Bacillus subtilis and application thereof

Abstract

The invention relates to bacillus subtilis, the bacillus subtilis is named as bacillus subtilis BS02, the bacillus subtilis is preserved in the China Center for Type Culture Collection, the preservation date is April 11, 2025, and the preservation number is CCTCC NO: M 2025753. The bacillus subtilis can be used for preparing a bacillus subtilis strain. According to the technical scheme, the bacillus subtilis BS02 is provided and can replace antibiotics for livestock after being processed, the number of viable bacteria of intestinal escherichia coli is reduced, the number of viable bacteria of beneficial bacteria is increased, then the intestinal health effect can be improved, and the incidence rate and the fatality rate of diarrhea can be reduced; meanwhile, the bacillus subtilis BS02 can also improve the average daily gain of the piglets to a certain extent and reduce the feed conversion ratio, and has objective application value in the field of feed additives.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Bacillus subtilis and applications thereof. Background Art

[0002] Key challenges in the development of the livestock industry lie not only in disease prevention, treatment, and promoting animal growth, but also in ensuring food safety and environmental protection. Non-infectious diarrhea in piglets can often be alleviated through improved feeding management, such as adjusting feed formulations and optimizing feeding times and methods. However, treatment for infectious diarrhea often relies on antimicrobial drugs. While these drugs can effectively control disease in the short term, the emergence of antimicrobial resistance is increasingly questioning this approach.

[0003] The widespread use of antimicrobials in food animal production is driving antimicrobial resistance (AMR) into a serious public health problem. Bacteria gradually acquire resistance to antibiotics through genetic mutations or the acquisition of foreign resistance genes, making some previously easily curable infections difficult to treat. Furthermore, the impact of antibiotics on livestock's intestinal flora cannot be ignored. They not only inhibit the growth of pathogenic bacteria but also affect beneficial bacteria in the gut, leading to an imbalance in the gut microbiome. This dysbiosis can further compromise the animals' health, reduce their immunity, and indirectly increase their reliance on antibiotics, creating a vicious cycle. Summary of the Invention

[0004] Based on the above description, the present invention provides a Bacillus subtilis and its application to solve the problem of how to reduce the number of pathogenic bacteria while increasing the number of beneficial bacteria.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: The present invention provides a Bacillus subtilis, which is named Bacillus subtilis BS02 ( Bacillus subtilis BS02), the Bacillus subtilis has been deposited in the China Center for Type Culture Collection on April 11, 2025, with a deposit number of CCTCC NO: M 2025753.

[0006] The present invention also provides an application of Bacillus subtilis in preparing a microbial agent, wherein the Bacillus subtilis is the aforementioned Bacillus subtilis BS02.

[0007] The present invention also provides an application of Bacillus subtilis in preparing an antibacterial agent, wherein the Bacillus subtilis is the aforementioned Bacillus subtilis BS02.

[0008] The present invention also provides a bacterial agent, comprising the aforementioned Bacillus subtilis.

[0009] Furthermore, in the bacterial agent, the concentration of Bacillus subtilis is 1×10 9 ~2×10 9 CFU / mL.

[0010] The present invention also provides a feed comprising the aforementioned bacterial agent.

[0011] Furthermore, the concentration of the bacterial agent in the feed is 1~3 L / t.

[0012] The present invention also provides an antibacterial agent, which includes the aforementioned Bacillus subtilis BS02.

[0013] The present invention also provides a pharmaceutical composition, which comprises the aforementioned Bacillus subtilis BS02.

[0014] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: (1) The Bacillus subtilis BS02 provided by the present invention can significantly improve the growth performance of weaned piglets. Compared with the group without any antibiotics, the group with probiotics, and the group with only antibiotics, the average daily weight gain increased by 14.81% and 8.77%, respectively, and the feed-to-meat ratio decreased by 0.2 and 0.1, respectively; the diarrhea rate decreased by 26.57% and 8.96%, respectively; (2) The Bacillus subtilis BS02 provided by the present invention can significantly increase the serum alkaline phosphatase activity and serum immunoglobulin concentration of piglets, thereby improving immune function; (3) The Bacillus subtilis BS02 provided by the present invention can reduce the number of viable Escherichia coli in the intestines of piglets, increase the number of viable beneficial bacteria Bifidobacterium and Lactobacillus, significantly increase the height of villi in the duodenum, jejunum, and ileum, reduce the depth of crypts, and significantly improve intestinal health; (4) The Bacillus subtilis BS02 provided by the present invention can reduce the mortality rate of piglets caused by diarrhea and has the effect of replacing antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram showing the enzyme production characteristics of strain N65 and strain N29 in Example 2 of the present invention; Figure 2 This is a colony diagram of Bacillus subtilis BS02 in Example 3 of the present invention; Figure 3 This is the Gram staining image of Bacillus subtilis BS02 in Example 3 of the present invention; Figure 4 This is a diagram showing the amplification results of the 16S rDNA gene fragment of Bacillus subtilis BS02 in Example 3 of the present invention; Figure 5This is a phylogenetic tree diagram constructed based on the 16S rDNA gene sequence in Example 3 of the present invention; Figure 6 This is a graph showing the temperature tolerance test results of Bacillus subtilis BS02 in Example 6 of the present invention; Figure 7 This is a graph showing the bile salt tolerance test results of Bacillus subtilis BS02 in Example 6 of the present invention; Figure 8 Graph showing the activity test results of Bacillus subtilis BS02 in artificial gastric juice and artificial intestinal juice in Example 6 of the present invention; Figure 9 This is a graph showing the pH tolerance test results of Bacillus subtilis BS02 in Example 6 of the present invention. DETAILED DESCRIPTION

