该研究以水解度为指标，仿生酶用量、提取时间、提取温度为条件因素，设计Box-Behnken响应方案，对珍珠母肽酶解条件进行优化；同时研究其不同质量浓度的珍珠母肽对HepG2肝癌细胞的抑制作用的影响。最佳酶解工艺条件为：温度55 ℃，调节pH值为2.0，加入胃蛋白酶质量分数1%，酶解2.6 h后，pH值调节为8.0，加入胰蛋白酶质量分数1.7%，酶解3 h，此时珍珠母肽水解度为31.21%；基于此酶解条件获得珍珠母肽进行对肝癌细胞抑制作用，研究实验结果显示不同质量浓度珍珠母肽（12.5、25、50、100、200 μg/mL）与氯化钴（CoCl2）诱导的HepG2肝癌细胞缺氧模型相比较，均可抑制细胞存活率，珍珠母肽中氨基酸结果显示，精氨酸可能参与抑制肝癌细胞增殖，从能量代谢的角度说明珍珠母肽可通过减少三磷酸腺苷的生成和降低乳酸、己糖激酶、丙酮酸激酶、乳酸脱氢酶能量代谢关键酶活力达到抑制HepG2细胞增殖的目的，可为珍珠母肽的制备工艺提供参考，为其开发利用提供一定的理论基础。

With the aim of enhancing the enzymatic hydrolysis of nacre peptide, we optimized enzymatic hydrolysis conditions, including the amount of biomimetic enzymes added and the extraction time and temperature, employing a Box-Behnken response design based on the degree of hydrolysis. The inhibitory effects of different mass concentrations of nacre peptide on HepG2 hepatoma cells were also investigated. The established optimal enzymatic hydrolysis conditions were as follows: a temperature of 55 ℃, pH adjusted to 2.0, and the addition of 1% (m/m) pepsin. Following enzymatic hydrolysis for 2.6 h, the pH was adjusted to 8.0 and 1.7% (m/m) trypsin was added for further enzymatic hydrolysis for 3 h. Under these conditions, percentage nacre peptide hydrolysis values of up to 31.21% were achieved. The nacre peptides obtained under the aforementioned conditions were assessed for their inhibitory effects on hepatoma cells. Experimental results revealed that nacre peptides, at different mass concentrations (12.5, 25, 50, 100, and 200 μg/mL), could reduce the rate of cell survival compared with the cobalt chloride (CoCl2)-induced hypoxia model of HepG2. Furthermore, results of an analysis of nacre peptide amino acids revealed that arginine might be involved in inhibiting the proliferation of hepatoma cells. From the perspective of energy metabolism, nacre peptides inhibited the proliferation of HepG2 cells by reducing the production of adenosine triphosphate and lactate, as well as by suppressing the activities of key energy metabolism-related enzymes, namely, hexokinase, pyruvate kinase, and lactate dehydrogenase. These findings can serve as a reference for the preparation of nacre peptides and provide a theoretical basis for their further development and utilization.

国家中医药公益性行业科研专项（201507002-4）；吉林省发改委产业技术研究与开发（2022C42-5）