Aitzizmania condensata gh01 and applications, products, and methods thereof

Abstract

The application discloses a kind of condensation Weitzman bacteria GH01 and its application, product and method, belong to the field of microorganism.The preservation number of the condensation Weitzman bacteria GH01 described is CGMCC No. 36505.The application also provides the application of the condensation Weitzman bacteria GH01 with preservation number CGMCC No. 36505 in preparation feed additive, and / or, bacteriostatic agent, and / or, diarrhea treatment drug, and / or, intestinal flora function food, and / or, non-therapeutic purpose in vitro bacteriostasis aspect.The application also provides microbial inoculant, bacteriostatic agent, feed additive and in vitro bacteriostasis method based on condensation Weitzman bacteria GH01.Through in vitro bacteriostasis experiment, and animal experiment confirm that the condensation Weitzman bacteria GH01 can significantly reduce salmonella adhesion invasion, and it is verified by bacteriostatic circle experiment that it has broad-spectrum antibacterial performance, can effectively relieve diarrhea, and can significantly improve the meat quality of broiler.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Weizmann's coagulans GH01 and its applications, products, and methods. Background Technology

[0002] salmonella( Salmonella *Ischemic mycotoxin* is a significant foodborne pathogen globally, infecting approximately 150 million people annually, with infants and young children, as well as those with weakened immune systems, being particularly vulnerable to severe illness. Traditional antibiotic treatments face challenges such as increasing drug resistance and disruption of the gut microbiota, necessitating the development of safe and sustainable alternatives.

[0003] In recent years, research has made progress in understanding how probiotics (such as lactic acid bacteria and bifidobacteria) antagonize pathogens through competitive inhibition and immune regulation. However, specific probiotic strains targeting Salmonella remain scarce. *Weizmannii coagulans* (…) Weizmannia coagulans Most studies have focused on its digestive function, while a systematic analysis of its anti-infection mechanism remains a gap.

[0004] Weizmann's coagulation bacteria ( Weizmannia coagulans *Weizmannii coagulans* is a Gram-positive, lactic acid-producing spore-forming bacterium with significant value in the field of industrial biotechnology. Currently, there are few reports of *Weizmannii coagulans* used as an agricultural feed additive, which offers broad-spectrum inhibition against common feed pathogens, alleviates diarrhea, and improves meat quality. Summary of the Invention

[0005] To address the technical problem that there is still a gap in the existing technology for *Weizmann's coagulans*, which has broad-spectrum antibacterial activity against common pathogens in feed, can alleviate diarrhea, and improve meat quality, this invention provides *Weizmann's coagulans* GH01 and its applications, products, and methods.

[0006] The technical solution of the present invention is as follows: A strain of Weizmannii coagulans GH01, with accession number CGMCC No. 36505.

[0007] The application of Weizmannii coagulans GH01 with accession number CGMCC No. 36505 in the preparation of feed additives, and / or, antibacterial agents, and / or, antidiarrheal drugs, and / or, functional foods for regulating intestinal flora, and / or, in vitro antibacterial activity for non-therapeutic purposes.

[0008] The antibacterial spectrum includes: Salmonella, Staphylococcus aureus, Listeria monocytogenes, Clostridium difficile, Aeromonas hydrophila, and Edwardsiella tarda. Preferably, the diarrhea refers to diarrhea caused by Salmonella; Preferably, the regulation of the intestinal flora includes: restoring the imbalance between Firmicutes and Bacteroidetes and increasing the abundance of Proteobacteria, decreasing the relative abundance of Helicobacter and Biliophilia, and increasing the abundance of Alternaria, Helicobacter tumefaciens NK4A136, Rumenococcus, Prevotellaceae UGG001, and Osmophilus. Muribaculum It belongs to the relative abundance.

[0009] A feed additive comprising: an active ingredient, said active ingredient comprising: Weizmannii coagulans GH01 with preservation number CGMCC No.36505.

[0010] The feed additive also includes: auxiliary materials.

[0011] An antibacterial agent comprising: an active ingredient, said active ingredient comprising: Weizmannii coagulans GH01 with accession number CGMCC No. 36505.

[0012] A drug for treating diarrhea includes: a pharmacodynamic active ingredient, said pharmacodynamic active ingredient including: Weizmannii coagulans GH01 with accession number CGMCC No. 36505.

[0013] A microbial inoculant, comprising: an active ingredient, said active ingredient comprising: Weizmannii coagulans GH01 with accession number CGMCC No.36505.

[0014] The aforementioned microbial inoculant also includes: excipients.

[0015] A non-therapeutic in vitro antibacterial method, which uses Weizmannella coagulans GH01 with accession number CGMCC No. 36505 for antibacterial activity.

