphingolipids play an essential role in the development, activation and regulation of the immune system (Cinque et al., 2003).
As described by Cinque et al. (2003), the breakdown products of
sphingolipid metabolism including ceramide, sphingosine, ceramide-1-phosphate and sphingosine-1-phosphate serve as second
lipid messengers, which are all involved in a common signalling
pathway that controls the main stages of immune cell development, differentiation, activation, proliferation and function. Interestingly, treatment of bone marrow-derived dendritic cells with the
human milk-derived gangliosides, GD3 and GM3, decreased the
production of interleukin-6 (IL-6), IL-10, IL-12 and tumor necrosis
factor-alpha. Furthermore, the expression of CD40, CD80, CD86 and
major histocompatibility complex class II on dendritic cells was
suppressed (Bronnum, Seested, Hellgren, Brix, & Frokiaer, 2005),
suggesting that dietary sphingolipids may also have a dramatic
effect on immune regulation.
4.5. Phospholipids and the central nervous system
Sphingolipids play a signicant role in neuronal cell function by
regulating rates of neuronal growth, differentiation and death
(Buccoliero & Futerman, 2003). Likewise, dietary sphingolipids may
also have the potential to exert dramatic effects on the activities of
the CNS. Interestingly, studies using L-cycloserine treated rats
(which lack the ability to produce serine palmitoyltransferase
(SPT), a rate-limiting enzyme for sphingolipid biosynthesis)
demonstrated that feeding the rats bovine sphingomyelin-supplemented diets contributed to CNS myelination with experimental
inhibition of SPT corrected (Oshida et al., 2003a). Given the potent
388
biological activities of sphingolipids, there is no doubt that these

food components could be categorized as functional food ingredients. However, while cell culture studies and a few animal studies
have provided promising results, more animal and human trials are
urgently required to thoroughly investigate the physiological efcacies of these agents.
While the nutritional and functional characteristics of the
MFGM are well known it should be recognised that raw milk is
exposed to various processing parameters to ensure safety and
prolong shelf-life, including heat treatments and homogenization,
which have profound effects on the physical structure of milk fat
(Michalski, 2007; Michalski & Januel, 2006). Homogenization, for
example, results in a dramatic reduction in fat globule size (Walstra,
2003). Using confocal laser scanning microscopy, a recent study
demonstrated that processing (centrifugation, pasteurizationhomogenisation and churning) changed the composition of the
MFGM through the loss of phospholipids and the adsorption of
caseins and whey proteins onto the surface (Gallier, Gragson,
Jimenez-Flores, & Everett, 2010). The effects of such structural
changes on the health properties associated with the MFGM are
still largely unknown. Therefore, in vitro and in vivo studies that
elucidate the effects of processing on the bioactivities of MFGM and
its substituents will provide valuable information which will
undoubtedly lead to dairy products/ingredients which harbour the
full complement of bioactivities found in unadulterated raw milk.
4.6. Polyunsaturated fatty acids
Both bovine and human milk fat contain PUFAs including low
levels of the essential n-6 and n-3 series, linoleic acid (LA, 18:2n-6)
and a-linolenic acid (ALA, 18:3n-3). LA and ALA concentrations
have been reported to range between 1e3 and 0.5e2 wt%,
respectively, in bovine milk (Jensen, 2002; Kaylegian & Lindsay,
1995), whereas concentrations of 10.75 4.22 and 0.35
0.05 wt%, respectively, have been reported in human milk (Gibson
& Kneebone, 1981). These fatty acids cannot be synthesised by the
body but are required for survival of humans and other mammals
and hence have to be obtained from the diet (Das, 2006). LA is the
most common PUFA incorporated into the dynamic phospholipids
required for membrane structure and lipoproteins [particularly
phospholipid-rich high density lipoprotein (HDL)] involved in lipid
transport (Wijendran & Hayes, 2004). Both LA and ALA can be
further metabolised by D6-desaturation, elongation and D5-desaturation to form arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5n-3), respectively, which form
precursors for the synthesis of prostaglandins and leukotrienes
which control numerous cell activities responsible for healthy
living (Innis, 2004, 2007b; Wall, Ross, Fitzgerald, & Stanton, 2010).
EPA is then further metabolised to form docosahexaenoic acid
(DHA, 22:6n-3) (Innis, 2003), which unlike the n-6 fatty acids, has
a specic distribution in tissues and phospolipids and is extremely
important in the development of the CNS (Innis, 2007a; Wurtman,
2008).
Interestingly, the n-6 and particularly the n-3 fatty acids have
demonstrated potential health benets both in vitro and in vivo
(Connor, 2000) by reducing the risk of cardiovascular disease (CVD)
(Fedacko et al., 2007; Siddiqui, Harvey, & Zaloga, 2008), type-2
diabetes, hypertension (Wijendran & Hayes, 2004; Willett, 2007;
Zhao et al., 2007), cancer (Dupertuis, Meguid, & Pichard, 2007;
Larsson, Kumlin, Ingelman-Sundberg, & Wolk, 2004) and certain
neurological disfunctions (Alessandri et al., 2004; Hamilton,
Hillard, Spector, & Watkins, 2007), and have recently been shown
to improve bone density in ovariectomized rats (Poulsen, Moughan,
& Kruger, 2007) and eye lens nuclear density in women aged 52e73
years (Lu et al., 2007).
The n-3 fatty acids have also exhibited therapeutic potential in

