酶解水牛奶制备抗凝血肽的工艺研究

Study on technology conditions of preparation for anticoagulant peptide from buffalo milk by enzymatic hydrolysis

目的:本文以新鲜水牛奶为原料,研究木瓜蛋白酶、中性蛋白酶及碱性蛋白酶复合酶解、混合酶解水牛奶的作用,比较不同水解方式的作用效果;并采用凝血酶滴定改进法测定水解液抗凝血活性。方法:分别进行三种蛋白酶单因素水解实验,确定各种酶的最适水解条件;在此基础上进行复合酶解与混合酶解实验,测定水解度和抗凝血活性。结果:木瓜蛋白酶的最适水解条件为:水解温度60℃、水解时间2.0 h、p H5.0、料水比1∶2(v/v)、酶用量6000 U/(m L底物);中性蛋白酶的最适条件为:水解温度40℃、水解时间3.0 h、料水比1∶2(v/v)、p H6.0、酶用量6000 U/(m L底物);碱性蛋白酶的最适条件为:水解温度50℃、水解时间4.0 h、料水比1∶2(v/v)、p H8.0、酶用量6000 U/(m L底物)。复合酶解法制备的水解液其抗凝血活性均高于混合酶解,最高抗凝血活性为52.0 ATU/m L。结论:本实验条件下,加酶组合为"中性蛋白酶-木瓜蛋白酶-碱性蛋白酶"的复合酶解制备的水解液具有最高的抗凝血活性,即52.0 ATU/m L。

Objective：Research on buffalo milk affected by the systems about mixed enzyme and composite enzyme including papain,neutral protease and alkaline protease, comparing with the result in different ways of hydrolysis. Method：First, the experiment went on with the single factor of three proteases hydrolyzing milk to explore the optimum condition of enzymatic hydrolysis.On this basis, the degree of hydrolysis was determinated through composite enzymatic hydrolysis and mixed enzyme and anticoagulant activity was determinated.Consequence：The results showed that optimum condition of papain were as follows： the ratio of material to water for 1 ： 2, pH = 5.0, temperature 60 -C, time 2 h, enzyme dosage 6000 u/mL.Neutral protease of were as follows ： the ratio of material to water for 1 ： 2, pH = 6.0, temperature 40 -C, time of 2 h, enzyme dosage 6000 u/mL.Optimum condition of alkaline protease were as follows ： the ratio of material to water for 1 ： 2, pH = 8.0, temperature 50 -C, time 4 h, enzyme dosage 6000 u/mL. Composite enzymatic hydrolysis anticoagulant activity was higher than by mixed enzymatic hydrolysis.The highest anticoagulant activity was 52 ATU/mL. Conclusion： In the experimental conditions, the degree and anticoagulant activity by composite enzymatic hydrolysis were higher than by mixed enzymatic hydrolysis. The enzymatic hydrolysis solution, which was prepared by the enzyme combination of the composite enzyme of neutral protease and papain protease and alkaline protease, had the highest anticoagulant activity, was 52.0 ATU/mL.