The Fermentation Conditions Of Exopolysaccharide Producing From Rahnella Sp. Pjt09 And The Structural Analysis

Bacterial exopolysaccharides (EPS) have a wide microbal sources, both Gram-negative bacterial and Gram-positive bacterial can prouduce EPS. Searching for EPS high production bacterial and optimizing the cultivation conditions to improve the production yield is very important to the industrial production for EPS. The strain and culture conditions influence the amount and the composition of EPS. The production of EPS is sensitive to many factors such as temperature, pH, incubation periods, carbon sources and nitrogen sources. In this paper, the EPS high producing strain is screened and identified. The fermentation medium and conditions for EPS production from the special strain are carried out and the average molecular weight and structure of the EPS is analyzed by high performance gel permeation chromatography (HPGPC), 1D NMR and 2D NMR technique. The contents of this paper are as following:1. The exopolysaccharide producing strain PJT09 is screened from fruiting bodies of large fungi and identified as Rahnella sp. by physiological and biochemical experiment and 16SrDNA sequence analysis.2. The best medium components of the Rahnella sp. PJT09 are determined by a single-factor test and response surface methodology (g/L):sucrose 40, yeast extract 5, NaCl 1.5, ZnCl2 0.3, K2HPO4 1; Based on the single-factor test, the optimal fermentation conditions are:250mL flask containing 100mL medium, inoculum volume 3%(v/v), initial pH 6.0, rotate speed of fermentation 160r/min, temperature 28℃, time 70h. In such optimal nutrition and environmental conditions, the maximum concentrations of the EPS is 24.05g/L, and the conversion rate can reach up to 64%.3. The optimal extracting process for exopolysaccharide is determined as following:At first, the filtrate is concentrated to 1/2 volume, and then it is adjusted to pH 8.0 and precipitated by 2 volume of 95% ethanol, the precipitate is solved in deionised water and precipitated again, then centrifuged, at last, the crude exopolysaccharide is obtained. The pure polysaccharide (P09) is obtained by using Sephacryl S-300HR gel filtration chromatographic technique. The average molecular weight of P09 is determined to be 320 kDa by high performance gel permeation chromatography (HPGPC).4. The structure of P09 is anylyzed by NMR technique. The P09 isβ-(2, 6)-D-Fruf(levan-type)Rahnella sp. PJT09 has many advantages for industrial production:easy to culture, simple nutritional requirements, short fermentation period, high levan yield, and low production costs. The study of this paper is to provide a theoretic foundation for the study of structure-activity relationship and to further exploitation and application of levan.