Weissella cibaria m1 and application thereof

Abstract

The present application relates to the technical field of microorganism, especially to a Weissella paramesenteroides M1 and application thereof.The Weissella paramesenteroides has a Latin name of Weissella Paramesenteroides, and a preservation number of CGMCC NO:26182.The Weissella paramesenteroides is negative to amino acid decarboxylase and nitrate reductase, is sensitive to chloramphenicol, is moderately resistant to penicillin, is resistant to two antibiotics of tetracycline and cefoxitin, and has no risk of drug resistance transfer, and has excellent bacteriostasis, and can be used as a preservative, and has a use effect obviously superior to conventional preservatives.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of enterobacterial Weissella M1 and its applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus is a Gram-positive bacterium and a common foodborne pathogen that can cause various acute and chronic infections. Among Staphylococcus aureus strains, methicillin-resistant Staphylococcus aureus (MRSA) is the most dangerous. Staphylococcus aureu MRSA (Mexican MRSA) has been spreading globally and causing numerous hospital outbreaks since its initial isolation in the early 1960s, making it one of the most common causes of hospital-acquired infections, leading to a range of problems from skin infections to bloodstream infections. MRSA infection has become a major threat to humanity, and the World Health Organization has identified it as one of the six "high priority" pathogens posing a significant threat to public health. Therefore, there is an urgent need to develop and identify new therapies and antimicrobial agents with novel mechanisms of action to combat this high-threat pathogen.

[0003] Lactic acid bacteria are a general term for a group of non-spore-forming, Gram-positive bacteria that can utilize glucose to produce large amounts of lactic acid. They are generally recognized as safe (GRAS) and widely used in the food, pharmaceutical, and animal feed industries. Bacteriologists classify lactic acid bacteria into four genera. Lactobacillus , Leuconostoc , Pediococcus and Streptococcus The latest taxonomy classifies lactic acid bacteria into the following genera: Aerococcu , Alloiococcus , Carnobacterium , Dolosigranulum , Enterococcus , Globicatella , Lactococcus , Oenococcus , Tetragenococcus , Vagococcus and Weissella Lactic acid bacteria can synthesize organic acids, such as acetic acid, lactic acid, lactobacillus, H2O2, hydroxy fatty acids, and cyclic peptides, which can inhibit the growth and reproduction of various pathogenic bacteria and other putrefactive bacteria. Studies have found... Lactobacillus rhamnosus SHA113 can effectively inhibit multidrug-resistant Staphylococcus aureus (MRSt), and studies have also found that... Lactiplantibacillus pantarum , L. acidophilus and L.caseivar Combined fermentation has a good inhibitory effect on clinically isolated MRSt.

[0004] Therefore, how to provide more types of lactic acid bacteria strains and apply them to the preservation or antiseptic purposes of food preparation or brewing is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention focuses on lactic acid bacteria isolated from Tibetan kefir grains. A strain of lactic acid bacteria with MRSA inhibitory activity was screened using the Oxford cup diffusion method. The strain was identified and its safety was evaluated. The properties of its antibacterial active substances were studied, providing a theoretical basis for the development of novel biological antibacterial agents.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a type of enteroblastic bacterium (Westernella enterica). Weissella Paramesenteroides M1, this bacterium is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 14, 2023, with accession number CGMCC NO: 26182, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] This invention provides the application of the aforementioned Enterobacter vesiculosus in the preparation of preservatives.

[0008] This invention provides the use of the aforementioned Enterobacter vesiculosus in the preparation of formulations for the prevention and treatment of diseases or pathogens caused by Staphylococcus aureus.

[0009] The present invention also provides a preservative comprising the aforementioned Enterobacter vesiculosus.

[0010] Preferably, the Enterobacter vesiculosus is a bacterial suspension of Enterobacter vesiculosus.

[0011] Preferably, the concentration of the bacterial suspension is 1×10⁻⁶. 6 ~1×10 10 CFU / mL.

[0012] The present invention also provides the application of the aforementioned preservative in food preservation.

[0013] Preferably, the 16S rDNA sequence of the Enterobacter vesiculosus is shown in SEQ ID NO: 1.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention screened a strain M1 with MRSA inhibitory activity from nine lactic acid bacteria isolated from Tibetan Ganoderma lucidum, and identified it as a Weissella-like enterobacter through morphological, physiological, biochemical, and molecular biological analysis. W. paramesenteroidesM1. Growth curve, pH and dynamic detection results of antibacterial activity showed that after 8 hours of fermentation, the supernatant of Enterobacter sieboldii M1 fermentation began to show an inhibitory effect on MRSA. At this time, the pH value of the fermentation broth was 4.62 and the diameter of the inhibition zone was 12.57±1.16 mm.

