Fermented lactobacillus muci-deus zy001 from cat and application and product thereof

Abstract

The application provides a cat-derived fermented lactobacillus muciadegus ZY001, application and product thereof, and relates to the technical field of biology. The fermented lactobacillus muciadegus ZY001 provided by the application is derived from a cat, and the inventor has found that the strain has good high-temperature resistance, acid resistance and bile salt resistance, can effectively inhibit the growth and proliferation of various diarrhea pathogenic bacteria, can be used for preventing or treating diarrhea, has antioxidant capacity, can relieve oxidative stress, is beneficial to animal anti-aging, and prolongs the life of a pet; has very strong self-aggregation and moderate hydrophobicity, and is beneficial to the strain in intestinal colonization and long-term exertion of the probiotic function.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a feline fermenting Lactobacillus mucinus ZY001 and its applications and products. Background Technology

[0002] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum, L. fermentum Classified under the genus *Lactobacillus* ( Limosilactobacillus *Lactobacillus fermentum* plays a vital role in milk fermentation, industrial lactic acid fermentation, and the health and pharmaceutical industries. Studies have shown that *Lactobacillus fermentum* has beneficial probiotic effects, such as regulating the gut microbiota, inhibiting harmful gut microbiota, reducing the activity of food allergens, decreasing mutagenic and carcinogenic activity, exhibiting immunomodulatory activity, and lowering cholesterol. Other studies have demonstrated that *Lactobacillus fermentum* plays an important role in enhancing the host's immune system and improving feed digestibility.

[0003] Pets serve as a bond and bridge for emotional communication among family members. The increasing awareness of scientific pet care has driven the upgrading of the pet market, with pet owners paying more attention to the scientific aspects of pet raising. Functional pet products have gained widespread attention, and market demand for these products continues to rise. A series of organic, natural, and health-beneficial functional pet foods have emerged. Probiotics, as a non-toxic, residue-free, and beneficial additive, are increasingly used in pet food. Examples include the use of Lactobacillus, Bifidobacterium, and Pediococcus lactis. However, most pet probiotic products currently on the market are not of host origin. The strains themselves have poor acid resistance, bile salt resistance, and heat resistance. After high-temperature processing and exposure to the gastrointestinal environment, the number of live bacteria reaching the intestines is significantly reduced; the effectiveness also varies. Considering the specificity of the host species and the tolerance of processing techniques and the gastrointestinal tract, using probiotics with high acid resistance, bile salt resistance, and heat resistance derived from the host species' intestines is essential to better adapt to the gastrointestinal environment and exert their beneficial functions. Currently, there are few reports on feline fermented Lactobacillus mucinus with excellent acid resistance, bile salt resistance, and high temperature resistance, and no related products have been found on the market.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The first objective of this invention is to provide a fermentation Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven ZY001) is used to solve the above-mentioned technical problems.

[0006] A second objective of this invention is to provide a microbial agent.

[0007] A third objective of this invention is to provide the aforementioned fermenting *Lactobacillus mucinus* ZY001 ( Limosilactobacillus leaven The application of ZY001 or microbial agents in the preparation of antibacterial products.

[0008] The fourth objective of this invention is to provide the above-mentioned fermenting *Lactobacillus mucinus* ZY001 ( Limosilactobacillus fermentum The application of ZY001 or bacterial agents in the preparation of products for the prevention or treatment of diarrhea.

[0009] The fifth objective of this invention is to provide the above-mentioned fermenting *Lactobacillus mucinus* ZY001 ( Limosilactobacillus leaven The application of ZY001 or microbial agents in the preparation of products that alleviate oxidative stress in animals.

[0010] The sixth objective of this invention is to provide a feed.

[0011] The seventh objective of this invention is to provide a drug.

[0012] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a fermentation of Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven ZY001), the fermenting Lactobacillus mucilaginosus ZY001 ( Limosilactobacillus fermentum ZY001 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251905, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on August 27, 2025.

[0013] As a further technical solution, the fermented Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven ZY001 is derived from cats.

[0014] Secondly, the present invention provides a microbial agent, comprising the aforementioned *Lactobacillus fermentans* ZY001 (… Limosilactobacillus fermentum ZY001).

[0015] Thirdly, the present invention provides the above-mentioned fermenting Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven The application of ZY001 or microbial agents in the preparation of antibacterial products.

[0016] As a further technical solution, the antibacterial product includes products that inhibit Escherichia coli, products that inhibit Staphylococcus aureus, products that inhibit Salmonella, products that inhibit Pseudomonas aeruginosa, or products that inhibit Listeria monocytogenes.

[0017] Fourthly, the present invention provides the above-mentioned fermenting Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven The application of ZY001 or bacterial agents in the preparation of products for the prevention or treatment of diarrhea.

[0018] Fifthly, the present invention provides the above-mentioned fermenting Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven The application of ZY001 or microbial agents in the preparation of products that alleviate oxidative stress in animals.

[0019] Sixthly, the present invention provides a feed comprising the aforementioned fermented Lactobacillus mucilaginosus ZY001 ( Limosilactobacillus fermentum ZY001) or the aforementioned microbial agent.

