Screening Of Strains For Producing Sulfated Polysaccharide And Purification And Identification Of Polysaccharide

Sulfated polysaccharide is formed by replacing the hydroxyl group on the polysaccharide molecule with sulfate.It has a variety of biological activities such as antioxidant,antitumor,antiviral,immunomodulatory and so on,and has a wide range of applications in the fields of biomedicine,food and cosmetics.At present,sulfated polysaccharides are mainly extracted from animals and plants or obtained by chemical modification,and there are problems such as complex production process,unstable product quality and environmental pollution.It is necessary to find new sources of sulfated polysaccharides.The production of sulfated polysaccharides by microbial method has short cycle and low cost,but the microbes producing sulfated polysaccharides found so far are few and the yield is low.In this paper,the isolated sulfated polysaccharide-producing bacteria were screened,the sulfated polysaccharides produced by the strains with high production were isolated and purified,structural analysis and antioxidant studies were studied,and the media composition was optimized to increase the yield of polysaccharides.The main contents are as follows:(1)Screening of strains producing sulfated polysaccharide.The sulfated polysaccharide production of 56 bacillus strains in two media were compared,and it was found that the sulfated polysaccharide production of 56 strains were consistent in two media.After protease treatment and dialysis treatment,the protein content of the fermentation broth was reduced,but the sulfated polysaccharide content was unaffected.The high-production strain BZ-S-TR-10-2 of sulfated polysaccharide was chosen.(2)The sulfated polysaccharides produced by strain BZ-S-TR-10-2 were isolated and purified.Firstly,the protease treatment conditions of the fermentation supernatant were optimized,and 62% of the protein could be removed by enzymatic treatment with 1 U/m L alkaline protease for 1h.The enzymatic treatment sample was separated by D201 macroporous resin ion exchange chromatography,and three elution peaks were obtained by gradient elution of sodium chloride solution,corresponding to 20,30,40 g/L sodium chloride concentrations.The sample obtained at 30 g/L sodium chloride elution has the maximum sulfated polysaccharide content and was purified by gel filtration chromatography.The pured sulfated polysaccharide has 31.98% recovery rate.(3)The isolated and purified polysaccharides were identified.UV spectroscopy showed the sample has high purity without proteins and nucleic acids.The infrared spectrum showed that the sample is a polysaccharide containing a sulfate group and has a characteristic signal of polysaccharide and sulfate.The NMR indicated that the sample is a α-configuration pyranoses.The analysis of monosaccharide composition showed that the glucose is the main monosaccharide,and galactose,mannuronic acid,glucosamine,N-acetyl-D glucosamine and a small amount of arabinose are also contained,the molar ratio was7.37:0.84:0.84:0.37:0.39:0.16.The weight average molecular weight(Mw)of the sample was74.375 k Da,and the average molecular weight(Mn)was 72.195 k Da.(4)The fermentation medium of strain BZ-S-TR-10-2 for producing sulfated polysaccharides was optimized.The optimized medium composed of 15 g/L sucrose,8 g/L yeast powder,3 g/L diammonium hydrogen phosphate,1 g/L magnesium sulfate,2 g/L dipotassium hydrogen phosphate,2 g/L potassium dihydrogen phosphate.When the liquid load was 50 m L/250 m L flask,the yield of sulfated polysaccharides was 1.69 g/L,which was4.2 times higher than before optimization.(5)The antioxidant properties of sulfated polysaccharides were preliminarily studied.The sulfated polysaccharide has antioxidant activity,and the radical scavenging activity of DPPH was 18.48%,the scavenging activity of hydroxyl radical was 97.62%,and the scavenging activity of ABTS radical was 38.92% at a concentration of 10 mg/m L.