Bacillus velezensis lw56-4 and applications thereof

Abstract

The application provides bacillus velezensis LW56-4 and application thereof. The bacillus velezensis is bacillus velezensis LW56-4, the classification name is Bacillus velezensis , and the preservation address is China.Wuhan.Wuhan University, and the preservation number is CCTCC NO: M 20251875. The bacillus velezensis LW56-4 has broad-spectrum antibacterial property, can reduce animal diarrhea, promote animal gastrointestinal development and improve antioxidant capacity, can effectively promote feed conversion and promote animal growth, and is an ideal substitute for aureomycin and other antibiotics.

Description

Technical Field

[0001] This invention relates to the screening and application of functional microorganisms, and more specifically, to a Bacillus belyssus LW56-4 and its application. Background Technology

[0002] Weaning stress syndrome in young animals is a common problem in large-scale farming, characterized by increased susceptibility to pathogens, low feed conversion efficiency, and severe oxidative stress, which seriously restricts farming efficiency. Young ruminants, such as calves and lambs, have stomachs that are not fully developed. Their rumen and reticulum are small, and their villi are not yet fully developed. Weaning stress leads to abnormalities such as delayed digestive tract development, loss of appetite, reduced feed intake, decreased feed conversion rate, and reduced rumination frequency, resulting in a significant slowdown in growth, poorer body condition, and difficulty in achieving expected subsequent production performance. Simultaneously, due to decreased immunity and oxidative stress, they are more susceptible to infectious diseases, exhibiting symptoms such as diarrhea, loose stools, coughing, and fever.

[0003] Currently, the prevention and treatment of weaning stress in lambs often relies on feed antibiotics such as chlortetracycline. However, the overuse of antibiotics often leads to the emergence of drug-resistant bacteria, antibiotic residues, and gastrointestinal flora imbalance. After long-term exposure to antibiotics, bacteria can gradually adapt and develop resistance through gene mutations and other means, significantly reducing the effectiveness of antibiotic treatment when infected with the same or similar bacteria in the future.

[0004] In aquaculture environments, drug-resistant bacteria can spread between different animal individuals and even be transmitted to humans through the food chain, posing a significant threat to public health and safety. Some antibiotics ingested by animals remain in their tissues and organs. When these animal products enter the human food chain, these residual antibiotics may have adverse effects on human health, such as causing allergic reactions and disrupting the balance of gut microbiota. Furthermore, antibiotic use may also produce other side effects in young ruminants, such as gut microbiota dysbiosis and abnormal liver and kidney function, affecting their normal growth and development.

[0005] Bacillus berleis is a novel biocontrol bacterium with broad-spectrum antibacterial activity, primarily used to inhibit plant pathogens. In recent years, it has also seen some application in aquaculture and poultry farming. However, there are no reports on Bacillus berleis possessing both broad-spectrum antibacterial activity and the ability to promote feed (especially fibrous roughage) digestion, or on its use as a feed additive to replace chlortetracycline in alleviating weaning stress (e.g., diarrhea) and promoting animal growth in ruminants. Therefore, screening for a Bacillus berleis species that simultaneously possesses broad-spectrum antibacterial activity, promotes feed (especially fibrous roughage) digestion, and enhances animal antioxidant capacity is of great significance in animal husbandry, particularly for ruminants. Summary of the Invention

[0006] The main objective of this invention is to provide a Bacillus belyss LW56-4 and its application, in order to solve the problem that there is a lack of Bacillus belyss that simultaneously has broad-spectrum antibacterial function, promotes feed (especially fibrous roughage) digestion and improves the antioxidant performance of animals.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a *Bacillus belyescens* strain is provided, which is *Bacillus belyescens* LW56-4, and its classification name is... Bacillus velezensis It was deposited on August 22, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20251875.

[0008] Furthermore, the 16S rRNA gene of the aforementioned Bacillus belyssus has the nucleotide sequence shown in SEQ ID NO: 1.

[0009] Furthermore, the gyrB gene of the aforementioned Bacillus belyssus has the nucleotide sequence shown in SEQ ID NO: 2.

[0010] To achieve the above objectives, according to a second aspect of the present invention, an antibacterial agent is provided, the antibacterial agent comprising the above-mentioned Bacillus belyes, or the fermentation broth of the above-mentioned Bacillus belyes, or the metabolites of the above-mentioned Bacillus belyes.

