Molecular Identification Of Lactobacillus Plantarum And The Deletion Of Gene Releated To Degradation Of Nitrite

The probiotics Lactobacillus planatrum have established close relationship with human beings, because they could improve the immunity, secrete bacteriocin to inhibit the growth of pathogen and spoilage organisms in fermented food and reduce the concentration of cholesterol in the blood serum of human beings. Recently, lactic bacteria Lactobacillus sp. DMDL9010 was isolated from fermented vegetables, which could effectively degrade the nitrite. And this strain had great potential to be applied into the industry. Based on the traditional method of bacteria identification, this research developed new molecular biology method of identification. Meanwhile, recombinant plasmids were constructed from p G+host9, and explored the method of gene knockout in Lactobacillus planatrum DMDL9010 with the recombinant plasmids.The method of 16 S r DNA and physiology and biochemistry was applied in identification of Lactobacillus sp. DMDL9010, but this method could not differentiate Lactobacillus plantarum and Lactobacillus pentosus. This study analyzed the up and down DNA sequences of L-lactate dehydrogenase1(L-ldh1) which was the conserved sequence in Lactobacillus plantarum and Lactobacillus pentosus. We successfully identified Lactobacillus sp. DMDL9010 as Lactobacillus plantarum.To apply p G+host9 into gene modification of L.plantarum DMDL9010, the reaction condition of SOE PCR was optimized to ligate the up and down sequences of L-ldh1. Meanwhile, the host strain, Escherichia coli TG1 and Escherichia coli MC1061 for the construction of recombinant plasmids with erythromycin resistant gene. The up-down frgment with cohesive end was cloned into p G+host9. After identification of colony PCR and double digestion, we successfully constructed double-crossover recombinant p GH-up-down. Then the single-crossover recombinant p GH-nir was constructed with the same method.The method of gene knockout and the mechanism of degrading nitrite by L.plantarum was investigated. And the method of preparing electro-transformation competent cells of Lactobacillus plantrum DMDL9010 was modified and optimized, and recombiant plasmid was effectively transformed into Lactobacillus plantrum DMDL9010, yielding 533 transformants per μg of plasmid DNA. Meanwhile, the method of colony PCR for lactic acid bacteria with thick cell wall was developed for identification of recombinant bacteria. The thermosensitivity of p G+host9 in Lactobacillus plantarum DMDL9010 was investigated and improved. In order to improve the thermosensitivity of p G+host9, the cells were maintained in exponential growth for 48 h by serial dilutions in MRS pre-heated at 42°C. The rate of strains with the plasmid reached 0.0076%. Due to the short size of DNA insert, the frequency of integration per cell was very low. The research found that the way of degrading nitrite by the strain was by enzyme and acid degradation, but how they cooperate to complete degradation need further research.