Caseins

Caseins are phosphoproteins precipitated from raw milk at pH 4.6 at 20°C. They comprise approximately 80% of the total protein content in milk. The principal proteins of this group are

classified according to the homology of their primary structures into αs1-, αs2-, β- and κ-caseins (Wong et al., 1996).

Caseins are conjugated proteins, most of them with phosphate group(s) esterified to serine residues. Calcium binding by the individual caseins is proportional to the phosphate content. The conformation of caseins is similar to denatured globular proteins. The high number of proline residues in caseins causes particular bending of the protein chain and inhibits the formation of close-packed, ordered secondary structures. The lack of tertiary structure accounts for the stability of caseins against heat denaturation, because there is very little structure to unfold.

Without a tertiary structure there is considerable exposure of hydrophobic residues. This results in strong association reactions of the caseins and renders them insoluble in water. Within the group of caseins, there are several distinguishing features, based on their charge distribution and sensitivity to calcium precipitation (Wong et al., 1996; Goff, 1995; Farrell et al., 2004).

4.2.1.1 αs1 caseins

αs1 caseins have five genetic variants, A, D, B, C and E. The B variant consists of 199 amino acid residues with a calculated molecular weight of 23,614 Da. The protein contains more acidic amino acids than basic ones. It has 17 proline residues which prevent the formation of certain types of secondary structures. Three hydrophobic regions are identified that contain all the proline residues. Seven of the eight phosphate groups are located in the hydrophilic region. αs1

caseins are calcium sensitive, they can be precipitated at very low levels of calcium (Wong et al., 1996; Goff, 1995; Farrell et al., 2004).

4.2.1.2 αs2 caseins

Four variants of αs2 are known, A, B, C and D. The amino acid sequence of αs2-CN A-11P consists of 207 residues, among them ten prolines and two cysteins, and the calculated molecular weight is 24,350 Da. Concentrated negative charges are found near N-terminus and positive charges near C-terminus. It can also be precipitated at very low levels of calcium (Wong et al., 1996; Goff, 1995; Farrell et al., 2004).

4.2.1.3 β-casein

β-casein constitutes 30-35% of the total caseins. Seven genetic variants of β-casein are known. The molecular weight of β-CN A1-5P is 23,982 Da; it is composed of 209 residues, among them 35 prolines. High negative net charge is around the N-terminal region, and the

C-terminal region is highly hydrophobic. β-casein is a very amphiphilic protein, and that’s why it acts like a detergent molecule. The protein’s self-association depends on temperature. It will form a large polymer at 20° C, but not at 4° C. This type of casein is less sensitive to calcium precipitation (Wong et al., 1996; Goff, 1995; Farrell et al., 2004).

4.2.1.4 κ-casein

κ-casein constitutes about 15% of the total caseins. The major casein, κ-CN B-1P contains 169 amino acid residues (twenty prolines) and its molecular weight is 19,023 Da. This protein is positioned on the outside of the casein micelle. Unlike the other caseins κ-casein is very resistant to calcium precipitation, stabilizing other caseins. Rennet cleavage at the Phe105-Met106 bond eliminates the stabilizing ability, leaving a hydrophobic portion, para-κ-casein, and a hydrophilic portion called κ-casein glycomacropeptide (GMP), or caseinomacropeptide (CMP). Cleavage of this bond is the first step in the coagulation of milk by aggregation of the casein micelles after the loss of the hydrophilic, negatively charged surface from the micelle (Farrell et al., 2004;

Goff, 1995; Wong et al., 1996).

4.2.1.5 Casein Micelles

The major part of milk proteins, together with calcium phosphate, occurs in the form of large colloidal particles, the casein micelles. An average micelle contains about 10⁴ caseins and its size ranges between 50 and 300 nm (Huppertz et al, 2006). Various different models have been proposed for micelle structure. One of them is the “sub-micelle model”. This model suggests that casein micelles are built of roughly spherical subunits or micelles. The composition of micelles is variable and the size is within the range of 12-15 nm in diameter, and each sub-micelle has 20-25 casein molecules. The sub-sub-micelles are kept together by hydrophobic interactions between proteins, and by calcium phosphate linkages. There are two main types of sub-micelles. One mainly consists of αs- and β-caseins, hydrophobic regions buried in the center of the sub-micelle. The other type consists of αs- and κ-caseins. The latter are more hydrophilic because of the sugar residues on them. The κ-caseins are located near the outside of the micelle with the hydrophilic part of the C-terminal end protruding from the micelle surface to form a 'hairy' layer that will avoid further aggregation of sub-micelles by steric and electrostatic repulsion. Consequently, micelles are stable, and they do not usually flocculate (Figure 1.) (Phadungath, 2005).

Another model has been evolved recently, especially from work by Carl Holt. This “internal structure” model shows a more or less spherical, highly hydrated, and fairly open particle. Holt’s model of the casein micelle shows a tangled web of flexible casein networks that form a gel-like

structure with micro-granules of colloidal calcium phosphate through the casein phosphate center. The C-terminal region of κ-casein extends to form a „hairy layer” (Figure 2.). The two main features of this model are the cementing role of colloidal calcium phosphate and the surface location of hairy layer, which confers steric and/or charge stability to native casein particles (Phadungath, 2005).

Figure 1. The sub-micelle model Figure 2. The internal structure model