A bacteria compound for dogs and cats and its preparation method and application

Abstract

The application provides a kind of dog cat pet bacterial agent compound and its preparation method and application, belong to microbial preparation technical field.The bacterial agent compound of the application includes fermented Lactobacillus mucus A6-3 and Pediococcus acidilactici PM-1;The preservation number of the fermented Lactobacillus mucus A6-3 is CGMCC No.27257, and the preservation number of the Pediococcus acidilactici PM-1 is CGMCC No.35007;A6-3 is combined with PM-1, which can optimize the intestinal flora structure, enrich beneficial metabolites, improve pet immunity and digestive and absorptive capacity.The application further screens freeze-dried powder of leech as bacterial body protective agent, which can improve the survival rate of probiotics, and also can cooperate with probiotics to enrich leech mucin degrading bacteria, and enhance the intestinal mucosal barrier function.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, and particularly relates to a canine and feline pet bacterial agent complex, its preparation method, and its application. Background Technology

[0002] With the rapid development of the pet industry, the intestinal health of dogs and cats has received increasing attention. Intestinal flora imbalance is one of the main causes of diarrhea, indigestion, and weakened immunity in pets, and probiotics, as important regulators of intestinal health, are widely used in pet food additives.

[0003] However, existing probiotics for pet food have many shortcomings: single probiotic strains have limited functions and cannot fully cover the needs of intestinal flora regulation; the combined use of different probiotics is not effective, and some combinations even have antagonistic effects; probiotics have poor tolerance to the pet's gastrointestinal environment (strong acid, digestive enzymes), have low survival rates, and are difficult to effectively colonize in the intestines; commonly used bacterial cell protectants (such as glycyrrhizin, maltodextrin, etc.) have limited protective effects and cannot synergistically enhance the intestinal health regulation effect of probiotics; some bacterial agents may produce trace amounts of harmful metabolites during metabolism, posing safety risks.

[0004] Therefore, developing a probiotic combination with synergistic effects, paired with a highly effective bacterial cell protectant, to achieve high survival rate of probiotics in the gastrointestinal tract, precise regulation of intestinal flora, and no accumulation of harmful metabolites in pet feed has significant practical significance and application value. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a canine and feline pet probiotic complex, its preparation method and application, which can improve pet immunity and digestive and absorptive capacity by optimizing the intestinal flora structure and enriching beneficial metabolites.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A compound probiotic for dogs and cats, comprising *Lactobacillus fermentum* A6-3 and *Pediococcus lactis* PM-1; the preservation number of *Lactobacillus fermentum* A6-3 is CGMCC No. 27257, and the preservation number of *Pediococcus lactis* PM-1 is CGMCC No. 35007.

[0007] Preferably, the total viable count of the fermenting *Lactobacillus mucinus* A6-3 and *Pediococcus lactis* PM-1 is ≥5 × 10⁻⁶. 9 CFU / g.

[0008] Preferably, the microbial agent complex further includes lyophilized leech powder.

[0009] More preferably, in the bacterial agent complex, the mass ratio of fermenting Lactobacillus mucinus A6-3, Pediococcus lactis PM-1 and lyophilized leech powder is 1~2:1~2:1~2.

[0010] The present invention also provides a method for preparing the above-mentioned microbial agent complex, comprising the following steps: Lactobacillus fermentum A6-3 and Pediococcus lactis PM-1 were activated and cultured separately, and the bacterial cells were obtained by centrifugation. The bacterial cells of Lactobacillus fermentum A6-3, Pediococcus lactis PM-1 and lyophilized leech powder were mixed.

[0011] Preferably, the method for preparing the fermenting *Lactobacillus mucinus* A6-3 cells includes: inoculating *Lactobacillus mucinus* A6-3 into MRS liquid medium, culturing at 37±2℃ with shaking for more than 24 hours, and collecting the cells by centrifugation.

[0012] Preferably, the preparation method of the *Pediococcus lactis* PM-1 includes: inoculating *Pediococcus lactis* PM-1 into MRS liquid medium, culturing at 37±2℃ with shaking for more than 24 hours, and collecting the bacterial cells by centrifugation.

[0013] Preferably, the method for preparing the leech freeze-dried powder includes: fasting healthy leeches for 1-2 weeks; anesthetizing the fasted leeches and collecting their salivary glands; homogenizing the obtained salivary glands with PBS buffer and collecting the supernatant by centrifugation; adding ammonium sulfate to the supernatant to achieve an ammonium sulfate saturation of 60%-80%; allowing it to stand for 4-6 hours; collecting the precipitate by centrifugation; dissolving the precipitate with PBS buffer, and removing ammonium sulfate and small molecule impurities from the resulting solution by dialysis; purifying A using an ion exchange column, eluting with PBS buffer containing 0-1M NaCl, and collecting the target protein peak; purifying B using a gel filtration column, eluting with PBS buffer, and collecting the target protein peak; purifying C using a specific affinity column, eluting with PBS buffer containing 0-1M NaCl, and collecting the high-purity leech extract; concentrating by ultrafiltration; and freeze-drying to obtain the leech freeze-dried powder.

