Bacillus subtilis for preventing and treating pet bacterial diarrhea and application thereof

Abstract

The present application relates to the field of functional microorganism screening and application technology, and particularly relates to a bacillus subtilis for preventing and treating pet bacterial diarrhea and application thereof. Bacillus subtilis The preservation number of the bacillus subtilis is CCTCC NO: M20231592, has strong acid resistance and bile salt resistance characteristics, can effectively inhibit common pathogenic bacteria causing bacterial diarrhea, significantly reduces the pet diarrhea rate, and can be widely applied to the production of pet food or medicine.

Description

Technical Field

[0001] This invention relates to the field of functional microbial screening and application technology, specifically to a Bacillus subtilis strain for the prevention and treatment of bacterial diarrhea in pets and its application. Background Technology

[0002] The pet's gut is a vital habitat for bacteria, boasting a rich diversity of microorganisms. Improper pet care and management can easily lead to bacterial enteritis. Mild cases of bacterial enteritis present with damp, hot diarrhea, loss of appetite, and loose stools, while severe cases can result in severe diarrhea, lethargy, and vomiting in puppies and kittens. Bacterial enteritis can also disrupt a pet's metabolism, making them more susceptible to secondary infections with highly pathogenic viruses (such as canine distemper virus and coronavirus), significantly increasing the risk of death. The bacteria that cause bacterial enteritis in pets are mostly opportunistic pathogens, primarily opportunistic Escherichia coli and Salmonella. When a pet is healthy, these pathogens are naturally eliminated by the body without causing illness. However, when a pet's immunity is weakened, it is malnourished, or infected with a virus, the original bacteria are difficult to eliminate, increasing the pet's chances of contracting bacterial enteritis.

[0003] Probiotics are defined as "live microorganisms that, when administered in a certain dose, are beneficial to the health of the host." Current research indicates that probiotics can compete with pathogens for nutrients and adhesion sites, improving the structure and function of the gut microbiota; promote the synthesis and secretion of digestive enzymes, enhancing the body's digestion, absorption, and utilization of nutrients; furthermore, they can strengthen the liver's function in nutrient metabolism; promote intestinal peristalsis and relieve constipation; improve intestinal mucosal structure and enhance physical barrier function; improve the body's immune function and prevent disease and death in animals; enhance stress resistance; reduce cell apoptosis; and promote intestinal mucosal repair. Studies in pet cats have shown that adding 5×10 [units of something] to their diet... 9 CFU / kg Lactobacillus acidophilus D2 / CSL can increase the number of lactobacilli and decrease the number of Escherichia coli in the feces of pet cats. Adding 2.1×10 9 CFU / kg Enterococcus faecalis SF68 can reduce the incidence of diarrhea in cats.

[0004] Lactobacillus is the most widely studied class of pet-derived probiotics, and it is a resident flora in humans and animals. Of the 34 species that can be used as feed additives, 22 are lactobacillus. Studies have found that the extracellular polysaccharides produced by lactobacillus have various biological effects, including promoting nutrient absorption, improving immunity, and maintaining intestinal flora balance. Bacillus is a Gram-positive bacterium and one of the probiotic species that can be directly fed to animals. In the intestine, Bacillus mainly maintains intestinal ecological balance by biologically depleting oxygen. After a short period of colonization, it can consume a large amount of oxygen to maintain an anaerobic environment and enhance the intestine's resistance to anaerobic bacteria. Compared with probiotic products made from other microorganisms, Bacillus can form spores under unfavorable conditions, and is resistant to acid, salt, high temperatures, and compression. It has high stability during feed processing and can be stored for a long time. Furthermore, Bacillus metabolites contain strong digestive enzyme activity and polypeptides that antagonize intestinal pathogens. Bacillus is frequently used in the selection of strains for microecological preparations.

[0005] Probiotic preparations are becoming an important alternative to growth-promoting antibiotics. With the continuous expansion of the pet industry, pet nutrition is receiving increasing attention. Numerous studies both domestically and internationally have confirmed that probiotics are effective in improving gastrointestinal function in dogs and cats, enhancing immunity, preventing oral diseases, and alleviating obesity. Therefore, developing probiotic preparations will be a key focus for the future development of the pet health industry. Summary of the Invention

[0006] The purpose of this invention is to provide a Bacillus subtilis strain for preventing and treating bacterial diarrhea in pets and its applications. The Bacillus subtilis strain exhibits strong acid and bile salt resistance, effectively inhibiting common pathogens that cause bacterial diarrhea, significantly reducing the rate of diarrhea in pets, and can be widely used in the production of pet food or pharmaceuticals.

