Bacillus subtilis and application thereof

Abstract

The invention relates to livestock feeding probiotics, in particular to bacillus subtilis and application, the bacillus subtilis is bacillus subtilis BW and is preserved in the China General Microbiological Culture Collection Center on April 10, 2025, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) 0.34165. The bacillus subtilis BW disclosed by the invention can be colonized in animal bodies, so that the diarrhea rate of piglets is remarkably reduced, the immunity of intestinal tracts is improved, and the health of the intestinal tracts is improved; after the bacillus subtilis BW is used for feeding ruminants, the feed conversion efficiency can be remarkably improved, the growth performance can be improved, and the bacillus subtilis BW has a good application prospect in livestock and poultry breeding.

Description

Technical Field

[0001] This invention relates to probiotics for livestock feed, and particularly to a strain of Bacillus subtilis (BW) and its applications. Background Technology

[0002] Bacillus subtilis is a probiotic widely found in the natural environment. It possesses strong biological activity, effectively improving the intestinal microecological balance of animals, increasing feed conversion rate, and thus promoting animal growth. In recent years, the application of Bacillus subtilis in the aquaculture industry has become increasingly widespread, and it has become an important means of improving aquaculture efficiency. Summary of the Invention

[0003] The purpose of this invention is to propose a strain of Bacillus subtilis (BW) and its applications.

[0004] To achieve the above objectives, the inventors isolated a probiotic (Bacillus subtilis) BW from the rhizosphere soil of pine trees in the Changbai Mountain Nature Reserve in Jilin Province. Through colony morphology observation and molecular biological identification, it was determined to be Bacillus subtilis, and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0005] The Bacillus subtilis provided by this invention, named Bacillus subtilis BW, was deposited on April 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34165 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The Bacillus subtilis BW strain of this invention grows well on LB agar plates. After 48 hours of incubation, single colonies are 5–8 mm in size, milky white, opaque, with a wrinkled surface, a concave center, and irregular edges. The colony morphology is as follows: Figure 1 Microscopic morphology such as Figure 2 Gram-positive, short rod-shaped, with spore formation but the spores do not swell.

[0007] The Bacillus subtilis BW strain of this invention exhibits strong adaptability to extreme environments, specifically:

[0008] The Bacillus subtilis BW strain of the present invention maintained a survival rate of over 96% after 3 hours of treatment in a low pH environment (pH 1.5–2.5), indicating that the Bacillus subtilis BW strain of the present invention has good acid resistance.

[0009] The Bacillus subtilis BW strain of the present invention exhibits significant tolerance in environments with bile salt concentrations ranging from 0.15% to 1.0%, especially at high bile salt concentrations (1.0%), where the survival rate of the strain is 96.63%. This demonstrates that the Bacillus subtilis BW strain of the present invention possesses excellent bile salt tolerance.

[0010] The Bacillus subtilis BW strain of the present invention exhibits good high-temperature resistance after being subjected to water baths at 80°C, 90°C, and 100°C for 30 minutes.

[0011] The present invention provides a microbial agent comprising Bacillus subtilis BW.

[0012] The present invention also provides feed additives or animal feeds containing the aforementioned Bacillus subtilis BW.

[0013] As an embodiment of the present invention, the live count of Bacillus subtilis BW in the feed additive of the present invention is 1.0 × 10⁻⁶. 9 ~9.0×10 11 CFU / g; preferably, the viable count of Bacillus subtilis BW is 1.0 × 10⁻⁶. 10 ~9.0×10 11 CFU / g.

[0014] As an embodiment of the present invention, the viable count of Bacillus subtilis BW in the animal feed of the present invention is 1.0 × 10⁻⁶. 7 ~1.0×10 9 CFU / kg; preferably, the viable count of Bacillus subtilis BW is 1.0 × 10⁻⁶. 8 C ~ 1.0 × 10 9 CFU / kg.

[0015] This invention also relates to the application of the aforementioned Bacillus subtilis BW or bacterial agent or feed or feed additive in increasing animal feed intake, promoting animal growth, promoting animal weight gain and / or improving feed conversion rate.

