Purification And Structural Characterization Of The Extracellular Polysaccharide Produced By Streptococcus Thermophilus

In this study,the extracellular polysaccharides(EPS)produced by a streptococcus thermophilus was purified and the structure was characterized.It is of great significance in theory and practice to improve the functionality and expand application area of EPS.A streptococcus thermophilus strain from Tibetan kefir Grain was cultured in IRIE liquid medium for fermentation to produce extracellular polysaccharides,and then the crude EPS were obtained after deproteinization,alcohol precipitation,dialysis,rotary evaporation and freeze-drying in order.The further separation and purification were completed by using DEAE cellulose 52 column and DEAE Sepharose CL-6B column,and the products were proved to be pure by HPLC.EPS are powders and soluble in water,insoluble in methanol,ethanol,acetone,ethyl acetate,butanol and other organic solvents.The pH of the extracellular polysaccharides aqueous solution(10mg/ml)is 6.5.The differential scanning calorimetric analysis(DSC)results showed that the EPS had two melting points at 120.83 ℃and 212.89 ℃.The thermogravimetric data revealed that the first decomposition temperature was 264.88 ℃ and the second one was 441.82 ℃.The determination of gel permeation chromatography showed that the average molecular weight(Mw)and the number average molecular weight(Mn)were 30092Da and 10121 Da.The infrared spectrum analysis indicated that the EPS had a long molecular chain and a configuration characteristic of P-glycosidic bond.Gas chromatography showed that the composition of its monosaccharides is mannose,glucose and trace galactose.The molar ratio of mannose and glucose is 2.5:1.Through infrared spectroscopy and nuclear magnetic resonance spectroscopy analysis,EPS showed a polysaccharide characteristics,moreover,showed that the component of mannose and glucose were mainly β-D-pyranose rings.GC-MS and NMR analysis showed that the mannose is connected mainly in 2,3 and 3,4 positions and the glucose mainly in 2,3,6positions;and the polysaccharide is composed of(2→3)-β-D-Manp,(3→4)-β-D-Manp,(2,3→6)-α-D-Glcp and the trace amounts of galactose with a molar ratio of three sugar residues is 1.26:1.5:1.