Screening Of High-yielding Strains,Separation,Purification And Identification Of Sulfated Polysaccharides

Sulfated polysaccharides,also known as polysaccharide sulfate,which are a type of sulfated derivatives formed by the substitution of sulfate radicals for partial hydroxyl groups of monosaccharide molecules in the sugar chain of neutral polysaccharides.It includes various natural sulfated polysaccharides extracted from animals and plants,sulfated modifications of natural neutral polysaccharides and various artificial and semi-synthetic sulfated polysaccharides.Sulfated polysaccharides have attracted much attention because of their anti-virus,anti-oxidantion,anti-tumor,anti-coagulation,immune-promoting functions and biological activities.In recent years,sulphated polysaccharides have been widely used in the fields of medicine,cosmetics and functional food.Morever,the market demand is getting bigger and bigger.At present,natural extraction methods and chemical synthesis methods are the main ways to prepare the sulfated polysaccharide.However,the former has cumbersome process,poor product uniformity and the risk of virus contamination;the latter has the disadvantages of low reaction rate and high degradation rate of polysaccharides.Therefore,in order to solve the shortcomings of the existing technology,this study aims to obtain a microbial strain with high yield of sulfated polysaccharide and a process to produce sulfated polysaccharide.The main research contents and conclusions are as follows:(1)Screening of high-yielding strains of sulfated polysaccharidesIn this study,we established and optimized the screening method for the high-yielding strains of sulfated polysaccharides:firstly,the samples were boiled at100? for 30 minutes.Secondly,spores were stained.Thirdly,strains were qualitatively screened by using the cetyl pyridinium chloride(CPC)on the test tube slant.Finally,strains were quantitatively re-screen by using dimethyl methylene blue(DMMB)in shake flasks.The CTAB-ethanol precipitation method is determined to be the method for extracting sulfated polysaccharides.A total of 128 samples from natto,natto strain powder,soil,rice straw and tap water were screened.A total of 1059strains were initially screened through the test tube slant.Among them,301 strains had a precipitation reaction with CPC.After fermented in shake flasks,the yield of sulfated polysaccharides was quantitatively determined by the DMMB method for re-screening.Bacillus subtilis GX10-35 with the highest yield and stable genetic performance(0.72±0.05 mg/mL)was used as the strain for subsequent study.(2)Optimization of fermentation process for strain GX10-35The optimal fermentation conditions for the strain GX10-35 were determined as follows:the initial pH of the fermentation medium was 7.5,the culture temperature was 37?,the liquid volume was 60 mL/250 mL,the rotation speed was 200 r/min and the inoculation amount was 10%.Through response surface test,the optimal fermentation medium for GX10-35 strain was determined to be sucrose 18.7 g/L,soy peptone 12 g/L,yeast powder 8 g/L,Mg SO₄0.468 g/L,K₂HPO₄0.4 g/L,KH₂PO₄0.4g/L.The yield of the high-yielding strain GX10-35 reached 0.803mg/mL after 48h shake-flask fermentation with the optimized medium,which was an increase of11.53%compared with the medium before optimization.And a 50 L fermenter scale-up experiment was carried out.(3)Separation and purification of sulfated polysaccharidesThe optimal conditions for the purification of sulfated polysaccharides produced by the fermentation of GX10-35 strain on IexCap DEAE 6FF anion exchange chromatography column were determined,that is,the elution method was linear-gradient elution,the buffer pH was 8,the Tris-HCl concentration was 10mmol/L and the sample load was 10mg.Under this separation condition,the GX10-35sulfated crude polysaccharide was purified and three obvious elution peaks appeared after identification.It was found that the GX10-35-P₁peak was sulfated polysaccharide with the highest recovery rate of 27.68%.The gel filtration chromatography of GX10-35-P₁showed a peak and the recovery rate was 66.72%.The freeze-dried GX10-35-P₁sample is a white powder with a total sugar content of68.70%,a sulfate content of 12.02%and a sulfate group content of 27.72%,indicating that GX10-35-P₁sample was a kind of sulfated polysaccharide.(4)Structure characterization and biological activity of sulfated polysaccharidesThe purified GX10-35-P₁samples were subjected to infrared spectrum,NMR spectra and monosaccharide composition analysis.The results showed that GX10-35-P₁mainly contained glucose and galactose,a small amount of arabinose,an unknown monosaccharide and an unknown uronic acid.GX10-35-P₁not only had good antioxidant activity,but also had significant immunomodulatory activity,including stimulating the proliferation of RAW264.7 macrophages,improving phagocytic activity,promoting NO production and increasing the expression of cytokines TNF-?,IL-1?,IL-6,IL-10 gene.In this study,a high-yield strain of Bacillus subtilis GX10-35 was screened.The structure,antioxidant activity and immunomodulatory activity of purified sulfated polysaccharides fermented by GX10-35 strain were analyzed.These results laid the foundation for the industrialization of sulfated polysaccharides produced by fermentation,and provided basic research data for the application of sulfated polysaccharides.