Aradicatrata lactobacillus TRM54999 with broad-spectrum antibacterial activity and application of Aradicatrata lactobacillus TRM54999
By isolating and identifying Lactobacillus argituratii TRM54999, the problem of lack of antibacterial activity against multiple pathogens in existing technologies has been solved, providing a safe and efficient antibacterial solution that achieves stable inhibition of multiple pathogens and alleviates drug resistance.
Patent Information
- Application Number
- CN202511463955.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-14
- Publication Date
- 2025-12-12
AI Technical Summary
There is a lack of Lactobacillus argituratii strains with significant antibacterial activity against a variety of pathogens, especially Salmonella, in the current technology. Chemical preservatives pose food safety risks, and the overuse of antibiotics has led to serious drug resistance problems. There is an urgent need for safe and efficient alternatives.
We provide a strain of Lactobacillus argituratii TRM54999, which has broad-spectrum antibacterial activity, can grow in acidic and bile salt environments, and its fermentation supernatant has a stable antibacterial effect on a variety of pathogens such as Salmonella, without widespread drug resistance.
The fermentation supernatant of Lactobacillus argituratii TRM54999 exhibits significant antibacterial effects against a variety of pathogens, with high stability. It is suitable for preparing biological antibacterial agents and intestinal probiotics to alleviate drug resistance problems and has broad application prospects.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microorganisms, in particular to a Lactiplantibacillus argentinus TRM54999 with broad-spectrum antibacterial activity and its application. BACKGROUND
[0002] Salmonella is a common and extremely harmful foodborne pathogenic bacteria, widely exists in nature, such as animal intestinal tract, water, soil, etc. In the field of food, it can contaminate various foods, including meat and meat products, eggs, dairy products, vegetables and fruits, etc. According to the statistics of the World Health Organization (WHO), there are millions of cases of foodborne diseases caused by Salmonella infection worldwide every year, and patients often have symptoms such as fever, abdominal pain, diarrhea, vomiting, etc. In severe cases, it can cause sepsis, meningitis, and even endanger life. Especially for children, the elderly and people with low immunity, the harm is more serious. At present, the main means to control its pollution depends on chemical preservatives and antibiotics, but the former has food safety hidden dangers, and the latter causes the problem of drug resistance to be aggravated due to abuse, destroys the ecological balance of microorganisms, and urgently needs a safe and efficient alternative. Natural antibacterial substances have become the focus of research, among which bacterial peptides and other metabolites produced by lactic acid bacteria are highly concerned due to their high safety and strong antibacterial activity. However, the existing resources of high-efficiency antibacterial lactic acid bacteria strains against Salmonella are still scarce.
[0003] Lactiplantibacillus argentinus, as a member of the genus Lactiplantibacillus, has gradually entered the researchers' field of vision in recent years. Studies have shown that the bacteria have certain potential in food fermentation and intestinal microecological regulation, but the research on their antibacterial activity, especially the inhibition of important foodborne pathogenic bacteria such as Salmonella, is relatively less. Developing Lactiplantibacillus argentinus strains with high-efficiency inhibition of Salmonella is of great significance to solve the problem of Salmonella contamination, protect food safety, and reduce the use of chemical preservatives and antibiotics. However, there is currently no report or application of Lactiplantibacillus argentinus strains with significant antibacterial activity against multiple pathogenic bacteria, and especially outstanding inhibition effect on Salmonella. SUMMARY
[0004] The purpose of the present application is to provide a Lactiplantibacillus argentinus TRM54999 with broad-spectrum antibacterial activity and its application, to solve the problems existing in the prior art. The Lactiplantibacillus argentinus TRM54999 has good acid and bile salt resistance, and its fermentation supernatant has inhibition effect on 9 kinds of pathogenic bacteria such as Salmonella, with stable antibacterial effect and wide application prospect.
