Pediococcus acidilactici K25-01 and antimicrobial peptide and application

Abstract

The application discloses a pediococcus acidilactici K25-01 and an antibacterial peptide and application, and belongs to the technical field of microorganisms. Pediococcus acidilactici The pediococcus acidilactici K25-01 is preserved in the China Center for Type Culture Collection on November 24, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20252686. The pediococcus acidilactici K25-01 has a strong inhibiting effect on escherichia coli and staphylococcus aureus, and the pediococcus acidilactici K25-01 also has excellent intestinal survival ability, rich functional genes and outstanding metabolic pathway characteristics; the screened antibacterial peptide GQ-15 has stable bacteriostatic activity and high sterilization efficiency, and has important application potential in the fields of natural food bacteriostatic agents and bacteriostatic drugs.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and particularly relates to a type of lactic acid cocci K25-01, its antimicrobial peptides, and their applications. Background Technology

[0002] With the continuous growth of the global population, the incidence of foodborne diseases is also increasing year by year. Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus, S.aureus ) and Escherichia coli ( Escherichia coli, E. coli These are two common and important foodborne pathogens. Escherichia coli can cause not only diarrhea and food poisoning, but also serious consequences such as kidney failure; while Staphylococcus aureus, with its various toxins, can damage the host's immune system, leading to widespread infection.

[0003] Lactic acid bacteria are a class of bacteria that metabolize carbohydrates and produce large amounts of lactic acid. They are widely distributed and diverse, with most being safe microorganisms closely related to human life. Their metabolism produces various antibacterial substances such as bacteriocins and organic acids, often forming beneficial flora in the animal intestines. These antibacterial substances do not affect the flavor of food, but inhibit the growth of pathogenic and spoilage bacteria, extending the shelf life of food. Among them, bacteriocins have the most prominent effect, possessing advantages such as heat stability, non-toxicity, easy decomposition by the human body, and low likelihood of inducing drug resistance, making them highly promising for applications in food preservation, aquaculture, and natural preservatives.

[0004] Currently, traditional treatment methods face the problem of increasing antibiotic resistance, and the use of chemical preservatives has raised public concerns about health. Therefore, the development of novel antimicrobial agents is imperative. Antimicrobial peptides, as an emerging type of antimicrobial agent, have become a research hotspot due to their excellent inhibitory activity against Staphylococcus aureus and Escherichia coli, as well as their advantages of low resistance development and rapid degradation. This makes antimicrobial peptides a promising candidate for preventing and treating foodborne illnesses caused by these bacteria. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a *Pediococcus lactis* K25-01 and an antimicrobial peptide and its application. The *Pediococcus lactis* K25-01 and the antimicrobial peptide GQ-15 provided by the present invention have a strong inhibitory effect on *Escherichia coli* and *Staphylococcus aureus*, and the antibacterial activity is stable and the bactericidal efficiency is high.

[0006] To achieve the above objectives, the present invention provides a *Pediococcus lactis* ( Pediococcus acidilactici The lactic acid cocci K25-01 was deposited on November 24, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252686.

[0007] The present invention also provides an antimicrobial peptide GQ-15 prepared from the *Pediococcus lactis* K25-01 described in the above scheme, wherein the amino acid sequence of the antimicrobial peptide GQ-15 is shown in SEQ ID NO.2.

[0008] The present invention also provides the application of Porphyromonas lactis K25-01 or antimicrobial peptide GQ-15 as described above in the preparation of products with preservative or antibacterial effects.

[0009] Preferably, the effective viable count of Pediococcus lactis K25-01 in the product is 1×10⁻⁶. 9 ~7.7×10 9 CFU / mL.

[0010] Preferably, the effective concentration of the antimicrobial peptide GQ-15 in the product is ≥5 mg / mL.

[0011] The present invention also provides the application of Porphyromonas lactis K25-01 or antimicrobial peptide GQ-15 as described above in the preparation of antimicrobial drugs.

[0012] Preferably, the antibacterial drug inhibits bacteria including Escherichia coli and Staphylococcus aureus.

[0013] The present invention also provides an antibacterial drug, wherein the active ingredient of the drug includes the *Pediococcus lactis* K25-01 or the antimicrobial peptide GQ-15 described in the above-mentioned scheme.

[0014] This invention also provides the application of Pyrococcus lactis K25-01 or antimicrobial peptide GQ-15 as described above in the preparation of food preservatives that inhibit Escherichia coli and Staphylococcus aureus.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The *Pediococcus lactis* and antimicrobial peptides provided by this invention have strong inhibitory effects on *Escherichia coli* and *Staphylococcus aureus*, and exhibit stable antibacterial activity and high bactericidal efficiency. Furthermore, based on *Pediococcus lactis* K25-01, food preservatives that inhibit *Escherichia coli* and *Staphylococcus aureus* can be prepared. These products not only enrich market choices but also provide consumers with healthier and safer options.

[0016] (2) The antimicrobial peptides in this invention are not prone to drug resistance, have low cytotoxicity and high cell selectivity, and can be used to prepare antimicrobial compositions for treating Escherichia coli and Staphylococcus aureus infections.