[0007] In one aspect, this invention provides a Bacillus subtilis strain, named Bacillus subtilis BS11, which was deposited on September 4, 2023, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231592.

[0008] This invention provides, in one aspect, the application of the Bacillus subtilis BS11 strain in pet food production.

[0009] This invention provides, in one aspect, the application of the Bacillus subtilis BS11 strain in the production of pet medicines.

[0010] The medicine in question is a medicine that has the function of preventing or treating diarrhea in pets.

[0011] The present invention also provides a probiotic preparation comprising the above-mentioned Bacillus subtilis BS11 strain.

[0012] The probiotic preparation further comprises any one or more combinations of Bacillus licheniformis, Bacillus coagulans, Enterococcus faecalis, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium thermophilum, Enterococcus faecalis, Pediococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and Clostridium butyricum.

[0013] The probiotic preparation contains at least 10 live bacteria of Bacillus subtilis strain BS11. 8 CFU / g.

[0014] The present invention also provides the application of the probiotic preparation in the production of pet food.

[0015] The present invention also provides the application of the probiotic preparation in the production of pet medicines.

[0016] The Bacillus subtilis BS11 provided by this invention is sensitive to common antibiotics, exhibits no drug resistance, and has good safety. It has strong acid and bile salt resistance; after treatment at pH 2.0 for 2 hours, the survival rate reaches 33.0%, and after treatment at a bile salt concentration of 0.5% for 2 hours, the survival rate reaches 67.8%. It has high protease production capacity, with a hydrolytic transparent zone diameter of up to 23 mm and a protease activity of 800 U / ml in the fermentation supernatant. It has significant deodorization effects, achieving a removal rate of 80.1% for ammonia and 60% for hydrogen sulfide.

[0017] Bacillus subtilis BS11 exhibits significant inhibitory effects against common pathogens causing bacterial diarrhea in cats, with the strongest inhibitory effects against Escherichia coli, Clostridium perfringens, and Staphylococcus aureus, showing inhibition zones exceeding 20 mm in diameter. This strain also demonstrates strong resistance to porcine epidemic diarrhea virus, significantly reducing its virulence.

[0018] Bacillus subtilis BS11 can effectively improve the growth performance of pet cats, increasing their food intake by 10%, weight gain by 0.15 kg, and significantly reducing the diarrhea rate, achieving unexpected technical results.

[0019] Bacillus subtilis BS11 can be added alone or in combination with other probiotics and prebiotics. It is widely used in pet food or health products, as well as in medicines for the prevention and treatment of pet diarrhea, and has a promising future. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of strain BS11;

[0021] Figure 2MALDI-TOF-MS protein profile of strain BS11;

[0022] Figure 3 This is the Ribo-Printer genetic fingerprint of strain BS11. Detailed Implementation

[0023] For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on existing technologies, and not be limited to the specific descriptions in the embodiments of the present invention.

[0024] The present invention will be further described below with reference to specific embodiments.

[0025] Example 1: Isolation and Screening of Strains

[0026] 1. Sample source: 10 samples of cat feces

[0027] 2. Strains Isolation and Purification

[0028] Weigh 1g of fecal sample and place it in 9mL of sterile physiological saline. Treat at 30℃ and 200rpm for 10min to obtain a bacterial suspension. Place the bacterial suspension in an 80℃ water bath for 10min, remove and cool rapidly. Dilute the treated bacterial suspension 10-fold to the appropriate concentration. Take 100uL and spread it evenly on a nutrient agar plate. Incubate at 37℃ for 16-24h. Pick different single colonies that have been cultured on the nutrient agar plate and streak them 3-4 times to purify and obtain 50 Bacillus strains, named YB01-YB50.

[0029] 3. Secondary screening of strains

[0030] Several common pathogens causing bacterial diarrhea in cats were selected: Escherichia coli, Salmonella, Clostridium perfringens, and Klebsiella pneumoniae. The inhibitory effect of the isolated spores on the above pathogens was evaluated by the size of the inhibition zone diameter (cm).