the treatment of inammatory diseases such as rheumatoid
arthritis, and in the alleviation of symptoms of mental health such
as depression and dementia (Ruxton, Reed, Simpson, & Millington,
2007). The n-3 fatty acid, DHA, has also shown promise as
a potential treatment for late-onset Alzheimers disease (Ma et al.,
2007). Indeed, genetic polymorphisms that reduce expression of
the protein LRII are associated with increased risk of late-onset
Alzheimers disease. However, DHA was shown to signicantly
increase LRII in multiple systems, including primary rat neurons,
aged non-Tg mice and an aged DHA-depleted APPsw AD mouse
model and in a human neuronal cell line (Ma et al., 2007).
4.7. Conjugated linoleic acid
In ruminant animals, dietary LA can be converted to CLA as
a consequence of biohydrogenation by the rumen bacteria coupled
with endogenous synthesis by the animals own tissues (Griinari
et al., 2000; Lock & Bauman, 2004) where the cis-9, trans-11
isomer accounts for the majority (over 90%) of total milk fat CLA
(Stanton, Murphy, McGrath, & Devery, 2003). CLA is thus produced
when dietary LA is converted to vaccenic acid by the microbial
enzyme linoleic acid isomerase, in the process called biohydrogenation (Stanton et al., 2003). Vaccenic acid is then converted to CLA in the mammary gland via the enzyme D9-desaturase
and is thus excreted in milk (Griinari & Bauman, 1999). CLA can also
be generated directly from LA as a result of microbial biohydrogenation, where it is formed as an intermediate in the
generation of vaccenic acid. Of the 1010e1011 bacteria residing in
the rumen of a dairy cow, only a few species have been shown to
perform these biohydrogenation reactions (Lock & Bauman, 2004).
Remarkably, compared with their parent fatty acids, CLA
isomers have demonstrated superior abilities in vitro and in vivo to
prevent various types of cancer, hypertension, atherosclerosis and
diabetes as well as an ability to improve immune function and body
composition, which have been discussed in detail in several
publications (Benjamin & Spener, 2009; Bhattacharya, Banu,
Rahman, Causey, & Fernandes, 2006; Churruca, FernandezQuintela, & Portillo, 2009; Kelley, Hubbard, & Erickson, 2007;
Mitchell & McLeod, 2008; Nagao & Yanagita, 2005; Pariza, Park, &
Cook, 2001; Silveira, Carraro, Monereo, & Tebar, 2007; Watras,
Buchholz, Close, Zhang, & Schoeller, 2007). Although there are 28
different CLA isomers, health benets have been mainly attributed
to two, namely, cis-9, trans-11 and trans-10, cis-12 CLA. In addition,
minute amounts of ALA biohydrogenation intermediates (conjugated alpha-linolenic acids, CLNA) have also been identied in milk
fat (Destaillats, Trottier, Galvez, & Angers, 2005; Plourde,
Destaillats, Chouinard, & Angers, 2007). Recent evidence has
highlighted the health-promoting properties of these conjugated
PUFAs (Nagao & Yanagita, 2005). These include C18:3 c9, t11, t13
CLNA (Tsuzuki, Tokuyama, Igarashi, & Miyazawa, 2004; Yasui,
Hosokawa, Kohno, Tanaka, & Miyashita, 2006; Yasui et al., 2005)
exhibiting anticancer properties, c9, t11, c13 CLNA that has
exhibited a hypolipidemic effect (Arao, Yotsumoto, Han, Nagao, &
Yanagita, 2004), and c9, t11, c15 CLNA that has been shown to
inhibit the growth of SW480 colon cancer cells (Coakley et al.,
2008; Hennessy et al., 2011).
4.8. Increasing the conjugated linoleic acid content of milk and
dairy foods
With regard to dietary CLA, it has been estimated that dairy
products and red meat provide about 70% and 25%, respectively of
total dietary intake in humans (Ritzenthaler et al., 2001). Understandably, however, the levels of these fatty acids in milk can vary
depending to a large extent on animal nutrition. Major research