[0016] This invention has the following characteristics and beneficial effects:

[0017] The Bacillus subtilis BW of this invention can withstand high temperatures of 100°C and has strong tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid. It can effectively inhibit major pathogenic bacteria in livestock and poultry such as Staphylococcus aureus, Escherichia coli, Salmonella, Clostridium perfringens, and Aeromonas hydrophila, and can be stored stably for a long time.

[0018] The Bacillus subtilis BW of the present invention can colonize in animals, significantly reduce the diarrhea rate in piglets, improve intestinal immunity, and improve intestinal health. When fed to ruminants, the Bacillus subtilis BW of the present invention can significantly improve feed conversion efficiency and growth performance, and has good application prospects in livestock and poultry farming. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Bacillus subtilis BW on LB medium, as shown in this embodiment of the invention.

[0020] Figure 2 This is a microscopic image of Bacillus subtilis BW, an embodiment of the present invention.

[0021] Figure 3 This is a diagram illustrating the antibacterial effect of Bacillus subtilis BW against Escherichia coli in an embodiment of the present invention. Detailed Implementation

[0022] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0023] Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0024] Example 1

[0025] Isolation, screening and identification of Bacillus subtilis BW

[0026] Step 1: Isolation and purification of Bacillus

[0027] The pine rhizosphere soil collected from the Changbai Mountain Nature Reserve was placed in a sterile bag and brought back to the laboratory. Under sterile conditions, 10g of the sample was weighed and placed in an Erlenmeyer flask containing 90mL of sterile physiological saline. After constant temperature shaking at 37℃ for 1 hour, it was placed in an 80℃ water bath for 10 minutes. The sample was serially diluted to 100,000 times using a 10-fold dilution method. Three dilutions of 1000, 10,000, and 100,000 times were selected, and 0.1mL of each was spread onto a nutrient agar plate. After spreading, the plates were incubated upside down at 37℃ for 24 hours. Colonies suspected to be Bacillus were picked with an inoculation loop and streaked onto nutrient agar plates for isolation and culture. After 24 hours of culture, colonies with good isolation results were picked and transferred to nutrient agar slants for pure culture. The pure culture was repeated 3 times. 113 strains of Bacillus were isolated from the sample. The bacterial cells were then suspended in 20% glycerol solution and stored at -80℃ for later use.

[0028] Step 2: Observation of colony morphology

[0029] The glycerol tube bacterial strains preserved in step 1 were activated 2-3 times on nutrient agar plates and then inoculated into nutrient broth medium. They were cultured at 37°C with shaking at 180 rpm for 18-20 hours. Clean glass slides were used for Gram staining and microscopic examination to observe the microscopic morphology of the strains. Gram-positive spore-forming bacilli were selected for later use.

[0030] Depend on Figure 1 It can be seen that the colonies of strain BW on LB solid medium are milky white, opaque, with wrinkled surfaces, an inwardly concave center, and irregular edges. Figure 2 It can be seen that the selected strain BW is Gram-positive, and the microscopic examination shows that it is short rod-shaped, with spore formation and no spore swelling.

[0031] Step 3: Preparation of Bacillus suspension and fermentation broth

[0032] The Bacillus obtained in step 1 was streaked on a nutrient agar plate and cultured at 37°C for 24 hours. Single colonies were picked from the plate and cultured in 100 mL of nutrient broth medium, and shaken at 37°C and 180 r / min for 24 hours to obtain a Bacillus suspension for later use. The suspension was then cultured at 37°C and 180 r / min for 48 hours, and heat-treated at 65°C for 15 minutes to terminate fermentation and promote spore release, thus obtaining a Bacillus fermentation broth for later use.

[0033] Step 4: Screening of antibacterial Bacillus

[0034] The concentration of pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Salmonella, Clostridium perfringens, Listeria monocytogenes) was 10. 9 Add 2 mL of CFU / mL bacterial suspension to 200 mL of sterile pathogenic bacteria culture medium cooled to approximately 45°C. Then, transfer 10 mL of the unconsolidated culture medium onto a nutrient agar plate containing a 10 mL bottom plate to prepare 10 pathogenic bacteria plates. On a clean bench, use sterile forceps to pick up one sterile Oxford cup (a small round tube with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 10 mm; the tube can hold 200 μL of liquid, and both ends should be smooth) from each pathogenic bacteria nutrient agar plate and place it on the plate, ensuring complete contact with the culture medium. After several minutes, transfer 200 μL of the suspected Bacillus strain fermentation broth (obtained in step 3) into each Oxford cup and incubate at 37°C for 24 hours. Perform at least three replicates for each strain, observe and measure the size of the inhibition zone. Eight strains with large inhibition zones were identified and labeled as S13, S22, S76, BA, SH, BP, BW, and BY.

