双酶水解芝麻11S蛋白制备抗氧化肽的工艺研究

STUDY ON PREPARATION OF ANTIOXIDANT PEPTIDES WITH TWO ENZYMES HYDROLYZING SESAME 11S PROTEIN

芝麻11S蛋白是芝麻蛋白的主体,研究其在水解过程中产生的多肽的抗氧化能力有利于深入了解水解芝麻蛋白制备抗氧化肽的作用机理,更好地指导实际加工生产过程。探索了混料酶(Alcalase和胰蛋白酶以酶活比为1∶1混合)水解芝麻11S蛋白产物抗氧化性的工艺条件。通过单因素试验讨论了水解过程中加酶量、p H、温度、时间、底物浓度这5个因素对水解多肽产率、DPPH自由基清除率、ABTS自由基清除率和总抗氧化能力的影响,确定了水解所需最适加酶量和底物浓度,并筛选出用于响应面优化试验的水解p H、温度、时间的范围。通过响应面优化试验,得到水解芝麻11S蛋白多肽产物抗氧化能力相对较高的工艺条件为:温度44.81℃,加酶量10 000 U/g,水解p H 8.6,底物浓度3%,时间1.96 h。在此条件下,实际测定多肽产率为(77.41±0.30)%,总抗氧化性为(1.40±0.02)mmol/L,DPPH自由基清除率IC50值为1.41 mg/m L,ABTS自由基清除率IC50值为1.06 mg/m L。结果表明,响应面所建立的回归模型方程准确可靠,而且与其他天然抗氧化肽相比,芝麻11S蛋白显示出较好的抗氧化活性,具有制备高活性抗氧化肽的能力,为进一步分离纯化芝麻11S蛋白抗氧化肽提供了参考。

Sesame 11S protein is the main body of sesame protein.Researching the antioxidative capacity of polypeptides produced during the hydrolysis of sesame 11S protein is in favor of an in-depth understanding of the mechanism of the preparation of sesame protein antioxidant peptides,and guiding the actual production processes and applications.The process conditions for the hydrolysis resistance of sesame 11S protein products by the mixed enzyme(Alcalase and trypsin are mixed in an enzyme activity ratio of 1:1)was investigated.The effects of five factors,including enzyme dosage,pH value,temperature,time,and substrate concentration,on the yield of hydrolyzed peptides,DPPH free radical scavenging rate,ABTS free radical scavenging rate and total antioxidant ability was studied,and the optimal amount of enzyme and substrate concentration required for hydrolysis,the range of hydrolysis pH,temperature,and time for the response surface optimization test were determined.The process conditions for the relatively high antioxidant ability of hydrolyzed sesame 11S polypeptide product was determined by response surface optimization experiment:temperature 44.81℃,enzyme amount 10 000 U/g protein,hydrolysis pH 8.6,substrate concentration 3%,time 1.96 h.Under this condition,the actual yield of polypeptide was(77.41±0.30)%,the total antioxidant activity was(1.40±0.02)mmol/L,the IC50 value of DPPH radical scavenging rate was 1.41 mg/mL,and the IC50 value of ABTS radical scavenging rate was 1.06 mg/mL.The results show that the regression model established by the response surface is accurate and reliable.Compared with other natural antioxidant peptides,sesame 11S protein showed better antioxidant activity and had the ability to prepare high-activity anti-oxidant peptides,which provided a reference for further isolation and purification of sesame 11S anti-oxidant peptides.