[0020] In a seventh aspect, the present invention provides a drug comprising the aforementioned *Lactobacillus fermentans* ZY001 ( Limosilactobacillus fermentum ZY001) or the aforementioned microbial agent.

[0021] As a further technical solution, the drug also includes excipients.

[0022] Compared with existing technologies, the fermented Lactobacillus mucinus ZY001 provided by this invention has the following beneficial effects: 1. Strain ZY001 has excellent high temperature resistance and can withstand the high temperature environment during processing, ensuring the survival rate of the strain after processing.

[0023] 2. Strain ZY001 has excellent acid resistance, which helps it resist the low pH environment in the stomach.

[0024] 3. Strain ZY001 has excellent bile salt tolerance and can adapt to the digestive tract environment with high bile salt concentration.

[0025] 4. Strain ZY001 and its metabolites can effectively inhibit the growth and proliferation of various diarrhea-causing bacteria and can be used to prevent or treat diarrhea.

[0026] 5. Strain ZY001 has good in vitro and in vivo antioxidant capacity, which can alleviate oxidative stress, benefit animals in anti-aging, and prolong the life of pets.

[0027] 6. The ZY0011 strain has strong self-cohesion and moderate hydrophobicity in the intestines, which facilitates its colonization in the intestines and allows it to exert its probiotic function for a long time. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 Colony morphology (left) and cell morphology (right) of fermenting Lactobacillus mucinus ZY001; Figure 2 Phylogenetic tree of Lactobacillus fermentans ZY001; Figure 3 The growth and acid production rate curves of fermented Lactobacillus mucilaginosus ZY001; Figure 4 To assess the acid tolerance of fermenting Lactobacillus mucilaginosus ZY001; Figure 5 Images showing the antibacterial activity of *Lactobacillus mucinus* ZY001 during fermentation (Note: A: *Escherichia coli*, B: *Staphylococcus aureus*, C: *Salmonella*, D: *Pseudomonas aeruginosa*, E: *Listeria monocytogenes*; a: bacterial suspension, b: cell-free supernatant, c: bacterial pellet, d: MRS liquid medium). Figure 6 The results of the hemolysis test for fermenting Lactobacillus mucinus ZY001; Figure 7 The growth and feed intake of ZY001 mice were shown (Note: A: Line graph of body weight over time, B: Bar graph of daily weight gain (ADG), C: Bar graph of average daily feed intake (ADFI)). Figure 8 A circos diagram of the genome; Figure 9 A classification chart of COG results; Figure 10 GO annotation results classification diagram (Note: BP: Biological Process; CC: Cellular Component; MF: Molecular Function) Figure 11 Classification diagram of KEGG pathway results. Detailed Implementation

[0030] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] In a first aspect, the present invention provides a fermentation of Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven ZY001), the strain is classified as: *Lactobacillus fermentatus*, Latin scientific name: Limosilactobacillus fermentum It is deposited at the China Center for Type Culture Collection, address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, date of deposit: August 27, 2025, accession number: CCTCC NO: M 20251905.

[0032] The fermenting Lactobacillus mucinus ZY001 provided by this invention is derived from cats. The inventors have found that this strain has good resistance to high temperature, acid, and bile salts, and can effectively inhibit the growth and proliferation of various diarrhea-causing bacteria, and can be used to prevent or treat diarrhea. It has antioxidant capacity, which can alleviate oxidative stress, benefit animals in anti-aging, and prolong the lifespan of pets. It has strong self-cohesion and moderate hydrophobicity, which is conducive to its colonization in the intestine and long-term exertion of its probiotic function.

[0033] Secondly, the present invention provides a microbial agent, comprising the aforementioned *Lactobacillus fermentans* ZY001 (… Limosilactobacillus fermentum ZY001).

[0034] The microbial agent provided by this invention includes *Lactobacillus fermentum* ZY001, or a mixture of *Lactobacillus fermentum* ZY001 with excipients or other microorganisms. For example, it can be a microbial agent prepared by mixing *Lactobacillus fermentum* ZY001 of this invention with other microorganisms, or it can be a microbial agent prepared by combining *Lactobacillus fermentum* ZY001 of this invention with excipients. This microbial agent possesses all the beneficial effects of *Lactobacillus fermentum* ZY001 of this invention.

[0035] Thirdly, the present invention provides the above-mentioned fermenting Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven The application of ZY001 or microbial agents in the preparation of antibacterial products.

[0036] According to the inventor's research, fermented Lactobacillus mucinus ZY001 has a good inhibitory effect on Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa and Listeria monocytogenes, and can be used to prepare antibacterial products.

[0037] Fourthly, the present invention provides the above-mentioned fermenting Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven The application of ZY001 or bacterial agents in the preparation of products for the prevention or treatment of diarrhea.

[0038] The inventors have discovered that the strain of this invention can effectively inhibit the growth and proliferation of various diarrhea-causing bacteria, and therefore can be used to prevent or treat diarrhea.

[0039] Fifthly, the present invention provides the above-mentioned fermenting Lactobacillus mucinus ZY001 ( Limosilactobacillus leaven The application of ZY001 or microbial agents in the preparation of products that alleviate oxidative stress in animals.

