牡蛎蛋白酶解条件优化及高活性体外抗凝血类蛋白修饰物制备

Optimization of conditions for enzymatic hydrolysis of oyster protein and preparation of highly active in vitro anticoagulant protein modified products

选取牡蛎肉为研究对象,以氨基酸态氮的含量和体外凝血因子Xa抑制活性为考核指标,考察木瓜蛋白酶、胃蛋白酶、胰蛋白酶、复合蛋白酶的酶解效果,其中胃蛋白酶对牡蛎的酶解效果最好。采用响应面法分析响应变量(酶添加量、温度、酶解时间)之间的交互影响,并确定最佳酶解工艺条件为:酶添加量2.5%、温度37.7℃、酶解时间4 h,氨基酸态氮的含量达到了3.60 g·mL^(-1)。采用超滤膜技术筛选四-六牡蛎肽组分,采用可控酶解技术,在抑制活性较高的牡蛎肽组分C-端,外结合位点结合一定量的天冬氨酸/谷氨酸。通过体外活性试验确定生成的牡蛎类蛋白修饰物具有体外凝血因子Xa抑制活性。

Selecting oyster meat as the research object,with the content of amino acid total nitrogen and in vitro coagulation factor Xa inhibitory activity as evaluation criteria,the enzymatic hydrolysis effects of papain,pepsin,trypsin and complex protease on oysters proteins were investigated,with pepsin showing the best hydrolysis effect on oysters.The interaction effects among response variables(enzyme addition amount,temperature,enzymatic hydrolysis time)were analyzed with response surface method,and the optimal enzymatic hydrolysis conditions were determined as follows:2.5%of enzyme addition amount,temperature of 37.7℃,and enzymatic hydrolysis time of 4 h,with the amino acid total nitrogen content reaching 3.60 g·mL^(-1).4-6 oyster peptide components were screened using ultrafiltration membrane technology,and controlled enzymatic hydrolysis technology was used to bind a certain amount of aspartic acid/glutamic acid to the outer binding site of the C-terminus of the oyster peptide component with high inhibitory activity.In vitro activity tests confirmed its in vitro coagulation factor Xa inhibitory activity.