Pediococcus acidilactici and application thereof

Abstract

The invention discloses pediococcus acidilactici and application thereof. Relates to the technical field of microorganisms. The invention provides a specific strain and application. The strain provided by the invention is good in growth condition, has good tolerance to strong acid, cholate and artificial gastrointestinal fluid, has certain sensitivity to antibiotics, and has good inhibition performance to escherichia coli and staphylococcus aureus, an antibacterial substance is antibacterial peptide, and the adhesion performance is superior to that of known animal lactobacillus with excellent probiotic performance. The strain can be used as a potential probiotic and is used for developing products related to daily feeding of pet cats and disease treatment.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a type of Pediococcus lactis and its applications. Background Technology

[0002] Probiotics are known to have many benefits, such as regulating the balance of intestinal flora, stabilizing the intestinal flora, helping the body to enhance immunity, and improving allergic reactions.

[0003] The use of probiotics in the feeding and disease treatment of production animals is relatively mature, but research on their application in pets is still relatively limited. As one of the most important companion animals, the health of pet cats is receiving increasing attention. Adding probiotics to pet food can improve gastrointestinal function and increase the abundance of beneficial bacteria.

[0004] Currently, human-derived probiotics appear to be the primary ingredient in pet food. However, host-derived microorganisms may be the most suitable source of probiotics.

[0005] Therefore, whether a *Pediococcus lactis* strain and its application can be provided to overcome the above-mentioned technical deficiencies is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a *Pediococcus lactis* strain and its applications. This strain exhibits good growth, good tolerance to strong acids, bile salts, and artificial gastrointestinal fluids, some sensitivity to antibiotics, and good inhibitory properties against *Escherichia coli* and *Staphylococcus aureus*. Its antibacterial substance is an antimicrobial peptide, and its adhesion properties are superior to those of known probiotic *Lactobacillus* species. Therefore, this *Pediococcus lactis* strain can serve as a potential probiotic for developing products related to the daily care and disease treatment of pet cats.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: Preservation information: Pediococcus lactis ( Pediococcus acidilactici FM135 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.31973 and deposit date of September 13, 2024.

[0008] A type of Pediococcus lactis, with accession number CGMCC NO.31973.

[0009] The present invention also provides a bacterial agent containing the above-mentioned Pediococcus lactis.

[0010] The present invention also provides the fermentation products of the above-mentioned Pediococcus lactis.

[0011] The present invention also provides the application of the above-mentioned Pediococcus lactis, or the above-mentioned bacterial agent, or the above-mentioned fermentation product in food processing and preparation of pet medicines.

[0012] Preferred: Food processing: pet food, or pet food additives.

[0013] Preferred: Medicine: Improves diarrhea symptoms in cats and has a protective effect against diarrhea-related dehydration and inflammation.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a *Pediococcus lactis* and its application, achieving the following technical effects: Hemolysis tests using Staphylococcus aureus as a control showed no obvious hemolytic zone. Drug susceptibility testing showed that *Pediococcus lactis* FM135 was sensitive to penicillins, tetracyclines, and β-lactams; but resistant to quinolones, aminoglycosides, and sulfonamides. *Pediococcus lactis* FM135 can be considered a safe strain with strong safety characterization.

[0015] An in vitro model was established using HCT-8 cells (human ileocecal cancer cells) that are similar in morphology and function to mature intestinal epithelial cells to evaluate the adhesion ability of probiotics. The results showed that the number of *Pediococcus lactis* cells adhering to the cells in this invention was higher than that of the control group *Lactobacillus animalis*, indicating that *Pediococcus lactis* has a strong adhesion effect on HCT-8 cells. Most research teams use in vitro cell models to evaluate the adhesion ability of probiotics, but this method has the drawback of large experimental errors. In contrast, the FM135 cell of this invention demonstrates good technical performance in terms of host-specific adhesion and colonization potential, achieving an adhesion number of (1138±140) CFU / 100 cells.

[0016] This invention simulates the gastrointestinal environment of a cat to test the tolerance of *Pediococcus lactis* FM135. Data shows that the bacteria maintained a 70.1% survival rate after 3 hours of culture in a pH 4.0 medium, an 80.1% survival rate after 2 hours in simulated gastric fluid, and an 89.3% survival rate after 3 hours in simulated intestinal fluid. Furthermore, after 3 hours of treatment with 0.3% bile salts, the survival rate reached 70.9%, indicating that this strain has strong tolerance to strong acid, gastrointestinal fluid, and concentrated bile. The experimental data also provides time-point, quantitative survival data.

[0017] This invention uses trypsin, proteinase K, catalase, and papain to treat the cell-free supernatant of this strain. The results show that the main antibacterial substances of this strain are bacteriocins such as antimicrobial peptides, but do not include hydrogen peroxide and papain. Therefore, it is speculated that *Pediococcus lactis* can produce antibacterial substances such as bacteriocins and other proteins or peptides. The antibacterial mechanism is clear.

