Lactobacillus brevis JS231 with high γ-aminobutyric acid production and its application

Abstract

The present invention discloses a Lactobacillus brevis JS231 with high γ-aminobutyric acid production and its applications, belonging to the field of biotechnology. The Lactobacillus brevis JS231 was deposited at the Guangdong Provincial Microbial Culture Collection Center on August 30, 2024. The deposit address is the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC 65079. The present invention also provides any one of the following applications of the Lactobacillus brevis JS231: (1) Application in γ-aminobutyric acid production; (2) Application in the preparation of fermentation products with antioxidant activity; (3) Application in inhibiting the activity of pathogenic bacteria; (4) Application in degrading nitrite; (5) Application in the development of lactic acid bacteria products rich in γ-aminobutyric acid. The Lactobacillus brevis JS231 provided by the present invention provides raw materials for the development of lactic acid bacteria resources.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Lactobacillus brevis JS231 with high yield of γ-aminobutyric acid and its application. Background Art

[0002] Lactobacillus brevis is a kind of lactic acid bacteria in the broad sense and is recognized as a generally recognized as safe (GRAS) food-grade microorganism. Lactic acid bacteria from food sources have higher safety. γ-aminobutyric acid (GABA) is a natural amino acid widely present in animals, plants and microorganisms, and is decarboxylated and transformed from glutamic acid by glutamic acid decarboxylase (GAD), and has various active functions. In the fields of functional foods and medicine, it has the effects of lowering blood pressure, promoting sleep and relieving anxiety. In the field of cosmetics, it has the effects of smoothing the skin and enhancing skin elasticity. In the agricultural field, it has the effects of regulating plant growth, development and stress response.

[0003] At present, the methods for synthesizing GABA mainly include two categories: chemical methods and biological methods. Biological methods include two methods: plant enrichment and microbial fermentation. In recent years, microorganisms have been widely used in the production of GABA due to their short growth cycle and fast reproduction speed. The production of GABA by fermentation method has been studied most on lactic acid bacteria that are generally recognized as safe and have various probiotic functions. A variety of studies have shown that lactic acid bacteria have glutamic acid decarboxylase activity and can catalyze the decarboxylation of glutamic acid to produce GABA. Compared with chemical methods, the GABA-rich food prepared by microbial fermentation is naturally fermented and does not require additional addition of chemically purified GABA, which is green and healthy and has good application prospects.

[0004] Although there have been many reports on lactic acid bacteria producing GABA, it is still very important for the food industry to carry out research on the isolation, identification, application, etc. of lactic acid bacteria with high yield of GABA in order to obtain stable and safe GABA for the research and development of GABA-rich functional foods. At present, there are few lactic acid bacteria strains with the potential for industrial production of GABA and that can be used for food fermentation, and the research on the probiotic functional characteristics of lactic acid bacteria with the potential for GABA production is still far from sufficient, resulting in the inability to directly use the strains in food, which limits the expansion of the market scale of GABA products. Therefore, screening lactic acid bacteria with high yield of GABA from food sources is of great significance for the development of γ-aminobutyric acid-rich functional foods. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a Lactobacillus brevis JS231 with high yield of γ-aminobutyric acid and its application. The Lactobacillus brevis JS231 provided by the present invention can efficiently convert glutamic acid into GABA.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A strain of Lactobacillus brevis JS231, characterized in that the Lactobacillus brevis JS231 was deposited at the Guangdong Provincial Microbial Culture Collection Center on August 30, 2024. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC 65079.

[0008] Technical solution 2: An application of the above-mentioned Lactobacillus brevis JS231, and the application includes any one of the following applications:

[0009] (1) Application in producing γ-aminobutyric acid;

[0010] (2) Application in preparing a fermentation product with antioxidant activity;

[0011] (3) Application in inhibiting the activity of pathogenic bacteria;

[0012] (4) Application in degrading nitrite;

[0013] (5) Application in developing a lactic acid bacteria product rich in γ-aminobutyric acid.

[0014] Further, the production of γ-aminobutyric acid is obtained by efficiently converting glutamic acid.

[0015] Further, the fermentation product includes Gastrodia elata fermentation extract.

[0016] Further, the pathogenic bacteria include Staphylococcus aureus, Bacillus subtilis and Escherichia coli.