[0016] The beneficial effects of this invention are as follows: This invention isolates a strain of *Weizmannii coagulans* from nature and demonstrates through in vitro antibacterial experiments and animal experiments that it can significantly reduce the adhesion and invasion of *Salmonella*. Inhibition zone experiments verify that it possesses broad-spectrum antibacterial properties, inhibiting common feed pathogens including *Staphylococcus aureus*, *Salmonella*, *Listeria monocytogenes*, *Clostridium difficile*, *Aeromonas hydrophila*, and *Edwards tarda*. Mouse experiments have confirmed that *Weizmannii coagulans* can effectively alleviate diarrhea. Specifically, it can alleviate Salmonella-induced diarrhea in mice, reduce colonic inflammation, restore colon length, significantly reduce serum levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α, restoring IL-6 and IL-1β to normal levels, while significantly increasing the level of anti-inflammatory factor IL-10. It also reduces inflammatory cell infiltration in the intestinal mucosa and submucosa, decreases mucosal epithelial shedding, and restores goblet cell numbers, thus enhancing the intestinal mucosal barrier and reducing ileal tissue damage. Furthermore, *Weizmannii coagulans* can increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria in the host gut, promoting gut microbiota homeostasis, improving the intestinal barrier, and playing an immunomodulatory role. Meanwhile, this coagulating Weizmannii can also significantly improve the meat quality of broilers, specifically by increasing the pH 45 min and pH 24 h of the breast and leg muscles, reducing the shear force of the breast and leg muscles, improving the post-slaughter muscle quality and increasing muscle tenderness; it also reduces the MDA content in the breast muscle of broilers and increases the levels of T-AOC, SOD and GSH-Px, thereby reducing the level of lipid oxidation in broiler muscle and improving the antioxidant capacity of the muscle.

[0017] The *Weizmannii* strain was named GH01 and deposited for preservation. Its preservation information is as follows: Accession number: CGMCC NO. 36505 Classification and nomenclature: Weizmann's coagulation bacteria Weizmannia coagulans Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Preservation date: November 5, 2025. Attached Figure Description

[0018] Figure 1 This is a morphological photograph of *Weizmannii coagulans* GH01, which is the subject of Experimental Example 1 of this invention.

[0019] Figure 2 Line graphs showing the body weight of mice in the NC, MC, and GH01 groups provided in Experimental Example 5 of this invention.

[0020] Figure 3The figures are bar charts showing the loose stool rate and diarrhea index of mice in the NC, MC and GH01 groups provided in Experimental Example 5 of this invention. Figure A is the loose stool rate bar chart and Figure B is the diarrhea index bar chart.

[0021] Figure 4 The graphs show the spleen test results of mice in the NC, MC and GH01 groups provided in Experiment Example 6 of this invention. A is a bar chart of spleen weight, B is a bar chart of spleen index, C is a bar chart of mouse colon length, and D is the actual colon condition of mice in each group.

[0022] Figure 5 The bar charts shown in Experiment Example 7 of this invention depict the effects of various immune factors on the serum of mice in the NC, MC, and GH01 groups. Figure A is a bar chart of IL-6, Figure B is a bar chart of IL-1β, Figure C is a bar chart of TNF-α, and Figure D is a bar chart of IL-10.

[0023] Figure 6 The images show pathological sections of the jejunum, ileum, and colon tissues of mice in the NC, MC, and GH01 groups provided in Experimental Example 8 of this invention.

[0024] Figure 7 The bar chart shows the results of the α-diversity analysis of the gut microbiota of mice in the NC, MC and GH01 groups provided in Experimental Example 9 of this invention; Figure A is the ACE index, Figure B is the Chao1 index, Figure C is the Simpson index, and Figure D is the Shannon index.

[0025] Figure 8 The figure shows the results of the β-diversity analysis of the intestinal flora of mice in the NC group, MC group and GH01 group provided in Experimental Example 9 of the present invention.

[0026] Figure 9 The bar charts showing the distribution of species diversity at the phylum level and the ratio of Firmicutes to Bacteroidetes in the gut microbiota of mice in the NC, MC and GH01 groups provided in Experiment Example 9 of this invention are shown. Figure A shows the distribution of species diversity at the phylum level, and Figure B shows the ratio of Firmicutes to Bacteroidetes.

[0027] Figure 10 The bar chart shows the genus-level species diversity analysis of mice in the NC, MC, and GH01 groups provided in Experimental Example 9 of this invention; the figures show the relative abundance of *Alternaria*, *Trichophyton* NK4A136 group, *Ruminococcus*, *Prevotella* UCG-001, *Ostomyces*, *Rhizobium*, *Helicobacter*, and *Bacillus*.

[0028] Figures 2-10 The labels are listed below: NC represents the normal control group, MC represents the diarrhea model group, and GH01 represents the Weizmann's coagulans GH01 treatment group. Detailed Implementation

[0029] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the experimental examples described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0030] Sources of biomaterials I. The Salmonella ATCC14028, Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923, Listeria monocytogenes ATCC19115, Clostridium difficile ATCC43593, Aeromonas hydrophila ATCC35654, and Edwardsiella tarda ATCC15947 used in Experiments 2-3 were all purchased from the Guangdong Microbial Culture Collection Center.

[0031] II. The BALB / c male mice used in Experiments 4-9 are commercially available.

