蚕蛹蛋白肽酶水解条件优化

Optimization of the peptide from silkworm pupa protein by enzymatic hydrolysis

为了降低蚕蛹过敏风险,提高蚕蛹优质蛋白利用价值并制备免疫活性肽,对蚕蛹蛋白进行了酶解优化。本文选用菠萝蛋白酶、碱性蛋白酶、中性蛋白酶、风味蛋白酶和木瓜蛋白酶这5种酶对脱脂蚕蛹蛋白进行酶解。通过对小鼠脾细胞增殖和水解度的检测,选取最佳蛋白酶。在此基础上对最佳蛋白酶的四个单因素(酶和底物量,初始p H,水解温度和底物浓度)进行优化,从而确定最佳的酶解条件。结果表明,5种蛋白酶中碱性蛋白酶对脱脂蚕蛹蛋白的酶解效果最好。对碱性蛋白酶进一步优化,通过Box-Behnken实验和回归分析获得碱性蛋白酶酶解脱脂蚕蛹蛋白的最佳条件为加酶量=6.0%,温度=55℃,酶解时间=2.00 h,p H=8.0,水∶底物=20∶1,测得的水解度为19.96%±1.02%,免疫活性OD490 nm为0.2512±0.0125。

In order to reduce the risk of silkworm allergic reaction, to improve the use of high- quality protein and the preparation of immunoactive peptide, the enzymatic hydrolysis of silkworm pupa protein was optimized. For the preparation of immunoaetive peptide, the five kinds of enzymes （ bromelain, alkaline protease, neutral protease, flavourzyme, papain） were selected to hydrolyze the defatted silkworm pupa protein. By the detection of spleen cell proliferation and the degree of hydrolysis, the optimal enzyme were determined.On the basis of that, the optimal condition was identified with fbur singe-factors of hydrolysis（ E0/S0 ,pH,temperature and substrate concentration）.The results showed that alkaline protease was the optimal enzyme and the optimum conditions of defatted silkworm pupa protein by alkaline protease enzyme was as that of the amount of enzyme = 6.0% , temperature = 55℃, enzymolysis time = 2.00 h, pH = 8.0, water： substrate = 20： 1.The degree of hydrolysis was 19.96% ± 1.02% and immunoreactive OD490 nm was 0.2512 ± 0.0125.