Screening and Characterization of Nitrite Degrading Lactobacillus plantarum in Chinese Traditional Pickles

A Gram-positive, non-spore, round ended, straight rod Lactic acid bacteria were screened. The strain was screened out from the traditional pickle jar in Yutang soy sauce garden of Jining. In order to degrade the nitrate content in the fermentation process of traditional pickles and improve the quality of pickles, it is necessary to screen out nitrite degrading strains from pickles, and preliminarily locate nitrite reductase, and find out the most suitable pH, temperature and culture time to degrade nitrite. Lactobacillus plantarum was screened by MRS medium in advance. After 48 hours of culture in a shaking table with Bacillus subtilis, the cell components were separated by centrifugation, wall breaking and other operations. After 20 hours at 30 ̊C, the content of nitrite in each component was determined by the naphthalene ethylenediamine hydrochloride method (NIR). The culture conditions were as follows: inoculation amount 3%, 6%, 9%, 12%, 15%; salinity 2%, 4%, 6%, 8%, 10%; temperature 15 ̊C, 20 ̊C, 25 ̊C, 30 ̊C, 35 ̊C for 20 h. The results showed that the best degradation effect of nitrite was obtained under the conditions of 9% inoculum, salinity 5% and 30 ̊C. Under the conditions of 9% inoculum, 5% salinity and 30 ̊C for 5 h, 10 h, 20 h, 48 h, 66 h and 78 h, the results showed that the degradation amount gradually increased with the extension of time, and gradually maintained a stable state. Lactobacillus plantarum JBA-3 is a new type of lactic acid bacteria which can degrade nitrite and produce nitrite reductase.

DOI: 10.4236/fns.2021. 1212094 1288 Food and Nutrition Sciences international cancer research. Nitrite is widely found in food, especially in pickled vegetables [1]. Nitrite will be produced in the process of vegetable pickling. When the human body ingests a certain amount of nitrite, a series of health problems can be induced. At present, the production method of pickle and pickle is relatively backward, the hygiene is not up to standard, the bacterial pollution is serious, and the content of nitrite is high [2]. Nitrite content has become a "stumbling block" to the progress and development of pickle industry.
Lactobacillus is one of the most common bacteria in nature, which exists in pickled vegetables [3]. Lactobacillus plantarum can degrade nitrite in pickles. It is of great significance for people's production and life to screen Lactobacillus plantarum strains with a strong ability to degrade nitrite. Jining pickle is famous for its good taste and high quality. The purpose of this study was to extract Lactobacillus plantarum with strong nitrite degradation ability from Jining pickles [4] and provide the reference for selecting suitable nitrite degrading Lactobacillus plantarum in the process of pickle production [5].
Nitrite is a potential carcinogen, which is produced in the process of vegetable fermentation, which brings potential problems to food safety. Intake of large amounts of nitrite can lead to methemoglobinemia and acute poisoning [6] [7].
Under appropriate conditions, nitrite reacts with amine, the product of protein decomposition, to form N-nitroso compounds. At present, more than 100 kinds of N-nitroso compounds have been synthesized, of which 80% are strong carcinogens in animals [8]. However, nitrite is also widely used in the meat industry to prevent the growth of Clostridium botulinum, and is also used as a colorant.
Therefore, limiting nitrite in food is the focus of food safety research [9]. It was found that inoculating lactic acid bacteria could inhibit the accumulation of high concentration nitrite in pickle fermentation [10]. It has also been reported that more than 10

Isolation of Lactobacillus plantarum
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.

Screening of Nitrite Degrading Lactobacillus plantarum
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 NaNO 2 , and blank control was made at the same time. The content of NaNO 2 in fermentation broth was detected after 24 hours of incubation in constant temperature incubator [17].

Physiological and Biochemical Identification of Nitrite Degrading Lactobacillus plantarum
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.

Phylogenetic Tree
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.

Parameter Optimization by Response Surface Methodology
In this study, RSM was used to identify the effects of optimum parameters of

Pickle Fermentation
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 10 7 -10 8 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 (CK 0 ) was fermented with conventional pickles and was not inoculated with Lactobacillus plantarum JBA-3.

Isolation of Lactobacillus Plantarum
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 ( 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.

Phylogenetic Tree
Neighbour-joining tree based on an almost complete 16S rRNA gene sequence

Response Surface Analysis
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:

Dynamic Change of Nitrite Concentration in Pickle Fermentation
The nitrite concentration of fermented pickles after different fermentation time is shown in Figure 4.    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.

Discussion
The most suitable pH value of lactic acid bacteria was obtained from this expe-    fermentation of pickles, Lactobacillus plantarum JBA-3 was not enriched to control the accumulation of nitrite in pickles. In our improved production process, we can increase the number of Lactobacillus plantarum JBA-3 ahead of time. In the process of pickle fermentation, we can establish the fermentation production system of Lactobacillus plantarum JBA-3 which can degrade nitrite as the dominant strain. In the whole process of pickle production, the synthesis and accumulation of nitrite can be controlled to make the pickle safer.