[0032] 3. The AA broiler chickens used in Experiment Example 10 are commercially available.

[0033] The following experimental example involves the preparation method of *Weizmannii coagulans* GH01 bacterial suspension: The desired strain is inoculated into MRS liquid medium and activated by culturing at 37°C for 16-18 hours, and activated twice to obtain an activated solution; the activated solution is inoculated into MRS liquid medium at an inoculation rate of 3% (v / v) and cultured at 37°C for 20-24 hours to obtain a bacterial suspension; the bacterial suspension is centrifuged at 4000 rpm for 5 minutes at 4°C, filtered, and the bacterial cells are obtained. The bacterial cells are resuspended in physiological saline to obtain the final product.

[0034] MRS medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L.

[0035] Group 1 Examples, the present invention's *Wietzmannii* GH01 This set of embodiments provides a *Weizmannii coagulans* GH01. All embodiments in this set share the following common feature: the *Weizmannii coagulans* GH01 has the accession number CGMCC No. 36505.

[0036] Any act of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, altering, selling, or offering for sale a strain of *Weizmannii coagulans* GH01 with accession number CGMCC No. 36505, and / or the act of using a strain of *Weizmannii coagulans* GH01 with accession number CGMCC No. 36505 in combination with other probiotics, and / or the use of *Weizmannii coagulans* GH01 with accession number CGMCC No. 36505 to antagonize various pathogens, and / or the preparation of antibacterial products, and / or the preparation of drugs to improve constipation or regulate intestinal flora, and / or the preparation of health foods with laxative or intestinal flora regulating functions, falls within the scope of protection of this invention.

[0037] Other probiotics include, but are not limited to: *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*, *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus delbrueckii* subsp. lactis, *Lactobacillus helveticus*, *Lactobacillus casei*, *Lactobacillus brevis*, *Lactobacillus johnsonii*, *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Lactobacillus salivarius*, *Saccharomyces cerevisiae*, *C. delbrueckii*, *C. candida*, *Wickhamia sacchari*, *Pichia pastoris*, *Blanctomyces boulardii*, *Pseudomonas leucosus*, *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, and *Schizosaccharomyces cerevisiae*. Genera including *Bacillus baumannii*, *Bacillus thuringiensis*, *Bacillus brevis*, *Bacillus megaterium*, *Bacillus mucilaginosus*, *Bacillus azoosus*, *Bacillus spheroides*, *Clostridium butyricum*, *Bifidobacterium adolescentis*, *Bifidobacterium angularis*, *Bifidobacterium animalis*, *Bifidobacterium astrum*, *Bifidobacterium bifidum*, *Bifidobacterium bovis*, *Bifidobacterium brevis*, *Bifidobacterium densiflorum*, *Bifidobacterium infantis* (i.e., *Bifidobacterium longum* subsp. *infantii*), *Bifidobacterium lactis* (i.e., *Bifidobacterium animalis* subsp. *milk fat*), *Bifidobacterium longum*, *Bifidobacterium pseudosporidis*, as well as *Bifidobacterium thermophilum* and *Bifidobacterium acidophilus*.

[0038] Those skilled in the art can, based on actual production needs and in conjunction with conventional technical means or basic common sense in pharmaceutical production processes (e.g., the Encyclopedia of Pharmaceutical Technology, Pharmaceutical Preparation Technology, etc.), routinely select or adjust pharmaceutical excipients, thereby producing products with different dosage forms, storage conditions, and shelf lives from a strain of Weizmannii coagulans GH01 with accession number CGMCC No. 36505. This is not a technical obstacle for those skilled in the art and is feasible and easy to do.

[0039] Group 2 Examples: Application of the present invention of *Wietzmannii coagulating* GH01 This set of embodiments provides the application of *Weizmannii coagulans* GH01 with accession number CGMCC No. 36505 in the preparation of feed additives, and / or, antibacterial agents, and / or, antidiarrheal drugs, and / or, functional foods for regulating intestinal flora, and / or, in vitro antibacterial activity for non-therapeutic purposes.

[0040] In a specific embodiment, the antibacterial spectrum includes: Salmonella, Staphylococcus aureus, Listeria monocytogenes, Clostridium difficile, Aeromonas hydrophila, and Edwardsiella tarda. Preferably, the diarrhea refers to diarrhea caused by Salmonella; Preferably, the regulation of the intestinal flora includes: restoring the imbalance between Firmicutes and Bacteroidetes and increasing the abundance of Proteobacteria, decreasing the relative abundance of Helicobacter and Biliophilia, and increasing the abundance of Alternaria, Helicobacter tumefaciens NK4A136, Rumenococcus, Prevotellaceae UGG001, and Osmophilus. Muribaculum It belongs to the relative abundance.

[0041] Group 3 Examples, Feed Additives of the Present Invention This set of embodiments provides a feed additive. All embodiments in this set share the following common feature: the feed additive comprises an active ingredient, which includes Weizmannii coagulans GH01 with preservation number CGMCC No. 36505.

[0042] In a further embodiment, the feed additive further includes: auxiliary materials.