[0015] The antibacterial active substance produced by *Westernella enterica* M1 is insensitive to proteases and temperature, but sensitive to pH; its antibacterial activity is completely lost when the pH is adjusted to 7.0. Ethyl acetate can effectively extract the antibacterial substance from the fermentation broth. Finally, the safety evaluation results show that *Westernella enterica* M1 is negative for amino acid decarboxylase and nitrate reductase, sensitive to chloramphenicol, moderately resistant to penicillin and streptomycin, and resistant to tetracycline and cefoxitin, with no risk of resistance transfer. This study provides a theoretical reference for the development of novel MRSA inhibitors.

[0016] 2. As can be seen from the data recorded in this invention, the suspension of Enterobacter sieboldii M1 can be used as a preservative, especially in the fermentation process of kimchi, which can significantly extend the shelf life of kimchi and meat. Its effect is significantly better than that of conventional preservatives. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The diagram shows the inhibitory effect of strain M1 in Example 1 on MRSA. Figure 2 This is a diagram showing the colony characteristics and cell morphology of strain M1 in Example 2. Figure 3 This is a phylogenetic tree diagram constructed based on the 16S rDNA sequence of strain M1 in Example 2; Figure 4 Example 3: Antimicrobial kinetic curve of Enterobacter malignancies M1; Figure 5 This is an electrophoresis image of plasmid extraction in Example 6.

[0019] Biological Preservation Instructions

[0020] A type of enterovirus (Westernella) Weissella ParamesenteroidesM1, this bacterium is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 14, 2023, with accession number CGMCC NO: 26182, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] The test materials used in the following examples

[0023] The lactic acid bacteria to be screened were isolated from *Ganoderma lucidum* (Tibetan shiitake mushroom) and preserved in the Food Quality and Safety Laboratory of Huainan Normal University; methicillin-resistant Staphylococcus aureus (BNCC326053) was obtained from Beijing Beina Chuanglian Biotechnology Research Institute; bacterial genomic DNA extraction kit (DP302), plasmid extraction kit, and Marker 2000 were obtained from Beijing Tiangen Biotech Co., Ltd.; BeyoRed DNA loading buffer was obtained from Beyotime Biotechnology Co., Ltd.; proteinase K was obtained from Saiguo Biotechnology; penicillin and streptomycin were obtained from Aladdin; pepsin, catalase, cefoxitin, and vancomycin were obtained from Yuanye Biotechnology; and ethyl acetate was obtained from Xilong Chemical Co., Ltd.

[0024] Lactic acid bacteria culture medium (Mann Rogosa and Sharpe broth, MRS): 10 g beef extract, 10 g peptone, 2 g diammonium citrate, 2 g dipotassium hydrogen phosphate, 5 g yeast extract, 5 g anhydrous sodium acetate, 20 g glucose, 0.2 g magnesium sulfate, 1 mL Tween-80, 0.05 g manganese sulfate, 1 L distilled water. Sterilize at 121 °C for 15 min, then add 18-20 g agar to obtain MRS solid culture medium.

[0025] Beef extract peptone agar medium: 3 g beef extract, 10 g peptone, 5 g sodium chloride, 20 g agar, 1 L distilled water, sterilized at 121 ℃ for 15 min.

[0026] Amino acid culture medium: 5 g peptone, 3 g yeast extract, 1 g glucose, 1 mL 1.6% bromocresol purple-ethanol solution, pH 6.8, with 0.5% (mass fraction) of tyrosine, ornithine, lysine, histidine and arginine added respectively to prepare 5 amino acid culture media.

[0027] Nitrate medium: Add 0.2 g KNO4 to 1000 mL of MRS medium, heat and stir until completely dissolved, adjust the pH to 7.4, and prepare nitrate medium.

