Pediococcus pentosaceus TRM54998 with broad-spectrum antibacterial activity and application thereof
By using Pediococcus pentosaceus TRM54998 and its fermentation supernatant, the problem of poor inhibitory effect on Salmonella in the existing technology has been solved, achieving safe and efficient broad-spectrum antibacterial effect and drug resistance mitigation, and providing applications for biological antibacterial agents and intestinal probiotics.
Patent Information
- Application Number
- CN202511410941.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-11-18
AI Technical Summary
There is a lack of Pediococcus pentosaceus strains with highly effective inhibitory activity against Salmonella in the current technology, chemical preservatives pose potential health risks, and the overuse of antibiotics has led to serious drug resistance problems. There is an urgent need to develop safe and effective alternatives.
We provide a strain of Pediococcus pentosaceus TRM54998 and its fermentation supernatant, which has broad-spectrum antibacterial activity and can grow in acidic and bile salt environments. The fermentation supernatant has a stable antibacterial effect against a variety of pathogens, including Salmonella, and is not prone to developing drug resistance.
The fermentation supernatant of Pediococcus pentosaceus TRM54998 exhibits stable antibacterial effects against a variety of pathogens, showing broad application prospects, alleviating drug resistance problems, and providing strain resources for biological antibacterial agents and intestinal probiotics.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, and in particular to a strain of Pediococcus pentosaceus TRM54998 with broad-spectrum antibacterial activity and its applications. Background Technology
[0002] Salmonella is a common and serious foodborne pathogen, widely distributed in nature, such as animal intestines, water bodies, and soil. In the food industry, it can contaminate a variety of foods, including meat and meat products, eggs, dairy products, vegetables, and fruits. According to the World Health Organization (WHO), millions of foodborne illnesses are caused by Salmonella infection globally each year. Patients often experience symptoms such as fever, abdominal pain, diarrhea, and vomiting, and in severe cases, it can develop into sepsis, meningitis, and even death. This bacterium poses a particularly significant threat to children, the elderly, and people with weakened immune systems. Currently, the control of Salmonella contamination mainly relies on chemical preservatives and antibiotics. However, the former may pose potential health risks, while the latter has led to increasingly serious problems of drug resistance due to overuse and disrupts the microbial ecological balance. Therefore, there is an urgent need to develop safe and effective alternatives. Natural antibacterial substances have become a research hotspot, among which antimicrobial peptides produced by lactic acid bacteria metabolism have attracted much attention due to their high safety and strong antibacterial activity. However, the resources of lactic acid bacteria strains with highly effective inhibitory activity against Salmonella are still relatively limited.
[0003] Pediococcus pentosaceus, a member of the Lactobacillus genus, has gradually attracted researchers' attention in recent years. Existing studies have shown that this bacterium has potential in food fermentation and intestinal microecological regulation, but research on its antibacterial activity, especially its inhibitory effect against important foodborne pathogens such as Salmonella, remains limited. Developing Pediococcus pentosaceus strains with highly effective inhibitory activity against Salmonella is of great significance for addressing Salmonella contamination, ensuring food safety, and reducing the use of chemical preservatives and antibiotics. Currently, there are no reports or applications of Pediococcus pentosaceus strains related to this invention that exhibit significant antibacterial activity against multiple pathogens (especially Salmonella). Summary of the Invention
[0004] The purpose of this invention is to provide a broad-spectrum antibacterial strain of Pediococcus pentosaccharide TRM54998 and its applications, in order to solve the problems existing in the prior art. Pediococcus pentosaccharide TRM54998 has good acid and bile salt resistance, and its fermentation supernatant has an inhibitory effect on nine pathogens, including Salmonella. The antibacterial effect is stable and has broad application prospects.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] This invention provides a strain of Pediococcus pentosaceus TRM54998 with broad-spectrum antibacterial activity, which was deposited at the China Center for Type Culture Collection on June 4, 2024, with accession number CCTCC NO: M20241142, and deposit address: Wuhan University, Wuhan, China.
[0007] The present invention also provides the fermentation supernatant of *Pediococcus pentosaceus* TRM54998, which is prepared by the following method:
[0008] After culturing the *Pediococcus pentosaceus* TRM54998, centrifuge to collect the supernatant, which is then vacuum dried, dissolved, and filtered to obtain the final product.
[0009] Optionally, the culture conditions are as follows: Pediococcus pentosaceus TRM54998 is inoculated into MRS liquid medium and cultured at 37°C and 120 r / min for 72 h.