[0035] Step 5: Stability screening of Bacillus subtilis

[0036] The Bacillus suspension prepared in step 3 was inoculated into sterile nutrient broth at an inoculation rate of 1% (v / v), with a volume of 300 mL / 1000 mL. The culture was incubated at 37°C with shaking at 180 rpm for 24 h. The fermentation broth was then transferred to a sealed bottle. The number of viable Bacillus bacteria in the fermentation broth was measured every day for 30 consecutive days. The BW fermentation broth was selected based on a 30-day survival rate of 100% for Bacillus bacteria, and this was chosen as the strain for further research.

[0037] Step 6: Identification of the strain species

[0038] Genomic DNA was extracted from strain BW using a bacterial DNA extraction kit. The 16S rDNA gene fragment of strain BW was sequenced using primers F and R, and the obtained sequence is shown in SEQ ID NO.1. BLAST analysis of the obtained sequence with 16S rDNA sequences in GenBank showed that strain BW shared 99.87% homology with Bacillus subtilis. Based on the morphological characteristics and 16S rDNA features of strain BW, it was confirmed that strain BW is Bacillus subtilis.

[0039] Step 7: Preservation of bacterial strains

[0040] The Bacillus subtilis BW obtained through isolation, purification, and screening was deposited on April 10, 2025, at the China General Microbiological Culture Collection Center (CGMCCN0.34165), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0041] Example 2

[0042] Preparation of Bacillus subtilis BW fermentation broth

[0043] (1) Take the seed culture of Bacillus subtilis BW (live bacteria concentration of 10) 9 1 mL of CFU / mL was inoculated into 70 mL of shake flask fermentation medium for shake flask fermentation. The shake flask fermentation conditions were: inoculum 1% (volume ratio), fermentation temperature 37℃, pH 7.0, 180 r / min, and fermentation time 8 h.

[0044] (2) After shake-flask fermentation, fermentation culture was carried out in a fermenter. 70 mL of the shake-flask fermentation seed liquid was inoculated into the fermentation medium in a 10 L fermenter for fermentation culture. The volume of the 10 L fermenter was 7 L of fermentation culture medium to obtain the primary seed of Bacillus subtilis BW. The fermentation culture conditions were: tank pressure 0.03-0.05 MPa, inoculation volume 70 mL, fermentation temperature 37℃, fermentation time 12 h, pH 7.0, stirring speed 200 r / min, and aeration rate 10 L / min.

[0045] (3) All of the above-mentioned primary seeds were transferred to the fermentation medium for fermentation culture. The fermentation culture conditions were as follows: 70L of fermentation medium was placed in a 100L fermenter, the tank pressure was controlled at 0.05-0.06MPa, the inoculum amount was 700mL, the fermentation temperature was 37℃, the fermentation time was 18h, the pH value was 6.0-8.0, the stirring speed was 200r / min, and the aeration rate was 30L / min.

[0046] (4) After fermentation, the viable bacteria count in the fermentation broth was measured to be 5.3 × 10⁻⁶. 10 The fermentation broth was prepared at CFU / mL with a spore count of 98.5% and stored at 4°C for later use.

[0047] The shake-flask fermentation medium and the primary seed fermentation medium have the same components, which can be composed of the following: sucrose 0.5-3.5%, glucose 0.5-3.0%, peptone 0.5-3.5%, yeast extract 0.5-2.5%, dipotassium hydrogen phosphate 0.02-2.0%, sodium chloride 0.01-0.5%, magnesium sulfate 0.01-0.5%, and the balance being water. In this embodiment: sucrose 2.0%, glucose 0.5%, peptone 1.0%, yeast extract 1.0%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate 0.4%, sodium chloride 0.2%, and the balance being water.