[0028] Instruments and equipment

[0029] SW-CJ-2F Double-person Double-sided Vertical Clean Workbench, Suzhou Weiye Air Conditioning & Purification Co., Ltd.; EX Zone2 Electronic Balance, Shenzhen Anput Electronic Technology Co., Ltd.; BSC-250 Constant Temperature and Humidity Incubator, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; LDZW-60KCS-Ⅱ Vertical Pressure Steam Sterilizer, Shanghai Shenan Medical Instruments; Unique-R20+UV Laboratory Ultrapure Water System, Xiamen Yuesijie Water Purification Technology Co., Ltd.; H0347 Electric Heating Constant Temperature Drying Oven, Shanghai Chengwei Instrument Technology Co., Ltd.; BCD-152 Meiling Refrigerator, Hefei Meiling Co., Ltd.; HZQ-F160A Constant Temperature Incubator Shaker, Shanghai Chengsi Intelligent Technology Co., Ltd.; RE-5286A Rotary Evaporator, Shanghai Yarong Biochemical Instrument Factory; HH-420 201 Digital Display Heating Water Bath, Hangzhou Youning Instrument Co., Ltd.

[0030] Example 1

[0031] 1. Screening of MRSA-inhibiting strains

[0032] Nine strains of *Ganoderma lucidum* were screened and purified from *Ganoderma lucidum*. These nine purified strains were then transferred to MRS liquid medium and incubated statically at 37°C for 48 h. The cultures were then centrifuged at 12,000 r / min at 4°C for 10 min, and the supernatant was obtained by filtration through a 0.45 μm sterile filter. The antibacterial activity of the test strains was detected using the Oxford cup diffusion method, with MRSA as the indicator bacterium. The OD... 600 A bacterial suspension of MRSA (methicillin-resistant Staphylococcus aureus), an indicator bacterium with a concentration of 0.2, was evenly spread onto beef extract peptone solid medium using a sterile cotton swab. Two Oxford cups were gently placed on each plate, and 100 µL of fermentation supernatant and sterile MRS liquid medium were added to the Oxford cups as controls, respectively. The plates were first incubated at 4°C for 3 h, and then incubated at 37°C for 18 h. The inhibition zone was observed and its diameter was measured.

[0033] Results: The inhibitory effect of nine test strains on the indicator bacterium MRSA was determined by the Oxford cup diffusion method. One strain, M1, was selected as having a significant inhibitory effect on MRSA, with an inhibition zone diameter of 18.42 ± 0.33 mm. Figure 1 Inhibitory effect of strain M1 on MRSA (left circle: inhibition zone of M1; right circle: control of MRS liquid culture medium).

[0034] Example 2

[0035] strain identification

[0036] (1) Morphological observation and physiological and biochemical reactions

[0037] The strain M1 selected in Example 1 was activated for 2 generations. The bacterial culture in the logarithmic phase was streaked on MRS solid medium and incubated at 37 °C. After colony formation, the colony morphology, color, and smoothness were observed. Then, single colonies were picked, Gram-stained, and cell morphology was observed under a biological microscope. All physiological and biochemical experiments were carried out according to the method of "Jin Hongxing, Yang Xiyin, Cheng Wenyu. Isolation and identification of Weissella bacteria containing endogenous plasmids in kimchi [J]. China Brewing, 2012, 32(1):77-79."

[0038] (2) 16S rDNA sequence analysis

[0039] The activated strain M1 was inoculated into MRS medium and incubated statically at 37°C for 16 h. The precipitate was collected by centrifugation, and total DNA of strain M1 was extracted according to the bacterial genomic DNA extraction kit. The extracted DNA template was used for PCR amplification, with the forward primer being 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (as shown in sequence 2) and the reverse primer being 1492R: 5'-ACGGCTACCTTGTTACGACTT-3' (as shown in sequence 3). After agarose gel electrophoresis, the PCR products were sent to General Biosystems (Anhui) Co., Ltd. for sequencing (PCR reaction system (50 µL): template DNA 3 µL, forward and reverse primers 1 µL each, premix 25 µL, ultrapure water to 50 µL. PCR reaction conditions: pre-denaturation 94 ℃ 5 min; denaturation 94 ℃ 1 min, annealing 52 ℃ 1 min; extension 72 ℃ 1.5 min, 30 cycles, final extension 72 ℃ 5 min). The obtained sequences were analyzed for sequence correlation using BLAST (http: / / www.ncbi.nlm.nih.gov / blast). Sequences with high similarity to the 16S rDNA sequence were obtained and homology analysis was performed. A phylogenetic tree was constructed using MEGA 4.0 software with the Neighbor-joining method.