[0005] To achieve the above purpose, the present application provides the following solutions:
[0006] The present application provides a Lactiplantibacillus argentoratensis TRM54999 with broad-spectrum antibacterial activity, which was preserved in the China Center for Type Culture Collection on June 4, 2024, with the preservation number CCTCC NO: M 20241143 and the preservation address being Wuhan, Wuhan University, China.
[0007] The present application also provides a fermentation supernatant of the Lactiplantibacillus argentoratensis TRM54999, which is prepared by the following method:
[0008] After the Lactiplantibacillus argentoratensis TRM54999 is cultured, the supernatant is obtained by centrifugation, vacuum drying, dissolution, and filtration.
[0009] Optionally, the culture conditions are that the Lactiplantibacillus argentoratensis TRM54999 is inoculated into MRS liquid medium and cultured at 37℃ and 120 r / min for 72 h.
[0010] Optionally, the centrifugation conditions are centrifugation at 4℃ and 8000 r / min for 5 min.
[0011] The present application also provides an application of the Lactiplantibacillus argentoratensis TRM54999 or the fermentation supernatant in inhibiting pathogenic bacteria.
[0012] The present application also provides an application of the Lactiplantibacillus argentoratensis TRM54999 or the fermentation supernatant in preparing a preparation for inhibiting pathogenic bacteria.
[0013] Optionally, the pathogenic bacteria include one or more of Staphyloccocus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Salmonella enterica, Shigella flexneri Castellani, and Erwinia amylovora.
[0014] The present application also provides a preparation for inhibiting pathogenic bacteria, which comprises the Lactiplantibacillus argentoratensis TRM54999 or the fermentation supernatant.
[0015] Optionally, the pathogenic bacteria include one or more of Staphyloccocus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Salmonella enterica, Shigella flexneri Castellani, and Erwinia amylovora.
[0016] The present application discloses the following technical effects:
[0017] The present application discloses a strain of Lactiplantibacillus arrentatus TRM54999 isolated from yogurt nodules in Jimunai County, Altay, which was preserved in China Center for Type Culture Collection on June 4, 2024, with a preservation number of CCTCC NO: M20241143. It has been confirmed that the strain has good acid and bile salt tolerance, and can grow in an environment with a bovine bile salt concentration of 1-2% (w / v) or in a pH range of 3-7. The fermentation supernatant has inhibitory effect on nine pathogenic bacteria (Staphyloccocus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Salmonella enterica, Shigella flexneri Castellani, and Erwinia amylovora), and the bacteriostatic effect is stable. Lactiplantibacillus arrentatus TRM54999 can secrete bacterial peptides. At the same time, it has been verified through experiments that Lactiplantibacillus arrentatus TRM54999 has no extensive drug resistance, can alleviate the problems of increasing drug resistance caused by antibiotic abuse and destroying the ecological balance of microorganisms, and provides a strain resource for preparing a biological bacteriostatic agent or an intestinal probiotic agent, which has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.
[0019] Figure 1 The growth of Lactiplantibacillus arrentatus TRM54999 colonies on MRS medium;
[0020] Figure 2Gram stain photo of Lactobacillus agitatus TRM54999;
[0021] Figure 3 Phylogenetic tree of Lactobacillus agitatus TRM54999;
[0022] Figure 4 Plate photo of Lactobacillus agitatus TRM54999 against 9 pathogenic bacteria;
[0023] Figure 5 Plate photo of Lactobacillus agitatus TRM54999 against 4 wild type Salmonella from sheep;
[0024] Figure 6 Minimum inhibitory concentration and inhibition curve of Lactobacillus agitatus TRM54999;
[0025] Figure 7 Whole genome gene cluster analysis of Lactobacillus agitatus TRM54999;
[0026] Figure 8 Whole genome bacterial peptide analysis of Lactobacillus agitatus TRM54999. DETAILED DESCRIPTION
[0027] Various illustrative embodiments of the present application are now described in detail below. The following description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In other instances, well-known structures and functions have not been described in detail in order to avoid obscuring the understanding of the present application.