[0010] Optionally, the centrifugation conditions are 4°C, 8000 r / min for 5 min.
[0011] The present invention also provides the application of the aforementioned Pediococcus pentosaceus TRM54998 or the aforementioned fermentation supernatant in inhibiting pathogenic bacteria.
[0012] The present invention also provides the use of the aforementioned Pediococcus pentosaceus TRM54998 or the aforementioned fermentation supernatant in the preparation of formulations that inhibit pathogenic bacteria.
[0013] Optionally, the pathogens include Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Shigella flexneri Castellani, or Erwinia amylovora.
[0014] The present invention also provides an inhibitory preparation for pathogenic bacteria, comprising the aforementioned Pediococcus pentosaceus TRM54998 or the aforementioned fermentation supernatant.
[0015] Optionally, the pathogens include Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enteritidis, Shigella, or Pythium spp.
[0016] The present invention discloses the following technical effects:
[0017] This invention isolated a strain of Pediococcus pentosaceus TRM54998 from yogurt curds in Mulei Kazakh Autonomous County, Changji City. It was deposited at the China Center for Type Culture Collection on June 4, 2024, with accession number CCTCC NO: M 20241142. This strain has been confirmed to have good acid and bile salt tolerance, and can grow in environments with bovine bile salt concentrations of 0.1-0.2% (w / v) or within a pH range of 3-7. Its fermentation supernatant has an inhibitory effect on nine pathogenic bacteria, including Salmonella (Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Shigella flexneri Castellani, and Erwinia amylovora), with stable antibacterial effects. Furthermore, Pediococcus pentosaceus TRM54998 can secrete bacterial peptides. Meanwhile, experimental verification showed that Pediococcus pentosaceus TRM54998 does not exhibit widespread drug resistance, which can alleviate problems such as increased drug resistance and disruption of microbial ecological balance caused by antibiotic abuse. This strain provides a strain resource for the preparation of biological antibacterial agents or intestinal probiotic agents and has broad application prospects. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 The colony growth of Pediococcus pentosacchari TRM54998 on MRS medium;
[0020] Figure 2 Gram-stained image of Pediococcus pentosaceus TRM54998;
[0021] Figure 3Phylogenetic tree of Pediococcus pentosaceus TRM54998;
[0022] Figure 4 Plate images showing the inhibition of four wild-type Salmonella species from sheep by Pediococcus pentosaceus TRM54998.
[0023] Figure 5 Minimum inhibitory concentration and inhibition curve of Pediococcus pentosaccharide TRM54998;
[0024] Figure 6 Genome cluster analysis of Pediococcus pentosaceus TRM54998;
[0025] Figure 7 Bacterial peptide analysis of the whole genome of Pediococcus pentosaceus TRM54998;
[0026] Figure 8 The results of antiSHMASH analysis of the whole genome of Pediococcus pentosaceus TRM54998;
[0027] Figure 9 The images show the protein structure of the antimicrobial peptide synthesis gene orf00038. The left image shows the protein structure after removing the leader sequence, and the right image shows the protein structure without removing the leader sequence.
[0028] Figure 10 The CAMPR4 prediction result for orf00038. Detailed Implementation
[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0034] Example 1: Isolation, purification and identification of Pediococcus pentosaceus TRM54998
[0035] MRS solid medium: 10 g peptone, 5 g beef extract, 4 g yeast extract, 20 g glucose, 2 g K2HPO4·3H2O, 5 g CH3COONa·3H2O, 2 g (NH4)3C6H5O7, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80, 16 g agar, and 1000 mL H2O, pH=6.2±0.2, sterilized at 121℃ for 15 min.
[0036] MRS liquid culture medium: 10 g peptone, 5 g beef extract, 4 g yeast extract, 20 g glucose, 2 g K2HPO4·3H2O, 5 g CH3COONa·3H2O, 2 g (NH4)3C6H5O7, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween-80 and 1000 mL H2O, pH=6.2±0.2, sterilized at 121℃ for 15 min.
[0037] 1. Isolation and purification of strains
[0038] The strain was derived from yogurt curds from Mulei Kazakh Autonomous County, Changji City, Xinjiang Uygur Autonomous Region.