[0018] This invention isolates *Pediococcus lactis* from the feces of healthy kittens, exhibiting excellent acid-producing capacity and growth characteristics. It also demonstrates good fermentation performance, ensuring stable formulation production.

[0019] This invention provides a strain source for the development of probiotic feed additives specifically for pet cats, and further lays the foundation for the development of microecological preparations. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 The attached figure shows an image of Pediococcus lactis FM135 provided by the present invention, wherein A: colony morphology; B: microscopic morphology.

[0022] Figure 2 The attached image is a photograph of hemolysis provided by this invention.

[0023] Figure 3 The attached figure shows the antibacterial activity of the supernatant provided by the present invention against different pathogenic indicator bacteria.

[0024] Figure 4 The attached figure is a phylogenetic tree diagram provided by the present invention.

[0025] Figure 5 The attached figure is a growth curve of Pediococcus lactis FM135 provided by the present invention.

[0026] Figure 6 The attached figure shows the effect of different treatment methods provided by the present invention on the antibacterial activity of the supernatant.

[0027] Figure 7 The attached figure is an acid production curve of Pediococcus lactis FM135 provided by the present invention.

[0028] Figure 8 The attached figure shows the bile salt tolerance survival rate of Pediococcus lactis provided by the present invention.

[0029] Figure 9 The attached figures show the resistance of *Pediococcus lactis* FM135 to artificial gastric fluid and artificial intestinal fluid provided by the present invention, wherein A: test results of resistance to artificial gastric fluid; B: test results of resistance to artificial intestinal fluid.

[0030] Figure 10 The attached figure shows the acid-resistant survival rate of Pediococcus lactis FM135 provided by the present invention.

[0031] Figure 11The attached figure is a comparative diagram of the adhesion of Pediococcus lactis FM135 and Lactobacillus animalis to HCT-8 cells provided by the present invention, wherein A: Pediococcus lactis FM135 adhesion to HCT-8; B: Lactobacillus animalis adhesion to HCT-8; C: Microscopic image of normal HCT-8 cells.

[0032] Figure 12 The attached figure is a comparison chart of HCT-8 cell adhesion data in Example 1 provided by the present invention.

[0033] Figure 13 The attached figure shows the emulsification phenomenon between the fermentation supernatant after shaking and ethyl acetate in Example 2 provided by the present invention.

[0034] Figure 14 The attached figure shows the layering phenomenon that occurred after the sample was left to stand for half an hour in Embodiment 2 of the present invention.

[0035] Figure 15 The attached figure is a diagram of the emulsion layer separated and collected in Example 2 of the present invention.

[0036] Figure 16 The attached figure is a diagram of the thick liquid obtained after rotary evaporation in Example 2 of the present invention.

[0037] Figure 17 The attached figure is a diagram of the liquid during drying in Embodiment 2 provided by the present invention.

[0038] Figure 18 The attached figure is a diagram of the semen powder obtained after complete drying in Example 2 of the present invention.

[0039] Figure 19 The attached figure shows the inhibition of Staphylococcus aureus by the stock solution in Example 2 provided by the present invention.

[0040] Figure 20 The attached figure shows the inhibition of Escherichia coli by the original solution in Example 2 provided by the present invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] This invention discloses a type of Pediococcus lactis and its applications.

[0043] In the examples, reagents and raw materials not mentioned were all commercially available. For example, MRS and LB media were purchased from Beijing Solarbio Science & Technology Co., Ltd.; TSA and TSB media were purchased from Haibo Biotechnology Co., Ltd.; lysozyme and bacterial genomic DNA extraction kits were purchased from Beijing TransGen Biotech Co., Ltd.; 17 antimicrobial susceptibility testing discs, including gentamicin, penicillin, and tetracycline, were purchased from Changde Bickman Biotechnology Co., Ltd.; proteinase K, trypsin, pepsin, porcine bile salts, papain, and catalase were all purchased from Beijing Solarbio Science & Technology Co., Ltd. Test strains: *Escherichia coli* ATCC 25922, CGMCC 1.2385; *Staphylococcus aureus* ATCC 25923, CGMCC 1.2386, both purchased from the China General Microbiological Culture Collection Center (CGMCC). The universal bacterial primers (27F, 1492R) and 16S rRNA were both prepared by Sequencing Sangon Biotech (Shanghai) Co., Ltd.

[0044] Data processing All experiments were performed in triplicate to avoid the possibility of randomness in the data. The results were presented using GraphPad Prism 8.0 software for statistical analysis and graphing.

[0045] Methods and instruments not mentioned are conventional and will not be described in detail here.

