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

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1.56?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。

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

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 Vibrio 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.