复合酶法制备鱿鱼皮胶原蛋白抗冻肽的工艺研究

Research on Preparation Technology for Collagen Antifreeze Peptide From Squid Skinby Composite Enzymatic Hydrolysis

为了获得具有较高活性的抗冻肽,提高水产加工副产物的综合利用率,本试验以水解度和过氧化氢酶低温保护活性为指标,优化鱿鱼皮胶原蛋白酶解工艺,并对水解产物进行分离纯化及结构鉴定。结果表明,最佳酶解条件为:当底物浓度为4%、加酶量为4000 U·g^(-1)时,先由碱性蛋白酶于pH值8、50℃条件下酶解2 h;灭酶后由木瓜蛋白酶于pH值6、45℃条件下酶解3 h。在此条件下所得酶解产物分子量主要在1000~5000 Da之间,经G-25凝胶柱分离后得到具有较高抗冻活性的F2组分,其主要成分的氨基酸序列可能为Gly-Pro-Try-Pro-Ala-Ala-Asn-Ser-Pro-Glu或Glu-Pro-Ser-Asn-Ala-Ala-Pro-Try-Pro-Gly。本研究为鱿鱼皮的精深加工和新型抗冻剂的开发提供了一定的理论依据。

In order to obtain the antifreeze peptide with higher activity and improve the utilization rate of byproducts from aquatic processing,the enzymatic hydrolysis of squid skin collagen was optimized according to the hydrolysis degree(DH)and cryoprotective effect on catalase,then the hydrolysates were purified and identified.The optimal condition of enzymatic hydrolysis was as follows:Alkaline protease was used to hydrolyze for 2 h at 50℃and pH 8 with substrate concentration of 4%and enzyme dosage of 4000 U·g^(-1),papain was used to hydrolyze subsequently for 3 h at 45℃and pH 6 after inactivation.Under this condition,the molecular weight of the hydrolysates was mainly distribution in 1000~5000 Da.The fraction F2 with high antifreeze activity was obtained by G-25 gel filtration chromatography,and its major amino acid sequence was tentatively identified as Gly-Pro-Try-Pro-Ala-Ala-Asn-Ser-Pro-Glu or Glu-Pro-Ser-Asn-Ala-Ala-Pro-Try-ProGly.This study provides a theoretical basis for the intensive processing of squid skin and the development of new antifreeze agents.