Bacillus subtilis for preventing and treating bacterial diarrhea of pets and application of bacillus subtilis

Abstract

The invention relates to the technical field of functional microorganism screening and application, in particular to bacillus subtilis for preventing and treating bacterial diarrhea of pets and application of the bacillus subtilis. The preservation number of the bacillus subtilis is CCTCC (China Center for Type Culture Collection) NO: M20231592, and the bacillus subtilis has relatively strong acid resistance and cholate resistance, can effectively inhibit common pathogenic bacteria causing bacterial diarrhea and remarkably reduce the diarrhea rate of pets, and can be widely applied to production of pet foods or medicines.

Description

Technical Field

[0001] The invention relates to the technical field of functional microorganism screening and application, and in particular to a Bacillus subtilis for preventing and treating bacterial diarrhea in pets and an application thereof. Background Art

[0002] The pet intestine is an important place for bacteria to survive, and the microbial flora is very rich. If the pet is not raised scientifically and managed properly, it is easy for the pet to be infected with bacterial enteritis. Pets infected with bacterial enteritis will have damp-heat diarrhea, loss of appetite, and loose stools in mild cases, severe diarrhea, mental depression in severe cases, and vomiting in young dogs and cats. At the same time, bacterial enteritis can cause metabolic disorders in pets, and it is easy to be infected with highly pathogenic viruses (such as canine distemper virus, coronavirus, etc.), which greatly increases the risk of death for pets. The bacteria that cause bacterial enteritis in pets are mostly conditional pathogens, among which the most important are conditional pathogenic Escherichia coli and Salmonella. When the pet's body is in a healthy state, the pathogenic bacteria will be eliminated spontaneously by the body and will not cause the disease. When the pet's immunity is reduced, nutrition is malnourished, and it is infected with viruses, the original bacteria are difficult to eliminate, thereby increasing the chance of pets being infected with bacterial enteritis.

[0003] Probiotics are defined as "live microorganisms that are beneficial to the health of the host when given in a certain dose." Current studies have shown that probiotics can compete with pathogens for nutrients and adhesion sites, improve the structure and function of intestinal flora; promote the synthesis and secretion of digestive enzymes, improve the body's digestion, absorption and utilization of nutrients; in addition, they can enhance the liver's function of 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 animal disease and death; improve stress resistance, reduce cell apoptosis and promote intestinal mucosal repair. Studies on pet cats have shown that adding 5×10 9 CFU / kg Lactobacillus acidophilus D2 / CSL can increase the number of Lactobacillus and reduce the number of Escherichia coli in pet cat feces. 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 type of pet-derived probiotics and is a resident flora in humans and animals. Of the 34 bacterial species that can be used as feed additives, 22 belong to lactic acid bacteria. Studies have found that the extracellular polysaccharides produced by lactobacilli have a variety of biological effects, which can not only promote the absorption of nutrients and improve the body's immunity, but also maintain the balance of intestinal flora and other probiotic functions. Bacillus is a Gram-positive bacterium and is one of the probiotic species that can be directly fed to animals. Bacillus maintains the ecological balance of the intestine mainly through biological oxygen deprivation in the intestine. After colonizing in the intestine for a short time, it can consume a large amount of oxygen, maintain the anaerobic environment of the intestine, and enhance the colonization resistance of the intestine to anaerobic bacteria. Compared with probiotic products made from other microorganisms, Bacillus can form spores under unfavorable conditions, is acid-resistant, salt-resistant, high-temperature-resistant and extrusion-resistant, has high stability during feed processing, and can be stored for a long time; and the metabolites of Bacillus contain strong digestive enzyme activity and polypeptide substances that antagonize intestinal pathogens. In the selection of microecological preparation strains, Bacillus is used more frequently.

[0005] Probiotic preparations have become an important alternative to growth-promoting antibiotics. With the continuous expansion of the pet industry, pet nutrition has gradually been valued. Many studies at home and abroad have confirmed that probiotics have certain effects in improving the gastrointestinal function of dogs and cats, improving the body's immunity, preventing oral diseases, and improving obesity. Therefore, the development of probiotic preparations will be the focus of the future development of the pet health industry. Summary of the invention

[0006] The present invention aims to provide a Bacillus subtilis for preventing and treating bacterial diarrhea in pets and its application. The Bacillus subtilis has strong acid resistance and bile salt resistance, can effectively inhibit common pathogenic bacteria that cause bacterial diarrhea, significantly reduce the diarrhea rate of pets, and can be widely used in the production of pet food or medicines.

