以苦杏仁醇溶蛋白为原料，采用甲醛电位滴定法以水解度和自由基清除率为评价指标对风味蛋白酶、碱性蛋白酶、木瓜蛋白酶、中性蛋白酶和胃蛋白酶五种生物酶在最适条件下对苦杏仁醇溶蛋白的酶解效果进行酶的优选。在单因素实验的基础上，以酶添加量、底物浓度、pH、时间为自变量，通过四因素三水平Box-Bohnken响应面分析法对生物酶解工艺进行优化。结果表明碱性蛋白酶为酶解苦杏仁醇溶蛋白的最优选择，预测最优工艺条件为时间：4.12 h、酶添加量5208.93 U/g、底物浓度3.21%、pH 9.22，根据最终试验目的，选取条件为：时间：4.0 h、酶添加量5000.0 U/g、底物浓度3.0%、pH 9.0。通过验证试验得出响应值DH%为26.74%±0.54%，DPPH自由基清除率为97.86%±0.58%，与预测值相差较小，故此条件下酶解苦杏仁醇溶蛋白抗氧化肽为最优工艺。

The degree of hydrolysis, as measured by potentiometric titration in the presence of formaldehyde, and free radical scavenging rate in five bioenzymes, namely flavor proteases, alkaline proteases, papains, neutral proteases, and pepsins were compared to identify the bioenzyme with the best proteolytic performance against the Prunus dulcis var. amara prolamins under optimal conditions. The effects of independent variables such as enzyme addition, substrate concentration, pH and reaction time were explored via single-factor experiments. Box-Behnken response surface methodology considering these four factors at three levels was then used to optimize the proteolysis of these five bioenzymes. The results demonstrate that alkaline proteases outperform the other enzymes when using the Prunus dulcis var. amara prolamins as substrate. The optimal conditions were determined to be as follows: a reaction time of 4.12 h; a total of 5208.93 U/g of the enzyme should be added; the substrate concentration should not exceed 3.21%; and the pH should be maintained at 9.22. Final evaluations using these conditions allowed for an additional round of refinement and we determined that the optimal conditions are as follows: a reaction time of 4.0 h; 5000.0 U/g of enzyme; substrate at 3.0%; and the pH at 9.0. These conditions were then validated by the evaluation of the end product with these processed petides increasing the DH% and DPPH free radical scavenging rate to 26.74%±0.54% and 97.86%±0.58%, respectively. These values are very similar to the predicted values from our in silico evaluation, confirming that these are the optimal conditions for the proteolysis of the prolamin proteins produced by Prunus dulcis var. amara.

国家重点研发计划项目（2019YFD1002300）