源于地龙蛋白的抗氧化肽鉴定与构效关系

Identification and Structure-activity Relationships of Antioxidant Peptides Derived from Earthworm Proteins

摘要:

为探究外源蛋白酶预酶解处理对地龙蛋白胃肠消化程度和胃肠消化后抗氧化活性的影响。该研究利用五种蛋白酶制备了不同的地龙蛋白酶解物，然后通过体外胃肠模拟消化探究这些酶解物的水解度、分子量分布和体外抗氧化活性的变化。五种蛋白酶酶解物中，碱性蛋白酶酶解物的水解度最高，为40.8%，其对DPPH、ABTS自由基的清除能力分别为27.36、175.46 μmol TE/g，ORAC值为0.70 μmol TE/mg。碱性蛋白酶酶解物经胃肠消化后的水解度、DPPH自由基清除率、ORAC值相比于地龙蛋白直接胃肠消化所得产物（EWP）分别提高了94.06%、72.21%及120.00%。GHEWP中鉴定出10 823条分子量<3 ku的肽，通过bioware.ucd.ie进行活性阈值评分筛选出活性评分最高的11个肽进行合成，验证活性，其中ATSGFFVY、HCFVLY、HLASGWY、HRYSDF、HYANMY的综合抗氧化能力较好，量子化学计算结果表明：ATSGFFVY、HCFVLY、HRYSDF、HYANMY的活性位点可能分别位于Tyr-8、Tyr-6、Tyr-3、Tyr-2上；HLASGWY的活性位点可能位于Trp-6上。该研究表明碱性蛋白酶预酶解可提高地龙蛋白的消化程度，增强其胃肠消化产物的抗氧化活性。

Abstract:

The effects of exogenous protease pre-enzymolysis treatment on the degree of gastrointestinal digestion and antioxidant activity of earthworm proteins were examined following gastrointestinal digestion. Five proteases were used to prepare different hydrolysates of earthworm proteins, and changes in the degree of hydrolysis, molecular weight distribution, and in vitro antioxidant activities of these enzymatic hydrolysates were investigated based on simulated gastrointestinal digestion in vitro. Among these hydrolysates, the degree of hydrolysis of the alkaline protease hydrolysate was the highest at 40.81%. The rates of DPPH and ABTS free radical scavenging were 27.36 and 175.46 μmol TE/g, respectively, and the ORAC value was 0.70 μmol TE/mg. Following gastrointestinal digestion, the degree of hydrolysis, rate of DPPH free radical scavenging, and ORAC value of the alkaline protease hydrolysate increased by 94.06%, 72.21%, and 120%, respectively, compared with the products obtained via direct gastrointestinal digestion of earthworm proteins. A total of 10 823 peptides with molecular weight <3 ku were identified in GHEWP, and 11 peptides with the highest activity scores were screened using bioware.ucd.ie to determine their activity threshold score for synthesis and verification. Among these, the peptides ATSGFFVY, HCFVLY, HLASGWY, HRYSDF, and HYANMY had the highest comprehensive antioxidant capacities. The quantum chemical calculation results revealed that the active sites of ATSGFFVY, HCFVLY, HRYSDF and HYANMY might be located at Tyr-8, Tyr-6, Tyr-3, and Tyr-2, respectively, and the active site of HLASGWY may be located at Trp-6. The findings of this study indicate that pre-enzymatic hydrolysis using alkaline protease can enhance the degree of digestion of earthworm proteins and also the antioxidant activity of their gastrointestinal digestion products.