Abstract: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.