The Pathway Of Nitrite Degradation By Lactobacillus Casei Subsp. Rhamnosus LCR6013and Its’ Nitrite Reductase Characterization

Nitrites are potentially strong carcinogens and can cause methemoglobin under excessiveintake of nitrites. It is very important to control the concentration of nitrite in food. Our groupscreened Lactobacillus casei subsp. rhamnosus6013(LCR6013) which could degrade nitrite.The effects and law of the nitrite degradation by LCR6013were determined in Chinesecabbage fermentation system and MRS system. Moreover, the products of nitrite reduced byLCR6013were detected by ECD-GC method to determine the degradation pathway. Then theinitial localization and several properties of nitrite reductase (NIRs) from LCR6013wereinvestigated. Main results are as follows:Compared to spontaneous fermentation group, nitrites of cabbage fermentation groupinoculated with LCR6013were degraded effectively. Changes in pH during fermentationindicated that pH reached approximately3.00after three days in both fermentation systems.The group of cabbage fermentation with LCR6013degraded nitrites faster than thespontaneous fermentation group. The nitrite depletion by LCR6013was not caused by theacid. In MRS system, the depleted nitrite concentration reached the highest to9.25μg/mL and9.70μg/mL when0.75%NaCl and0.02%VC was added, respectively. The OD600valuereached highest to3.05when0.1%FOS was added, but it could not improve the nitritedepletion ability of LCR6013. Moreover, LCR6013could degrade no more than50.00μg/mLnitrite in this study.ECD-GC method was used to measure changes of the concentration of N2O in headspaceof MRS system. When the initial concentration of nitrites was10.00μg/mL, the degradationproducts contained28.81×10-6(v/v) N2O, while CK1and CK2exhibited no absorption peakof N2O.The trial group, the other two control groups showed no significant differences in theproduction of TAN which demonstrated TAN was not produced after nitrite reduced by LCR6013. Overall, LCR6013deplete nitrite through denitrification rather than ammonificationand the possible pathway was NO2-NO N2O N2. Both enzymes from periplasm andcytoplasm could reduce nitrite and the nitrite reductase activity was312.64U and124.69U,respectively, the former was2.5times of the latter. To summary, the intracellular nitritereductase in LCR6013degraded nitrite and was mostly located in periplasm.Crude periplasm nitrite reductase was purified through DEAE Sepharose CL-6B andG-100and the enzyme activity of purified enzyme was improved35.08times. Relativemolecular mass was41.43kD through SDS-PAGE. The mass spectrometry properties of nitrite reductase from LCR6013were similar with that of flagellin from Bacillus cereusAH187by means of MS/MS.