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目的优化牡蛎酶解制备抗氧化肽的工艺。方法通过正交试验法确定胃蛋白酶、胰蛋白酶及这两种蛋白酶的酶解液抗氧化(Fenton体系)的最佳工艺条件;采用Sephadex G-25葡聚糖凝胶柱层析法对酶解液中抗氧化肽进行分离纯化。结果胃蛋白酶最佳酶解条件:温度35℃、加酶量2.5%、时间6h、pH 2.0,在此条件下清除率达到50%时的酶解液浓度(EC50)为1.929mg.mL-1;胰蛋白酶最佳酶解条件:温度45℃、加酶量2%、时间8h、pH 7.5,EC50为1.165mg.mL-1;胰蛋白酶、胃蛋白酶分步酶解法的酶解液EC50为0.869mg.mL-1。双酶酶解液经纯化后,得相对分子质量为751的抗氧化活性肽组分(BPO-Ⅱ),其EC50为0.529mg.mL-1。结论双酶酶解法优于单酶酶解法,从双酶酶解液中分离纯化得抗氧化肽BPO-Ⅱ。
Objective To optimize the process of enzymatic hydrolysis of oyster to prepare antioxidant peptide. Methods The optimum conditions for the oxidation of pepsin, trypsin and enzymatic hydrolyzate of these two proteases were determined by orthogonal test. Sephadex G-25 gel filtration chromatography Antioxidant peptides in liquid were isolated and purified. Results The optimum enzymatic hydrolysis conditions of pepsin were as follows: the temperature was 35 ℃, the amount of enzyme was 2.5%, the time was 6h, the pH was 2.0. The EC50 of the pepsin at this condition was 1.929mg.mL-1 ; Trypsin optimal enzymolysis conditions: temperature 45 ℃, enzyme dosage 2%, time 8h, pH 7.5, EC50 1.165mg.mL-1; trypsin, pepsin enzymatic hydrolysis step by step EC50 of 0.869 mg.mL-1. After purification by double enzyme digestion, the antioxidant activity peptide component (BPO-II) with a relative molecular mass of 751 was obtained with an EC50 of 0.529 mg.mL-1. Conclusion The double enzyme digestion method is superior to the single enzyme digestion method, and the antioxidant peptide BPO-Ⅱ is isolated and purified from the double enzyme digestion solution.