efforts have therefore focused on methods to achieve higher CLA
content in milk through animal nutrition that have been reviewed
by Lock and Bauman (2004) and Stanton et al. (2003). Normally,
diets consumed by lactating dairy cows are low in fat content,
containing only about 4e5% lipid (Lock & Bauman, 2004).
Increasing the CLA content of milk, in particular the cis-9, trans-11
isomer, generally requires an increase in rumen vaccenic acid
output, thus allowing for increased endogenous synthesis in the
mammary gland (Lock & Bauman, 2004; Stanton et al., 2003).
Increasing rumen vaccenic acid concentration can be achieved by
increasing the supply of 18-carbon PUFA precursors. Likewise,
inhibiting the reductase enzymes in the rumen responsible for the
conversion of vaccenic acid to stearic acid will also ensure the
increased accumulation and absorption of vaccenic acid by
the animal that can be endogenously converted to CLA in the
mammary gland via D9-desaturase (Griinari & Bauman, 1999).
Plant oils and seeds, marine oils, animal fats, forage, ionophores,
synthetic CLA supplements and combination diets have all been
investigated as means of increasing milk fat CLA concentrations
(Chilliard, Ferlay, & Doreau, 2001; Hennessy, Ross, Stanton, Devery,
& Murphy, 2007; Murphy, Coakley, & Stanton, 2008; Stanton et al.,
2003). Physiological factors also affect milk fat content of CLA, even
between individual animals, presumably related to individual
variations in expression of the D9-desaturase gene and rumenic
outow of vaccenic acid and CLA.
The ultimate aim of increasing the CLA content of milk fat is to
generate dairy products with increased CLA concentration and thus
improved nutritive and therapeutic value when consumed. In
a recent study, CLA-enriched cheese was successfully manufactured
using milk from cows that had been fed on pasture with sunower
oil (Table 4 and Fig. 1, adapted from Coakley et al., 2007). The content
of cis-9, trans-11 CLA in the cheese derived from the pasture-based
sunower oil milk was 1.93 g 100 g1 of fatty acid methyl esters
(FAME), which remained stable throughout the 6 months of
ripening, as opposed to 0.78 g 100 g1 of FAME for the control cheese.
Moreover, sensory evaluation of CLA-enriched milk and cheese by an
open and trained panel clearly demonstrated that the control and
test samples were comparable in terms of mouth-feel, colour, avour
and quality (Khanal et al., 2005). Likewise, manufacture of UHT milk,
butter and cheese with CLA-enriched milk demonstrated that the
high content of CLA did not negatively affect the overall impression
and avour of the three products (Jones et al., 2005a). Processing has
little effect on CLA, so its content in dairy products is directly related
to the CLA concentration of the starting milk (Parodi, 2003).
However, a more recent study has demonstrated that thermal
treatment and refrigerated storage resulted in signicant decreases
or disappearances of the minor CLA isomers coupled with an
increase in trans, trans isomers (Rodriguez-Alcala & Fontecha, 2007).
In terms of the effects of ingestion of CLA-enriched foods on plasma
fatty acid proles, a recent animal study using growing pigs indicated that consumption of butter enriched with cis-9, trans-11 CLA
over a three week period resulted in increased serum concentrations
of ALA and decreased myristic and palmitic acid, with no signicant
effect on the lipoprotein prole (Haug et al., 2008).
In addition, milk-fat CLA must also retain its biological activity.
In a study by Miller, Stanton, Murphy, and Devery (2003), milk-fat
CLA was shown to be as effective as synthetic isomers of CLA at
modulating synthetic CLA-responsive biomarkers in MCF-7 and
SW480 cell lines. However, nutritional strategies to increase the
CLA content of milk also result in milk fat containing increased
concentrations of trans-18:1 (particularly trans-11 18:1, vaccenic
acid), lowered saturated fatty acid concentrations and slightly
increased n-3 PUFA content (Jones et al., 2005b). The greater vaccenic acid content of the CLA-enriched products reects increased
389
Table 4
Effect of treatment on the milk fatty acid compositiona of cows indoors on the
control diet and cows outdoors on pasture receiving sunower oil supplementation
at day 14 (adapted from Coakley et al., 2007).
Fatty acid
Indoors
control
diet
Outdoors on
pasture with
sunower oil
supplementation
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0
C14:1 total
C15:0
C16:0
C16:1 total
C17:0
C17:1
C18:0
C18:1 trans-9 18:trans-11
C18:1 trans-13 18:cis-9
C18:2 cis-9, cis-12 linoleic acid
C20:0
C18:3 cis-9, cis-12, cis-15 linolenic acid
C18:2 cis-9, trans-11 CLA
Other fatty acids
1.46
1.50
1.18
2.92
3.56
12.82
1.45
1.66
35.59
2.30
0.60
0.52
7.51
1.37
18.27
1.14
0.12
0.53
0.46
4.38
1.51
1.22
0.85
1.86
2.30
9.35
1.41
1.13
21.62
2.09
0.61
0.35
12.63
8.05
25.63
2.53
0.10
0.53
2.22
4.50
Values are g 100 g1 fatty acid methyl ester.
ruminal biohydrogenation required to increase synthesis of cis-9,

trans-11 CLA. However, dietary trans fatty acids have been associated with atherogenic risk factors including increased plasma
cholesterol and triglyceride levels as well as plasma markers of
inammation and endothelial dysfunction (Lopez-Garcia et al.,
2005; Mensink & Katan, 1990). A comparison of the effects of
vaccenic and elaidic acid (trans-9 18:1) consumption by male
hamsters indicated that the ratio of LDL/HDL-cholesterol in plasma
was signicantly higher in those fed vaccenic acid (Meijer, van Tol,
van Berkel, & Weststrate, 2001). In contrast to this Bassett et al.
(2010) found that a vaccenic acid-rich butter protected against
atherosclerosis in LDL receptor decient (LDLr/) mice, whereas
a hydrogenated elaidic acid-rich diet stimulated the condition.
Likewise, cholesterol-fed hamsters supplemented with a vaccenic
acid-enriched butter resulted in a plasma lipoprotein cholesterol
prole that is associated with a reduced risk of atherosclerosis
(Lock, Horne, Bauman, & Salter, 2005) Moreover, humans can
convert vaccenic acid to cis-9, trans-11 CLA (Turpeinen et al., 2002).
In a double-blind, cross-over study, Burdge et al. (2005) examined
the level of cis-9, trans-11 CLA and the vaccenic acid content of
plasma and leukocyte lipids in healthy men following consumption
of dairy products naturally enriched with these fatty acids. While
the cis-9, trans-11 CLA content of both plasma and cellular lipids
was increased, a signicant increase was also observed in vaccenic
acid content. However, in a more recent study, it was demonstrated
that consumption of dairy products naturally enhanced with cis-9,
trans-11 CLA and trans-11 18:1 by healthy middle-aged men did not
cause signicant changes in CVD risk variables, as assessed by
investigating blood lipid proles, the atherogenicity of LDL,
markers of inammation and insulin resistance, following a 6-week
consumption period (Tricon et al., 2006).
4.9. Increasing eicosapentaenoic acid and docosahexaenoic
acid content of milk
Enhancing the EPA and DHA concentration of milk fat is a worthy
approach to enhance the fatty acid prole of dairy products. Both
EPA and DHA are either absent or present at minimal levels in
Cis-9, trans-11 CLA (g 100 g-1 of FAME)
390
2.0
1.5
1.0
0.5
0.0
0
50
100
150
Ripening time (days)
200
250
Fig. 1. Cis-9, trans-11 conjugated linoleic acid (CLA) concentration in cheese manufactured from (>) milk from cows fed on pasture and receiving sunower oil supplementation at
day 14 and (B) milk from cows fed indoors with a control diet (adapted from Coakley et al., 2007).
traditional dairy cow diets. One potential means of increasing the