[0040] The inventors have discovered that the strain of this invention has good in vitro and in vivo antioxidant capacity, which can alleviate oxidative stress, benefit animals in anti-aging, and prolong the lifespan of pets.

[0041] Sixthly, the present invention provides a feed comprising the aforementioned fermented Lactobacillus mucilaginosus ZY001 ( Limosilactobacillus fermentum ZY001) or the aforementioned microbial agent.

[0042] In a seventh aspect, the present invention provides a drug comprising the aforementioned *Lactobacillus fermentans* ZY001 ( Limosilactobacillus fermentum ZY001) or the aforementioned microbial agent.

[0043] In some alternative embodiments, the drug may also include excipients.

[0044] The feed and medicine provided by this invention contain the fermented myxobacterium ZY001 of this invention, and therefore have all the beneficial effects of the fermented myxobacterium ZY001.

[0045] In some preferred embodiments, the drug further includes excipients. The selection of excipients is not specifically limited in this invention; pharmaceutical excipients well-known to those skilled in the art can be used.

[0046] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0047] Example 1 1. Isolation and identification of feline fermenting Lactobacillus mucinus 1.1 Culture medium for isolation MRS culture medium, the specific components of which are shown in Table 1: Table 1. Composition of MRS culture medium (g / L)

[0048] Sterilize at pH 6.5±0.2 at 121℃ for 15 min.

[0049] 1.2 Separation Method (1) Sample collection and microbial isolation: Three healthy adult tabby cats from the experimental farm of the College of Animal Science and Technology, Qingdao Agricultural University were selected. Rectal feces were collected by sterile swabs and quickly placed into 50 mL sterile screw-capped test tubes containing physiological saline. The tubes were transported to the Special Economic Animal Nutrition Laboratory of Qingdao Agricultural University under low temperature refrigeration conditions, and serial dilutions were immediately performed. Three plates were used for each dilution, and the microorganisms were isolated and cultured by pouring method at 37℃.

[0050] (2) Isolation and purification: Take the plates containing various bacteria, select single colonies of lactic acid bacteria with different colony morphologies, and isolate and purify them using the streak method. Repeat 3-4 times and examine under a microscope until a pure culture is obtained.

[0051] (3) Observe the colony characteristics and observe the bacterial morphology using Gram staining and microscopic examination.

[0052] (4) Bacterial DNA was extracted according to the instructions of the Bacterial Genome Extraction Kit (D1600) from Beijing Solarbio Science & Technology Co., Ltd. The obtained conserved sequences of lactic acid bacteria were amplified and sequenced using universal bacterial primers: 27F, 1492R. Primer synthesis and sequencing were performed by Beijing Qingke Biotechnology Co., Ltd. Homology comparison of the 16S rRNA sequence was conducted. The 16S rDNA amplification system is as follows, with a total volume of 50 μl:

[0053] PCR reaction program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 45 s, 56℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 30 cycles, with a final extension at 72℃ for 10 min. Save as C.

[0054] 1.3 Isolation, identification, and screening of fermenting *Lactobacillus mucinus* Based on morphological observation, microscopic examination, enzyme analysis, and carbohydrate utilization experiments, combined with 16S rRNA sequencing comparison, the strain was identified as *Lactobacillus fermentatus*, named ZY001. The 16S rRNA gene sequence is shown below. After 24 h of culture, the colonies showed a raised center, regular edges, and a smooth surface; they were facultative anaerobic, Gram-positive, and rod-shaped. The colony morphology of ZY001 is as follows: Figure 1 As shown in A, the morphology under a microscope is as follows: Figure 1 As shown in B in the diagram.

[0055] 16S rRNA gene sequence:

[0056] Phylogenetic analysis of the 16S rRNA genes of ZY001 confirmed their close phylogenetic relationship with *Pediococcus lactis*. The phylogenetic tree is shown below. Figure 2 As shown.

[0057] 2. Growth characteristics 2.1 Physiological and Biochemical Characteristics After 18 hours of activation and culture, the isolated bacterial culture was used to identify the physiological and biochemical characteristics of lactic acid bacteria, referring to Bergey's Manual of Bacterial Identification, the Manual of Systematic Identification of Common Bacteria, and the Classification, Identification and Experimental Methods of Lactic Acid Bacteria. Biochemical characteristics such as sugar fermentation test, VP test, methyl red (MR) test, and gelatin test were used for identification.

[0058] 2.2 Determination of growth performance and acid production performance The isolated bacterial culture, after 18 h of activation culture, was inoculated into MRS medium at a 2.0% inoculum and incubated at 37℃ for 24 h. The control group consisted of liquid culture medium without inoculum. OD values ​​of the culture medium were measured at 0, 1, 2, 3, 6, 9, 12, 18, 24, 30, 36, and 48 h. 600nm Absorbance values ​​were measured, and pH was determined at 0, 1, 2, 3, 6, 9, 12, 18, 24, 30, 36, and 48 h. The values ​​of pH and OD of the culture medium were plotted on the x-axis as time. 600nm Using absorbance as the ordinate, plot its growth curve and acid production curve.