[0048] The fermentation medium in the 100L fermenter can be composed of the following components: 0.5-3.5% soybean meal extract, 0.2-2.0% corn flour, 0.2-2.0% yeast extract, 0.5-2.5% glucose, 0.05-2.0% dipotassium hydrogen phosphate, 0.01-1.0% magnesium sulfate, 0.01-0.5% ammonium sulfate, 0.02-0.2% manganese sulfate, 0.01-0.1% calcium carbonate, and the balance being water. In this embodiment: 1.0% soybean meal extract, 1.0% corn flour, 0.5% yeast extract, 1.5% glucose, 0.5% dipotassium hydrogen phosphate, 0.5% magnesium sulfate, 0.1% ammonium sulfate, 0.1% manganese sulfate, 0.1% calcium carbonate, and the balance being water.

[0049] Example 3

[0050] Bacillus subtilis BW probiotic verification

[0051] The concentration of pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Salmonella, Clostridium perfringens, Listeria monocytogenes) was 10. 9 2 mL of CFU / mL bacterial suspension was added to 200 mL of sterile pathogenic bacteria culture medium cooled to approximately 45°C. Then, 10 mL of the unconsolidated culture medium was transferred to a nutrient agar plate containing a 10 mL bottom plate, preparing 10 pathogenic bacteria plates. On a clean bench, a sterile Oxford cup (a small round tube with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 10 mm, containing 200 μL of liquid, with smooth ends) was placed on each pathogenic bacteria nutrient agar plate using sterile forceps and placed on the plate, ensuring complete contact with the culture medium. After 10 minutes, 200 μL of the preserved fermentation broth prepared in Example 2 was added to each tube, taking care not to spill. The plates were incubated at 37°C for 18–48 hours, and the diameter of the inhibition zone was measured. Each experiment was repeated in triplicate, and the average value was taken. The results are shown below. Figure 3 As shown in Table 1.

[0052] The culture media for the pathogenic bacteria are as follows: Escherichia coli, Staphylococcus aureus and Salmonella are nutrient agar medium, Clostridium perfringens is tryptone-sulfite-cycloserine agar (TSC) medium, and Listeria monocytogenes is TSA+5% defibrinated sheep blood agar.

[0053] The nutrient agar medium consists of: 1.0% peptone, 0.4% beef extract, 2.0% agar, 0.6% sodium chloride, and the remainder is water, with a pH of 7.0 ± 0.2.

[0054] The composition of the tryptone-sulfite-cycloserine agar (TSC) medium is as follows: 1.5% tryptone, 0.5% soybean peptone, 0.5% yeast extract, 0.1% sodium metabisulfite, 0.1% ferric ammonium citrate, 2.0% agar, with the balance being water. The pH value is 7.6±0.2. When using, 0.03% D-cycloserine is added after cooling to 50°C.

[0055] The TSA+5% defibrinated sheep blood agar composition is: 1.5% tryptone, 0.5% soybean peptone, 0.5% sodium chloride, 2.0% agar, with the balance being water, and a pH value of 7.3±0.2. When using, add 5% defibrinated sheep blood after cooling to 50°C.

[0056] Table 1. Antibacterial effect of Bacillus subtilis BW against pathogens.

[0057]

[0058]

[0059] Example 4

[0060] Preparation of Bacillus subtilis BW formulation

[0061] The fermentation medium consists of: 1.5% corn starch, 1% glucose, 1% peptone, 2% soybean meal, 0.5% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.05% manganese sulfate, 0.03% magnesium sulfate, 1% calcium carbonate, 0.05% defoamer, and the remainder is water.

[0062] The above culture medium was sterilized by high-temperature steam at 121℃ for 30 min, and then inoculated with 3% (v / v) of Bacillus subtilis BW primary seed liquid (prepared according to the method described in Example 2). Fermentation was carried out at 37℃ with stirring at 200 rpm, pH 6.5–7.0, and an aeration rate of 30 L / min for 18 h. The culture was then transferred to a container to obtain Bacillus subtilis BW active bacterial sludge (viable count greater than 1.0 × 10⁻⁶). 10 (CFU / mL)

[0063] The above-mentioned bacterial sludge was placed in a low-temperature vacuum drying oven for drying, sieved, and the product was collected to obtain Bacillus subtilis preparation.