[0017] (3) The Lactococcus lactis K25-01 of the present invention can be used to prepare drugs for adjuvant treatment or prevention of intestinal infections caused by pathogens such as Escherichia coli or Staphylococcus aureus. 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 inhibition zone experiments of Pediococcus lactis K25-01 against Escherichia coli and Staphylococcus aureus are shown in Figure A, where A is the inhibition zone experiment of Pediococcus lactis K25-01 against Escherichia coli and B is the inhibition zone experiment of Pediococcus lactis K25-01 against Staphylococcus aureus. Figure 2 To exclude the effects of acid inhibition, CFS in the figure represents the cell-free supernatant of the strain, K25-01 represents Pyrococcus lactis K25-01, and different lowercase letters indicate significant differences between groups. P <0.05); Figure 3 To exclude the effects of hydrogen peroxide, CFS in the figure represents the cell-free supernatant of the strain, K25-01 represents Pyrococcus lactis K25-01, and different lowercase letters indicate significant differences between groups. P <0.05); Figure 4 This is a graph of a protease sensitivity test. In the graph, CFS is the cell-free supernatant of the strain, K25-01 is Pediococcus lactis K25-01, and different lowercase letters indicate significant differences between groups. P <0.05); Figure 5 This is a graph showing the optimal growth temperature of the strain. In the graph, K25-01 represents Pediococcus lactis K25-01, and different lowercase letters indicate significant differences between groups. P <0.05); Figure 6 This is a graph showing the optimal pH value for the growth of the strain. In the graph, K25-01 represents Pediococcus lactis K25-01, and different lowercase letters indicate significant differences between groups. P <0.05); Figure 7 The figure shows the growth curve of the strain under optimal conditions. K25-01 in the figure is Pietrococcus lactis K25-01. Figure 8 The results are based on the morphological characteristics of the strain. Figure 9 Phylogenetic tree of Pediococcus lactis K25-01; Figure 10 Functional annotation and classification diagram of strain KEGG; Figure 11 Functional annotation and classification diagram of strain CAZy; Figure 12 Volcano diagram of differential metabolites before and after fermentation of Pediococcus lactis K25-01; Figure 13 These are upregulated metabolites from *Pediococcus lactis* K25-01 fermentation. In the figure, K25-01 represents *Pediococcus lactis* K25-01, and MK25-01 represents the initial inoculum for fermentation. Significant differences were observed between representative groups ( P <0.05), The differences between the representative groups were highly significant. P <0.01), The differences between the representative groups were highly significant ( P <0.001); Figure 14 The time-dependent killing curves of antimicrobial peptide GQ-15 against Escherichia coli and Staphylococcus aureus are shown. Figure 15 The figure shows the changes in the inhibition rate of antimicrobial peptide GQ-15 against Escherichia coli and Staphylococcus aureus after enzyme treatment. GQ-15 in the figure represents antimicrobial peptide GQ-15. Detailed Implementation

[0020] This invention provides a type of Pietrococcus lactis ( Pediococcus acidilactici Pediococcus lactis K25-01 was deposited at the China Center for Type Culture Collection on November 24, 2025, and classified as Pediococcus lactis. Pediococcus acidilactici, The deposit address is Wuhan University, Wuhan, China, and the accession number is CCTCC NO: M20252686.

[0021] In this invention, the *Pediococcus lactis* K25-01 was isolated from fermented mare's milk in Xilingol League, Inner Mongolia. After Gram staining, K25-01 appears as purple spherical or nearly elliptical spherical specimens, approximately 0.6–1.0 μm in diameter, mainly arranged in pairs or quadrupeds, without long chain structures. Molecular biological identification based on 16S rDNA sequencing confirmed that the gene sequence of K25-01 is related to… Pediococcus acidilactici

[0022] The present invention also provides an antimicrobial peptide GQ-15 prepared from the *Pediococcus lactis* K25-01 described in the above scheme, wherein the amino acid sequence of the antimicrobial peptide GQ-15 is shown in SEQ ID NO.2.

[0023] In this invention, the amino acid sequence of the antimicrobial peptide GQ-15 is shown in SEQ ID NO.2, SEQ ID NO.2: GPIGPVGARGPAGPQ. The antimicrobial peptide GQ-15 has a CAMPR3 score > 0, indicating predicted antibacterial activity; a net charge of 1; a GRAVY index < 0; a molecular weight of 1330.73 Da; and a basic pI.

[0024] In this invention, the preparation method of the antimicrobial peptide GQ-15 includes the following steps: 1) Centrifuge the lactic acid bacteria fermentation broth, collect the supernatant, and filter to obtain the filtrate; adjust the pH of the filtrate to 6.0~7.0, slowly add ammonium sulfate powder, stir overnight at 4℃; centrifuge again to discard the supernatant, resuspend the precipitate, desalt and freeze dry to obtain crude polypeptide extract; 2) The crude peptide extract was separated using a reversed-phase C18 column. Each elution peak was collected and freeze-dried. The purified peptide was dissolved in 50% acetonitrile aqueous solution and mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) at a volume ratio of 1:1. 1 μL of the mixture was spotted onto a target plate and allowed to air dry. The molecular weight of the peptide was obtained by mass spectrometry, and target components were screened. 3) The target component was digested with sequencing-grade trypsin. The digested product was desalted by a C18 nano-column and then loaded onto the sample. Liquid chromatography-tandem mass spectrometry was used for detection. Data-dependent scanning mode (DDA) was selected for mass spectrometry to acquire secondary mass spectra. The mass spectrometry data was compared with the lactic acid bacteria genome database (NCBI, EnsemblBacteria). The amino acid sequence was matched using Mascot software to obtain the target polypeptide sequence. 4) The target peptide sequence was converted into FASTA format. Then, the potential of the given peptide sequence to become AMPs was analyzed using online prediction software such as CAMPR4 and APD6. PeptideRanker predicted the bioactivity of the peptide. ProtParam analyzed the average coefficients of charge and hydrophilicity of the candidate peptide. ToxinPred was used to analyze the toxicity of the candidate peptide online. The sequence with the higher score was selected for chemical solid-phase synthesis to obtain the antimicrobial peptide GQ-15.