[0031] 3.1 Preparation of test bacterial solutions

[0032] The isolated spore-forming bacteria were cultured on plates, and then one inoculation loop was transferred to nutrient broth medium and incubated at 37°C and 220 rpm for 16 h to prepare the test bacterial suspension with a bacterial count of 10. 8 -10 9 CFU / ml available for use.

[0033] 3.2 Preparation of pathogens

[0034] Glycerol tubes containing pathogens such as Escherichia coli, Clostridium perfringens, Salmonella, and Klebsiella pneumoniae were inoculated into nutrient broth medium and cultured at 37°C and 220 rpm for 16-18 hours for later use. Clostridium perfringens was inoculated into nutrient broth medium and cultured anaerobically at 42°C for 24 hours for later use.

[0035] 3.3 Antibacterial test

[0036] Take a sterile Petri dish and pour in 18-20 mL of sterile nutrient agar medium, spreading it evenly. Place the dish on a horizontal plate to solidify, forming the bottom layer. Invert the plate to divide the area into equal sections and label them with the substances to be added. Separately, take an appropriate amount of sterile semi-solid nutrient agar medium (1% agar content) and cool it to 48-50℃. Add 1-2 mL of pathogen suspension to every 50-100 mL of medium. Add 5 mL of semi-solid nutrient agar medium to each Petri dish, spreading it evenly on the bottom layer to form the bacterial layer. After cooling on a horizontal plate, evenly place 4-6 Oxford cups at equal intervals in each Petri dish. Add 0.2 mL of expanded Bacillus spore culture to each Oxford cup in each double-layer Petri dish. Incubate at 37℃ for 24 hours, then measure the diameter of each inhibition zone.

[0037] Comparative analysis showed that strain YB05 had the best overall antibacterial effect, and the specific results are shown in Table 1.

[0038] Table 1. Antibacterial effect of YB05 against pathogens.

[0039] Escherichia coli ATCC 20±1.0 Salmonella ATCC 18±1.0 Clostridium perfringens ATCC 23±1.0 Klebsiella pneumoniae 16±1.0 Staphylococcus aureus 20±1.0

[0040] As shown in Table 1, the YB05 strain screened in this invention has a significant inhibitory effect on common pathogens causing bacterial diarrhea in cats, with the strongest inhibitory effect on Escherichia coli, Clostridium perfringens and Staphylococcus aureus, and the inhibition zone diameter exceeds 20 mm.

[0041] The applicant named the YB05 strain BS11 and conducted further evaluation on it.

[0042] Example 2 Identification of BS11 strain

[0043] 1. Colony morphology identification

[0044] The colony morphology of strain BS11 is as follows: Figure 1 As shown, the colonies are round with regular, slightly raised edges, milky white in color, Gram-positive under an optical microscope, and the bacteria are rod-shaped with spores.

[0045] 2. 16S rRNA molecular identification

[0046] The genome of strain BS11 was extracted using a kit. Then, using this genome as a template, its 16S rRNA was amplified using specific primers 27F and 1492R.

[0047] 27F: 5'-AGAGTTTGATCATGGCTCAG-3';

[0048] 1492R: 5'-TAGGGTTACCTTACGACTT-3'.

[0049] The PCR system consisted of: 0.7 μl 27F, 0.7 μl 1492R, 4 μl template DNA, 17.5 μl SuperMiX, and 12.1 μl water. The PCR reaction conditions were set as follows: (1) 94℃, 5 min; (2) 94℃, pre-denaturation for 30 s; (3) 55℃, 30 s; (4) 72℃, 1 min; repeat steps (2) to (4) for 35 cycles; (5) 72℃, 10 min. The amplified PCR products were detected by 1% agarose gel electrophoresis, and the results showed that the PCR product size met the requirements.

[0050] The PCR amplification product was sent to a sequencing company for sequencing, and the results showed that the 16S rRNA sequence of strain BS11 was EU883786.1. BLAST alignment of this sequence in the NCBI database showed the highest similarity to *Bacillus subtilis*. Therefore, the strain was preliminarily identified as *Bacillus subtilis*.

[0051] 3. Identification using MALDI-TOF-MS spectra

[0052] Take a small amount of BS11 single colony and spread it on the target plate in the form of a thin film; add 1 μL of the lysis buffer from the mass spectrometry sample pretreatment kit and let it air dry at room temperature; add 1 μL of the matrix solution from the mass spectrometry sample pretreatment kit to cover the sample and let it air dry at room temperature; put the sample target into the mass spectrometer for identification.

