响应面优化南极磷虾蛋白酶解工艺及蛋白肽组分分析

Response Surface Optimization of Proteolytic Process and Protein Peptide Composition Analysis of Antarctic Krill

以南极磷虾蛋白酶解液中的氨基氮含量为指标,采用甲醛电位滴定法测定胰蛋白酶、木瓜蛋白酶、碱性蛋白酶、胃蛋白酶、风味蛋白酶、枯草杆菌蛋白酶6种生物酶在其最适酶解条件下对南极磷虾蛋白酶解效果,筛选出酶解南极磷虾蛋白最佳生物酶。在单因素试验基础上,以酶解液中氨基氮含量为响应值,选择酶解温度、时间、pH为自变量,通过三因素三水平Box-Bohnken响应面分析法对筛选出来的生物酶解工艺进行优化。在优化酶解工艺的基础上,利用SDS-PAGE凝胶电泳方法分析南极磷虾酶解蛋白肽及南极磷虾蛋白的分子量分布范围。结果表明:胰蛋白酶为酶解南极磷虾蛋白的理想蛋白酶;响应面分析法建立二次多项回归方程:Y=51.07-0.83A+1.72B-2.06C+0.25AC+1.15BC-3.06A2-2.77B2-3.69C2,模型p<0.0001,而失拟项p>0.05,表明模型极显著且比较稳定,通过模型分析得最佳条件:酶解温度44.48℃、酶解时间8.52 h、pH 7.88,考虑实际应用可行性,我们在酶解温度45.0℃、酶解时间8.5 h,pH 7.9条件下实验得到的氨基氮含量为50.96±1.07mg/g,与理论预测值51.57mg/g基本一致。通过电泳分析得到南极磷虾蛋白分子量范围在15 ku以上,主要集中分子量范围为15 ku～45 ku;胰蛋白酶解多肽分子量在25 ku以下,主要分子量分布在5 ku以下。

The amino nitrogen content in the antarctic krill hydrolysate was used as an indicator.The hydrolysis results of trypsin,papain,alkaline protease,pepsin,flavor protease and subtilisin were evaluated by formaldehyde potentiometric titration.The enzymatic hydrolysis of Antarctic krill protein was optimized for the enzymatic hydrolysis of Antarctic krill.On the basis of single factor experiment,the amino acid content in the enzymatic hydrolysate was used as the response value,and the enzymatic hydrolysis temperature,time and pH were selected as independent variables.The three-factor and three-level Box-Bohnken response surface analysis method was used to screen the bio-enzymatic solution.The process is optimized.Based on the optimized enzymatic hydrolysis process,the molecular weight distribution range of Antarctic krill enzymatic protein peptide and Antarctic krill protein was analyzed by SDS-PAGE gel electrophoresis.The results showed that trypsin was the ideal protease for enzymatic hydrolysis of Antarctic krill protein;the response surface analysis method was used to establish a quadratic multiple regression equation:Y=51.07-0.83A+1.72B-2.06C+0.25AC+1.15BC-3.06A^2-2.77B^2-3.69C^2,the model p<0.0001,and the missing term p >0.05,indicating that the model is extremely significant and stable,the best condition obtained by model analysis is:Enzymatic hydrolysis temperature was 44.48℃,the enzymatic hydrolysis time was 8.52 h,and the pH was 7.88.Considering the practical application feasibility,the amino nitrogen content of the experiment was 50.96±1.07 mg/g under the conditions of enzymatic hydrolysis temperature of 45.0℃,hydrolysis time of 8.5 h and pH 7.9,which is basically consistent with the theoretical prediction value of 51.57 mg/g.The molecular weight of Antarctic krill protein was over 15 ku by electrophoresis analysis,and the main molecular weight range was 15 ku^45 ku.The molecular weight of trypsin peptide was below 25 ku,and the main molecular weight distribution was below 5 ku.