响应面法优化黄粉虫抗菌肽酶解工艺

目的：确定黄粉虫抗菌肽的最佳酶解工艺。方法：以黄粉虫为原料，碱提酸沉法提取黄粉虫蛋白质，再利用筛选出的蛋白酶进行水解，并用琼脂平板扩散法(打孔法)表征制得抗菌肽的抑菌活性。在单因素试验的基础上，应用Box-Behnken中心组合试验设计和响应面(RSM)分析法，探讨酶解温度、pH值、酶解时间、加酶量及底物质量浓度对酶解产物抑菌活性的影响。结果：碱性蛋白酶最适合水解黄粉虫蛋白制备抗菌肽，其最佳工艺条件为：底物质量浓度810g/100mL、酶解时间4.4h、加酶量440U/g pro、酶解温度54℃、pH9.5；酶解物抑菌圈大小理论预测值为15.17mm，实际测量值为15.04mm。结论：碱性蛋白酶水解黄粉虫蛋白能够得到抑菌活性较强的酶解物，优化的工艺条件与理论预测拟合程度较高。

Optimization of Enzymatic Preparation of Antibacterial Peptides by Response Surface Methodology

Objective: To determine the optimal conditions for enzymatic preparation of Tenebrio molitor antibacterial peptides.Methods: Antibacterial peptides were prepared by hydrolyzing the protein extracted from Tenebrio molitor by alkaline extractionand acid precipitation. The antibacterial activity of the peptides was evaluated by agar diffusion method. One-factor-at-a-timemethod and response surface analysis based on Box-Behnken design were used to explore the effects of hydrolysis temperature,pH, hydrolysis time, enzyme dosage and substrate concentration on antibacterial activity of Tenebrio molitor antibacterialpeptides. Results: Alkaline protease was the most efficient protease for the preparation of Tenebrio molitor antibacterial peptides,and the optimum hydrolysis conditions were found to be hydrolysis at 54 ℃ and pH 9.5 for 4.4 h with an enzyme dosage of 440U/g pro and a substrate concentration of 810 g/100 mL. The predicted maximum inhibition circle diameter was 15.17 mm, closeto the actual value of 15.04 mm. Conclusion: Peptides with high antibacterial activity can be prepared by hydrolyzing Tenebriomolitor protein with alkaline protease, and the optimized process conditions allow accurate theoretical prediction.