Yoshitaka Kako

Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate

Artificial casein micelles were prepared by adding 30 mM calcium, 22 mM phosphate and 10 mM citrate to sodium caseinate solutions, and the content of the casein aggregates cross-linked by colloidal calcium phosphate was determined by high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. The content of the casein aggregates cross-linked by colloidal calcium phosphate in artificial whole casein micelles was 48% of total casein, and their relative casein composition determined by high-performance ion-exchange chromatography was 53.1% for alpha s1-casein, 15.8% for alpha s2-casein, 31.1% for beta-casein and 0% for kappa-casein. The order of cross-linking by colloidal calcium phosphate agreed with that of the ester phosphate content of casein constituents. The content of the casein aggregates cross-linked by colloidal calcium phosphate was higher in alpha s1-kappa-casein micelles than in beta-kappa-casein micelles. kappa- and gamma-caseins and dephosphorylated alpha s1-casein were not cross-linked by colloidal calcium phosphate. Although kappa-casein was not cross-linked, chemically phosphorylated kappa-casein, of which the average phosphate content was 8.5 per molecule, was cross-linked. It is concluded that caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate.

Dissociation during dialysis of casein aggregates cross-linked by colloidal calcium phosphate in bovine casein micelles

Casein micelles separated by ultracentrifugation of raw skim milk were dispersed at a casein concentration of 2·5% in simulated milk ultrafiltrate and dialysed against 10 mM-imidazole buffer (pH 7·0) at 5 °C. The amounts of colloidal Ca and inorganic P decreased from 77 to 11 mg and from 31 to 2 mg respectively in 100 ml during 72 h of dialysis. Micellar casein content was reduced to 43 and 11% after 48 and 72 h of dialysis respectively. In high-performance gel chromatography of casein micelles in the presence of 6 M-urea, fraction 1, consisting of the casein aggregates cross-linked by colloidal Ca phosphate (CCP) decreased during dialysis and the retention time of the peak of fraction 1 was prolonged, suggesting that the cross-linkage between CCP and casein molecules was disrupted. The dissociation rates of the individual casein constituents from the casein aggregates cross-linked by CCP during dialysis were in the order β-> α s1 - > α s2 -casein. The higher the ester phosphate content, the slower was the dissociation rate of the individual casein constituent. It is suggested that the strength of interaction between CCP and casein molecules depends on the ester phosphate content.

Cleavage of the linkage between colloidal calcium phosphate and casein on heating milk at high temperature.

In order to examine the effect of heating on the changes in the linkage between colloidal Ca phosphate (CCP) and casein, high-performance gel chromatography of casein micelles disaggregated was carried out using 6 M-urea simulated milk ultrafiltrate as the effluent. The cleavage of the linkage between CCP and casein on heating was considered to occur without liberaction of ester phosphate groups. It was suggested that the transformation of CCP to another form was responsible for the cleavage between CCP and casein on heating milk at high temperature

Relation between micelle size and formation of soluble casein on heating concentrated milk

In order to examine the influence of micelle size on the formation of soluble casein brought about by heating concentrated milk, casein micelles were separated into 3 fractions by differential centrifugation. The micelles were dispersed in the ultrafiltrate of skim-milk and the preparations were concentrated to 1/2·5 of the original volume. When the concentrated milk samples were heated at 135–140 °C, a larger amount of soluble casein was formed from smaller micelles than from larger micelles. Only a slight effect on the formation of soluble casein on heating was observed when colloidal calcium phosphate (CCP) was removed from the large micelles to a level lower than that associated with the small micelles or when the CCP in the small micelles was increased to a level higher than that of the large micelles. The soluble caseins formed on heating from different sizes of micelles were similar in composition. The fact that the formation of soluble casein on heating was affected by micelle size may be ascribed to variation in the κ-casein content of micelles rather than to variation in CCP content.

Cross-linking of caseins by colloidal calcium phosphate in the presence of urea

Abstract Cross-linked casein was formed when calcium, phosphate and citrate were added to sodium caseinate solution containing 6 M urea, although the content of cross-linked casein was somewhat lower than when artificial casein micelles were formed in the absence of urea. The colloidal phosphate content was also lower in the presence of 6 M urea than in the absence of urea. The extent of incorporation of individual casein constituents into cross-linked casein in the presence of 6 M urea was very similar to that in the absence of urea. Results suggest that the submicelle structure and ordered structures of caseins are unnecessary for caseins to be cross-linked by colloidal calcium phosphate.

Influence of 2-mercaptoethanol on heat stability of concentrated whey-protein-free milk and formation of soluble casein

The heat coagulation time (HCT) of concentrated whey-protein-free (WPF) milk measured at 120 and 130 °C was reduced to by addition of 5–20 mM-2-mercaptoethanol (ME). However, although the amount of soluble casein formed on heating was doubled by addition of ME, the shape of the HCT–pH profile was affected only slightly. The proportion of κ-casein in the soluble casein from heated concentrated WPF milk containing ME was very high, though it was somewhat lower than that of the soluble casein from heated concentrated WPF milk containing no ME. No solubilization of colloidal Ca phosphate was observed in either unheated or heated concentrated WPF milk on addition of ME. These facts suggest that ME probably promotes the formation of soluble casein with release of κ-casein from micelles on heating, thus destabilizing the casein micelles.