[0011] Furthermore, the above-mentioned antibacterial agents can inhibit any one or more of the following pathogens: Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, or Streptococcus dolphinus.

[0012] To achieve the above objectives, according to a third aspect of the present invention, a microecological preparation is provided, comprising the aforementioned Bacillus berleis, or the fermentation broth of the aforementioned Bacillus berleis, or the metabolites of the aforementioned Bacillus berleis, or the aforementioned antibacterial agent.

[0013] To achieve the above objectives, according to a fourth aspect of the present invention, a feed additive is provided, comprising the aforementioned Bacillus berleis, or the fermentation broth of the aforementioned Bacillus berleis, or the metabolites of the aforementioned Bacillus berleis, or the aforementioned antibacterial agent, or the aforementioned microecological preparation.

[0014] To achieve the above objectives, according to a fifth aspect of the present invention, the application of the above-described Bacillus berberis, or the above-described antibacterial agent, or the above-described microecological preparation, or the above-described feed additive in animal husbandry is provided.

[0015] Furthermore, the above applications are selected from any one or more of the following: preventing animal diarrhea, promoting animal growth, promoting animal digestion, or improving animal antioxidant capacity.

[0016] Furthermore, the animals mentioned above are selected from any one or more of the following: ruminants, pigs, broilers, laying hens, dogs, or cats.

[0017] Feeding animals with Bacillus vesiculosus LW56-4 can prevent diarrhea, promote animal growth, improve feed (especially fibrous roughage) conversion rate, and enhance antioxidant capacity. Bacillus vesiculosus LW56-4 shows promise as an ideal alternative to antibiotics such as chlortetracycline and is of significant importance in preventing weaning stress syndrome in animals. Attached Figure Description

[0018] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 An evolutionary tree of Bacillus belye LW56-4 according to an embodiment of the present invention is shown.

[0020] Figure 2 A colony diagram of Bacillus berberis LW56-4 according to an embodiment of the present invention is shown.

[0021] Figure 3 HE staining images of the rumen and intestinal villi of a weaned Hu sheep according to an embodiment of the present invention are shown. Detailed Implementation

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0023] Terminology Explanation:

[0024] Microecological preparations, also known as probiotic preparations, are bioactive products made using normal microorganisms (probiotics) or their promoting substances that are beneficial to the host. Their core principle is to adjust, maintain, or rebuild the balance of the host's (including humans, animals, and plants) microecological system by supplementing with exogenous beneficial bacteria or promoting the growth and activity of existing beneficial bacteria, thereby exerting beneficial effects such as disease prevention and treatment, health promotion, and improved productivity.

[0025] Animal microecological preparations are a specific branch of microecological preparations, referring to preparations specifically designed for use in animal husbandry, aquaculture, and other fields. Based on the principles of microecology, and targeting the physiological characteristics and intestinal flora structure of animals, they are live bacterial preparations or their metabolites produced through artificial methods of isolating, culturing, purifying, and revitalizing beneficial microorganisms, and then using special processes. The selection of strains and the design of the formulation are based on the digestive physiology and common pathogens of specific animals (such as pigs, chickens, cattle, fish, and shrimp).

[0026] Artificial rumen biomimetic digestion: Artificial rumen biomimetic digestion refers to the technology of constructing a similar digestive system in vitro by simulating the structure and function of the rumen of ruminants, the microbial community and its metabolic characteristics, so as to achieve efficient degradation and transformation of substrates such as cellulose biomass.

[0027] 16S rRNA gene: This gene encodes part of the small subunit RNA of the bacterial ribosomal molecule and is a highly conserved region in the bacterial genome. Due to its high conservation and variability in bacterial classification, the 16S rRNA gene is often used in bacterial phylogenetic studies and identification. However, because different bacterial species can have very similar 16S rRNA gene sequences, it may not be precise enough in distinguishing very closely related bacterial species.

[0028] The gyrB gene encodes the B subunit of DNA gyrase, a component of an enzyme that plays a role in bacterial DNA replication. The gyrB gene exhibits higher variability across different bacterial genera and species, providing greater resolution for distinguishing closely related species or different strains within the same species. The 16S rRNA gene is more suitable for intraspecific and interspecific bacterial identification.