[0059] 2.3 Acid resistance test After 18 h of activation culture, the isolated bacterial suspension was inoculated at a rate of 2.0% into MRS liquid medium with initial pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, and incubated at 37°C. The absorbance at OD600 nm was measured at 0, 1, 2, 3, 6, 9, 12, 18, 24, 30, 36, 42, and 48 h, with three replicates per group. The average value of the three parallel results was calculated, and a curve was plotted based on the calculation results.

[0060] 2.4 High Temperature Resistance Test After 18 h of activation culture, the isolated bacterial culture was treated in water baths at 50℃, 60℃, 70℃, 80℃, and 90℃ for 5 min each, and immediately placed in an ice box after treatment. 100 μL of culture medium was serially diluted and spread on a plate, and the viable cell count was calculated using the plate count method. The viable cell count at 0 h was used as a control to calculate the survival rate.

[0061] (1); in, and These represent the number of surviving bacteria at 0 min and 5 min (log CFU / mL).

[0062] 2.5 Gastrointestinal colonization capacity 2.5.1 Acid resistance test After 18 h of activation culture, the isolated bacterial culture was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was discarded, and the culture was washed twice with sterile neutral PBS buffer (pH 7.0), then resuspended in sterile PBS buffer (pH 2.5). After incubation at 37 °C for 0 and 3 h, 10× serially diluted cultures were plated on MRS agar plates and incubated at 37 °C for 24 h. The acid tolerance of the isolated strain was assessed by observing viable colonies. The experiment was repeated three times.

[0063] (2); in, and The values ​​represent the number of surviving bacteria (log CFU / mL) before incubation (0 h) and after incubation (3 h).

[0064] 2.5.2 Bile salt tolerance test After 18 h of activation culture, the isolated bacterial culture was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was discarded, and the culture was washed twice with sterile neutral PBS buffer (pH 7.0). The culture was then resuspended in PBS buffer containing 0.1% and 0.3% (w / v) porcine bile salts (Solarbio, China). After incubation at 37 °C for 0 and 4 h, 10× serially diluted cultures were plated on MRS agar plates and incubated at 37 °C for 24 h. The tolerance of the isolated strain to bile salts was assessed by viable colonies. The experiment was repeated three times.

[0065] (3); in, and The values ​​represent the number of surviving bacteria (log CFU / mL) before incubation (0 h) and after incubation (4 h).

[0066] 2.5.3 Artificial gastrointestinal fluid tolerance test The preparation of artificial gastric fluid and artificial intestinal fluid should refer to the 2010 edition of the Chinese Pharmacopoeia.

[0067] After 18 h of activation and culture, the isolated bacterial suspension was centrifuged at 10,000 rpm for 10 min at 4 °C, the supernatant was discarded, and the bacterial pellet was washed twice with sterile neutral PBS buffer (pH 7.0). The pellet was then resuspended in simulated gastric fluid and incubated at 37 °C and 200 rpm for 1.5 h. After incubation, the bacterial pellet was washed twice with sterile neutral PBS buffer (pH 7.0), centrifuged at 10,000 rpm for 10 min at 4 °C, and then resuspended in simulated intestinal fluid. The pellet was incubated at 37 °C and 200 rpm for 2 h. Bacterial pellet viability was calculated using the plate count dilution method before and after incubation in simulated gastric and intestinal fluids. The experiment was repeated three times.

[0068] (4); in, and The values ​​represent the number of surviving bacteria (log CFU / mL) before and after incubation in artificial gastric or intestinal fluids.

[0069] 2.5.4 Surface hydrophobicity test The bacterial adhesion to hydrocarbons (BATH) method was used to assess the hydrophobic properties of the bacterial strain surface by measuring the affinity of lactic acid bacteria for hydrocarbons. After 18 h of activation culture, the isolated bacterial culture was centrifuged at 10,000 rpm for 10 min at 4°C, washed three times with sterile neutral PBS buffer (pH 7.0), and the OD of the bacterial culture was adjusted. 630 The absorbance value was 0.25 ± 0.05 (A1) and used for later use. Then, the bacterial culture and chloroform were added at a 1:1 ratio, vortexed for 3 min, and incubated at 37°C for 10 h. Finally, the aqueous phase was carefully aspirated, and the OD was measured. 630 Absorbance value (A2). Repeat the test three times. The hydrophobicity calculation formula is as follows: (5); A1 and A2 are the OD630 absorbance values ​​before and after 10 hours of incubation, respectively.

[0070] 2.5.5 Self-cohesion test Take the bacterial culture that has been cultured overnight for 18 h, centrifuge at 10,000 rpm for 10 min at 4 °C, wash twice with sterile neutral PBS buffer (pH 7.0), then resuspend in an equal volume of sterile neutral PBS buffer (pH 7.0), measure the OD630 absorbance (A3), then vortex for 10 s, incubate at 37 °C for 8 h without stirring, carefully absorb the upper part, and measure the OD630. 630 Absorbance value (A4), repeated 3 times. The self-condensation rate is calculated using the following formula: (6); Among them, A3 and A4 are the OD values ​​before and after 8 hours of incubation, respectively. 630 Absorbance value.