[0017] Technical solution 3: A fermentation product rich in γ-aminobutyric acid, and the γ-aminobutyric acid in the fermentation product is obtained by fermenting the above-mentioned Lactobacillus brevis JS231.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] The present invention provides a Lactobacillus brevis JS231 strain that can efficiently convert glutamic acid into GABA. The strain has a relatively slow growth rate in the early stage of fermentation, but has a high cell density after reaching the stationary phase, showing the potential for high-density culture; it can produce acid using glucose and generate gas at the same time; it has the ability to metabolize a variety of carbohydrates; the fermentation broth of the strain has good antibacterial properties and is inhibitory to Escherichia coli, Staphylococcus aureus, and Bacillus subtilis; at the same time, the strain has excellent nitrite degradation ability; it can efficiently convert glutamic acid to produce GABA; it has good tolerance to artificial gastrointestinal fluids and non-hemolytic characteristics, providing raw materials for the development of lactic acid bacteria resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is the colony morphology (a) and microscopic examination picture (b) of the JS231 strain;

[0022] Figure 2 It is the growth curve (a) and acid production curve (b) of the JS231 strain;

[0023] Figure 3 It is the inhibitory ability of the JS231 strain against pathogenic bacteria;

[0024] Figure 4 It is the peak diagram of each standard product detected by high performance liquid chromatography; among them, a is the blank group; b is the GABA standard product; c is the L-Glu standard product;

[0025] Figure 5 It is the peak diagram of the cell-free supernatant of lactic acid bacteria detected by high performance liquid chromatography. Among them, a is the control group; b is the cell-free supernatant of the JS231 strain;

[0026] Figure 6 It is the hemolytic test result of the JS231 strain; among them, (a) is the JS231 strain; (b) is Staphylococcus aureus;

[0027] Figure 7 It is the identification of the antioxidant ability of the cell-free supernatant of Gastrodia elata fermented by the JS231 strain. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementation manners of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0032] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0033] In the present invention, unless otherwise specified, the raw materials, equipment, etc. used can be purchased from the market or are commonly used in the art. The methods in the following examples are conventional methods in the art unless otherwise specified.

[0034] MRS broth medium, MRS agar medium, and MRS agar medium containing 1% CaCO3: Both MRS broth medium and MRS agar medium are purchased from Qingdao Haibo Biotechnology Co., Ltd. For MRS agar medium, 1 g of agar is supplemented per 100 ml of the medium when in use. The MRS agar medium containing 1% CaCO3 is prepared by supplementing 1 g of agar and 1 g of CaCO3 per 100 ml of the MRS agar medium when in use.

[0035] MH broth (MHB) medium: Purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0036] Gastrodia elata fermentation medium: Prepared according to the ratio of 1 g of Gastrodia elata extract, 4 g of sucrose, 1 g of peptone, and adding 100 ml of water.

[0037] The above media were sterilized at 118 °C for 15 min.

[0038] Preparation of simulated gastric juice: Simulated gastric juices with pH 2.0 and pH 3.0 were prepared respectively. Take 10 g of pepsin, dissolve it in water and adjust the pH, make up the volume to 1 L to make its final mass concentration 0.01 g / mL, sterilize with a 0.22 μm microporous filter membrane, and store at 4 °C for later use.

[0039] Preparation of simulated intestinal juice: Take 6.8 g of KH2PO4, dissolve it in water, adjust the pH to 6.8 with NaOH solution; take another 10 g of trypsin, dissolve it in water, mix the two solutions and make up the volume to 1 L, sterilize with a 0.22 μm microporous filter membrane, and store at 4 °C for later use.

[0040] Method for activating strains and preparing seed liquid: Inoculate the preserved glycerol tube of bacteria into MRS solid medium, activate at 35 - 37 °C for 14 - 18 h, pick 1 loop of single colony into a centrifuge tube containing 1 mL of MRS medium, and culture statically at 35 - 37 °C for 12 - 14 h to obtain the seed liquid.

[0041] Example 1

[0042] 1. Screening, isolation and identification of strains

[0043] Strain JS231 was isolated and screened from Hanzhong pickled vegetables. The specific scheme is as follows: After mixing the pickled vegetable samples, dilute them step by step with physiological saline to 10 -3 、10 -4 、10 -5 , pipette 100 μL and spread it on MRS agar medium containing 1% CaCO3, and culture at 37 °C for 24 h. Pick the single colonies that can produce CaCO3 dissolution circles and purify them by streaking on MRS-CaCO3 medium multiple times. Pick the purified single colonies into MRS liquid medium and culture statically at 37 °C for 24 h, and store them in a -80 °C refrigerator with 15% glycerol.