[0025] In this invention, the preparation method of lactic acid bacteria fermentation broth in step 1) preferably includes the following steps: taking the strain with antibacterial activity obtained from the initial screening, inoculating it into MRS liquid culture medium, and anaerobically culturing it at 37°C for 48 h; the centrifugation conditions are preferably 4°C, 10000 r / min for 5 min; the filtration is preferably carried out using a filter membrane, and the pore size of the filter membrane is preferably 0.22 μm; the saturation of the solution after adding ammonium sulfate powder is preferably 40%~80%; the re-centrifugation conditions are preferably 4°C, 12000×g for 20 min; the buffer used for resuspension is preferably PBS buffer, and the pH value of the PBS buffer is preferably 7~8, more preferably 7.4; the desalting is preferably carried out using a C18 solid-phase extraction column or dialysis bag, and the molecular weight cutoff of the solid-phase extraction column or dialysis bag is preferably 3~5 kDa.

[0026] In step 2), the preferred length of the reversed-phase C18 column is 250 mm, the preferred inner diameter of the column is 4.6 mm, and the preferred particle size of the packing material is 5 μm. The preferred separation conditions are: mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, the gradient elution program is preferably set to 0~60 min, and the proportion of phase B increases from 5% to 60%. The flow rate is set to 1 mL / min, the column temperature to 30℃, and the detection wavelength to 214 nm. The preferred conditions for the mass spectrometry are positive ion mode, accelerating voltage to 20 kV, and scan range to 500~10000 Da.

[0027] The preferred conditions for enzymatic hydrolysis in step 3) are a mass ratio of trypsin to target polypeptide of 1:50, and enzymatic hydrolysis at 37°C for 12 h; detection is performed using liquid chromatography-tandem mass spectrometry, with gradient elution of 0.1% formic acid aqueous solution and acetonitrile solution as the mobile phase.

[0028] The sequence with the higher score mentioned in step 4) is preferably chemically synthesized by solid-phase synthesis at Jier Biochemical (Shanghai) Co., Ltd.; the purity and molecular weight of the synthesized peptide are preferably determined by high performance liquid chromatography and mass spectrometry; the purity of the synthesized peptide is preferably >95%; the synthesized peptide is preferably frozen and stored at -20℃.

[0029] The present invention also provides the application of Porphyromonas lactis K25-01 or antimicrobial peptide GQ-15 as described above in the preparation of products with preservative or antibacterial effects.

[0030] In this invention, the product preferably includes preservatives and antibacterial agents. The effective viable count of *Pediococcus lactis* K25-01 in the product is preferably 1 × 10⁻⁶. 9 ~7.7×10 9 CFU / mL, more preferably 1×10⁻⁶ 9CFU / mL; the preferred effective concentration of the antimicrobial peptide GQ-15 in the product is ≥5 mg / mL. At a concentration of 10 mg / mL, the antimicrobial peptide exhibits 100% inhibition against both Escherichia coli and Staphylococcus aureus, effectively inhibiting both Gram-negative and Gram-positive foodborne pathogens simultaneously.

[0031] The present invention also provides the application of Porphyromonas lactis K25-01 or antimicrobial peptide GQ-15 as described above in the preparation of antimicrobial drugs.

[0032] In this invention, the pathogenic bacteria inhibited by the antibacterial drug preferably include Escherichia coli and Staphylococcus aureus.

[0033] The present invention also provides an antibacterial drug, wherein the active ingredient of the drug includes the *Pediococcus lactis* K25-01 or the antimicrobial peptide GQ-15 described in the above-mentioned scheme.

[0034] In this invention, the dosage form of the medicine preferably includes an oral preparation, which preferably includes powder, tablets, granules, capsules, or decoction. In this invention, the *Pediococcus lactis* K25-01 possesses acid- and bile salt-resistant properties, enabling it to survive in the body through the gastrointestinal tract of mammals. In this invention, the *Pediococcus lactis* K25-01 exerts its effect as a live bacterium.

[0035] This invention also provides the application of Pyrococcus lactis K25-01 or antimicrobial peptide GQ-15 as described above in the preparation of food preservatives that inhibit Escherichia coli and Staphylococcus aureus.

[0036] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a *Pediococcus lactis* K25-01 strain, its antimicrobial peptides, and their applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0037] The culture medium formulations used in the examples are shown in Table 1.

[0038] Table 1 Culture medium formulation

[0039] Example 1 Isolation, screening, and basic characteristics of lactic acid bacteria.

[0040] 1. Isolation and screening of lactic acid bacteria.

