Food Biochemistry and Food Processing (2 edition)

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500 Part 4: Milk

Table 26.4.Properties of Equine, Bovine and Human Para-κ-Casein and Caseinomacropeptide

Protein Species Residues

Amino

Acid

Residues

Molecular

Weight (Da) pI GRAVYa

Para-κ-casein Equine 1–97 97 11,693.3 8.96 −0.675

Bovine 1–105 105 12,285.0 9.33 −0.617

Human 1–105 97 11,456.9 9.63 −1.004

CMP Equine 98–165 68 7,169.3 4.72 0.203

Bovine 106–169 63 6,707.4 4.04 −0.370

Human 106–162 65 6,723.7 4.24 0.182

aGrand average hydropathy (GRAVY) score using the scale of Kyte and Doolittle (1982).

Source: Modified from Uniacke et al. 2010.

CMP,C-terminal caseinomacropeptide.

summarised in Table 26.4, the CMPs released from equine and

humanκ-caseins are considerably less hydrophilic than bovine

CMP. The sequence 97–116 ofκ-casein is highly conserved

across species, suggesting that the limited proteolysis ofκ-casein

and subsequent coagulation of milk are of major biological sig-

nificance (Mercier et al. 1976, Martin et al. 2011)

A grouping system for mammals based onκ-casein structure

and the site of cleavage by chymosin has been suggested

(Mercier et al. 1976, Nakhasi et al. 1984). Group I species (cow,

goat, sheep and buffalo) have a higher content of dicarboxylic

amino acids and low hydrophobicity and carbohydrate content

andκ-casein is cleaved at Phe 105 –Met 106 , while Group II species

(horse, human, mouse, pig, rat) have a high proline content, less

dicarboxylic amino acids and a much higher hydrophobicity

and carbohydrate content and are cleaved at Phe 97 –Ile 98 or

Phe 105 –Leu 106. Marsupialκ-casein appears to form a separate

group with a cleavage site different from that in eutherian

mammals (Stasiuk et al. 2000). Cleavage of equine milk at

Phe 97 –Ile98,as well as other characteristics of itsκ-casein,

place the horse in Group II. The divergence between species

into Groups I and II could account for differences in the clotting

mechanisms of ruminant and non-ruminant milks (Herskovits

1966). In addition to the differences in cleavage site, the

grouping system also divides species based on the number of

O-glycosylation sites in κ-caseins. As equine and human

κ-casein are considerably more highly glycosylated than bovine

κ-casein and non-glycosylatedκ-casein has not been found in

equine milk (Egito et al. 2001), equine and humanκ-caseins

belong to the same group. The level of glycosylation does not

affect micelle structure but it does affect the susceptibility ofκ-

casein to hydrolysis by chymosin, with susceptibility decreasing

as the level of glycosylation increases (Doi et al. 1979, Addeo

et al. 1984, Van Hooydonk et al. 1984, Vreeman et al. 1986,

Zbikowska et al. 1992). Therefore, equine milk probably has a

different clotting mechanism by chymosin than bovine milk.

Equid Casein Micelles

In the milk of all species studied in sufficient detail, the caseins

exist predominantly as micelles, which are hydrated spheri-

cal structures with dimensions in the sub-micron range. The

dry matter of casein micelles consists predominantly (>90%)

of proteins, with small amounts of inorganic matter, collec-

tively referred to as micellar calcium phosphate (MCP). The

structure and sub-structure of bovine casein micelles has been

studied in detail and reviews include: Holt and Horne (1996),

Horne (1998, 2006), De Kruif and Holt (2003), Phadungath

(2005), Farrell et al. (2006), Qi (2007), Fox and Brodkorb

(2008).

Equine casein micelles are larger than bovine or human mi-

celles (Table 26.2) (Welsch et al. 1988, Buchheim et al. 1989)

while those of asinine milk are similar in size to bovine micelles

(Salimei 2011). Electron microscopy shows that bovine and

equine micelles have a similar ‘spongy’ appearance, while hu-

man micelles seem to have a much ‘looser’, more open structure

(Jasi ́nska and Jaworska 1991). Such a loose open structure may

affect the susceptibility to hydrolysis by pepsin. Jasinska and ́

Jaworska (1991) reported that human micelles are much more

susceptible to pepsin hydrolysis than either equine or bovine

micelles. There are no specific reports on the sub-structure of

equine casein micelles although equine milk does contain ap-

proximately 10.1 mmol.L−^1 micellar calcium and approximately

2.6 mmol.L−^1 micellar inorganic phosphate, suggesting a micel-

lar calcium:casein ratio of>20:1 which, on a molar basis, far ex-

ceeds the calcium-binding capacity of equine casein molecules.

Hence, it may be assumed that equine micelles, like bovine ca-

sein micelles, contain nanoclusters of calcium phosphate. Since

equine milk contains little or noκ-casein, unphosphorylatedβ-

casein may play a role in micellar stability (Ochirkhuyag et al.

2000, Doreau and Martin-Rosset 2002). A similar conclusion

was reported by Dev et al. (1994) for the stabilisation of human

casein micelles.

Both equine αs1-casein (residues 75–81) and β-casein

(residues 23–28) contain a phosphorylation centre, which is

required for the formation of nanoclusters; furthermore, both

proteins also contain distinct hydrophobic regions through

which solvent-mediated protein–protein interactions may oc-

cur. Equineαs2-casein may have similar properties to equine

αs1-casein, pending further characterisation. The ratio of micel-

lar calcium:micellar inorganic phosphate is 2.0 in equine milk,