用中性蛋白酶酶解苦荞蛋白制备抗菌肽,以抑菌率和肽质量浓度为响应值,在底物质量分数、加酶量、pH值、酶解温度和酶解时间5个单因素试验基础上,采用Plackett-Burman试验选出其中影响显著的因素,再以肽质量浓度为评价指标,用Box-Behnken试验优化抗菌肽制备工艺.最佳工艺为:底物质量分数3.22％、加酶量4 000 U/g、pH 6.86.在此条件下,苦荞蛋白酶解产生的肽质量浓度为32.41 mg/mL(预测值为32.12 mg/mL),对大肠杆菌抑菌率为(27.88±2.78)％,对金黄色葡萄球菌的抑菌率为(94.56±0.74)％.结果表明,优化中性蛋白酶酶解苦荞蛋白制备抗菌肽工艺合理.%The enzymatic hydrolysis of tartary buckwheat protein with neutral protease to produce antibacterial peptides was optimized using Plackett-Burman and Box-Behnken designs.One-factor-at-a-time experiments were carried out to investigate the effect of substrate concentration,enzyme-to-substrate ratio,pH,hydrolysis temperature and time on the antibacterial activity and peptide concentration of hydrolysates.The factors with a significant influence on the response variables were selected using Plackett-Burman design and optimized for maximum peptide concentration using Box-Behnken design.The optimum parameters for enzymatic hydrolysis were determined as follows:substrate concentration 3.22％,enzyme-to-substrate ratio 4 000 U/g and pH 6.86.Under these conditions,the peptide concentration was 32.41 mg/mL,in good agreement with the predicted value (32.12 mg/mL),and the percentage inhibition of antimicrobial peptides was (27.88 ± 2.78)％ against E.coli and (94.56 ± 0.74)％ against S.aureus.In conclusion,this optimized procedure was reasonable.
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