[0041] Lactic acid bacteria derived from fermented mare's milk were inoculated into MRS agar medium and cultured at 37°C for 24–48 h to enrich the lactic acid bacteria. The enriched lactic acid bacteria were then inoculated onto MRS solid medium using the streak plate method and cultured at 37°C for 24–48 h. Single colonies were then picked for purification culture.

[0042] The Oxford cup method was used for initial screening. The concentrations of known indicator bacteria, *Escherichia coli* and *Staphylococcus aureus*, were adjusted to 10⁻⁶. 6 After CFU / mL concentration, the sample was spread onto MRS solid medium plates, and then Oxford cups were placed on the plates. After a period of incubation, if the strain could produce antibacterial substances, a clear inhibition zone would form outside the Oxford cup. The diameter of the inhibition zone was measured to preliminarily screen strains with antibacterial activity.

[0043] The results show (e.g.) Figure 1 The inhibition zones of *Pediococcus lactis* K25-01 against *Escherichia coli* and *Staphylococcus aureus* were 21.30±0.72 mm and 21.50±0.85 mm, respectively.

[0044] 2. Basic characteristics of lactic acid bacteria.

[0045] Preparation method of cell-free supernatant (CFS) of the strain: The strain with antibacterial activity obtained from the initial screening was inoculated into MRS liquid medium and anaerobic cultured at 37℃ for 48h; after the culture was completed, it was centrifuged at 4℃ and 10000r / min for 5min, the supernatant was collected, and filtered through a 0.22μm filter membrane to remove bacteria to obtain cell-free supernatant (CFS).

[0046] 1) Using Escherichia coli as an indicator bacterium, a CFS treatment group and an organic acid (lactic acid, acetic acid) control group were set up to eliminate the interference of fermentation acid production on the antibacterial effect.

[0047] CFS treatment group: cell-free supernatant containing antibacterial strains; Lactic acid treatment group: After measuring the pH value of CFS, lactic acid solution was prepared using sterile PBS buffer as solvent, and its pH value was adjusted to be consistent with that of CFS. After being filtered through a 0.22μm filter membrane for sterilization, it was ready for use.

[0048] Acetic acid treatment group: After measuring the pH value of CFS, acetic acid solution was prepared using sterile PBS buffer as solvent, and its pH value was adjusted to be consistent with that of CFS. After being filtered through a 0.22μm filter membrane for sterilization, it was ready for use.

[0049] The same volume of antibacterial agent was used in each treatment group to compare the inhibitory effects of CFS and organic acid (lactic acid, acetic acid) simulated solutions on Escherichia coli. If the antibacterial effects of the lactic acid treatment group and the acetic acid treatment group were weaker than those of the CFS treatment group, it indicates that the antibacterial activity of CFS is not entirely caused by fermentation acid production, but may be related to other antibacterial active substances produced by the strain.

[0050] The results show (e.g.) Figure 2The strain exhibited strong antibacterial activity, with an inhibition zone diameter of approximately 20 mm, while the antibacterial activity of the acidic control group was significantly lower than that of the lactic acid bacteria group. These results indicate that the antibacterial effect of lactic acid bacteria is not solely dependent on fermentation and acid production, but is closely related to other active substances contained in its CFS (Continuous Flavor Surface).

[0051] 2) The cell-free supernatant (CFS) was treated with catalase to eliminate the antibacterial effect of hydrogen peroxide.

[0052] CFS treatment group: cell-free supernatant containing antibacterial strains; Catalase treatment group: Catalase was dissolved in sterile PBS buffer at pH 7.0 to prepare an enzyme solution with a final concentration of 10 mg / mL. The catalase solution was added to cell-free supernatant (CFS) at a volume ratio of 1:100 and incubated at 37°C for 1 hour.

[0053] The results show (e.g.) Figure 3 After treatment with catalase, the antibacterial activity of *Pediococcus lactis* decreased only slightly, but the diameter of the inhibition zone remained at a relatively high level of 19-21 mm, and there was no significant difference between the CFS treatment group and the catalase treatment group. These results indicate that hydrogen peroxide is not the main active substance responsible for the antibacterial effect of *Pediococcus lactis*, and its antibacterial effect depends more on other components in the fermentation supernatant.

[0054] 3) Prepare solutions of trypsin, pepsin, and proteinase K under different pH conditions. After adjusting the CFS to match the corresponding reaction pH, add different proteases and incubate at a constant temperature. After inactivation by boiling water bath, pH adjustment and sterilization by filter membrane, the changes in antibacterial activity are finally detected by Oxford cup method to explore the protease sensitivity of antibacterial active substances.

[0055] CFS treatment group: cell-free supernatant containing antibacterial strains; Trypsin treatment group: Dissolve an appropriate amount of trypsin in PBS buffer at pH 7.0 for later use. Adjust the pH of CFS to 7.0. Add the trypsin solution to CFS until the final concentration of trypsin is 1 mg / mL. React at 37℃ for 2 h. Then, inactivate the enzyme by boiling in a water bath for 5 min. Adjust the pH of CFS to the initial pH. Filter with a 0.22 μm aqueous filter membrane and conduct antibacterial experiments. Pepsin treatment group: Dissolve an appropriate amount of pepsin in PBS buffer at pH 2.0 for later use. Adjust the pH of CFS to 2.0. Add the pepsin solution to CFS until the final pepsin concentration is 1 mg / mL. React at 37℃ for 2 h, then inactivate the enzyme by boiling in a water bath for 5 min. Adjust the pH of CFS to the initial pH, filter with a 0.22 μm aqueous filter membrane, and conduct antibacterial experiments. Proteinase K treatment group: Dissolve an appropriate amount of proteinase K in PBS buffer at pH 7.0 for later use. Adjust the pH of CFS to 7.0. Add proteinase K solution to CFS until the final concentration of proteinase K is 0.5 mg / mL. React at 37℃ for 2 h. Then boil in water for 5 min to inactivate the enzyme. Adjust the pH of CFS to the initial pH. Filter with a 0.22 μm aqueous filter membrane and conduct antibacterial experiments.