[0007] On the one hand, the present invention provides a Bacillus subtilis, named Bacillus subtilis BS11 strain, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 4, 2023, with a deposit number of CCTCC NO: M20231592.

[0008] In one aspect, the present invention provides the use of the Bacillus subtilis BS11 strain in the production of pet food.

[0009] On the one hand, the present invention provides the use of the Bacillus subtilis BS11 strain in the production of pet medicines.

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

[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 thermophilus, Enterococcus faecium, Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and Clostridium butyricum.

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

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

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

[0016] The Bacillus subtilis BS11 provided by the invention is sensitive to common antibiotics, has no drug resistance and has good safety; has strong acid resistance and bile salt resistance, and has a survival rate of 33.0% after being treated for 2 hours under the condition of pH 2.0, and a survival rate of 67.8% after being treated for 2 hours under the condition of bile salt concentration of 0.5%; has a high protease production ability, a hydrolysis transparent zone diameter of 23 mm, and a protease activity of 800 U / ml in the fermentation supernatant; has a significant deodorization effect, and can achieve a removal rate of 80.1% for ammonia and a removal rate of 60% for hydrogen sulfide.

[0017] Bacillus subtilis BS11 has a significant inhibitory effect on common pathogens that cause bacterial diarrhea in cats, among which Escherichia coli, Clostridium perfringens and Staphylococcus aureus have the strongest inhibitory effect, with an inhibition zone diameter of more than 20mm. This strain has a strong resistance to porcine epidemic diarrhea virus and can significantly reduce the virulence of the virus.

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

[0019] Bacillus subtilis BS11 can be added alone or in combination with other probiotics, prebiotics, etc. It is widely used in pet feed or health products, as well as in medicines for preventing and treating pet diarrhea, and has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2This is the MALDI-TOF-MS protein spectrum of the BS11 strain;

[0022] Figure 3 This is the Ribo-Printer gene fingerprint of the BS11 strain. DETAILED DESCRIPTION

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

[0024] The present invention is further described below in conjunction with specific embodiments.

[0025] Example 1 Isolation and screening of strains

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

[0027] 2. Strain Isolation and Purification

[0028] Weigh 1 g of fecal sample and place it in 9 mL of sterile saline, and treat it at 30°C, 200 rpm for 10 min to obtain a bacterial suspension; place the bacterial suspension in an 80°C water bath, treat it for 10 min, take it out, and cool it quickly; dilute the treated bacterial suspension to an appropriate concentration in a 10-fold gradient, take 100uL on a nutrient agar plate, spread it evenly, and culture it at 37°C for 16-24h; pick different single colonies that have been cultured on the nutrient agar plate, further streak 3-4 times, and purify 50 strains of Bacillus, which were named YB01-YB50 respectively.

[0029] 3. Strain rescreening

[0030] Several common pathogens that cause bacterial diarrhea in cats were selected: Escherichia coli, Salmonella, Clostridium perfringens, and Klebsiella pneumoniae. The antibacterial ability of the isolated spore-forming bacteria against the above pathogens was evaluated by the diameter of the inhibition zone (cm).

[0031] 3.1 Preparation of test bacterial solution

[0032] The isolated spore-forming bacteria were cultured on plates, and then one inoculation loop was inoculated into nutrient broth medium, cultured at 37°C, 220 r / min for 16 h to prepare the test bacterial solution. The bacterial count was 10 8 -10 9 CFU / ml for future use.

[0033] 3.2 Preparation of pathogens

[0034] The pathogenic bacteria such as Escherichia coli, Clostridium perfringens, Salmonella, Klebsiella pneumoniae, etc. were inoculated in glycerol tubes into nutrient broth medium, cultured at 37°C, 220r / min for 16-18h and set aside; Clostridium perfringens was inoculated into nutrient broth medium and cultured anaerobically at 42°C for 24h and set aside.

