响应面法优化紫贻贝免疫活性肽的制备工艺

Optimization of preparation technology of immunomodulatory peptides from Mytilus edulis by response surface methodology

目的探讨紫贻贝免疫活性肽的酶解制备工艺。方法本研究以紫贻贝(Mytilus edulis)为原料,选用5种不同的蛋白酶对其进行酶解,以小鼠RAW264.7巨噬细胞的相对增殖率和酶解液中活性肽的浓度为指标,在单因素实验基础上通过响应面法优化制备紫贻贝免疫活性肽(Mytilus edulis immunomodulatory peptides, MIP),同时采用CCK-8和中性红吞噬实验对其免疫活性进行初步研究。结果实验数据表明,胰蛋白酶为最适蛋白酶,最佳酶解条件为:酶解温度43.7℃,p H 8.9,时间3.2 h,加酶量3 000 U/g。在此条件下,100μg/mL MIP对RAW264.7巨噬细胞的相对增殖率为92.4%。CCK-8结果显示,MIP对RAW264.7细胞无毒性作用且能显著促进细胞增殖。当MIP浓度为100μg/mL时,中性红吞噬指数可提高至1.22。同时,形态学观察结果显示MIP可以刺激细胞分化形成伪足,细胞形态以不规则多边形为主。结论 MIP对RAW264.7细胞具有较好的免疫调节活性。

Objective To study the enzymatic hydrolysis process of immunomodulatory peptides from Mytilus edulis. Methods Mytilus edulis was used as the raw material, and five different enzymes were applied for enzymatic hydrolysis in this study. The relative proliferation rate of enzymatic peptides on macrophage RAW264.7 cells and the concentration of active peptides in the hydrolysate were used as indicators. On the basis of single-factor experiment, response surface methodology(RSM) was adopted to optimize the hydrolysis process of Mytilus edulis immunomodulatory peptides(MIP). At the same time, CCK-8 and neutral red phagocytic experiment were adopted to evaluate its immunomodulatory activity. Results The results showed that trypsin was the most suitable enzyme, and the optimum hydrolysis conditions were as follows: hydrolysis temperature 43.7 ℃, pH 8.9, hydrolysis time 3.2 h, enzyme substrate ratio 3 000 U/g. The relative proliferation rate under the optimal condition was 92.4%. CCK-8 assay results showed that MIP had no cytotoxicity and could significantly promote the proliferation of RAW264.7 cells. The phagocytic index of RAW264.7 cells was up to 1.22 at the MIP concentration of 100 μg/mL. Furthermore, morphological observation showed that RAW264.7 cells formed multiple pseudopodia and were characterized with irregular polygon. Conclusion MIP has an immunomodulatory effect on RAW264.7 cells.