[0039] Crush 3 g of yogurt curds and add them to a bottle of sterile milk containing 20 mL of liquid. Enrich at 37 °C and 120 r / min for 6 h. Then, take 1 mL of the enriched solution and serially dilute it. Select 10... -3 10 -4 and 10 -5 Three dilution gradients were plated on MRS plates and anaerobically incubated at 37°C for 1-3 days. Single colonies of different morphologies and colors were picked and streaked multiple times, followed by 2-3 generations of subculturing to obtain purified Pediococcus pentosaceus strain TRM54998 with consistent growth morphology. The cell morphology of the pure isolated bacteria was observed by Gram staining and microscopic examination, and the acid-producing capacity of the strain was tested using MRS medium containing 5% CaCO3.
[0040] Figure 1 The colony morphology of Pediococcus pentosaceus TRM54998 is shown. It can form calcium phosphate in MRS medium containing 5% CaCO3 and has the ability to produce acid.
[0041] 2. Classification and identification of strains.
[0042] 2.1 Biological identification
[0043] After culturing *Pediococcus pentosaceus* TRM54998 on MRS solid agar plates at 37°C for 2 days, the initial growth colonies appeared as small, opaque, milky-white dots. Later, the colonies became round, milky-white, with smooth, slightly raised edges, and a mucous-like substance adhering to the surface. The colony growth pattern is as follows: Figure 1 and Figure 2 As shown.
[0044] 2.2 System Classification and Identification
[0045] Colony PCR was used for identification. *Pediococcus pentosaceus* TRM54998 was picked from MRS solid medium plates using an inoculation loop. The bacterial block was diluted with 15 μL of sterile water, centrifuged at 12000 r / min for 5 min, the supernatant was discarded, and the bacterial suspension was resuspended in 15 μL of sterile water. Centrifugation was repeated once, and the suspension was incubated at 95℃ for 5 min, followed by placement on ice for 15–20 min. The treated bacterial suspension was used as a template for PCR identification. The target gene fragment was amplified using universal bacterial primers 27F and 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1); 1492R: 5'-GGCTACCTTGTTACGACTT-3' (SEQ ID NO.2)). The PCR reaction system was as follows: 1 μL each of 27F and 1492R primers (10 mmol / L), 3 μL of *Pediococcus pentosaceus* TRM54998 bacterial suspension, 12.5 μL of 2×PCR mix, and 7.5 μL of sterile water, totaling 25 μL; a control system was used without bacterial suspension. The amplification program was: 95℃ for 3 min; 95℃ for 1 min, 56℃ for 1 min, 72℃ for 1 min, 25 cycles; 72℃ for 10 min. The PCR products were submitted for sequencing (Sangon Sequencing (Xi'an) Co., Ltd.), and the 16S rDNA of *Pediococcus pentosaceus* TRM54998 was detected.
[0046] The obtained 16S rDNA was compared with the nucleic acid data in Ezbiocloud (https: / / www.ezbiocloud.net / identify). The results showed that *Pediococcus pentosaceus* TRM54998 and *Pediococcus pentosaceus* DSM 20336 strains had 99.39% homology. A phylogenetic tree was constructed using MOLECULAR EVOLUTIONARYGENETIC ANALYSIS software (MEGA 7.0). Figure 3 As shown, the sequences of Pediococcus pentosaceus TRM54998 and Pediococcus pentosaceus DSM 20336 can form a stable evolutionary branch and have a high degree of similarity.
[0047] Therefore, the above-mentioned strain was identified as Pediococcus pentosaceus, named Pediococcus pentosaceus TRM54998, which was deposited at the China Center for Type Culture Collection on June 4, 2024, with accession number CCTCC NO: M 20241142, and deposit address: Wuhan University, Wuhan, China.
[0048] Example 2 Evaluation of the acid and bile salt resistance of Pediococcus pentosaceus TRM54998
[0049] 1. Evaluation of acid resistance
[0050] Pediococcus pentosaceus TRM54998 was cultured overnight in MRS medium at 37°C, followed by centrifugation at 12,000 r / min for 5 min to collect bacterial cells. The bacterial cells were washed with sterile physiological saline and then 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, and 7.0 (using 1M hydrochloric acid). The culture was then incubated at 37°C under microaerophilic conditions for 24 h, and the OD was measured. 600 Value (with no bacteria added as a negative control, and with bacteria added to MRS medium at normal pH as a positive control).