[0043] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0044] According to the present invention, and in accordance with the different needs of actual production applications, and combined with conventional technical means in the field of drug preparation (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare an antibacterial agent containing Weizmannii coagulans GH01 with preservation number CGMCC No.36505 into different dosage forms, such as powder, tablets, gels, sprays, granules, capsules, liquids, etc.

[0045] In a specific embodiment, the dosage form of the feed additive is selected from one or more of the following: powder, tablet, gel, spray, granule, capsule, and liquid.

[0046] Group 4 Examples, the antibacterial agent of the present invention This set of embodiments provides an antibacterial agent. All embodiments in this set share the following common feature: the antibacterial agent comprises an active ingredient, which includes Weizmannii coagulans GH01 with accession number CGMCC No. 36505.

[0047] In a further embodiment, the antibacterial agent further includes: excipients.

[0048] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0049] According to the present invention, and in accordance with the different needs of actual production applications, and combined with conventional technical means in the field of drug preparation (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare an antibacterial agent containing Weizmannii coagulans GH01 with preservation number CGMCC No.36505 into different dosage forms, such as powder, tablets, gels, sprays, granules, capsules, liquids, etc.

[0050] In a specific embodiment, the dosage form of the antibacterial agent is selected from one or more of the following: powder, tablet, gel, spray, granule, capsule, and liquid.

[0051] Group 5 Examples, the Drug of the Present Invention This set of embodiments provides a drug for treating diarrhea. All embodiments in this set share the following common feature: the drug for treating diarrhea comprises a pharmacodynamic ingredient, which includes: *Weizmannii coagulans* GH01 with accession number CGMCC No. 36505.

[0052] In some embodiments, the drug is a veterinary drug.

[0053] In a further embodiment, the drug further includes excipients.

[0054] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0055] According to the present invention, and in accordance with the different needs of actual production applications, and combined with conventional technical means in the field of drug preparation (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare an antibacterial agent containing Weizmannii coagulans GH01 with preservation number CGMCC No.36505 into different dosage forms, such as powder, tablets, gels, sprays, granules, capsules, liquids, etc.

[0056] In a specific embodiment, the dosage form of the drug is selected from one or more of the following: powder, tablet, gel, spray, granule, capsule, and liquid.

[0057] Group 6 Examples, Microbial Agents of the Present Invention This set of embodiments provides a microbial inoculant. All embodiments in this set share the following common feature: the microbial inoculant comprises an active ingredient, which includes Weizmannii coagulans GH01 with accession number CGMCC No. 36505.

[0058] In a further embodiment, the microbial agent further includes: excipients.

[0059] In a more specific embodiment, the excipients are selected from: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants, anti-flocculation agents, filter aids, release inhibitors, etc.

[0060] According to the present invention, and in accordance with the different needs of actual production applications, and combined with conventional technical means in the field of drug preparation (e.g., "Encyclopedia of Pharmaceutical Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare an antibacterial agent containing Weizmannii coagulans GH01 with preservation number CGMCC No.36505 into different dosage forms, such as powder, tablets, gels, sprays, granules, capsules, liquids, etc.

[0061] In a specific embodiment, the dosage form of the microbial agent is selected from one or more of the following: powder, tablet, gel, spray, granule, capsule, and liquid.

[0062] Group 7 Examples, In Vitro Antibacterial Method of the Present Invention This set of embodiments provides a non-therapeutic in vitro antibacterial method. All embodiments in this set share the following common feature: the antibacterial activity is achieved using *Weizmannii coagulans* GH01 with accession number CGMCC No. 36505.

[0063] In a specific embodiment, the antibacterial spectrum includes: Staphylococcus aureus, Salmonella, Listeria monocytogenes, Clostridium difficile, Aeromonas hydrophila, and Edwardsiella tarda.

[0064] Experimental Example 1: Isolation and Identification of Weizmannii GH01 1) Strain strain isolation Take the sample and place it in a centrifuge tube containing sterile physiological saline. Shake to mix thoroughly. After standing, take 1 ml of the sample suspension and place it in an 80°C water bath for 10 min. Perform a 10-fold serial dilution. Take the appropriate dilution gradient and spread it on MRS agar medium containing 1.5% calcium carbonate. Incubate at 42°C for 72 h. After the colonies grow, select single colonies that are white, shiny, round, with neat edges, slightly raised, and with a large clear zone. Perform Gram staining and cell morphology observation. Then, select single colonies and repeatedly streak them on Weizmann's coagulation solid plates for purification to obtain pure culture strains.

[0065] 2) Strain identification Morphological characteristics: Purified *Weizmannii coagulans* appears as round colonies on PCA agar plates, with a diameter of 2-5 mm, off-white color, neat edges, and a moist surface; Gram staining reveals single or paired rods; when forming spores, one end swells, resembling a drumstick shape. Figure 1 As shown.

