Bacillus velezensis LW56-4 and application thereof

Abstract

The invention provides bacillus velezensis LW56-4 and an application of the bacillus velezensis LW56-4. The bacillus velezensis is bacillus velezensis LW56-4, the classification name is bacillus velezensis, the bacillus velezensis is preserved in the China Center for Type Culture Collection on August 22, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20251875. The bacillus velezensis LW56-4 has a broad-spectrum antibacterial property, can relieve animal diarrhea, promote gastrointestinal tract development of animals and improve oxidation resistance, can also effectively promote feed conversion and promote animal growth, and is an ideal substitute for antibiotics such as aureomycin and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the screening and application of functional microorganisms, in particular to a Bacillus velezensis LW56-4 and application thereof. BACKGROUND

[0002] Weaning stress syndrome of young animals is a common problem in large-scale breeding, which is characterized by increased susceptibility to pathogens, low feed conversion efficiency, and severe oxidative stress, which seriously restricts the breeding benefit. Young ruminants, for example, calves and lambs, have an immature stomach. The volume of rumen and reticulum is small, and the villi are not perfect. Weaning stress leads to abnormal conditions such as delayed development of the digestive tract, loss of appetite, reduced feed intake, decreased feed conversion rate, and reduced rumination, which results in a significant slowdown in the growth rate of young ruminants, poor body condition, and difficulty in achieving the expected production performance. At the same time, due to decreased immunity and oxidative stress, it is more susceptible to infectious diseases, such as diarrhea, soft and loose stools, coughing, and fever.

[0003] Current prevention and treatment of weaning stress in lambs often relies on chlortetracycline and other feed antibiotics. However, the misuse of antibiotics often leads to the emergence of drug-resistant bacteria, antibiotic residues, and gastrointestinal flora disorders. After long-term exposure to antibiotics, bacteria gradually adapt to antibiotics through genetic mutation and other methods, and develop resistance to antibiotics, which greatly reduces the therapeutic effect of antibiotics when infected with the same or similar bacteria in the future.

[0004] In the breeding environment, drug-resistant bacteria can also spread between different animal individuals and even be transmitted to humans through the food chain, posing a great hidden danger to public health safety. A portion of the antibiotics ingested by animals will remain in their tissues and organs, and when these animal products enter the human food chain, the residual antibiotics can have adverse effects on human health, such as causing allergic reactions and disrupting the balance of the human intestinal microbiota. In addition, the use of antibiotics can also have other side effects on young ruminants, such as intestinal flora disorders, abnormal liver and kidney function, and affect their normal growth and development.

[0005] Bacillus velezensis is a new type of biological control bacteria with broad-spectrum antibacterial activity, mainly used to inhibit plant pathogens. In recent years, it has been applied in aquaculture and poultry feeding. However, there is no report on Bacillus velezensis with both broad-spectrum antibacterial function and promoting the digestion of feed (especially fiber roughage) as a feed additive to replace chlortetracycline to alleviate weaning stress (such as diarrhea) in ruminants and promote animal growth. Therefore, screening a Bacillus velezensis with both broad-spectrum antibacterial function, promoting the digestion of feed (especially fiber roughage), and improving the antioxidant performance of animals (especially ruminants) is of great significance in animal (especially ruminant) breeding. SUMMARY

[0006] The main purpose of the present application is to provide a Bacillus velezensis LW56-4 and application thereof, so as to solve the problem that there is no Bacillus velezensis with broad-spectrum antibacterial function, promoting feed (especially fiber roughage) digestion and improving animal antioxidant performance in the prior art.

[0007] In order to achieve the above-mentioned purpose, according to the first aspect of the present application, a Bacillus velezensis is provided, which is Bacillus velezensis LW56-4, the classification name is Bacillus velezensis , and it was preserved in China Center for Type Culture Collection on August 22, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20251875.

[0008] Further, the 16s rRNA gene of the above-mentioned Bacillus velezensis has the nucleotide sequence shown in SEQ ID NO: 1.