content of both EPA and DHA in milk fat is to exploit sh oils, sh byproducts and marine algae as dairy feedstuffs, since these are rich
sources of both fatty acids. The term transfer efciency refers to the
extent to which dietary supplementation enhances the secretion of
benecial fatty acids in milk. However, the transfer efciency of EPA
and DHA to milk fat is low (Lock & Bauman, 2004). For example,
before sh oil supplementation, the content of EPA and DHA in milk
fat averaged less than 0.1% of total fatty acids but only increased to
0.2e0.3% of total fatty acids after supplementation (Chilliard et al.,
2001). However, transfer efciencies can be improved when sh
oil is directly administered postruminally or fed in a rumen-protected form (Lock & Bauman, 2004). Indeed, McConnell (2004)
reported transfer efciencies of 30 and 25% for EPA and DHA,
respectively when 150 g d1 of fractionated sh oil was infused into
the abomasum. Transfer efciencies can also be improved by
feeding sh meal as opposed to sh oils, the latter which are protected from rumen biohydrogenation by the matrix within which
the oil is encased (Lock & Bauman, 2004).
Overall, two hypothesis have been put forward for the low
transfer efciencies for EPA and DHA into milk (Lock & Bauman,
2004). The rst hypothesis suggests that EPA and DHA are biohydrogenated by rumen bacteria; the second hypothesis suggests
that EPA and DHA partition into plasma lipids that are less available
to the mammary gland.
4.10. Butyrate
Milk fats contain a large variety of SCFAs (Christie, 1995) with 1e6
carbon atoms in the acyl chain. Butyric acid (C4:0), exerts an array of
effects on the colonic mucosa and represents approximately 10% of
all fatty acids found in bovine milk, being present at approximately
2e5 wt % (Jensen, 2002; Kaylegian & Lindsay, 1995). Interestingly,
butyrate is generated by bacteria in the rumen from carbohydrates
and then transported via the blood to the mammary gland where it is
reduced to butanoic acid (Jensen, 1999). In humans, butyrate
provides the principal source of energy for colonic epithelial cells but
also regulates a number of genes associated with cell differentiation,
proliferation and apoptosis (Hamer et al., 2008; Scheppach et al.,
1992). Butyrate is also produced in the intestine following the

microbial breakdown of prebiotic substances and non-digestible
carbohydrates. Several studies have yielded results demonstrating
the anti-proliferative, anti-inammatory and apoptotic properties of
butyrate, these have been reviewed by Mills, Ross, Fitzgerald, and
Stanton (2009).
While ingested butyrate does not reach the large intestine,
studies suggest that dietary butyrate can exhibit biological effects
at locations other than the colon. Orally ingested milk butyrate
undergoes lipase-mediated hydrolysis in the stomach which is
complete on reaching the proximal small intestine (Parodi, 1997).
Here, butyrate is immediately absorbed, processed and released
into the blood stream for transport to the liver where most is
metabolized (Parodi, 1997; Smith, Yokoyama, & German, 1998).
Interestingly, Yanagi, Yamashita, and Imai (1993) demonstrated
that margarine supplemented with sodium butyrate signicantly
reduced the incidence of mammary tumors in a rat model. Ingestion of butyrate as a natural component of anhydrous milk fat
(containing 0.8% butyrate) was found to be effective at inhibiting
mammary tumorigenesis in rats (Belobrajdic & McIntosh, 2000).
Such studies provide promising evidence that dietary sources of
butyrate can inuence carcinogenesis at other sites in the body.
4.11. Medium chain fatty acids

Medium chain fatty acids (MCFA) that contain 8e12 carbons and
are saturated are constituents of milk and have been associated
with benecial effects linked with metabolic activities. It has been
postulated that MCFA may help to reduce the risk of developing
features of metabolic syndrome (Pfeuffer & Schrezenmeir, 2002;
2007), a cluster of metabolic disorders including dyslipidaemia,
hypertension, obesity and glucose intolerance, where insulin
resistance is the core phenomenon and co-occurrence is associated
with increased CVD risk (Pfeuffer & Schrezenmeir, 2007). Earlier
studies have shown that dietary substitution of medium chain
triglycerides (MCT) for long chain triglycerides (LCT) can inuence
energy balance and thus it was postulated that they may promote
weight reduction (Dulloo, Fathi, Mensi, & Girardier, 1996; Hill et al.,
1989).
Unlike long chain fatty acids (LCFAs), MCFAs are hydrolysed