[0016] To facilitate understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings. The accompanying drawings provide embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0018] The widespread use of antimicrobials in food animal production is driving antimicrobial resistance (AMR) into a serious public health problem. Bacteria gradually acquire resistance to antibiotics through genetic mutations or the acquisition of foreign resistance genes, making some previously easily curable infections difficult to treat. Furthermore, the impact of antibiotics on livestock's intestinal flora cannot be ignored. They not only inhibit the growth of pathogenic bacteria but also affect beneficial bacteria in the gut, leading to an imbalance in the gut microbiome. This dysbiosis can further compromise the animals' health, reduce their immunity, and indirectly increase their reliance on antibiotics, creating a vicious cycle.

[0019] Functional probiotics are commonly used antibiotic alternatives. Acting as "live" probiotics, they inhibit the growth and reproduction of intestinal pathogens through competitive niche occupation and oxygen deprivation, helping animals establish a beneficial intestinal microbial ecosystem. Furthermore, their metabolites or "dead" bacterial cell components can be used in the animal's intestines to enhance disease resistance, thereby promoting growth and improving production performance. Bacillus subtilis is a microbial feed additive approved for use in my country's feed additive catalog. Its production requirements are simple, it forms highly stress-resistant spores, exhibits strong environmental tolerance, and is easy to store and transport. It also possesses a rich enzyme-producing system and abundant active metabolites. Bacillus subtilis not only produces antimicrobial active ingredients that inhibit the colonization of pathogens in the intestine and maintain intestinal microecological balance, but also secretes a variety of hydrolytic enzymes that improve the digestive tract environment and enhance feed utilization. It also boosts intestinal immunity and enhances disease resistance. Bacillus subtilis has been widely used as a functional probiotic in livestock, poultry, and aquaculture. Bacillus subtilis from different sources varies significantly in their colonization ability, metabolite functions, and stress resistance, significantly limiting their practical application in production. Generally speaking, microorganisms are most active in their natural habitats. Intestinal symbiotic microorganisms serve as a natural defense for animals, living in a mutually beneficial symbiosis with their hosts and co-evolving with the intestinal immune system and internal environment. They promote nutrient absorption, vitamin synthesis, and immune regulation, making them crucial for maintaining intestinal health.

[0020] In view of this, the present invention provides a Bacillus subtilis, which is named Bacillus subtilis BS02 ( Bacillus subtilis BS02), the Bacillus subtilis has been deposited in the China Center for Type Culture Collection on April 11, 2025, in Wuhan, China, with a deposit number of CCTCC NO: M 2025753.

[0021] In the technical solution of the present invention, a Bacillus subtilis BS02 is proposed, which can replace veterinary antibiotics after processing, reduce the number of live Escherichia coli in the intestine, and increase the number of live beneficial bacteria, thereby improving intestinal health and reducing the incidence and mortality of diarrhea; at the same time, Bacillus subtilis BS02 can also increase the average daily weight gain of piglets to a certain extent and reduce the feed-to-meat ratio, and has objective application value in the field of bacterial agents.

[0022] The present invention also provides an application of Bacillus subtilis in preparing a microbial agent, wherein the Bacillus subtilis is the aforementioned Bacillus subtilis BS02.

[0023] The present invention also provides an application of Bacillus subtilis in preparing an antibacterial agent, wherein the Bacillus subtilis is the aforementioned Bacillus subtilis BS02.

[0024] The present invention also provides a bacterial agent, comprising the aforementioned Bacillus subtilis.

[0025] Furthermore, in the bacterial agent, the concentration of Bacillus subtilis is 1×10 9 ~3×10 9 CFU / mL. Preferably, the concentration of Bacillus subtilis is 2×10 9 CFU / mL.

[0026] The present invention also provides a feed comprising the aforementioned bacterial agent.

[0027] Furthermore, the concentration of the bacterial agent in the feed is 1~3 L / t.

[0028] The present invention also provides an antibacterial agent, which includes the aforementioned Bacillus subtilis BS02.

[0029] The present invention also provides a pharmaceutical composition, which comprises the aforementioned Bacillus subtilis BS02.

[0030] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] 1. Sample The intestinal contents of healthy adult Duchang pigs were collected from a large-scale pig farm in Ningde, Fujian.

[0033] 2. Main Reagents Pfu PCR Mix, DNA gel extraction kit, agarose, pepsin (1:1200), and trypsin (1:250) were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; high maltose syrup was purchased from Jinan Xinshuaiyuan Chemical Co., Ltd.; bacitracin zinc (USP grade, 70 U / mg), colistin sulfate (USP grade, 19,000 IU / mg), defibrinated sheep blood, and porcine bile salts (bile acid content ≥60%) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; drug susceptibility test strips were purchased from Hangzhou Binhe Microbiological Reagent Co., Ltd.; and other reagents were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0034] Artificial gastric juice: Take 1.64 mL of 1.0 mol / L dilute hydrochloric acid, add about 80 mL of water and 1.0 g of pepsin, shake well, and then add water to make up to 100 mL.

[0035] Artificial intestinal fluid: Take 0.68 g of potassium dihydrogen phosphate, add 50 mL of water, and adjust the pH to 6.8 with 0.4% sodium hydroxide solution; take 1.0 g of trypsin, add appropriate amount of water to dissolve; mix the above two solutions and adjust the volume to 100 mL.