[0009] Further, the gyrB gene of the above-mentioned Bacillus velezensis has the nucleotide sequence shown in SEQ ID NO: 2.

[0010] In order to achieve the above-mentioned purpose, according to the second aspect of the present application, an antibacterial agent is provided, which comprises the above-mentioned Bacillus velezensis or the fermentation broth of the above-mentioned Bacillus velezensis or the metabolite of the above-mentioned Bacillus velezensis.

[0011] Further, the above-mentioned antibacterial agent can inhibit any one or more of the following pathogenic bacteria: Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium or Streptococcus iniae.

[0012] In order to achieve the above-mentioned purpose, according to the third aspect of the present application, a microecological preparation is provided, which comprises the above-mentioned Bacillus velezensis or the fermentation broth of the above-mentioned Bacillus velezensis or the metabolite of the above-mentioned Bacillus velezensis or the above-mentioned antibacterial agent.

[0013] In order to achieve the above-mentioned purpose, according to the fourth aspect of the present application, a feed additive is provided, which comprises the above-mentioned Bacillus velezensis or the fermentation broth of the above-mentioned Bacillus velezensis or the metabolite of the above-mentioned Bacillus velezensis or the above-mentioned antibacterial agent or the above-mentioned microecological preparation.

[0014] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present application, the above-mentioned Bacillus velezensis or the above-mentioned antibacterial agent or the above-mentioned microecological preparation or the above-mentioned feed additive is applied in animal breeding.

[0015] Further, the above-mentioned application is selected from any one or more of the following: preventing diarrhea of the animal, promoting growth of the animal, promoting digestion of the animal, or improving antioxidant capacity of the animal.

[0016] Further, the above-mentioned animal is selected from any one or more of the following: a ruminant, a pig, a broiler, a laying hen, a dog, or a cat.

[0017] Feeding the animal with Bacillus velezensis LW56-4 can prevent diarrhea of the animal, promote growth of the animal, improve feed conversion rate of the animal (especially for fiber roughage), and improve antioxidant capacity of the animal. Bacillus velezensis LW56-4 is expected to be an ideal substitute for antibiotics such as aureomycin, and has important significance in preventing weaning stress syndrome of the animal. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which form a part of the present description, illustrate the present application and, together with the written description, serve to explain the application. In the drawings:

[0019] Figure 1 A phylogenetic tree of Bacillus velezensis LW56-4 according to an embodiment of the present application is shown.

[0020] Figure 2 A colony map of Bacillus velezensis LW56-4 according to an embodiment of the present application is shown.

[0021] Figure 3 An HE staining map of rumen and intestinal villi of weaned Hu sheep according to an embodiment of the present application is shown. DETAILED DESCRIPTION

[0022] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0023] Explanation of terms:

[0024] Microecological preparation, also known as probiotic preparation, is a bioactive preparation prepared by using normal microorganisms (probiotics) or promoting substances beneficial to the host. The core principle is to adjust, maintain or rebuild the balance of the microecological system in the host (including humans, animals and plants) by supplementing exogenous beneficial bacteria or promoting the growth and activity of the original beneficial bacteria in the body, so as to play a beneficial role in preventing and treating diseases, promoting health and improving production performance.

[0025] Animal microecological preparation is a specific branch of microecological preparation, which refers to microecological preparations specifically used in the fields of livestock farming, aquaculture, etc. It is based on the principles of microecology, targeting the physiological characteristics and intestinal flora structure of animals, and through artificial methods of isolation, culture, purification, and rejuvenation of beneficial microorganisms, and special process to produce live bacterial preparations or their metabolites. The selection of bacterial strains and formula design are based on the digestive physiology and common pathogenic bacteria of specific animals (such as pigs, chickens, cows, fish, and shrimp).

[0026] Artificial rumen biomimetic digestion: Artificial rumen biomimetic digestion refers to simulating the structure and function of the rumen of ruminants, microbial community and its metabolic characteristics, etc., to construct a similar digestion system in vitro to achieve efficient degradation and conversion of substrates such as cellulose.

