鮰鱼蛋白ACE抑制肽的酶法制备及分离纯化

Preparation of ACE Inhibitory Peptide from Longsnout Catfish Protein by Enzymatic Method and Its Separation and Purification

以脱脂后的鮰鱼肉为原料,采用分步酶解法制备血管紧张素转化酶(ACE)抑制肽,通过正交试验优化酶解条件。最佳酶解工艺:调节pH为8,温度为37℃,底物浓度为0.17 g/mL,先加胰蛋白酶,酶活/底物量为6 U/mg,水解1 h后再调节pH为9,温度为55℃,再加入碱性蛋白酶,酶活/底物量为6 U/mg,水解4 h。在此条件下,鮰鱼肉蛋白水解度为12.63%,酶解液ACE抑制率为18.7%。分子量分布分析发现酶解产物分子量主要分布在500 Da以下,占多肽的98.23%。酶解产物经超滤(3 000 Da)分离后ACE抑制率提高至35.4%,再用DA201-C大孔吸附树脂脱盐后ACE抑制率为56.6%,最后用葡聚糖凝胶G-15柱层析进行纯化后得到3个组分,组分G2的ACE抑制率为66.9%。经过分离纯化后的ACE抑制肽活性相比原酶解液提高了3.6倍。

In this paper,skimmed longsnout catfish meat is used as the raw material to prepare angiotensin I-converting enzyme(ACE)inhibitory peptide by stepwise enzymatic hydrolysis method.The enzymatic hydrolysis conditions are optimized by orthogonal test.The optimal enzymatic hydrolysis conditions are as follows:pH is adjusted to 8,temperature is 37℃,substrate concentration is 0.17 g/mL,trypsin is added firstly,enzyme activity/substrate content is 6 U/mg,being hydrolyzed for 1 h,and then pH is adjusted to 9,temperature is 55℃,and then alkaline protease is added,enzyme activity/substrate content is 6 U/mg,being hydrolyzed for 4 h.Under such conditions,the hydrolysis degree of longsnout catfish meat protein is 12.63%,and the ACE inhibition rate of enzymatic hydrolysate is 18.7%.The molecular weight distribution analysis results show that the molecular weight distribution of the hydrolysate is mainly below 500 Da,accounting for 98.23%of the polypeptides.After separation by ultrafiltration(3000 Da),the ACE inhibitory rate of enzymatic hydrolysate increases to 35.4%,and after desalination with DA201-C macroporous adsorption resin,the ACE inhibitory rate is 56.6%.Finally,three components are obtained after purification with Sephadex G-15 column chromatography,and the ACE inhibitory rate of component G 2 is 66.9%.After separation and purification,the activity of ACE inhibitory peptide is 3.6 times higher than that of the original enzymatic hydrolysate.