[0028] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of, for example, a parameter, an intermediate value of the parameter is encompassed if the intermediate value is within the range of values. Also, it is to be understood that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" includes a plurality of such components.
[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.
[0030] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the principles of the application, and such variations are of no concern to the inventors. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.
[0031] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.
[0032] Example 1 Isolation, purification and identification of Lactobacillus argutus TRM54999
[0033] MRS solid medium: Proteose peptone 10 g, beef extract powder 5 g, yeast extract powder 4 g, glucose 20 g, K2HPO4·3H2O 2 g, CH3COONa·3H2O 5 g, (NH4)3C6H5O7 2 g, MgSO4·7H2O 0.2 g, MnSO4·4H2O 0.05 g, Tween-80 1 mL, agar 16 g and H2O 1000 mL, pH = 6.2 ± 0.2, sterilized at 121℃ for 15 min.
[0034] MRS liquid medium: Proteose peptone 10 g, beef extract powder 5 g, yeast extract powder 4 g, glucose 20 g, K2HPO4·3H2O 2 g, CH3COONa·3H2O 5 g, (NH4)3C6H5O7 2 g, MgSO4·7H2O 0.2 g, MnSO4·4H2O 0.05 g, Tween-80 1 mL and H2O 1000 mL, pH = 6.2 ± 0.2, sterilized at 121℃ for 15 min.
[0035] 1. Isolation and purification of strains
[0036] The strain was derived from yogurt nodules in Jimunai County, Altay, Xinjiang Uygur Autonomous Region.
[0037] 3 g of yogurt nodules were crushed and placed in sterile milk containing 20 mL / bottle, and then enriched at 37℃ and 120 r / min for 6 h. Then 1 mL of the enrichment liquid was gradiently diluted, and 10 -3 , 10 -4 and 10 -5Three dilution gradients were coated on MRS plates, and incubated anaerobically at 37°C for 1-3 d. Single colonies with different morphologies and colors were picked and subcultured for 2-3 times, and then the purified and morphologically consistent L. argutum strain TRM54999 was obtained. The cell morphology of the purified bacteria was observed by Gram staining and microscopy, and the acid-producing ability of the strain was tested by MRS medium containing 5% CaCO3.
[0038] Figure 1 The colony morphology of L. argutum TRM54999 is shown, which can form calcium whole in MRS medium containing 5% CaCO3 and has acid-producing ability.
[0039] 2. Classification and identification of the strain
[0040] 2.1 Biological identification
[0041] After L. argutum TRM54999 was cultured on MRS solid medium plates at 37°C for 2 d, the colonies were initially small and opaque, and then became round, milky white, smooth-edged and slightly raised, with mucous material attached to the surface of the bacteria. The colony growth is shown in Figure 1 and Figure 2 .
[0042] 2.2 Systematic classification and identification
[0043] The Lactiplantibacillus argentoratensis TRM54999 was picked up from the MRS solid medium plate by using a colony PCR identification method. The bacterial mass was diluted with 15 μL sterile water, centrifuged at 12000 r / min for 5 min, the supernatant was discarded, and then 15 μL sterile water was used for suspension, and the centrifugation was repeated once. The bacterial suspension was treated at 95℃ water bath for 5 min, and then placed on ice for 15-20 min. The treated bacterial suspension was used as the PCR identification template, and the bacterial universal primers 27F and 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1); 1492R: 5'-GGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2)) were used for amplification to obtain the target gene fragment. The PCR reaction system was as follows: 27F and 1492R primers (10 mmoL) were 1 μL each, Lactiplantibacillus argentoratensis TRM54999 bacterial suspension was 3 μL, 2x PCR mix was 12.5 μL, sterile water was 7.5 μL, and the total volume was 25 μL. The control system was without bacterial suspension. The amplification program was as follows: 95℃ for 3 min; 95℃ for 1 min, 56℃ for 1 min, 72℃ for 1 min, 25 cycles; 72℃ for 10 min. The PCR product was submitted for sequencing (Shengong Sequencing (Xi'an) Co., Ltd.), and the 16S rDNA of Lactiplantibacillus argentoratensis TRM54999 was measured.
