模拟酶解海参蛋白制备抗氧化肽及其抗疲劳活性研究

Preparation of antioxidant peptides from simulated enzymatic hydrolysis of sea cucumber protein and their anti-fatigue activity

中文关键词:  海参，虚拟，抗氧化肽，酶解，抗疲劳

英文关键词:sea cucumber  virtual  antioxidant peptides  enzymatic hydrolysis  anti fatigue

中文摘要:

      目的 采用虚拟酶解和传统酶法相结合制备海参抗氧化肽, 并进一步考察其抗疲劳功效。方法 以海参为原料, 借助在线数据库进行虚拟酶解, 对水溶性、生物毒性、致敏性等特性预测, 筛选制备抗氧化肽的最佳蛋白酶, 通过单因素和响应面实验考察反应温度、时间、加酶量、pH、底物浓度对海参蛋白水解度、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除率的影响, 并确定最佳的抗氧化肽制备工艺参数, 进一步采用负重游泳实验小鼠模型探讨其抗疲劳功效。结果 经过多轮筛选最终获得亲水性、安全性、稳定性较好的蛋白酶为蛋白酶K, 在反应时间2.0 h, 加酶量350 U/g的条件下, 最佳的工艺参数为温度50℃、pH 6.4、底物浓度1.0%, 此条件下的DPPH自由基清除率为64.2%1.1%, 与理想值接近。抗疲劳功效研究表明海参抗氧化肽对小鼠体重影响不大, 能够明显延长负重游泳时间(P<0.05), 显著降低血清尿素氮、乳酸含量, 增加肝糖原、肌糖原储备量(P<0.05), 提升运动耐力。结论 通过虚拟酶解和实际酶解相结合的方法制备的海参抗氧化肽具有明显的抗疲劳作用。

英文摘要:

      Objective To prepare sea cucumber antioxidant peptides by combining virtual enzymatic hydrolysis and traditional enzymatic methods, and further investigate their anti fatigue effects. Methods Using sea cucumber as raw material, virtual enzymatic hydrolysis was carried out using an online database to predict and screen the best protease for preparing antioxidant peptides based on characteristics such as water solubility, biological toxicity, and allergenicity. The effects of reaction temperature, time, enzyme dosage, pH, and substrate concentration on sea cucumber protein hydrolysis degree and 1,1-diphenyl-2-picrylhydrazyl (DPPH) clearance rate were investigated through single factor and response experiments. The optimal process parameters for preparing antioxidant peptides were determined, and their anti fatigue effects were further explored using a load swimming mouse model. Results After multiple rounds of screening, the protease K with good hydrophilicity, safety, and stability was finally obtained. Under the conditions of reaction time of 2.0 hours and enzyme addition of 350 U/g, the optimal process parameters were temperature 50℃, pH 6.4, and substrate concentration of 1.0%. The DPPH clearance rate obtained under these conditions was 64.2%1.1%, which was close to the ideal value, indicating that the feasibility of the process was good. Research on the anti fatigue effect showed that sea cucumber antioxidant peptides had little effect on mouse body weight, could significantly prolong weight-bearing swimming time (P<0.05), significantly reduced serum urea nitrogen and lactate content, increased liver glycogen and muscle glycogen reserves (P<0.05), and improved exercise endurance. Conclusions The sea cucumber antioxidant peptide prepared by combining virtual enzymatic hydrolysis and actual enzymatic hydrolysis has a significant anti fatigue effect.