more rapidly and metabolized more completely after absorption
across the epithelial barrier (Babayan, 1987; Bach & Babayan, 1992).
They are transported directly to the liver via the portal circulation
unlike the LCFA that are preferentially incorporated into chylomicrons as LCT and transported via lymph (Guillot, Vaugelade,
Lemarchal, & Rerat, 1993; Papamandjaris, MacDougall, & Jones,
1998). Once transported to the liver, the MCFA can follow various
catabolic pathways including beta-oxidation, omega-oxidation, and
peroxisomal oxidation (Papamandjaris et al., 1998). Thus, few MCFA
are recovered in triglyceride (Christensen, Gronn, Hagve, &
Christophersen, 1991), phospholipid or cholesterol ester fractions
(Mascioli et al., 1989).
Tsuji et al. (2001) demonstrated that a daily intake of 10 g of
MCT for a 12-week period signicantly reduced body weight, body
fat, abdominal subcutaneous fat and waist and hip sizes in subjects
with a body mass index (BMI) of 23 kg m2, suggesting that intake
of MCT could be effective for preventing obesity in subjects with
a high BMI. In follow-up studies the differences in the dynamics of
postprandial serum lipids between two groups of subjects with
BMI 23 kg m2 and <23 kg m2 after intake of MCT or LCT at
a single dose of 10 g demonstrated that the response of triglyceride
after LCT intake was greater for subjects with BMI 23 kg m2 than
for those with BMI < 23 kg m2 (Kasai et al., 2003a). Moreover,
subjects with BMI 23 kg m2 showed a smaller triglyceride
response after receiving MCT than after receiving LCT and remnantlike lipoprotein cholesterol levels were lower in subjects who
received MCT (Kasai et al., 2003b). Interestingly, the use of
a mixture of both medium- and long-chain triacylglycerols as
substitutes for common edible vegetable oils has resulted in the
FOSHU (Food for Specied Health Use) product with the trade
name Healthy Resseta which has been shown to have a suppressing effect on body fat accumulation and to increase diet induced
thermogenesis and is now widely sold in Japan (Aoyama, Nosaka, &
Kasai, 2007; Ogawa et al., 2007).
4.12. Antimicrobial lipids
Components of human and bovine milk fat have been shown to
exert antimicrobial properties (German & Dillard, 2006; Isaacs,
2005). For example, a number of milk fatty acids were highly
active against the enveloped viruses, where the antiviral fatty acids
were shown to affect the viral envelope, causing leakage, and at
higher concentrations, complete envelope disintegration (Thormar,
Isaacs, Brown, Barshatzky, & Pessolano, 1987). Likewise, synthetic
lipids adapted from naturally occurring compounds found in
human breast milk demonstrated an ability to kill the sexually
transmitted Chlamydia trachomatis by disrupting the chlamydial
inner membrane (Lampe, Ballweber, Isaacs, Patton, & Stamm, 1998).
In another study, lauric acid (C12:0), and linoleic and linolenic
acids were shown to be bactericidal and decreased the invasiveness
of the food-borne pathogen L. monocytogenes in a Caco-2 enterocyte-like cell line (Petrone et al., 1998). Capric acid (C10:0) and
lauric acid and digestion products of sphingolipids from bovine
milk triglycerides and membrane lipids were also shown to be
effective bactericidal agents against a variety of pathogens as well
as L. monocytogenes, such as E. coli 0157, Salmonella enteritidis,
Campylobacter jejuni, and Clostridium perfringens (Sprong, Hulstein,
& Van der Meer, 2001). In a follow-up study, L. monocytogenes and
C. jejuni were shown to be very sensitive to capric acid and lauric
acid and the C18:0 fatty acids, together with digestion products of
sphingolipids, whereas E. coli 0157 and S. enteritidis were less
vulnerable (Sprong, Hulstein, & Van der Meer, 2002). However, the
antimicrobial effect of these fatty acids may be limited to C10:0 and
C12:0 fatty acids in vivo as determined following testing in rats
391
(Sprong et al., 2002). Analysis of the anti-microbial properties of

