Analysis of calcium phosphate cross-linkage in casein micelles by radioactive isotope Professor

虽然牛酪蛋白胶束中的钙和无机磷酸盐（Pi）与可溶形式的钙和无机磷酸盐处于平衡状态，但它们的性质尚未得到充分的阐明。本研究采用^<45>Ca和^<32>P，通过超滤、平衡透析和凝胶渗透高效液相色谱法研究了在6M尿素存在下钙和Pi在酪蛋白胶束中的行为。据估计，牛酪蛋白胶束中约有30-40%的钙和π难以与可溶性相交换。然后，用^<45>Ca和^<32>P，在酪蛋白浓度为2.5%的条件下，用30-40 mM钙、22-27 mM Pi和10mM柠檬酸制备人工酪蛋白胶束。人工酪蛋白胶束与模拟牛奶超滤液在4‰的温度下透析72h后，胶束中钙和Pi的交换率分别为75%和65%。胶束磷酸钙交联的酪蛋白聚集体中^<45>Ca和^<32>P所占的比例分别为26.9%和27.6%，经过模拟牛奶超滤液在4‰的温度下平衡透析72h后，这一比例降至10%左右。透析72h时，人工酪蛋白胶束中交换钙和Pi的比例在25ºC时高于4ºC，表明钙和Pi在扩散相和胶束相之间的交换与温度有关。利用响应面分析法考察了钙和π对酪蛋白胶束形成和性质的影响。结果表明，钙和磷酸盐在扩散相和胶束相之间的交换过程缓慢，酪蛋白胶束中有一定比例的钙和Pi难以交换。

Although calcium and inorganic phosphate (Pi) in bovine casein micelles were at equilibrium with those in soluble form, their properties have not yet been elucidated sufficiently. In this study, the behavior of calcium and Pi in casein micelles was studied using ^<45>Ca and ^<32>P by ultrafiltration, equilibrium dialysis and gel permeation HPLC in the presence of 6M urea. Approximately 30-40% of the calcium and Pi in bovine casein micelles were estimated to be hard to exchanged with those in soluble phase. Then, artificial casein micelles were prepared at a casein concentration of 2.5% with 30-40 mM calcium, 22-27 mM Pi and 10mM citrate using ^<45>Ca and ^<32>P.About 75 and 65% of micellar calcium and Pi in artificial casein micelles were exchanged after dialysis of artificial casein micelles against a simulated milk ultrafiltrate at 4ﾟC for 72h.The percentage of ^<45>Ca and ^<32>P in the fraction of casein aggregates cross-linked by micellar calcium phosphate were 26.9 and 27.6%, which decreased to about 10% after equilibrium dialysis against a simulated milk ultrafiltrate at 4ﾟC for 72h. The proportion of exchanged calcium and Pi in artificial casein micelles during dialysis for 72h were higher at 25ﾟC than at 4ﾟC,suggesting that the exchange of calcium and Pi between diffusible and micellar phases depended on temperature. The effects of calcium and Pi on the formation and properties of casein micelles were also examined using response surface analyzes. The results suggest that the exchange of calcium and phosphate between diffusible and micellar phases proceeds slowly, and a proportion of calcium and Pi in casein micelles is difficult to exchage.