响应面试验优化双酶酶解法制备鱼鳞抗菌肽工艺及其抑菌性能分析

Optimization of Preparation of Antimicrobial Peptides by Two-Step Enzymatic Hydrolysis of Fish Scales Using Response Surface Methodology and Antimicrobial Activity of Purified Antimicrobial Peptide

采用双酶酶解法制备鱼鳞抗菌肽,进行酶筛选并通过响应面法优化酶解条件;通过葡聚糖凝胶G-25分离纯化酶解液,研究有效抑菌组分对不同菌的最小抑菌浓度(minimum inhibitory concentration,MIC)。结果表明,当采用碱性蛋白酶结合酸性蛋白酶分步酶解鱼鳞,底物质量浓度为30 g/100 m L时,最适酶解条件为碱性蛋白酶在pH 9.5时首次酶解,酶解时间62 min,酶解温度55℃;酸性蛋白酶在pH 3.0时再次酶解,酶解时间3 h,酶解温度34.4℃。此条件下制备的酶解液对副溶血性弧菌的抑菌圈直径为27.72 mm,与预测值基本相符。酶解液经层析后,其抑菌性G2组分对假单胞菌和希瓦氏菌的MIC为1.56μg/m L,对金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌、沙门菌及副溶血性弧菌的MIC为6.25μg/m L。可见,双酶酶解法制备的鱼鳞抗菌肽具有较强的抑菌性。

This study aimed to prepare antimicrobial peptides(AMPs)by two-step enzymatic hydrolysis of crucian carp scales and to evaluate the antimicrobial activity of purified AMPs.Hydrolysis conditions were optimized by one-factorat-a-time method and response surface methodology based on the diameter of inhibition zones against tested bacteria.The enzymatic hydrolysate prepared using optimized conditions was then purified by Sephadex G-25column chromatography,yielding only a single peak(G2)with antimicrobial activity.The minimum inhibitory concentration(MIC)of G2was tested.The results indicated that a solid-to-liquid ratio of30g/100mL and sequential hydrolysis with alcalase at pH9.5and55℃for62min followed by acid protease at pH3.0and34.4℃for another3h were found to be the optimal conditions to obtain a greater diameter of inhibition zone against Vibrio parahemolyticus of27.72mm,which was well matched with the predicted value(27.37mm).The MIC of G2was1.56μg/mL against Pseudomonas and Shewanella putrefaciens,and6.25μg/mL against Escherichia coli,Staphylococcus aureus,Salmonella choleraescens,V.parahemolyticus and Bacillus subtilis.In conclusion,AMPs derived from freshwater fish scales by stepwise enzymatic hydrolysis possess strong antimicrobial activity.