[0053] The identification results showed that strain BS11 was Bacillus subtilis, and its protein spectrum peak diagram is as follows. Figure 2 As shown.

[0054] 4. Ribo-Printer Fully Automated Microbial Gene Fingerprint Identification

[0055] The BS11 strain was identified using the fully automated microbial gene fingerprinting system according to its operating instructions, and its rRNA gene fingerprint was obtained, as follows: Figure 3 As shown.

[0056] By comparing the fingerprints with those of a known standard strain library, it was found that strain BS11 had a similarity of over 90% with Bacillus subtilis. Therefore, this strain was identified as Bacillus subtilis.

[0057] In summary, the applicant used three molecular biology methods—16S rRNA identification, MALDI-TOF-MS protein proteometry, and Ribo-Printer automated microbial genetic fingerprinting—to identify strain BS11, and the results were consistent. Combined with the colony morphology characteristics of this strain, the applicant identified it as Bacillus subtilis and named it Bacillus subtilis BS11.

[0058] The applicant deposited the above-mentioned Bacillus subtilis BS11 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China on September 4, 2023, with accession number CCTCC NO: M20231592.

[0059] Example 3: Safety evaluation of Bacillus subtilis BS11

[0060] 1. Hemolytic:

[0061] Activated Bacillus subtilis BS11 was inoculated onto blood agar plates and incubated at 37°C for 24 hours. The presence of clear hydrolysis zones around the colonies was then observed. The results showed no hydrolysis zones, indicating that Bacillus subtilis BS11 does not possess hemolytic activity and can be used in pet food.

[0062] 2. Drug resistance:

[0063] Antibiotic resistance in strains can pose safety hazards during production and application. To prevent antibiotic resistance in Bacillus subtilis BS11, its antibiotic susceptibility was studied. The minimum inhibitory concentration (MIC) of Bacillus subtilis BS11 among seven common antibiotics was determined using the CLSI antibiotic susceptibility testing gradient dilution method. Sigma standards were used as antibiotics, and its susceptibility to antibiotics was assessed according to the EFSA (2012) standards. Specific results are shown in Table 2.

[0064] Table 2. Minimum inhibitory concentration (MIC) of antibiotics

[0065] 0.75(S) 0.25(S) 0.25(S) 1(S) 0.75(S) 0.75(S) 2(S)

[0066] Note: Unit is ug / ml. According to the EFSA (2012) standard for evaluating the susceptibility of strains to antibiotics, S indicates susceptibility and R indicates resistance.

[0067] As can be seen from the results in Table 2, the Bacillus subtilis BS11 provided by this invention is sensitive to all seven common antibiotics and does not exhibit resistance, and can be used in pet breeding.

[0068] Example 4: Enzyme Production Characteristics Analysis of Bacillus subtilis BS11

[0069] Protein, as one of the six essential nutrients for dogs and cats, plays a vital role in promoting growth, development, and health. Cats are obligate carnivores and have a high minimum requirement for crude protein. To evaluate the protein utilization ability of Bacillus subtilis BS11, the applicant used a spot inoculation method to evaluate the protease production capacity of this strain and determined its fermentation enzyme activity.

[0070] 1. Preparation of the test bacterial solution

[0071] Bacillus subtilis BS11 was first cultured on a plate. One inoculation loop of the cultured Bacillus subtilis BS11 was then inoculated into LB medium and cultured at 37°C and 220 r / min for 16 h to prepare the test bacterial solution for later use.

[0072] 2. Evaluation of the protease production capacity of Bacillus subtilis BS11

[0073] The cultured Bacillus subtilis BS11 was inoculated onto an agar medium containing skim milk powder and incubated at 37°C for 28 hours. A clear zone was observed around the culture, indicating that Bacillus subtilis BS11 can produce certain proteases.

[0074] Bacillus subtilis BS11 was inoculated at a ratio of 1% into protease-producing liquid medium (0.8g casein, 0.2g Na2HPO4, 0.05g MgSO4, 0.5g NaCl, 0.3g beef extract powder, 1.5g agar, 100mL ultrapure water, pH 7.0) and cultured at 37℃ and 150rpm for 24h. The fermentation broth was then centrifuged at 4500rpm for 20min, and the supernatant was collected for protease activity determination.

[0075] The results showed that the Bacillus subtilis BS11 provided by the present invention has a high protease production capacity, with a hydrolysis clear zone diameter of up to 23 mm and a protease activity of 800 U / ml in the fermentation supernatant.