[0064] Example 5

[0065] Stress resistance verification of Bacillus subtilis BW formulation

[0066] 1. Storage stability verification

[0067] The Bacillus subtilis BW preparation was placed in a sealed bag, and then the sealed bag containing the Bacillus subtilis preparation was placed in a constant temperature and humidity incubator set at 37℃ and 60% relative humidity. Samples were taken weekly to test the effective viable Bacillus subtilis count. Comparison with the initial effective viable count showed that the survival rate of Bacillus subtilis after 21 days of accelerated storage at 37℃ was 100%, and the survival rate after 28 days was 93%. This indicates that the BW strain preparation has high storage stability and meets the requirements for use.

[0068] 2. Tolerance to artificial gastric juice

[0069] Preparation of artificial gastric fluid: According to the preparation method in the 2010 edition of the Pharmacopoeia of the People's Republic of China, take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, dilute with water to 1000 mL, adjust the pH value to 1.5, 2.0 and 2.5 respectively, and sterilize with a microporous filter membrane (0.22 μm) for later use.

[0070] Accurately weigh 10g of Bacillus subtilis BW preparation (prepared according to the method described in Example 4) and place it in a 90mL (250mL Erlenmeyer flask) of artificial gastric fluid. Incubate at 37℃ with constant shaking at 200rpm for 120min. After shaking, take 10mL of the sample solution, adjust the pH to 7.0, add 90mL of physiological saline, and incubate at 37℃ with constant shaking at 200rpm for 30min. Then, perform dilution plate culture and colony counting. The results are shown in Table 2. Table 2 shows that the survival rate of Bacillus subtilis BW preparation after treatment in artificial gastric fluid (containing enzymes) at pH 1.5, pH 2.0, and pH 2.5 for 3 hours was all above 96%, indicating that this strain has strong tolerance to artificial gastrointestinal fluid.

[0071] Table 2 Survival of Bacillus subtilis BW 3 h after treatment in artificial gastric fluid

[0072] deal with pH 1.5 pH 2.0 pH 2.5 Initial activity CFU / mL <![CDATA[1.56×10 10 ]]> <![CDATA[1.56×10 10 ]]> <![CDATA[1.56×10 10 ]]> Post-treatment activity CFU / mL <![CDATA[1.50×10 10 ]]> <![CDATA[1.53×10 10 ]]> <![CDATA[1.56×10 10 ]]> Survival rate after treatment % 96.46 98.38 100.00

[0073] 3. Tolerance to artificial intestinal fluid

[0074] Preparation of artificial intestinal fluid: Refer to the preparation method in the 2010 edition of the Pharmacopoeia of the People's Republic of China. Phosphate buffer (containing pancreatic enzyme, pH 6.8): Take 6.8g of potassium dihydrogen phosphate, add 500mL of water to dissolve, and adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution; Separately, take 10g of pancreatic enzyme, add an appropriate amount of water to dissolve, mix the two solutions, add water to dilute to 1000mL and shake well, and then sterilize with a microporous membrane (0.22μm) for later use.

[0075] 1 g of Bacillus subtilis BW preparation (prepared according to the method described in Example 4) was accurately weighed and placed in 99 mL (250 mL Erlenmeyer flask) of artificial intestinal fluid. The mixture was incubated at 37°C and 200 rpm for 5 h with constant shaking. After shaking, 1 mL of the sample was added to 99 mL of physiological saline, and the mixture was incubated at 37°C and 200 rpm for 30 min with constant shaking. Then, a dilution plate culture was performed for colony counting. The results showed that the survival rate of Bacillus subtilis BW in the artificial intestinal fluid was 100%, indicating that Bacillus subtilis BW can successfully reach the intestine, colonize, and exert its effects.

[0076] 4. Test of bile salt tolerance

[0077] (1) Preparation of reagents:

[0078] Preparation of 5% bile salt solution: Accurately weigh 5.0g of bile salt, dissolve and bring to volume with 100mL of PBS solution, and sterilize at 121℃ for 20min.