[0044] The obtained 16S rDNA was subjected to homology comparison with the nucleic acid data in Ezbiocloud (https: / / www.ezbiocloud.net / identify), and the results showed that the homology of Lactiplantibacillus argentoratensis TRM54999 and Lactiplantibacillus argentoratensis DSM 16365 strain was 99.59%. The phylogenetic tree was constructed by using software MOLECULAR EVOLUTIONARY GENETIC ANALYSIS software (MEGA 7.0), as shown in Figure 3 Lactiplantibacillus argentoratensis TRM54999 and Lactiplantibacillus argentoratensis DSM 16365 can form a stable evolutionary branch and have high similarity.
[0045] Therefore, the above strain is identified as Lactiplantibacillus argentoratensis, named Lactiplantibacillus argentoratensis TRM54999, which has been preserved in the China Center for Type Culture Collection on June 4, 2024, with the preservation number CCTCC NO: M20241143, and the address of the preservation center is Wuhan, Wuhan University, China.
[0046] Example 2 Evaluation of the acid resistance and bile salt resistance of Lactiplantibacillus argentoratensis TRM54999
[0047] 1. Evaluation of acid resistance
[0048] After Lactiplantibacillus argentoratensis TRM54999 was cultured overnight at 37°C in MRS medium, the bacterial cells were collected by centrifugation at 12,000 r / min for 5 min. The bacterial cells were washed with sterile normal saline and resuspended in 10 mL of fresh MRS medium. 100 μL of the culture was inoculated into 10 mL of MRS medium, and the MRS medium was adjusted to pH 3.0, 4.0, 5.0, 7.0 (with 1 M hydrochloric acid). The culture was incubated at 37°C under microaerobic conditions for 24 h to detect OD 600 value (with no bacteria as negative control, and normal pH MRS medium with bacteria as positive control).
[0049] 2. Evaluation of bile salt resistance
[0050] After Lactiplantibacillus argentoratensis TRM54999 was cultured overnight at 37°C in MRS medium, the bacterial cells were collected by centrifugation at 12,000 r / min for 5 min. The bacterial cells were washed with sterile normal saline and resuspended in 10 mL of fresh MRS medium. 100 μL of the culture was inoculated into 10 mL of MRS medium, and 0%, 0.1%, 0.2%, 0.3% bovine bile salt was added. The culture was incubated at 37°C under microaerobic conditions for 24 h to detect OD 600 value (with no bacteria as negative control, and normal pH MRS medium with bacteria as positive control).
[0051] The experimental results show that TRM54999 can grow under acidic conditions of pH 3-7 and bile salt 0.1-0.2%, and can continuously grow in MRS medium with pH 3-7 and bile salt 0.1-0.2% (w / v).
[0052] Example 3 Detection of the antibacterial activity of Lactiplantibacillus argentoratensis TRM54999 on 9 strains of pathogenic bacteria
[0053] The nine pathogens identified were: Staphylococcus aureus (S. aureus, Sa, ATCC 25923), Enterococcus faecalis (E. faecalis, Ef, ATCC 29212), Pseudomonas aeruginosa (P. aeruginosa, Pa, ATCC 27853), Escherichia coli (E. coli, Ec, ATCC 25922), Klebsiella pneumoniae (K. pneumoniae, Kp, ATCC 10031), Acinetobacter baumannii (A. baumannii, Ab, ATCC 19606), Salmonella enterica (S. enterica, Sal, CICC 10982), and Shigella. Flexneri Castellani (Shigella, S. Castellani, Sc, ATCC 12022) and Erwinia amylovora (Fire Plague Bacteria, E. amylovora, Ea, ATCC 15357) were purchased from the respective ATCC or CICC.