[0014] More preferably, in the method for preparing the leech extract: The pH of the PBS buffer is 7.0~7.4; The homogenate temperature is 1~6℃; the ratio of salivary gland to PBS buffer used in homogenization is 1g:3~8mL; The centrifugation speed is 8000~12000 rpm; the centrifugation temperature is 1~6℃; The dialysis time is 24-48 hours; the dialysis solution is PBS buffer. The ion exchange columns are DEAE-Sepharose and / or Q-Sepharose ion exchange columns; The gel filter column is a Sephadex G-75 and / or Superdex 75 gel filter column; The specific affinity column is a heparin column; Ultrafiltration has a molecular weight cutoff of 5~10 kDa. This invention also provides the application of the above-mentioned microbial agent complex in the preparation of pet feed products for dogs and cats.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The fermented *Lactobacillus mucinus* A6-3 strain of this invention exhibits excellent gastrointestinal tolerance and can stably survive in saliva, gastric juice, and intestinal juice, significantly enriching anti-inflammatory and immunomodulatory metabolites; *Pediococcus lactis* PM-1 can effectively inhibit the proliferation of harmful intestinal bacteria and enrich cardiovascular and cerebrovascular protective metabolites; when used in combination, the number of viable bacteria is significantly higher than that of a single strain, and the coverage of beneficial metabolites is broader, exhibiting a synergistic effect of metabolic regulation and inhibition of harmful bacteria.

[0016] This invention uses freeze-dried leech powder as a bacterial cell protectant. When mixed with Lactobacillus fermentum A6-3 and Pediococcus lactis PM-1, it can significantly improve the survival rate of probiotics in the intestine, which is significantly better than traditional protectants such as licorice polysaccharide. At the same time, freeze-dried leech powder can synergistically enrich leech mucin-degrading bacteria with probiotics, enhance the protection of intestinal mucosal homeostasis, and prevent the accumulation of harmful metabolites.

[0017] The bacterial agent of this invention can increase the proportion of beneficial bacteria (Lactobacillus spp. + Pediococcus spp., etc.) in the intestines of dogs and cats to >90%, while the abundance of harmful bacteria such as Escherichia coli and Shigella spp. is <2%; it also enriches beneficial metabolites such as hexadecylsphingosine (intestinal barrier protection), D-(+)-phenyllactic acid (antibacterial and anti-inflammatory), and conjugated linoleic acid (lipid-regulating and anti-inflammatory). Attached Figure Description

[0018] Figure 1 Survival rates of *Lactobacillus fermentatus* A6-3 and its protectant in simulated saliva-gastric-intestinal fluid were determined. In the simulated saliva-gastric-intestinal fluid mixture, A represented 5 min of artificial saliva treatment, B 3 h of artificial gastric fluid treatment, and C 6 h of artificial intestinal fluid treatment. A6-3 represented *Lactobacillus fermentatus*, A6-3+G represented *Lactobacillus fermentatus* A6-3 plus glycyrrhizic acid, and A6-3+S represented *Lactobacillus fermentatus* A6-3 plus lyophilized leech powder. Statistical differences were expressed as follows: .

[0019] Figure 2 Summary results of the abundance of pet fecal metabolites fermented by A6-3 cells and their protectant (Group 1).

[0020] Figure 3 Summary results of the abundance of metabolites in pet feces fermented by PM-1 cells and its protectant (Group 3).

[0021] Figure 4 Summary results of the abundance of pet fecal metabolites fermented by A6-3+PM-1 cells and their protectant (Group 5).

[0022] Figure 5 Annotation results for species at the genus and species level in each group of A6-3 cells and their protectants (Group 1).

[0023] Figure 6 Annotation results for species at the genus and species level in each group of PM-1 cells and its protectant (Group 3).

[0024] Figure 7 Annotation results for species at the genus and species level in each group of A6-3+PM-1 cells and its protectant (Group 5).

[0025] Figure 8 Metabolite enrichment analysis for the optimal treatment groups in Groups 1, 3, and 5.

[0026] Figure 9 Annotate enrichment analysis at the genus and species levels for the optimal treatment groups in Groups 1, 3, and 5.

[0027] Instructions for the Preservation of Biological Materials The strain of this invention is named A6-3, belongs to the genus Lactobacillus, and is a fermenting mucinous Lactobacillus ( Limosilactobacillus fermentum The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 27257, and the deposit date is May 4, 2023. It was published as invention patent CN 119033827 A on November 29, 2024.

[0028] Another strain of this invention is named PM-1, belonging to the genus Pediococcus, specifically *Pediococcus lactis*. Pediococcus acidilactici The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 35007, and the deposit date is June 25, 2025. Detailed Implementation

[0029] This invention provides a canine and feline pet probiotic complex comprising *Lactobacillus fermentum* A6-3 and *Pediococcus lactis* PM-1; the preservation number of *Lactobacillus fermentum* A6-3 is CGMCC No. 27257, and the preservation number of *Pediococcus lactis* PM-1 is CGMCC No. 35007. *Lactobacillus fermentum* A6-3 can enhance the tolerance of probiotics in the gastrointestinal environment and significantly enrich beneficial metabolites with anti-inflammatory and immunomodulatory properties; *Pediococcus lactis* PM-1 can effectively inhibit the proliferation of harmful intestinal bacteria (such as *Clostridium* and *Streptococcus*) and promote the production of intestinal homeostatic metabolites. The combined use of these two probiotics achieves functional complementarity and synergistically enhances the intestinal flora regulation effect, with both the beneficial bacteria proliferation efficiency and harmful bacteria inhibition ability significantly superior to those of a single strain.

