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 extraction and acid precipitation. The antibacterial activity of the peptides was evaluated by agar diffusion method. One-factor-at-a-time method 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 antibacterial peptides. 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 440 U/g pro and a substrate concentration of 810 g/100 mL. The predicted maximum inhibition circle diameter was 15.17 mm, close to the actual value of 15.04 mm. Conclusion: Peptides with high antibacterial activity can be prepared by hydrolyzing Tenebrio molitor protein with alkaline protease, and the optimized process conditions allow accurate theoretical prediction.