牡蛎蛋白酶解肽制备工艺优化及其对小鼠睾丸间质细胞睾酮分泌和氧化应激的影响

刘璐; 李晶峰; 兰梦; 李冬冰; 张凯月; 王跃龙; 申嘉明; 李春楠; 张辉; 孙佳明

doi:10.13386/j.issn1002-0306.2023050205

牡蛎蛋白酶解肽制备工艺优化及其对小鼠睾丸间质细胞睾酮分泌和氧化应激的影响

Optimization of the Preparation Process of Oyster Peptide by Enzymatic Hydrolysis and Its Effects on Testosterone Secretion and Oxidative Stress in Mouse Testicular Interstitial Cells

摘要

摘要: 为探究牡蛎在药食同源领域的研究与开发，本实验采用响应面法优化牡蛎蛋白酶解肽酶法制备工艺，并研究其对小鼠睾丸间质细胞睾酮分泌和氧化应激的影响。以水解度为考察指标，采用仿生酶解法，在单因素实验的基础上，根据响应面分析法优化牡蛎蛋白酶解肽的制备工艺。同时构建过氧化氢（H₂O₂）诱导的小鼠睾丸间质细胞（TM3）氧化损伤模型，通过细胞存活率、DAPI染色、睾酮（Testosterone，T）分泌量，超氧化物歧化酶（SOD）活力和丙二醛（MDA）含量研究牡蛎蛋白酶解肽对TM3细胞睾酮分泌和氧化应激的影响。结果表明：牡蛎蛋白酶解肽的最佳酶解条件为：料液比1:10 g/mL、胃蛋白酶量为1.1%、酶解时间为1.0 h、胰蛋白酶量为2.1%、酶解时间3.1 h，此条件下的水解度为39.43%±0.42%。牡蛎蛋白酶解肽对H₂O₂诱导的TM3细胞均有不同程度的增殖活性，可显著（P<0.05）增加TM3细胞睾酮分泌量、SOD酶活力和降低（P<0.05）MDA含量，并且在牡蛎蛋白酶解肽物质量浓度为200 μg/mL时效果最好。综上所述，响应面法优化酶解工艺有效可行，牡蛎蛋白酶解肽可极显著的促进TM3细胞增殖，促进睾酮分泌量，增加SOD酶活力，降低MDA含量（P<0.01)。

Abstract: In order to explore the research and development of oyster in the field of medicine and food homology, response surface methodology was used to optimize the preparation process of oyster protease-depeptidase, and its effects on testosterone secretion and oxidative stress in mouse leydig cells were studied. Based on the investigation of hydrolysis degree as the evaluation index, a biomimetic enzymatic hydrolysis method was employed to optimize the preparation process of oyster protein enzymolysis peptides using response surface analysis, building upon the foundation of single-factor experiments. Simultaneously, a hydrogen peroxide (H₂O₂)-induced oxidative damage model was established using mouse testicular interstitial cells (TM3), and the effects of oyster protein enzymolysis peptides on testosterone (T) secretion and oxidative stress were investigated through assessments of cell viability, DAPI staining, testosterone secretion level, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in TM3 cells. The results showed that the optimal enzymatic hydrolysis conditions for oyster protein enzymolysis peptides were as follows: Substrate-to-solvent ratio of 1:10 g/mL, gastric protease concentration of 1.1%, hydrolysis time of 1.0 h, pancreatic protease concentration of 2.1%, and hydrolysis time of 3.1 h. Under these conditions, the degree of hydrolysis was determined to be 39.43%±0.42%. Oyster protein enzymolysis peptides exhibited varying degrees of proliferative activity on H₂O₂-induced TM3 cells, significantly (P<0.05) increasing testosterone secretion, SOD enzyme activity, and reducing MDA levels in TM3 cells. The most pronounced effects were observed at a concentration of 200 μg/mL of oyster protein enzymolysis peptides. In conclusion, the optimization of enzymatic hydrolysis process using response surface methodology proved to be effective and feasible. Oyster protein enzymolysis peptides was found to extremely significant promote TM3 cell proliferation, increase testosterone secretion, enhance SOD enzyme activity, and reduce MDA levels (P<0.01).