Mrs solid medium [14] [15]: glucose 10 g, agar 20 g, peptone 5 g, KH₂PO₄ 2 g, yeast extract 2G, calcium carbonate powder 15 g, distilled water 1000 mL, pH 7.0. Liquid medium: glucose 10 g, peptone 5 g, KH₂PO₄ 2 g, yeast extract 2G, distilled water 1000 mL, pH 7.0.

1 g of pickle is randomly cut into a mortar, added with 1 mL normal saline, fully ground, and left for 15 minutes, then put into the ultra clean table. The pickle samples of Yutang soy sauce were diluted with normal saline according to the concentration gradient of 10⁻¹ - 10⁻³: Six EP tubes were prepared, numbered 1, 2, 3, 4, 5 and 6 on the tube rack, and the bacterial solution of different concentrations was prepared in EP [16]. Colony observation: under the microscope, the direct microscopic examination was carried out first. Gram staining was performed before microscopic examination. The strains with large calcium dissolving circle were selected from the plate and purified repeatedly on Mrs solid medium. Lactobacillus plantarum was screened and inoculated into liquid medium.

According to the method of naphthalene ethylenediamine hydrochloride, the nitrite degradation ability of Lactobacillus plantarum was measured. The selected Lactobacillus plantarum was inoculated into Mrs liquid medium containing the appropriate amount of NaNO₂, and blank control was made at the same time. The content of NaNO₂ in fermentation broth was detected after 24 hours of incubation in constant temperature incubator [17].

Lactobacillus plantarum, which was screened in 2.3, was activated for 2 generations and inoculated into Mrs agar medium. After 24 hours of culture, the morphology of the strain was observed. The physiological and biochemical characteristics of Lactobacillus plantarum were identified according to “Berger’s Bacteria Identification Manual” and “plant lactobacillus classification and identification and experimental methods” [18]. The carbon source utilization test was conducted by biochemical identification tube.

According to the 16S rDNA gene sequence, a universal primer 27F was designed: 5’-agagttttgatcctggctcag-3’, 1492r: 5’-cggttacttgttacgcactt-3’. The whole genome of nitrite degrading Lactobacillus plantarum was extracted and amplified by 16S rRNA bacteria with primers. The amplification system was 25ul PCR reaction system. The amplification reaction conditions were as follows: pre denaturation at 94˚C for 3 min, denaturation at 94˚C for 30 s, annealing at 52˚C for 1.5 min, extending for 2 min and 30 s at 72˚C for 35 cycles and extending for 10 min at 72˚C. Blast was used for homology comparison. PCR products were sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequence was compared by blast in GenBank and identified to species [19].

The PCR product was purified and sequenced as described previously [20]. BLAST analysis for 16S rRNA gene sequences was done on National Center for Biotechnology Information (NCBI) database. The sequence was multiply aligned with the selected sequences of type strains obtained from GenBank/EMBL/DDBJ databases by using Clustal W version 1.81. A phylogenetic tree was reconstructed using MEGA5.0 software, based on the neighbour-joining, maximum-parsimony and maximum likelihood methods.

In this study, RSM was used to identify the effects of optimum parameters of four variables, Inoculation quantity (A), salinity (B), temperature (C), and Nitrite degradation rate (D), on the yield of Nitrite degradation. To estimate the model coefficients, a three-factor-three-level Box Benhnken experiment was performed (29 runs), and the order of the experiments was randomized, analysis of the experimental design data and the calculation of the predicted responses were computed by using SPSS Statistics 22.0. Additional confirmatory experiments were subsequently conducted to verify the reliability of the statistical experimental design [21].