[0027] 16S rRNA gene: This is a part of the bacterial ribosomal small subunit RNA, which is a highly conserved region in the bacterial genome. Due to its high conservation and variation interval characteristics in bacterial classification, the 16S rRNA gene is commonly used for phylogenetic research and identification of bacteria. However, different species of bacteria may have very similar 16S rRNA gene sequences, so it may not be accurate enough to distinguish very similar bacterial species.

[0028] gyrB gene: encodes the B subunit of DNA gyrase, which is a component of the enzyme that plays a role in bacterial DNA replication. The gyrB gene has higher variability among different bacterial genera and species, and it can provide higher resolution for distinguishing closely related species or different strains within the same species. The 16S rRNA gene is more suitable for intra- and inter-species identification of bacteria.

[0029] Fiber roughage: refers to feed containing high cellulose, hemicellulose, and lignin, etc. components, mainly used for feeding ruminants such as cattle, sheep, etc. Roughage is an important component of animal diets, providing essential fiber to maintain the health of the animal's digestive system. Fiber roughage usually includes grass, hay, silage, straw, etc. These feeds not only provide basic energy and nutrients, but also promote normal rumination and rumen function in ruminants. The characteristics of fiber roughage are large volume, low density, and slow digestion, but they are an important basis for maintaining the health and production performance of ruminants.

[0030] As mentioned in the background, weaning stress syndrome in animals is a common problem in large-scale farming, which is characterized by increased susceptibility to pathogens, low feed conversion efficiency, severe oxidative stress, and delayed rumen development, etc., which seriously hinders the efficiency of farming. The main measure to alleviate the symptoms is to use antibiotics such as aureomycin, but the use of antibiotics often leads to the emergence of drug-resistant bacteria, antibiotic residues, and animal intestinal flora disorders.

[0031] In recent years, Bacillus velezensis has been applied in aquaculture and poultry feeding as a new biological control bacteria with broad-spectrum antibacterial activity. However, there is no report on Bacillus velezensis with both broad-spectrum antibacterial function and promoting feed (especially fiber roughage) digestion function, and its use as a feed additive to replace aureomycin to relieve the stress (such as diarrhea) of ruminants during weaning and promote the growth of animals.

[0032] In the present application, the inventors attempt to screen Bacillus velezensis strains with both broad-spectrum antibacterial and promoting feed (especially fiber roughage) digestion from the rumen of ruminants. The feed added with the bacterial agent can effectively reduce animal diarrhea, improve animal feed conversion rate, promote animal growth, and improve animal antioxidant performance. The bacteria have green and safe benefits, and are an ideal substitute for antibiotics such as aureomycin in the prior art, thus the protection scheme of the present application is proposed.

[0033] In a first typical embodiment of the present application, a Bacillus velezensis is provided, which is Bacillus velezensis LW56-4, the classification name is Bacillus velezensis , and was preserved in the China Center for Type Culture Collection on August 22, 2025, the preservation address is Wuhan, Wuhan University, China, and the preservation number is CCTCC NO: M20251875.

[0034] Bacillus velezensis LW56-4 ( Bacillus velezensis LW56-4), which has a clear classification status and belongs to the genus Bacillus, specifically Bacillus velezensis. The strain is selected due to its significant potential in animal health and breeding benefit improvement, and is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number CCTCC NO: M20251875, ensuring the stability and traceability of microbial resources.

[0035] The 16s rRNA gene is a highly conserved region in the bacterial genome. In a preferred embodiment of the present application, the 16s rRNA gene of the above-mentioned Bacillus velezensis has the nucleotide sequence shown in SEQ ID NO: 1. The strain with the above-mentioned 16s rRNA gene, for example, the Bacillus velezensis LW56-4 of the present application, has both broad-spectrum antibacterial function and promoting feed digestion function.

[0036] The gyrB gene has higher variability in different bacterial genera and species, which can provide higher resolution for distinguishing between closely related species or different strains within the same species. In a preferred embodiment of the present application, the gyrB gene of the above-mentioned B. velezensis has the nucleotide sequence shown in SEQ ID NO: 2. After the strain or metabolite thereof with the above-mentioned gyrB gene is ingested by animals, it can effectively reduce animal diarrhea, improve animal feed conversion rate, promote animal growth, and improve animal antioxidant performance.