bovine milk fat after partial hydrolysis by calf pregastric lipase
demonstrated that lauric acid was most potent against the Gram
positive enterococci, while caprylic acid (C8:0) was most potent
against the Gramnegative coliforms (Sun, OConnor, & Roberton,
2002). More recently, the products from lipase-catalysed hydrolysis of bovine milk fat were shown to kill H. pylori in vitro (Sun,
OConnor, MacGibbon, & Roberton, 2007).
Interestingly, free fatty acids from bovine whey cream have been
shown to inhibit the germination of C. albicans in vitro, which was
mainly attributed to lauric acid, myristoleic acid (C14:1n-5), LA and
ARA (Clement, Tremblay, Lange, Thibodeau, & Belhumeur, 2007). A
more recent study demonstrated that capric acid, lauroleic acid
(C12:1), 11-methyldodecanoic acid (iso-C13:0), myristoleic acid
(C14:1n-5), and g-linolenic acid (C18:3n-6) from bovine whey cream
also exhibited antifungal activities against Aspergillus fumigates as
well as C. albicans (Clement, Tremblay, Lange, Thibodeau, &
Belhumeur, 2008).
5. Dairy calcium
Both animal and human studies have clearly indicated that
dietary calcium can play a signicant role in weight management;
remarkably, however, the effect from dairy sources is distinctly
more dramatic than the effect from non-dairy sources (Zemel, 2003;
2004; 2005). It has been suggested that dietary calcium exerts this
effect on weight through the calcitrophic hormones, parathyroid
hormone and 1,25(OH)2D (Zemel, 2004). These hormones have
been shown to respond to low-calcium diets and exert coordinated
regulatory effects on human adipocyte lipogenic and lipolytic
systems (Zemel, 2004). In addition, high calcium diets have been
shown to increase fecal fat excretion (Jacobsen, Lorenzen, Toubro,
Krog-Mikkelsen, & Astrup, 2003; Papakonstantinou, Flatt, Huth, &
Harris, 2003). These fat-reducing effects have been observed in
both animal (Causey & Zemel, 2003; Metz, Karanja, Torok, &
McCarron, 1988; Papakonstantinou et al., 2003; Parra, Bruni,
Palou, & Serra, 2008; Shi, Dirienzo, & Zemel, 2001; Sun & Zemel,
2004; Zemel & Geng, 2001; Zemel & Morgan, 2002; Zemel, Shi,
Greer, Dirienzo, & Zemel, 2000; Zemel, Sun, & Geng, 2001) and
human trials (Zemel, Thompson, Milstead, Morris, & Campbell,
2004) that have been discussed in detail in the following reviews
by Zemel (2003; 2004, 2005).
Dairy sources of calcium have been reported to be 50e100%
more effective than supplemental calcium (Zemel, 2005), which
can probably be best appreciated from the results of the following
clinical trials. In the rst clinical trial (Zemel et al., 2004), 32 obese
adults were maintained on balanced caloric-decit diets
(500 kcal day1 decit) and randomized to control diet
(400e500 mg of calcium day1), high calcium (control diet supplemented with 800 mg of calcium day1) or high dairy (3e4
servings of milk, yoghurt and/or cheese day1, representing a total
calcium intake of 1200e1300 mg day1). Over a 24-week study,
control subjects lost 5.4% of their body weight, subjects on the high
calcium diet lost 8.6%, while those on the dairy diet lost 10.9%
(p < 0.01). Fat loss followed a similar trend. In addition, there was
notable change in the distribution of body fat loss, as fat loss from
the trunk region represented 19% of the total fat lost on the lowcalcium diet, which increased to 50% and to 66% on the high
calcium and high dairy diets, respectively (Zemel et al., 2004). A
follow-up study of 34 obese subjects also supported these ndings
(Zemel et al., 2005a) which was replicated in a six-month clinical
trial in obese African Americans (Zemel, Richards, Milstead, &
Campbell, 2005b). Likewise, dairy calcium intake over 18 months
by young female subjects resulted in reduced fat mass accumulation, although the effect was small (Eagan, Lyle, Gunther, Peacock, &
392
Teegarden, 2006). Moreover, a diet rich in dairy calcium was also

shown to enhance weight loss in type-2 diabetic patients (Shahar,
Abel, Elhayany, Vardi, & Fraser, 2007).
A study involving 49 Caucasian postmenopausal women indicated that increased calcium intake was associated with lower body
fat and higher dairy intake was associated with lower fat mass
(Heiss, Shaw, & Carothers, 2008). More recently, a meta-analysis of
randomized controlled trials indicated that increasing dairy
calcium intake by 1241 mg day1 increased faecal fat excretion by
5.2 g day1, which could have signicant consequences for the
prevention of weight (re-)gain (Christensen et al., 2009).
The greater effect observed for dairy calcium has been attributed
to additional bioactive components which have been assigned to
the whey portion of milk (Causey & Zemel, 2003; Ha & Zemel, 2003;
Zemel, 2005). ACE-inhibitory activity (previously discussed) is one
such component which has been postulated as increasing the fatreducing effects of dairy calcium, since angiotensin-II regulates, in
part, adipocyte lipogenesis (Pfeuffer & Schrezenmeir, 2007).
Moreover, branched chain amino acids (BCAA, leucine, isoleucine
and valine) have also been implicated, since in addition to protein
synthesis, these amino acids also play specic metabolic roles as
energy substrates and in the regulation of muscle protein synthesis
(Layman, 2003). As already discussed in this review, MCFAs have
been shown to have a positive effect on weight reduction. Thus, the
weight-reducing effects of milk possibly result from a collection of
effects rather than being exerted from a single mechanism.
However, not all studies have concluded that dietary calcium or
indeed dairy calcium has a positive effect on obesity and this
controversy relating to results from clinical trials has been discussed in the following reviews; Barba and Russo (2006), Barr
(2003) and Teegarden (2005). Indeed, some studies have failed to
identify the benecial effects associated with reductions in weight
loss following calcium consumption (supplemental or dairy). For
example, in a study of an obesity-prone population consisting of 65
Indian Pima adults (35 men and 30 women aged 33 8 years) and
78 Pima Indian children (36 boys and 42 girls, aged 10 0.3 years),
no association was observed between dietary calcium intake and
body size or adiposity (Venti, Tataranni, & Salbe, 2005). The authors
concluded that the high-fat energy diet consumed by the volunteers possibly overwhelmed the anti-obesity effect of calcium. A
one-year intervention trial of 155 young (18e30 years), healthy,
normal weight women consuming a medium dairy diet (calcium
intake of w1000e1100 mg day1) or high dairy diet (calcium intake
of 1300e1400 mg day1) indicated that the increased intake of
dairy products did not alter body weight or fat mass (Gunther et al.,
2005). Longer term trials are required to thoroughly investigate the
effects of dietary calcium as changes in fat mass may be too small to
detect in short periods of time. In addition, several other dietary
factors must be considered such as total energy intake, dietary
protein content and vitamin D status. Moreover, nutrigenomics is
proving that different populations and indeed different individuals
exert variable responses to the same dietary components. Barba
and Russo (2006) have suggested that the complex composition
of dairy foods may be responsible for this biological variability.
Overall, the evidence suggests that dairy foods have an important
role to play in weight regulation. In the future, a better understanding of the mechanisms involved in dairy calcium and weight
loss will enable reliable recommendations for dairy calcium
consumption which will potentially help reduce the development
of obesity for many individuals.
6. Microbial diversity inscribed in human milk
The origin of lactobacilli and bidobacteria that colonise the
neonatal gut has in the past been attributed to contamination of the
infant with maternal microorganisms upon passage through the

