BLBS102-c26 BLBS102-Simpson March 21, 2012 13:51 Trim: 276mm X 219mm Printer Name: Yet to Come
26 Equid Milk: Chemistry, Biochemistry and Processing 499Equine, bovine and humanβ-casein have a very hydrophilic
N-terminus, followed by a relatively random hydropathy distri-
bution in the rest of the protein, leading to an amphiphilic protein
with a hydrophilicN-terminus and a hydrophobicC-terminus.
In equine sodium caseinate, the Lys 47 –Ile 48 , bond ofβ-casein
is hydrolysed readily by bovine plasmin, whereas no cleavage
of the corresponding bond, Lys 48 –Ile 49 in bovineβ-casein has
been shown (Egito et al. 2003). In bovineβ-casein, Lys 28 -Lys 29
is readily cleaved by plasmin but the equivalent, Lys 28 –Leu 29 ,
in equineβ-casein is insensitive (Egito et al. 2002). Other
plasmin cleavage sites in equineβ-casein are Lys 103 –Arg 104 ,
Arg 104 –Lys 105 and Lys 105 –Val 196 (Egito et al. 2002). Equine
β-casein is readily hydrolysed by chymosin at Leu 190 –Tyr 191
(Egito et al. 2001).
Equine β-casein and equineα-La undergo spontaneous
deamidation under physiological conditions at Asn 135 –Gly 136
and Asn 45 –Gly 46 , respectively (Girardet et al. 2004), which has
been reported also for canine milk Lyz (Nonaka et al. 2008) and
human lactoferrin (Lf) (Belizy et al. 2001) but not, to our knowl-
edge, for bovine or humanβ-casein orα-la. Recent research has
shown that temperature may be an important factor control-
ling the spontaneous deamidation process and at 10◦C, the phe-
nomenon is strongly reduced (Mat ́eos et al. 2009b). Spontaneous
deamidation represents an important modification of equine milk
proteins under certain conditions where bovine milk proteins,
which do not contain a potential site for deamidation, remain
unaffected. Equine Lf also contains the Asn–Gly sequence and
may be susceptible to spontaneous deamidation (Girardet et al.
2006).
Unique to equine milk and apparently absent from the milk
of other species, including ruminants, is a low-molecular weight
(MW) multi-phosphorylatedβ-casein variant which accounts
for 4% of the total casein (Miclo et al. 2007). This short pro-
tein (94 amino acid residues) is the result of a large deletion
(residues 50–181) from full-length equineβ-casein. No spon-
taneous deamidation of this low-MW form ofβ-casein has
been found. Multi-phosphorylated isoforms ofβ-casein, approx-
imately 34–35.4 kDa, have been identified in asinine milk but
no further characterisation has been reported to date (Criscione
et al. 2009).κ-CaseinThe presence ofκ-casein in equine milk was an issue of de-
bate for several years, with several authors (Visser et al. 1982,
Ono et al. 1989, Ochirkhuyag et al. 2000) reporting its absence.
However, other studies (Kotts and Jenness 1976, Malacarne et al.
2000, Iametti et al. 2001, Egito et al. 2001) showed its presence,
albeit at a low concentration. The primary structure of equine
κ-casein has been derived (Iametti et al. 2001, Lenasi et al. 2003,
Miranda et al. 2004); it contains 165 amino acids residues, that
is four less than bovineκ-casein but three more than human
κ-casein (Table 26.3). The MW of equineκ-casein, prior to
post-translational modification, is 18,844.7 Da. Equine and hu-
manκ-casein have a considerably higher isoelectric pH than
bovineκ-casein (Table 26.3), and they have a net positive charge
at physiological pH, whereas bovineκ-casein has a net negativecharge. The GRAVY score of bovineκ-casein is considerably
lower than that of equineκ-casein (Table 26.3), indicating that
the latter is more hydrophilic. Bovineκ-casein is characterised
by a hydrophilicC-terminus, which is very important for the
manner in which bovine casein micelles are stabilised, but a
comparison of the hydropathy distribution of bovine and equine
κ-caseins indicates that theC-terminus of equineκ-casein is
far less hydrophilic, particularly as a result of the absence of a
strong hydrophilic region at residues 110–120. Humanκ-casein
appears to be more like equine than bovineκ-casein in terms
of the distribution of hydropathy along the polypeptide chain.
Studies on asinine milk have not foundκ-casein (Vincenzetti
et al. 2008, Chianese et al. 2010).Glycosylation ofκ-Casein κ-Casein, the only glycosylated
member of the casein family, exhibits microheterogeneity due
to the level of glycosylation (Saito and Itoh 1992). Tri- or tetra-
saccharides consisting ofN-acetylneuraminic acid (NANA),
galactose andN-acetylgalactosamine are attached toκ-casein
via O-glycosidic linkages to threonine residues in theC-terminal
portion of the molecule (the glycomacropeptide region). About
two-thirds of bovineκ-casein molecules are glycosylated at one
of six threonyl residues, that is Thr 121 ,Thr 131 ,Thr 133 ,Thr 135 ,
Thr 136 (only in bovineκ-casein variant A) or Thr 142 (Pisano et al.
1994); Ser 141 is also a potential glycosylation site (Kanamori
et al. 1981). Humanκ-casein has seven glycosylation sites,
Thr 113 ,Thr 123 ,Thr 128 ,Thr 131 ,Thr 137 ,Thr 147 and Thr 149 (Fiat
et al. 1980). Although no direct information is available, lectin-
binding studies indicate that equineκ-casein is glycosylated
(Iametti et al. 2001), possibly at residues Thr 123 ,Thr 127 ,Thr 131 ,
Thr 149 and Thr 153 (Lenasi et al. 2003) (these glycosylation sites
are not fully in agreement with those proposed by Egito et al.
(2001)). To date, no non-glycosylatedκ-casein has been identi-
fied in equine milk (Martuzzi and Doreau 2006).
κ-Casein is located mainly on the surface of the casein mi-
celles and is responsible for their stability (Walstra 1990). The
presence of a glycan moiety in theC-terminal region ofκ-casein
enhances its ability to stabilise the micelle, by electrostatic repul-
sion, and may increase the resistance by the protein to proteolytic
enzymes and high temperatures (Minkiewicz et al. 1993, Dzi-
uba and Minkiewicz 1996). Biologically, NANA residues have
antibacterial properties and act as a bifidogenic factor (Dziuba
and Minkiewicz 1996).κ-Casein is thought to play a major role
in preventing the adhesion ofHelicobacter pylorito human gas-
tric mucosa (Stromqvist et al. 1995). It is likely that heavily gly- ̈
cosylatedκ-casein provides some protection to breast-feeding
infants due to its carbohydrate content which may be important
especially asH. pyloriinfection is occurring at an increasingly
younger age (Lonnerdal 2003). ̈Hydrolysis ofκ-Casein The hydrolysis of bovineκ-casein
by chymosin at Phe 105 –Met 106 leads to the production of
the hydrophobicN-terminal para-κ-casein and the hydrophilic
C-terminal caseinomacropeptide (CMP) (Walstra and Jenness
1984). Chymosin hydrolyses the Phe 97 –Ile 98 bond of equineκ-
casein (Egito et al. 2001) and slowly hydrolyses the Phe 105 –Ile 106
bond of humanκ-casein (Plowman et al. 1999). However, as