[0030] In this invention, the total viable count of *Lactobacillus fermentum* A6-3 and *Pediococcus lactis* PM-1 is preferably ≥5 × 10⁻⁶. 9 CFU / g.

[0031] In this invention, the preferred microbial agent complex also includes lyophilized leech powder. As a cell protectant for *Lactobacillus mucinus* A6-3 and *Pediococcus lactis* PM-1, lyophilized leech powder can significantly improve the survival rate of probiotics in artificially simulated saliva-gastric juice-intestinal juice, especially achieving a survival rate of over 95% in intestinal juice. Simultaneously, it can synergistically enrich leech mucin-degrading bacteria with probiotics, enhancing the intestinal mucosal barrier function. Compared with traditional protectants such as licorice polysaccharide, it can reduce the formation of potentially harmful metabolites, resulting in superior safety.

[0032] In this invention, the preferred mass ratio of fermented Lactobacillus mucinus A6-3, Pediococcus lactis PM-1, and lyophilized leech powder is 1~2:1~2:1~2, and the more preferred mass ratio is 1:1:2.

[0033] The present invention also provides a method for preparing the above-mentioned microbial agent complex, comprising the following steps: Lactobacillus fermentum A6-3 and Pediococcus lactis PM-1 were activated and cultured separately, and the bacterial cells were obtained by centrifugation. The Lactobacillus fermentum A6-3 cells, Pediococcus lactis PM-1 cells, and lyophilized leech powder were then mixed. Further optimization involved mixing the Lactobacillus fermentum A6-3 cells and Pediococcus lactis PM-1 cells, and then mixing the mixed bacterial cells with the lyophilized leech powder.

[0034] In this invention, a preferred method for preparing *Lactobacillus fermentatus* A6-3 cells includes: inoculating *Lactobacillus fermentatus* A6-3 into MRS liquid medium, culturing at 37±2℃ with shaking for at least 24 hours, and collecting the cells by centrifugation; a further preferred method includes: inoculating activated *Lactobacillus fermentatus* A6-3 into MRS liquid medium, culturing at 37℃ and 180 rpm with shaking for 24 hours; then centrifuging at 5000 rpm for 5 minutes, discarding the supernatant, and collecting the cells; resuspending and washing with the same volume of PBS, centrifuging again at 5000 rpm for 5 minutes, discarding the supernatant, and obtaining the cells.

[0035] In this invention, a preferred method for preparing Pediococcus lactis PM-1 includes: inoculating Pediococcus lactis PM-1 into MRS liquid medium, culturing at 37±2℃ with shaking for at least 24 hours, and collecting the bacterial cells by centrifugation; a further preferred method includes: inoculating activated Pediococcus lactis PM-1 into MRS liquid medium, culturing at 37℃ and 180 rpm with shaking for 24 hours; then centrifuging at 5000 rpm for 5 minutes, discarding the supernatant, and collecting the bacterial cells; resuspending and washing with the same volume of PBS, centrifuging again at 5000 rpm for 5 minutes, discarding the supernatant, and obtaining the bacterial cells.

[0036] In this invention, a preferred method for preparing leech freeze-dried powder includes: (1) Take healthy leeches and fast them for 1-2 weeks; anesthetize the leeches after fasting and collect their salivary glands; (2) The obtained salivary gland is homogenized with PBS buffer and the supernatant is collected by centrifugation; the pH value of the PBS buffer is more preferably 7.0~7.4, more preferably 7.2~7.3; the homogenization temperature is more preferably 1~6℃, more preferably 4℃; the ratio of salivary gland to PBS buffer used in homogenization is 1g:3~8mL, more preferably 1g:4~6mL, more preferably 1g:5mL; (3) Add ammonium sulfate to the supernatant to make the ammonium sulfate saturation reach 60%~80%, more preferably 70%; let stand for 4~6h, more preferably 5h; collect the precipitate by centrifugation; more preferably the centrifugation speed is 8000~12000rpm, more preferably 10000rpm; more preferably the centrifugation temperature is 1~6℃, more preferably 4℃; (4) The precipitate was dissolved in PBS buffer, and the resulting solution was dialyzed to remove ammonium sulfate and small molecule impurities; the dialysis time was preferably 24-48 h, more preferably 36 h; the dialysis solution was PBS buffer. (5) Purify A using an ion exchange column, elute with PBS buffer containing 0-1M NaCl, and collect the target protein peak; further optimize the ion exchange column as DEAE-Sepharose and / or Q-Sepharose ion exchange column; (6) B was purified by gel filtration column, eluted with PBS buffer, and the target protein peak was collected; the gel filtration column was further preferred to be Sephadex G-75 and / or Superdex 75 gel filtration column. (7) C was purified using a specific affinity column, eluted with PBS buffer containing 0-1M NaCl, and high-purity leech extract was collected; the specific affinity column was further optimized to be a heparin column; (8) Ultrafiltration concentration; further preferably, the molecular weight cutoff of ultrafiltration is 5~10kDa; freeze-dry to obtain the leech freeze-dried powder.