[0066] Molecular identification: Genomic DNA was extracted from the strain using a genomic DNA extraction kit. The 16S rDNA sequence of the strain was amplified using universal bacterial 16S primers to obtain PCR products. The PCR products were then sequenced. The PCR reaction mixture consisted of: 20 μL 10×Buffer, 4 μL primer dNTPs, 1 μL each of forward and reverse primers, 2 μL DNA template, 0.5 μL Taq enzyme, and 34.5 μL ddH2O. PCR conditions were: pre-deformation at 95℃ for 10 min, deformation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 2 min, for 35 cycles, and a final extension at 72℃ for 10 min.

[0067] The upstream and downstream primers for PCR amplification were 27F and 1492R, and their sequences were described in Chinese patent ZL202210478937.4.

[0068] The PCR products were detected by gel electrophoresis and then sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The identified gene sequences were submitted to the NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis and comparison. Based on the molecular biological identification results, the strain was identified as *Weizmannii coagulans* and named *Weizmannii coagulans* GH01. The strain was deposited for preservation, and its preservation information is as follows: Accession number: CGMCC NO. 36505 Classification and nomenclature: Weizmann's coagulation bacteria Weizmannia coagulans Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Preservation date: November 5, 2025.

[0069] Experimental Example 2: Evaluation of the co-aggregation ability of *Weizmannii* GH01 and *Salmonella*. Co-aggregation ability refers to the ability to prevent pathogens from attaching to host tissues, thereby eliminating the colonization of gastrointestinal pathogens. Salmonella culture was inoculated into LB medium and incubated in shake flasks at 30°C for 16-18 hours for later use.

[0070] The OD600 value of the *Weizmannii* GH01 bacterial suspension was adjusted to 0.6, and the OD600 value of the *Salmonella* bacterial suspension was adjusted to 0.4. The suspensions were thoroughly mixed by shaking, and the initial OD600 values ​​(Ax and Ay) were measured. 20 mL of each of the *Weizmannii* and *Salmonella* bacterial suspensions were transferred to equal volumes in 50 mL centrifuge tubes, mixed, vortexed for 20 s, and then incubated at 37°C. At 2 h, 5 h, 21 h, and 24 h of incubation, 2 mL of the supernatant was carefully aspirated, and the absorbance (A) at 600 nm was measured. The cohesive strength between strain GH01 and *Salmonella* was calculated. Cohesive strength % = [(Ax + Ay) / 2 - A(x + y)] / [(Ax + Ay) / 2] × 100, where x and y represent the two bacterial strains, and (x + y) represents the mixture. The results are shown in Table 1.

[0071] Table 1. Co-aggregation ability of *Weizmannii* GH01 and *Salmonella*

[0072] As shown in Table 1, the strain GH01 involved in this invention exhibits a co-aggregation capacity of 67.55% with Salmonella within 24 hours, demonstrating excellent co-aggregation ability with Salmonella.

[0073] Example 3: Evaluation of the broad-spectrum antibacterial activity of *Weizmannii coagulans* GH01 MRS medium containing 1.5% agar was cooled to 55°C and then mixed with suspensions of Salmonella, Staphylococcus aureus, Listeria monocytogenes, Clostridium difficile, Aeromonas hydrophila, and Edwardsiella tarda, respectively, to ensure that the viable count of each of the seven indicator bacteria was 10-1. 6 The concentration was on the order of CFU / mL. The culture was then rapidly poured into plates pre-placed with Oxford cups. After the culture medium cooled and solidified, the Oxford cups were removed, and 200 μL of *Weizmannii coagulates* was injected into each well (the total viable count of the bacterial suspension in each group was 10⁻⁶). 8 After incubation at 37℃ for 24-48 hours (on the order of CFU / mL), the diameter of the inhibition zone (mm) was measured. Results are as follows: Figure 1 As shown in Table 2.

[0074] Table 2. Evaluation of the broad-spectrum antibacterial activity of *Weizmannii coagulans* GH01

[0075] As shown in Table 2, the GH01 strain involved in this invention has an inhibition zone of 25 mm against Salmonella and exhibits antibacterial activity against six pathogens, including Salmonella, indicating that GH01 of Weizmannii coagulans has good broad-spectrum antibacterial activity.

[0076] Experiment Example 4: Mouse Diarrhea Model Experiment and Grouping Male BALB / c mice (20-22g) without specific pathogens were housed in the animal room of the Hubei Provincial Center for Food and Drug Safety Evaluation (No.: Anping Center Animal (Fujian) No. 202510109). The temperature was maintained at 22±2℃, humidity at 50±5%, with a 12-hour light-dark cycle, and free access to water and food was provided. Mice were acclimatized for one week before the experiment, and then randomly divided into three groups: normal control group (NC), diarrhea model group (MC), and *Weizmannii coagulans* GH01 treatment group (GH01). Throughout the experiment, mice in the NC and MC groups were administered 0.2mL of sterile saline by gavage daily, while mice in the *Weizmannii coagulans* group were administered an equal volume of *Weizmannii coagulans* GH01 bacterial suspension (live count 10-1). 9 CFU / mL). From day 8 to 11 of the experiment, mice in the MC group and the *Weizmannii coagulans* GH01 group were continuously administered Salmonella (live count 10⁻⁶ CFU / mL) via gavage. 9 A mouse model of diarrhea was established on day 4 (CFU / mL). The NC group was administered an equal volume of sterile saline by gavage. Mice were sacrificed on day 15, and samples were collected for subsequent analysis. Detailed animal experiments are shown in Table 3.