[0029] Fiber-rich roughage refers to feeds containing high levels of cellulose, hemicellulose, and lignin, primarily used for feeding ruminants such as cattle and sheep. Roughage is an important component of animal diets, providing essential fiber to maintain the health of the animal's digestive system. Fiber-rich roughage typically includes pasture, hay, silage, and straw. These feeds not only provide basic energy and nutrients but also promote normal rumination and rumen function in ruminants. Fiber-rich roughage is characterized by its large volume, low density, and slow digestibility, but it is a crucial foundation for maintaining the health and production performance of ruminants.

[0030] As mentioned in the background section, weaning stress syndrome is a common problem in large-scale animal farming. This disease is characterized by increased susceptibility to pathogens, low feed conversion efficiency, severe oxidative stress, and delayed rumen development, significantly hindering farming profitability. Current main measures to alleviate related symptoms involve the use of antibiotics such as chlortetracycline. However, antibiotic use often leads to problems such as the emergence of drug-resistant bacteria, antibiotic residues, and intestinal flora imbalance in animals.

[0031] In recent years, Bacillus vesiculosus, as a novel biocontrol bacterium with broad-spectrum antibacterial activity, has been used in aquaculture and poultry farming. However, there are no reports on Bacillus vesiculosus, which simultaneously possesses broad-spectrum antibacterial function and promotes feed (especially fibrous roughage) digestion, or on its use as a feed additive to replace chlortetracycline in alleviating weaning stress (such as diarrhea) in ruminants and promoting animal growth.

[0032] In this invention, the inventors attempted to screen for *Bacillus belyceae* strains from the rumen of ruminants that simultaneously possess broad-spectrum antibacterial properties and promote the digestibility of feed (especially fibrous roughage). Feed containing this bacterium effectively reduces animal diarrhea, improves feed conversion ratio, promotes animal growth, and enhances antioxidant capacity. This bacterium has green and safe beneficial effects and is an ideal alternative to antibiotics such as chlortetracycline in the prior art; therefore, this invention provides a protective scheme.

[0033] In a first typical embodiment of the present invention, a *Bacillus belye* strain is provided, which is *Bacillus belye* LW56-4, and its classification name is [missing information]. Bacillus velezensis It was deposited on August 22, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20251875.

[0034] Bacillus belyssus LW56-4 ( Bacillus velezensis LW56-4), with a clearly defined taxonomic position, belongs to the genus Bacillus, specifically Bacillus belesii. This strain underwent rigorous screening and was selected due to its significant potential for improving animal health and aquaculture efficiency. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251875, ensuring the stability and traceability of the microbial resource.

[0035] The 16S rRNA gene is a highly conserved region in the bacterial genome. In a preferred embodiment of the present invention, the 16S rRNA gene of the above-mentioned Bacillus belyss has the nucleotide sequence shown in SEQ ID NO: 1. Strains possessing the above-mentioned 16S rRNA gene, such as the Bacillus belyss LW56-4 of this application, simultaneously exhibit broad-spectrum antibacterial function and the function of promoting feed digestion.

[0036] The gyrB gene exhibits higher variability across different bacterial genera and species, providing greater resolution for distinguishing closely related species or different strains within the same species. In a preferred embodiment of the present invention, the gyrB gene of the aforementioned Bacillus belyssus has the nucleotide sequence shown in SEQ ID NO: 2. When strains possessing the aforementioned gyrB gene or their metabolites are ingested by animals, they can effectively alleviate animal diarrhea, improve feed conversion ratio, promote animal growth, and enhance the antioxidant capacity of animals.

[0037] In a second typical embodiment of the present invention, an antibacterial agent is provided, comprising the aforementioned *Bacillus berberis*, its fermentation broth, or its metabolites. The antibacterial agent of the present invention has broad-spectrum antibacterial activity, aiming to overcome the problems of drug resistance, residues, and intestinal flora imbalance caused by traditional antibiotics, and is expected to be developed into an ideal alternative to antibiotics such as chlortetracycline.

[0038] The aforementioned antibacterial agents not only effectively inhibit a range of important pathogens, including but not limited to *Clostridium perfringens*, *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, and *Streptococcus dolphinus*, but also exhibit good compatibility and safety during use, making them suitable for various animal husbandry environments. In a preferred embodiment of the present invention, the aforementioned antibacterial agent can inhibit any one or more of the following pathogens: *Clostridium perfringens*, *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, or *Streptococcus dolphinus*.