[0035] 3.3 Antibacterial test

[0036] Take a sterile plate, pour 18-20mL of sterile nutrient agar medium, spread it evenly in the plate, place it on a horizontal surface to solidify, as the bottom layer, turn the plate upside down to evenly divide the area and mark the material to be added. Take an appropriate amount of sterile semi-solid nutrient agar medium (agar content is 1%), cool it to 48-50℃, add 1-2mL of pathogen suspension to every 50-100mL of medium, add 5mL of semi-solid nutrient agar medium to each plate, spread it evenly on the bottom layer, as the bacterial layer. After cooling on a horizontal platform, place 4-6 Oxford cups evenly at equal distances in each plate. Add 0.2mL of the expanded spore-forming bacteria solution to the Oxford cup in each double-layer plate, and measure the diameter of each inhibition zone after culturing at 37℃ for 24h.

[0037] Through comparative analysis, the comprehensive antibacterial effect of strain YB05 was the best. The specific results are shown in Table 1.

[0038] Table 1 Antibacterial effect of YB05 on pathogenic bacteria

[0039] Pathogens Diameter of inhibition zone mm 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] From the data in Table 1, it can be seen that the YB05 strain screened by the present invention has a significant inhibitory effect on common pathogenic bacteria that cause bacterial diarrhea in cats, among which the inhibitory effect on Escherichia coli, Clostridium perfringens and Staphylococcus aureus is the strongest, and the diameter of the inhibition zone 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 BS11 strain is as follows Figure 1 As shown, the colonies are round, with regular edges, slightly raised, milky white, Gram-positive under an optical microscope, and the bacteria are rod-shaped and have spores.

[0045] 2. 16S rRNA molecular identification

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

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

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

[0049] The PCR system included: 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°C, 5 min; (2) 94°C, pre-denaturation for 30 s; (3) 55°C, 30 s; (4) 72°C, 1 min; perform steps (2) to (4) 35 cycles; (5) 72°C, 10 min. The amplified PCR product was detected by 1% agarose gel electrophoresis, and the results showed that the size of the PCR product 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 the BS11 strain was EU883786.1. The sequence was compared by BLAST in the NCBI database, and it had the highest similarity with Bacillus subtilis. Therefore, the strain was preliminarily determined to be Bacillus subtilis.

[0051] 3. MALDI-TOF-MS spectrum identification

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

[0053] The identification results showed that the BS11 strain was Bacillus subtilis. Its protein spectrum peaks were as follows: Figure 2 shown.

[0054] 4. Ribo-Printer fully automatic microbial gene fingerprint identification

[0055] According to the operating instructions of the fully automatic microbial gene fingerprint identification system, the BS11 strain was identified on the machine and its rRNA gene fingerprint was obtained. Figure 3 shown.

[0056] By comparing the fingerprints with those of the known standard strain library, it was found that the similarity between the BS11 strain and Bacillus subtilis was as high as more than 90%, so the strain was identified as Bacillus subtilis.

[0057] In summary, the applicant used three molecular biological methods, namely 16S rRNA identification, MALDI-TOF-MS protein spectrum identification, and Ribo-Printer fully automatic microbial gene fingerprint identification, to identify the BS11 strain, and the identification results were consistent. Combined with the colony morphology characteristics of the strain, the applicant determined that it was Bacillus subtilis and named it Bacillus subtilis BS11.

[0058] The applicant deposited the above-mentioned Bacillus subtilis BS11 (Bacillus subtilis BS11) with the China Type Culture Collection of Wuhan University, Wuhan, China on September 4, 2023, with the deposit number CCTCC NO: M20231592.

[0059] Example 3 Safety evaluation of Bacillus subtilis BS11

[0060] 1. Hemolytic:

[0061] The activated Bacillus subtilis BS11 was spotted onto the blood plate and cultured at 37°C for 24 hours to observe whether a transparent hydrolysis zone was produced around the colony. The results showed that no hydrolysis zone was produced, indicating that Bacillus subtilis BS11 was not hemolytic and could be used in pet feed.

[0062] 2. Drug resistance:

[0063] The drug resistance of strains may lead to safety hazards in the production and application process. In order to prevent the existence of drug resistance in Bacillus subtilis BS11, its antibiotic sensitivity was studied. The minimum inhibitory concentration (MIC value) of Bacillus subtilis BS11 in 7 common antibiotics was determined according to the CLSI antibiotic sensitivity test gradient dilution method. The antibiotics used were Sigma standards, and the sensitivity of Bacillus subtilis BS11 to antibiotics was evaluated in accordance with the EFSA (2012) standard. The specific results are shown in Table 2.

