Mechanism And Application Of Nitrite Enzymatic Degradation By Microorganisms In Saukraut In Northeast China

The microbial-driven nitrite enzymatic and acid degradation can reduce nitrite concentration in fermented fruits and vegetables.The research on acid degradation is relatively thorough and is widely used in the direct-type fermentation by lactic acid bacteria.However,the abuse of nitrogen fertilizer in agriculture has resulted in the accumulation of high concentrations of nitrate in vegetables and fruits that cannot be eliminated by acid degradation,which can be reduced to nitrite by intestinal microorganisms after they are carried out in the human body where the acid degradation will be ineffective,leading to second hazard.Therefore,the analysis and application of microbial enzymatic degradation mechanism of nitrate and nitrite in the fermentation process of pickles of vegetables and fruits are of great significance.In this paper,the saukraut in Northeast China was carried out as the type sample,with reference to the biochemical earth cycle pathways and characteristics of nitrogen.Through the dynamic monitoring of the physicochemical properties of the sauerkraut solid and fermentation broth,the functional gene abundance of microbial transformed nitrogen and the diversity of bacterial community structure,the microbial pathways and groups of nitrite enzymatic degradation during the fermentation process of sauerkraut in Northeast China were identified.Simultaneously,the corresponding microbial specific culture medium was designed to isolate microbial strains with enzymatic degradation function,with reference to the type of substrate in fermentation broth.Finally,a preliminary experiment of multi-strain direct-type fermentation was carried out.The main contents of the study are as follows:?1?The study on the pathway of nitrite enzymatic degradation in the fermentation process of Northeast saukraut.Continuous dynamic monitoring was carried out in the laboratory for 31 days under simulated fermentation conditions of saukraut in Northeast China.According to the dynamic monitoring of pH in fermentation broth of saukraut in Northeast China,the nitrite degradation was divided into enzymatic degradation period?1-15 days?and acid degradation period?17-37 days?.The concentration of ammonium,nitriteand nitrate in the fermentation broth were determinedthrough N-?1-naphtyl?-ethylenediamine dihydrochloride spectrophoto-metric method and Ion chromatography.The nitrite concentration of the fermentation broth increased first and then decreased,reaching the peak value?31.29 mg/kg?on the 7th day and then decreased to 0.42mg/kg.It was found that the concentration of ammonium salt changed slightly and its concentration was much lower than that of nitrite in the whole fermentation process.So we speculated that the nitrite enzymatic degradation pathway was dominated by denitrification.Then,Real-time quantitative PCR detection of functional gene nir confirmed the speculation and defined the types and abundance of gene nir at different fermentation stages.The results showed that denitrification was the main pathway of nitrite degradation in the early stage of saukraut fermentation,and the microbial groups with nirSC₁ genotype,nirKC₅ genotype and nirKC₂ genotype played important roles in the degradation process.The determination of denitrification substrate organic acid by high-performance liquid phase showed that the most likely carbon sources for denitrifying microbial compositions in the early stage of sauerkraut fermentation included oxalic acid,malic acid,citric acid,fumaric acid,succinic acid,propionic acid,tartaric acid and lactic acid.?2?The analysis on microbial groups of nitrite degradation in the fermentation process of saukraut in Northeast China.A high-throughput sequencing library of fermentation broth microorganisms was constructed at different fermentation stages and the corresponding microbial groups were annotated.The results of rarefacation curve,Chao1 index,Shannon index,PCoA analysis of community structure and species annotation based on OTU showed that the bacterial community structure had successed distinctly in the fermentation process of saukraut in Northeast China.At the stage of nitrite enzymatic degradation,the genus Pseudomonas,Leuconostoc,weissella,as well as the unknown new species of the order Enterobacteriales,were the most dominant.These are the key microflora in the stage of nitrite enzymatic degradation in the early fermentation stage of saukraut in Northeast China.?3?The isolation and identification of nitrate and nitrite enzymatic degradation of microflora.According to the 8 kinds of organic acids in fermentation broth,nitrate and nitrite were used as substrates for nitrogen source,the culture medium was prepared under the conditions of aerobic and anaerobic.The denitrifying strains which can degrade nitrate and nitrite in fermentation broth were cultured and isolated.A total of 133 strains were isolated.By means of denitrification ability test,the strains of Enterobacter,Chryseobacterium,Kluyvera,Raoultella,Enterococcus and Leuconostoc were determined to possess the ability of degrading nitrate and nitrite simultaneously.Two new strains of the genus Enterobacter and one new strain of the genus Chryseobacterium were obtained.?4?The application of direct-type fermentation of functional strains with the ability of both nitrate and nitrite reduction.We chose Leuconostoc citreum FXLB-3S2 and Enterococcus casseliflavus NXBC-1S2,which can degrade both nitrate and nitrite,from the perspective of safety,and Lactobacillus plantarum subsp.The results showed that Enterococcus casseliflavus NXBC-1S2 combined with Lactobacillus plantarum subsp.Plantarum PXLB-1S1 and Leuconostoc citreum FXLB-3S2,can degraded nitrate signifantly with the nitrate degradation rate of solid could be increased from 75%to 84%and the degradation rate of nitrate infermentation broth reached 48%with no nitrite accumulation during fermentation.