Screening, Mutagenesis Of Nitrite Degaration Lactobacillius And Optimization For Nitrite Complex Degradation

Nitrite has much serious damage to human body. At present, certain health risks exist inaquatic product due to nitrite component, and lactic acid bacteria is the most ideal microbe todegrade nitrite. Lactobacillus not only produces nitrite reductase to degrade nitrite but alsoimproves the flavor of pickles. Therefore, the research is devoted to screen lactic acid bacteriawith effective ability to degrade nitrite from pickled food, establish and optimize the method ofcompound lactobacillus to degrade nitrite and apply to practical production. The contents andresults of this study are as follows:1、To determine the optimal time of screening lactobacillusSauerkraut was used for bacteria source, screening from initial, middle and finalfermentation stage respectively. According to the degradation rate of sodium nitrite to determinethe best screening period. Experiment shows that the middle fermentation stage, that was,"nitrite peak" period was the best screening stage. The highest degradation rate of the strainswas80%.2、Identify of the screened strainsThe lactic acid bacteria B1、C1、D2screened from the preliminary work were identified.The bacteria were sequenced after PCR amplification using16S rDNA gene method, and theresults were compared with NCBI standard sequence. Using the MEGA (version5.0),Application of the Neighbour-Joining method to construct phylogenetic tree. Eventually confirmthat the B1was lactobacillus pentosus, C1、D2were lactobacillus plantarum.3、Ultraviolet-nitrosoguanidine composite mutagenesis strainsAfter screening, the screened strains were mutated by being exposed to ultraviolet (15W,last for60s) and nitrosoguanidine (0.5mg/mL, last for60mins), then we get efficient nitritedegration lactic acid bacteria B1, C1, D2, these bacteria would be cultivated in MRS with0.02%(mg/ml) NaNO2under37℃for24h. Finally the nitrite degradation rate was81.5%、88.1%、91.4%, which increased by5.8%、8%、12.7%respectively compared with original strains.4、Extract nitrite reductase from degradation bacteria and enzymatic property study By means of ultrasonic crushing(5mg/mL) and being dissolved in lysozyme(200w, work5s,pause10s，cycle99), nitrite reductase was extracted from lactic acid bacteriaD2. According tothe enzymatic activity of nirtrite reductase in different growth periods of the bacteria todetermine the production way. The result shows that the production way was continuationsynthetic. The optimum pH was5.5, the optimum temperature was35℃. Enzymatic Km valuewas20.7mmol/L, Vmax was30.7μg/min. Using acid-base precipitation method measured theoelectric point of enzyme was about4.0. Through Sephadex G-75gel chromatography andSDS-PAGE gel electrophores, the tested molecular weight was about35.8kDa, belongs to thecopper nitrite reductase.5、Condition optimization for nitrite degradation using mixed bacteriaOrthogonal method was used to optimize the degradation condition. The research showsthe optimum reaction conditions were: bacteria ratio B1: C1: D2=1:2:1, the temperature was35℃and the adding proportion was5%(v/v).The nitrite degradation rate was94.2%.