[0037] In a second typical embodiment of the present application, an antibacterial agent is provided, which comprises the above-mentioned B. velezensis or the above-mentioned fermentation broth of B. velezensis or the above-mentioned metabolite of B. velezensis. The antibacterial agent of the present application has a broad spectrum of antibacterial activity, and is intended to overcome the problems of drug resistance, residues and intestinal flora disorders caused by traditional antibiotics, and is expected to be developed into an ideal substitute for antibiotics such as aureomycin.

[0038] The above-mentioned antibacterial agent can not only effectively inhibit a series of important pathogenic bacteria, including but not limited to Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Streptococcus iniae, but also exhibits good compatibility and safety during use, and is suitable for various animal breeding environments. In a preferred embodiment of the present application, the above-mentioned antibacterial agent can inhibit any one or more of the following pathogenic bacteria: Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium or Streptococcus iniae.

[0039] Among them, metabolites refer to a series of chemical substances produced, secreted or released into the environment by B. velezensis LW56-4 through its own metabolic mechanism during growth, reproduction and life activities. Fermentation broth refers to a mixed liquid system containing bacterial cells, metabolites and unconsumed medium components obtained after B. velezensis LW56-4 grows, reproduces and metabolizes in a liquid medium under artificial or industrial control conditions (such as a fermentation tank) for a period of time.

[0040] In a third typical embodiment of the present application, a microecological preparation is provided, which comprises the above-mentioned B. velezensis or the above-mentioned fermentation broth of B. velezensis or the above-mentioned metabolite of B. velezensis or the above-mentioned antibacterial agent. The present application further integrates B. velezensis LW56-4 and its fermentation broth and metabolites into a 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 application, a feed additive is provided, which comprises the above-mentioned Bacillus velezensis or the above-mentioned fermentation broth of Bacillus velezensis or the above-mentioned metabolite of Bacillus velezensis or the above-mentioned antibacterial agent or the above-mentioned microecological preparation.

[0042] The Bacillus velezensis LW56-4 and its derivatives (e.g. fermentation broth or metabolite) can also be used as a feed additive, which is added to the feed as a natural bioactive agent, to optimize the feed formula, to improve the feed conversion rate, and thus to promote the healthy growth of animals, while reducing environmental pollution and improving animal welfare.

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

[0044] The use of the Bacillus velezensis LW56-4, the antibacterial agent, the microecological preparation or the feed additive of the present application in animal breeding covers multiple dimensions of health care measures. They not only can prevent the occurrence of animal diarrhea, improve the growth rate, and optimize the function of the digestive system, but also can enhance the antioxidant capacity of animals, resist the pressure of external environment and the influence of internal diseases, so as to comprehensively improve the health status of animals, and improve the breeding efficiency and economic benefits.

[0045] In a preferred embodiment of the present application, the above-mentioned use is 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 use of the Bacillus velezensis LW56-4 and its derivative products of the present application in animal breeding covers a wide range of target animal groups, including but not limited to ruminants (such as cattle, sheep), pigs, poultry (such as broilers, laying hens), pets (such as dogs, cats). This means that the present application has broad application prospects in the livestock industry, breeding industry and pet health care industry. In a preferred embodiment of the present application, the above-mentioned animal is selected from any one or more of the following: ruminants, pigs, broilers, laying hens, cats or dogs.

[0047] The present application will be further described in detail below in conjunction with specific examples, which should not be understood as limiting the scope of the present application.