[0077] Table 3. Animal Experiment Design Grouping

[0078] After one week of acclimatization, the mice were randomly divided into three groups. Following oral administration of Salmonella, the mice's weight was measured daily. All feces were collected within one hour the day before dissection, and the rate of loose stools within that hour was recorded, along with the diarrhea index. At dissection, the spleen and colon were collected, the colon length was measured, and the spleen index was calculated.

[0079] Spleen index = Spleen mass (mg) / Body weight (g) Loose stool rate = Number of loose stools in mice / Total number of defecations in mice Stool grade = Sum of stool grades / Number of loose stools Diarrhea Index = Stool Rate × Stool Grade Grade 0 / Normal stool (stool shape is normal on tissue paper, no watermarks) Grade 1 / Wet (stool shape is normal, watermarks on toilet paper do not extend beyond the edge of the stool) Grade 2 / Watery stool (the stool is irregular in shape, the watermark on the paper towel extends beyond the edge of the stool or watery diarrhea with few formed components).

[0080] Experiment Example 5: Effects of Weizmannii coagulans GH01 on body weight and diarrhea in mice Experimental results are as follows Figure 2As shown, compared with the NC group, the body weight of mice in the model group decreased significantly after Salmonella gavage (P<0.05). Compared with the model group, the body weight loss trend of mice in the GH01 group was slower after Salmonella gavage. On day 14, the body weight of mice in the GH01 group was significantly higher than that in the MC group (P<0.05), indicating that GH01 significantly inhibited Salmonella-induced diarrhea in mice and alleviated the body weight loss. Figure 3 As shown, compared with the NC group, the rate of loose stools and the diarrhea index of mice in the model group significantly increased after gavage administration of Salmonella (P<0.05). Compared with the model group, the rate of loose stools and the diarrhea index of mice in the GH01 group significantly decreased after gavage administration of Salmonella (P<0.05). This indicates that gavage administration of GH01 can alleviate Salmonella-induced diarrhea in mice.

[0081] Experimental Example 6: Effects of Weizmannii coagulans GH01 on the spleen and colon of mice. Experimental results are as follows Figure 4 As shown, compared with the NC group, after gavage with Salmonella, the spleen weight and spleen index of mice in the MC group were significantly increased (P<0.05), indicating that the spleen of the MC group mice underwent an inflammatory response, leading to spleen swelling. Compared with the MC group, the spleen weight and spleen index of mice in the GH01 group were significantly decreased (P<0.05), indicating that gavage with GH01 can alleviate Salmonella-induced spleen inflammation and reduce spleen swelling in mice. Figure 4 As shown in Figure C, compared with the NC group, the colon of mice in the MC group was significantly shortened due to the infiltration of inflammatory cells caused by Salmonella infection, which damaged the normal intestinal structure (P<0.05). After GH01 was administered to the mice, the inflammatory response in the colon was reduced, and the length of the mice's colon was restored to a level close to that of the NC group.

[0082] Experimental Example 7: Effects of Weizmannii coagulans GH01 on mouse serum cytokines Mouse blood was collected into 1.5 mL centrifuge tubes and centrifuged at 4000 rpm for 10 minutes. The supernatant was stored at -20°C for subsequent experiments. The levels of IL-6, IL-10, IL-1β, and TNF-α in mouse serum were measured according to the ELISA kit. The results are as follows. Figure 5As shown, Salmonella can induce an inflammatory response in mice, significantly increasing the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in mouse serum (P<0.05), while significantly decreasing the level of anti-inflammatory cytokines IL-10. Compared with the model group, gavage administration of GH01 significantly reduced the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in serum (P<0.05), restoring IL-6 and IL-1β to normal levels, while significantly increasing the level of anti-inflammatory cytokines IL-10 (P<0.05), bringing IL-10 levels close to those of the normal group.

[0083] Experiment Example 8: Effects of Weizmannii coagulans GH01 on mouse jejunum, ileum, and colon tissues After euthanizing the mice, 0.5 cm sections of jejunum, ileum, and colon were collected, fixed in 10% formaldehyde solution, stained with hematoxylin and eosin (HE), and finally observed under an optical microscope to examine the structure of the jejunum, ileum, and colon. Figure 6 As shown, the MC group had a large number of inflammatory cells infiltrating the jejunal / ileal / colonic mucosa, with mucosal epithelial cell shedding and a significant reduction in the number of goblet cells. The GH01 group showed reduced inflammatory cell infiltration in the intestinal mucosa and submucosa, decreased mucosal epithelial shedding, and a recovery in the number of goblet cells, which could enhance the intestinal mucosal barrier and reduce ileal tissue damage.