[0056] The results show (e.g.) Figure 4 The CFS treatment group showed the highest antibacterial activity. After treatment with trypsin and pepsin, the antibacterial activity was significantly reduced (P<0.05). Although the antibacterial activity decreased after treatment with proteinase K, it still remained at a relatively high level of 17-19 mm. Based on this, it can be preliminarily determined that the antibacterial active substances in the lactic acid bacteria fermentation broth are polypeptides or proteins.

[0057] 4) K25-01 bacterial suspensions that have been continuously activated for two generations were inoculated into MRS medium at an inoculation rate of 2% (v / v) and cultured for 24 h at different temperatures of 28℃, 32℃, 37℃, 41℃ and 45℃ before the viable count was determined.

[0058] The results show (e.g.) Figure 5 K25-01 showed the highest viable bacterial count at 40℃, at 1×10⁻⁶. 9 The CFU / mL concentration showed an optimal growth temperature of 40℃ and maintained a high growth level even at 45℃, demonstrating good heat resistance.

[0059] 5) K25-01 bacterial suspensions that have been continuously activated for two generations were inoculated into MRS medium with pH values ​​of 4.5, 5.5, 6.5, 7.5, and 8.5 at an inoculation rate of 2% (v / v) and cultured for 24 hours before the viable count was determined.

[0060] The results show (e.g.) Figure 6 K25-01 grows best under pH conditions of 6.5 to 7.5, with the highest number of viable bacteria, showing adaptability to slightly acidic to neutral environments.

[0061] 6) Under the optimal growth temperature of 40℃ and pH conditions, K25-01 was inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured. The absorbance value (OD value) at 600nm was measured every 2 hours, and the growth curve was plotted.

[0062] The results show (e.g.) Figure 7 The growth curve of K25-01 exhibits typical microbial growth cycle characteristics, including the lag phase, logarithmic growth phase, and stationary phase. During the logarithmic growth phase, the microorganism proliferates rapidly, while the OD value remains at a high level during the stationary phase, indicating that the strain is actively growing and has sufficient biomass.

[0063] 7) After K25-01 was cultured to the third generation, it was inoculated into MRS liquid medium at pH=3 at a 2% (v / v) inoculum and cultured for 3 hours, and the OD value was measured.

[0064] The results (as shown in Table 2) showed that the OD value of K25-01 under acidic culture conditions was 0.266±0.009, which was significantly increased compared with the initial value, indicating that the strain has good acid resistance.

[0065] 8) K25-01 was inoculated at a rate of 2% into MRS liquid medium containing 0.3% bovine bile salt and cultured for 36 h. The OD value was then measured.

[0066] The results (as shown in Table 2) showed that the OD value of K25-01 in bile salt medium was 0.315±0.010, which was significantly increased compared with the initial value, indicating that the strain has good bile salt tolerance.

[0067] Table 2. Acid and bile salt tolerance of bacterial strains (OD value)

[0068] Note: 0h in the table represents the initial level OD value, and different lowercase letters indicate significant differences between groups (P<0.05).

[0069] 9) After centrifuging and washing the K25-01 bacterial culture, add it to artificial gastric fluid with pH=2, incubate at 37℃ for 3 hours, and then determine the number of viable bacteria.

[0070] The results (as shown in Table 3) indicate that K25-01 had a survival rate of 79.4% in simulated gastric fluid, demonstrating its high activity under strongly acidic conditions. After inoculating the K25-01 culture in simulated intestinal fluid at pH 8 for 4 hours, the viable cell count was determined. The results showed that K25-01 had a survival rate of 75.3% in simulated intestinal fluid, indicating that the strain can also maintain a high survival rate in alkaline environments.

[0071] Table 3. Simulated gastrointestinal fluid survival rate

[0072] Example 2 Characteristics of Pediococcus lactis K25-01.

[0073] Morphological observation of the strains was performed, including colony morphology (such as colony size, shape, color, and edge) and cell morphology (observation of cell shape, size, and arrangement under a microscope). These findings were compared with known morphological characteristics of lactic acid bacteria to preliminarily determine the strain's category. A series of physiological and biochemical tests were then conducted to further determine the strain's physiological and biochemical characteristics, and these were compared with a database of known physiological and biochemical characteristics of lactic acid bacteria to accurately identify the strain's species.

[0074] I. Morphological observation of the strain.

[0075] Bacterial morphology.

[0076] The strain was streaked onto an MRS plate and incubated at 37°C for 24 hours. Single colonies were picked and placed on a glass slide in a clean bench, and the smear was held above an alcohol lamp flame with forceps. Gram staining was then performed, and the morphological characteristics of the strain were observed and recorded under a microscope (100x oil immersion).