[0036] 3. Main instruments The PCR instrument (model EasyCycler 96) was purchased from Fuzhou Keyuan Trading Co., Ltd.; the electrophoresis instrument (model DYY-6C) was purchased from Beijing Liuyi Instrument Factory; the fully automatic gel imaging system (model JS-3000) was purchased from Shanghai Peiqing Technology Co., Ltd.; the electronic balance (model BS223S) was purchased from Sartorius (Shanghai) Trading Co., Ltd.; the ultra-low temperature preservation box (model DW-86L388J) was purchased from Qingdao Haier Biomedical Co., Ltd.; the electric constant temperature incubator (model DNP-9052) was purchased from Shanghai Jinghong Laboratory Equipment Co., Ltd.; the desktop high-speed refrigerated centrifuge (model TGL-16M) was purchased from Xiangyi Centrifuge Instrument Co., Ltd.; the constant temperature incubation shaker (model ZWYR-211C) and the ultra-clean workbench (model ZHJH-C1118B) were both purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; and the automatic steam sterilizer (model GI54TW) was purchased from Zhiwei (Xiamen) Instrument Co., Ltd.

[0037] 4. Culture Medium Isolation medium: 12.0 g tryptone, 8.0 g yeast extract, 2.0 g potassium dihydrogen phosphate, 0.1 g magnesium sulfate, 20.0 g agar powder, 1000 mL deionized water, pH 7.0 ± 0.2, sterilized at 121°C for 20 min.

[0038] Bile salt tolerance medium: Add 0.5% porcine bile salts to the isolation medium.

[0039] Enzyme production screening medium: On the basis of the separation medium, add 1.0% skim milk powder (protease screening), 0.5% soluble starch (amylase screening), 0.5% sodium carboxymethyl cellulose (cellulase screening) or 0.5% xylan (xylanase screening).

[0040] Indicator culture medium: (1) LB liquid medium, the formula is 10.0 g tryptone, 5.0 g yeast powder, 10.0 g sodium chloride, 1000 mL deionized water, pH 7.0 ± 0.2, sterilized at 121°C for 20 min. (2) LB solid medium, based on LB liquid medium, add 2.0% agar powder.

[0041] Shake flask culture medium: 25.5 g soybean meal powder, 45.0 g high maltose syrup, 5.0 g corn steep liquor powder, 6.0 g ammonium sulfate, 3.0 g potassium dihydrogen phosphate, 1.0 g manganese sulfate, 1.0 g calcium carbonate, 1000 mL deionized water, pH 7.0 ± 0.2, sterilized at 121°C for 20 min.

[0042] 5. Indicator bacteria Gram-positive bacteria included Staphylococcus aureus, Clostridium perfringens GDMCC 1.307, and Streptococcus agalactiae GDMCC 1.408, and Gram-negative bacteria included Escherichia coli GDMCC 1.355 and Salmonella typhimurium, all of which were obtained from Guangdong Provincial Microbiological Culture Collection Center.

[0043] Example 1 Sample Collection The duodenum, jejunum, and ileum of healthy adult pigs were cut into approximately 5.0 cm segments, and the two ends were tied with sterile cotton ropes after cutting. The intestinal contents of each segment were squeezed out and mixed in a sterile environment. About 5.0 g of intestinal contents were weighed and placed in a 50 mL Erlenmeyer flask with sterile glass beads and sterile phosphate buffered saline (PBS, pH 7.4). The flask was incubated in a water bath at 80°C for 10 min. 1 mL of the supernatant was taken and diluted 10-fold with sterile PBS to 1 × 10 -3 , 1×10 -4 With 1×10 -5 There are 3 gradients in total to obtain treatment solutions with different dilution multiples.

[0044] Example 2 Isolation and screening of strains The three gradients of treatment solutions in Example 1 were spread on bile salt tolerance culture medium and incubated upside down at 37°C for 48 hours; a single colony with obvious Bacillus characteristics was selected and re-streaked onto separation medium and incubated at 37°C for 24 hours; and different colonies were selected for further screening.

[0045] Initial screening of antibacterial activity: Staphylococcus aureus was used as the indicator bacteria, and the indicator bacteria suspension was prepared in LB liquid medium (the number of viable bacteria was about 1.0×10 9 cfu / mL), and 0.5 mL of indicator bacteria suspension was added to 100 mL of indicator culture medium to prepare opposing plates containing indicator bacteria. The individual colonies separated by streaking were then spotted on the opposing plates and incubated at 37°C for 24 h. Strains with obvious inhibition zones were selected, and the inhibition zone diameter (B) and colony diameter (C) were measured, and their ratio was calculated. Strains with a larger ratio (>2) were rescreened for enzyme production characteristics.

[0046] Rescreening for enzyme production: Strains with distinct inhibition zones were plated onto different enzyme screening media and incubated at 37°C for 24 hours. Cellulase, xylanase, and amylase activities were determined by adding 0.1% Congo red or iodine solution, respectively. Protease plates can be observed without staining. The diameter of the clearing zone (H) and colony diameter (C) were measured, and their ratios were calculated. Simultaneously, isolated strains with distinct functionalities were selected, added to 25% glycerol, and stored at -80°C until ready for use.

[0047] Individual colonies of varying sizes were visible on the isolation medium. Most colonies were irregularly round, pale yellow, with small white protrusions and wrinkles in the center. They were viscous, opaque, and had neat edges. Using Staphylococcus aureus as an indicator bacteria, 33 strains were found to form distinct zones of inhibition. Twelve strains with a large ratio (B / C) of inhibition zone diameter to colony diameter (>2) were selected for rescreening of enzyme production. The results are shown in Table 1.