[0037] This invention also provides the application of the above-mentioned microbial agent complex in the preparation of pet feed products for dogs and cats.

[0038] As one possible implementation method, the microbial compound of the present invention can be added to the daily diet of pet dogs / cats, which can significantly optimize the intestinal flora structure, increase the proportion of beneficial bacteria, reduce the abundance of harmful bacteria, enrich anti-inflammatory and intestinal barrier protective metabolites, improve the pet's digestion and absorption efficiency, enhance immunity, and reduce the occurrence of intestinal problems such as diarrhea and indigestion.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 A compound for disinfecting canine and feline pets, prepared by the following method: (1) Preparation of fermented Lactobacillus mucinus A6-3 cells: Fermented Lactobacillus mucinus A6-3 was inoculated into MRS liquid medium and cultured at 37℃ and 180rpm for 12h, and activated twice; 5mL of bacterial solution was centrifuged at 5000rpm and 4℃ for 5min and the supernatant was discarded. The supernatant was washed twice with the same volume of sterile PBS buffer, and centrifuged at 5000rpm for 5min each time and the supernatant was discarded to obtain A6-3 cells.

[0041] (2) Preparation of P. 1 lactic acid cocci PM-1 cells: P. 1 lactic acid cocci PM-1 were inoculated into MRS liquid medium and cultured at 37℃ and 180 rpm for 12 h, and activated twice; 5 mL of bacterial solution was centrifuged at 5000 rpm and 4℃ for 5 min and the supernatant was discarded. The supernatant was washed twice with the same volume of sterile PBS buffer, and centrifuged at 5000 rpm for 5 min each time and the supernatant was discarded to obtain PM-1 cells.

[0042] (3) Preparation of lyophilized leech powder: Healthy leeches were fasted for 1.5 weeks and their salivary glands were collected after anesthesia. PBS buffer at pH 7.2 was added at a ratio of 1g:5mL and homogenized at 4℃. The supernatant was collected by centrifugation at 10000rpm and 4℃ for 15min. Ammonium sulfate was added to 70% saturation and allowed to stand for 5h. The precipitate was collected by centrifugation at 10000rpm and 4℃ for 15min. The precipitate was dissolved in PBS buffer at pH 7.2 and dialyzed for 36h. The leech powder was purified sequentially by passing it through a DEAE-Sepharose ion exchange column, a Sephadex G-75 gel filter column, and a heparin affinity column, and the target protein peak was collected. The leech powder was concentrated by ultrafiltration membrane with a molecular weight cutoff of 8kDa and then lyophilized to obtain lyophilized leech powder.

[0043] (4) Mixing of bacterial agents: Mix A6-3 cells and PM-1 cells at a ratio of 1:1 to obtain mixed bacterial cells (total viable count ≥ 5 × 10⁻⁶). 9 (CFU / g); then mix the mixed bacteria and leech freeze-dried powder at a mass ratio of 1:1, and stir evenly under aseptic conditions to obtain a dog and cat pet bacterial agent complex.

[0044] Example 2 A compound for disinfecting canine and feline pets, prepared by the following method: (1) Preparation of fermented Lactobacillus mucinus A6-3 cells: Same as step 1 in Example 1, to obtain A6-3 cells.

[0045] (2) Preparation of Pietrococcus PM-1 cells: Same as step 2 in Example 1, to obtain PM-1 cells.

[0046] (3) Preparation of freeze-dried leech powder: Same as step 3 in Example 1.

[0047] (4) Mixing of bacterial agents: Mix A6-3 bacterial cells, PM-1 bacterial cells, and leech freeze-dried powder at a mass ratio of 2:1:2, and stir evenly under aseptic conditions to obtain a dog and cat pet bacterial agent complex with a total viable count ≥5×10⁻⁶. 9 CFU / mL.

[0048] Example 3 A compound for disinfecting canine and feline pets, prepared by the following method: (1) Preparation of fermented Lactobacillus mucinus A6-3 cells: Same as step 1 in Example 1, to obtain A6-3 cells.

[0049] (2) Preparation of Pietrococcus PM-1 cells: Same as step 2 in Example 1, to obtain PM-1 cells.

[0050] (3) Preparation of freeze-dried leech powder: Same as step 3 in Example 1.

[0051] (4) Mixing of bacterial agents: Mix A6-3 bacterial cells, PM-1 bacterial cells, and lyophilized leech powder at a mass ratio of 1:2:1, and stir evenly under aseptic conditions to obtain a dog and cat pet bacterial agent complex with a total viable count ≥5×10⁻⁶. 9 CFU / mL.