[0039] Metabolites refer to a series of chemical substances produced, secreted, or released into the environment by Bacillus belyssus LW56-4 through its own metabolic mechanisms during growth, reproduction, and maintenance of life activities. Fermentation broth refers to a mixed liquid system containing bacterial cells, metabolic products, and unconsumed culture medium components obtained after Bacillus belyssus LW56-4 has grown, reproduced, and metabolized in a liquid culture medium for a period of time under artificial or industrially controlled conditions (such as a fermenter).

[0040] In a third typical embodiment of the present invention, a microecological preparation is provided, comprising the aforementioned *Bacillus berberis*, its fermentation broth, its metabolites, or its antibacterial agent. The present invention further incorporates *Bacillus berberis* LW56-4, its fermentation broth, and its metabolites into the microecological preparation, aiming to adjust the intestinal microecological balance of animals, promote digestion and absorption, and enhance animal immunity by supplementing beneficial microorganisms.

[0041] In a fourth typical embodiment of the present invention, a feed additive is provided, which includes the above-mentioned Bacillus berleis, or the fermentation broth of the above-mentioned Bacillus berleis, or the metabolites of the above-mentioned Bacillus berleis, or the above-mentioned antibacterial agent, or the above-mentioned microecological preparation.

[0042] Bacillus berberis LW56-4 and its derivatives (such as fermentation broth or metabolites) can also be used as feed additives. They can be added to feed as natural bioactive agents to optimize feed formulation, improve feed conversion rate, thereby promoting healthy animal growth, while reducing environmental pollution and improving animal welfare.

[0043] In a fifth typical embodiment of the present invention, the application of the above-mentioned Bacillus berberis, or the above-mentioned antibacterial agent, or the above-mentioned microecological preparation, or the above-mentioned feed additive in animal breeding is provided.

[0044] The application of Bacillus vesiculosus LW56-4, antibacterial agents, microecological preparations, or feed additives in animal husbandry covers multiple dimensions of health and wellness measures. They can not only prevent diarrhea in animals, improve their growth rate, and optimize digestive system function, but also enhance their antioxidant capacity, resist external environmental stress and the effects of internal diseases, thereby comprehensively improving animal health and increasing breeding efficiency and economic benefits.

[0045] In a preferred embodiment of the present invention, the above-mentioned applications are selected from any one or more of the following: preventing and treating animal diarrhea, promoting animal growth, promoting animal digestion, or improving animal antioxidant capacity.

[0046] The application of Bacillus belyceca LW56-4 and its derivatives in animal husbandry covers a wide range of target animal groups, including but not limited to ruminants (such as cattle and sheep), pigs, poultry (such as broilers and laying hens), and pets (such as dogs and cats). This means that the present invention has broad application prospects in animal husbandry, breeding, and pet health industries. In a preferred embodiment of the present invention, the animals are selected from any one or more of the following: ruminants, pigs, broilers, laying hens, cats, or dogs.

[0047] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0048] Example 1: Isolation and Identification of Bacillus belyssus strain LW56-4

[0049] 1.1 Isolation and purification of strains

[0050] The contents of the rumen of healthy Hu sheep (from the sheep farm of Yichang Angel Bio-Agriculture Technology Co., Ltd.) were collected in sterile physiological saline and thoroughly vortexed. The mixture was then incubated at 85°C for 10 min, serially diluted 10-fold, and spread onto LB agar medium. The culture was incubated at 37°C for 12-14 h. Single colonies exhibiting Bacillus characteristics were streaked onto LB agar medium and subcultured at 37°C for 12 h. This process was repeated until the colonies on LB agar exhibited a uniform morphology, yielding purified single colonies. Different purified single colonies were inoculated into LB liquid medium and cultured at 37°C with shaking at 200 rpm for 24 h to obtain the fermentation broth.

