酶法制备联合Plastein反应修饰牡蛎ACE抑制肽工艺优化

Optimized Preparation of ACE Inhibitory Peptides from Oyster by Enzymatic Hydrolysis Coupled with Plastein Reaction

以牡蛎为原料,采用酶解联合Plastein反应修饰的方法,获得高活性血管紧张素转换酶(angiotensin converting enzyme,ACE)抑制肽。以ACE抑制率和水解度为指标,对比胃蛋白酶、木瓜蛋白酶、碱性蛋白酶、中性蛋白酶、胰蛋白酶这5种蛋白酶对牡蛎肉的酶解效果,筛选出木瓜蛋白酶最佳。通过单因素试验和正交试验对酶解工艺进行优化,得到最佳酶解工艺为料液比1∶8(g/m L)、加酶量2.0%、温度65℃、时间1.0 h、pH6.0,此条件下酶解产物的ACE抑制率可达到63.30%,在此基础上采用Plastein反应对酶解产物进行修饰,以游离氨基酸减少量和ACE抑制率为指标,考察反应过程中酶种类、底物质量分数、加酶量、时间和温度对修饰结果产生的影响。通过该反应的修饰,得到选用中性蛋白酶、底物质量分数40%、加酶量1.0%、温度30℃、时间2.5h、pH7.0时,ACE抑制率最高可达82.31%,比修饰前提高了19%。

Angiotensin converting enzyme(ACE)inhibitory peptides from oyster were prepared by the combined use ofenzymatic hydrolysis and Plastein reaction.Papain,pepsin,alcalase,neutural protease and trypsin were tested for theirefficiencies in hydrolyzing oyster based on ACE inhibitory activity and degree of hydrolysis and it turned out that papainwas the optimal choice.The hydrolysis conditions were optimized by one-factor-at-a-time method and orthogonal arraydesign as follows:solid-to-liquid ratio(g/mL),1:8;concentration of papain,2.0%;hydrolysis time,1.0h;hydrolysistemperature,65℃;and initial pH,6.0.Under these conditions,the percentage inhibition of ACE activity by oysterhydrolsate was63.30%.The hydrolysate was modified via the Plastein reaction.Based on ACE inhibitory activity andreduction in free amino acid content,the optimal values of Plastein reaction parameters including the type of enzyme,enzyme dosage,substrate concentration,temperature and reaction time were determined to be papain,1.0%,40%,2.5hand30℃for hydrolysis at an initial pH of7.0,respectively.The modified product showed maximum percentage inhibitionagainst ACE activity of82.31%,which was increased by19%compared to that before modification.