The changes of bovine casein micelles during cold storage were investigated from the standpoint of the size of micells. Large, medium, and small micelles (>50, 50-30, and <30nmin radius) were obtained from skim-milk by differential centrifugation. After the definite-sized micelles were equilibrated at 4°C and 37°C, they were fractionated by differential centrifugation and gel chromatography. The casein contents of these fractions were measured by SDS-polyacrylamide gel electrophoresis. A high quantitative separation of caseins was achieved by this method. The liberation of /i-casein by cooling occurred easily from the larger-sized micelles. A larger amountof ic-casein was liberated from the smaller-sized micelles. The mediummicelle fraction was increased by the supply from large micelles at 4°C, and the medium micelles degraded partly to the small micelle fraction and soluble casein. Small micelles degraded partly to soluble casein. The small micelle fraction was not produced from large micelles. The stability of micelles under cooling was related closely to the miceller size.After milking, milk is usually stored at low temperatures. The effects of low temperature on milk have been studied in connection with casein micellar structure. Part of the minerals and caseins dissociate from micelles upon storage of milk overnight at low temperatures. The liberated protein is mostly /?-casein,1} and the released minerals are calcium phosphate.2) The solubility of colloidal calcium phosphate bound to a micelle increases at low temperatures.^The casein and calcium phosphate are liberated from micelles without influencing their size,3) and the voluminosity of casein micelles increased.4) This increase was due to loosening of the micelle. Casein micelles were dissociated by desalting with the addition of calcium complexing agents.5) The equilibrium between the micellar and soluble caseins was due to the content of colloidal calcium. Pierre and Brule6)
Materials and MethodsChemicals. All chemicals were of the highest purity available, used without further purification.Preparation of samples. Casein micelles were prepared from skim-milk by the following method.9) Skim-milk was prepared from bulk milk of Holstein cows by centrifugation at 2000xg for 15min. Sodium azide (to 0.02%) and e-aminocaproic acid (to 0.1 him) were added to skimmilk to inhibit microbial growth and proteinase activity, respectively.10) Skim-milk was then kept at 37°C for 2hr to allow the equilibration of micelles.11* Whole micelles were obtained from the skim-milk by centrifugation at 100,000xg for 1 hr at 35°C. The definite-sized micelles were prepared at 35°C by differential centrifugation as follows. Large micelles (>50nm in radius) were prepared as pellets by centrifugation of skim-milk at 15,000 x g for 30min. Medium micelles (50-30nm) were obtained by centrifugation of the above supernatant at 60,000 x g for 30min and then small micelles ( <30nm) were obtained by centrifugation of the successive supernatant at 100,000 x g for 1 hr. These pellets w...
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