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26 Equid Milk: Chemistry, Biochemistry and Processing 505has been determined and differs from those of equine and bovine
proteins with 39 and 40 amino acid substitutions, respectively
(Godovac-Zimmermann et al. 1987).ImmunoglobulinsThe concentration of whey proteins is significantly elevated in
the colostrum of all ruminants and equids as maternal Igs are
passed from mother to neonate after birth when the small in-
testine is capable of absorbing intact proteins. After a few days,
the gut ‘closes’ and further significant passage of proteins is pre-
vented and within 2–3 days, the serum level of IgG in the neonate
is similar to adult levels (Widdowson 1984). In contrast,in utero
transfer of Igs occurs in humans and in some carnivores Igs are
passed to the newborn both before and after birth. The milk of
species that provide prenatal passive immunisation tends to have
relatively small differences in protein content between colostrum
and mature milk compared to species that depend on post-natal
passage of maternal Igs. In the latter cases, of which all un-
gulates are typical, colostrum is rich in Igs and there are large
quantitative differences in protein content between colostrum
and mature milk (Langer 2009).
Three classes of Igs, which form part of a mammal’s natural
defense against infection, are commonly found in milk, IgIgG,
IgA and IgM; IgG is often sub-divided into two sub-classes, IgG 1
and IgG 2 (Hurley 2003, Madureira et al. 2007). All monomeric
Igs consist of a similar basic structure of four polypeptides, two
heavy chains and two light chains, linked by disulphide bridges,
yielding a sub-unit with a molecular mass of approximately 160
kDa. IgG consists of one sub-unit, while IgA and IgM consist of
two or five sub-units, with a molecular mass of approximately
400 or approximately 1000 kDa, respectively. The relative pro-
portions of the Igs in milk differ considerably between species
(Table 26.2). IgG is the principal Ig in equine colostrum, but
IgA is the principal form in equine milk. In bovine milk and
colostrum, IgG is the principal immunoglobulin, while IgA is
the predominant Ig in human colostrum and milk.LactoferrinLf is an iron-binding glycoprotein, comprising of a single
polypeptide chain of MW approximately 78 kDa (Conneely
2001). Lf is structurally very similar to transferrin (Tf), a plasma
iron transport protein, but has a much higher (∼300-fold) affin-
ity for iron (Brock 1997). Lf is not unique to milk although it is
especially abundant in colostrum, with small amounts in tears,
saliva and mucus secretions and in the secondary granules of
neutrophils. The expression of Lf in the bovine mammary gland
is dependent on prolactin (Green and Pastewka 1978); its con-
centration is very high during early pregnancy and involution
and is expressed predominantly in the ductal epithelium close
to the teat (Molenaar et al. 1996). Human, equine, asinine and
bovine milk contain 1.65 , 0.58 , 0.37 and 0.1 g Lf/kg, respec-
tively (Table 26.2). The concentration of Lf in asinine milk,
which comprises approximately 4% of total whey protein, is
significantly lower than in equine milk (Table 26.2).Shimazaki et al. (1994) purified Lf from equine milk and
compared its iron-binding ability with that of human and bovine
Lfs and with bovine Tf. The iron-binding capacity of equine Lf
is similar to that of human Lf but higher than that of bovine Lf
and Tf. Various biological functions have been attributed to
Lf but the exact role of Lf in iron-binding in milk is unknown
and there is no relationship between the concentrations of Lf
and Tf and the concentration of iron in milk (human milk is very
rich in Lf but low in iron) (Masson and Heremans 1971).
Lf is a bioactive protein with nutritional and health-promoting
properties (Baldi et al. 2005). Bacterial growth is inhibited by its
ability to sequester iron and also to permeabilise bacterial cell
walls by binding to lipopolysaccharides through itsN-terminus.
Lf can inhibit viral infection by binding tightly to the envelope
proteins of viruses and is also thought to stimulate the establish-
ment of a beneficial microflora in the gastrointestinal tract (Baldi
et al. 2005). Ellison and Giehl (1991) suggested that Lf and
Lyz work synergistically to effectively eliminate Gram-negative
bacteria; Lf binds oligosaccharides (OSs) in the outer bacterial
membrane, thereby opening ‘pores’ for Lyz to hydrolyse glyco-
sidic linkages in the interior of the peptidoglycan matrix. This
synergistic process leads to inactivation of both Gram-negative,
for exampleE.coli(Rainhard 1986) and Gram-positive bacte-
ria, for exampleStaphylococcus epidermidis(Leitch and Will-
cox 1999) bacteria. Furthermore, a proteolytic digestion product
of bovine and human Lf, lactoferricin, has bactericidal activity
(Bellamy et al. 1992). Bovine and human Lf are reported to
have antiviral activity and a role as a growth factor (Lonnerdal ̈
2003). The specific biological function of equine Lf has not been
studied, but is likely to be similar to that of bovine and human Lf.
Equine Lf contains 689 amino acid residues, which is sim-
ilar to bovine Lf and two more than human Lf (Table 26.5).
Compared to most other milk proteins, Lf has a high isoelec-
tric point, that is, at pH 8.32, 8.67 or 8.47 for equine, bovine
or human Lf (Table 26.5). As a result, the protein is positively
charged at the pH of milk and may associate with negatively
charged proteinsviaelectrostatic interactions. GRAVY scores
are comparable for equine, bovine and human Lf (Table 26.5).
Equine and human Lf contain 17 and 16 intra-molecular disul-
phide bonds, respectively (Table 26.5). On the basis of structural
similarities with human Lf, it has been assumed that bovine
Lf contains 16 intra-molecular disulphide bonds (Table 26.5).
The iron-binding capacity of equine, bovine and human Lfs are
equivalent, although the pH-dependence of the iron-binding ca-
pacity of bovine Lf differs from that of equine and human Lf
(Shimazaki et al. 1994).
All Lfs studied to date are glycosylated, but the location and
number of potential glycosylation sites, as well as the number
of sites actually glycosylated, vary. In bovine Lf, four out of five
potential glycosylation sites, that is, Asn 223 ,Asn 368 ,Asn 476 and
Asn 545 , are glycosylated (Moore et al. 1997), whereas in hu-
man Lf, two of three potential glycosylation sites, that is, Asn 137
and Asn 478 , are glycosylated (Haridas et al. 1995). Glycosyla-
tion of equine Lf has not been studied, but using the consensus
sequence, Asn-Xaa-Ser/Thr (where Xaa is not Pro), for glyco-
sylation, three potential glycosylation sites are likely in equine
Lf, that is Asn 137 ,Asn 281 and Asn 476.