[0076] Example 5: Analysis of the acid and bile salt resistance of Bacillus subtilis BS11

[0077] 1. Acid resistance test

[0078] The pH of the nutrient broth was adjusted to 2.0, 3.0, and 4.0 respectively using NaOH and HCl at concentrations of 2 mol / L.

[0079] Bacillus subtilis BS11 in the logarithmic growth phase was inoculated at a volume ratio of 10% into nutrient broth media at pH 2.0, pH 3.0, and pH 4.0, respectively. After mixing, the media were placed in a water bath at 37°C and incubated for 2 hours. The culture medium without acid or alkali treatment served as the control group. The viable cell counts before and after treatment were compared by plate counting. Each sample was repeated 3 times.

[0080] 2. Bile salt tolerance test

[0081] The porcine bile salts were diluted to concentrations of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% by weight using nutrient broth medium.

[0082] Bacillus subtilis BS11 in the logarithmic growth phase was inoculated at a volume ratio of 10% into nutrient broth media with bile salt concentrations of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% (w / w). After mixing, the media were placed in a water bath at 37°C and incubated for 2 hours. The untreated medium served as a control group. Viable bacterial counts were compared before and after treatment using plate counting. Each sample was tested in triplicate.

[0083] The above results show that the Bacillus subtilis BS11 provided by the present invention has strong acid resistance and bile salt resistance. After treatment at pH 2.0 for 2 hours, the survival rate reached 33.0%, and after treatment at a bile salt concentration of 0.5% for 2 hours, the survival rate reached 67.8%.

[0084] Example 6: Analysis of the antiviral properties of Bacillus subtilis BS11

[0085] Bacillus subtilis BS11 at a determined safe concentration was mixed with a 6-fold dilution of porcine epidemic diarrhea virus (PEDV) in 1 mL of virus dilution buffer (each mL containing 5 μg trypsin). The mixture was incubated in a CO2 cell culture incubator for 1 hour, with mixing every half hour. Vero cells (24-well plate) that had grown to 80% confluence were washed twice with PBS and once with virus dilution buffer. 500 μL of the incubated sample and PEDV virus mixture was added to each well. Positive and negative control groups were also included. Virus was harvested after 17 hours.

[0086] Collect the virus-harvested samples and determine TCID50. Perform 10-fold serial dilutions of the samples using viral dilution buffer. -1 ~10 -8 Each dilution was performed in quadruplicate. Vero cells (96-well plate) that had grown to 80% confluence were washed twice with PBS, once with viral diluent, and then 100 μL of the diluted sample was added to each well. Positive and negative controls were also included. Cytopathic effects were observed. TCID50 was calculated.

[0087] Table 3 Antiviral properties of Bacillus subtilis BS11

[0088] PEDV (Poisonous Price TCID50) <![CDATA[10 1.67 / mL]]> <![CDATA[10 6.67 / mL]]>

[0089] As shown in Table 3, Bacillus subtilis BS11 has a strong resistance effect against porcine epidemic diarrhea virus and can significantly reduce the virulence of the virus.

[0090] Case Study 6: Deodorization Performance Analysis of Bacillus subtilis BS11

[0091] Take 300g of fresh pig manure and place it in a 1000mL wide-mouth bottle. Inoculate with Bacillus subtilis BS11 bacterial suspension at a 5% inoculation rate and mix thoroughly with the pig manure. Place two 50mL beakers in the wide-mouth bottle, one containing 20mL of 2% boric acid solution and the other containing 20mL of 0.2% zinc ammonium complex salt solution. Seal the wide-mouth bottle with rubber stoppers and cover with double layers of plastic wrap, then incubate at 30℃. Use the uninoculated group as a control, and set up three replicates for each experiment. On the 7th day of incubation, take out the absorbent and measure the release of ammonia and hydrogen sulfide, respectively, and calculate the removal rate of ammonia and hydrogen sulfide by Bacillus subtilis BS11.

[0092] Removal rate = (Release amount in control group - Release amount in experimental group) / Release amount in control group × 100%.

[0093] The results showed that Bacillus subtilis BS11 could remove 80.1% of ammonia and 60% of hydrogen sulfide, achieving unexpected technical results.

[0094] Example 7: Effects of Bacillus subtilis BS11 on growth performance and diarrhea in pet cats.

