鱼鳔类肝素的分离纯化与结构鉴定  

Isolation, Purification and Structural Identification of Heparinoids from Fish Swim Bladder

作　　者：周斯仪 钟赛意 苏伟明[1,3] 杜振兴 陈建平 洪鹏志 章超桦 ZHOU Siyi;ZHONG Saiyi;SU Weiming;DU Zhenxing;CHEN Jianping;HONG Pengzhi;ZHANG Chaohua(Shenzhen Research Institute,Guangdong Ocean University,Shenzhen 518108,China;College of Food Science and Technology,Guangdong Ocean University,Zhanjiang 524088,China;Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety,Zhanjiang 524088,China;Guangdong Province Engineering Laboratory for Marine Biological Products,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学深圳研究院,广东深圳518108 [2]广东海洋大学食品科技学院,广东湛江524088 [3]广东省水产品加工与安全重点实验室,广东湛江524088 [4]广东省海洋生物制品工程实验室,广东湛江524088

出　　处：《食品科学》2019年第15期84-91,共8页Food Science

基　　金：深圳市科技计划项目(JCYJ20170818111335796);广东省渔港建设和渔业产业发展专项资金项目(A201708C13);湛江市海洋经济创新发展示范市建设项目(湛海创2017C8B1);广东普通高等学校水产品高值化加工与利用创新团队项目(GDOU2016030503)

摘　　要：本研究以鱼鳔为原料,采用酶法提取类肝素化合物,采用阴离子交换树脂吸附、氯化钠溶液梯度洗脱和醇沉法进行分离纯化;并进一步分析其结构特征,分别通过紫外光谱法、高效凝胶色谱分析其纯度和分子质量,醋酸纤维素薄膜电泳初步鉴定其种类,柱前衍生-高效液相色谱法分析其单糖组成,傅里叶变换红外光谱分析其官能团结构,酶裂解-质谱/质谱法测定其二糖组成,核磁共振鉴定其一级结构。结果显示:鱼鳔类肝素的得率为(2.21±0.03) mg/g,纯度较高,类肝素含量为(85.79±0.63)%,分子质量约为84 000 u;单糖组成为葡萄糖醛酸(glucuronic acid,GlcA)和N-乙酰基半乳糖胺(N-acetylgalactosamine,GalNAc),同时含有少量的艾杜糖醛酸、半乳糖;电泳位移与硫酸软骨素相似,且具有羧基、乙酰氨基、硫酸基轴向伸缩等特征吸收峰。最终确定鱼鳔类肝素主要组成是由重复的二糖单位[→4GlcUAβ1→3GalNAc(4S)β1→]构成的硫酸软骨素A。In the present study, heparin-like compounds were isolated from fish swim bladder by enzymatic hydrolysis,and they were purified and fractionated into four fractions(F1–F4) by anion exchange resin adsorption followed by sodium chloride gradient elution and alcohol precipitation. Each fraction was identified by cellulose acetate electrophoresis.Furthermore, the structural characteristics of F4 were determined, and the homogeneity and molecular mass were analyzed by UV spectroscopy and high performance gel chromatography. Its monosaccharide composition was analyzed by high performance liquid chromatography(HPLC) after pre-column derivatization, functional group structure by Fourier transform infrared(FTIR) spectroscopy, disaccharide composition by enzymatic cleavage and tandem mass spectrometry(MS/MS),and primary structure by nuclear magnetic resonance. The results showed that the yield of F4, with a purity of up to(85.79 ± 0.63)% was(2.21 ± 0.03) mg/g of dried fish swim bladder. Its molecular mass was about 84 000 u. Monosaccharide analysis revealed that F4 was composed of glucuronic acid(GlcA) and N-acetylgalactosamine(GalNAc) with a small amount of aldoluronic acid and galactose. Its electrophoretic shift was similar to that of chondroitin sulfate and it had the characteristic absorption peaks of carboxyl, acetyl amino and sulfate groups(axial stretching). Finally, F4 was identified as chondroitin sulfate-A composed of repeated disaccharide units of [→4 GlcUA β1→3 GalNAc(4 S)β1→].

关 键 词：鱼鳔 类肝素 分离纯化 结构鉴定 

分 类 号：TS254.1[轻工技术与工程—水产品加工及贮藏工程]