Colloidal calcium phosphate association with casein

The inorganic colloidal calcium phosphate associated with casein in normal milk dissolves on acidification of milk to pH 4.6 so that if sufficient time is allowed for solution, isoelectric casein is essentially free of calcium phosphate. In the laboratory, best results are obtained by acidifying skim milk to pH 4.6 at 2°C, holding for about 30 min and then warming to 30-35°C. The fine precipitate formed at 2°C allows time for the colloidal calcium phosphate to dissolve (Chapter 5). A moderately dilute acid (1 M) is preferred, since concentrated acid may cause localized coagulation. Acid production by a bacterial culture occurs slowly and allows time for colloidal calcium phosphate to dissolve. The casein is recovered by filtration or centrifugation and washed repeatedly with water to free the casein of lactose and salts. Thorough removal of lactose is essential since even traces of... 

In principle, it would be logical to combine plots of the buffer index curves of each of the buffer components of milk and thus obtain a plot which could be compared with that actually found for milk. It is not difficult, of course, to conclude that the principal buffer components are phosphate, citrate, bicarbonate, and proteins, but quantitative assignment of the buffer capacity to these components proves to be rather difficult. This problem arises primarily from the presence of calcium and magnesium in the system. These alkaline earths are present as free ions as soluble, undissociated complexes with phosphates, citrate, and casein and as colloidal phosphates associated with casein. Thus precise definition of the ionic equilibria in milk becomes rather complicated. It is difficult to obtain ratios for the various physical states of some of the components, even in simple systems. Some concentrations must be calculated from the dissociation constants, whose... 

Figure 5.10 Association of colloidal calcium phosphate (CajfPOjj) with the serine phosphate groups of casein (from Schmidt, 1982).

Skim milk can be considered as a two-phase system consisting of casein-colloidal calcium phosphate micelles in quasi-equilibrium with an aqueous solution of salts and proteins the phase boundary is ill-defined because of the intimate association between the calcium phosphate and the caseins (phosphoproteins). 

Certain of the milk salts (e.g. chlorides, and the salts of sodium and potassium) are sufficiently soluble to be present almost entirely in the dissolved phase. The concentration of others, in particular calcium phosphate, is higher than can be maintained in solution at the normal pH of milk. Consequently, these exist partly in soluble form and partly in an insoluble or colloidal form associated with casein. The state and distribution of these salts has been extensively reviewed by Pyne (1962) and Holt (1985). 

Association with casein. The colloidal calcium phosphate is closely associated with the casein it does not precipitate out of solution and is considered to be protected against precipitation by the casein. Two possible forms of protection are suggested ... 

Minerals The main mineral constituents in milk are calcium and chlorine, magnesium chloride, phosphate, and citrate. Minerals in milk are mainly present as soluble salts or in colloidal form associated with caseins. Their concentrations may vary enormously. Thus, the minerals present in milk can be classified according to their concentration level as major and minor elements, with small quantitative contributions from trace and ultratrace elements. The total content of minerals in mammalian milks should correspond to the growth requirements of each biological species. Accordingly, the mineral total content in cow milk is four times higher than in human milk. 

About 80% of the total milk protein is in casein, 19% in serum and 1.5% in the fat globules. The casein accounts for about 25% and the calcium phosphate about 65% of the total P in milk. Nearly all of the latter is in colloidal form, closely associated with the casein. Above pH 6, the casein phosphoproteins are among the most stable of all food proteins. Phospholipids constitute -30% of the fat globule membranes. 

The butter fat is a coarse dispersion readily removable on standing or by a centrifuging operation. The casein will be present in the skimmed milk as colloidally dispersed micelles of diameter of the order of 10 cm, and is associated with calcium and phosphate ions. 

The caseins exist in milk as polydisperse aggregates ranging in size from ca. 40 to 220nm (3), but the size distribution of micelles depends upon the method of measurement. These casein micelles scatter light and are responsible for the whitish, opaque nature of skim milk. The casein micelles are also associated with a colloidal apatite comprised of calcium-phosphate-citrate (CPC) which has a stabilizing influence on the micelle structure. The colloidal CPC is in equilibrium with soluble CPC in the milk serum phase and is solubilized as the pH is reduced. Thus, as the pH is reduced to the isoelectric point of the caseins (4.6), the colloidal CPC solubilizes, and the caseins precipitate (143). This phenomenon should be kept in mind during some of the following discussions. 

The colloidal casein micelles are formed from an intimate association of the phosphoprotein with calcium phosphate. Minor quantities of other cations, citrate anions, ATP and some enzymes are also incorporated. The exact nature of the calcium phosphate is not yet known with certainty. It may be Ca3(P04)2, Caio(P04)g(OH)2 or CaHP04 2H2O. Various models have been proposed as, for example, in Figure 12.9. 

---