[0046] Sample collection The samples involved in the examples were all collected from five healthy kittens that were one month old, growing well, not suffering from diarrhea, and not receiving any medication, from a cattery in Hohhot. Fresh feces from the kittens were collected, and immediately placed in a cryopreservation box at 4°C after excretion. The kittens were labeled with their sampling numbers and dates and immediately taken back to the laboratory for further processing.

[0047] Example 1 Aseptically extract approximately 5 g of the center of each kitten feces sample and add it to a centrifuge tube containing 45 mL of sterile physiological saline (with 15 glass beads). Vortex thoroughly until no obvious solid particles remain. After standing, divide the supernatant into 10 mL portions. -1 The diluted solution was then prepared into 10 solutions using a 10-fold dilution method. -2 10 -3 10 -4 10 -5 10 -6The fecal sample was diluted. 100 μL of the diluted sample was evenly spread on MRS agar medium and incubated at 37°C for 24 h. Single colonies were picked and streaked multiple times for pure culture until the colonies on the plate were of uniform size, color, and morphology and no contaminants were observed under a microscope. Then, single colonies were picked and placed in MRS liquid medium and incubated at 37°C for 18 h. 40% glycerol was added to the bacterial culture, shaken to mix, and stored at -80°C for later use.

[0048] Morphological characteristics observation Select a single colony, prepare a smear, and perform Gram staining. Observe the bacterial morphology and staining characteristics under an optical microscope.

[0049] Based on morphological observation, physiological and biochemical experiments, and microscopic examination, three strains of *Pediococcus lactis* were preliminarily identified and named FM134, FM135, and FM136, respectively. FM135 colonies are milky white, with a raised center, regular edges, and a smooth surface; they are facultative anaerobic, Gram-positive, and spherical. The colony morphology of FM135 is as follows: Figure 1 As shown in Figure A, the morphology under a microscope is as follows: Figure 1 As shown in B.

[0050] Hemolysis test First, prepare Columbia blood agar, sterilize and cool to 50°C, then add 5% (w / w) of defibrinated sheep blood, mix well, and pour into plates. Streak the test strain onto Columbia blood agar plates and incubate at 37°C for 24 h. Use Staphylococcus aureus as a positive control and observe the hemolysis zone of the strain.

[0051] Staphylococcus aureus was used as a positive control. Figure 2 The colonies of the isolated bacteria showed obvious β-hemolysis, but there was no hemolytic zone around the colonies, which preliminarily indicates that strain FM135 is safe and non-pathogenic.

[0052] Determination of antibacterial activity The test strains were inoculated into MRS liquid medium and cultured at 37℃ for 48 h. After centrifugation at 12,000 r / min for 2 min, the supernatant was filtered through a 0.22 μm microporous membrane to obtain cell-free supernatant (CFS), and its pH was measured using a pH meter. The antibacterial activity of the cell-free supernatant after fermentation was determined using the Oxford cup method. Pathogenic *Escherichia coli* and *Staphylococcus aureus* were selected as indicator bacteria. *Escherichia coli* and *Staphylococcus aureus* culture solutions were inoculated at a 1% inoculum into LB broth and tryptone soybean broth (TSB) medium, respectively. The test tubes containing the indicator strains were placed at a 45° angle in a 37℃ constant temperature shaker at 200 r / min for amplification culture. The absorbance (OD) of the bacterial solution was measured every 2 h using a UV-Vis spectrophotometer (T6 New Century, Beijing Purkinje). 600When the absorbance of *E. coli* reached 0.8-1.1 and that of *Staphylococcus aureus* reached 1.4-1.8, the culture was stopped. Then, 100 μL of each medium was evenly spread onto LB agar and tryptone-soybean agar (TSA). After complete absorption, a sterile Oxford cup (6 mm inner diameter) was placed at an appropriate position on the medium using sterile forceps, ensuring close contact between the cup and the medium. 150 μL of cell-free supernatant from the test strain was added to each Oxford cup, and the cup was labeled with the strain number. Kanamycin (100 mg / L) was used as a positive control. The plates were placed in a 4°C refrigerator for 12 hours for slow infiltration, and then incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured and recorded using calipers.

[0053] Using *Escherichia coli* and *Staphylococcus aureus* as pathogen indicator bacteria, an antibacterial test was conducted on the three isolated strains using the Oxford cup method. The results are as follows: Figure 3 As shown, compared with the inhibition zone of Kanamycin, the three strains exhibited different antibacterial effects against the two pathogenic indicator bacteria, and the diameter of the inhibition zones was >14 mm for all three strains, indicating that they all possessed strong antibacterial activity. The test bacterium with the best antibacterial effect against Escherichia coli and Staphylococcus aureus was FM135, with inhibition zone diameters of (18.74±0.58) mm and (17.09±0.61) mm, respectively. This bacterium will be used for subsequent research.

