罗非鱼鱼皮胶原蛋白降血压酶解液的制备与活性研究

Study on Preparation and Activity of Lower the Blood Pressure Enzymatic Hydrolysate from Oreochromis niloticus Skin Collagen

本文研究了单酶和复合酶水解罗非鱼鱼皮胶原蛋白制备抑制血管紧张素转换酶抑制剂(ACEI)的工艺条件。通过正交试验确定了菠萝蛋白酶和Alcalase2.4L蛋白酶单独水解鱼皮胶原蛋白的最适酶解条件。菠萝蛋白酶和Alcalase2.4L蛋白酶的最适酶解温度,pH,酶/底物,时间,底物浓度分别为45℃,pH4.5,4000U/g,4h,6%和55℃,pH7.5,6000μ/g,2h,4%。在单酶水解的基础上,进行复合酶水解实验,结果表明:先采用菠萝蛋白酶水解,再用Alcalase2.4L蛋白酶水解,效果更佳。采用该双酶复合水解工艺,所得水解产物的水解度为30.43%,体外对ACE的抑制率达68.6%。采用SephadexG-25,HPLC的分离方法对酶解液进行分离纯化,得到了高抑制活力的组分。所得水解原液,SephadexG-25过柱所得高活力组分的IC50分别为2.82mg/ml和0.65mg/ml。

The single and compound enzymatic hydrolysis conditions of tilapia （Oreochromis niloticus） skin collagen were studied. Based on the assay of inhibitory activity of single enzymatic hydrolysates to ACE, Bromelain and Alcalase 2.4L were selected to be compound enzyme. The optimal hydrolytic conditions of skin collagen with the two enzymes were determined by orthogonal test. The results show that the optimal hydrolytic conditions of Bromelaln and Alcalase 2.4L are temperature 45 ℃, pH4.5, E/S 4000U/g, time 4h and concentration of substrate 6% and temperature 55 ℃, pH7.5, E/S 6000U/g,time 2h and concentration of substrate 4%, respectively. Thereafter, hydrolytic experiments of compound enzyme were done. The results indicate that it is better to hydrolysis by Bromelain at first, then by Alcalase 2.4L.The hydrolytic degree of the prepared hydrolysates is 30.43%, the ACE inhibitory ratio is as high as 68.6% in vitro. The high activity ACE inhibitory fractions were separated and purified using the Sephadex G-25 and the Reverse-Phase High Performance Liquid Chromatography （RP-HPLC）. The concentrations of the hydrolysates to inhibit 50% of ACE activity （IC50） were measured. The results showed that the IC50 of the original hydrolysate and the Sephadex G-25 filtrate were 2.82mg/ml and 0.65mg/ml, respectively.