[0052] Experimental Example 1 Artificial simulated gastrointestinal tolerance test of fermenting Lactobacillus mucinus A6-3 1. Test materials Strain: Lactobacillus fermentum A6-3 (CGMCC No. 27257); Protective agents: licorice polysaccharide, leech freeze-dried powder; Reagents: NaCl, KCl, CaCl2, NaHCO3, HCl, α-amylase, pepsin, and trypsin, all of analytical grade; Culture media: MRS liquid medium, MRS solid medium; Instruments: constant temperature shaker, sterile ultra-clean workbench, constant temperature incubator, high-speed centrifuge.

[0053] 2. Test methods Artificial preparation of saliva-gastric juice-intestinal juice Saliva electrolyte solution: 0.7644g NaCl, 1.491g KCl, and 0.1332g CaCl2 were dissolved in 1L distilled water, and the pH was adjusted to 6.9±0.05 with 1mol / L HCl and 1mol / L NaHCO3. The solution was filtered through a 0.22μm filter and stored at 4℃. Simulated saliva: 0.3450g α-amylase was dissolved in 400mL of salivary electrolyte solution, and the mixture was magnetically stirred for 20min. After filtration through a 0.22μm filter, 400mL of salivary electrolyte solution was added and mixed well.

[0054] Gastric electrolyte solution: 3.19g NaCl, 1.10g KCl, 0.15g CaCl2, and 0.60g NaHCO3 were dissolved in 1L distilled water, and the pH was adjusted to 3 with 1mol / L HCl; Simulated gastric juice: 0.265g pepsin was dissolved in 800mL of gastric electrolyte solution, and the pH was adjusted to 3 with 1mol / L HCl. The solution was filtered through a 0.22μm filter and stored at 4℃.

[0055] Intestinal electrolyte solution: 5.40g NaCl, 0.65g KCl, and 0.33g CaCl2 were dissolved in 1L distilled water, and the pH was adjusted to 7 with 1mol / L NaHCO3; Simulated intestinal fluid: Trypsin was added at 1g / 100mL, filtered through a 0.22μm filter, and stored at 4℃.

[0056] Strain activation and suspension preparation: After activating A6-3 for two generations, the bacterial concentration was adjusted to 1×10⁻⁶. 9CFU / mL; 0.02g of licorice polysaccharide, leech lyophilized powder, A6-3+ licorice polysaccharide, and A6-3+ leech lyophilized powder were weighed and inoculated into 5mL of MRS liquid culture medium to prepare suspensions. A pure A6-3 suspension control group was set up at the same time.

[0057] 3. Tolerance testing Saliva treatment: Each suspension was inoculated into 3 mL of simulated saliva at a 4% inoculation rate and cultured on a shaker at 37°C and 180 rpm. Samples were taken at 0 min and 5 min. Gastric juice treatment: Take the bacterial culture after saliva treatment for 5 min, inoculate it into 3 mL of simulated gastric juice at a 4% inoculation rate, and incubate at 37℃ and 180 rpm for 3 h before taking samples; Intestinal fluid treatment: Take the bacterial culture after 3 hours of gastric fluid treatment, inoculate it into 3 mL of simulated intestinal fluid at a 4% inoculation rate, and incubate it in a shaker at 37℃ and 180 rpm for 6 hours before taking samples.

[0058] Viable bacterial count and survival rate calculation: Samples at each time point were serially diluted to 10⁻⁶ with sterile physiological saline. -7 Three dilutions were selected, with three replicates for each dilution. 10 μL of each sample was spotted onto MRS solid medium and incubated at 37°C for 24 hours. Viability was then counted. The survival rate was calculated using the formula: Viability = A i / A0×100%(A i (A0 represents the initial number of viable bacteria, where A0 is the number of viable bacteria after treatment).

[0059] 4. Test Results like Figure 1 As shown, *Lactobacillus fermentum* A6-3, *Lactobacillus fermentum* A6-3 + glycyrrhiza polysaccharide, and *Lactobacillus fermentum* A6-3 + leech freeze-dried powder can all survive in saliva-gastric juice-intestinal juice. The survival rate can reach over 99% after 5 minutes of treatment in artificial simulated saliva. Figure 1 A). Treatment in simulated gastric fluid for 3 hours ( Figure 1 B), the survival rate of *Lactobacillus fermentum* A6-3 decreased to 79.29%, the survival rate of *Lactobacillus fermentum* A6-3 + glycyrrhiza polysaccharide decreased to 66.82%, and the survival rate of *Lactobacillus fermentum* A6-3 + leech lyophilized powder decreased to 60.21%, but all could survive in gastric juice. After treatment in artificial simulated intestinal fluid for 6 hours ( Figure 1 C), the survival rate of fermented Lactobacillus mucin A6-3 increased to 95.15%, the survival rate of fermented Lactobacillus mucin A6-3 + licorice polysaccharide increased to 91.71%, and the survival rate of fermented Lactobacillus mucin A6-3 + leech freeze-dried powder increased to 98.05%.

[0060] Lactobacillus fermentum A6-3 has good tolerance to artificially simulated gastrointestinal tract and can survive stably in saliva, gastric juice and intestinal juice. When combined with lyophilized leech powder, its survival rate in intestinal juice is further improved, and lyophilized leech powder has a significant intestinal protective effect on A6-3.