[0048] Table 1 Results of re-screening of enzyme production characteristics of different strains

[0049] Note: ND means no clear ring was detected or the clear ring was not obvious As shown in Table 1, all 12 strains showed good protease and amylase activities, and there were large differences between the strains. Among them, the N65 strain (i.e., Bacillus subtilis BS02) had the best protease and amylase activities, followed by the N29 strain. Seven strains did not show cellulase activity, six strains did not show xylanase activity, and three strains showed neither cellulase activity nor xylanase activity. Only the N65 and N29 strains showed the activities of four enzymes, namely, protease, amylase, cellulase and xylanase, at the same time, reflecting a good comprehensive enzyme production capacity, especially the N65 strain, which not only had the best antibacterial activity, but also had the most outstanding comprehensive enzyme production capacity. The enzyme production characteristics of the N65 and N29 strains are shown in Figure 1 .

[0050] Example 3 Molecular identification of strains According to the above results, the strain with the best antibacterial activity and enzyme production ability was inoculated into the separation medium and cultured for 48 hours. The colony morphology was observed and Gram staining and microscopic examination were performed.

[0051] Colony PCR was used to amplify the 16S rDNA gene fragment of the superior strain. The pre-amplified fragment size was approximately 1446 bp. The universal primers for 16S rDNA PCR amplification were 27F 5'-AGAGTTTGA-TCCTGGCTCAG-3' and 1492R 5'GGTTACCTTGTTA-CGACTT-3', synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0052] PCR amplification system: 25 μL of 2× Pfu PCR Mix, 2 μL of upstream and downstream primers, and a small number of single colonies as templates were added to a total volume of 50 μL with ddH2O. The PCR amplification procedure included pre-denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 30 s, and extension at 72°C for 90 s; an additional extension at 72°C for 10 min, and storage at 4°C. The target product was detected and recovered by agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with 16S rDNA sequences in GenBank by BLAST, and a phylogenetic tree was constructed using the N-J method in MEGA 4.0 software.

[0053] The N65 strain was cultured on the isolation medium for 48 h, and the resulting single colony was milky yellow, opaque, and had a raised wrinkle in the middle (see Figure 2 ), Gram staining is positive, the bacteria are long rod-shaped, solitary or opposite, with obvious oval spores, and the spores almost fill the entire body (see Figure 3 ), with typical characteristics of Bacillus. PCR amplified the 16S rDNA gene fragment to obtain a target fragment of approximately 1446 bp (see Figure 4 ), which was consistent with the expected result. The target fragment was recovered by tapping and submitted to Sangon Biotech Co., Ltd. for sequencing. The 16S rDNA gene sequence was subjected to BLAST comparison analysis in GenBank. 13 different Bacillus sequences were selected and compared with the N65 strain sequence, and a phylogenetic tree was constructed, as shown in Figure 5. Figure 5 Strain N65 clusters with strains of the genus Bacillus and is most closely related to the Bacillus subtilis clade, indicating that strain N65 and Bacillus subtilis are evolutionarily closely related. BLAST analysis reveals that strain N65 is most closely related to Bacillus subtilis NBRC 13719, with a similarity of 99.79%. Combined with its colony morphology, strain N65 is essentially a member of the genus Bacillus subtilis.

[0054] Example 4 Detection of biological characteristics of strains The strain was activated by subculture in LB liquid medium and a bacterial suspension was prepared (the number of viable bacteria was about 1×10 10 cfu / mL), and the bacterial suspension was transferred to a conical flask at an inoculum size of 0.2%, cultured at 37°C and 220 rpm for 48 h, and centrifuged at 10,000 rpm for 5 min. The fermentation supernatant was collected and sterilized by filtration using a 0.22 μm filter membrane, and the sterile supernatant was collected for biological characteristic detection.

[0055] 1) Antibacterial spectrum Several common pathogens in animal husbandry, including Gram-positive bacteria such as Staphylococcus aureus, Clostridium perfringens, and Streptococcus agalactiae, and Gram-negative bacteria such as Escherichia coli and Salmonella, were selected as indicator bacteria. The agar diffusion method was used to investigate the inhibitory effects of sterile supernatant on different indicator bacteria: the indicator culture medium was heated and then cooled to 45-55°C at room temperature. An appropriate amount of indicator bacteria was added, mixed, and poured into a plate. A hole was punched in the plate with a sterile hole puncher with a diameter of 6.0 mm, and the agar block was removed. 50 μL of the sterile supernatant was added to the sample wells and the plate was incubated at 4°C for approximately 1 h. The plate was then incubated at 37°C for 24 h. The plate was removed, and the diameter of the transparent zone was measured with a vernier caliper. The diameter of the inhibition zone was then calculated.

[0056] Diameter of inhibition zone (mm) = diameter of transparent zone (mm) - 6.0 (aperture).

[0057] The results are shown in Table 2.

[0058] Table 2 Detection results of the antibacterial activity of the sterile supernatant of strain N65 against different indicators

[0059] As shown in Table 2, the sterile supernatant of strain N65 has a relatively broad-spectrum antibacterial activity. However, overall, its antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, Clostridium perfringens, and Streptococcus agalactiae is stronger than that against Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium. Among them, the antibacterial activity against Streptococcus agalactiae is slightly better than that against Staphylococcus aureus and Clostridium perfringens, and the antibacterial activity against Escherichia coli is better than that against Salmonella typhimurium.