[0048] Example 1 Isolation and identification of Bacillus velezensis strain LW56-4

[0049] 1.1 Isolation and purification of the strain

[0050] Take healthy lake sheep rumen content (from Yichang Angel Biotechnology Co., Ltd. sheep breeding farm) in sterile saline, using vortex instrument vortex mix well, 85℃ water bath 10 min, 10 times gradient dilution, coating in LB agar medium, 37℃ culture 12h-14h, pick up the single colony with bacillus characteristics on LB agar medium for streak 37℃ subculture 12h, pick up single colony on LB agar medium for streak, 37℃ culture 12h, until the colony morphology is single in LB agar medium, obtain the purified single colony. Pick up different purified single colonies inoculated in LB liquid medium, 37℃, 200r / min shaking culture 24h to obtain the fermentation broth.

[0051] 1.2 Screening of strains with antibacterial activity

[0052] The indicator bacteria (Escherichia coli ATCC25922, Salmonella typhimurium ATCC14028, Clostridium perfringens CVCC2030, Staphylococcus aureus ATCC6538, Streptococcus iniae ATCC29178, the above strains are purchased from China General Microbiological Culture Collection Center) were coated on LB agar medium with sterile cotton swab, and sterile Oxford cups were placed at fixed positions in the medium. 100µL of indicator bacteria were added and poured into the LB agar base medium, which was cooled to solidification at room temperature. The Oxford cups were gently pulled out using sterile hemostatic forceps, and the supernatant obtained by centrifuging the fermentation broth obtained in step 1.1 at 8000r / min for 10min was added to the wells at 100µL / well. The culture was incubated at 37℃ for 16h in an incubator, and sterile PBS solution was used as a negative control. The diameter of the antibacterial circle was measured. Six strains of bacillus with good antibacterial effect were obtained, among which the strain named LW56-4 had the best antibacterial effect, and the antibacterial results are shown in Table 1.

[0053] Table 1 Diameter of antibacterial circle of strain LW56-4

[0054]

[0055] 1.3 Strain identification

[0056] The strain LW56-4 that produced an antibacterial circle after preliminary screening was cultured at 37℃ for 2d on NA medium plates, and its colony morphology and color were observed. It grew well on NA medium, with a cream-colored, flat colony with a flame mountain-shaped ridge in the middle, an irregular edge, no pigment production, and dryness (see Figure 2 ); Gram staining was purple, which was a Gram-positive bacterium; spore staining showed spores.

[0057] The whole genome DNA of the target strain was extracted by using bacterial genome DNA kit, and the 16S rDNA gene universal primers 27F and 1492R and housekeeping gene (gyrB) primers gyrB-f and gyrB-r were used for PCR amplification. After the amplification product was sequenced, bioinformatics analysis was performed. The 16S rDNA gene sequence of the LW56-4 strain obtained by sequencing is shown as SEQ ID NO: 1 (1443 bp):

[0058]

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

[0060]

[0061] The results of BLAST analysis with related sequences in the NCBI database showed that the 16S rDNA sequence of the LW56-4 strain had 100% similarity with strains of the Bacillus genus, and the housekeeping gene gyrB reached the species determination standard with B. velezensis. The gyrB sequence of the LW56-4 strain was subjected to BLAST comparison with known sequences in the NCBI, and the sequence was downloaded, and tools such as MEGA were used for multiple sequence alignment and construction of a phylogenetic tree (see Figure 1 ). Therefore, based on the above morphological and homology analysis results of the 16S rRNA sequence and the gyrB gene of the LW56-4 strain and related strains, the LW56-4 strain was identified as B. velezensis.

[0062] Example 2 Artificial rumen simulation digestion test

[0063] The obtained strain LW56-4 of Example 1.1 was evaluated together with other 5 screened bacteriostatic Bacillus samples.

[0064] Measurement procedure: An AGRS-III in vitro fermentation system (Beijing Boxingxingwang Technology Co., Ltd., AGRS-III) - artificial rumen was used, and the fermentation bottle specification was 120 mL of a Henry anaerobic fermentation bottle (containing a specially designed screw cap and a white chitin plug). Each bottle was added with a Bacillus fermentation liquid to be tested (the viable cell count of the Bacillus to be tested was 2 x 10 8 CFU / mL), and the addition amount was 0.5% (volume of fermentation liquid / volume of total fermentation bottle liquid, ml / ml). It should be noted that the fermentation method of each strain was the same as that of Example 1.1.

