Abstract: 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.

Key words: enzymatic hydrolysis, fish scales, antimicrobial peptides, response surface methodology, antimicrobial activity