[0077] The results show (e.g.) Figure 8 After Gram staining, the bacteria are spherical or nearly elliptical, with a diameter of 0.6–1.0 μm, mainly arranged in pairs or quadrupeds, without long chain structures. Based on the typical morphological characteristics of *Pediococcus* lactic acid bacteria, the strain was preliminarily identified as *Pediococcus lactis*.

[0078] II. Molecular biological identification of the strain.

[0079] 1) Genomic DNA was extracted from the strain, and specific gene fragments, such as the 16S rRNA gene, were amplified using PCR technology and sequenced. The molecular sequence was compared with the NCBI database BLAST, confirming that K25-01 is *Pediococcus lactis*. The phylogenetic tree of K25-01 is as follows: Figure 9 As shown.

[0080] 2) The whole genome of *Pediococcus lactis* K25-01 was sequenced using the Illumina HiSeq sequencing platform. After assembly and alignment, the genome length was 1997314 bp, and the G+C content was 42.22%.

[0081] 3) Functional annotation of the entire genome of *Pediococcus lactis* K25-01 was performed using the KEGG database. KEGG is a professional database for comparing and analyzing bacterial genes and metabolic pathways at the molecular level, enabling exploration at the bacterial molecular level and studying the relationship between functional genes and biomolecules in various biological pathways. The KEGG database classifies microbial biological pathways into six categories: metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases.

[0082] The results show (e.g.) Figure 10 As shown in the figure, 1486 functional genes of *Pediococcus lactis* K25-01 were annotated using KEGG. The most prevalent gene function in this strain was metabolism, while the least prevalent was cellular processes. Among the metabolic pathways, there were 198 genes related to carbohydrate metabolism, 111 genes related to membrane transport, 89 genes related to cofactor and vitamin metabolism, and 81 genes related to nucleotide metabolism, indicating that this strain possesses strong capabilities in carbohydrate utilization, substance transport, and nutrient metabolism.

[0083] 4) The Carbohydrate Active Enzyme Database (CAZy) is a database that compares and annotates complex enzymes that bacteria use to synthesize or utilize carbohydrates and other sugars, including helper oxidoreductases, carbohydrate esterases, glycoside hydrolases, and glycosyltransferases.

[0084] The results show (e.g.) Figure 11 As shown in the figure, 66 genes encoding carbohydrate-active enzymes were successfully annotated using the CAZy database in Pediococcus lactis K25-01. Based on the similarity of amino acid sequences in the encoded protein domains, they were divided into 4 protein families, including 5 helper oxidoreductase genes, 7 carbohydrate esterase genes, 34 glycoside hydrolases (GH) genes, and 20 glycosyltransferase (GT) genes.

[0085] 5) Annotation of tolerance-related functional genes in Pediococcus lactis K25-01 was performed, and 12 genes related to acid tolerance (gadC, nhaC, arcA, arcC, atpE, atpG, atpD, atpF, atpH, atpA, atpC, atpB); 4 genes related to bile salt tolerance (opuA, dltA, recA, groEL); 4 genes related to antioxidants (npr, gor, trxA, trxB); 6 genes related to antibacterial activity (nisE, nisF, agrA, ldhA, ackA, pta); and 4 genes related to adhesion (acm, map, mapA, lspA).

[0086] Genes related to antibacterial function were detected and annotated in the genome of Pediococcus lactis K25-01. The specific genes are shown in Table 4.

[0087] Table 4. Genes and annotations related to antibacterial function of *Pediococcus lactis* K25-01.

[0088] Example 3 Metabolic characteristics of Pyotrophic Lactococcus K25-01.

[0089] I. Experimental Methods.

[0090] After lactic acid bacteria fermentation, the bacterial culture was centrifuged at low temperature to remove bacterial cells, and the fermentation supernatant was collected. For ease of sample preservation and transportation, the fermentation supernatant was freeze-dried into a lyophilized powder before testing. Before testing, the lyophilized powder was reconstituted with ultrapure water to the original volume of the fermentation supernatant before lyophilization, and thoroughly mixed as the sample to be tested. An appropriate amount of sample was then pipetted into a sterile EP tube, and a freshly prepared methanol-acetonitrile mixture of equal volumes was added at a 1:4 volume ratio. The mixture was vortexed for 30 seconds to mix thoroughly, ultrasonically extracted in an ice-water bath for 10 minutes, allowed to stand at -40℃ for 1 hour, thawed at 4℃, centrifuged at 12000×g for 15 minutes, and the supernatant was collected and placed in a sample vial. Subsequently, detection was performed using a Vanquish ultra-high performance liquid chromatograph with a BEH Amide column. An aqueous solution containing 25 mmol / L ammonium acetate and ammonia, and acetonitrile were used as mobile phases A and B, respectively. The sample pan temperature was controlled at 4℃, and the injection volume was 2 μL to complete the chromatographic separation and detection of the target substances.

[0091] II. Experimental Results.

