SummaryThe dissociation of caseins of different types from casein micelles in milk, acidified to different pH values in the range 4·9–6·7, and at temperatures of 4, 20 and 30 °C, has been studied. In contrast to a number of previous findings, it was shown that caseins of all types were dissociated from the micelles, although in all cases β-casein was in highest concentration. The amounts and proportions of all of the caseins were found to be pH- and temperature-dependent, especially the former. Studies of the proportions of the different caseins liberated suggested that, at a defined temperature, the proportions of κ;- and αs2-caseins were independent of pH, while the proportions of β- and αsl-caseins were variable, changes in one being compensated by changes in the other. The manner in which the proportions of the αsl-casein and β-caseins changes with pH was found to be temperature-dependent.

SummaryMeasurements of the release of Ca, Mg and inorganic phosphate(Pi) from the casein micelles of bovine milk have been made, as functions of the pH, in the range 4·9–6·7, and at temperatures of 4, 20 and 30 °C. The results are in general agreement with earlier published studies in giving a value of 1·75–1·84 for the micellar Ca:Pi ratio. Mg appeared to behave similarly to Ca, although the amounts of micellar material were much smaller. The results on the acid-solvation of calcium phosphate are considered in relation to published quantitative studies of the pH-induced dissociation of the different types of caseins from the micelle, and of the micellar dissociation caused when micellar calcium phosphate is dissolved at neutral pH. It is evident from this that at present it is not possible to derive a universal relation between the dissociation of minerals and of caseins from the micelles at different temperatures and under different conditions.

The effect of pH on thermal denaturation of four main whey protein fractions in skim milk was examined by gel permeation FPLC. On heating skim milk at 80 degrees C for 0.5-20.0 min over the pH range 5.2-8.8, the extent of denaturation, based on loss of solubility at pH 4.6, increased with heating time and was usually in the order immunoglobulins > serum albumin/lactoferrin > beta-lactoglobulin > alpha-lactalbumin. Rates of denaturation of the immunoglobulins and the serum albumin/lactoferrin fraction were highest at the lower end of this pH range, whereas those of beta-lactoglobulin and alpha-lactalbumin increased over most of the pH range. The effects of pH, addition of Ca, and reduction of disulfide bonds on the rates of the unfolding and aggregation stages of denaturation are discussed.

The caseinate complex in bovine milk was partitioned by differential centrifugation at both 20 and 4 °C into 4 micellar fractions and a fraction representing serum casein, and the protein composition of the fractions determined. At both temperatures the relative amount of K-casein in the micellar caseins increased markedly and that of /?-casein decreased appreciably with decreasing micelle size. The relative amount of a s2 -casein also tended to decrease with decreasing micelle size, but the relative amounts of a sl -and y-caseins, and an unidentified casein fraction, showed little systematic variation. The serum casein differed appreciably in composition from the micellar caseins, being very rich in /?-casein and comparatively poor in a sl -and a s2 -caseins, and the amount present at 4 °C was considerably greater than at 20 °C, with the increase being due almost entirely to /?-casein, but with y-casein also making a significant contribution. The changes in the composition and distribution of micellar and serum caseins induced by cooling milk at 4 °C were completely reversible when the milk was re-equilibrated at 20 °C for 18 h.

SummaryThe strength of binding of the individual caseins and the nature of the bonding within bovine casein micelles were examined through dissociation of the micelles by dialysis of skim milk either against phosphate-free buffers containing 3 or 6 mm-CaCl2, or against buffers that were nearly saturated with respect to micellar calcium phosphate, but which had a free Ca2+ concentration in the range 0·4–5·9 mm. Dissociation was followed by ultracentrifuging the dialysed milks and determining the partition of the total and the individual caseins between the pellet and serum. During dialysis against the phosphate-free buffers both colloidal Ca and Pi in the milks decreased and about 30 % of the Pi could be removed without significant casein dissociation. With further loss of Pi, however, increasing dissociation occurred and the proportions of the individual caseins retained in the casein pellet were in the order αs2- > αs1- > β- ≈ κ-casein. Dialysis against the calcium phosphate buffers resulted in no loss of colloidal Pi but colloidal Ca increased with the free Ca2+ concentration of the buffer. Little change in the casein partition occurred in the presence of more than 1 mm free Ca2+, but serum casein increased markedly at lower levels, and the strength of binding of the individual caseins in the pelleted casein was in the order αs2-> αs1- > β- > κ-casein. In both types of buffer, dissociation is considered to occur through the breaking of linkages between the caseins and inorganic constituents. Analysis of the amino acids in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles suggests that these linkages involve the phosphate centres of the caseins.