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 mL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3h,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1.56 μg/mL,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6.25 μg/mL.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-factor-at-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-25 column chromatography,yielding only a single peak (G2) with antimicrobial activity.The minimum inhibitory concentration (MIC) of G2 was tested.The results indicated that a solid-to-liquid ratio of 30 g/100 mL and sequential hydrolysis with alcalase at pH 9.5 and 55 ℃ for 62 min followed by acid protease at pH 3.0 and 34.4 ℃ for another 3 h were found to be the optimal conditions to obtain a greater diameter of inhibition zone against Hbrio parahemolyticus of 27.72 mm,which was well matched with the predicted value (27.37 mm).The MIC of G2 was 1.56 μg/mL against Pseudomonas and Shewanella putrefaciens,and 6.25 μg/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.
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