[0040] Results: Strawberry M1 was streaked on MRS solid medium and cultured at 37 ℃ for 24 h. Its morphological characteristics were observed, and the results are as follows: Figure 2 As shown. By Figure 2It can be seen that on MRS solid medium, strain M1 forms white, round colonies with regular edges. The colonies are slightly raised, smooth, and opaque. Under a microscope, they are Gram-positive, rod-shaped, non-spore-forming, non-motile, and lack flagella. The physiological and biochemical characteristics of strain M1 are shown in Table 1. Table 1 shows that strain M1 cannot utilize hydrogen peroxide, lactose, raffinose, mannitol, or sorbitol, but can utilize sucrose, salicin, inulin, maltose, etc. According to the "Classification and Identification of Lactic Acid Bacteria" and "Bergey's Manual of Bacteriology," strain M1 is similar to *Enteromorpha vesicularis* (…). Weissella Paramesenteroides Their physiological and biochemical characteristics are highly similar. Figure 2 Colony characteristics and cell morphology of strain M1.

[0041] Further PCR amplification and sequencing yielded the 16S rDNA sequence fragment of strain M1, which was then compared online using BLAST with... W. paramesenteroides The similarity to strain 3151 (MT515963.1) reached 99.73%, and a phylogenetic tree was constructed with similar species using MEGA software. Figure 3 Based on a phylogenetic tree constructed from the 16S rDNA sequence of strain M1, strain M1 was identified as *Westernella enteritidis* and named... W. paramesenteroides M1. Enterobacter vesiculosus is an important lactic acid bacterium. Although it is rarely isolated from Tibetan kefir or kefir, it is distributed in other fermented foods such as fermented vegetables, cheese, sausages, and fermented black beans. Some strains can produce bacteriocins and non-bacteriocin antibacterial substances during fermentation, which can inhibit a variety of pathogenic bacteria, including Gram-negative bacteria, Gram-positive bacteria, and fungi.

[0042] Table 1. Physiological and biochemical characteristics of strain M1

[0043] Note: A "+" sign indicates a positive result; a "-" sign indicates a negative result.

[0044] Example 3

[0045] Antibacterial kinetic curve of strain M1

[0046] The activated strain M1 from Example 2 was inoculated into MRS liquid medium at a 3% inoculum and incubated statically at 37°C for 48 h. OD was measured every 4 h. 600 The pH value was measured. Simultaneously, the fermentation broth was collected and centrifuged at 12,000 r / min, 4 ℃ for 10 min, and the supernatant was obtained by filtration through a 0.45 μm sterile filter membrane. The inhibitory effect of the supernatant on MRSA at different fermentation times was determined using the Oxford cup diffusion method.

[0047] Results: The growth curve, pH, and antibacterial activity results of Enterobacter sieboldii M1 are as follows: Figure 4 As shown. By Figure 4 It can be seen that during the 48 h culture period, the biomass of *Westernella enterica* M1 increased from 0.02 to 2.43 (OD200). 600 The pH of the fermentation broth decreased from 5.36 to 3.52. Enterobacter sieboldii M1 began to show inhibitory effects on MRSA after 8 h of fermentation, at which point the pH of the fermentation broth was 4.62 and the inhibition zone diameter was 12.57 ± 1.16 mm. After 36 h of fermentation, entering the stationary phase, the fermentation supernatant showed the strongest inhibitory effect on MRSA, with an inhibition zone diameter reaching 24.41 ± 0.60 mm, after which it remained stable. Figure 4 Antimicrobial kinetics of Enterobacter sieboldii M1.

[0048] Example 4

[0049] Characteristic analysis of antibacterial substances

[0050] (1) Effects of enzymes on antibacterial substances

[0051] The fermentation supernatant obtained from strain M1 in Example 3 was treated with bromelain, catalase, pepsin, and proteinase K to achieve a final enzyme concentration of 2 mg / mL, and the optimal pH values ​​were adjusted: bromelain pH 7.0, catalase pH 7.0, pepsin pH 2.0, and proteinase K pH 7.5. After enzymatic hydrolysis in a 37 °C water bath for 2 h, the pH of the fermentation supernatant was adjusted back to the initial pH value. The effect of the enzymes on the activity of the antibacterial substances produced by strain M1 was then tested. The fermentation supernatant of the untreated strain was used as a blank control group, and MRSA was used as an indicator bacterium.