birth canal. However, recent studies have provided clear evidence
that human milk is a direct source of both lactobacilli (Martin,
Heilig, Zoetandal, Smidt, & Rodriguez, 2007a) and bidobacteria
(Gueimonde, Laitinen, Salminen, & Isolauri, 2007; Martin et al.,
2009), and LAB colonisation may not be signicantly related to
the mode of delivery (Martin et al., 2007b). Indeed, Martn et al.
(2006) successfully tracked a Lactobacillus salivarius isolate, which
was shown to have potential probiotic properties, from the faeces
of a one-month-old breast-fed infant to the breast milk of the
respective mother through the use of DNA nger-printing. Moreover, in a related study, analysis of the probiotic potential of three
more Lactobacillus isolates from breast milk indicated that each
strain harboured probiotic properties comparable to strains
commonly used in commercial probiotic products (Martn et al.,
2005) and all four strains were shown to possess potent antimicrobial activities (Oliveras, Diaz-Ropero, Martn, Rodriguez, & Xaus,
2006). In the case of bidobacteria, B. longum was found to be the
dominant species in human breast milk followed by Bidobacterium animalis, B. bidum and Bidobacterium catenulatem following
the analysis of 20 human milk samples (Gueimonde et al., 2007).
The source of such bacteria in the milk perhaps represents
a unique form of mother-infant communication (Perez et al., 2007).
Indeed, in a compelling study by Perez et al. (2007), it was
demonstrated that human breast milk cells contain a limited
number of viable bacteria but a range of bacterial DNA signatures
which are also found in maternal peripheral blood mononuclear
cells. The results of the study suggest that intestinally derived
bacterial components are transported to the lactating breast within
mononuclear cells. The authors proposed that this directs the
neonatal immune system to recognize specic bacterial molecular
patterns and to respond appropriately to pathogens and
commensals (Perez et al., 2007). In support of this, bacterial DNA
has been shown to stimulate innate immunity in pregnant mice,
improve maternal survival rates and prevent pathogen transmission to the fetus (Ito, Ishii, Shirota, & Klinman, 2004). Thus, it
appears that through human milk the infant is immediately
equipped with a strategy for immune surveillance at the earliest
stages of life.
7. Conclusion and outlook for the future
Milk provides a plethora of bioactive ingredients for incorporation into functional food products. This has come at a time when
consumers want more from food than just basic nutrition, but
rather a capacity to alleviate the onset of lifestyle diseases through
diet. Using milk as a model, system could prove invaluable for
developing designer foods or even designer milk, a concept that
has been discussed in a review by Sabikhi (2007). However, it
should be noted that many of the physiological effects observed for
the bioactive components of milk have only been proven in vitro or
in animal models and have yet to be proven in humans. Another
major challenge facing food scientists and manufacturers alike is
the cost-effective large-scale production of milk bioactive ingredients. For example, while the potential for milk proteins and
peptides as ingredients in functional food products has been well
documented on a vast scale, their large-scale production and
commercialisation is still limited. Major efforts are now underway
to develop methods to ensure optimal activity of these agents in
food systems and their subsequent utilisation in the body. One
group has recently cloned the 11-residue antimicrobial peptide
from bovine lactoferrin (BL-11) and the 12-residue hypotensive
peptide from as1-casein (C-12) in the dairy starter culture Streptococcus thermophilus, which is utilised in the production of yoghurt
and various cheeses (Renye & Somkuti, 2008). Multiple repeats of
the hypocholesterolemic peptide IIAEK, derived from bovine milk