[0095] 1. Preparation of mycelium powder

[0096] Bacillus subtilis BS11 was liquid fermented in a 5-ton fermenter. Fermentation was stopped when the spore count reached over 90% under microscopic examination. The fermentation broth was then centrifuged and spray-dried to obtain a bacterial powder with a bacterial count of 10 billion / g.

[0097] 2. Test Plan

[0098] (1) Experimental location: Animal room of Qingdao Agricultural University;

[0099] (2) Experimental design: Forty adult Ragdoll cats were selected according to the following criteria: age 1-2 years, average weight 4kg, and randomly divided into 4 groups, with 10 replicates in each group and 1 cat in each replicate, where the ratio of male Ragdoll cats to female Ragdoll cats was 4:6.

[0100] Negative control group: fed antibiotic-free basal diet;

[0101] Experimental group 1: fed with basal diet + 0.5 / (kg BW·d) Bacillus subtilis BS11 bacterial powder;

[0102] Experimental group 2: fed with basal diet + 1.0 g / (kg BW·d) Bacillus subtilis BS11 powder.

[0103] Prior to the experiment, all necessary immunizations and deworming treatments were administered, and the cats had not received any medications (such as antibiotics) that could alter their gut microbiota within one month prior to the start of the experiment. The litter box was cleaned twice daily, morning and evening, and the litter was changed weekly. The cat enclosures were cleaned and disinfected daily to maintain cleanliness, and the cats were played with for at least 30 minutes each day. A fixed amount of food was weighed for each cat at a set time each day. The food bowls were cleaned thoroughly before weighing in the morning. The amount of food given to each cat and the amount remaining the following day were accurately recorded daily. The condition of the feces was observed and recorded during the afternoon litter box cleaning. The cats had free access to water and food throughout the entire experiment.

[0104] (3) Indicator measurement: growth performance, diarrhea rate

[0105] Cats were weighed on an empty stomach one day before and one day after the experiment. Throughout the experiment, food intake was recorded at fixed times each day, and fecal scores (FS) were calculated according to the criteria in Table 4. The number of diarrhea episodes was also recorded. The diarrhea rate and soft stool rate for each group were calculated using the following formulas:

[0106] Diarrhea rate (%) = Number of times the cats experienced diarrhea during the experimental period / Total number of fecal scores of the cats during the experimental period;

[0107] Soft stool rate (%) = Number of times the cats had soft stools during the experimental period / Total number of fecal scores of the cats during the experimental period.

[0108] Table 4. Stool Scoring Criteria

[0109]

[0110] 1≤FS<2 indicates constipation, 2≤FS≤3 indicates normal stool, 3<FS<4 indicates soft stool, and 4≤FS≤5 indicates diarrhea.

[0111] Table 5. Effects of Bacillus subtilis BS11 on body weight and diarrhea rate in pet cats.

[0112]

[0113]

[0114] As shown in Table 5, the Bacillus subtilis BS11 powder provided by this invention can effectively improve the growth performance of pet cats, increasing their feed intake by 10%, increasing their weight by 0.15 kg, and significantly reducing the diarrhea rate, achieving unexpected technical effects.

Claims

1. A Bacillus subtilis strain, characterized in that, The Bacillus subtilis has the accession number CCTCC NO: M20231592.

2. The application of Bacillus subtilis as described in claim 1 in the preparation of pet food.

3. The application of Bacillus subtilis according to claim 1 in the preparation of pet medicines, characterized in that, The medicine in question is a medicine that has the function of preventing or treating diarrhea in cats.

4. A probiotic preparation, characterized in that, The probiotic preparation comprises Bacillus subtilis as described in claim 1.

5. The probiotic preparation as described in claim 4, characterized in that, The probiotic preparation further comprises any one or more combinations of Bacillus licheniformis, Bacillus coagulans, Enterococcus faecalis, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium thermophilum, Enterococcus faecalis, Pediococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and Clostridium butyricum.

6. The probiotic preparation as described in claim 5, characterized in that, The probiotic preparation contains at least 10 live Bacillus subtilis bacteria. 8 CFU / g.

7. The use of the probiotic preparation according to any one of claims 4-6 in the preparation of pet food.

8. The use of the probiotic preparation according to any one of claims 4-6 in the preparation of pet medicines, characterized in that, The medicine in question is a medicine that has the function of preventing or treating diarrhea in cats.

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