Isolation Entophytes and Study on the Fermentation of Bacillus Subtilis tiD7 from Rehmannia
|School||Henan Agricultural University|
|Keywords||Rehmannia glutinosa Bacillus subtilis entophytic bacteria HPLC MS catalpol polysaccharide|
30 strains of entophytic bacteria and fungi were isolated from roots, stems and leaves of Rehmannia by strict disinfection of the surface. Observing the Morphological characteristics (texture, color, moisture degree, transparency, viscosity, edge character) of the entophytic bacteria, and using Gram’s staining and Physiological and biochemical tests to identify and classify the bacteria. Analysis the distribution characteristics of the entophytic bacteria which is separated form different organization (the organization is from different growth period) of Rehmannia. It showed that with the growth of Rehmannia, the entophytic bacteria in roots is more than stems and leaves, in maturity period is more than seedling stage, and more of the bacteria is Gram-negative bacteria, and more can produce spores.A novel strain tjD7 which produces Catalpol,one of main substances of Rehmannia, was isolated. The strain was identified as Bacillus subtilis by Gram’s staining,biochemical characteristics and 16S RNA sequence analysis. Liquid fermentation product by Bacillus subtilis tjD7 was extracted by ethyl acetate and the crude extract spectrum was obtained, and then separated by column chromatography. Catalpol was sured by TLC,HPLC and MS specially.A fermentation medium for catalpol by Bacillus subtilis tjD7 had been optimized. In shake flask, catalpol yields 37mg/mL under 2% of the inoculation quantity bacteria in 32℃, pH7, 220rpm and casing fermentation 7days.Products screen showed that Bacillus subtilis tjD7 could produce somekainds of polysaccharide. To use digitalis raw materials from the extracted polysaccharide rehmannia glutinosa method, cannot satisfy the market demand. Polysaccharide from Rehmannia glutinosa endogenous bacteria by fermentation may be an effective way to solve this problem. This experiment is designed to preliminary exploration of whether there is origin yellow polysaccharide produced bacteria, through polysaccharide method for the cultivation of microorganism fermentation digitalis polysaccharide, to overcome the defects of polysaccharide plants. Through the paper chromatography and the TLC method digitalis polysaccharides and qualitative analysis with digitalis polysaccharide, endogenous bacteria for so digitalis polysaccharide qualitative analysis of endogenous bacteria polysaccharide, whether endogenous bacteria testing producing yellow polysaccharide. Results: for digitalis polysaccharide, hydrolysis heteropoly sugar can be mainly detected glucose, d-galactose, rat lee sugar etc. Endogenous bacteria polysaccharide can mainly digitalis detected glucose, half of lactose, etc. TLC etc qualitative detection method is simple, feasible, and but this experiment still need to take other more effective method to do further research.