[0054] Strain identification Centrifuge 1 mL of the bacterial culture (24 h incubation) at 12,000 r / min for 2 min, discard the supernatant, and retain the bacterial pellet. Extract bacterial 16S rRNA using a bacterial genomic DNA extraction kit (Beijing TransGen Biotech Co., Ltd.). Using bacterial DNA as a template, PCR amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492 R (5'-GGTTACCTTGTTACGACTT-3'). The reaction mixture (50 μL) consisted of 25 μL Green Taq Mix, 2 μL each of forward and reverse primers, 4 μL DNA template, and ddH2O to a final volume of 50 μL. A blank control was used without added template DNA. The PCR amplification program was as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 2 min, 30 cycles; 72 °C extension for 10 min. The amplified products were detected by 1% agarose gel electrophoresis, and the bands were observed and analyzed using a gel imaging system. The amplified products were sent to Sangon Biotech Co., Ltd. (Shanghai) for 16S rRNA sequencing. The sequencing results were compared with the GenBank sequence database of NCBI (National Center for Biotechnology Information) using BLAST analysis, and a 16S rRNA phylogenetic tree was constructed.

[0055] Phylogenetic analysis of the 16S rRNA gene of FM135 confirmed its close relationship with *Pediococcus lactis*. The phylogenetic tree is shown below. Figure 4 As shown.

[0056] Growth curve determination of strains Single colonies of the test strain were picked and inoculated into MRS broth medium and incubated at 37 ℃ for 24 h. Then, 1% (v / v) of the bacterial culture was inoculated into fresh MRS broth medium and incubated at 37 ℃. Every 2 h, the culture was vortexed and mixed. 2 mL of the bacterial culture and MRS broth medium were transferred to a cuvette, and the absorbance was measured at 600 nm using a UV-Vis spectrophotometer. A total of 13 absorbance values ​​were measured within 0–24 h. The x-axis represents time, and the y-axis represents OD. 600 Plot the growth curve of the strain.

[0057] like Figure 5 As shown, the strain exhibited a lag phase within 2 hours after inoculation; from 2 to 10 hours, it entered the logarithmic growth phase, with the fastest proliferation rate; and after 10 hours, it reached the plateau phase. The FM135 strain showed good growth, consistent with bacterial growth characteristics.

[0058] Analysis of antibacterial substances Prepare a 5 mmol / L CaCl2 buffer solution. Dissolve proteinase K, catalase, papain, and trypsin powders in the buffer solution to a concentration of 10 mg / ml for each enzyme. Prepare 1 mol / L NaOH and 1 mol / L HCl solutions and store at 4°C for later use. For the assay of antibacterial activity, obtain 900 μL of cell-free supernatant (CFS) of the test bacteria. Adjust the pH of the cell-free supernatant to the optimal pH values ​​for each enzyme (proteinase K pH 7.5, catalase pH 7.0, papain pH 5.0, and trypsin pH 7.0) with NaOH solution, and add 100 μL of each enzyme solution to bring the final enzyme concentration to 1 mg / ml. Incubate at 37°C for 1 h, and after cooling, adjust the pH back to the initial pH value of the cell-free supernatant with HCl solution. Use *E. coli* as the indicator bacterium and the untreated cell-free supernatant as the control group. Measure the diameter of the inhibition zone using the Oxford cup method.

[0059] The main antimicrobial substances that may be produced by strain FM135, such as antimicrobial peptides and hydrogen peroxide, were analyzed, and the results are as follows: Figure 6 As shown, treatment of the cell-free supernatant of strain FM135 with trypsin significantly reduced its antibacterial activity (P<0.01). Treatment with proteinase K significantly reduced the antibacterial activity of the strain (P<0.05). Treatment with catalase and papain did not significantly reduce the antibacterial activity of the strain (P>0.05), indicating that the main antibacterial substance of this strain is antimicrobial peptide.

[0060] acid production performance 100 μl of the bacterial culture solution was inoculated into 5 ml of Mrs broth medium and incubated at 37 ℃ for 24 h. Then, 1% of the culture solution was inoculated into fresh MRS broth medium and incubated at 37 ℃. The pH value of the bacterial culture solution in each test tube was measured every 12 h using a pH meter. A total of 6 pH values ​​were measured within 0–60 h. The acid production capacity curve of the strain was plotted with time on the x-axis and pH value on the y-axis.

[0061] As the culture time increased, the pH value of the FM135 strain culture medium also decreased. Figure 7 After 48 hours of cultivation, the pH value dropped to 4.05 and gradually stabilized, indicating that the strain has strong acid-producing ability.

