Research On Mutagenesis Breeding And Optimization Of Lactobacillus Lactis

Lactic acid bacterium exopolysaccharides is produced with Lactic acid bacteria growing and penetrates into culture medium. Exopolysaecharides by lactic acid bacteria plays an important role in the theological properties of dairy products, as stabilizers and thickeners. Important features of the starter are rapid acidification, microbial preservation of the milk, formation of specific flavours, texturing capacities and health benefits. In recent years, There has been a upsurge in interest in immunostimulating activity, enhance lymphocyte mitogenicity and macrophage cytostaticity. The following are main results:1. Two strains were obtained by sceening, isolate and identify Lactic acid bacterium from yoghurt from market. Based on results of the characteristics of morphology, physiology, and biochemistry, sugar fermentation, they are identified by API bacterial identifiation system as Lactobacillus lactis and Lactobacillus bulgaricus.2. After the characteristic study of strain L. lactis, we studied physical, chemic, biologic mutantation to enhance the yield of exopolysaccarides. Two mutant strains were obtained by ultraviolet irradiation, the high yield of which was 2.85g/L,3.15 g/L. Four mutant strains were obtained by NTG mutagenesis, the high yield of which was 2.413g/L,2.841 g/L,2.743g/L,2.694g/L. Three mutant strains were obtained by ultraviolet irradiation and NTG mutagenesis, the high yield of which was 5.219g/L, 4.926g/L,4.805g/L. The mutant strains with high yield, which were obtained by ultraviolet irradiation and NTG mutagenesis, as a parent library in recursive fusion. Through two rounds of genome shuffling, a mutant strains with highest yield were obtained, the yield of which was 9.38g/L,3. After the characteristic study of strain L. lactis in the fermentation, the results showed that Elliker was the optimal medium. Three statistically significant ingredients, yeast extract, the initial pH and fermentation time, which were determined by Plackett-Burman design. Based on the results of the first phase, the five-level three-factor central composite design was employed to optimize the above critical factors. It showes that the maxmium yield of exopolysaccharid was 3.910g/L, with yeast extract 17.35g/L, the initial pH5.85, fermentation time 23.18h.4. After the characteristic study of strain L. lactis in deffierent yeast extract concentration medium, the results indicated high yeast extract The high yield mutant strains with improved yeast extract tolerance, which were obtained by ultraviolet irradiation and NTG mutagenesis, as a parent library in recursive fusion. The fusants were screened under YE plates. Through two rounds of genome shuffling, the mutants obtained the compatibility of two optimal phenotypes of yeast extract tolerance and EPS enhancement, the highest yield was 10.021g/L. The results indicated application of the genome shuffling in improving the yeast extract tolerance of L. lactis and enhance EPS production.