[0052] (2) Effect of temperature on antibacterial substances

[0053] The fermentation supernatant obtained from strain M1 in Example 3 was treated at 65 ℃, 85 ℃, 100 ℃, and 121 ℃ for 40 min each, and the effect of temperature on the inhibition of MRSA by the fermentation supernatant was measured.

[0054] (3) Effect of pH on antibacterial substances

[0055] The pH of the fermentation supernatant obtained in Example 3 was adjusted to 7.0 using 1 mol / L NaOH (the original pH of the fermentation supernatant was 3.52), with a supernatant containing an equal volume of sterile water used as a control. The inhibitory effect of the alkali-treated fermentation supernatant on MRSA was determined.

[0056] Results: The inhibitory effects of different enzyme treatments on MRSA in the fermentation supernatant of Enterobacter sieboldii M1 are shown in Table 2. As can be seen from Table 2, compared with the control, there was no significant difference in the inhibitory effects of the fermentation supernatant treated with proteinase K, pepsin, and catalase on MRSA. P <0.05, indicating that the antibacterial active substance produced by *Enterobacter sieboldii* M1 is insensitive to both proteases and catalase. Temperature sensitivity experiments showed no significant difference in the inhibitory activity of fermentation supernatants against MRSA after treatment at 65–121 °C for 40 min. P >0.05 (Table 3), indicating that the antibacterial active substance is heat-stable. When the pH value was adjusted to 7.0, the inhibitory activity of the fermentation supernatant against MRSA was completely lost (Table 4).

[0057] Table 2 Effects of different enzymes on antibacterial activity

[0058] Note: Different letters in the same column indicate significant differences. P <0.05).

[0059] Table 3 Effect of different temperatures on antibacterial activity

[0060] Table 4 Effect of pH on antibacterial substances

[0061] Note: "--" indicates no antibacterial properties.

[0062] Example 5

[0063] Identification of antibacterial active substances

[0064] (1) Extraction of antibacterial active substances

[0065] Following the method described in "Li Weina, Huang Wenyu, Liu Chenjian, et al. Main organic acids and their antibacterial properties in Lactobacillus casei fermentation broth [J]. Food Industry Technology, 2019, 40(3):66-70+6," the fermentation broth obtained in Example 3 after 48 h of culture was centrifuged at 12,000 r / min and 4 ℃ for 15 min. The supernatant was then mixed with ethyl acetate at a volume ratio of 1:1 and extracted three times with the aqueous phase to obtain an upper ethyl acetate phase, an intermediate flocculent phase, and a lower aqueous phase. The ethyl acetate phase and the intermediate white flocculent phase were concentrated at 45 °C and 150 r / min, and the aqueous phase was concentrated by rotary evaporation at 60 °C and 150 r / min. Each phase was concentrated by 10 times its volume. The concentrated phases were the aqueous rotary evaporation phase, the aqueous rotary evaporation residue phase, the intermediate rotary evaporation phase, the intermediate rotary evaporation residue phase, the ethyl acetate rotary evaporation residue phase, and the ethyl acetate rotary evaporation phase. The antibacterial effect of each phase was determined by the Oxford cup method using MRSA as the indicator bacterium.

[0066] Table 5. Inhibitory effect of Enterobacter mesenchymal stem cell M1 extract relative to MRSA (mm)

[0067] Note: "--" indicates no antibacterial properties.

[0068] Example 6

[0069] Safety evaluation of strains

[0070] (1) Detection of amino acid decarboxylase

[0071] Strain M1 was inoculated at a 3% inoculation rate into four different amino acid decarboxylase detection media and cultured statically at 37 °C for 48 h. The color changes were observed, with MRS medium used as the control group.

[0072] (2) Nitrate reductase detection experiment

[0073] Strain M1 was inoculated into nitrate medium at a 3% inoculation rate and incubated at 37 °C for 48 h. Then, 10 drops of 5% (w / w) potassium iodide solution were added, followed by 10 drops of 5% (w / w) starch solution. After thorough mixing, the color change of the medium was observed, with the uninoculated medium serving as the control group.

[0074] (3) Drug sensitivity test

[0075] The diameter of the inhibition zones of five antibiotics—tetracycline, cefoxitin, penicillin, streptomycin, chloramphenicol, and vancomycin—against bacterial strain M1 was determined using the Kirby-Bauer (KB) disk diffusion method recommended by the Clinical Laboratory Standards Institute (CLSI) to assess drug resistance. After overnight activation, the bacterial suspension of strain M1 was diluted with sterile MRS liquid medium to allow it to reach OD200. 625 A bacterial suspension with a value between 0.08 and 0.13 (zeroed using sterile MRS liquid medium as a blank) was then evenly spread onto MRS solid medium using a sterile cotton swab. Antimicrobial susceptibility testing discs were placed on the surface of the medium using sterile forceps, and incubated at 37 °C for 21 h. The discs were then removed, and the diameter of the inhibition zone was measured using calipers. Each experiment was repeated three times.