[0062] Tolerance testing Bile salt tolerance test: MRS broth culture media with bile salt concentrations of 0.1%, 0.2%, and 0.3% were prepared using porcine bile salts. The media were autoclaved at 121°C for 15 min, cooled, and dispensed. The bacterial solutions to be tested were centrifuged at 12,000 r / min for 1 min, the supernatant was discarded, and the media were washed three times with sterile physiological saline. Under sterile conditions, the media were inoculated into bile salt solutions of different concentrations at a 5% inoculum. After incubation at 200 r / min and 37°C for 3 h, the media were serially diluted and plated. After incubation at 37°C for 24 h, the viable cell count was recorded. The viable cell count at 0 h was used as a control. Each group had three replicates, and the survival rate of the strains at different bile salt concentrations for 3 h was calculated according to formula (1).

[0063] Survival rate (%) = (lg N1 / lg A) × 100 Equation (1) Note: N1 represents the number of surviving bacteria (CFU) in bile salt solution after 3 h; A represents the number of surviving bacteria (CFU) in bile salt solution after 0 h.

[0064] The test results are as follows Figure 8 As shown, when strain FM135 was treated with 0.3% bile salt concentration for 3 hours, its survival rate reached 70.9%, indicating that the strain has strong tolerance to bile salts.

[0065] Test for resistance to artificial gastrointestinal fluid: Preparation of artificial gastric fluid: A 10 g / L pepsin (1:3000 U) solution was prepared using pre-adjusted and sterilized PBS (pH 2.0), filtered through a 0.22 μm filter membrane, and prepared fresh for each use. After activating the test bacteria, 2% was inoculated into MRS liquid medium and incubated overnight at 37°C. The cells were collected by centrifugation, washed three times with PBS (pH 7.4), and the cell concentration was adjusted to 10 g / L. 9 CFU / mL. Take 1 mL of bacterial cells resuspended in PBS and add it to 5 mL of artificial gastric fluid. Incubate in a shaker at 200 r / min and 37℃ for 1 h, 2 h, and 3 h. Then, perform serial dilutions and plate the bacteria. Incubate at 37℃ for 24 h and record the number of viable bacteria. Use the number of viable bacteria at 0 h as a control. Set up 3 replicates for each group and calculate the survival rate of the strain in artificial gastric fluid according to formula (2).

[0066] Survival rate (%) = (lg N2 / lg B) × 100 Equation (2) Note: N2 represents the number of surviving bacteria (CFU) in the artificial gastric fluid; B represents the number of surviving bacteria (CFU) in the artificial gastric fluid at 0 h.

[0067] Preparation of artificial intestinal fluid: A 10 g / L solution of trypsin (1:250 U) was prepared using pre-mixed and sterilized PBS (pH 8.0), filtered through a 0.22 μm filter membrane, and prepared fresh for each use. The preparation of bacterial suspension was the same as that of artificial gastric fluid. 1 mL of resuspended bacterial cells was added to 5 mL of artificial intestinal fluid, and cultured in a shaker at 200 r / min and 37 °C for 3 h. After that, the suspension was serially diluted and plated. After incubation at 37 °C for 24 h, the number of viable bacteria was recorded. The number of viable bacteria at 0 h was used as a control. Each group had 3 replicates, and the survival rate of the strain at 3 h was calculated according to formula (3).

[0068] Survival rate (%) = (lg N3 / lg C) × 100 (Equation 3) Note: N3 represents the number of surviving bacteria (CFU) in the artificial intestinal fluid after 3 hours; C represents the number of surviving bacteria (CFU) in the artificial intestinal fluid after 0 hours.

[0069] Results of the artificial gastric fluid resistance test are as follows Figure 9 As shown in Figure A, the survival rates of this strain after treatment with artificial gastric fluid for 1 hour and 2 hours were 93% and 80.1%, respectively. After 3 hours of treatment, the survival rate remained as high as 68.8%, indicating that the strain has strong tolerance to artificial gastric fluid; the results of the artificial intestinal fluid tolerance test are as follows... Figure 9 As shown in Figure B, the survival rate of this strain reached 89.3% after being treated with artificial intestinal fluid for 3 hours, indicating that the strain has a strong tolerance to artificial intestinal fluid.

[0070] Acid resistance test: MRS broth culture media with pH values ​​of 2.0, 3.0, 4.0, and 5.0 were prepared using HCl solution, autoclaved at 121℃ for 15 min, cooled, and dispensed for use. Under aseptic conditions, the bacterial suspension was inoculated into the culture media with different pH values ​​at a 5% inoculum, and cultured in a constant temperature shaker at 200 r / min and 37℃ for 3 h. After that, the culture was serially diluted and plated, and cultured at 37℃ for 24 h. The viable count was recorded, with the viable count at 0 h as the control. Each group was divided into 3 replicates, and the survival rate of the strains in the culture media with different pH values ​​for 3 h was calculated according to formula (4).