[0060] 2) Temperature tolerance The sterile supernatant was treated in a water bath at 60, 80, and 100 ℃ and in a high temperature and high pressure at 120 ℃ for 30 min, respectively. The supernatant was shaken every 5 min during the water bath treatment. After cooling to room temperature, the antibacterial activity was detected by agar diffusion method. The diameter of the inhibition zone was measured. The untreated sterile supernatant was used as a control (CK, antibacterial activity was 100%). The indicator bacteria was Staphylococcus aureus. The effects of different temperatures on the antibacterial properties of the sterile supernatant were investigated.

[0061] Test results are shown in Figure 6 .Depend on Figure 6 Compared with CK, the antibacterial activity gradually decreased with increasing temperature, but the decrease was relatively small. The antibacterial activity of the sterile supernatant treated at 100°C for 30 minutes remained above 90%, and the antibacterial activity of the sterile supernatant treated at 120°C for 30 minutes remained above 75%, demonstrating excellent temperature tolerance.

[0062] 3) Bile salt tolerance To 0.5 mL of sterile supernatant, 4.5 mL of bile salt solutions with concentrations of 0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% were added, respectively. Considering that high-concentration bile salts themselves may have an inhibitory effect on indicator bacteria, sterile water was used instead of sterile supernatant to prepare bile salt solutions of the same concentration as solvent controls. The solutions were incubated in a water bath at 37°C and 100 rpm for 3 h. The indicator bacteria was Staphylococcus aureus. The antibacterial activity was detected by the agar diffusion method. The diameters of the inhibition zones were measured, and the difference between the diameters of the inhibition zones of the sterile supernatant and the solvent was calculated.

[0063] Test results are shown in Figure 7 .Depend on Figure 7 It can be seen that the antibacterial activity of the sterile supernatant was maintained above 95% after being treated with 0.5% bile salt for 3 h, indicating that the antibacterial performance of the sterile supernatant was basically unaffected by the bile salt concentration, reflecting a strong bile salt tolerance.

[0064] 4) Stability of artificial gastric juice and intestinal juice To each of the 0.5 mL of sterile supernatant, 4.5 mL of artificial gastric juice, artificial intestinal juice, and sterile purified water were added, respectively, and the mixture was incubated in a water bath at 37°C and 100 rpm for 3 h. After treatment, the pH value of each treatment solution was adjusted back to 6.5-7.0 with 0.4% sodium hydroxide. The antibacterial activity was determined by the agar diffusion method, and the diameter of the inhibition zone was measured. The indicator bacteria was Staphylococcus aureus. This study investigated the tolerance of the sterile supernatant to gastric and intestinal juices.

[0065] See the results Figure 8 .Depend on Figure 8 It can be seen that the antibacterial activity of the sterile supernatant remained above 95% after being treated in artificial gastric juice and intestinal juice for 3 hours respectively, which reflects the very good stability of the artificial gastric juice and intestinal juice environment and has a certain potential for tolerance to the animal gastrointestinal environment.

[0066] 5) pH stability The pH values of the sterile supernatant were adjusted to 2, 4, 6, 8, and 10 using 0.4% sodium hydroxide or 1.0 mol / L hydrochloric acid for 2 h, respectively. After treatment, the pH value of each treatment solution was adjusted to 6.5-7.0 (the original pH value of the sterile supernatant was approximately 7.0) using 0.4% sodium hydroxide or 1.0 mol / L hydrochloric acid. The antibacterial activity was detected by the agar diffusion method, and the diameter of the inhibition zone was measured. The indicator bacteria was Staphylococcus aureus. The effects of different pH values on the antibacterial properties of the sterile supernatant were investigated.

[0067] Test results are shown in Figure 9 .Depend on Figure 9 It can be seen that the sterile supernatant is more stable in an acidic environment, and the antibacterial activity remains above 85% at a pH value of ≤ 8. At a pH value of 10, the antibacterial activity decreases significantly, but still remains above 80%, demonstrating a relatively wide pH tolerance and the potential to adapt to the dynamic changes in pH in the animal gastrointestinal tract.

[0068] 6) Hemolytic activity The hemolysis rate of the sterile supernatant was determined by ultraviolet spectrophotometry. A 2.0% defibrinated sheep red blood cell suspension was prepared with normal saline, and the sterile supernatant was diluted 5-fold and 10-fold with normal saline, respectively. 2.5 mL of the sterile supernatant stock solution and the dilution were added to a test tube containing 2.5 mL of the 2.0% defibrinated sheep red blood cell suspension. The tubes were incubated at 37°C for 3 h and centrifuged at 2000 rpm for 5 min. The supernatant was collected and tested for OD. 576 Calculate the hemolysis rate [Hemolysis rate (%) = (sample tube OD 576 Value-negative tube OD 576 Value - blank sample OD 576 value) / (positive tube OD 576 Value-negative tube OD 576 The results were shown in Table 3.

[0069] Table 3 Hemolysis rate test results

[0070] As shown in Table 3, the hemolytic rate of the sterile supernatant of the N65 strain, whether it is the original solution or the diluted solution, on red blood cells is below 1.0% (the hemolytic rate is <5%, which is considered to have no hemolytic activity). It can be basically considered that it has no hemolytic reaction on red blood cells, has a low hemolytic risk, and is safe.

[0071] Example 6 Safety of Bacillus subtilis BS02 in piglet farming Experimental subjects: 1600 healthy four-week-old weaned piglets with body weight close to (7.36±0.1kg), half male and half female.