[0054] Lactobacillus argentifer TRM54999 strain was inoculated into 50 mL / bottle of MRS liquid medium and cultured at 37°C and 120 r / min for 72 h. The supernatant was collected by centrifugation (4°C, 8000 r / min, 5 min), vacuum dried using a freeze dryer, and quantitatively dissolved in distilled water. The supernatant was filtered through a 0.22 μm microporous membrane to obtain cell-free fermentation supernatant (referred to as fermentation supernatant). Using the Oxford cup method, 200 μL of the fermentation supernatant was added to LB agar plates containing 1% of nine pathogens: *S. aureus*, *E. faecalis*, *P. aeruginosa*, *E. coli*, *K. pneumonia*, *A. baumannii*, *S. enterica*, *S. castellani*, and *E. amylovora*. The plates were cultured at 37°C for 24 h, and the size of the inhibition zones was measured and recorded.
[0055] like Figure 4 As shown in Table 1, the fermentation supernatant of Lactobacillus argituratii TRM54999 showed good antibacterial effects against all nine pathogens.
[0056] Table 1. Antibacterial effect of Lactobacillus argyrate TRM54999 against 9 pathogens
[0057]
[0058] Example 4: Detection of the antibacterial activity of Lactobacillus argulatus TRM54999 against four strains of wild-type Salmonella ovis.
[0059] Four wild-type Salmonella strains from sheep, Sal D58-1, Sal D58-2, Sal D62-3, and Sal F21, have been published in the literature Liu, L., Liu, C., Wang, H., Tang, H., Chen, Z., Dou, X., Chang, J., Li, Z., Wang, Z., Mei, Y., & Ren, M. (2025). Probiotic potential of enterococcuslactis in improving egg production and quality in quails during late egg-laying period. Poultry science, 104(2), 104765, and Liu, Shengchen, Tao, Dayong, Qi, Meng, et al. Detection and analysis of drug resistance genes in Salmonella sheep from the southern foothills of the Tianshan Mountains [J]. Journal of Tarim University, 2023, 35(01): 9-18.
[0060] Using the Oxford cup method, 200 μL of fermentation supernatant of Lactobacillus argitulat TRM54999 was added to LB plates containing 1% of four wild-type Salmonella strains from sheep (D58-1, D58-2, D62-3, and F21). After incubation at 37°C for 24 h, the size of the inhibition zone was measured and recorded.
[0061] like Figure 5 As shown in Table 2, the fermentation supernatant of Lactobacillus argituratii TRM54999 showed good antibacterial effects against four wild-type Salmonella ovis strains.
[0062] Table 2. Antibacterial effect of Lactobacillus argentiferus TRM54999 against four wild-type Salmonella strains from sheep.
[0063]
[0064] Example 5: Whole genome data analysis of Lactobacillus argyrulatus TRM54999
[0065] Lactobacillus argentifer TRM54999 was inoculated into MRS liquid medium and cultured at 37°C with shaking at 120 r / min until OD540. 600The bacterial culture was set at 1.0. The culture was transferred to 50 mL screw-capped centrifuge tubes and centrifuged at 4°C and 8000 r / min for 10 min to collect the cells. The cells were then sent to Shanghai Paisennong Biotechnology Co., Ltd. for whole-genome sequencing and assembly annotation. Gene clusters of the *Lactobacillus gentius* TRM54999 genome were predicted using the Antismash online software (https: / / antismash.secondarymetabolites.org / ), and bacterial peptides of the *Lactobacillus gentius* TRM54999 genome were predicted using the Bagel5 online software (http: / / bagel5.molgenrug.nl / ).
[0066] like Figure 7 As shown, Antismash analysis of the whole genome of *Lactobacillus argentaria* TRM54999 revealed that this strain contains six gene clusters, including one T3PKS, two RiPP, two terpene, and one cyclic. Analysis of the encoded bacterial peptides and (non-)bactericidal post-translational modified peptides in *Lactobacillus argentaria* TRM5499 using Bagel 5.0 indicated that TRM5499 may possess the Epidermicin NI0 bacterial peptide gene cluster.
