Genome Shuffling Improved The Ability Of Nitrite Degradation In Lactic Aicd Bacteria And Their Characteristics

Due to the large number of use with nitrogen fertilizer in argriculture, questions caused by nitrite become more often than before. Nitrite could result in digestive system cancer, reduce hemoglobin oxygen abilities, and cause the body’s oxygen poisoning.Biological method is more easer and harmless to degrade nitrite compare to other methods. It was found that Lactic acid bacteria could reduce the nitrite in vegetables during the fermentation. In this thesis it was aimed at improving the nitrite degradation ability of lactic aicd bacteria by Genome shuffling.Then the proteins of different strains was extracted and prepared for 2D electrophoresis.2DE gels of the wild strains mutant strains and shuffled strains were analysis by PDQuest software and the proteins of differential expression were identified by MALDI-TOF-MS. Datum were were blasted in NCBI protein database to identify the proteins and the nitrite degradation pathay was predicted preliminarily. The following is main conclusion:(1) As a mutagenic source,60Coγirradiation rays was used on Lactobacillus plantarum CICC20765. According to the strains of fatality rate and the relationship between mutation rate and radiation dose, the optimal radiation was determined in 800Gy. Two mutant strains (named mut-1 and mut-2) which have a better nitrite degradation ability than original strain and genetic stability were obtained from those mutants by 60Coyirradiation rays in 800Gy repeatly.These strains whose nitrite degradation were 53% and 50% separately were used as the start strains for Genome shuffling.(2) It was showed that protoplastion and regeneration rate were 94.5% and 31% separately after lactobacillus plantarum had been mixed by adding mutanolysin (15 ug/mL) and lysozyme (30 mg/mL) for 1h.(3) After protoplast prepared by the method were inactivated by heating in 60 ℃ for 60min and inactivated by ultraviolet irradiation for 15s, the fatality rate achieved 100%. When PEG6000 was added to 40% content and treated for 10 min at room temperature, the inactivated protoplast fusion rate was maximum.(4) Mut-1 and mut-2 were used as the start strain for genome shuffling, five mustants were obtained from two rounds fusion. Mutant of F2-12 could reduce 86% nitrite in MRS culture medium (contain 1.25g/L) after 24 hours.(5) The nitrite degradation rate of F2-12 was higher than times of the original strain and start strains, the glucose consumption rate and lactic acid production has increased too. In the process Kimchi fermentation inoculated with original strain, srart strain and shuffled mutant, nitrite degradation was faster than other groups.(6) The method of protein extraction and the pH range of PG strips was determined.2-D electrophoresis Maps of the original strain, start strain and shuffled mutant were analysis by PDUest,19 different expression protein spots were analysised by using MALDI-TOF-MS analysis, and 14 spots were identified.(7) Compared with the original strain and start strain, the expression level of shuffled mutant incressed in NIRS and ATP synthase. Since more ATPs were synthesized and a larger number of NAD+s were generated, the shufflied mutant had a better ability of nitrite absorption and reduction. In addition since the glycolytic rate of the shuffled mutant increased, the formation rate of lactic acid was increased and the shuffled mutant had a better ability of nitrite by aicd degradation.