[0065] Rumen fluid sampling was selected from fresh rumen fluid of a 200 kg or so fattening cow (purchased from the Three Gorges Livestock Industry Park), and the rumen fluid was weighed, and an anaerobic sterile physiological saline solution with a temperature of 39°C was added in an anaerobic condition according to a weight to volume ratio of 1:5. After stirring uniformly and thoroughly mixing, the filtered rumen fluid was obtained.

[0066] Inoculation: 50 mL of buffer solution + 25 mL of rumen fluid was weighed for each fermentation bottle. Each bottle was added with a Bacillus fermentation liquid to be tested, and the addition amount was 0.5%. Each group was added with 0.5 g of an in vitro test culture substrate, and 5 parallels were set for each sample. The composition of the substrate referred to the basic daily ration of high-yield dairy cows, and the specific composition and nutritional level were shown in Table 2, and the formula of the buffer solution was shown in Table 3.

[0067] After connecting the fermentation system through an intravenous infusion needle, fermentation was carried out at 39°C for 48 h, and the total gas production was automatically recorded.

[0068] The premix in Table 2 is formulated as follows: 240000 IU vitamin A, 370000 IU vitamin D, 1100 mg vitamin E, 270 mg Fe, 370 mg Cu, 1900 mg Zn, 1800 mg Mn, 16 mg I, 17 mg Co, 11 mg Se per kg of premix.

[0069] Table 2 Composition of in vitro fermentation substrate

[0070]

[0071] The formulation of the above buffer solution is as follows: according to the needs of the test design, the buffer solution is prepared by referring 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, 10.0 g MnCl2 4H2O, 1.0 g CoCl2 6H2O, 6.78 g FeCl2 4H2O, distilled water to 100 mL; artificial saliva B includes 35 g NaHCO3, 4 g NH4HCO3, distilled water to 1000 mL; macroelement solution C includes 5.7 g Na2HPO4, 6.2 g KH2PO4, 0.6 g MgSO4 7H2O, distilled water to 100 mL; 0.1% (w / v) resazurin solution D: weigh 100 mg resazurin, and make up to 100 mL; reducing agent solution: weigh 625 mg Na2S 9H2O, add 4.0 mL of 1.0 M NaOH solution, and make up to 100 mL with distilled water.

[0072] Table 3 Buffer formulation

[0073]

[0074] After fermentation, the ice bath was used to terminate fermentation. TVFA (volatile fatty acid) was determined according to the method in T / NAIA 005-2020: 1 mL of fermentation liquid was taken after fermentation, 250 µL of metaphosphoric acid was added, 4°C incubation for 10 min, 15000 x g centrifugation for 10 min, and the supernatant was determined by 0.22 µm filter membrane and gas chromatograph to determine the TVFA (volatile fatty acid) concentration. The dry matter digestion rate was calculated by the difference method before and after fermentation. The dry matter digestion rate and TVFA concentration are shown in Table 4.

[0075] Table 4 Dry matter digestion rate and TVFA concentration

[0076]

[0077] As shown in Table 4, the Bacillus velezensis LW56-4 of the present application can significantly improve the dry matter digestibility, and is higher than other strains. Higher dry matter digestibility indicates that the Bacillus velezensis LW56-4 of the present application can promote animal digestion and improve the utilization rate of feed, thereby further promoting animal growth and saving the use of feed.

[0078] The Bacillus velezensis LW56-4 strain of the present application can significantly improve the concentration of butyric acid, isobutyric acid and branched-chain fatty acid in the fermentation system. Butyric acid, isobutyric acid and branched-chain fatty acid are the main energy supply substances for ruminants, and their increase indicates that the feed conversion rate is improved and the energy supply substance is increased, which is helpful to promote the growth of ruminants.

[0079] Example 3:

[0080] The present embodiment provides a fermentation culture method of the Bacillus velezensis LW56-4, wherein the fermentation scale is a 100L fermenter.

