胶束酪蛋白复酶水解肽与钙形成螯合物的制备、表征及螯合机制

Preparation,Characterization and Formation Mechanism of Calcium-Chelating Peptide Derived from Micellar Casein by Enzymatic Hydrolysis

本研究利用胶束酪蛋白为原料制备酪蛋白水解肽(casein hydrolytic peptide,CHP),并与氯化钙进行螯合(肽钙质量比2∶1),制备得到酪蛋白肽钙螯合物CHP-Ca。以水解度(hydrolysis degree,DH)和钙螯合量为指标,对5种酶(风味蛋白酶、碱性蛋白酶、胰蛋白酶、木瓜蛋白酶、中性蛋白酶)进行筛选,筛选出风味蛋白酶和胰蛋白酶作为复酶,采用响应面法用进一步优化复酶酶解条件,结果表明最佳酶解条件为酶底比6000 U/g、复酶比1∶1、酶解时间90 min、pH 6.9、温度42℃,在此条件下肽钙螯合量为(90.46±0.72)μg/mg。最后通过X射线衍射、热重分析、Zeta电位和粒径分析以及红外光谱对CHP-Ca的结构特性及螯合机制进行研究。结果表明CHP-Ca主要通过羧基、氨基和磷酸基团相互作用的方式结合,螯合后Zeta电位和粒径减小,CHP-Ca结构变得更加紧密。

In this study,calcium-chelating casein hydrolytic peptide(CHP-Ca)was prepared by the chelation reaction between CHP,which was prepared from enzymatic hydrolysis of micelle casein,and calcium chloride at a mass ratio of 2:1.Five enzymes(flavourzyme,alcalase,trypsin,papain and neutral protease)were screened based on hydrolysis degree(DH)and calcium-chelating capacity.A mixture of flavourzyme and trypsin was found to be the best enzyme for the enzymatic preparation of CHP.Furthermore,response surface methodology(RSM)was used to optimize the enzyme hydrolysis conditions.The results showed that the optimal conditions were enzyme-to-substrate ratio 6000 U/g,flavourzyme-to-trypsin ratio 1:1,hydrolysis time 90 min,pH 6.9,and temperature 42℃.The calcium chelation capacity of the peptide prepared under these conditions was(90.46±0.72)μg/mg.Finally,the structural properties and formation mechanism of CHP-Ca were investigated by X-ray diffraction(XRD),thermogravimetry(TG),zeta potential,particle size analysis and infrared(IR)spectroscopy.The results showed that CHP-Ca bound mainly by the interaction of carboxyl,amino and phosphate groups,and the zeta potential and particle size decreased after chelation,and the structure of CHP-Ca became more compact.