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.

A description is given of various methods for the ultrafiltration and dialysis of milk and of the composition of the sera obtained. Ultrafiltrate prepared by the procedure recommended is reasonably representative of the aqueous phase of milk, but its content of lactose and citric acid, and consequently also of calcium, is determined to a slight degree by the sieving phenomenon known to occur often in ultrafiltration. The composition of diffusate obtained from milk at 20 °C is not thought to be controlled to any significant extent by a Donnan effect and is regarded as identical with that of the aqueous phase of milk. The lactose content of diffusate suggests that about 2 % of the water in milk is bound to protein, and allowance should be made for this when calculating the concentrations of the soluble constituents in milk from the composition of diffusate. Diffusate prepared from milk at 3°C contains slightly more total calcium, ionized calcium and phosphorus than diffusate prepared at 20 °C. These differences are attributed to a change in the partition of calcium and phosphorus between the disperse and aqueous phases at the lower temperature, an explanation that is supported by the reversibility of the change. The composition of diffusate prepared by the procedure recommended indicates that about 5 % of the sodium and about 6 % of the potassium and citric acid in milk are in the. disperse phase.It is sometimes desirable to know not only the total concentration in milk of a given constituent but also the amounts of it in the disperse and aqueous phases, i.e. how much can be regarded as ' colloidal' and how much as ' soluble'. This information is usually obtained by determining the total amount of the constituent and the amount in solution; the colloidal fraction is then obtained by difference. Such a fractionation depends on the preparation of a suitable serum for estimating the soluble constituents. This serum should have the same composition as the aqueous phase of milk and therefore in its preparation the equilibria in milk should be disturbed as little as possible. Several methods of preparing a serum are available and of these the most suitable would appear to be ultrafiltration or dialysis. Other methods of estimating the soluble constituents of milk are the analysis of rennet whey or the serum obtained by high-speed centrifuging, and the treatment of milk with ionexchange resins.

SummaryA subjective test for the determination of the stability of milk protein to heat is described. In the test, the time required for particles of coagulated protein to become visible throughout a 2·5-ml sample of separated milk maintained at 135°C in a glass tube rocking at 8 c/min is taken as a measure of stability. The precision of the test was such that single determinations were generally adequate.Coagulation time decreased by about 12% as rocking speed was increased over the range 4–12 c/min and increased by a factor of about 3 for a decrease in heating temperature of 10 degC over the range 140–105 °C; with some milks the Q10 °C value increased to 5–8 a temperature decreased. As sample volume was increased over the range 1–3 ml coagulation time increased, especially With milks whose coagulation was poor (initial clots small). This volume effect appeared to be a consequence of the accompanying decrease in the proportion of headspace oxygen to volume of milk.

A method for the quantitative analysis of casein mixtures is described. The method uses ion-exchange chromatography at 4 °C on DEAE cellulose, Whatman DE52 in the presence of tris-chloride-urea buffer (pH 8-6), and a NaCl gradient to fractionate the alkylated casein mixtures and a micro-biuret technique to determine protein concentrations. Values for fractions representative of (1) y-caseins, (2) /c-casein plus some unknown proteins, (3) /?-casein, (4) minor a g -caseins and (5) a 8l -plus a 80 -caseins were obtained, and whole casein from herd bulk milk was found to consist on average of about 2, 13, 36, 11 and 38% respectively of these fractions. Agreement between duplicate fractionations was satisfactory and the recovery of material varied between 94 and 98%.Rose, Davies & Yaguchi (1969) described a method for the quantitative determination of the major components of casein mixtures which was based on the use of column chromatography on DEAE cellulose. The method gave a more extensive fractionation than that obtained by moving-boundary electrophoresis (Rolleri, Larson & Touchberry, 1956;Larson & Kendall, 1957), and yielded results which were more accurate than those derived from sialic acid (Marier, Tessier & Rose, 1963) and turbidimetric measurements (Tessier, Rose & Marier 1963) and also probably more reliable than those obtained from scanning patterns obtained by gel electrophoresis (Morr, Lin & Josephson, 1971). In the method of Rose et al. fractionation was done on DEAE cellulose, Whatman DE11, an anion exchanger with low proteinbinding capacity and relatively poor kinetic properties which has now been superseded by more advanced ion-exchange materials. In an attempt to improve on their method the improved exchanger Whatman DE52 has been used for casein fractionation and certain aspects of the procedure, such as the methods used for protein determination and for the measurement of the recovery of material, have been more critically examined. These studies have produced a system giving greater resolution of casein mixtures than achieved previously and have shown that agreement between duplicate fractionations and recovery of material is satisfactory. However, complete separation of /c-casein from other proteins was not possible and values for /c-casein are therefore somewhat overestimated.