[0071] Survival rate (%) = (lg N4 / lg D) × 100 Equation (4) Note: N4 represents the number of bacterial cells that survived for 3 hours in culture media with different pH values ​​(CFU); D represents the number of bacterial cells that survived for 0 hours in culture media with different pH values ​​(CFU).

[0072] Experimental data show that in a relatively acidic environment (pH 4.0), this strain still has a 70.1% survival rate after 3 hours of culture. Figure 10 This indicates that the strain has strong acid resistance.

[0073] Drug sensitivity testing Antimicrobial susceptibility testing of isolated strains was conducted using the disk diffusion method (KB method). 100 μL of the test bacterial suspension was evenly spread onto MRS solid medium, and antimicrobial susceptibility discs were placed in appropriate positions, gently pressed down with tweezers to ensure tight adhesion. The medium was incubated at 37°C inverted for 24 h, and the diameter of the inhibition zone was measured and recorded using calipers. The antimicrobial susceptibility discs included: ampicillin, ceftriaxone, cefoxitin, gentamicin, neomycin, tetracycline, ciprofloxacin, azithromycin, erythromycin, amoxicillin, enrofloxacin, kanamycin, amikacin, doxycycline, ambroxol, azithromycin, clindamycin, trimethoprim-sulfamethoxazole, cefotaxime, cefuroxime, and chloramphenicol. The resistance level of the tested strains was determined according to the NCCLS standards.

[0074] As shown in Table 1, this bacterium is sensitive to antibiotics such as doxycycline, amoxicillin, erythromycin, ampicillin, chloramphenicol, cefotaxime, cefuroxime, and clindamycin, but not sensitive to azithromycin, tetracycline, neomycin, and ceftriaxone. It has developed resistance to enrofloxacin, kanamycin, amikacin, compound sulfamethoxazole, streptomycin, gentamicin, ciprofloxacin, spectinomycin, cefoxitin, and ofloxacin.

[0075]

[0076] Note: Criteria for determining the diameter of the inhibition zone: Sensitive (S), Intermediate (I), Resistant (R) Adhesion ability of HCT-8 cells Cell culture: Resuscitate HCT-8 cells (human ileocecal carcinoma cells) and transfer them to cell culture flasks. Add RPMI 1640 complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. Incubate overnight at 37°C in a 5% CO2 incubator. When the cells are in good condition and the adhesion rate reaches 70%–80%, passage them several times with 0.25% trypsin. Once the cells are in good condition, adhesion assays can be performed.

[0077] Preparation of the bacterial suspension for testing: FM135 bacterial suspension was inoculated at a 1% inoculum onto MRS liquid medium and incubated at 37 °C for 24 h. The bacterial suspension was then centrifuged at 12000 r / min for 2 min to collect the bacterial cells, and washed three times with sterile PBS. The bacterial cells were then resuspended in RPMI 1640 basal medium without fetal bovine serum and the concentration was adjusted to 1×10⁻⁶. 9 An adhesion test was conducted using CFU / mL.

[0078] Stably passaged HCT-8 cells were then used at 10 5Cells were seeded into 6-well plates (with autoclaved smears added to wells 2, 3, and 4), and incubated at 37 ℃ with 5% CO2. Once a dense monolayer of cells had formed, the plates were washed twice with sterile PBS (pH 7.4). Cells in the first well were digested with 0.25% trypsin and counted using a hemocytometer. 1 mL of sterile saline was added to the second well as a control, and 1 mL of the above-mentioned bacterial suspension was added to the remaining four wells. The plates were incubated at 37 ℃ for 2 h. Wells 2, 3, and 4 were washed three times with sterile PBS to remove any unattached bacterial strains. The smears were then removed, fixed with 0.4% paraformaldehyde for 20 min, air-dried, stained with crystal violet for 1 min, and observed and photographed under a microscope. Wash wells 5 and 6 five times with sterile PBS to remove unadhered test strains. Add 1 mL of 1% Triton X-100 to lyse the cells for 20 min. Collect the contents of the wells using a cell scraper, perform serial dilutions, and select appropriate dilution factors for plate counting. Use *Lactobacillus animalis* as a positive control. Adhesion rate is calculated using the following formula: Adhesion number = Number of bacteria to be tested × 100 / Number of cells (Equation 5) Note: The number of bacteria to be tested is the number of bacteria adhering to the cells in the well; the number of cells is the number of cells read from the first well using a hemocytometer.

[0079] Using HCT-8 cells as an adhesion model and *Lactobacillus animalis* as a control strain, the in vitro adhesion ability of strain FM135 was evaluated (see [link to relevant documentation]). Figure 11 ).