Lactobacillus plantarum JBA-3 was inoculated into sterilized MRS medium and incubated at 30˚C for 24 hours without shaking to produce seed solution. Then, 5% of the seed liquid was added to the liquid MRS medium to bring the bacteria to 10⁷ - 10⁸ CFU/mL, and then incubated at 30˚C for 24 hours without shaking to produce a secondary seed solution [22]. The fermentation process of pickles is carried out according to the following procedures: fresh cucumber cleaning, drying, segmentation, blanching. Add 5% secondary seed liquid and soy sauce. Fermentation was carried out at 37˚C in sealed bottles for 120 hours. Samples of fermented pickles were collected regularly under aseptic conditions to detect the concentration of nitrite [23]. Fermentation is also carried out under sterile conditions. In order to obtain the best growth and anaerobic fermentation of lactic acid bacteria, pickles were not exposed to the air in a large number of secondary seed liquid. The control group (CK₀) was fermented with conventional pickles and was not inoculated with Lactobacillus plantarum JBA-3.

The supernatant of ground pickle was extracted and prepared into different concentrations of bacterial solution in EP tube. The higher the dilution concentration was, the higher the number of colonies was detected (Table 1). Morphological

ND: Not Detected.

observation was carried out to observe the morphology of the better growing colonies. After Gram staining, Lactobacillus plantarum was dyed blue purple, which proved that Lactobacillus plantarum was Gram-positive (Figure 1). The strains with large calcium dissolving circle were selected from the plate and purified repeatedly on Mrs solid medium. Finally, the Lactobacillus plantarum JBA-3 strains with the largest calcium dissolving circle were selected. More than 300 strains were isolated, and one strain with high nitrite degradation activity was found, and inoculated into liquid medium to screen Lactobacillus plantarum.

Neighbour-joining tree based on an almost complete 16S rRNA gene sequence (1492 nt) showing the relationships among strain Lactobacillus plantarum JBA-3 and related type strains (Figure 2). Phylogenetic tree was constructed by using related strains of Lactobacillus. Asterisks (*) and hashes (#) indicate that the corresponding nodes were also found in trees generated with the maximum-likelihood and maximum-parsimony algorithms, respectively. The numbers at branch nodes indicate bootstrap percentages derived from 1000 replications; only values > 50% are shown. Bar, 0.01 substitutions per single nucleotide position.

The response surface 3D map (Figure 3) showed the combined effects of different factors on the response (Nitrite degradation rate). The experimental data were fitted to equation, with the following optimal operating parameters: (a): Inoculation quantity, 9.13%; (b): salinity, 5.41%; (c): temperature, 30.19˚C; (d): Nitrite degradation rate, 91.47%.

The nitrite concentration of fermented pickles after different fermentation time is shown in Figure 4. The content of nitrite in fermented pickles without Lactobacillus plantarum JBA-3 18.35 mg/Kg and 51.26 mg/Kg, which decreased to 1.75 mg/Kg and 0.89 mg/Kg after 24 h and 48 h fermentation by Lactobacillus plantarum JBA-3. The content of nitrite after 72 hours of fermentation is higher than that of after 48 hours from Figure 4. Compared with the conventional fermentation, the nitrite concentration in the fermentation system with Lactobacillus plantarum JBA-3 was significantly lower (P < 0.01). The nitrite concentration

in fermented pickles without Lactobacillus plantarum JBA-3 increased first and then decreased, and the highest concentration reached 51.26 mg/Kg after 48 h fermentation, which was consistent with the previous report. Therefore, Lactobacillus plantarum JBA-3 could significantly inhibit the abnormal accumulation

of nitrite during the fermentation of pickles. For a long time, high concentration of nitrite in food has been a research hotspot in the field of food safety. The potential reason of nitrite accumulation in Chinese pickles was studied. The results showed that nitrate accumulated in the process of bacterial fermentation, and lactic acid fermentation reduced nitrate concentration. High concentration of nitrite was maintained for a long time. The abnormal accumulation of nitrite in pickles was related to the following factors: 1) The number of coliforms was higher than that of the control group; 2) The concentration of soluble nitrogen compounds was higher than that of the control group; 3) The buffering capacity of the control group was higher than that of the control group. These results suggest that the abnormal accumulation is caused by the long-term survival of coliforms that promote nitrate reduction. Lactobacillus delbrueckiicidca 133 inhibited the nitrate reductase activity of Escherichia coli. The nitrate reductase activity was closely related to the number of viable cells of Lactobacillus, rather than low pH value. This indicates that some substances are directly transferred from Lactobacillus to Escherichia colis.