b-lactoglobulin, have been introduced into the ve variable regions
of soybean proglycinin A1aB1b and when expressed in E. coli have
demonstrated the large-scale production of a small peptide of
fewer than 10 amino acids (Prak, Maruyama, Maruyama, & Utsumi,
2006; Prak & Utsumi, 2009). The potent antihypertensive peptide
derived from ovalbumin, RPLKPW (novokinin), has recently been
incorporated into the soybean through transgenesis (Yamada et al.,
2008). Through such approaches milk bioactive peptide sequences
may even be incorporated into food proteins of non-dairy origin.
However, using molecular genetic approaches to increase
bioavailability or production of therapeutic peptides is perhaps an
unrealistic goal, considering the strict regulations governing the
use of genetically modied organisms in the food industry in
certain parts of the world. The existing modern technologies
applicable for the isolation of bioactive native proteins and peptides
from milk are beyond the scope of this review but have been discussed in detail in reviews by Korhonen and Pihlanto (2003, 2007).
HMOs are essential components for the correct functioning of
numerous physiological systems: they modulate the growth of the
intestinal microbiota; they prevent adhesion of pathogenic microorganisms to the intestinal mucosa; through selectin binding, they
behave as anti-inammatory mediators by reducing the binding of
platelets to neutrophils; they may play a role in the development of
the infant brain, through sialylated oligosaccharides. However, the
variation between bovine milk oligosaccharides and HMOs is
extensive and studies are indicating that the diversity and vast
number of HMOs are essential to the correct overall development of
the infant. Strategies to emulate the oligosaccharide content of
human milk are ongoing (Espinosa, Tamez, & Prieto, 2007),
although to date FOS and GOS have proven successful as prebiotic
ingredients for use in infant formula. Goats milk may also hold
potential as a natural source of lactose-derived oligosaccharides as
supplements for infant formulas and for the development of
functional foods, having been successfully isolated by membrane
technology (Martinez-Ferez et al., 2006). The production of HMOs
on a large-scale has been achieved in the past through the use of
metabolically engineered bacteria by direct fermentation of inexpensive carbon sources (Priem, Gilbert, Wakarchuk, Heyraud, &
Samain, 2002). Future research must now focus on means of
determining which structures exert an advantageous biological role
and an economic means of harvesting such oligosaccharides, which
are currently nding themselves at the core of a new food additives
generation (Barreteau, Delattre, & Michaud, 2006) and as potential
ingredients in drug development (Kobata, 2003).
While milk fat serves as a source of energy for the neonate of
each species, it is also a source of bioactive agents which can
inuence all aspects of physiology from the immune system to the
CNS. In addition, its components exert both antibacterial and
antiviral activities. Transgenesis may in the future provide an
approach towards altering the fatty acid composition of milk.
Indeed, the expression of a rat stearoyl-CoA desaturase gene under
the control of the bovine b-lactoglobulin promoter in transgenic
dairy goats altered the fatty acid composition of milk, resulting in
a less saturated and more monounsaturated prole (Reh et al.,
2004). However, milk fat, unlike other components, is more
pliable in that its content can be manipulated through diet. Indeed,
the ability to manipulate the content of certain fatty acids, such as
CLA, has enabled scientists to directly enhance the therapeutic
properties of milk and dairy products as a result.
In conclusion, it appears that almost every component of milk
provides benecial therapeutic effects. This may not be surprising
given that milk is the natural food having evolved under selective
pressure to meet the nutritional needs of its species. Now scientists
are going beyond the components of milk itself and examining the
393
genomics of milk, the genes that code the composition of milk in

the Milk Genomics Consortium (German et al., 2006), with the
expectation that such an approach will reveal the principal biological denition of mammalian nutrition. The future for milk
research therefore looks brighter than ever and no doubt will
continue in this direction as scientists learn more about this
complex, yet highly organized food which should serve as a model
system for creating superior functional food products.
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International Dairy Journal 21 (2011) 377e401

Contents lists available at ScienceDirect

International Dairy Journal

Milk intelligence: Mining milk for bioactive substances associated

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

manufactured and regulated and how they achieve their specic

Ion carrier (Ca, PO4, Fe, Zn, Cu),

Total whey protein

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

research has concentrated on the therapeutic potential of milk

VPP, IPP from b-casein and k-casein

Reduction of blood pressure

Calpis Co., Japan

Reduction of blood pressure

Casein-derived dodecapeptide FFVAPFPEVFGK

DMV, The Netherlands

as1-casein f (91e100) YLGYLEQLLR

Reduction of blood pressure

CSPHP ProDiet F200

Helps mineral absorption

Arla Foods, Denmark

as1-casein f (1e6) RPKHPI, f (1e7)

Reduction of blood pressure

RPKHPIK, f (1e9) RPKHPIKHQ,

Boost energy, improve sleep quality

Borcula Domo Ingredients

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

2.2. Antihypertensive peptides

Indeed, enzymatic hydrolysis of k-casein has resulted in some of the

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

ELISA studies (Zoghbi et al., 2006). Since intestinal mucins play

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

cysteine to the glutathione antioxidant system (GSH), one of the

2.10. Mammary-associated serum amyloid A3

Bovine milk Bovine colostrum

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

(2007) demonstrated that the infant gut isolate B. longum biovar

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

the authors suggest, may provide guidance for optimizing formula

responses whereas GPR43-decient mice (Gpr43/) demonstrated

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

loss of chloride and bicarbonate transport, culminating in the efux

2002). Milk oligosaccharides have been shown to harbour

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

gangliosides (65%) followed by glycoproteins (32%) and the

effectiveness and safety of sialic acid supplemented formulas.

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

antimicrobial effects, with some of these effects attributable to the

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

biological activities of sphingolipids, there is no doubt that these

The n-3 fatty acids have also exhibited therapeutic potential in

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

depending to a large extent on animal nutrition. Major research

Values are g 100 g1 fatty acid methyl ester.

ruminal biohydrogenation required to increase synthesis of cis-9,

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

Cis-9, trans-11 CLA (g 100 g-1 of FAME)

traditional dairy cow diets. One potential means of increasing the

1992). Butyrate is also produced in the intestine following the

4.11. Medium chain fatty acids

S. Mills et al. / International Dairy Journal 21 (2011) 377e401

Unlike long chain fatty acids (LCFAs), MCFAs are hydrolysed