[0072] The 1,600 weaned piglets were randomly divided into five groups, including one control group and four experimental groups. Each group consisted of 320 pigs, divided into five parallel groups, with 64 pigs in each parallel group. The control group (CK group) drank ordinary water, group A used amoxicillin soluble powder / cefotaxime sodium for injection as daily antibiotics according to the drug instructions, group B drank a low-dose BS02 preparation (1 L / ton of drinking water), group C drank a medium-dose BS02 preparation (3 L / ton of drinking water), and group D drank a high-dose BS02 preparation (5 L / ton of drinking water). The concentration of Bacillus subtilis BS02 in the BS02 preparation was 2×10 12 The experimental groups are shown in Table 4, and the feed composition of each experimental group is shown in Table 5. The experimental time was 90 days.

[0073] Table 4 Grouping of breeding experiments

[0074] Table 5 Composition and nutritional level of common feed

[0075] Note: Each kilogram of feed contains the following provided by premix: iron 120mg, zinc 115mg, copper 10mg, manganese 10mg, selenium 0.3mg, iodine 0.3mg, vitamin A 6450IU, vitamin D3 4350IU, vitamin E 20IU, vitamin K3mg, choline 500mg, niacin 25mg, biotin 2.5mg, folic acid 0.5mg.

[0076] 1) Growth performance Each pig was weighed on an empty stomach at the beginning of the experiment and at the end of the experiment. The average daily weight gain was calculated based on the obtained figures. During the experiment, the feed consumption was recorded every day. At the end of the experiment, the skin color and coat condition of each pig were evaluated. The feed intake was counted in units of repetitions, and the average feed intake of each repetition was calculated. The feed conversion rate was calculated based on the daily weight gain and the average feed intake.

[0077] Average daily gain (ADG) = total weight gain / (number of piglets × number of test days), Average daily feed intake (ADFI) = total weight of feed consumed / (number of piglets × number of experimental days), Feed to Grain Ratio (F / G) = Average Daily Feed Intake (ADFI) / Average Daily Gain (ADG).

[0078] The results of one-way ANOVA on growth performance are shown in Table 6.

[0079] Table 6 Effects of BS02 on growth performance of weaned piglets

[0080] Note: Different lowercase letters after the same data indicate significant differences (P<0.05), and different uppercase letters indicate extremely significant differences (P<0.01), the same below.

[0081] There were no significant differences in initial body weight among the four groups (P>0.05). Final body weights of groups A, B, C, and D were significantly higher than those of the CK control group (P<0.05), with group C achieving the highest final body weight, at 63.28±0.63 kg. Average daily gain (ADG) of groups A, B, C, and D was significantly higher than that of the control group (P<0.05), with both groups C and D achieving extremely significant ADG (P<0.01), and group C achieving the highest ADG, at 0.62±0.22 kg / day. Group C had the lowest feed-to-meat ratio and the highest feed conversion rate, both significantly different from those of the control group. The experimental data showed that the addition of a medium-dose BS02 preparation (3 L / t) to drinking water significantly improved the growth performance of weaned piglets. Compared with the groups receiving no antibiotics, the probiotic preparation, and the antibiotics-only group, the ADG increased by 14.81% and 8.77%, respectively, and the feed-to-meat ratio decreased by 0.2 and 0.1, respectively.

[0082] 2) Diarrhea rate From the beginning to the end of the experiment, the health and defecation patterns of the pigs were observed and recorded daily. The number of individuals with diarrhea and their duration were recorded. Diarrhea was determined by unformed feces and visible dry or wet feces adhering to the anus. Finally, the number of piglets with diarrhea was calculated per group (one pig with diarrhea for one day was counted as one episode of diarrhea).

[0083] Diarrhea rate (%) = (total number of diarrhea episodes per day in a group of pigs during the experimental period / (total number of pigs in a group during the experimental period x number of days of feeding)).

[0084] The experimental results of piglet diarrhea rate are shown in Table 7.

[0085] Table 7 Effect of BS02 on diarrhea rate in piglets

[0086] Note: Different lowercase letters after the same data indicate significant differences (P<0.05), and different uppercase letters indicate extremely significant differences (P<0.01), the same below.

[0087] Compared with the control group, adding BS02 to drinking water significantly reduced the diarrhea rate in piglets (P<0.05). Groups C and D also showed significantly lower diarrhea rates compared to the antibiotic group (Group A) (P<0.05). Group C had the lowest diarrhea rate in piglets, demonstrating a strong anti-diarrhea effect. Experimental data showed that adding a medium dose of BS02 probiotics (3 L / t) to drinking water significantly reduced diarrhea rates in piglets, with diarrhea rates decreasing by 26.57% and 8.96% compared to the no antibiotic group, the probiotic group, and the antibiotic group alone, respectively.

[0088] 3) Biochemical indicators At the end of the experiment, two pigs from each replicate were randomly selected and fasted. Approximately 10 ml of blood was collected from the anterior vena cava for immune marker determination. A 10 ml tube of blood was placed in a culture dish in a 37°C water bath for 30 minutes. The separated serum was aspirated into a centrifuge tube and centrifuged at 3000 rpm for 15 minutes to obtain serum samples. Each serum sample was aliquoted into a centrifuge tube, immersed in liquid nitrogen, rapidly frozen, and stored in a -80°C freezer until further use. Serum alkaline phosphatase and urea nitrogen levels were measured using an automated biochemical analyzer.

[0089] The effects of BS02 on the biochemical indices of piglets are shown in Table 8.