[0092] The results showed that the differential metabolite volcano diagram of *Pediococcus lactis* K25-01 before and after fermentation was as follows: Figure 12 As shown, the volcano plot integrates univariate significance analysis based on the T-test with multi-group metabolite fold variation analysis, visually presenting the statistical significance of metabolite differences among different samples, providing an intuitive visualization method for screening potential biomarker metabolites. Each scatter point corresponds to one metabolite, and the size of the scatter point is positively correlated with the variable projection importance (VIP) value of the orthogonal partial least squares discriminant analysis (OPLS-DA) model; that is, the larger the scatter point, the higher its corresponding VIP value. The figure shows that the strain underwent significant metabolic changes during fermentation. Specifically, 756 differentially expressed metabolites were screened before and after fermentation of K25-01, with 209 upregulated and 547 downregulated.

[0093] From the metabolites that were significantly upregulated after K25-01 fermentation, five core beneficial metabolites that promote the growth and metabolism of lactic acid bacteria were screened (such as...). Figure 13 The following are listed as phenylacetic acid, indole-3-acetaldehyde, 5'-N-methylcarboxamidoadenosine, DL-4-hydroxyphenyllactic acid, and N-lactoyl-Phenylalanine, respectively.

[0094] Example 4 The inhibitory effect of GQ-15, an antimicrobial peptide synthesized by Pediococcus lactis K25-01, on Escherichia coli infection and Staphylococcus aureus.

[0095] I. Preparation of antimicrobial peptides.

[0096] Take the lactic acid bacteria fermentation broth, centrifuge at 4℃ and 8000×g for 10 min, collect the supernatant and filter it through a 0.22μm filter membrane; adjust the pH of the filtrate to 6.0~7.0, slowly add ammonium sulfate powder to saturation of 40%~80%, stir overnight at 4℃; centrifuge again at 4℃ and 12000×g for 20 min, discard the supernatant, resuspend the precipitate in PBS buffer (pH 7.4), desalt it through a C18 solid phase extraction column (molecular weight cutoff 3~5kDa), and freeze dry to obtain crude peptide extract; The crude peptide extract was separated using a reversed-phase C18 column (250 mm × 4.6 mm, 5 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The gradient elution program was 0–60 min, with the proportion of phase B increasing from 5% to 60%. The flow rate was set at 1 mL / min, the column temperature at 30 °C, and the detection wavelength at 214 nm. Each elution peak was collected and freeze-dried. The purified peptide was dissolved in 50% acetonitrile aqueous solution and mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) at a volume ratio of 1:1. 1 μL of the mixture was spotted onto a target plate and allowed to air dry. Mass spectrometry was performed in positive ion mode with an accelerating voltage of 20 kV and a scan range of 500–10000 Da to obtain the precise molecular weight of the peptide and screen for target components. The target component was enzymatically digested with sequencing-grade trypsin (enzyme / substrate mass ratio 1:50) at 37℃ for 12 h. The digestion product was desalted using a C18 nano-column and then loaded onto the sample. Detection was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) with a gradient elution of 0.1% formic acid aqueous solution and acetonitrile solution. Data-dependent scanning (DDA) mode was selected for mass spectrometry to acquire secondary mass spectra. The mass spectrometry data were compared with lactic acid bacteria genome databases (NCBI, EnsemblBacteria), and amino acid sequence matching was performed using Mascot software to obtain the target polypeptide sequence. Candidate antimicrobial peptides were screened from the target polypeptide sequence. The screening criteria for candidate antimicrobial peptides were as follows: CAMPR4 prediction results showed that the peptide was identified as AMP by all three models (RF, SVM, and ANN); APD6 prediction results showed antimicrobial peptide characteristics; PeptideRanker prediction score > 0.5, indicating potential biological activity; ProtParam analysis results showed that the peptide had the common physicochemical characteristics of antimicrobial peptides; and ToxinPred prediction results indicated no toxicity. Based on the above prediction results, candidate antimicrobial peptides that met the above screening criteria and had the best overall evaluation were selected for chemical solid-phase synthesis, ultimately obtaining the antimicrobial peptide GQ-15.

[0097] II. Safety evaluation of antimicrobial peptide GQ-15.

[0098] The antibacterial activity of peptides was predicted using the online software CAMPR3. Physicochemical properties of the peptides, including net charge number, GRAVY total average hydrophilicity, Wimley-White total residue hydrophobicity scale, and Boman index, were calculated using the online software APD3; isoelectric point and instability index were predicted using ProtParam in ExPASy.

[0099] Based on the above indicators, the peptide (SEQ ID NO.2: GPIGPVGARGPAGPQ) with a CAMPR3 score >0 was predicted to have antibacterial activity; the net charge number was 1, the GRAVY index was <0, the molecular weight was 1330.73 Da, and the pI was alkaline (as shown in Table 5).

[0100] Table 5. Prediction of physicochemical properties and antibacterial activity of peptides

[0101] III. Activity analysis of antimicrobial peptide GQ-15.

[0102] 1. Determination of minimum inhibitory concentration.