[0051] 1.2 Screening of strains with antibacterial activity

[0052] Indicator bacterial suspensions (Escherichia coli ATCC25922, Salmonella Typhimurium ATCC14028, Clostridium perfringens CVCC2030, Staphylococcus aureus ATCC6538, and Streptococcus dolphinii ATCC29178, all purchased from the China General Microbiological Culture Collection Center) were spread onto LB agar medium using sterile cotton swabs. Sterile Oxford cups were placed in fixed positions within the medium, and 100 µL of indicator bacterial suspension was added. The mixture was then poured into LB agar basal medium and cooled to room temperature until solidified. The Oxford cups were gently removed using sterile hemostatic forceps. The supernatant obtained after centrifuging the fermentation broth obtained in step 1.1 at 8000 rpm for 10 min was added to each well at 100 µL / well. The wells were incubated at 37°C for 16 h. Sterile PBS solution was used as a negative control, and the diameter of the inhibition zone was measured. Six Bacillus strains with good antibacterial effects were obtained, among which the strain named LW56-4 had the best antibacterial effect. The antibacterial results are shown in Table 1.

[0053] Table 1. Diameter of inhibition zone of strain LW56-4

[0054]

[0055] 1.3 Strain Identification

[0056] The strain LW56-4, which produced inhibition zones after initial screening, was cultured on NA agar plates at 37°C for 2 days. Colony morphology and color were observed. It grew well on NA agar, with milky-white, flat colonies featuring a raised, flame-like center and slightly irregular edges. No pigment was produced. (See [link to NA agar plate]). Figure 2 Gram staining is purple, indicating Gram-positive bacteria; spore staining shows the presence of spores.

[0057] Whole-genome DNA was extracted from the target strain using a bacterial genomic DNA kit. PCR amplification was performed using universal primers 27F and 1492R for the 16S rDNA gene and primers gyrB-f and gyrB-r for the housekeeping gene (gyrB). The amplified products were sequenced and then analyzed using bioinformatics. The 16S rDNA gene sequence of strain LW56-4 obtained by sequencing is shown in SEQ ID NO: 1 (1443 bp).

[0058]

[0059] The gyrB gene sequence of strain LW56-4 obtained by sequencing is shown in SEQ ID NO: 2 (1137bp):

[0060]

[0061] BLAST analysis of sequences in the NCBI database showed that the 16S rDNA sequence of strain LW56-4 had 100% similarity to strains of the genus *Bacillus*, and its housekeeping gene *gyrB* met the species criteria for *Bacillus belyesense*. The *gyrB* sequence of strain LW56-4 was BLAST-aligned with known sequences in NCBI, and the sequences were downloaded. Multiple sequence alignment was performed using tools such as MEGA, and a phylogenetic tree was constructed (see [link to documentation]). Figure 1 Therefore, based on the above morphological analysis and the homology analysis of the 16S rRNA sequence and gyrB gene of strain LW56-4 and related strains, strain LW56-4 was identified as Bacillus belyes.

[0062] Example 2: Artificial Rumen Biomimetic Digestion Experiment

[0063] The strain LW56-4 obtained in Example 1.1 was evaluated with five other screened antibacterial Bacillus samples.

[0064] Assay Procedure: The AGRS-III in vitro fermentation system (Beijing Boxiang Xingwang Technology Co., Ltd., AGRS-III) – an artificial rumen – was used. The fermentation flasks were 120mL Heinz anaerobic fermentation flasks (with specially designed screw caps and white chitin stoppers). Each flask was supplemented with the Bacillus spp. fermentation broth to be tested (the viable count of the Bacillus spp. to be tested was 2 × 10⁻⁶). 8 The addition amount is 0.5% (CFU / mL) (volume of fermentation broth / total volume of inoculum in the fermentation bottle, ml / ml). It should be noted that the fermentation method for each strain is the same as the method in 1.1 of Example 1.

[0065] Rumen fluid samples were taken from fresh rumen fluid of fattening cattle weighing approximately 200 kg (purchased from the Three Gorges Livestock Industrial Park). The rumen fluid was weighed and then, under anaerobic conditions, a 39°C, sterile saline solution was added at a weight-to-volume ratio of 1:5. After stirring and mixing thoroughly, the mixture was filtered through four layers of gauze, and the filtered rumen fluid was collected.