[0064] Table 2 Minimum inhibitory concentration (MIC value) in antibiotics

[0065] penicillin Clindamycin Gentamycin Norfloxacin Ampicillin Vancomycin tetracycline 0.75(S) 0.25(S) 0.25(S) 1(S) 0.75(S) 0.75(S) 2(S)

[0066] Note: The unit is ug / ml. According to the EFSA (2012) standard for evaluating the sensitivity of strains to antibiotics, S indicates sensitive and R indicates resistant.

[0067] It can be seen from the results in Table 2 that the Bacillus subtilis BS11 provided by the present invention is sensitive to 7 common antibiotics and has no drug resistance, and can be used in pet breeding.

[0068] Example 4 Analysis of enzyme production characteristics of Bacillus subtilis BS11

[0069] Protein, as one of the six major nutrients for dogs and cats, plays an important role in promoting the growth, development and health of the body. Cats are obligate carnivores and have a high demand for the minimum crude protein. In order to evaluate the ability of Bacillus subtilis BS11 to utilize protein, the applicant used the spot inoculation method to evaluate the protease production ability of the strain and measured its fermentation enzyme activity.

[0070] 1. Preparation of test bacterial solution

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

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

[0073] The cultured Bacillus subtilis BS11 was inoculated onto an agar medium containing skimmed milk powder and cultured at 37°C for 28 hours. A transparent circle was observed around the culture solution, indicating that Bacillus subtilis BS11 could produce a certain amount of protease.

[0074] Bacillus subtilis BS11 was inoculated into a protease enzyme production liquid medium (casein 0.8g, Na2HPO4 0.2g, MgSO4 0.05g, NaCl 0.5g, beef extract powder 0.3g, agar 1.5g, ultrapure water 100mL, pH7.0) at a ratio of 1%, and cultured at 37°C, 150rpm for 24h. The fermentation broth was then centrifuged at 4500rpm for 20min, and the supernatant was taken for protease enzyme activity determination.

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

[0076] Example 5 Analysis of acid and bile 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 using 2 mol / L NaOH and HCl, respectively.

[0079] Bacillus subtilis BS11 in the logarithmic growth phase was inoculated into nutrient broth medium at pH 2.0, pH 3.0, and pH 4.0 at a volume ratio of 10%, mixed and placed in a 37°C water bath for 2 hours. The culture medium that had not been treated with acid or alkali was used as the control group, and the number of viable bacteria before and after was compared by plate counting. Each sample was repeated 3 times.

[0080] 2. Bile salt tolerance test

[0081] The pig bile salt was diluted with nutrient broth culture medium to a mass percentage concentration of 0.1%, 0.2%, 0.3%, 0.4% and 0.5% respectively.

[0082] Bacillus subtilis BS11 in the logarithmic growth phase was inoculated into nutrient broth medium with a mass percentage concentration of bile salt of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% at a volume ratio of 10%, mixed and placed in a 37°C water bath for 2 hours. The culture medium not treated with bile salt was used as the control group, and the number of viable bacteria before and after was compared by plate counting, and each sample was repeated 3 times.

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

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

[0085] Bacillus subtilis BS11 was mixed with 6-fold diluted porcine epidemic diarrhea virus (PEDV) at a safe concentration and added to 1 mL of virus dilution solution (containing 5 μg of trypsin per mL) and placed in a CO 2 Incubate in a cell culture incubator for 1 hour and mix once every half an hour. Wash Vero cells (24-well plate) grown to 80% twice with PBS and once with virus diluent, add 500 μL of the incubated sample and PEDV virus mixture to each well, set up a positive and negative control group, and collect the virus after 17 hours.

[0086] Take the collected samples to determine TCID50. Use virus diluent to dilute the samples 10 times. -1 ~10 -8 4 replicates were made for each dilution. Vero cells (96-well plate) grown to 80% were washed twice with PBS and once with virus dilution solution, and then 100 μL of diluted sample was added to each well. At the same time, positive and negative controls were set up. The cytopathic effect was observed. TCID50 was calculated.