[0061] Experimental Example 2 Detection of viable bacteria count in pet feces using single or combined probiotics and cell protectants. 1. Test materials Strains: Lactobacillus fermentum A6-3 (CGMCC No. 27257), Pediococcus lactis PM-1 (CGMCC No. 35007); Sample: Mixed feces from 3 healthy dogs and cats; Protective agents: Glycyrrhiza polysaccharide (G), leech freeze-dried powder (S); Culture media: MRS liquid / solid medium and Bifidobacterium medium, both aseptically treated; Reagents: Sterile physiological saline, PBS buffer; Instruments: grinder, constant temperature shaker, high-speed centrifuge, constant temperature incubator, sterile ultra-clean workbench.

[0062] 2. Test methods Preparation of fecal suspension: Weigh 0.03g of feces from 3 pets and place them in a sterile grinding tube. Add 1 4mm grinding bead, 3 3mm grinding beads and 900μL of physiological saline. Set the grinder to 45s / cycle, 5s interval, 45HZ, for 3 cycles. After grinding, the fecal suspension is obtained and stored at -80℃ for later use.

[0063] 3. Preparation of bacterial cells and protective agents Single bacterial cells: A6-3 and PM-1 were activated at 37℃ and 180rpm for 24h, respectively. After centrifugation at 5000rpm for 5min, the supernatant was discarded, and the cells were washed twice with PBS to obtain pure bacterial cells. Combined bacterial cells: A6-3 and PM-1 bacterial cells were mixed in a 1:1 ratio to prepare A6-3+PM-1 combined bacterial cells; Composite protectant: A6-3, PM-1, and A6-3+PM-1 bacterial cells were mixed with G and S in a 1:1 ratio to prepare composite protectants A6-3+G, A6-3+S, PM-1+G, PM-1+S, A6-3+PM-1+G, and A6-3+PM-1+S.

[0064] 4. Experimental Grouping and Culture: The experiment included a blank control group (NC, fecal suspension + MRS only), single-bacterial groups (A6-3, PM-1), single-bacterial + protectant groups (A6-3 + G, A6-3 + S, PM-1 + G, PM-1 + S), combined groups (A6-3 + PM-1), combined groups + protectant groups (A6-3 + PM-1 + G, A6-3 + PM-1 + S), and pure protectant groups (G, S), with three replicates per group. For each treatment group, 0.03 g of bacterial cells / protectant / combined protectant was weighed, added to 5 mL of MRS liquid medium, and then 500 μL of fecal suspension was added. The mixture was incubated at 37℃ and 180 rpm for 0 h and 24 h, respectively. Data from 48 h were discarded (as it showed no advantage and easily produced acid, inhibiting bacterial growth).

[0065] Viable bacteria count: Bacterial suspensions were collected at each time point and serially diluted with physiological saline to 10-1. -7 Three dilutions were selected, with three replicates for each dilution. 10 μL of each product was spotted onto MRS solid medium and Bifidobacterium medium. After incubation at 37°C for 24 h, the dilutions with colony counts of 30 to 300 were counted. No viable bacteria were observed on MacConkey plates, so they were not counted.

[0066] 5. Test Results The core results of the viable bacterial counts in MRS and Bifidobacterium culture medium after 24 hours of culture for each treatment group are shown in Table 1. As shown in Table 1, the viable bacterial counts in all probiotic treatment groups were significantly higher than those in the blank control group. Although the viable bacterial counts in the pure protectant group increased, they were still much lower than those in the probiotic group. In the single bacterial group, the viable bacterial count of A6-3 (10 11 The level was significantly higher than PM-1 (10). 10 (Level); the viable cell counts after synergistic use of A6-3 and S, and PM-1 and G were both higher than those in the pure single-cell group; the viable cell count in the A6-3+PM-1 combination group reached 1.89×10 on MRS plates. 11 The CFU / mL count on the Bifidobacterium plate was 3.97 × 10⁻⁶. 10 The CFU / mL was significantly higher than that of the PM-1 single-strain group, and the viable count in the combined treatment + protectant group remained stable without significant decrease.

[0067] Table 1. Viable bacterial count (CFU / mL) of fermented pet feces in each treatment group after 24 hours.

[0068] Experimental Example 3 Metabolomics analysis of the effects of single probiotic strains / combined probiotics + bacterial cell protectants on pet feces 1. Test materials Samples and groups: Supernatant of each treatment group after 24h fermentation (flash-frozen in liquid nitrogen and stored at -80℃); each treatment group is shown in Table 2.

[0069] Table 2. Sample names and treatment categories

[0070] Reagents: Methanol, acetonitrile, and formic acid, all of chromatographic grade; ultrapure water (prepared by Millipore). Instruments: Ultra-high performance liquid chromatograph (UPLC), high resolution mass spectrometer (Q-TOF / MS), data analysis software (Metabo Analyst, SIMCA-P).

[0071] 2. Test methods Sample pretreatment: Take 200 μL of supernatant from each treatment group, add 800 μL of pre-cooled methanol, vortex to mix, centrifuge at 4℃ and 12000 rpm for 15 min, take the supernatant, blow dry with nitrogen, redissolve with 100 μL of acetonitrile-water (1:1, v / v), filter through a 0.22 μm filter membrane, and wait for instrument detection.