[0067] Figure 8 The whole genome bacterial peptide prediction of Lactobacillus argitulatus TRM54999 shows the Epidermicin NI0 bacterial peptide gene cluster.
[0068] Example 6: Study on the characteristics of fermentation supernatant of Lactobacillus argentaria TRM54999
[0069] The physicochemical properties of antibacterial active substances were studied from three aspects: acid and alkali tolerance, thermal stability, and metal ion sensitivity.
[0070] The pH values of the fermentation supernatant of Lactobacillus argyrulatus TRM54999 and MRS liquid medium were adjusted to 2, 3, 4, 5, 6, 7 and 8, respectively, and the effect of pH value on the fermentation supernatant was determined.
[0071] The fermentation supernatant was heated in water baths at 65℃, 85℃, and 100℃ for 10 min and 30 min, respectively, and the effect of temperature on the antibacterial active substances in the fermentation supernatant was measured.
[0072] Sterile solutions of NaCl, KCl, CaCl2, MgCl2, FeCl3, FeSO4, CuCl2, MnCl2, AlCl3, and ZnSO4 were added to the fermentation supernatant to final concentrations of 0.02 mol / L, 0.03 mol / L, and 0.05 mol / L, respectively. The effects of metal ions on the antibacterial activity in the fermentation supernatant were determined. The diameter of the inhibition zone against *S. enterica* in different treatment groups was tested using the Oxford cup method.
[0073] As shown in Table 3, the relative antibacterial activity of *Lactobacillus argentaria* TRM54999 decreased with increasing pH, reaching zero at pH 7, indicating that the fermentation supernatant of *Lactobacillus argentaria* TRM54999 exhibits acid tolerance. Overall, temperature had little effect on the relative antibacterial activity of *Lactobacillus argentaria* TRM54999, indicating high thermal stability of the fermentation supernatant. Using Na... + K + Ca 2+ Mg 2+ and Mn 2+ When treating the fermentation supernatant of *Lactobacillus argyroderma* TRM54999, the relative antibacterial activity was significantly different from the control group, exhibiting an inhibitory effect on antibacterial activity, and the inhibitory effect increased with increasing metal ion concentration; Cu 2+ Fe 3 + And Al 3+ When treating the fermentation supernatant of *Lactobacillus argentaria* TRM54999, the relative antibacterial activity was significantly different compared to the control group, and it had a promoting effect on antibacterial activity. (Cu) 2+ and Fe 3+ The promoting effect on antibacterial activity increases with increasing concentration, while different concentrations of Al 3+ Similar promoting effects on antibacterial activity; using Zn 2+ and Fe 2+ When treating the fermentation supernatant of Lactobacillus argituratii TRM54999, there was no significant difference in relative antibacterial activity, indicating that the two had no significant effect on antibacterial activity (Table 4).
[0074] Table 3. Effects of different treatments on the relative antibacterial activity of fermentation supernatant of Lactobacillus argytii TRM54999
[0075]
[0076] Table 4. Effects of different metal ion treatments on the relative antibacterial activity of fermentation supernatant of Lactobacillus argytii TRM54999
[0077]
[0078] Example 7: Detection of the antibacterial curve of fermentation supernatant of Lactobacillus argyrulatus TRM54999
[0079] To determine the minimum inhibitory concentration (MIC) of the fermentation supernatant of *Lactobacillus enterica* TRM54999 against *S. enterica*, *S. enterica* strain was inoculated into LB medium and incubated overnight at 37°C and 180 r / min. 200 μL of fermentation supernatant containing antimicrobial substances at concentrations of 132 mg / mL, 64 mg / mL, 32 mg / mL, 16 mg / mL, 8 mg / mL, 4 mg / mL, 2 mg / mL, and 0 mg / mL were added to 96-well plates, followed by the addition of 1% *S. enterica* pathogen test solution. A control without pathogen test solution was used. The plates were incubated overnight at 37°C, and OD was measured. 600 Numerical value.
