动物双歧杆菌是一种高产胞外多糖(exopolysaccharide,EPS)的益生菌,在食品工业中应用广泛。该研究以动物双歧杆菌亚种M8为研究对象,发酵提取EPS,分别选用乙醇分级沉淀(ethanol gradient precipitation,EGP)法和阴离子交换色谱(anion-exchange chromatography,AEC)法对其进行分离纯化,并通过化学成分和高效分子排阻色谱分析评价纯化效果。结果表明,EGP法分离得到的多糖级分,在糖纯度和分子质量分布均一性方面均未达到纯化要求;而AEC法可分离纯化得到高纯度的酸性杂多糖F0.2、蛋白聚糖F0.5,其工艺为:柱填料DEAE-Sepharose Fast Flow,依次采用纯水、0.1、0.2、0.5 mol/L NaCl溶液洗脱,制备得到纯多糖级分F0.2和F0.5,样品总回收率达80.85%。分子结构特征分析表明,F0.2是由葡萄糖、半乳糖、甘露糖和葡萄糖醛酸组成的一种酸性杂多糖,重均分子质量(Mw)为21.4 kDa,在水溶液中呈刚性较强的松散线团结构;而F0.5是缀合有蛋白组分的蛋白聚糖,Mw为28.3 kDa,在水溶液中呈紧凑的柔性卷曲状结构。该研究确定的分离纯化方法具有高效、回收率高等特点,为动物双歧杆菌乳亚种M8产EPS的结构与功能研究及其在食品工业中的应用奠定科学依据。
Bifidobacterium animalis is a kind of probiotics with high exopolysaccharide (EPS) production, and has been widely used in food industry. In this study, two methods, ethanol gradient precipitation (EGP) and anion exchange chromatography (AEC), were compared to fractionate the crude EPSs extracted from the fermentation broth of Bifidobacterium animalis subsp. lactis M8. The chemical components and high performance size exclusion chromatography (HPSEC) analysis were carried out to evaluate the effects of fractionation. Results showed that the saccharide purity and molecular homogeneity of fractions obtained by EGP method did not meet the requirement for purification. However, the AEC method by DEAE-Sepharose Fast Flow resin with gradient eluents of pure water, 0.1, 0.2 and 0.5 mol/L NaCl solutions was successful to separate and obtain two purified fractions of F0.2 and F0.5 with high recovery of 80.85%. The molecular characteristic analysis indicated that F0.2 was an acidic heteropolysaccharide composed of glucose, galactose, mannose and glucuronic acid, while F0.5 was a proteoglycan conjugated with protein moiety. The weight-averaged molecular weights (Mw) of F0.2 and F0.5 were determined as 21.4 and 28.3 kDa, respectively. The conformational analysis revealed that F0.2 presented as relative rigid and loose coil, while F0.5 was a compact and flexible random coil chain in aqueous solution. This study established a fractionation protocol for the EPS from M8 with high efficiency and recovery, and settled a foundation for its further study on structure and function, as well as its application in food industry.
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