[0071] 2.6 Antibacterial test The antibacterial activity of the strain was determined using the Oxford cup agar diffusion method. The isolated bacterial culture, after 18 h of activation culture, was centrifuged at 10,000 rpm for 10 min at 4°C. Cell-free supernatant (CFS) was removed, and the bacterial pellet (BP) was resuspended in an equal volume of sterile neutral PBS buffer (pH 7.0). A sterile Oxford cup was placed on an LB agar plate, and 200 μL of a 1×10⁻⁶ concentration was taken. 7 Pathogenic bacteria (Escherichia coli ATCC25922; Salmonella ATCC14028; Staphylococcus aureus ATCC25923; Pseudomonas aeruginosa ATCC27853; Listeria monocytogenes ATCC19115) at CFU / mL were spread on LB agar plates. After the bacterial culture was completely absorbed, 200 μL of BS, CFS, BP, and MRS liquid medium of the isolated strains were inoculated into each well. The LB plates were incubated upright in a 37°C incubator. After 24 h, the presence of inhibition zones was observed and the diameter of the inhibition zones was accurately measured.

[0072] 2.7 Antibiotic susceptibility testing Antibiotic susceptibility of the strains was determined using the disk diffusion method. Based on recommendations for evaluating probiotic safety and considering commonly available antibiotics, 30 antibiotics were selected and tested using 6 mm disks (BIO-KONT, China): penicillin, ampicillin, piperacillin, vancomycin, chloramphenicol, minocycline, doxycycline, erythromycin, clindamycin, lincomycin, florfenicol, cefoperazone, cefazolin, cefuroxime sodium, imipenem, gentamicin, tetracycline, azithromycin, ceftriaxone, vancomycin, cephalexin, oxacillin, streptomycin, amikacin, levofloxacin, kanamycin, polymyxin B, trimethoprim-sulfamethoxazole, norfloxacin, and ciprofloxacin.

[0073] After 18 h of activation culture, the isolated bacterial suspension was centrifuged at 10,000 rpm for 10 min at 4 °C. The suspension was washed twice with sterile neutral PBS buffer (pH 7.0) and then resuspended in sterile neutral PBS buffer (pH 7.0) to obtain a bacterial solution in the logarithmic growth phase (0.5 μL McFarland suspension). 100 μL of the McFarland suspension was spread onto an MRS agar plate. Finally, antibiotic discs were affixed to the surface of the MRS agar plate within 15 min. The plate was incubated at 37 °C for 24 h, and the formation of inhibition zones was observed. The diameter of the inhibition zones was measured using calipers.

[0074] 2.8 Free radical scavenging test 2.8.1 DPPH free radical scavenging test The DPPH scavenging ability of the isolated strains was determined using the Grace DPPH assay kit. Following the instructions, briefly, 150 μL of the isolated bacterial culture after 18 h of culture was added to 150 μL of working solution, mixed well, and incubated at room temperature in the dark for 30 min. Then, it was centrifuged at 12000 rpm for 5 min at room temperature. 200 μL of the mixture was added to a 96-well plate, and the absorbance was recorded at 517 nm using a microplate reader. Blank and control samples were also prepared. The formula for calculating the DPPH free radical scavenging rate is as follows: DPPH free radical scavenging rate (%) = [(1-(A5-A6) / A7)]×100% (7); Where: A5 is the absorbance of the sample to be tested; A6 is the absorbance of the control well; A7 is the absorbance of the blank well.

[0075] 2.8.2 ABTS Free Radical Scavenging Test The ABTS scavenging ability of the isolated strain was determined using the Greens ABTS assay kit. Following the instructions, briefly, 10 μL of the isolated bacterial culture after 18 h of incubation was added to 190 μL of working solution, mixed thoroughly, and incubated at room temperature in the dark for 6 min. The absorbance at 734 nm was measured using a microplate reader. Blank and control samples were also prepared. The formula for calculating the ABTS free radical scavenging rate is as follows: ABTS radical scavenging rate (%) = [(1-(A8-A9) / A] 10 )]×100%(8; Where: A8 is the absorbance of the sample to be tested; A9 is the absorbance of the control well; A 10 The absorbance of the blank aperture is denoted as .

[0076] 2.8.3 Superoxide anion radical scavenging test The superoxide anion scavenging ability of the isolated strains was determined using the Greens Superoxide Anion Scavenging Ability Assay Kit. Following the instructions, briefly, 500 μL of the isolated bacterial culture after 18 h of incubation was centrifuged at 10,000 rpm for 10 min at room temperature, and the supernatant was collected. 630 μL of Reagent I was added to an EP tube and incubated at room temperature for 20 min. Then, 30 μL of sample and 40 μL of Reagent II were added, and incubated at room temperature for 4 min. Finally, 20 μL of Reagent III was added, mixed well, and the absorbance was measured at 320 nm. Blank and control samples were also prepared. The formula for calculating superoxide anion radical scavenging ability is as follows: Superoxide anion radical scavenging rate (%) = [1-(A 12 -A 11 ) / A 13 ]×100%(9; Among them, A 11 A 12 and A 13 These are the absorbance values ​​of the control group, the measurement group, and the blank group, respectively.