[0080] Depend on Figure 12 It can be seen that the adhesion number of animal lactobacillus can reach (1045±80) CFU / 100 cells, while the adhesion number of lactococcus FM135 can reach (1138±140) CFU / 100 cells. It can be seen that the adhesion performance of lactococcus FM135 is better than that of animal lactobacillus, so FM135 has the potential to colonize the intestine.

[0081] Example 2 Crude extraction of antibacterial substances (1) Activation and scale-up of strain: Activate strain FM135 on solid LB medium and confirm that there is no contamination; pick a single colony and inoculate it into liquid LB medium for shaking culture to complete the activation; scale up the inoculation into multiple bottles of liquid LB medium according to the ratio and culture at constant temperature (37℃, 150r / min for 24 hours) to obtain fermentation broth.

[0082] (2) Centrifugation of fermentation broth and collection of supernatant: Centrifuge the fermentation broth at low temperature and high speed (4℃, 12000r / min for 20min); remove the bacterial precipitate and collect the fermentation supernatant; take a portion of the supernatant for Oxford cup antibacterial experiment to verify the antibacterial activity.

[0083] (3) Extraction of antibacterial substances with organic solvents: The fermentation supernatant was mixed with ethyl acetate in equal volumes and extracted by shaking (shaking at 150 r / min for 1.5 hours at 20℃). Figure 13 ); let stand for 0.5 hours to separate into layers, forming an ethyl acetate layer, an emulsion layer, and an aqueous phase layer. Figure 14 Collect the ethyl acetate layer and the emulsion layer, and repeat the extraction multiple times to improve the recovery rate. Figure 15 ).

[0084] (4) Rotary evaporation concentration: The collected ethyl acetate phase is placed in a rotary evaporator; the ethyl acetate solvent is removed under low temperature conditions (42℃); an oily or concentrated crude extract containing antibacterial substances is obtained. Figure 16 ).

[0085] (5) Drying, dissolving and storing: The concentrated product is air-dried to form a crystalline or powdery crude extract. Figure 17 , 18 Dissolve in an appropriate amount of physiological saline to prepare a crude extract of antibacterial substances (1µg / mL); after aliquoting, store at -80 ℃.

[0086] Identification of antibacterial effects of crude extract The antimicrobial activity of the antimicrobial peptides was determined using the Oxford cup method. Concentrated antimicrobial peptide solutions were prepared as stock solutions and serially diluted (2, 4, 8, 16, 32, and 64 times), with the supernatant from centrifuged fermentation broth serving as a control. A total of eight samples were collected. The antimicrobial effects against Staphylococcus aureus and Escherichia coli were determined. Staphylococcus aureus was diluted 1000-fold, and Escherichia coli was diluted 100-fold. 100 μL of the sample was added to each Oxford cup and incubated at 4 ℃ for 12–18 h to promote diffusion, followed by incubation at 37 ℃ for 12 h. The diameter of the inhibition zone was measured using calipers after a clear inhibition zone appeared.

[0087] The results are shown in Table 2 (Oxford cup antibacterial test results, unit mm): Some experimental results are shown in the figure below, for example, the original solution inhibits Staphylococcus aureus and Escherichia coli. Figure 19 , 20 .

[0088]

[0089] The results showed that the crude extract of antimicrobial peptide stock solution and its gradient dilutions both had antimicrobial effects on Staphylococcus aureus and Escherichia coli. The antimicrobial activity decreased with increasing dilution factor and was concentration-dependent.

[0090] The preventive effect of P. 135 lactococcus FM135 on feline diarrhea Experimental animals: 18 healthy Chinese domestic cats of similar weight at 6 months of age, half male and half female.

[0091] Experimental Design: After 5 days of feeding basal cat food with free access to water, 18 healthy cats were randomly divided into 3 groups: a blank control group, a diarrhea model group, and a Pediococcus FM135 group, with 6 cats in each group. The blank control group received no treatment and was fed regular cat food. During the experiment, the Pediococcus FM135 group received 1.0 × 10⁻⁶ cat food in addition to the treatment given to the blank control group. 9 5 mL of CFU / mL Pediococcus lactis FM135 was administered to the diarrhea model group, who were fed regular cat food. On day 8 of the experiment, both the diarrhea model group and the Pediococcus lactis FM135 group were given 1 g / mL senna leaves (200 μL / kg / cat) to establish the feline diarrhea model. During the experiment, fecal morphology, weight changes, diet, mental state, and other clinical symptoms were recorded for each cat. Blood samples were collected after the experiment to test complete blood count indicators.

[0092] The fecal characteristics scoring table is shown in Table 3.