[0084] Experiment Example 9: Effects of Weizmannii coagulans GH01 on the intestinal flora of mice Gut microbiota diversity analysis was conducted using the Illumina Novaseq sequencing platform, employing paired-end sequencing to construct small fragment libraries for sequencing. Read splicing and filtering, clustering or denoising, and species annotation and abundance analysis revealed the species composition of the samples. Further alpha diversity analysis, beta diversity analysis, and species diversity analysis were performed to uncover differences between samples. (1) α-diversity analysis, β-diversity analysis α-diversity analysis of mouse gut microbiota showed Figure 7 Compared with the control group, the ACE, Chao1, Shannon, and Simpson indices of the gut microbiota in the model group mice were significantly decreased (P<0.05), indicating that Salmonella infection in mice led to gut microbiota dysbiosis and a decrease in gut microbiota diversity. After feeding GH01, the ACE, Chao1, Shannon, and Simpson indices of the gut microbiota in mice were significantly higher than those in the model group (P<0.05), and there was no significant difference compared with the NC group, indicating that GH01 can regulate gut microbiota and increase the richness of gut microbiota in diarrheal mice.

[0085] β-diversity measures the similarity of microbial community composition among different samples, focusing on the differences in microbial community composition between samples. Experimental results are as follows: Figure 8 As shown, the MC group and the NC group were clearly separated, indicating that the microbial composition of the MC group was significantly different from that of the NC group. The GH01 group was adjacent to the NC group, indicating that gavage administration of GH01 brought the composition of the intestinal microbiota in the diarrheal mice closer to that of the normal group.

[0086] (2) Phylum-level species diversity analysis Experimental results are as follows Figure 9 As shown, compared with the NC group, the relative abundance of Firmicutes and the Firmicutes / Bacteroidetes ratio were significantly decreased in the MC group (P<0.05). Firmicutes and Bacteroidetes are the main microbiota in the gut, and the Firmicutes / Bacteroidetes ratio is considered an important indicator of gut microbiota balance. Imbalance in this ratio is often associated with disease. Compared with the MC group, the relative abundance of Firmicutes and the Firmicutes / Bacteroidetes ratio were significantly increased in the GH01 group, but there was no significant difference compared with the NC group. Since Salmonella belongs to the Proteobacteria phylum, the relative abundance of Proteobacteria in the MC group increased significantly after mouse infection (P<0.05), but the relative abundance of Proteobacteria in the GH01 group was not significantly different from that in the NC group. This indicates that GH01 supplementation can restore the imbalance between Firmicutes and Bacteroidetes caused by Salmonella and the increase in Proteobacteria abundance, thereby maintaining gut microecological balance.

[0087] (3) Genus-level species diversity analysis Figure 10 This shows the relative abundance of *Alternaria* genus in the cecal contents of mice in each group. Compared with the NC group, the MC group had a higher abundance of *Alternaria* genus. Alistipes ), Helicobacter pylori_NK4A136_ group ( Lachnospiraceae _NK4A136_group), Rumenococcus spp. ( Ruminococcus Prevotellaceae_UCG_001, Odorbacterium genus ( Odoribacter )and Muribaculum The relative abundance of the genus *Helicobacter* was significantly reduced (P<0.05), while the abundance of *Helicobacter* genus was significantly reduced. Helicobacter ), bilirubin Bilophila The relative abundance of *Otherbacteria* spp. was significantly increased (P<0.05). Compared with the MC group, the abundance of *Otherbacteria* spp. in the *Weizmannii* GH01 group was significantly higher (P<0.05). Alistipes ), Helicobacter pylori_NK4A136_ group ( Lachnospiraceae _NK4A136_group), Rumenococcus spp. ( Ruminococcus Prevotellaceae_UCG_001, Odorbacterium genus ( Odoribacter )and MuribaculumThe relative abundance of *Helicobacter* spp. increased. Furthermore, compared to the MC group, the abundance of *Weizmannii* GH01 group was significantly higher. Helicobacter ), bilirubin Bilophila The relative abundance of *Ostomyces* was significantly reduced. Odoribacter )and Muribaculum This genus can produce short-chain fatty acids (SCFAs), mainly succinic acid and acetic acid, which can improve the intestinal epithelial barrier and prevent inflammation. *Helicobacter pylori* NK4A136 group ( Lachnospiraceae _NK4A136_group), Rumenococcus spp. ( Ruminococcus Prevotellaceae GH01 can inhibit the proliferation of pathogenic bacteria, and its metabolites can interact with immune receptors, leading to the production of immunomodulatory cytokines to resist the invasion of foreign pathogens. The results showed that *Weizmannii coagulans* GH01 can increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria in the host gut, promote the restoration of gut microbiota homeostasis, improve the intestinal barrier, and exert an immunomodulatory effect.