[0077] 2.9 Safety Test 2.9.1 Hemolysis test After 18 h of activation culture, the isolated bacterial suspension was streaked onto trypsin-soybean agar (TSA) containing 5.0% (w / v) sheep blood (Oxoid, Germany). The plates were incubated at 37°C for 48 h, and the presence of β-hemolysis (a clear area around the colony), α-hemolysis (a green area around the colony), or γ-hemolysis (no area around the colony) was examined on the blood agar plates. Staphylococcus aureus (ATCC25923) strain was used as a positive control.

[0078] 2.9.2 Safety test in mice To evaluate in vivo safety, 56 healthy Kunming mice of similar weight were randomly divided into 4 groups of 14 animals each. The control group (CK) mice were administered 0.2 mL of sterile saline, while the other three groups were administered 0.2 mL of the isolated bacterial strain at a dose of 1×10⁻⁶ ml. 8 (LA), 1×10 9 (MA) and 1×10 10(HA) CFU / mL. Body weight and food intake were measured every 3 days, and the health status of the mice was recorded. After 28 days, the mice were fasted for 12 hours and then anesthetized with 1% sodium pentobarbital (50 mg / kg). Blood was collected from the abdominal aorta, and serum was collected by centrifugation (4000 rpm, 4°C, 10 min) for further analysis. After euthanasia, the visceral toxicity of each group of mice was observed by dissection, and organs such as the liver, kidneys, spleen, and thymus were collected and weighed. The organ coefficient was calculated as organ weight / body weight × 100%.

[0079] 2.10 strain whole genome sequencing Co-sequencing was performed using the Illumina NovaSeq 6000 high-throughput sequencing platform, and genome assembly was performed using SOAPdenovo2 (http: / / soap.genomics.org.cn / ). The assembled genome was then used to predict coding genes using Prodigal-2.6.3 (https: / / github.com / hyattpd / Prodigal). MinCED (Version: 0.2.0) was used to predict clustered, regularly spaced short palindromic repeats (CRISPR) sequences. Gene islands were predicted using the IslandPath-DIMOB website (http: / / www.pathogenomics.sfu.ca / islandviewer / ), and prophages were predicted using the Phigario (https: / / github.com / bobeobibo / phigaro) website. The predicted gene sequences were analyzed using COG, KEGG, and GO, and compared with these functional databases to obtain gene function annotation results. Genome loops were constructed using R packages.

[0080] 3. Results and Analysis 3.1 Growth characteristics 3.1.1 Physiological and biochemical identification Table 2 shows some of the physiological and biochemical characteristics of *Lactobacillus fermentans* ZY001. *Lactobacillus fermentans* ZY001 can utilize glucose, arabinose, maltose, xylose, fructose, mannose, and raffinose, and is motile.

[0081] Table 2 Physiological and biochemical characteristics of Lactobacillus fermentans ZY001

[0082] +: Positive reaction; -: Negative reaction.

[0083] 3.1.2 Determination of growth performance and acid production performance like Figure 3 As shown, *Lactobacillus mucinus* ZY001 grows slowly from 0-3 h, indicating a slow growth phase, and its growth rate is 3-12 hOD. 600nm The absorbance value rose rapidly, indicating the logarithmic growth phase. After 12 hours, the absorbance of the bacterial solution stabilized, entering the stationary growth phase. The pH curve of the bacterial solution's acid production was consistent with the growth curve. The pH decreased slowly within 3 hours; the rate of pH decrease accelerated after 3 hours; and after 12 hours, the pH change of the bacterial solution leveled off and stabilized at around 4.4.

[0084] 3.1.3 Acid resistance of the strain Depend on Figure 4 It can be seen that the growth of strain ZY001 was completely inhibited at pH ≤ 3.0, and the OD decreased with prolonged culture time. 600nm No significant change was observed in absorbance values. Strain ZY001 grew normally at pH ≥ 4.0, but OD values ​​decreased upon reaching the stationary phase. 600nm There are certain differences in absorbance values, with OD values ​​showing the following order: pH=7.0 > pH=6.0 > pH=5.0 > pH=8.0 > pH=9.0 > pH=4.0. The viable cell count is related to the OD value. 600nm The absorbance values ​​show the same trend. 3.1.4 High-temperature tolerance of the strain As shown in Table 3, the survival rate of strain ZY001 exceeded 90% at 50℃, 50% at 60℃, 50% at 70℃, 40% at 80℃, and 10% at 90℃, indicating that fermenting Lactobacillus mucilaginosus ZY001 has good heat resistance.

[0085] Table 3. High-temperature tolerance of Lactobacillus fermentum ZY001

[0086] 3.2 Colonization ability of the strain in the gastrointestinal tract 3.2.1 Strain tolerance to acid and bile salts Table 4 shows that strain ZY001 exhibits strong tolerance to acid and bile salts. After 3 hours of inoculation in MRS liquid medium at pH 2.5, the survival rate of the strain was 80.96%, with a viable count of approximately 2.36 × 10⁻⁶. 7 CFU / mL. After 4 h of inoculation in MRS liquid medium with a bile salt concentration of 0.1%, the survival rate of the strain was 80.25%, with a viable count of approximately 1.29 × 10⁻⁶. 7 CFU / mL; after inoculation into MRS liquid medium with a bile salt concentration of 0.3% for 4 h, the survival rate of the strain was 69.08%, and the viable count was approximately 8 × 10⁻⁶. 4After inoculation with CFU / mL in MRS liquid medium containing 0.5% bile salts for 4 h, the survival rate of the strain was 61.06%, with a viable count of approximately 3.67 × 10⁻⁶. 3 CFU / mL.