[0093] Experimental Results: Table 4 shows that on day 0, the fecal characteristics scores of the cats in each group were similar, with no significant differences between groups, indicating that the intestinal state of the animals in each group was consistent before the experiment began. By day 9, the fecal characteristics score of the diarrhea model group was significantly higher (6.17), significantly higher than that of the blank control group (2.83, P<0.05), indicating that senna leaves successfully induced significant diarrhea. The fecal characteristics score of the P. FM135 lactococcus group was 5.17, which was significantly higher than that of the blank control group (P<0.05), but lower than that of the diarrhea model group, suggesting that probiotic intervention had a certain alleviating effect on the severity of diarrhea. By day 11, the fecal characteristics score of the diarrhea model group remained at a high level (6.00), significantly different from that of the blank control group (3.00) (P<0.05). The score of the Lactococcus FM135 group decreased to 4.17, which was between the blank control group and the diarrhea model group. The difference was not significant compared with the diarrhea model group, but it showed a significant downward trend, suggesting that probiotics have a promoting effect on improving stool characteristics during the diarrhea recovery period.

[0094]

[0095]

[0096] Note: Different lowercase letters in the same row indicate significant differences between groups. P< 0.05).

[0097] Furthermore, Table 5 shows that on day 0 of the experiment, no cats in any group experienced diarrhea, with a diarrhea rate of 0%, indicating a consistent baseline condition. On day 9, the diarrhea rate in the diarrhea model group rose to 100%, while the rate in the blank control group remained at 0%, indicating that the diarrhea model was successfully established. The diarrhea rate in the *Pediococcus lactis* FM135 group was 66.67%, significantly lower than that in the diarrhea model group. By day 11, the diarrhea rate in the diarrhea model group remained at 100%, showing no trend of spontaneous remission; while the diarrhea rate in the *Pediococcus lactis* FM135 group further decreased to 33.33%, indicating that the diarrhea symptoms of some animals were significantly relieved.

[0098] Although intervention with P. FM135 of Lactococcus lactis did not completely prevent diarrhea, it significantly reduced the incidence of diarrhea and markedly improved stool characteristics in the later stages of diarrhea, suggesting that it has a certain preventive and recovery-promoting effect on diarrhea.

[0099]

[0100] Table 6 shows that senna leaves successfully induced a feline diarrhea model, characterized by a significant increase in white blood cells and neutrophils, a significant decrease in the proportion of lymphocytes, and an increase in erythrocytes and hematocrit, suggesting an acute inflammatory response and dehydration stress. After intervention with *Pediococcus lactis* FM135, the excessive increase in neutrophils was significantly suppressed, while the absolute number and proportion of lymphocytes were significantly upregulated, indicating that this probiotic can reshape the distribution of immune cells, promote adaptive immune responses, and alleviate stress-induced inflammatory blood counts in the context of diarrhea. Simultaneously, erythrocyte and platelet-related indicators tended to normalize, suggesting a certain protective effect against diarrhea-related dehydration and inflammation.

[0101] Pietrococcus FM135 did not suppress the immune response under diarrheal stress, but rather improved diarrhea-related hematologic disorders by inhibiting excessive neutrophil inflammatory response and significantly enhancing lymphocyte-mediated adaptive immunity.

[0102]

[0103] Note: WBC (White blood cell count), Neu# (Neutral cell count), Lym# (Lymphocyte count), Mon# (Monocyte count), Eos# (Eosinophil count), Bas# (Basophil count), Neu% (Neutral percentage), Lym% (Lymphocyte percentage), Mon% (Monocyte percentage), Eos% (Eosinophil percentage), Bas% (Basophil percentage), RBC (Red blood cell count), HGB (Hemoglobin concentration), HCT (Hematocrit), MCV (Mean red blood cell volume), MCH (Mean red blood cell hemoglobin content), MCHC (Mean red blood cell hemoglobin concentration), RDW-CV (Red blood cell distribution width coefficient of variation), RDW-SD (Red blood cell distribution width standard deviation), PLT (Platelet count), MPV (Mean platelet volume), PDW (Platelet distribution width), PCT (Plateletcrit).

[0104] Note: Different lowercase letters in the superscript of the same data indicate statistically significant differences (P < 0.05).

[0105] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0106] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of Pediococcus lactis, characterized in that, The accession number is CGMCC NO.31973.

2. An inoculum containing the *Pediococcus lactis* as described in claim 1.

3. The fermentation product of Pediococcus lactis according to claim 1.

4. The use of the *Pediococcus lactis* of claim 1, or the bacterial agent of claim 2, or the fermentation product of claim 3 in food processing and the preparation of pet medications.

5. The application as described in claim 4, characterized in that, The food processing mentioned: pet food, or pet food additives.

6. The application as described in claim 5, characterized in that, The medication described improves diarrhea symptoms in cats and has a protective effect against diarrhea-related dehydration and inflammation.

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