酪蛋白抗凝血肽的分离纯化及其抗凝血活性测定  

Separation and purification of casein anticoagulant peptide and determination of its anticoagulant activity

作　　者：刘丽莎 苗艳丽 陈绍红[2] 张刘 卢晓 LIU Lisha;MIAO Yanli;CHEN Shaohong;ZHANG Liu;LU Xiao(College of Chemistry and Environment,Guangdong Ocean University,Zhanjiang 524088,China;College of Food Science and Technology,Guangdong Ocean University,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学化学与环境学院,广东湛江524088 [2]广东海洋大学食品科技学院,广东湛江524088

出　　处：《药学研究》2022年第2期79-82,共4页Journal of Pharmaceutical Research

基　　金：湛江市科技计划项目(No.2019A01020);广东海洋大学大学生创新创业项目(No.CXXL2021288)。

摘　　要：目的本论文采用复合酶解法制备酪蛋白抗凝血肽,然后对其进行了分离及抗凝血活性测试。方法本实验通过比较混合酶解和复合酶解酪蛋白的水解液活性,获得抗凝血活性最高的组合,为“菠萝蛋白酶+胰蛋白酶+中性蛋白酶+木瓜蛋白酶”复合酶解酪蛋白组。然后选择截留分子量为1 kD和3 kD的超滤膜对抗凝血活性最高的组合进行超滤,实验结果表明截留分子量为1 kD的超滤浓缩液抗凝血活性最高。结果超滤浓缩液进行抗凝血实验,实验结果表明,兔血︰1 kD浓缩液(V︰V)=6︰4和兔血︰1kD浓缩液(V︰V)=1︰1组合都是48 h后血液没有凝固;体外溶栓实验表明,置于浓缩液中的血栓条24 h后红色褪去,证明1 kD超滤浓缩液具有较好的溶栓和抗凝血作用。1 kD超滤浓缩液进行脱盐,然后采用凝胶渗透色谱法进行分子量分布测定,由数据分析可知分子量主要集中在500~180、1000~500、2000~1000范围。结论按照本实验方法制备的酪蛋白抗凝血肽具有较好的溶栓和抗凝血作用。Objective In this paper,casein anticoagulant peptide was prepared by complex enzymatic hydrolysis,and then its separation and anticoagulant activity were tested.Methods In this experiment,by comparing the hydrolysate activity data of mixed enzymolysis and composite enzymolysis of casein,the combination with the highest anticoagulant activity was obtained,which is the"bromelain+trypsin+neutral protease+papain"composite enzymolysis casein group.Then the ultrafiltration membranes with molecular weight cutoffs of 1 kD and 3 kD were selected for ultrafiltration.Results An anticoagulation experiment was carried out on the ultrafiltration concentrate,and the experimental results showed that the combination of rabbit blood︰1 kD concentrate(V∶V)=6∶4 and rabbit blood︰1 kD concentrate(V∶V)=1∶1 had no coagulation after 48 hours.In vitro thrombolysis experiments showed that the thrombus strips placed in the concentrate disappeared after 24 hours,proving that the 1 kD ultrafiltration concentrate has good thrombolytic and anticoagulant effects.The experimental results showed that the ultrafiltration concentrate with molecular weight cutoff of 1 kD had the highest anticoagulant activity.After desalting the ultrafiltration concentrate,the molecular weight distribution was determined by gel permeation chromatography.From the data analysis,the molecular weight is mainly concentrated in the range of 500~180,1000~500,2000~1000.Conclusion The casein anticoagulant peptide prepared according to this experimental method has good thrombolytic and anticoagulant effects.

关 键 词：酪蛋白 抗凝血肽 超滤 体外溶栓 分子量分布 

分 类 号：R543[医药卫生—心血管疾病]