Research On Stability Mechanism Of Casein Micell Based On Salt Equilibrium And Calcium Phosphate Nanocluster

Milk stability is an important scientific issue in dairy industry,and milk stability is dependent on the structural stabilization of casein micelles.Casein micelles are composed by countless caseins and calcium phosphate salts,in which calcium phosphate salts are sequestered by phosphopeptides forming calcium phosphate nanoclusters that is the core of sub-structure of casein micelles,maintaining the structural integrity and stability of casein micelles.Knowledge is limited on calcium phosphate nanoclusters and their important role within casein micelles at home and abroad.In this study,calcium phosphate nanoclusters were synthesized similar to native ones in chemical composition and structure,and changes of calcium phosphate nanoclusters induced by heating and acidification/alkalization were explored,then the instability mechanism of casein micelle or milk was revealed from the aspects of salt equilibrium and calcium phosphate nanoclusters.Firslty,casein phosphopeptides and milk salt system were used to synthesize calciumphosphate nanoclusters similar to native ones in milk,and the structure of calcium phosphate nanoclusters were analyzed.In natural milk system,～70%of total calcium and～48%of total inorganic phosphate were associated to be amorphous calcium phosphate salts,which were sequestered by casein phosphorylated groups forming spherical calcium phosphate nanoclusters within casein micelles,and the chemical formula of calcium phosphate salts was proved to be Ca（HPO₄）_0.7（PO₄）_0.2.Casein phosphopeptides and calcium salts at a ratio of 8:1 were used to synthesize calcium phosphate nanoclusters at physiological temperature（～38℃）of mammary gland and native pH of milk（6.6）.Compared with native calcium phosphate nanoclusters,their chemical formula was similar,and both were amorphous.The core of amorphous acidic calcium phosphate salts（diameter of～2.4 nm）were bound to casein phosphopeptide forming core-shell spherical nanoclusters with casein phosphopeptide as the shell（thickness of～0.8 nm）.For the nanocluster system,the chemical composition and structure of calcium phosphate nanoclusters induced by heating and acidification/alkalization were investigated,separately.Heating of 70-80℃/15 min hardly altered salt equilibrium in both the aqueous phase and nanoclusters,the chemical formula of calcium phosphate salts kept unchanged,and calcium phosphate salts were still bound to casein phosphopeptide as spherical nanoclusters that remained stable.Heating of 90-100℃/15 min caused soluble calcium and inorganic phosphate in the aquoues phase transferred into nanoclusters,and the calcium phosphate within nanoclusters was transformed to be a basic salt.The spherical calcium phosphate nanoclusters began to disappear because calcium phosphate salts couldn’t be stabilized by phosphopeptides,but part of amorphous calcium phosphate salts grew and matured into plate-like apatite crystalline particles with casein phosphopeptide as nucleation sites,whose size and crystallinity were positively correlated with heating intensity.At pH 6.2-6.4,some of calcium phosphate nanoclusters dissociated.The calcium phosphate salts within nanoclusters were dissolved as ions,while the chemical formula(Ca（HPO₄）_0.6（PO₄）_0.267)and structure of remaining calcium phosphate nanoclusters kept unchanged and the nanocluster system kept stable.At pH7.0-7.4,soluble calcium and inorganic phosphate in aqueous phase were transferred into nanoclusters,while calcium phosphate salts kept amorphous,but the solubility decreased.The core-shell spherical calcium phosphate nanoclusters re-assembled into metastable core-shell rod-like nanoclusters,and further aggerated into unstable worm-like aggregates with linkage of salt bridge.Based on the deeper understanding of calcium phosphate nanoclusters,the instability mechanism of casein micelles was further investigated induced by heating and acidification/alkalization.Heating of 70-80℃/15 min did not significantly alter salt equilibrium between milk serum and casein micelles（p>0.05）,the chemical composition and structure of calcium phosphate salts within casein micelles remained unchanged,and the aggregation between casein micelles was small,so that casein micelle system kept stable.Heat treatment at 90℃/15 min caused soluble calcium and inorganic phosphate transferred from milk serum to casein micelles,calcium phosphate salts within casein micelles remained as amorphous acidic salts,but the solubility decreased and became metastable.Besides,an aggregation occurred between casein micelles crosslinked by salt bridge,which did not cause the structure of casein micelle destroyed.Heat treatment at 100℃/15 min caused soluble calcium and soluble inorganic phosphate shifting from milk serum to casein micelles,and calcium phosphate salts were transformed to be Ca（HPO₄）_0.3（PO₄）_0.47.Part of amorphous calcium phosphate salts was transformed into plate-like apatite crystallinity that was still bounded to caseins,they were reaggregated into large aggregates,leading to a decreased heat stability.Acidification（pH 6.2-6.4）caused a release of micellar calcium and inorganic phosphate dissolved as ions into milk serum,and calcium phosphate within casein micelles remained as amorphous acidic salts that were bound to caseins as nanoclusters.A decrease of calcium phosphate nanoclusters caused a decrease in size of casein micelles,but the intergrity of casein micelles was not destroyed.At pH 7.0-7.4,soluble calcium and inorganic phosphate in milk serum were shifted to casein micelles,calcium phosphate salts within casein micelles were still amorphous and the chemical formula kept unchanged,but became metastable with a lower solubility.An aggregation between casein micelles occurred by a linkage of salt bridge,and the size of casein micelle increased,which did not destroy the structural stability of casein micelles.Heating of 100℃/15 min at pH range of 6.4-7.0 caused salt equilibrium between two phases was disrupted,soluble calcium and inorganic phosphate in milk serum were transferred into casein micelles,leading to an increase in micellar calcium phosphate salts.Besides,the chemical formula of calcium phosphate was transformed to be Ca（HPO₄）_0.4（PO₄）_0.4 after heated at pH 6.4/100℃,while that was transformed to be Ca（HPO₄）_0.2（PO₄）_0.533after heated at pH 7.0/100℃,and some amorphous calcium phosphate salts were transformed to be plate-like apatite crystalline,the degree of phase transformation was dependent on pH upon heating.Phase transformation of calcium phosphate salts caused a disruption of the internal structure of casein micelles,and apatite crystalline was not sedimented but was interacted with caseins forming larger aggregates.Compared to pH 6.6-7.0,a larger aggregation between casein micelles occurred at a lower pH（6.4）upon high-heating,and sediment percentage of casein micelle solution induced by heating significantly increased（p<0.05）,showing a poorer heat stability.Heating and acidification/alkalization caused an alteration in salt equilibrium in milk system,high-heating induced phase transition that was dependent on pH,leading to changes in the interstructure of casein micelle,and aggregations between casein micelles occurred by linkage of calcium salt,the stability of milk decreases.The findings provide support for designing and optimizing parameters in dairy manufacture.