[0066] Inoculation: Weigh 50 mL of buffer solution + 25 mL of rumen fluid into each fermentation bottle. Add 0.5% of the Bacillus spp. fermentation broth to be tested to each bottle, and add 0.5 g of the culture substrate for the in vitro test to each group. Set up 5 replicates for each sample. The composition of the substrate is based on the basal diet of high-yielding dairy cows. The specific composition and nutrient levels are shown in Table 2. The buffer solution formula is shown in Table 3.

[0067] After being connected to the fermentation system via an intravenous infusion needle, fermentation was carried out at 39°C for 48 hours, and the total gas production was automatically recorded.

[0068] The formula of the premix in Table 2 is as follows: each kilogram of premix contains 240,000 IU of vitamin A, 370,000 IU of vitamin D, 1,100 mg of vitamin E, 270 mg of Fe, 370 mg of Cu, 1,900 mg of Zn, 1,800 mg of Mn, 16 mg of I, 17 mg of Co, and 11 mg of Se.

[0069] Table 2. Substrate composition for in vitro fermentation

[0070]

[0071] The formulation of the above buffer solution is as follows: Based on the experimental design requirements, the buffer solution was prepared according to the addition order and preparation ratio shown in Table 3 (Menke and Steingass, 1988). In Table 3, trace element solution A includes 9.97 g CaCl2 and 10.0 g MnCl2. 4H₂O, 1.0g CoCl₂ 6H2O, 6.78g FeCl2 4H₂O, diluted to 100mL with distilled water; Artificial saliva B, including 35g NaHCO₃, 4g NH₄HCO₃, diluted to 1000mL with distilled water; Macro-element solution C, including 5.7g Na₂HPO₄, 6.2g KH₂PO₄, 0.6g MgSO₄. 7H2O, distilled water to a final volume of 100 mL; 0.1% (w / v) resazurin solution D: weigh 100 mg of resazurin and dilute to a final volume of 100 mL; reducing agent solution: weigh 625 mg of Na2S Add 4.0 mL of 1.0 M NaOH solution to 9H2O, and then distilled water to bring the volume to 100 mL.

[0072] Table 3 Buffer Formulation

[0073]

[0074] After fermentation, the fermentation was terminated by an ice bath. TVFA (volatile fatty acids) was determined according to the method in T / NAIA 005-2020: After fermentation, 1 mL of fermentation broth was extracted, 250 µL of metaphosphoric acid was added, incubated at 4 °C for 10 min, centrifuged at 15000 × g for 10 min, and the supernatant was filtered through a 0.22 µm filter membrane. The TVFA concentration was then determined by gas chromatography. The dry matter digestibility was calculated using the difference method to determine the change in dry matter before and after fermentation. Dry matter digestibility and TVFA concentration are shown in Table 4.

[0075] Table 4 Dry matter digestibility and TVFA concentration

[0076]

[0077] As shown in Table 4, the *Bacillus belyssus* LW56-4 of this invention can significantly improve dry matter digestibility, and is higher than other strains. The higher dry matter digestibility indicates that the *Bacillus belyssus* LW56-4 of this application can promote animal digestion and improve feed utilization, thereby further promoting animal growth and saving feed usage.

[0078] The *Bacillus belycera* strain LW56-4 of this invention can significantly increase the concentrations of butyric acid, isobutyric acid, and branched-chain fatty acids in the fermentation system. Butyric acid, isobutyric acid, and branched-chain fatty acids are the main energy-providing substances for ruminants; their increase indicates improved feed conversion rate and increased energy-providing substances, which helps promote the growth of ruminants.

[0079] Example 3:

[0080] This embodiment provides a fermentation culture method for Bacillus belyss LW56-4, wherein the fermentation scale is a 100L fermenter.

[0081] 3.1 Preparation of Seed Liquid

[0082] A single colony of *Bacillus belyssiensis* strain LW56-4, cultured for 14 h in one loop of NA medium, was inoculated into LB liquid medium and cultured at 37°C with shaking at 180 rpm for 16 h to obtain *Bacillus belyssiensis* strain LW56-4 seed culture with a viability of 5 × 10⁻⁶. 8 CFU / mL.

[0083] 3.2 Fermentation Process

[0084] The above-mentioned Bacillus subtilis seed liquid was inoculated into a fermentation medium for fermentation culture at a temperature of 37°C and dissolved oxygen of not less than 20% until the spore formation rate was above 90%.

