Study On Mutation Breeding And High Cell Density Culture Of High EPS-producing Streptococcus Thermophilus

As a long history of industrial strain, Lactic acid bacteria can produce extracellular polysaccharides (exopolysaccharide, EPS), which have important applications in vegetable products, dairy products, meat products. At present there is low EPS production, poor stability, high degradation rate in the production process and other issues, limiting its application in fermentation industry. Some microbial EPS, having high yield, good stability and excellent performance are obtained by mutation breeding research. It is of great significance in theory and practice to improve the market competing capacity and expand application area of EPS.In this study, a EPS-producing streptococcus thermophilus strain was isolated from Tibetan Saussurea, and was mutated individually by single physical and chemical experiments. The results showed that the optimal dose of UV mutation was120s, the optimal dose of nitrous acid mutation was15min, then the original strain was mutated by UV and HNO2environment. And thin layer chromatography and phenol sulfuric acid method were adopted for screening mutant strains. EPS production of the mutant strains by physical, chemical or combined method was increased respectively by29.7%,24.8%and33.8%compared with original strain, and the results showed that the effect of compound mutation was better than that of single mutation of the physical or chemical, and its high EPS-producing ability remained stable.Their growth and fermentation characteristics showed that mutant strains were superior to original strain in growth characteristics and fermentation characteristics (viscosity, titration acidity and pH).MRS, modified IRIE and Elliker were selected as medium for the growth of lactic acid bacteria, and the optimum medium for EPS production was modified IRIE medium. Then the fermentation conditions were optimized by single factor and orthogonal experiments. And the results indicated the optimum EPS production conditions were temperature46℃, inoculation volume4%, initial medium pH6.4and incubation time12h, and under the above conditions, EPS production was452mg/L. High-density cultivation experimental research indicated that the suitable initial concentration of lactose is20g/L for the mutant strain, the substrates limiting cells continous growth in the logarithm growth late stage were carbon source, nitrogen source and inorganic salt, and their optimal volume proportion was5:10:10.The mutant strain was cultured through the chemical neutralization and fed-batch cultivation method, and the research showed that once fed-batch was better than no fed-batch, the viable number of mutant strain was increased by15.9%and its EPS production was increased by2.4%. But second fed-bacth fermentation was better than once fed-batch, the viable number of mutant strain was increased by8.8%and its EPS production was increased by12.8%. The results showed that chemical neutralization combined with fed-batch cultivation method can effectively improve the viable number of mutant strain and its EPS yield.