[0087] Table 3 Antiviral properties of Bacillus subtilis BS11

[0088] Bacillus subtilis BS11 Positive control group PEDV toxicity TCID50 <![CDATA[10 1.67 / mL]]> <![CDATA[10 6.67 / mL]]>

[0089] From the data in Table 3, it can be seen that Bacillus subtilis BS11 has a strong resistance to porcine epidemic diarrhea virus and can significantly reduce the virulence of the virus.

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

[0091] Take 300g of fresh pig manure and put it into a 1000mL wide-mouth bottle. Inoculate Bacillus subtilis BS11 at a 5% inoculation rate and stir it evenly with the pig manure. Place 2 50mL beakers in the wide-mouth bottle, each containing 20mL of 2% boric acid solution and 20mL of 0.2% zinc ammonium complex salt solution. The wide-mouth bottle is sealed with a rubber stopper and covered with a double layer of plastic wrap and then cultured at 30°C. The non-inoculated group was used as the control, and 3 replicates were set for each group of experiments. Take out the absorption liquid on the 7th day of culture, measure the release of ammonia and hydrogen sulfide, and calculate the removal rate of ammonia and hydrogen sulfide by Bacillus subtilis BS11.

[0092] Removal rate = (release amount of control group - release amount of test group) / release amount of control group × 100%.

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

[0094] Example 7 Effect of Bacillus subtilis BS11 on growth performance and diarrhea of ​​pet cats

[0095] 1. Preparation of bacterial powder

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

[0097] 2. Experimental 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: aged 1-2 years old, with an average weight of 4 kg, and randomly divided into four groups, with 10 replicates in each group and one cat in each replicate, where the ratio of male ragdoll cats to female ragdoll cats was 4:6.

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

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

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

[0103] Necessary immunization and deworming treatments had been carried out before the experiment, and no drugs (such as antibiotics) that would change the intestinal microflora had been received within 1 month before the start of the experiment. The cats were shoveled once in the morning and evening, the cat litter was changed once a week, the cat house was cleaned and disinfected every day, the pet pen was kept clean, and the cats were played with for more than 30 minutes every day. A fixed amount of feed was weighed for each cat at regular intervals every day, and the food bowl was cleaned before weighing in the morning. The amount of feed for each cat and the remaining amount on the second day were accurately recorded every day. The fecal status was observed and recorded when the feces were shoveled in the afternoon. The cats were guaranteed free access to water and food during the entire experiment.

[0104] (3) Index determination: growth performance, diarrhea rate

[0105] The cats were weighed on an empty stomach one day before and one day after the experiment. The food intake was recorded at a fixed point every day during the entire experimental period, and the feces score (FS) and the number of diarrhea were recorded according to the standards in Table 4. The diarrhea rate and soft stool rate of each group were calculated using the following calculation formula:

[0106] Diarrhea rate (%) = number of cats with diarrhea during the test period / total number of cat feces scores during the test period;

[0107] Soft stool rate (%) = number of soft stools in cats during the test period / total number of cat feces scoring statistics during the test period.

[0108] Table 4 Fecal scoring criteria

[0109]

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

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

[0112]

[0113]

[0114] From the results in Table 5, it can be seen that the Bacillus subtilis BS11 powder provided by the present invention can effectively improve the growth performance of pet cats, increase their feed intake by 10%, increase their body weight by 0.15 kg, and significantly reduce the diarrhea rate, achieving unexpected technical effects.

Claims

1. A Bacillus subtilis, characterized in that The deposit number of the Bacillus subtilis is CCTCC NO: M20231592.

2. Use of the Bacillus subtilis according to claim 1 in the production of pet food.

3. Use of the Bacillus subtilis according to claim 1 in the production of pet medicines.

4. The use according to claim 3, characterized in that The medicine is a medicine with the function of preventing or treating diarrhea.

5. A probiotic preparation, characterized in that: The probiotic preparation comprises the Bacillus subtilis according to claim 1.

6. The probiotic preparation according to claim 5, 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 thermophilus, Enterococcus faecium, Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and Clostridium butyricum.

7. The probiotic preparation according to claim 6, characterized in that The amount of live bacteria of Bacillus subtilis in the probiotic preparation is at least 10 8 CFU / g.

8. Use of the probiotic preparation according to any one of claims 5 to 7 in the production of pet food.

9. Use of the probiotic preparation according to any one of claims 5 to 7 in the production of pet medicines.

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