该研究在酶解法和响应面法的基础上通过增加响应面法中的响应值来优化碱性蛋白酶酶解蚕蛹蛋白的工艺，以DPPH和ABTS+自由基清除率作为水解肽评估指标筛选并找到了一条抗氧化活性较强的酶解工艺流程。最优酶为碱性蛋白酶，最佳条件为pH值为9.0、温度为48 ℃、底物质量分数为4.1%、反应时间为2 h、酶质量分数为3.0%，与现有的工艺流程相比较缩短且降低了反应的时间和温度，且提升了体外抗氧化活性，其体外抗氧化活性达到了Vc的89.10%。在此基础上使用D-半乳糖引起的氧化应激小鼠模型评价了该蚕蛹肽在体内的抗氧化活性，低剂量组的蚕蛹肽即可使血清中CK、SOD、T-AOC、GSH-PX因子恢复至0.31、105.44、3.51、308.36 U/mL肝脏中MDA、SOD因子恢复至0.71 nmol/mL、77.40 U/mL均与模型组有显著差异（P<0.05）。综上通过双响应面法制备而得的蚕蛹肽不仅优化了工艺流程而且使得其抗氧化活性有所提升且在体内外均能发挥抗氧化活性，为蚕蛹蛋白肽的开发利用提供了实验依据。

This study sought to optimize the process of the enzymatic hydrolysis of silkworm pupa protein by alkaline protease by increasing the response value in response surface method. DPPH and ABTS free radical scavenging rates were employed as the evaluation indices of hydrolyzed peptides. This allowed for the screening and identification of enzymatic hydrolysis processes exhibiting strong antioxidant activity. As a result, the optimal enzyme was identified as alkaline protease with pH value 9.0, a temperature of 48 ℃, a substrate mass fraction of 4.1%, a reaction time of 2 h, and an enzyme mass fraction of 3.0%. Compared with the existing process, the reaction time and temperature were reduced, and the antioxidant activity in vitro was improved, reaching 89.10% of Vc. On this basis, the antioxidant activity of the silkworm pupa peptide in vivo was evaluated using an oxidative stress mouse model induced by D-galactose. The low-dose group of silkworm pupa peptide was able to restore the CK, SOD, T-AOC and GSH-PX factors in serum to 0.31, 105.44, 3.51 and 308.36 U/mL, respectively. In addition, the MDA and SOD factors in the liver were restored to 0.71 nmol/mL and 77.40 U/mL, respectively, which were significantly different from the model group (P<0.05). In summary, silkworm pupa peptide prepared using the double response surface method optimized the process flow and improved the antioxidant activity, enhancing antioxidant activity both in vitro and in vivo. This provides an experimental basis for the development and utilization of silkworm pupa protein peptides.

国家自然科学基金面上项目（32172311）；广东省自然科学基金项目项目（2021A1515012413）；深圳市科技计划项目（JCYJ20220531102205012；GJHZ20210705141803010）