[0076] (4) Plasmid extraction test

[0077] Take 3-5 mL of activated strain M1 suspension and extract the plasmid of the target strain using a plasmid extraction kit and lysozyme according to the manufacturer's instructions. Detect using a 1% agarose gel, and then use an automated gel imaging system to observe for the appearance of electrophoretic bands.

[0078] Results: Nitrate reductase can catalyze the reduction of nitrate to nitrite, which in turn produces nitrosamines. Nitrosamines can cause various diseases such as stomach cancer and liver cancer. The results of this invention show that the bacterial solutions did not turn blue, indicating a negative result (see Table 6), suggesting that the metabolites of *Enterobacter sieboldii* M1 contain no nitrate reductase activity or no nitrate reductase at all.

[0079] Some lactic acid bacteria possess amino acid decarboxylase activity, capable of decarboxylating and reducing amino acids to amines. Excessive accumulation of amines in the body can cause toxicological effects and make the culture environment alkaline. When bromocresol purple indicator is added, a yellow color on the culture medium indicates a negative result, while a purple color indicates a positive result. A yellow result indicates that the metabolites of *Enterobacter sieboldii* M1 do not contain amino acid decarboxylase, meaning they are harmless to human health.

[0080] Table 6 Detection Results of Harmful Metabolites

[0081] Table 7 shows the results of the drug susceptibility test. The results indicate that chloramphenicol showed a high sensitivity to *Enterobacter vesiculosus* M1 with an inhibition zone diameter of 27.12 ± 0.29 mm, moderate sensitivity to penicillin and streptomycin, but resistance to tetracycline and cefoxitin. In recent years, the presence of transferable resistance genes in lactic acid bacteria has been a major focus of research on their resistance. Many studies have shown that lactic acid bacteria resistance originates from two sources: inherent resistance and resistance acquired from foreign DNA. Research suggests that most lactic acid bacteria resistance is not transferable, but it can become transferable after binding to plasmids. To further investigate the risk of transferable antibiotic resistance in *Enterobacter vesiculosus* M1, plasmids were extracted from the strain. The plasmid extraction electrophoresis image is shown in Figure 5. After detecting the plasmid of Enterobacter vesiculosus M1, no band was found. It can be inferred that Enterobacter vesiculosus M1 does not contain drug-resistant plasmids or the drug-resistant gene is only present on the genomic DNA. Therefore, it can be preliminarily determined that Enterobacter vesiculosus M1 has no risk of drug resistance transfer. Figure 5 Electrophoresis image of plasmid extraction; M-DL2000 Marker, 1-W. Paramesenteroides M1 plasmid.

[0082] Table 7 Results of drug susceptibility testing

[0083] Note: R (Resistant) – drug resistant, I (Insensitive) – intermediate, S (Sensitivity) – sensitive; "--" indicates no zone of inhibition; Judgment criteria: Clinical & Laboratory Standards Institutions (CLSI) criteria.

[0084] Example 7

[0085] Comparative experiment: (1) Prepare 9 kg of radish pickles according to the method of Example 1 in CN201510250096.1, divide them into 9 portions, and put them into pickle jars for later use. Three jars are randomly selected from each experimental group.

[0086] (2) Experimental group 1 was a bacterial suspension of the aforementioned Enterobacter sieboldii, with a bacterial concentration of 1×10⁻⁶. 8 CFU / mL; the addition ratio is 50g / kg.

[0087] Control group 1 consisted of potassium sorbate, with an addition ratio of 0.5 g / kg.

[0088] The blank control group 1 received no treatment.

[0089] Preservatives from different experimental groups were added to the kimchi jars and mixed thoroughly. The kimchi from each experimental group was then stored at 5℃, 15℃, and 25℃, respectively. After 60 days of storage, the appearance, flavor, and taste of the kimchi were assessed, and the number of miscellaneous bacteria was measured and averaged. The results are shown in Tables 8-10.

