Primary Research Of An Exopolysaccharide Produced By Geobacillus TS3-9

The diversity in chemical composition of microbial polysaccharides results in a variety of properties that could not be found in plant polysaccharides and animal polysaccharides, simple chemical structure; short production cycle; not affected by seasonal, geography and pests diseases, all these factors provided an opportunity for the successful replacement of plant and animal polysaccharides with microbial polysaccharides in many industrial areas. Extreme thermophiles, as one special species of bacteria could also produce extracellular polysaccharides, and aroused wide interest in many food, medicine field because of its novel structure characteristics and chemical properties. Further study on extreme thermophiles extracellular polysaccharide will develop more considerable potential application value in the near future. This experiment describes the effect of medium components on exopolysaccharide production from radioactive-radon hot spring as Geobacillus TS3-9and Extraction, isolation, structure identification and bioactivity of the polymer. The major results obtained in this study were as follows:Response surface method (RSM) was applied to optimize extractions of polysaccharides from Geobacillus TS3-9, including carbon source, nitrogen source and inorganic salt. Through the single factor analysis method was first adopt to obtain the obviously factors on carbon source, nitrogen source and inorganic salt components, and based on this, the Plackett-Burman design method and the center combination experiment were used to optimize the culture medium composition, final optimization conditions was achieved as follows:lactose (2.1%), tryptone (1.3%), yeast extract (1.1%). Extracellular polysaccharide production up to91.6mg/L. therefore, Response surface method (RSM) was good method for greatly improve the polysaccharide production.Ethanol precipitation, Sevage deproteinization method, dialysis method, column chromatography method were used to optimize the purification process. The experimental results showed that in the process of extracting, the adding amount of ethanol and fermentation medium was in a ratio of3:1; three times was the best for deproteinization using Sevage method, the lowest protein content is16.3%, and the sugar content was41.0%eventually; Dialysis can effectively remove small molecular impurity in the polysaccharide; The obtained polysaccharides were further applied to a DEAE Sepharose Fast Flow column, eluted with a gradient of NaCl and got an acidic polysaccharides AEPS, then a Sephadex G-200chromatography column was used to purify the AEPS, and it was pure with the molecular weight3.2X106Da determined by high performance liquid gel permeation chromatography.Chemical method analysis showed the pure polysaccharide contain the uronic acid content18.6%and acetyl content6.1%, Fourier infrared transformation analysis preliminarily determines the AEPS has the functional groups as polysaccharide substances, and also reveal the pyranose ring, acetyl amino (-NHCOCH3) substituent; Gas chromatograph analysis showed that the AEPS contains three kinds of monosaccharide heteropoly sugars, composition of mannose, glucose and rhamnose, and the molar ratio of16.7:2.4:1.0; Combined with1H NMR and13C NMR spectrum determined the a configuration with glucose type and pyranose residue and the existence of acetyl amino substituent.Determination of the Scavenging activity in vitro showed that AEPS was able to eliminate free radicals, especially the hydroxyl radicals and the superoxide radicals. Besides, the AEPS also showed a certain inhibitory activity of hepatoma carcinoma cell (HepG2).