[0051] 2. Evaluation of bile salt tolerance
[0052] Pediococcus pentosaceus TRM54998 was cultured overnight in MRS medium at 37°C, followed by centrifugation at 12,000 r / min for 5 min to collect bacterial cells. The bacterial cells were washed with sterile physiological saline and then resuspended in 10 mL of fresh MRS medium. 100 μL of the culture was inoculated into 10 mL of MRS medium, with the addition of 0%, 0.1%, 0.2%, and 0.3% ox bile salts, and incubated at 37°C under microaerophilic conditions for 24 h to detect OD. 600 Value (with no bacteria added as a negative control, and with bacteria added to MRS medium at normal pH as a positive control).
[0053] Experimental results showed that Pediococcus pentosaceus TRM54998 could grow under acidic conditions of pH 3-7 and bile salts of 0.1-0.2%, and could continue to grow in MRS medium with pH 3-7 and bile salts of 0.1-0.2% (w / v).
[0054] Example 3: Detection of the antibacterial activity of Pediococcus pentosaceus TRM54998 against 9 pathogenic strains
[0055] 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.
[0056] Pediococcus pentosaceus strain TRM54998 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.
[0057] As shown in Table 1, the fermentation supernatant of Pediococcus pentosaceus TRM54998 showed good antibacterial effects against all nine pathogens.
[0058] Table 1. Antibacterial effects of Pediococcus pentosaceus TRM54998 against 9 pathogens.
[0059]
[0060] Example 4: Detection of the antibacterial activity of Pediococcus pentosaceus TRM54998 against four wild-type Salmonella strains from sheep.
[0061] 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.
[0062] Using the Oxford cup method, 200 μL of fermentation supernatant was added to LB plates containing 1% of four wild-type Salmonella strains from sheep (D58-1, D58-2, D62-3, and F21), and incubated at 37°C for 24 h. The size of the inhibition zone was then measured and recorded.
[0063] like Figure 4 As shown in Table 2, the fermentation supernatant of Pediococcus pentosaceus TRM54998 showed good antibacterial effects against four wild-type Salmonella ovis strains.
[0064] Table 2. Antibacterial effect of Pediococcus pentosaceus TRM54998 against four wild-type Salmonella strains from sheep.
[0065]
[0066] Example 5: Whole genome data analysis of Pediococcus pentosaceus TRM54998
[0067] Pediococcus pentosaceus strain TRM54998 was inoculated into MRS liquid medium and cultured at 37°C with shaking at 120 r / min until OD500. 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 Paisenuo Biotechnology Co., Ltd. for whole-genome sequencing and assembly annotation. Gene clusters of the whole genome of *Pediococcus pentosaccharis* TRM54998 were predicted using the Antismash online software (https: / / antismash.secondarymetabolites.org / ), and bacterial peptides of the whole genome of *Pediococcus pentosaccharis* TRM54998 were predicted using the Bagel5 online software (http: / / bagel5.molgenrug.nl / ).
[0068] like Figure 6 As shown, Antismash analysis of the whole genome of Pediococcus pentosaceus TRM54998 revealed the presence of three gene clusters in this strain: one T3PKS, one RiPP, and one terpene. Figure 7 As shown, Bagel 5.0 was used to analyze the encoding antimicrobial peptides and (non)bactericidal post-translational modified peptides in *Pediococcus pentosaceus* TRM5499. The results showed that TRM5499 has four antimicrobial peptide synthesis genes: coagulinA, plantaricin_s, and orf00038, among which orf00038 may be a novel antimicrobial peptide. Figure 8 As shown, antiSHMASH analysis also identified the gene orf00038. Figure 9 As shown, the left image is the protein structure of gene orf00038 after removing the leader sequence, and the right image is the protein structure of gene orf00038 without removing the leader peptide; both structures are typical α-helical structures. Figure 10 The results shown are the CAMPR4 prediction results for orf00038. The higher the score, the more likely it is to be an antimicrobial peptide (maximum score is 1).
[0069] Example 6: Study on the characteristics of fermentation supernatant of Pediococcus pentosaceus TRM54998
[0070] The physicochemical properties of antibacterial active substances were studied from three aspects: acid and alkali tolerance, thermal stability, and metal ion sensitivity.
[0071] The pH values of the fermentation supernatant of Pediococcus pentosacchari TRM54998 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.
[0072] 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.
[0073] 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.