[0081] 3.1 Preparation of seed liquid

[0082] A single colony of the Bacillus velezensis LW56-4 strain cultured in NA medium for 14h was picked and inoculated in LB liquid medium, and then cultured at 37℃ and 180r / min for 16h to obtain the Bacillus velezensis LW56-4 seed liquid, and the bacterial activity was 5×10 8 CFU / mL.

[0083] 3.2 Fermentation process

[0084] The seed liquid of the above Bacillus subtilis was inoculated into the fermentation medium for fermentation culture, the culture temperature was 37℃, the dissolved oxygen was not less than 20%, and the culture was carried out until the spore formation rate was more than 90%;

[0085] The fermentation medium includes, in terms of mass fraction, 30-50 parts of soybean meal powder, 3-5 parts of glucose, 20-35 parts of corn starch, 3-6 parts of yeast powder, 4-6 parts of peptone, 1-5 parts of calcium carbonate, 1-5 parts of sodium chloride, 0.5-1 part of magnesium sulfate, 1-5 parts of dipotassium hydrogen phosphate, and 0.1-0.3 parts of manganese sulfate, and 0.2 parts of polyoxypropylene glycerol.

[0086] During the fermentation culture process, when the spore formation rate (using malachite green staining method) does not reach 60%, the culture temperature is 37℃; when the spore formation rate reaches 70%, the culture temperature is reduced to 32℃.

[0087] In this embodiment, the fermentation broth of the lower tank was diluted and coated on NA medium plates, and the number of bacteria in the fermentation broth of the lower tank was 15 billion CFU / mL and the spore concentration was 14.5 billion CFU / mL after overnight culture at 37°C.

[0088] Example 4: Weaning lake sheep breeding test

[0089] This test adopts a single factor randomized block design scheme, selects 60 healthy weaned female lake sheep (from Yichang Angel Biotechnology Co., Ltd.) with similar age and weight of 13.5±1 kg, and randomly divides them into 3 groups, 5 replicates in each group, and 4 in each replicate. The specific design is shown in Table 5, wherein the formal test period is 30 days. During the test period, the lake sheep diarrhea rate, feed intake and daily gain are counted. After the test, 7 samples are selected from each group for slaughter, and the rumen and intestinal tissues are fixed with formaldehyde and stained with HE (see Figure 3 ), and the development of the villi is analyzed. The rumen fluid is taken to detect the change of SCFAs content in the rumen fluid; the serum is taken to detect the serum biochemical index. The feces are taken to detect the neutral detergent fiber and crude protein digestibility in the feces by acid-insoluble ash method. The specific animal test grouping scheme is shown in Table 5 (T in Table 5 represents ton). The average daily gain (ADG) and feed to gain ratio (FG, which represents the ratio of animal intake to body weight, reflecting the feed conversion level) of weaned lake sheep are shown in Table 6. The diarrhea rate of weaned lake sheep is shown in Table 7. The analysis of serum biochemical index of weaned lake sheep is shown in Table 8. The analysis of short-chain fatty acid content in the rumen fluid of weaned lake sheep is shown in Table 9. The analysis of rumen and intestinal villus length of weaned lake sheep is shown in Table 10. The neutral detergent fiber and crude protein digestibility in the feces of weaned lake sheep are shown in Table 11.

[0090] Table 5 Test grouping scheme of weaned lake sheep

[0091]

[0092] Table 6 Average daily gain (ADG) and feed to gain ratio (FG) of weaned lake sheep

[0093]

[0094] Note: a-c same row with different notations means significant difference (P<0.05). No notations in the table means no significant difference between the three groups in the same row; different lower case letters means significant difference (P<0.05) between the two; capital letters means extremely significant difference (P<0.01).

[0095] “ADG14” and “ADG28” in Table 6 respectively represent the average daily gain of the animal in the 14-day period and in the 28-day period. “FG14” and “FG28” respectively represent the feed to gain ratio of the animal in the 14-day period and in the 28-day period.