[0085] The fermentation medium, calculated by weight, comprises: 30 to 50 parts soybean meal powder, 3 to 5 parts glucose, 20 to 35 parts corn starch, 3 to 6 parts yeast powder, 4 to 6 parts peptone, 1 to 5 parts calcium carbonate, 1 to 5 parts sodium chloride, 0.5 to 1 part magnesium sulfate, 1 to 5 parts dipotassium hydrogen phosphate, 0.1 to 0.3 parts manganese sulfate, and 0.2 parts polyoxypropylene glycerol.

[0086] During the fermentation process, the culture temperature was 37℃ when the spore formation rate (using malachite green staining method) was less than 60%; when the spore formation rate reached 70%, the culture temperature was reduced to 32℃.

[0087] In this embodiment, the fermentation broth from the lower tank was diluted and spread onto NA medium plates. After overnight incubation at 37°C, the cells were counted. The count showed that the number of cells in the fermentation broth from the lower tank was 15 billion CFU / mL and the spore concentration was 14.5 billion CFU / mL.

[0088] Example 4: Experiment on raising weaned Hu sheep

[0089] This experiment employed a single-factor randomized block design, selecting 60 healthy weaned female Hu sheep (from Yichang Angel Bio-Agriculture Technology Co., Ltd.) of similar age and weighing 13.5±1 kg. They were randomly divided into 3 groups, with 5 replicates per group and 4 sheep per replicate. They were housed in separate pens, as shown in Table 5. The formal experimental period was 30 days. During the experiment, diarrhea rate, feed intake, and daily weight gain were recorded. After the experiment, 7 samples from each group were slaughtered, and rumen and intestinal tissues were collected for formaldehyde fixation and HE staining (see Table 5). Figure 3 The development of villi in weaned sheep was analyzed. Changes in SCFAs content in rumen fluid were detected; serum biochemical indicators were analyzed. Fecal samples were analyzed using the acid-insoluble ash method to determine the digestibility of neutral detergent fiber and crude protein. Specific animal experiment grouping schemes are shown in Table 5 ("T" in Table 5 represents tons). Average daily gain (ADG) and feed conversion ratio (FG, representing the ratio of feed intake to body weight, reflecting feed conversion level) of weaned sheep are shown in Table 6. Diarrhea rate of weaned sheep is shown in Table 7. Serum biochemical indicators of weaned sheep are analyzed in Table 8. Short-chain fatty acid content in rumen fluid of weaned sheep is analyzed in Table 9. Rumen and intestinal villi length analysis of weaned sheep is shown in Table 10. Neutral detergent fiber and crude protein digestibility in feces of weaned sheep is shown in Table 11.

[0090] Table 5 Grouping scheme for weaned Hu sheep

[0091]

[0092] Table 6. Average Daily Weight Gain (ADG) and Feed Conversion Ratio (FG) of Weaned Lake Sheep

[0093]

[0094] Note: Different shoulder marks for ac indicate significant differences (P<0.05). No shoulder mark in the table indicates no significant difference between the three groups of data in the same row; different lowercase letters mean significant differences between the two groups (P<0.05); uppercase letters mean extremely significant differences (P<0.01).

[0095] In Table 6, “ADG14” and “ADG28” represent the average daily weight gain of the animals in a 14-day cycle and a 28-day cycle, respectively. “FG14” and “FG28” represent the feed conversion ratio of the animals in a 14-day cycle and a 28-day cycle, respectively.

[0096] As shown in Table 6, compared with the control group, the microecological preparation prepared by Bacillus vesicle LW56-4 of the present invention can significantly increase the average daily weight gain of weaned Hu sheep for 14 and 28 days, and its effect is not significantly different from that of chlortetracycline, indicating that it can effectively replace chlortetracycline to promote the growth of weaned Hu sheep.

[0097] Table 7 Diarrhea rate in weaned Hu sheep

[0098]

[0099] As shown in Table 7, feeding weaned Hu sheep with the microecological preparation made from Bacillus vesicle LW56-4 of the present invention can effectively reduce their diarrhea rate.

[0100] Table 8. Analysis of serum biochemical indicators of weaned sheep from Huzhou

[0101]

[0102] Note: Different shoulder marks in the same row (a, b) indicate significant differences (P<0.05).

