Analysis And Application Of The Nitrite Degradation Ability Of Lactobacillus Kefiranofaciens ZW3

Nitrite can be accumulated in large amounts in the process of vegetable fermentation,it can bring a variety of hazards to human health when overeating.Lactobacillus Kefirano-faciens ZW3 have the ability to degrade nitrite,and the rate of nitrite degradation could be 100%.To analysis the molecular mechanism of nitrite degradation,we found three genes might be related to nitrite degradation in Lactobacillus Kefiranofaciens ZW3,WANG₀769,WANG₀780,WANG₁034 respectively.Bioinformatics analysis showed that WANG₀769 and WANG₁034 belong to Ni-tro_FMN_reductase superfamily,and transmembrane domains and signal peptide were not found in the proteins,which suggested that they were not trans-membrane proteins and could not be secreted into the extracellula.a-helices,β-sheets and random coils were the major structural elements in secondary structure of proteins.Intracellular localization anal-ysis showed that they were existent in the cytoplasm of the great possibility.To identify the function,they were expressed in E.coil BL21（DE3）with plasmid pET-30a as vector,and purified with Ni2+-NTA affinity chromatogram.Then we detect the activity of nitrite deg-radation,the results showed that both proteins had the ability of nitrite degradation.The activity were 3.16 U/mg,1.47 U/mg（U=μmol nitrite/minute）respectively.Those results suggested that the enzymes play a key role in the process of nitrite degradation.Semi-quantitative RT-PCR technique was also used to analyze the expression of different time point of genes,we observed that in enzyme degradation stage the enzymes show the key role under high nitrite content of stimulation.WANG₀780 belongs to Form_Nir_trans superfamily.There were six transmembrane regions in the protein,which consisted with the result that WANG₀780 belong to transport protein,and it should be associated with the cell membrane.Secondary structure also show that the protein contained α-helices,β-sheets and random coils,accounting for 65.37%,10.51%and 24.12%respectively.The results of signal peptide and localization prediction were the same as WANG₀769 and WANG₁034.After successfully expressed in E.coil BL21（DE3）,the protein was purified to detect the activity of nitrite degradation,and weak activity was observed.This phenomenon may be due to that the protein was related to the cell membrane,and belongs to nitrite transport protein.In the process of nitrite degradation,it play a key role of the transport of nitrite,which directly related to degrade nitrite.Gene expression analysis suggested that both high and low concentration of nitrates were not conducive to the expression of WANG₀780.When Nitrite concentration was appropriate,gene expression quantity increases sharply.Although with low nitrite reductase activity,WANG₀780 still play a key role in the process of nitrite degradation.Above all the experiments,we draw a conclusion that ZW3 has a strong ability to de-grade nitrite.So we applied the strain to the fermentation of cowpea.By single factor test and orthogonal test,the optimal fermentation conditions were determined.Quality analysis results show that the ZW3 could promote the generation of acid in fermentation process,and make greatly improved in color,aroma,taste at the same time.So ZW3 has great con-tribution to the formation of flavor.Then nitrite content of fermentation product was tested,the fermentation with ZW3 was only 0.150mg/kg which suggest that Lactobacillus Kefiranofaciens ZW3 have the effect of degradation of nitrite in fermentation vegetables.