高效阴离子交换色谱-脉冲安培法分析嗜热链球菌胞外多糖的单糖组成  

Determination of monosaccharide composition of Streptococcus thermophilus exopolysaccharides using high performance anion exchange chromatography with pulsed amperometric detector

作　　者：聂彩清 艾连中 熊智强 张汇 NIE Caiqing;AI Lianzhong;XIONG Zhiqiang;ZHANG Hui(Shanghai Engineering Research Center of Food Microbiology,School of Medical Instrument and Food Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China)

机构地区：[1]上海理工大学医疗器械与食品学院,上海食品微生物工程技术研究中心,上海200093

出　　处：《食品与发酵工业》2021年第15期255-261,共7页Food and Fermentation Industries

基　　金：国家杰出青年科学基金项目(32025029);国家自然科学基金面上项目(31771956)。

摘　　要：为建立稳定的以阴离子交换色谱分析嗜热链球菌胞外多糖单糖组成的方法,以淋洗液浓度为切入点,优化了梯度洗脱程序;同时,对比分析了不同方法除蛋白对多糖单糖组成分析的影响。优化后的色谱分析条件为:CarboPac PA20色谱柱(3 mm×150 mm),流速0.5 mL/min,0~20 min使用1.75 mmol/L NaOH溶液洗脱中性糖和氨基糖,20~30 min使用1.75 mmol/L NaOH溶液结合50~200 mmol/L NaOAc溶液洗脱糖醛酸,30~40 min使用200 mmol/L NaOH溶液冲洗色谱柱,40~60 min重新平衡系统。基于上述方法,12种混合单糖标准可得到理想分离,各单糖质量浓度标准曲线的相关系数>0.99,检出限(S/N=3)为2.5~21.1μg/L,平均回收率在77.3%~118.5%。此外,样品预处理分析表明,最佳的胞外多糖除蛋白方法为三氯乙酸法。利用上述优化条件对不同嗜热链球菌产胞外多糖的单糖组成进行分析,结果表明,胞外多糖均由半乳糖胺、鼠李糖、葡糖胺、半乳糖、葡萄糖、甘露糖、半乳糖糖醛酸和葡萄糖醛酸组成。研究建立的色谱分离和胞外多糖预处理方法可为乳酸菌胞外多糖的单糖组成分析提供参考。This work aimed to establish a stable method to determine monosaccharide composition of Streptococcus thermophilus exopolysaccharides by anion exchange chromatography.The gradient elution procedure was optimized with different concentration and the different deproteinization method was analyzed.The optimal chromatographic conditions were as follows:CarboPac PA20 column(3 mm×150 mm)was used with elute rate at 0.5 mL/min,the eluent was 1.75 mmol/L NaOH for the first 20 min,followed by a linear NaAc gradient(50-200 mmol/L)with fixed 1.75 mmol/L NaOH for the next 10 min to analyze the neutral with 1.75 mmol/L NaOH.Based on the optimal method,twelve mixed monosaccharide standards were separated.The correlation coefficients of each standard were greater than 0.99,the limits of detection were 2.5-21.1μg/L,and the recoveries ranged were 77.3%-118.5%.In addition,the optimal deproteinization method was trichloroacetic acid.The monosaccharide composition of exopolysaccharides from four S.thermophilus strains were analyzed with optimized method.The results revealed that the exopolysaccharides were mainly composed of galactosamine,rhamnose,glucosamine,galactose,glucose,mannose,galacturonic acid,and glucuronic acid.This study provides a reliable method for the monosaccharide composition analysis of exopolysaccharides from S.thermophilus.

关 键 词：嗜热链球菌 胞外多糖 脱蛋白 高效阴离子交换色谱 单糖组成 

分 类 号：O657.7[理学—分析化学] TQ920.1[理学—化学]