[0103] As shown in Table 8, feeding weaned Hu sheep with the microecological preparation prepared by Bacillus belysus LW56-4 of the present invention can effectively increase the level of superoxide dismutase in serum, indicating that it can effectively improve the antioxidant capacity of weaned Hu sheep.

[0104] Table 9. Analysis of short-chain fatty acid content in rumen fluid of weaned sheep.

[0105]

[0106] As shown in Table 9, the microecological preparation made from Bacillus belye LW56-4 of this invention can effectively increase the levels of acetic acid, propionic acid, and butyric acid in the rumen of weaned Hu sheep. In ruminant nutrition research, the correlation between the production of short-chain fatty acids and feed digestibility and animal production performance is often studied. An increase in this indicator means improved feed digestibility, which ultimately helps promote animal growth.

[0107] Table 10 Analysis of rumen and intestinal villus length in weaned Hu sheep

[0108]

[0109] Note: Different shoulder marks in the same row (a, b) indicate significant differences (P<0.05).

[0110] As shown in Table 10, the microecological preparation prepared by Bacillus vesicle LW56-4 of the present invention can effectively promote the development of rumen and intestinal villi when fed to weaned Hu sheep, which helps to promote animal digestion and thus further promotes animal growth.

[0111] Table 11 Digestibility of Neutral Detergent Fiber and Crude Protein in Weaned Lake Sheep Dung

[0112]

[0113] Note: Different shoulder marks in the same row (a, b) indicate significant differences (P<0.05).

[0114] As shown in Table 11, feeding weaned Hu sheep with the microecological preparation made from Bacillus vesicle LW56-4 of the present invention can effectively promote the digestibility of neutral detergent fiber and crude protein in Hu sheep, which is consistent with the results of increasing daily weight gain.

[0115] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: Bacillus berberis LW56-4 provided by the present invention has broad-spectrum antibacterial properties, can alleviate animal diarrhea, promote the development of animal gastrointestinal tract and improve antioxidant capacity, and can also effectively promote feed (especially fibrous roughage) conversion and promote animal growth.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Bacillus belesii, characterized in that, The *Bacillus belyssus* mentioned is *Bacillus belyssus* LW56-4, and its classification name is [not specified]. Bacillus velezensis It was deposited on August 22, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251875.

2. The Bacillus belye according to claim 1, characterized in that, The 16S rRNA gene of the *Bacillus belyssus* has the nucleotide sequence shown in SEQ ID NO:

1.

3. The Bacillus belesiensis according to claim 1, characterized in that, The gyrB gene of the Bacillus belyssus has the nucleotide sequence shown in SEQ ID NO:

2.

4. An antibacterial agent, characterized in that, The antibacterial agent comprises Bacillus berleisi according to any one of claims 1-3 or the fermentation broth of Bacillus berleisi according to any one of claims 1-3; wherein the fermentation broth contains Bacillus berleisi.

5. The antibacterial agent according to claim 4, characterized in that, The antibacterial agent can inhibit any one or more of the following pathogens: Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, or Streptococcus dolphinus.

6. A microecological preparation, characterized in that, The microecological preparation includes Bacillus bellisi according to any one of claims 1-3, or the fermentation broth of Bacillus bellisi according to any one of claims 1-3, or the antibacterial agent according to claim 4 or 5.

7. A feed additive, characterized in that, The feed additive includes Bacillus bellisi according to any one of claims 1-3, or the fermentation broth of Bacillus bellisi according to any one of claims 1-3, or the antibacterial agent according to claim 4 or 5, or the microecological preparation according to claim 6.

8. The application of *Bacillus belye* according to any one of claims 1-3, or the antibacterial agent according to claim 4 or 5, or the microecological preparation according to claim 6, or the feed additive according to claim 7, in animal husbandry; wherein, The application is to promote animal growth.

9. The application according to claim 8, characterized in that, The animal is selected from any one or more of the following: ruminants, pigs, broilers, laying hens, dogs, or cats.

10. The use of Bacillus vesiculosus of any one of claims 1-3, or the antibacterial agent of claim 4 or 5, or the microecological preparation of claim 6, or the feed additive of claim 7, in the preparation of a medicament for preventing animal diarrhea or promoting animal digestion.

11. The application according to claim 10, characterized in that, The animal is selected from any one or more of the following: ruminants, pigs, broilers, laying hens, dogs, or cats.

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