胶原三肽的酶法制备工艺研究

Study on Preparation Process of Collagen Tripeptide by Enzymic Method

以罗非鱼鱼皮为实验原料,研究不同蛋白酶处理对其蛋白质回收率、水解度以及酶解液风味的影响;以罗非鱼鱼皮内切蛋白酶酶解产物为底物,经解淀粉芽孢杆菌蛋白酶二次酶解后测定酶解液的肽分子量分布、胶原三肽种类和特征胶原三肽GPH含量。结果表明,罗非鱼鱼皮经碱性蛋白酶和木瓜蛋白酶于55℃下水解3.5 h,其蛋白回收率和水解度分别为87.59%和7.50%、88.59%和9.45%,酶解液有轻微苦味。在碱性蛋白酶和木瓜蛋白酶酶解产物中加入解淀粉芽孢杆菌蛋白酶于55℃二次水解胶原蛋白肽12 h后,其水解度分别为18.40%和14.64%,水解产物中分子量<500 Da的肽段分别占比54.28%和48.90%。UPLC-MS/MS分析显示碱性蛋白酶酶解后经解淀粉芽孢杆菌蛋白酶二次酶解,鉴定发现酶解液中主要的胶原三肽有GPH、GPA和HAG,其中分步酶解产物中GPH含量达到1.46%。

Taking the fish skin of Tilapia mossambica as the experimental raw material,the effect of different protease treatments on protein recovery rate,hydrolysis degree and flavor of hydrolyzate is studied.Using the hydrolysate of Tilapia mossambica fish skin endoprotease as the substrate,the distribution of peptide molecular weight,the type of collagen tripeptide and the content of characteristic collagen tripeptide GPH are determined after secondary enzymolysis by Bacillus amyloliquefaciens protease.The results show that Tilapia mossambica fish skin is hydrolyzed by alkaline protease and papain at 55℃for 3.5 h,the protein recovery rate and the hydrolysis degree are 87.59%and 7.50%,88.59%and 9.45%respectively,and the hydrolysate has slight bitterness.Adding Bacillus amyloliquefaciens protease into alkaline protease and papain hydrolysate,after secondary hydrolysis of collagen peptide at 55℃for 12 h,the hydrolysis degree of collagen peptide is 18.40%and 14.64%respectively,and the peptide fragments with molecular weight<500 Da in the hydrolysate account for 54.28%and 48.90%respectively.UPLC-MS/MS analysis shows that GPH,GPA and HAG are the main collagen tripeptides of the hydrolysate after hydrolysis by alkaline protease and secondary hydrolysis by Bacillus amyloliquefaciens protease,among which,the GPH content in the hydrolysate of stepwise enzymolysis reaches 1.46%.