[0044] Morphological identification: Observe the color, size, and edge morphology of the purified single colonies, and conduct Gram staining to observe the microscopic cell morphology and its Gram identification results, and record the colony morphology and microscopic pictures (see Figure 1 ).

[0045] 16S rRNA sequencing identification: Pick the preserved glycerol bacteria and streak them on MRS agar medium, culture at 37℃ for 24h, pick a single colony in MRS liquid medium at 37℃ and culture it statically for 24h. Use a bacterial genomic DNA purification kit to extract genomic DNA. Use 16S rRNA universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) and 1429R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2) to amplify the full-length sequence of strain 16S rRNA. The PCR amplification system is shown in Table 1. The PCR amplification program is: 95℃ pre-denaturation for 5min, 30 cycles (95℃ denaturation for 1min, 50℃ annealing for 30s, 72℃ extension for 90s), and 72℃ full extension for 10min. The amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0046] The sequencing results were compared with the NCBI database Blast to confirm the biological classification of the strain, and the identification showed that the bacteria was Lactobacillus brevis. Lactobacillus brevis JS231 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 30, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC 65079.

[0047] Table 1 PCR amplification program

[0048] Reagent Volume (μL) 2× Mix 20 Genomic DNA 1 Primer 27F (10 μM) 2 Primer 1429R (10 μM) 2 <![CDATA[ddH2O]]> Make up to 40 μL

[0049] Example 2

[0050] The growth ability, acid production ability, physiological and biochemical identification, gastrointestinal fluid tolerance, cell surface characteristics, probiotic properties and safety of the Lactobacillus brevis JS231 identified above were evaluated. The specific scheme is as follows:

[0051] 1. Growth and acid production capacity

[0052] The activated Lactobacillus brevis JS231 was inoculated into MRS liquid medium at 1% inoculation amount and cultured at 37°C. The OD was measured within 0 to 28 hours. 600 and pH value, with culture time as the horizontal axis, OD 600 The growth curve is drawn on the ordinate, and the results are shown in Figure 2 a. The acid production curve was drawn with the strain culture time as the horizontal axis and the pH value as the vertical axis. The results are shown in Figure 2 b.

[0053] 2. Carbohydrate fermentation characteristics

[0054] The carbohydrate fermentation pattern of Lactobacillus brevis JS231 was determined using API 50CHL reagents (BioMérieux, Marcyl’Etoile, France). The results showed that Lactobacillus brevis JS231 had a wide range of carbon sources utilized, good carbohydrate fermentation performance, and was able to metabolize 14 carbon sources, namely L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, methyl-α-D-glucopyranoside, N-acetylglucosamine, D-maltose, D-sucrose, potassium gluconate, 2-ketogluconate potassium, and 5-ketogluconate potassium (Table 2).

[0055] Table 2 Identification results of carbohydrate metabolism of Lactobacillus brevis JS231

[0056]

[0057]

[0058] Note: "+" indicates positive; "-" indicates negative.

[0059] 3. Tolerance to artificial gastric and intestinal fluids

[0060] The activated seed liquid of Lactobacillus brevis JS231 was inoculated into simulated gastric juice at an inoculation amount of 10%, mixed well, and then placed in a static culture at 37°C. Samples were taken at 0 h and 3 h respectively for viable cell plate counting. After culturing in simulated gastric juice for 3 h, it was inoculated into simulated intestinal juice at an inoculation amount of 10%, mixed well, and then placed in a static culture at 37°C. Samples were taken at 0, 3, and 6 h respectively for viable cell plate counting, and parallel experiments were carried out 3 times. The survival rates are shown in Table 3.

[0061] Table 3 Survival rates of JS231 in simulated gastric and intestinal fluids

[0062]

[0063] Generally, the pH of gastric juice in humans is about 2.0 on an empty stomach, and it can rise to 3.0 after appropriate eating. The passage time of food in the stomach is generally 0 - 3 h. The present invention found that Lactobacillus brevis JS231 had relatively weak tolerance to gastric juice with a pH of 2.0, and the survival rate was 40.84%. It had stronger tolerance to gastric juice with a pH of 3.0, and the survival rate after 3 h was higher than 100%, indicating that not only did the bacterial cell mass not decrease under the stress of gastric juice with a pH of 3.0, but there was also proliferation. At the same time, the bacterial cell mass of Lactobacillus brevis JS231 increased to 2 times after 6 h of stress in simulated intestinal juice. The results showed that Lactobacillus brevis JS231 had good tolerance to gastric and intestinal fluids.