[0103] The lowest concentration of an antimicrobial peptide that can completely inhibit bacterial growth is called the minimum inhibitory concentration (MIC). Activated *Escherichia coli* and *Staphylococcus aureus* were cultured at 37°C and 200 rpm to the logarithmic growth phase. The bacterial concentration was then adjusted to 10-1 using fresh LB medium. 5CFU / mL. Dissolve the antimicrobial peptide in sterile water to a concentration of 10 mg / mL. Add 50 μL of the antimicrobial peptide solution to column 1 of a sterile 96-well plate. Add 25 μL of PBS to columns 2-10. Transfer 25 μL of the antimicrobial peptide solution from column 1 to column 2 and mix. Transfer 25 μL of the solution from column 2 to column 3. Continuous serial dilutions are performed up to column 10. Discard the 25 μL from column 10. Add the diluted 25 μL of bacterial solution to columns 1-10 to obtain final antimicrobial peptide concentrations of 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, 0.15625 mg / mL, 0.078125 mg / mL, 0.0390625 mg / mL, and 0.01953125 mg / mL. A positive control was prepared by adding 25 μL of bacterial culture to 25 μL of ampicillin solution, and a negative control was prepared by adding 25 μL of bacterial culture to 25 μL of PBS without antimicrobial peptides. The 96-well plates were incubated at 37°C and 120 rpm for 12 h, and the absorbance was measured at 600 nm using a microplate reader. Each experiment was performed in triplicate.

[0104] The results of the antimicrobial activity of antimicrobial peptide GQ-15 against Escherichia coli and Staphylococcus aureus showed that the minimum inhibitory concentrations (MICs) of antimicrobial peptide GQ-15 against Staphylococcus aureus and Escherichia coli were 10.0 mg / mL and 5.0 mg / mL, respectively.

[0105] 2. Time-based lethality curve.

[0106] Staphylococcus aureus and Escherichia coli indicator bacteria were cultured using the same method described above. The indicator bacterial suspension was mixed with antimicrobial peptide solution or PBS at a 1:1 volume ratio to achieve final antimicrobial peptide concentrations of 0MIC and 1MIC, with 0MIC serving as a blank control. Samples were taken at 0h, 0.5h, 1h, 1.5h, 2h, and 3h, and viable bacteria were counted using the pour plate method. After incubation at 37℃ for 24h, the viable bacterial count was recorded, and a time-dependent killing curve of the antimicrobial peptide against the indicator bacteria was plotted.

[0107] The results show (e.g.) Figure 14 As shown in the figure, the antimicrobial peptide GQ-15 has an inhibitory effect on Staphylococcus aureus and Escherichia coli, and the inhibitory effect increases with the extension of treatment time.

[0108] 3. Protease treatment.

[0109] Staphylococcus aureus and Escherichia coli indicator bacteria were cultured using the same method described above. Trypsin, pepsin, and papain, each weighed to a final concentration of 1 mg / mL, were thoroughly mixed with the antimicrobial peptide GQ-15 at a final concentration of 1 MIC. Simultaneously, the untreated antimicrobial peptide GQ-15 at a concentration of 1 MIC served as a control group for the enzyme-treated experimental group. The mixture was incubated at 37°C for 4 hours to allow for complete reaction. Afterward, the reacted sample was mixed with an equal volume of bacterial suspension and treated for 2 hours. The OD values ​​were measured. 600 Its antibacterial rate was calculated to verify its antibacterial effect.

[0110] The results show (e.g.) Figure 15 As shown in the figure, the control group consisted of indicator bacteria treated with 1 MIC of antimicrobial peptide, while the experimental group consisted of indicator bacteria treated with protease-treated antimicrobial peptide GQ-15. The higher the viable count of the indicator bacteria, the weaker the antibacterial activity of the sample. After treatment with trypsin, pepsin, and papain, the antibacterial activity of antimicrobial peptide GQ-15 against Staphylococcus aureus and Escherichia coli was reduced to varying degrees. The enzyme treatment experiment indicates that the substance producing antibacterial activity is the antimicrobial peptide, and this sensitivity to proteases can prevent the accumulation of antimicrobial peptides in vivo, improving their safety.

[0111] 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 type of lactic acid cocci ( Pediococcus acidilactici K25-01, characterized in that, The *Pediococcus lactis* K25-01 was deposited on November 24, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252686.

2. An antimicrobial peptide GQ-15 prepared from *Pediococcus lactis* K25-01 as described in claim 1, characterized in that, The amino acid sequence of the antimicrobial peptide GQ-15 is shown in SEQ ID NO.

2.

3. The application of the Lactococcus lactis K25-01 as described in claim 1 or the antimicrobial peptide GQ-15 as described in claim 2 in the preparation of products with preservative or antibacterial effects.

4. The application according to claim 3, characterized in that, The effective viable count of *Pediococcus lactis* K25-01 in the product is 1×10⁻⁶. 9 ~7.7×10 9 CFU / mL.

5. The application according to claim 3, characterized in that, The effective concentration of the antimicrobial peptide GQ-15 in the product is ≥5 mg / mL.

6. The use of Pyrococcus lactis K25-01 as described in claim 1 or the antimicrobial peptide GQ-15 as described in claim 2 in the preparation of antimicrobial drugs.

7. The application according to claim 6, characterized in that, The pathogenic bacteria inhibited by the antibacterial drug include Escherichia coli and Staphylococcus aureus.

8. An antibacterial drug, characterized in that, The active ingredient of the drug includes the *Pediococcus lactis* K25-01 as described in claim 1 or the antimicrobial peptide GQ-15 as described in claim 2.

9. The application of the Lactococcus lactis K25-01 as described in claim 1 or the antimicrobial peptide GQ-15 as described in claim 2 in the preparation of food preservatives that inhibit Escherichia coli and Staphylococcus aureus.

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