[0087] Table 4. Survival rate of Lactobacillus fermentum ZY001 in acid and different bile salt concentrations.

[0088] 3.2.2 Tolerance of the strain to artificial gastrointestinal fluid Table 5 shows that *Lactobacillus fermentum* ZY001 has a strong survival ability in simulated gastric and intestinal fluid. After 1.5 h of inoculation into simulated gastric fluid, the survival rate of the strain was 78.39%, with a viable count of approximately 2.99 × 10⁻⁶. 6 CFU / mL; after 3 hours of inoculation into artificial intestinal fluid, the survival rate of the strain was 99.26%, with a viable count of approximately 1.14 × 10⁻⁶. 8 After co-digestion with artificial gastrointestinal fluid for 4.5 h at CFU / mL, the survival rate was 71.89%, and the viable bacterial count was approximately 6.7 × 10⁻⁶. 5 CFU / mL.

[0089] Table 5 Survival rate of Lactobacillus fermentum ZY001 in artificial gastrointestinal fluid

[0090] 3.3 Surface hydrophobicity and self-aggregation properties of the strain Based on the definition of surface hydrophobicity, bacteria are generally classified as highly hydrophobic (CSH% > 50%), moderately hydrophobic (CSH% between 20% and 50%), and non-hydrophobic (CSH% < 20%). Self-aggregation ability is generally categorized into three types: low (16%-35%), medium (35%-50%), and high (above 50%). Table 6 shows that *Lactobacillus fermentans* ZY001 exhibits moderate hydrophobicity and high self-aggregation ability.

[0091] Table 6. Surface hydrophobicity and self-aggregation rate of *Lactobacillus fermentatus* ZY001

[0092] 3.4 Antibacterial properties of the strain Depend on Figure 5It was found that the bacterial suspension and cell-free supernatant of *Lactobacillus myxitis* ZY001 could effectively inhibit the growth and proliferation of five pathogenic bacteria, while the bacterial pellet and MRS medium showed no antibacterial effect. Table 7 shows that the inhibition zone diameter of this strain was greater than 12 mm against *Escherichia coli*, greater than 13 mm against *Staphylococcus aureus*, greater than 11 mm against *Salmonella*, greater than 17 mm against *Pseudomonas aeruginosa*, and greater than 19 mm against *Listeria monocytogenes*.

[0093] Table 7 Results of antibacterial test of Lactobacillus fermentum ZY001

[0094] 3.5 Antibiotic susceptibility of strains The diameter of the susceptibility testing tablet is 6 mm. Therefore, a zone of inhibition greater than 7 mm is considered antibacterial, while a zone of inhibition less than or equal to 7 mm is considered non-antibacterial. Table 8 shows that strain ZY001 exhibits varying susceptibility to common antibiotics. It is highly sensitive to penicillin, ampicillin, piperacillin, vancomycin, chloramphenicol, minocycline, doxycycline, erythromycin, clindamycin, lincomycin, florfenicol, cefoperazone, cefuroxime sodium, and cefazolin, with a zone of inhibition greater than 23 mm. It is moderately sensitive to imipenem, gentamicin, tetracycline, azithromycin, ceftriaxone, cephalexin, and ceftazidime, with a zone of inhibition greater than 15 mm. It exhibits a zone of inhibition between 7-14 mm for oxacillin, streptomycin, amikacin, kanamycin, levofloxacin, and polymyxin B. It is not sensitive to trimethoprim-sulfamethoxazole, norfloxacin, and ciprofloxacin.

[0095] Table 8 Antibiotic sensitivity of Lactobacillus fermentum ZY001

[0096] Note: Diameter of inhibition zone (mm): +++: 23 30 mm; ++: 15 22 mm; +: 7 14 mm; No inhibition zone.

[0097] 3.6 Free radical scavenging ability of strains As shown in Table 9, the scavenging rates of fermenting Lactobacillus myxitis ZY001 against DPPH, ABTS and superoxide anion free radicals were 66.28%, 70.93% and 54.87%, respectively.

[0098] Table 9. Free radical scavenging rate of *Lactobacillus mucinus* ZY001 during fermentation.

[0099] 3.7 Safety Test Results 3.7.1 Results of the hemolysis test The results are as follows Figure 6 (A: Front view of hemolysis test, B: Back view of hemolysis test, a: Staphylococcus aureus, b: strain ZY001) As shown, with Staphylococcus aureus as a positive control, obvious β-hemolysis (clear area around the colony) is visible, but strain ZY001 shows γ-hemolysis (no area around the colony). It can be preliminarily judged that strain ZY001 is safe and non-pathogenic.