[0064] 4. Evaluation of the probiotic characteristics of Lactobacillus brevis JS231

[0065] 4.1 Detection of the inhibitory ability against common pathogenic bacteria

[0066] Escherichia coli (ATCC 25922), Bacillus subtilis (CMCC(B)63501), and Staphylococcus aureus (CMCC(B)26003) were selected as indicator bacteria. The activated Lactobacillus brevis JS231 was cultured for 24 h, and then centrifuged at 6000 r / min for 15 min to obtain cell-free supernatant (CFS). The indicator bacteria suspension cultured for 12 h was diluted to OD 600 of 0.5, and inoculated and cultured with JS231 CFS and MHB medium at a ratio of 1:2:17. The OD 600 value of the bacterial solution was recorded as Ax, and the OD 600 value of the indicator bacteria without adding Lactobacillus brevis JS231 CFS was recorded as A0.

[0067]

[0068] It was found that the cell-free supernatant of Lactobacillus brevis JS231 had good inhibitory ability against Staphylococcus aureus and Bacillus subtilis within 6 h, and had certain inhibitory ability against Escherichia coli. With the increase of the culture time, the inhibitory ability decreased to varying degrees (see Figure 3 ).

[0069] 4.2 Detection of the ability of Lactobacillus brevis JS231 to degrade sodium nitrite

[0070] One loop of the glycerol-preserved tube of JS231 bacteria was taken with an inoculation loop and inoculated into MRS liquid medium, and cultured at 37 °C for 10 h. The OD 600 value of the bacterial solution was measured and adjusted to 0.6 as the seed solution. Then JS231 was inoculated into MRS liquid medium with a sodium nitrite mass concentration of 125 mg / L at an inoculation amount of 1%, and cultured statically at 37 °C for 24 h. 0.1 mL of the bacterial solution was placed in a 10 mL centrifuge tube, and 0.25 mL of saturated borax solution (50 g / L), 0.1 mL of potassium ferrocyanide solution (106 g / L), 0.1 mL of zinc acetate solution (220 g / L), and 4 mL of ultrapure water were added in sequence. After mixing, it was centrifuged at 8000 r / min for 5 min. 2.5 mL of the supernatant was added to a 10 mL centrifuge tube, 0.50 mL of sulfanilic acid solution (4 g / L) was added, vortexed and mixed well, and then left to stand in the dark for 5 min. Then 0.25 mL of hydrochloric acid naphthyl ethylenediamine solution (2 g / L) and 1.75 mL of ultrapure water were added, vortexed and mixed well, and left to stand in the dark for 10 min. The absorbance value was measured at a wavelength of 538 nm, with water as the control group.

[0071] The group of the present invention was denoted as C1, and the control group was denoted as C0.

[0072] Excessive intake of nitrite can cause symptoms such as acute poisoning. Microorganisms such as lactic acid bacteria and a few Bacillus species have been proven to have the effect of degrading nitrite. Among them, lactic acid bacteria are the most common microorganisms for degrading nitrite at present. The present invention finds that the degradation rate of 125 mg / L NaNO2 added to the culture medium by JS231 is close to 100% within 24 hours.

[0073] 4.3 Detection of γ-aminobutyric acid content produced by Lactobacillus brevis JS231

[0074] Inoculate the activated Lactobacillus brevis JS231 into MRS liquid medium containing 10 g / L L-Glu at 1%, and incubate statically at 37 °C for 48 h. Centrifuge at 8000 r / min for 5 min to remove the cells, and collect the fermentation supernatant for the determination of γ-aminobutyric acid (GABA) content. The detection method of GABA content is carried out according to QBT5633.7-2022. Sample derivatization reaction: Take 1 mL of the fermentation broth sample, add 24 mL of ultrapure water, then add 5 mL of derivatization buffer (42 g / L sodium bicarbonate), 2.5 mL of derivatization reagent solution (10 g / L FDNB), water bath at 60 °C for 1 h, and after cooling, add the volume-fixing solution (1.36 g / L potassium dihydrogen phosphate) to make the volume up to 50 mL. Filter with a 0.22 μm filter membrane, collect the filtrate, and use it as the sample solution. Standard derivatization reaction: Weigh 0.05 g of GABA and L-Glu reference substances respectively and weigh 0.05 g of each as a mixed reference substance, make the volume up to 100 mL with pure water, measure 25 mL and place it in a volumetric flask, add 5 mL of derivatization buffer, 2.5 mL of derivatization reagent solution, water bath at 60 °C for 1 h, and after cooling, add the volume-fixing solution to make the volume up to 50 mL. Filter with a 0.22 μm filter membrane, collect the filtrate, and use it as the standard solution. The elution program is shown in Table 4. High performance liquid chromatography conditions: C18 chromatographic column (250 mm × 4.6 mm × 5 μm), mobile phase A is sodium acetate with 60 μL of glacial acetic acid added, mobile phase B is acetonitrile, flow rate is 0.8 mL / min, column temperature is 40 °C, injection volume is 10 μL, and detection wavelength is 360 nm.