[0080] To detect the inhibition curve of fermentation supernatant of Lactobacillus enterica TRM54999, 1% S. enterica pathogen was inoculated into LB medium containing 32 mg / mL, 16 mg / mL, 8 mg / mL, 4 mg / mL and 0 mg / mL, respectively, and then incubated at 37℃ and 800 r / min for 48 h using a high-throughput real-time microbial growth analyzer.
[0081] The results are as follows Figure 6 As shown, the fermentation supernatant of Lactobacillus argituratii TRM54999 maintained complete antibacterial activity for more than 50 hours at a concentration of 32 mg / mL and for more than 30 hours at a concentration of 16 mg / mL, indicating that the fermentation supernatant of Lactobacillus argituratii TRM54999 had a stable antibacterial effect against Salmonella.
[0082] Example 8: Drug susceptibility testing of Lactobacillus argituratii TRM54999.
[0083] According to the standard operating procedures and judgment criteria of the Clinical and Laboratory Standards Institute (CLSI, 2017), the disk diffusion method was used to test the susceptibility of Lactobacillus argentifer TRM54999 to 10 drugs in seven classes. The 10 drugs were: ampicillin (AMP), cefazolin (KZ), and cefoxitin (FOX) of the β-lactam class; tetracycline (TE) of the tetracycline class; ciprofloxacin (CIP) of the quinolone class; chloramphenicol (C) of the chloramphenicol class; gentamicin (CN) and streptomycin (S) of the aminoglycoside class; rifampicin (RD) of the rifamycin class; and erythromycin (E) of the macrolide class.
[0084] As shown in Table 5, Lactobacillus argentifer TRM54999 is sensitive to five major classes of antibiotics, namely ampicillin, cefoxitin, chloramphenicol, rifampin and tetracycline, and moderately sensitive to cefazolin. It can be seen that Lactobacillus argentifer TRM54999 does not have extensive drug resistance, which is convenient for its later application and promotion.
[0085] Table 5. Results of drug susceptibility testing for Lactobacillus arginulatae TRM54999
[0086] AMP C E S CIP CN FOX RD KZ TE TRM54999 S S R R R R S S I S
[0087] Note: S: Sensitive; I: Moderately sensitive; R: Drug resistant.
[0088] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A strain of *Lactiplantibacillus sargentoratensis* TRM54999 with broad-spectrum antibacterial activity, characterized in that... It was deposited at the China Center for Type Culture Collection on June 4, 2024, with accession number CCTCC NO: M 20241143.
2. The fermentation supernatant of *Lactobacillus argentifer* TRM54999 as described in claim 1, characterized in that, Prepared by the following method: After culturing the *Lactobacillus argyratis* TRM54999, centrifuge to collect the supernatant, which is then vacuum dried, dissolved, and filtered to obtain the final product.
3. The fermentation supernatant as described in claim 2, characterized in that, The culture conditions were as follows: Lactobacillus argentulatus TRM54999 was inoculated into MRS liquid medium and cultured at 37°C and 120 r / min for 72 h.
4. The fermentation supernatant as described in claim 2, characterized in that, The centrifugation conditions were 4℃, 8000 r / min for 5 min.
5. The application of *Lactobacillus argyrutilatus* TRM54999 as described in claim 1 or the fermentation supernatant as described in claim 2 in inhibiting pathogenic bacteria.
6. The use of Lactobacillus argyrutilatus TRM54999 as described in claim 1 or the fermentation supernatant as described in claim 2 in the preparation of formulations that inhibit pathogenic bacteria.
7. The application as described in claim 5 or 6, characterized in that, The pathogens include one or more of the following: Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Shigella flexneri Castellani, and Erwinia amylovora.
8. A preparation for inhibiting pathogenic bacteria, characterized in that, It contains either Lactobacillus argituratii TRM54999 as described in claim 1 or the fermentation supernatant as described in claim 2.
9. The formulation as described in claim 8, characterized in that, The pathogens include one or more of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enteritidis, Shigella, and Pythium bacillus.