[0100] 3.7.2 Results of mouse experiments Clinical observations showed that all mice were in good spirits, able to eat and drink normally, and their feces showed no abnormalities. No mice died during the experiment. Post-experimental necropsy revealed no abnormal histopathological changes or bacterial translocation in their internal organs, indicating that the strain has no toxic side effects for clinical application in mice.

[0101] like Figure 7 As shown, body weight, food intake, and organ coefficient are commonly used indicators for evaluating animal health, and we used them to assess the safety of the isolated strain. First, we observed the effects of ZY001 supplementation on mouse body weight and food intake. There were no significant differences in body weight, average daily gain (ADG), and average daily food intake (ADFI) between mice supplemented with different doses of ZY001 and the control group. These results indicate that ZY001 supplementation has no adverse effects on mouse body weight and food intake.

[0102] We also investigated how ZY001 affects organ coefficients in mice. Table 10 shows the organ weight data of mice supplemented with ZY001. There were no significant differences in the organ weights of the heart, liver, spleen, kidney, and thymus between the CK and experimental groups. This indicates that strain ZY001 has no adverse effects on animal growth and development and is safe with no toxic side effects.

[0103] Table 10 Effects of feeding ZY001 mice on organ index

[0104] The results of biochemical analysis of mouse serum are shown in Table 11. The AST enzyme activity level in the CK group was significantly higher than that in the L, M, and H groups. P <0.05); In terms of antioxidant activity, the T-AOC enzyme activity levels in groups L, M, and H were significantly higher than those in the CK group ( P <0.05); the SOD activity level in the CK group mice was significantly lower than that in the L, M and H groups ( P <0.05); indicating that ZY001 supplementation may enhance the antioxidant capacity of the animal body.

[0105] Table 11 Effects of ZY001 feeding on serum biochemical parameters in mice

[0106] Note: Data in the same row with no letter or the same letter above the header indicates that the difference is not significant. P >0.05), different lowercase letters indicate significant differences ( P <0.05).

[0107] 3.8 Whole genome sequencing results of strain 3. To understand the characteristics of probiotics and explore the probiotic potential of ZY001, whole-genome sequencing was performed. Figure 8 The results show the complete circular genome map of ZY001. The complete genome of ZY001 consists of a 2.20M circular chromosome with a (G+C) content of 51.03% and a genome size of 2,205,605 bp, which is considered medium-sized. These bacteria typically exhibit strong metabolic activity, tolerance, and adaptability. A total of 5 prophages and 18 gene islands were predicted. The COG, GO, and KEGG results of the ZY001 genome are shown below. Figure 9-Figure 11 As shown. COG annotation results show that ZY001 is mainly enriched in ribosome structure and biosynthesis, carbohydrate transport and metabolism, and its main predicted functions are transcription and amino acid transport metabolism. GO annotation results show that the top three enriched biological processes are translation, DNA integration, and DNA translocation; the top three enriched cellular components are cytosol, cytoplasm, and cytoplasmic membrane; and the top three enriched molecular functions are ATP binding, DNA binding, and metal ion binding. KEGG annotation revealed the top five signaling pathways as: global and overview maps (490), carbohydrate metabolism (140), amino acid metabolism (139), metabolism of cofactors and vitamins (96), and translation (88).

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermenting Lactobacillus mucinus ZY001 ( Limosilactobacillus fermentum ZY001), characterized in that, The fermented Lactobacillus mucinus ZY001 ( Limosilactobacillus fermentum ZY001 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20251905.

2. The fermenting Lactobacillus mucinus ZY001 according to claim 1 ( Limosilactobacillus fermentum ZY001), characterized in that, The fermented Lactobacillus mucinus ZY001 ( Limosilactobacillus fermentum ZY001 is derived from cats.

3. A microbial agent, characterized in that, Including the fermenting Lactobacillus mucinus ZY001 as described in claim 1 ( Limosilactobacillus fermentum ZY001).

4. The fermenting Lactobacillus mucinus ZY001 as described in claim 1 ( Limosilactobacillus fermentum The application of ZY001 or the bacterial agent according to claim 3 in the preparation of antibacterial products.

5. The application according to claim 4, characterized in that, The antibacterial products include products that inhibit Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, or Listeria monocytogenes.

6. The fermenting Lactobacillus mucinus ZY001 as described in claim 1 ( Limosilactobacillus fermentum The use of ZY001 or the bacterial agent according to claim 3 in the preparation of products for the prevention or treatment of diarrhea.

7. The fermenting Lactobacillus mucinus ZY001 as described in claim 1 ( Limosilactobacillus fermentum The use of ZY001 or the microbial agent according to claim 3 in the preparation of products that alleviate oxidative stress in animals.

8. A feed, characterized in that, Including the fermenting Lactobacillus mucinus ZY001 as described in claim 1 ( Limosilactobacillus fermentum ZY001) or the bacterial agent according to claim 3.

9. A drug, characterized in that, Including the fermenting Lactobacillus mucinus ZY001 as described in claim 1 ( Limosilactobacillus fermentum ZY001) or the bacterial agent according to claim 3.

10. The medicament according to claim 9, characterized in that, The drug also includes excipients.

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