[0088] Experimental Example 10: The effect of *Weizmannii coagulans* GH01 on improving the meat quality of broilers. Three hundred and twenty one-day-old healthy AA broilers were randomly divided into two groups: a control group and a *Weizmannii coagulans* GH01 group, with eight replicates per group and twenty chickens per replicate. The control group was fed a basal diet, while the *Weizmannii coagulans* GH01 group received *Weizmannii coagulans* GH01 added to their basal diet at a concentration of 1 × 10^8 CFU / kg of diet. The experiment lasted for 42 days. During the experiment, the broilers in both groups had free access to feed and water and were fed according to standard broiler feeding and management procedures.

[0089] At 42 days of age, one broiler chicken close to its average weight was randomly selected from each replicate for slaughter and sampling, with a total of 8 chickens per group. After slaughter, samples of pectoral and leg muscles were collected. The pH at 45 min and 24 h, shear force, muscle antioxidant indices, and routine muscle nutrients were measured in the pectoral muscle. The muscle antioxidant indices included MDA, T-AOC, SOD, and GSH-Px; the routine muscle nutrients included moisture, crude protein, crude fat, and ash. The results are shown in the table.

[0090] Table 4. Effects of *Weizmannii coagulans* GH01 on pH and shear force in broiler breast muscle

[0091] As shown in Table 4, compared with the control group, the pH 45 min and pH 24 h of the breast and leg muscles of broilers in the GH01 group were increased, and the shear force of the breast and leg muscles was decreased. This indicates that the coagulating Weizmannii GH01 can improve the quality of broiler muscle after slaughter and increase muscle tenderness.

[0092] Table 5. Effects of *Weizmannii coagulans* GH01 on antioxidant indices of broiler breast muscle

[0093] As shown in Table 5, compared with the control group, the MDA content in the breast muscle of broilers in the GH01 group was reduced, while the levels of T-AOC, SOD and GSH-Px were increased, indicating that *Weizmannii coagulans* GH01 can reduce the lipid oxidation level in broiler muscle and improve the antioxidant capacity of muscle.

[0094] Table 6. Effects of *Weizmannii coagulans* GH01 on routine nutrient composition of broiler breast muscle

[0095] As shown in Table 6, compared with the control group, the crude protein content in the breast and leg muscles of broilers in the GH01 group was increased, the crude fat content was moderately increased, the moisture content was slightly decreased, and the ash content was slightly increased, indicating that *Weizmannii coagulans* GH01 can improve the nutritional quality of broiler muscle.

[0096] In summary, the addition of Weizmannii coagulans GH01 to the diet can improve the quality of broiler breast and leg muscles, specifically by stabilizing muscle pH, reducing muscle shear stress, increasing muscle antioxidant capacity, and improving the routine nutritional components of the muscle, indicating that Weizmannii coagulans GH01 has the effect of improving the quality of broiler meat.

Claims

1. A type of *Weizmannii* GH01, characterized in that, Its accession number is CGMCC No. 36505.

2. The application of Weizmannii coagulans GH01 with accession number CGMCC No. 36505 in the preparation of feed additives, and / or, antibacterial agents, and / or, antidiarrheal drugs, and / or, functional foods for regulating intestinal flora, and / or, in vitro antibacterial activity for non-therapeutic purposes.

3. The application of *Weizmannii coagulans* GH01 with accession number CGMCC No. 36505 according to claim 2 in the preparation of feed additives, and / or, antibacterial agents, and / or, antidiarrheal drugs, and / or, functional foods for regulating intestinal flora, and / or, in vitro antibacterial activity for non-therapeutic purposes, characterized in that... The antibacterial spectrum includes: Salmonella, Staphylococcus aureus, Listeria monocytogenes, Clostridium difficile, Aeromonas hydrophila, and Edwardsiella tarda. And / or, the diarrhea refers to diarrhea caused by Salmonella; And / or, the regulation of the intestinal flora includes: restoring the imbalance between Firmicutes and Bacteroidetes and increasing the abundance of Proteobacteria, decreasing the relative abundance of Helicobacter and Biliophilia, and increasing the abundance of Alternaria, Helicobacter tumefaciens NK4A136, Rumenococcus, Prevotellaceae UGG001, Osmotherium, and Muribaculum It belongs to the relative abundance.

4. A feed additive, comprising: The active ingredient is characterized in that it comprises: Weizmannii coagulans GH01 with accession number CGMCC No. 36505.

5. A feed additive according to claim 4, characterized in that, Also includes: Auxiliary materials.

6. An antibacterial agent, comprising: The active ingredient is characterized in that it comprises: Weizmannii coagulans GH01 with accession number CGMCC No. 36505.

7. A medication for treating diarrhea, comprising: The active pharmaceutical ingredient is characterized in that it comprises: Weizmannii coagulans GH01 with preservation number CGMCC No. 36505.

8. A microbial inoculant, comprising: The active ingredient is characterized in that it comprises: Weizmannii coagulans GH01 with accession number CGMCC No. 36505.

9. A microbial inoculant according to claim 8, characterized in that, Also includes: Auxiliary materials.

10. A non-therapeutic in vitro antibacterial method, characterized in that, Weizmannii coagulans GH01 with accession number CGMCC No. 36505 was used for antibacterial treatment.

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