Isolation,Purification,Structural Characteristics And Bioactivity Of Exopolysaccharides From Candida Ethanolica SN-2

Yeast exopolysaccharides(YEPS)with many physiological functions get more and more attention from the researchers for their superiorities such as continuous fermentation,being easy to separation,no affection of regional,season and so on.YEPS can be used in production and potential application value with very broad application prospect.The studies of our work indicate that laboratory northeast natural fermented soybean paste in the presence of large amounts of yeast,especially in the middle and later stage of fermented soybean paste.In this study,68 naturally fermented soybean paste samples are collected from the northeast China,139 suspected yeast strains are isolated.And a yeast strain SN-2 with high yield of exopolysaccharide is isolated from the sample.Then using gel permeation chromatography(GPC),ultraviolet wavelength scanning(UV),the analysis method of high performance liquid chromatography(HPLC)and methylation,KBr tabletting method,nuclear magnetic resonance(NMR)analysis,scanning electron microscopy(SEM)and thermogravimetric analysis(TGA)on the structure of pure component has carried on the preliminary analysis,at the same time,through determined by MTT method,inverted microscope observation and for the clearance of free radicals on the potential biological activities are analyzed,aimed at laying a foundation for the further research and application of the polysaccharides.The research main results are listed as follows:1.One hundred and thirty-nine strains of suspected yeast are isolated from northeast 68 traditionally fermented soybean paste.Then the viscosity and sugar content of the fermentation are measured,and a higher exopolysaccharide-producing yeast stain from SN-2 is obtained by phenol-sulfuric acid method.The strain is identified as Candida ethanolica by morphological observation,physiological and biochemical test and 26 S r DNA sequence analysis.In order to increase the yield of its YEPS,the medium and conditions of Candida ethanolica SN-2 are tested.Under the optimum conditions,yield of YEPS can reach to 796 mg/L,which is 24.6 % higher than the initial yield.After purified,two pure components are obtained,named as EPS 1 and EPS 2,and the yield are 42.6 % and 11.3 %.2.The chemical composition of the polysaccharide is analyzed by colomassie bright blue method,phenol-sulfuric acid method and barium chloride turbidimetric method.The results reveal the crude EPS contained 2.36 % of protein,while the purified fractions have no protein.The sulfated group content of crude EPS and EPS 1 are 0.25 % and 0.09 %,respectively.It has a negative response to the Bradford test and no absorption at 260 nm or 280 nm in the UV spectrum,indicating the absence of protein or nucleic acid in both of samples.The Mw are 223.8 k Da and 202.67 k Da,respectively.Monosaccharide composition analysis demonstrates that both EPS 1 and EPS 2 are composed of mannose,glucose,and galactose,but the composition content is different.The results of methylation analysis show that the skeleton of EPS 1 is composed of 1,4-connected Man and 1,4-connected Glc residues,and the skeleton of EPS 2 is composed of 1,4-connected Gal residues,1,4-connected Glc residues and 1,6-connected Glcresidues.The FT-IR spectra shows all the peaks detected are in agreement with the typical absorption peaks of polysaccharides.NMR analysis show that polysaccharide have two kinds of oligosaccharide bonds.SEM analysis indicates the surface of two polysaccharide samples have differences in size and shape.TGA analysis shows that both of them have good thermal stability,and the thermal stability of EPS 1 is better than that of EPS 2.3.The antioxidant activities of exopolysaccharides from EPS 1 and EPS 2 are investigated.EPS 1shows·OH(55.52 %),DPPH·(62.88 %)and ABTS· +scavenging activities(57.76 %),reducing power(0.236)at a concentration of 10 mg/m L.EPS 2(10 mg/m L)shows remarkable·OH(64.90 %),DPPH·scavenging activity(68.71 %),ABTS· +scavenging activity(63.96 %).Furthermore,the reducing power is0.337.So,we can see that the antioxidant activities of EPS 2 are better than these of EPS 1.MTT assay shows that EPS 1 and EPS 2 have inhibition effects on the proliferation of Hep G-2 cells,and the inhibition activities depend on polysaccharide concentrations and action time.The inhibitory activity of two purified polysaccharide components on Hep G-2 cells is: EPS 2 > EPS 1.Inverted microscope shows that EPS can cause cells shrinkage and decrease cells density.The results indicate that EPS 1 and EPS 2 from Candida ethanolica SN-2 after isolation and purification have the typical characteristics of pure polysaccharides and have potential application value in the food industry,cosmetics industry and the research and development of anti-tumor drugs.