中性蛋白酶水解鸡骨泥制备短肽工艺优化

为了探究酶法制备鸡骨泥短肽的最佳工艺,该文研究了以中性蛋白酶酶解鸡骨泥时各因素对短肽得率和羟自由基清除率的影响,以及鸡骨泥肽清除超氧阴离子自由基和1,1-二苯基-2-三硝基苯肼自由基(2,2-diphenyl-1-picrylhydrazyl)的能力。试验探究了酶解过程中不同底物浓度、酶浓度、反应温度和反应时间对短肽得率和羟自由基清除率的影响,采用响应曲面优化得到了以短肽得率为目标的中性蛋白酶酶解鸡骨泥的最优工艺,当反应液p H值为7.2,反应温度42℃,反应时间2 h,底物质量分数5%,酶添加量200 mg/g,该条件下制备鸡骨泥肽,测得其短肽得率为56.16%,羟自由基清除率为54.12%。采用该酶解工艺制备鸡骨泥肽,测得其对超氧阴离子自由基最高清除率为56.01%,对DPPH自由基最高清除率为81.57%,说明鸡骨泥肽具有一定的抗氧化活性,研究结果为酶法制备鸡骨泥肽提供参考。

Optimization of enzymatic hydrolysis of chicken bone protein for production of antioxidant peptides using nutrase

Chicken bone is the main by-product of chicken slaughtering and processing. In order to explore new ways to use chicken bone and promote the high added value of chicken bone, the preparing technology and antioxidant activity of chicken bone peptides are investigated systematically in this paper, so as to provide theory basis for the industrial preparation and utilization of chicken bone peptides. This experiment uses the nutrase to hydrolyze chicken bone residue, investigates the effect of different substrate concentration, enzyme concentration, reaction temperature and reaction time on the yield of oligopeptide of product, and evaluates the antioxidant activity of the product according to the clearance rate of hydroxyl free radicals, superoxide anion free radicals and DPPH free radicals. The single factor experiment is aimed to explore the influence of different substrate concentration, enzyme concentration, reaction temperature and reaction time during the enzymolysis on the oligopeptide yield and the clearance rate of hydroxyl free radical. The results show: 1) Lower substrate concentration can affect the combination of enzyme and substrate molecules, on the other hand, higher substrate concentration will cause the phenomenon of substrate inhibition and enzyme inactivation; 2) Lower enzyme concentration will affect the combination of enzyme and substrate molecules, and higher enzyme concentration also will make part of the short peptides hydrolyzed to free amino acid, which can lead to oligopeptide yield decline; 3) Lower reaction temperature will affect the diffusion motion of enzyme and substrate molecules in the solution, and higher temperature will cause the structural changes of enzyme molecules and then can make a part of enzyme lose activity; 4) Shorter reaction time will affect the combination of enzyme and substrate molecules, and longer reaction time will lead to the fluctuating phenomenon of oligopeptide yield and clearance rate of hydroxyl free radical. Aiming at higher oligopeptide yield, using the response surface optimization, the best process using nutrase to hydrolyze chicken bone residue is obtained: the pH value of reaction solution is 7.2, the reaction temperature is 42℃, the reaction time is 2 h, the substrate concentration is 5%, and the enzyme concentration is 200 mg/g. When preparing the peptides of chicken bone residue under these conditions, the oligopeptide yield and clearance rate of hydroxyl free radicals are measured as 56.16% and 54.12%, respectively. Also the regression equation is gotten by using the response surface optimization, which can explain how substrate concentration and enzyme concentration affect the oligopeptide yield. By determining the antioxidant activity index of the product with optimal enzymatic hydrolysis conditions, it is concluded that the chicken bone residue peptide has some clearance ability on superoxide anion free radicals and DPPH free radicals. The clearance rate of ultra oxygen anion free radical is as high as 56.01%, which is a common removal ability, and the clearance rate of DPPH free radical is as high as 81.57%, which is a good removal ability. The test result shows that the product hydrolyzed by nutrase from chicken bone residue has biological antioxidant activity from a certain extent.