[0079] Preparation of PBS solution: 0.363% disodium hydrogen phosphate, 0.024% potassium dihydrogen phosphate, 0.02% potassium chloride, 0.8% sodium chloride, with the remainder being water. Adjust the pH to 7.4 with 6 mol / L HCl, sterilize at 121℃ for 20 min, and set aside.

[0080] Preparation of bile salt solutions of different concentrations: 9 mL, 18 mL, 30 mL, and 60 mL of 5% bile salt solution were added to PBS solution at pH 7.4, and the volume was adjusted to 300 mL. The solutions were mixed evenly to obtain PBS solutions containing 0.15%, 0.3%, 0.5%, and 1.0% bile salts, respectively.

[0081] (2) Performance Measurement

[0082] 1.000 g of Bacillus subtilis BW preparation sample was accurately weighed and placed in 99 mL (250 mL Erlenmeyer flask) solutions with different bile salt concentrations: 0.15%, 0.3%, 0.5%, and 1.0%. The solution was then incubated at 37°C with shaking at 200 rpm for 2 h. After shaking, 1 mL of the sample solution was added to 99 mL of physiological saline, and the solution was incubated at 37°C with shaking at 200 rpm for 30 min. Finally, a plate culture was performed for colony counting. The results are shown in Table 3. The survival rate of Bacillus subtilis BW preparation treated in a 0.5% bile salt solution for 2 h was 100%, indicating that this strain has high bile salt tolerance and can withstand bile salts in duodenal fluid, allowing the strain to reach the intestine and exert its effects.

[0083] Table 3 Survival of Bacillus subtilis BW preparation after treatment with different concentrations of bile salt solution for 2 hours

[0084] deal with 0.15％ 0.3％ 0.5％ 1.0％ Initial activity CFU / mL <![CDATA[3.45×10 10 ]]> <![CDATA[3.45×10 10 ]]> <![CDATA[3.45×10 10 ]]> <![CDATA[3.45×10 10 ]]> Post-treatment activity CFU / mL <![CDATA[3.45×10 10 ]]> <![CDATA[3.45×10 10 ]]> <![CDATA[3.45×10 10 ]]> <![CDATA[3.33×10 10 ]]> Survival rate after treatment % 100 100 100 96.63

[0085] Example 6

[0086] Safety evaluation of Bacillus subtilis BW

[0087] In this embodiment, mice were used as experimental animals. An acute toxicity test was conducted, and the safety of Bacillus subtilis BW was evaluated according to the maximum tolerated dose method in the national standard GB15193.3-2003. The specific method is as follows:

[0088] 1. The lyophilized powder of Bacillus subtilis inoculant prepared by the method in Example 4 was analyzed by plate counting, and the viable Bacillus subtilis BW count was 2.0 × 10⁻⁶. 10 CFU / g.

[0089] 2. Fifty 3-week-old SPF-grade mice, half male and half female, with an average weight of 19.43±0.02g, were selected. After one week of routine feeding, the mice were administered 0.25g / mL Bacillus subtilis BW bacterial solution (equivalent to 15000mg / kg body weight) by gavage three times a day for two consecutive weeks. The mice were then observed for signs of poisoning or death.

[0090] 3. During the experiment, the mice were in good mental condition and there were no signs of poisoning or death. It can be seen that the maximum tolerated dose (MTD) of the strain of this invention in the acute toxicity test is >15000mg / kg. According to the grading standard, the strain can be determined to be non-toxic and has high safety.

[0091] Example 7

[0092] Application of Bacillus subtilis BW preparation in weaned piglets

[0093] This experiment selected 72 Duroc × Landrace × Large White crossbred weaned piglets at 28 days of age, and divided them into two groups of 36 piglets each, with 6 replicates per group and 6 piglets per replicate. The control group received a corn-soybean meal basal diet, while the experimental group received a basal diet supplemented with 500g / ton of Bacillus subtilis BW preparation prepared in Example 4 (with an effective viable count of 1.5 × 10⁻⁶). 10 CFU / g).

[0094] During the trial, piglets were housed in fully enclosed nursery pens at a temperature maintained between 25 and 27°C. They were fed three times daily with free access to food and water. The pens were cleaned daily for 60 days. The basal diet did not contain any antibiotics, and piglets were immunized according to standard immunization procedures.

