Screening Of Exopolysaccharide-producing Lactic Acid Bacteria And Structral Characterization Of Exopolysaccharide

Lactic acid bacteria(LAB)exopolysaccharides(EPS)are extracellular polysaccharides which are produced and secreted into the extracellular environment in the form of capsules or slime during the growth and metabolic process of LAB.The EPS produced by LAB not only has good rheological properties,but also has important physiological activities,such as antioxidant,antitumor,immunomodulatory,prebiotic and so on.Our country has abundant foods fermented by LAB,but the LAB resource including the EPS-producing strains has not been effectively exploited.The objectives of this work were to isolate and identify EPS-producing LAB strains,to isolate and purify the EPS and study the structure and physico-chemical characterization of the EPS.Two EPS-producing strains named NM105 and 2-19 were screened from homemade manchurian sauerkraut by combining with the appearance of viscous colony formed on the screening plate and the polysaccharide production.They were all identified as Leuconostoc according to morphological,physiological,biochemical and 16 S rDNA tests,and were named as L.citreum NM105 and L.mesenteroides subsp.dextranicum 2-19,respectively.L.citreum NM105 and L.mesenteroides subsp.dextranicum 2-19 were cultured in MRS medium with 5% sucrose at 25? for 48 h,respectively,to get the fermented liquid containing EPS.The crude EPS were prepared by centrifuging,removing proteins,followed by ethanol precipitation and dialysis,respectively.The crude EPS were further purified by gel filtration chromatography.The elution profile presented a single peak,which indicated the correspond fraction was homogenous.The collected fraction was then lyophilized and weighted.The yield of the purified EPS achieved to23.5 g/L and 11.4 g/L,respectively.The analysis of Ultraviolet and high perfomance size-exclusion chromatography showed that the purified EPS contained no protein and no nucleic acid,and the weight-average molecular weight of EPS was 1.01×108 Da and 8.79×107 Da,respectively.The EPS produced by L.citreum NM105 and L.mesenteroides subsp.dextranicum 2-19 all consisted exclusively of glucose according to Gas chromatography(GC)and Thin-layer chromatography(TLC).The structural characterization of the purified EPS determined by FT-IR,1H,13 C and two-dimensional NMR spectroscopy demonstrated that the EPS produced by L.citreum NM105 was a highly ?-(1?2)-branched dextran,which contained?-(1?6)-linked D-glucopyranose unit,2,6-linked D-glucopyranse unit and terminal?-D-glucopyranse unit at a ratio of 1:1:1.And the EPS produced by L.mesenteroides subsp.dextranicum 2-19 was a highly linear dextran with ?-(1?6)glycosidic bonds.The physical and chemical properties analysis of the EPS produced by L.citreum NM105 revealed that a high water solublity of 72.0 g/L and excellent water retention of 23.5% were showed from the EPS.A sheet-like smooth gliteering and highly branched microstructure of the EPS was observed by scanning electron microscope(SEM).In summary,we obtained two high EPS-producing LAB strains,purified and structurally analyzed the EPS with good application prospect.This will help to provide important gist for the development of functional foods and other industrial applications.