[0072] 3. Non-targeted metabolomics detection UPLC conditions: C18 column (2.1×100mm, 1.7μm); mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution; gradient elution: 0~2min, 5% B; 2~10min, 5%~95% B; 10~12min, 95% B; flow rate 0.3mL / min; column temperature 40℃; injection volume 5μL.

[0073] MS conditions: Electrospray ionization (ESI), simultaneous detection in positive and negative ion modes; scan range m / z 50~1000; capillary voltage 3.0kV; cone voltage 40V; desolvation gas temperature 400℃; desolvation gas flow rate 800L / h.

[0074] Data analysis: The detection data were imported into the software for peak identification, peak matching and normalization. The top 15 metabolites in total abundance and differential metabolites (VIP>1, P<0.05) were screened. The enrichment characteristics of metabolites in each treatment group were analyzed, with a focus on beneficial metabolites with anti-inflammatory and antioxidant properties, intestinal barrier protection and cardiovascular protection, as well as potentially harmful metabolites with hepatotoxicity, drug contamination and lipid peroxidation.

[0075] 4. Test Results like Figures 2-4As shown, the abundance of beneficial metabolites in all probiotic treatment groups was significantly higher than that in the blank control group, and all groups significantly reduced oxidative stress-related harmful metabolites. The single-strain + S group was enriched with specific beneficial metabolites and had a low abundance of harmful metabolites. In the combination group, the beneficial metabolites enriched by APSF5 (A6-3+PM-1+S) covered multiple dimensions such as anti-inflammatory and antioxidant effects, intestinal barrier protection, cardiovascular protection, and energy metabolism, and the abundance of potentially harmful metabolites was the lowest among all treatment groups, which was significantly better than APGF5 (combination + G).

[0076] Both single-strain and combined use of probiotics can significantly regulate the metabolic profile of pet feces, promoting the accumulation of beneficial metabolites and reducing the abundance of harmful metabolites. The effect is even better when combined with leech freeze-dried powder. The combination of A6-3 + PM-1 + leech freeze-dried powder exhibits the best metabolic regulation effect, accumulating the broadest range of beneficial metabolites and having the lowest abundance of potentially harmful metabolites, providing multifaceted protection for pet gut health at the metabolic level. Test Example 4 Amplicon sequencing analysis of the effects of single probiotic strains / combined probiotics + protectants on pet feces 1. Test materials Samples: Same as in Experiment 3, bacterial suspensions after 24 hours of fermentation in each treatment group (flash-frozen in liquid nitrogen and stored at -80°C). Reagents: DNA extraction kit, PCR amplification kit, agarose, and nucleic acid dyes were all commercially available reagents; Instruments: Nucleic acid extractor, PCR instrument, agarose gel electrophoresis instrument, high-throughput sequencer (PacBioSequel), bioinformatics analysis software (QIIME2, R language).

[0077] 2. Test methods Microbial DNA extraction: Total DNA was extracted from the bacterial suspensions of each treatment group using a DNA extraction kit according to the instructions. The purity and concentration of DNA were detected by agarose gel electrophoresis and stored at -20℃ for later use.

[0078] 16S rRNA gene amplification and sequencing: Using the extracted total DNA as a template, PCR amplification was performed using universal primers for the full-length 16S rRNA gene; after purification and quantification of the PCR products, a sequencing library was constructed and subjected to Pac Bio Sequel high-throughput sequencing.

[0079] Bioinformatics analysis: Quality control, OTU clustering, and species annotation were performed on sequencing data, down to the genus level (core analysis level), and the top 20 genera in relative abundance were screened. LEfSe analysis was used to screen for biomarkers with significant differences (LDAscore>3.0, q-value<0.05), with a focus on analyzing the abundance changes of beneficial bacteria such as Lactobacillus and Pediococcus, and harmful / opportunistic pathogens such as Escherichia coli-Shigella, Clostridium, and Streptococcus.

[0080] 3. Test Results like Figures 5-7 As shown, the abundance of beneficial bacteria in all probiotic treatment groups was significantly higher than that in the blank control group, while the abundance of harmful / opportunistic pathogens was significantly reduced. The proportion of opportunistic pathogens in the blank control group was as high as 28%. The single-strain + S group could significantly enrich the corresponding probiotic genera, and the PM-1 + S group specifically enriched leech mucin-degrading bacteria, which is beneficial to intestinal mucosal homeostasis. In the combination group, the total abundance of beneficial bacteria in APSF5 was >90% (Lactobacillus spp. + Pediococcus spp. were the absolute dominant bacteria), while the abundance of harmful bacteria such as Escherichia coli and Shigella spp. was <2%, which was the lowest among all treatment groups. At the same time, it specifically enriched leech mucin-degrading bacteria, which was significantly better than APGF5.

[0081] Experimental Example 5 Comprehensive comparative verification of different optimal treatment groups Based on the experimental examples 3-4, AGF1 in Group 1, PSF3 in Group 3, and APSF5 in Group 5 were selected and compared to determine the final optimal treatment group.