[0096] As shown in Table 6, compared with the blank group, the microecological preparation prepared from the Bacillus velezensis LW56-4 of the application can significantly improve the average daily gain of the weaned Hu sheep fed for 14 and 28 days, and the effect has no significant difference with aureomycin, indicating that the microecological preparation can effectively replace aureomycin to promote the growth of weaned Hu sheep.

[0097] Table 7 Diarrhea rate of weaned Hu sheep

[0098]

[0099] As shown in Table 7, the microecological preparation prepared from the Bacillus velezensis LW56-4 of the application can effectively reduce the diarrhea rate of weaned Hu sheep.

[0100] Table 8 Analysis of serum biochemical indexes of weaned Hu sheep

[0101]

[0102] Note: a-b, different in the same row, indicates significant difference (P<0.05).

[0103] As shown in Table 8, the microecological preparation prepared from the Bacillus velezensis LW56-4 of the application can effectively improve the level of superoxide dismutase in serum of weaned Hu sheep, indicating that the microecological preparation 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 Hu sheep

[0105]

[0106] As shown in Table 9, the microecological preparation prepared from the Bacillus velezensis LW56-4 of the application can effectively improve the levels of acetic acid, propionic acid and butyric acid in the rumen of weaned Hu sheep. In the research of ruminant nutrition, the correlation between the generation amount of short-chain fatty acids and the feed digestion efficiency and animal production performance is often concerned. The increase of the index means that the feed digestion efficiency is improved, which ultimately helps to promote the growth of animals.

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

[0108]

[0109] Note: a-b, different in the same row, indicates significant difference (P<0.05).

[0110] As shown in Table 10, the microecological preparation prepared from the Bacillus velezensis LW56-4 of the application can effectively promote the development of rumen and intestinal villi of weaned Hu sheep, which helps to promote the digestion of animals, and thus further promotes the growth of animals.

[0111] Table 11 Digestibility of neutral detergent fiber and crude protein in feces of weaned Hu sheep

[0112]

[0113] Note: a-b The same column shoulder mark different means significant difference (P < 0.05).

[0114] As shown in Table 11, the microecological preparation prepared from the bacillus velezensis LW56-4 of the present application can effectively promote the Hu sheep to digest the neutral detergent fiber and crude protein, which is consistent with the results of increasing daily weight gain and the like.

[0115] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: the bacillus velezensis LW56-4 provided by the present application has broad-spectrum antibacterial properties, can reduce animal diarrhea, promote the development of the gastrointestinal tract of animals, and improve the antioxidant capacity, and can also effectively promote the conversion of feed (especially fiber roughage) and promote the growth of animals.

[0116] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A Bacillus velezensis, characterized in that, The bacillus velezensis is bacillus velezensis LW56-4, the classification name is Bacillus velezensis , and was preserved in the China Center for Type Culture Collection on August 22, 2025, the preservation address is Wuhan, Wuhan University, China, and the preservation number is CCTCC NO: M 20251875.

2. The B. velezensis according to claim 1, characterized in that, The 16s rRNA gene of the Bacillus velezensis has the nucleotide sequence shown in SEQ ID NO:

1.

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

2.

4. An antibacterial agent, characterized by, The antibacterial agent comprises the Bacillus velezensis of any one of claims 1-3 or the fermentation broth of the Bacillus velezensis of any one of claims 1-3 or the metabolite of the Bacillus velezensis of any one of claims 1-3.

5. The antimicrobial agent of claim 4, wherein, The antibacterial agent is capable of inhibiting any one or more of the following pathogenic bacteria: Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium or Streptococcus iniae.

6. A microecological preparation, characterized by, The microecological preparation comprises the Bacillus velezensis of any one of claims 1-3 or the fermentation broth of the Bacillus velezensis of any one of claims 1-3 or the metabolite of the Bacillus velezensis of any one of claims 1-3 or the antibacterial agent of claim 4 or 5.

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

8. Use of the Bacillus velezensis 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 animal breeding.

9. Use according to claim 8, characterized in that, The use is selected from any one or more of the following: preventing diarrhea of animals, promoting growth of animals, promoting digestion of animals or improving antioxidant capacity of animals.

10. Use according to claim 8 or 9, 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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