[0075] Table 4 Mobile phase gradient elution program

[0076] Time / min Mobile phase A / % Mobile phase B / % 0 90 10 10 70 30 15 70 30 16 90 10 30 90 10

[0077] The present invention establishes a high performance liquid chromatography detection method by derivatizing FDNB with GABA and L-Glu. The present invention finds that the utilization rate of L-Glu by Lactobacillus brevis JS231 reaches 97.7%, and the GABA production amount reaches 4.7 g / L in 48 h, indicating that Lactobacillus brevis JS231 has good GABA production ability (see Figure 4 and Figure 5 ).

[0078] 5. Safety identification of Lactobacillus brevis JS231

[0079] 5.1 Hemolytic Identification of Lactobacillus brevis JS231

[0080] The activated strain was streaked on Columbia blood agar medium and cultured at 37 °C for 24 h to observe whether hemolysis occurred. Staphylococcus aureus (CMCC(B)26003) was used as a positive control strain. When a greenish hemolytic zone appears around the colony, it is α-hemolysis, that is, incomplete hemolysis; when a well-defined and completely transparent hemolytic zone appears, it is β-hemolysis, that is, complete hemolysis of red blood cells; when there is no hemolysis around the colony, it is γ-hemolysis, that is, non-hemolysis. It was found by identification on Columbia blood agar medium that the positive control Staphylococcus aureus was β-hemolytic, and JS2-3 was γ-hemolytic (see Figure 6 ).

[0081] 5.2 Antibiotic Sensitivity Detection of Lactobacillus brevis JS231

[0082] The disk diffusion method (K-B method) was used. The Lactobacillus brevis JS231 bacterial solution was evenly spread on MRS agar medium, and antibiotic disks were placed in a plum blossom shape and cultured at 37 °C for 24 h. The diameter of the inhibition zone was measured and recorded. In this invention, 14 kinds of 7 classes of antibiotics were selected to detect the antibiotic sensitivity of Lactobacillus brevis JS231. S, I, and R represent sensitive, moderately sensitive, and resistant respectively. It was found in this invention that Lactobacillus brevis JS231 was resistant to kanamycin, norfloxacin, vancomycin, sulfisoxazole, and enrofloxacin, and showed moderate sensitivity to cefotaxime (Table 5).

[0083] Table 5 Antibiotic Sensitivity of JS231

[0084]

[0085] Example 3

[0086] The antioxidant capacity of the cell-free supernatant of Lactobacillus brevis JS231 fermented Gastrodia elata was detected. Lactobacillus brevis JS231 was inoculated into the Gastrodia elata fermentation medium for fermentation culture and its antioxidant capacity was detected. The specific scheme is as follows:

[0087] The glycerol preservation tube of Lactobacillus brevis JS231 was streaked and activated on MRS solid medium and cultured overnight at 37 °C. A single colony was picked and inoculated into 1 ml of MRS liquid medium and cultured at 37 °C for 8 - 12 h. It was inoculated into 1% Gastrodia elata fermentation medium at an inoculation amount of 5%, sampled at 0, 12, and 24 h respectively, centrifuged at 12000 r / min for 5 min, and the fermentation supernatant was taken to detect the DPPH free radical scavenging ability. The results showed that after fermentation by JS231, the antioxidant capacity of the supernatant was significantly enhanced after 12 h of fermentation compared with that at 0 h (see Figure 7 ).

[0088] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Lactobacillus brevis ( Lactobacillus brevis ) The application of JS231 is characterized by: The application includes any of the following applications: (1) Application in the production of γ-aminobutyric acid from glutamate fermentation; (2) Application in the preparation of a fermentation product having antioxidant activity, wherein the fermentation product comprises a Gastrodia elata fermentation supernatant; (3) Application in inhibiting the activity of pathogenic bacteria, including Staphylococcus aureus, Bacillus subtilis and Escherichia coli; (4) Application in degradation of nitrite; The Lactobacillus brevis JS231 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 30, 2024, with the storage address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC 65079.

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