[0074] As shown in Table 3, the relative antibacterial activity of *Pediococcus pentosaceus* TRM54998 decreased with increasing pH, reaching zero at pH 7, indicating that the fermentation supernatant of *Pediococcus pentosaceus* TRM54998 exhibits acid tolerance. Overall, temperature had little effect on the relative antibacterial activity of *Pediococcus pentosaceus* TRM54998, indicating high thermal stability of the fermentation supernatant. Using Na... + K + Ca 2+ Mg 2+ and Mn 2+ When treating the fermentation supernatant of *Pediococcus pentosaceus* TRM54998, the relative antibacterial activity was significantly different from the control group, exhibiting an inhibitory effect on antibacterial activity. Furthermore, the inhibitory effect increased with increasing metal ion concentration. Using Cu... 2+ Fe 2+ Fe 3+ And Al 3+ When treating the fermentation supernatant of *Pediococcus pentosaceus* TRM54998, the relative antibacterial activity was significantly different compared to the control group, indicating a promoting effect on antibacterial activity. (Cu) 2+ Fe 3+ And Al 3+ The promoting effect on antibacterial activity increases with increasing concentration; using Zn 2+ When treating the fermentation supernatant of *Pediococcus pentosaceus* TRM54998, there was no significant difference in relative antibacterial activity, indicating that Zn... 2+ It had no significant effect on antibacterial activity (Table 4).
[0075] Table 3. Effects of different treatments on the relative antibacterial activity of the fermentation supernatant of *Pediococcus pentosaceus* TRM54998.
[0076]
[0077] Table 4. Effects of different metal ion treatments on the relative antibacterial activity of the fermentation supernatant of *Pediococcus pentosaceus* TRM54998.
[0078]
[0079] Example 7: Detection of Antibacterial Curve of Fermentation Supernatant of Pediococcus Pentosaccharide TRM54998
[0080] To determine the minimum inhibitory concentration (MIC) of the fermentation supernatant of *Pediococcus pentosaceus* TRM54998 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.
[0081] To detect the inhibition curve of the fermentation supernatant of *Pediococcus pentosaceus* TRM54998, 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.
[0082] The results are as follows Figure 5 As shown, the fermentation supernatant of Pediococcus pentosaceus TRM54998 can maintain complete antibacterial activity for more than 50 h at a concentration of 32 mg / mL and for more than 18 h at a concentration of 8 mg / mL, indicating that the fermentation supernatant of Pediococcus pentosaceus TRM54998 has a stable antibacterial effect against Salmonella.
[0083] Example 8 Drug susceptibility testing of Pediococcus pentosaceus TRM54998.
[0084] 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 Pediococcus pentosaceus TRM54998 to 10 drugs in seven classes. The 10 drugs were: ampicillin (AMP), cefazolin (KZ), and cefoxitin (FOX) of β-lactams; tetracycline (TE) of tetracyclines; ciprofloxacin (CIP) of quinolones; chloramphenicol (C) of chloramphenicols; gentamicin (CN) and streptomycin (S) of aminoglycosides; rifampicin (RD) of rifamycins; and erythromycin (E) of macrolides.
[0085] As shown in Table 5, Pediococcus pentosaceus TRM54998 is sensitive to five major classes of antibiotics, namely ampicillin, cefazolin, chloramphenicol, rifampin and tetracycline, and moderately sensitive to cefoxitin. It can be seen that Pediococcus pentosaceus TRM54998 does not have extensive drug resistance, which is convenient for its later application and promotion.
[0086] Table 5. Drug susceptibility test results of Pediococcus pentosaceus TRM54998
[0087] AMP C E S CIP CN FOX RD KZ TE TRM54998 S S R R R R I S S S
[0088] Note: S: Sensitive; I: Moderately sensitive; R: Drug resistant.
[0089] 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 Pediococcus pentosaceus TRM54998 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: M20241142, and the deposit address is Wuhan University, Wuhan, China.
2. The fermentation supernatant of *Pediococcus pentosaceus* TRM54998 as described in claim 1, characterized in that, Prepared by the following method: After culturing the *Pediococcus pentosaceus* TRM54998, 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: Pediococcus pentosaceus TRM54998 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 Pediococcus pentosaceus TRM54998 as described in claim 1 or the fermentation supernatant as described in claim 2 in inhibiting pathogenic bacteria.
6. The use of Pediococcus pentosaceus TRM54998 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 Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Shigella flexneri Castellani, or Erwinia amylovora.
8. A preparation for inhibiting pathogenic bacteria, characterized in that, It contains Pediococcus pentosaceus TRM54998 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 Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enteritidis, Shigella, or Pythium bacillus.