[0090] Table 8 Effects of BS02 on biochemical parameters of piglets

[0091] The results in Table 8 show that adding BS02 to drinking water significantly increased serum alkaline phosphatase activity in piglets (P < 0.05). Compared with the control and antibiotic groups, serum alkaline phosphatase activity in piglets in Groups C and D was significantly higher (P < 0.05). Furthermore, with increasing BS02 dosage, blood alkaline phosphatase activity in piglets gradually increased. In this experiment, the 3 L / t BS02 drinking water group had the highest blood alkaline phosphatase activity. Compared with the control group, the BS02-added drinking water groups significantly reduced serum urea nitrogen concentrations in weaned piglets (P < 0.05). The 3 L / t BS02 drinking water group had the lowest serum urea nitrogen concentration. Compared with the antibiotic group, serum urea nitrogen concentrations in Groups C and D were extremely significantly lower (P < 0.01). There was no significant difference between the 1 L / t BS02 drinking water group and the antibiotic group. Serum urea nitrogen concentrations varied significantly among the BS02 drinking water groups, with the 3 L / t BS02 drinking water group having the lowest concentration.

[0092] 4) Immune indicators On the evening of the 117th day of the experiment, food was stopped at 6:00 PM. After fasting for 24 hours, 10 mL of blood was collected from the anterior vena cava of one pig randomly selected from each replicate. The tube was tilted, allowed to rest for 30 minutes, and then centrifuged at 3000 rpm for 10 minutes. 1–1.5 mL of the supernatant was aspirated and transferred to a 1.5 mL centrifuge tube, labeled with the group date, and stored in a refrigerator at −20°C. Serum immunoglobulin A, immunoglobulin G, and immunoglobulin M activity were measured using a semiautomatic biochemical analyzer.

[0093] The effects of BS02 on immune indicators of piglets are shown in Table 9.

[0094] Table 9 Effects of BS02 on immune parameters of piglets

[0095] The results in Table 9 show that the serum immunoglobulin concentration of piglets in the group using BS02 added to drinking water was significantly increased (P<0.05), and increased with the increase of the amount of BS02 additive. The IgA, IgM, and IgG concentrations of each BS02 group and antibiotic group were higher than those of the control group, among which Group C and Group D were extremely significantly higher than those of the control group and antibiotic group (P<0.01), and the immune index coefficient was the highest in the 3L / t BS02 drinking water group.

[0096] 5) Intestinal microbial population At the end of the experiment, each group of experimental pigs were randomly selected and one pig was killed for each repetition. The whole body was disinfected and the intestinal contents were dissected under aseptic operation. About 2g of intestinal contents were collected and placed in a 1.5ml sterile centrifuge tube. The intestinal contents were diluted step by step with diluent to 10 -8 After inoculation, the samples were incubated under appropriate temperature and humidity conditions to determine the number of Escherichia coli, Bifidobacterium, and Lactobacillus. Microorganisms in the intestinal contents were cultured and counted, using a dilution that produced 30-300 colonies on the plate. Bacterial counts were calculated using the plate colony count method and expressed as the logarithm of the number of bacteria per gram of intestinal contents (1g CFU) / gram.

[0097] Culture of E. coli: Use a pipette to take the intestinal contents culture medium 10 -6 , 10 -7 and 10 -8 The diluted solution was spread on MacConkey medium plates (three replicates were set for each dilution stage and numbered simultaneously). After the coated plates were placed in a 37°C constant temperature incubator for aerobic culture for 24 hours, colonies that were pink or red, with a smooth and raised surface, neat and opaque edges, a soft and sticky texture, and a diameter of 0.9-3.1 mm were selected for counting.

[0098] Lactobacillus: Use a pipette to remove the intestinal contents culture medium 10 -6 , 10 -7 and 10 -8 0.1 ml of the dilution solution was spread on an MRS agar plate and cultured aerobically in a 37°C constant temperature incubator for 48 hours. Colonies with a milky white, slightly yellowish color, smooth and convex surface, neat and opaque edges, soft texture and a diameter of 0.5-2.8 mm were selected for counting.

[0099] Bifidobacterium: Use a pipette to remove the intestinal contents culture medium 10 -6 , 10 -7 and 10 -8 The diluted solution was spread on a plate of bifidobacterium isolation selective culture medium, and after anaerobic culture in a 37°C constant temperature incubator for 48 hours, milky white colonies with smooth and raised surfaces, neat and opaque edges, soft texture, and a diameter of 0.6-2.6 mm were selected for counting.

[0100] The results of BS02 on piglet intestinal microorganisms are shown in Table 10.

[0101] Table 10 Effects of BS02 on intestinal microorganisms in piglets

[0102] Note: The results are expressed as log10 CFU / g. Different lowercase letters after the same data indicate significant differences (P<0.05), and different uppercase letters indicate extremely significant differences (P<0.01). The same below.

[0103] Table 10 shows that the addition of BS02 promotes the growth of bifidobacteria in the piglet intestine and increases the number of viable bifidobacteria. Compared with the control group, the groups supplemented with 1L / t, 3L / t, and 5L / t of BS02 significantly increased the number of bifidobacteria in the piglet intestine (P < 0.05). Compared with the antibiotic group, the addition of BS02 to drinking water significantly increased the number of bifidobacteria in the intestine (P < 0.05). In particular, the group supplemented with 3L / t of BS02 significantly increased the number of bifidobacteria in the intestine (P < 0.01). Compared with the control group, the number of bifidobacteria decreased in the antibiotic group, indicating that the antibiotic treatment may damage beneficial intestinal bacteria.