[0095] The experiment lasted for 60 days. Piglets were weighed on days 0, 30, and 60. Feed intake was recorded daily, and average daily weight gain, average daily feed intake, and feed conversion ratio were calculated. The frequency of diarrhea in piglets was recorded daily, and the diarrhea rate was calculated.

[0096] The experimental results are shown in Table 4: Bacillus subtilis BW preparation significantly improved the final weight and average daily weight gain of piglets (P<0.05), significantly reduced the feed conversion ratio (P<0.05), and effectively reduced the diarrhea rate of piglets (P<0.05). This indicates that Bacillus subtilis BW can effectively improve piglet production performance, reduce piglet diarrhea, and promote piglet intestinal health.

[0097] Table 4. Effects of Bacillus subtilis BW formulation on growth performance of weaned piglets

[0098] project control group experimental group Initial weight (kg / head) 7.36±0.26 7.38±0.31 Final weight (kg / head) <![CDATA[33.21±0.13 b ]]> <![CDATA[36.54±0.21 a ]]> Average daily weight gain (g / d / head) <![CDATA[430.54±0.51 b ]]> <![CDATA[485.92±0.38 a ]]> Average daily feed intake (g / d / head) 727.12±0.45 733.51±2.12 Material weight ratio <![CDATA[1.69±0.06 a ]]> <![CDATA[1.51±0.05 b ]]> Diarrhea rate (%) 13.88±0.15 6.43±0.20

[0099] Note: Differences are represented by different letters in the same column, with lowercase letters indicating significant differences (P<0.05).

[0100] Example 8

[0101] Application of Bacillus subtilis BW preparation in broilers

[0102] One hundred and eighty healthy one-day-old broiler chickens of similar weight were randomly divided into two groups, with six replicates per group and 15 chickens per replicate. The control group was fed a basal diet, while the experimental group was fed a corn-soybean meal basal diet supplemented with 500 g / ton of Bacillus subtilis BW preparation (effective viable count of 1.5 × 10⁻⁶). 10(CFU / g). The experiment lasted 42 days, with weighings taken on days 0, 21, and 42. After 21 days of age, the chickens were switched to a late-stage feed. The chickens had free access to feed and water. The experiment was conducted according to routine chicken house management procedures. The results are shown in Table 5: Bacillus subtilis BW preparation effectively increased feed intake in broiler chickens, significantly improved daily weight gain (P<0.05), and significantly reduced feed conversion ratio (P<0.05). This indicates that adding the Bacillus subtilis preparation of this invention provides better feed efficiency.

[0103] Table 5 Effects of Bacillus subtilis BW formulation on broiler growth performance

[0104] project control group experimental group Average daily feed intake (ADFI / g) 92.38±2.69 101.91±3.15 Average daily weight gain (ADG / g) <![CDATA[56.25±2.41 b ]]> <![CDATA[65.33±2.88 a ]]> Material weight ratio F / G <![CDATA[1.64±0.04 a ]]> <![CDATA[1.56±0.03 b ]]>

[0105] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Bacillus subtilis, characterized in that, The bacterium is Bacillus subtilis BW, which was deposited at the China General Microbiological Culture Collection Center on April 10, 2025, with the accession number CGMCCN0.34165.

2. A microbial agent, characterized in that, It includes Bacillus subtilis BW as described in claim 1.

3. A feed, characterized in that, The feed contains Bacillus subtilis BW as described in claim 1 or is prepared from Bacillus subtilis BW as described in claim 1.

4. A feed additive, characterized in that, The feed additive comprises Bacillus subtilis BW as described in claim 1 or is prepared from Bacillus subtilis BW as described in claim 1.

5. The application of Bacillus subtilis BW as described in claim 1 in increasing animal feed intake, promoting animal growth, promoting animal weight gain and / or improving feed conversion ratio.

6. The use of the microbial agent according to claim 2 in increasing animal feed intake, promoting animal growth, promoting animal weight gain and / or improving feed conversion rate.

7. The use of the feed according to claim 3 in increasing animal feed intake, promoting animal growth, promoting animal weight gain and / or improving feed conversion rate.

8. The use of the feed additive of claim 4 in increasing animal feed intake, promoting animal growth, promoting animal weight gain and / or improving feed conversion rate.

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