[0090] Note: "Miscellaneous bacteria" refers to Gram-negative microorganisms such as *Escherichia coli*, *Acinetobacter*, *Vibrio*, and *Proteus*, which are detrimental to food fermentation, human health, or flavor production. Cells are stained with crystal violet solution and iodine solution, destained with 95% ethanol, and stained again with fuchsin solution. After this treatment, microscopic observation reveals that the microorganisms stained red are Gram-negative bacteria. They are then counted using a hemocytometer.

[0091] Table 8. Survey Results of Storage at 5℃

[0092] Table 9. Survey Results of Storage at 15℃

[0093] Table 10. Survey Results of Storage at 25℃

[0094] As can be seen from Tables 8-10, the storage results of kimchi differ under different storage conditions, which may be related to the inhibition of microbial activity at low temperatures. However, regardless of the storage temperature, experimental group 1 showed the best results, followed by control group 1. This indicates that the *Enterobacter vesiculosus* bacterial solution described in this invention can be used as a preservative for kimchi, and its effect is significantly better than that of preservatives in existing technologies.

[0095] Example 8

[0096] Comparative experiment: (1) Select a piece of chilled pork and divide it into 9 pieces. Three pieces are randomly selected for each experimental group.

[0097] (2) Experimental group 2 was a bacterial suspension of the aforementioned Enterobacter sieboldii, with a bacterial concentration of 1×10⁻⁶. 8 CFU / mL; the addition ratio is 50g / kg, and the bacterial suspension is sprayed onto the surface of chilled meat using a small sprayer.

[0098] Control group 2 was YC9-6-1 chilled meat preservative (Wuhan Fresh Preservation Biotechnology Co., Ltd.), and the usage method was as per the instruction manual.

[0099] The blank control group 2 received no treatment.

[0100] The processed chilled meat from each experimental group was wrapped in plastic wrap and stored at 4°C. Meat quality was measured on days 7 and 14, with three measurements taken as the average. The results are shown in Table 11.

[0101] (3) Measured data: 1) The determination of 2-thiobarbituric acid (TBA) was performed using the direct method for determining the 2-thiobarbituric acid value of animal and vegetable oils.

[0102] The total fatty acid oxidation (TBA) value in meat products reflects the degree of final fat oxidation in muscle tissue. A higher TBA value indicates a greater degree of rancidity and poorer meat quality. Evaluation standard: TBA > 1 mg / kg indicates spoiled meat.

[0103] 2) Determination of volatile basic nitrogen (TVB-N) using a semi-micro nitrogen determination method.

[0104] Evaluation criteria: A TVB-N value of ≤15mg / 100g indicates Grade 1 freshness, 15mg / 100g~25mg / 100g indicates Grade 2 freshness, and >25mg / 100g indicates spoiled meat.

[0105] 3) The total number of colonies was determined using the plate count method.

[0106] Evaluation criteria: Meat products with a total bacterial count log value less than 4.0 are considered Grade 1 freshness, those with a total bacterial count log value between 4.0 and 6.0 are considered Grade 2 freshness, and those with a total bacterial count log value greater than 6.0 are considered spoiled meat.

[0107] 4) Use centrifugation to determine the water holding capacity of meat.

[0108] Table 11 Test Results of Fresh Chilled Meat

[0109] As can be clearly seen from Table 11, the *Enterobacter vesiculosus* described in this invention has excellent effects on meat preservation. Its TBA, TVB-N, and total bacterial count are significantly better than the control group, and its water retention rate is significantly higher than other control groups. Therefore, the *Enterobacter vesiculosus* described in this invention can significantly extend the shelf life of chilled meat and is significantly superior to existing products. This is sufficient to prove that the *Enterobacter vesiculosus* provided by this invention can be used as a food preservative.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of enterobacterial Weissella ( Weissella Paramesenteroides M1, this bacterium is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 14, 2023, with accession number CGMCC NO: 26182, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The use of the Enterobacter vesiculosus of claim 1 in the preparation of preservatives.

3. A preservative, characterized in that, Includes the enteromembranous Weissella as described in claim 1.

4. The preservative according to claim 3, characterized in that, The aforementioned Enterobacter vesiculosus is a bacterial suspension of Enterobacter vesiculosus.

5. The preservative according to claim 4, characterized in that, The concentration of the bacterial suspension is 1×10⁻⁶. 6 ~1×10 10 CFU / mL.

6. The use of the preservative according to any one of claims 3 to 5 in food preservation.

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