仿刺参不同部位ACE抑制活性分析及活性肽制备工艺优化

王扬铎; 苏永昌; 王晓燕; 施文正; 刘智禹

doi:10.13386/j.issn1002-0306.2023070015

仿刺参不同部位ACE抑制活性分析及活性肽制备工艺优化

Analysis of ACE Inhibitory Activity in Different Parts of Sea Cucumber (Apostichopus japonicus) and Optimization of Preparation Process of Active Peptides

摘要

摘要: 本文对仿刺参具有高降血压活性部位进行筛选并优化其活性肽制备工艺。采用酶解法对仿刺参不同部位（体壁、肠、卵）进行水解，以血管紧张素转换酶（Angiotensin converting enzyme，ACE）抑制率为指标筛选最适蛋白酶，通过对各酶解物ACE抑制率的半数抑制浓度（IC₅₀）测定比较筛选出最优抑制活性部位。经单因素实验与响应面试验优化确定活性肽最佳酶解制备条件，对蛋白酶解物进行分子量测定确定其分布范围，经超滤膜分离后对不同组分的ACE抑制活性分析。结果显示，选用碱性蛋白酶为最适水解酶，体壁、肠、卵各蛋白酶解物的ACE抑制率的IC₅₀分别为1.11、4.02、0.65 mg/mL，仿刺参卵具有更好的ACE抑制效果，为最优抑制活性部位。其最佳的酶解制备工艺参数为：酶解时间5 h，加酶量3.5 U/mg，酶解温度65.26 ℃，底物浓度3.51%，酶解pH9.02，在该条件下仿刺参卵酶解产物的ACE抑制率为80.65%±0.52%，与预测值接近。蛋白酶解产物的分子量集中分布在3000 Da以下，占总含量的98.37%，其中1000~3000 Da占比9.50%，小于1000 Da占比88.87%。超滤膜分离所得低聚肽组分ACE抑制活性（IC₅₀=0.30 mg/mL）显著（P<0.05）强于经工艺优化后酶解物及截留液组分。本研究结果为仿刺参副产物高值化利用提供理论依据，可作为分离纯化制备降血压肽的优质资源。

Abstract: In this paper, the active parts of sea cucumber (Apostichopus japonicus) with high antihypertensive activity were screened and the preparation process of active peptides was optimized. Different parts (body wall, intestine, and ovum) of A. japonicus were hydrolyzed by enzymolysis, and the ACE inhibition rate was used as an indicator to screen the optimal protease. The optimal active site for inhibition was selected by comparative screening of the half maximal inhibitory concentration (IC₅₀) determination of ACE inhibitory rate of each lysate. Single factor and response surface tests were used to determine the optimum enzymatic hydrolysis conditions of the active peptides. The relative molecular weight of the protease hydrolysates was determined to determine its distribution range. The ACE inhibition activity of different components was analyzed after separation by ultrafiltration membrane. Search results, alkaline protease was selected as the optimal hydrolytic enzyme, and the IC₅₀ values of ACE inhibition of each protease lysate from body wall, intestine and ovum were 1.11, 4.02, 0.65 mg/mL, respectively, so that A. japonicus ovum had a better ACE inhibition effect and were the optimal active site for inhibition. Its optimal preparation process parameters for enzymatic hydrolysis were as follows: 5 h enzymatic hydrolysis time, 3.5 U/mg enzyme added, 65.26 °C enzymatic hydrolysis temperature, 3.51% substrate concentration, pH9.02 enzymatic hydrolysis, and ACE inhibition rate of A. japonicusr ovum was 80.65%±0.52% under these conditions, which was close to the predicted value. The molecular weight of proteolytic products was concentrated under 3000 Da, accounting for 98.37% of the total content, of which 1000~3000 Da accounted for 9.50%, and less than 1000 Da accounted for 88.87%. The ACE inhibitory activity of oligopeptide components (IC₅₀=0.30 mg/mL) isolated by ultrafiltration membrane was significantly higher than that of hydrolysates and trapped liquid components after process optimization. The results of this study would provide a theoretical basis for high-quality utilization of the by-products of A. japonicus, which could be used as high-quality resources for the isolation and purification of antihypertensive peptides.