[0082] Metabolite enrichment analysis results are as follows Figure 8 As shown, under the Pos mode, AGF1, PSF3, and APSF5 were all significantly enriched in the metabolite Hexadecasphinganine, which has anti-inflammatory and barrier-protective functions. Furthermore, the abundance of D-erythro-Sphingosine and D-Ribo-phytosphingosine was increased in APSF5 compared to the other two groups. The abundance of other beneficial metabolites showed little difference among the groups. Notably, the potentially harmful metabolite Glycidyl stearate was slightly lower in APSF5 than in the AGF1 group, and the potentially harmful metabolites Octadecanoic acid were slightly lower in APSF5 than in PSF3. Under the Neg mode, D-(+)-Phenyllactic acid was the significantly enriched metabolite in all three treatment groups, while the types and abundance of other beneficial metabolites were similar and did not differ significantly.

[0083] Amplicon sequencing analysis of the three treatment groups showed significant enrichment of bacterial genera and species, as follows: Figure 9 As shown, at the genus level, the proportion of beneficial bacteria in APSF5 was >90%, followed by PSF3, and then AGF1. The abundance of opportunistic pathogens and harmful bacteria, Escherichia coli-Shigella, in APSF5 was significantly lower than that in the AGF1 and PSF3 groups, therefore the APSF5 group had higher safety.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound for disinfecting canine and feline pets, characterized in that, It includes *Limosilactobacillus fermentum* A6-3 and *Pediococcus acidilactici* PM-1; the preservation number of *Limosilactobacillus fermentum* A6-3 is CGMCC No. 27257, and the preservation number of *Pediococcus acidilactici* PM-1 is CGMCC No. 35007.

2. The microbial agent complex according to claim 1, characterized in that, The total viable count of *Lactobacillus fermentum* A6-3 and *Pediococcus lactis* PM-1 is ≥5 × 10⁻⁶. 9 CFU / g.

3. The microbial agent complex according to claim 1, characterized in that, The microbial agent complex also includes lyophilized leech powder.

4. The microbial agent complex according to claim 3, characterized in that, In the bacterial agent complex, the mass ratio of fermenting Lactobacillus mucinus A6-3, Pediococcus lactis PM-1, and lyophilized leech powder is 1~2:1~2:1~2.

5. A method for preparing the microbial agent complex according to any one of claims 1-4, characterized in that, Includes the following steps: Lactobacillus fermentum A6-3 and Pediococcus lactis PM-1 were activated and cultured separately, and the bacterial cells were obtained by centrifugation. The bacterial cells of Lactobacillus fermentum A6-3, Pediococcus lactis PM-1 and lyophilized leech powder were mixed.

6. The preparation method according to claim 5, characterized in that, The method for preparing the fermenting *Lactobacillus mucinus* A6-3 cells includes: inoculating *Lactobacillus mucinus* A6-3 into MRS liquid medium, culturing at 37±2℃ with shaking for more than 24 hours, and collecting the cells by centrifugation.

7. The preparation method according to claim 5, characterized in that, The preparation method of the *Pediococcus lactis* PM-1 includes: inoculating *Pediococcus lactis* PM-1 into MRS liquid medium, culturing at 37±2℃ with shaking for more than 24 hours, and collecting the bacterial cells by centrifugation.

8. The preparation method according to claim 5, characterized in that, The method for preparing the leech freeze-dried powder includes: Healthy leeches were fasted for 1-2 weeks. The fasted leeches were anesthetized, and their salivary glands were collected. The collected salivary glands were homogenized with PBS buffer, and the supernatant was collected by centrifugation. Ammonium sulfate was added to the supernatant to achieve 60%-80% saturation. The mixture was allowed to stand for 4-6 hours. The precipitate was collected by centrifugation. The precipitate was dissolved in PBS buffer, and the resulting solution was dialyzed to remove ammonium sulfate and small molecule impurities. Purification A was performed using an ion exchange column, eluted with PBS buffer containing 0-1M NaCl, and the target protein peak was collected. Purification B was performed using a gel filtration column, eluted with PBS buffer, and the target protein peak was collected. Purification C was performed using a specific affinity column, eluted with PBS buffer containing 0-1M NaCl, and the high-purity leech extract was collected. The extract was concentrated by ultrafiltration and lyophilized to obtain the leech lyophilized powder.

9. The preparation method according to claim 8, characterized in that, In the preparation method of the leech extract: the pH value of the PBS buffer is 7.0~7.4; The homogenate temperature is 1~6℃; the ratio of salivary gland to PBS buffer used in homogenization is 1g:3~8mL; The centrifugation speed is 8000~12000 rpm; the centrifugation temperature is 1~6℃; The dialysis time is 24-48 hours; the dialysis solution is PBS buffer. The ion exchange columns are DEAE-Sepharose and / or Q-Sepharose ion exchange columns; The gel filter column is a Sephadex G-75 and / or Superdex 75 gel filter column; The specific affinity column is a heparin column; Ultrafiltration has a molecular weight cutoff of 5~10 kDa.

10. The use of the microbial agent complex according to any one of claims 1 to 4 in the preparation of pet feed products for dogs and cats.

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