[0104] The average Lactobacillus counts in the groups supplemented with BS02 were higher than those in the control group, indicating that BS02 supplementation in drinking water promotes the growth of Lactobacillus in the piglets' intestines and increases the number of viable Lactobacillus. Compared with the control group, groups B, C, and D significantly increased the number of Lactobacillus in the piglets' intestines (P < 0.05). Compared with the antibiotic group, the group supplemented with 3 L / t of BS02 significantly increased the number of Lactobacillus in the intestines (P < 0.05). Compared with the control group, the number of Lactobacillus in the antibiotic group decreased, suggesting that antibiotics may damage beneficial intestinal bacteria.

[0105] 6) Intestinal structure After the experiment, five pigs were selected from each group and slaughtered. Tissue samples approximately 2 cm long were carefully excised from the duodenum, jejunum, and mid-ileum. The samples were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE) for mounting. The samples were observed, imaged, and photographed using a bio-optical microscope combined with the NIS-Elements high-definition color image analysis system. The height (VH) and crypt depth (CD) of 10 complete villi were measured, and the ratio of villus height to crypt depth (VH / CD) was calculated.

[0106] The results of the effects of BS02 on the intestinal structure of weaned piglets are shown in Table 11.

[0107] Table 11 Effects of BS02 on intestinal structure of weaned piglets

[0108] Note: Different lowercase letters after the same data indicate significant differences (P<0.05), and different uppercase letters indicate extremely significant differences (P<0.01), the same below.

[0109] As shown in the results of Table 11, compared with the control group, Group B, Group C and Group D could significantly increase the height of villi in the duodenum, jejunum and ileum (P<0.05), while reducing the crypt depth (P<0.05). There was no significant difference between the antibiotic group and the control group (P>0.05). Among them, the group with 3L / t BS02 added to drinking water could significantly improve the intestinal structure and had the best effect (P<0.01).

[0110] 7) Mortality rate A total of 1,600 piglets were sampled in the clinical trial, with 320 in each test group. During the trial, 29 piglets died of viral diarrhea and bacterial dysentery. See Table 12 for details.

[0111] Table 12 Effect of BS02 on piglet mortality due to diarrhea

[0112] Experimental data show that adding 3L / t of BS02 proecological preparation to drinking water can reduce the mortality rate of piglet diarrhea.

[0113] In summary, the experimental data show that adding 3L / t of BS02 proecological preparations to drinking water can ① significantly improve the growth performance of weaned piglets. Compared with the group without any antibiotics, the group with proecological preparations and the group with only antibiotics, the average daily weight gain increased by 14.81% and 8.77%, respectively, and the feed-to-meat ratio decreased by 0.2 and 0.1, respectively; the diarrhea rate decreased by 26.57% and 8.96%, respectively; ② significantly increased the serum alkaline phosphatase activity and serum immunoglobulin concentration of piglets, and improved the immune function; ③ reduced the number of live Escherichia coli in the piglet intestine, increased the number of live beneficial bacteria Bifidobacterium and Lactobacillus, significantly increased the villus height of the duodenum, jejunum and ileum, reduced the crypt depth, and significantly improved intestinal health; ④ reduced the mortality rate of piglets caused by diarrhea.

[0114] The BS02 probiotic developed in this project has a significant alternative to antibiotics. Test data show that compared with the group receiving only antibiotics, the piglet group receiving 3L / t of BS02 probiotic in drinking water experienced an 8.77% increase in average daily weight gain, a 0.1% decrease in feed-to-meat ratio, and an 8.96% decrease in diarrhea rate. BS02 also demonstrated significant advantages in serum alkaline phosphatase activity and immunoglobulin concentrations, live Escherichia coli counts, the number of beneficial bifidobacteria and lactobacilli, and villus height and crypt depth in the duodenum, jejunum, and ileum.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0116] In summary, the technical solution of this application has the following beneficial technical effects: (1) The Bacillus subtilis BS02 provided by the present invention can significantly improve the growth performance of weaned piglets. Compared with the group without any antibiotics, the group with probiotics, and the group with only antibiotics, the average daily weight gain increased by 14.81% and 8.77%, respectively, and the feed-to-meat ratio decreased by 0.2 and 0.1, respectively; the diarrhea rate decreased by 26.57% and 8.96%, respectively; (2) The Bacillus subtilis BS02 provided by the present invention can significantly increase the serum alkaline phosphatase activity and serum immunoglobulin concentration of piglets, thereby improving immune function; (3) The Bacillus subtilis BS02 provided by the present invention can reduce the number of viable Escherichia coli in the intestines of piglets, increase the number of viable beneficial bacteria Bifidobacterium and Lactobacillus, significantly increase the height of villi in the duodenum, jejunum, and ileum, reduce the depth of crypts, and significantly improve intestinal health; (4) The Bacillus subtilis BS02 provided by the present invention can reduce the mortality rate of piglets caused by diarrhea and has the effect of replacing antibiotics.

Claims

1. A Bacillus subtilis, characterized in that The Bacillus subtilis was named Bacillus subtilis BS02 ( Bacillus subtilis BS02), the Bacillus subtilis has been deposited in the China Center for Type Culture Collection on April 11, 2025, with a deposit number of CCTCC NO: M 2025753.

2. An application of Bacillus subtilis in preparing a microbial agent, characterized in that: The Bacillus subtilis is the Bacillus subtilis BS02 according to claim 1.

3. An application of Bacillus subtilis in preparing an antibacterial agent, characterized in that: The Bacillus subtilis is the Bacillus subtilis BS02 according to claim 1.

4. A bacterial agent, characterized in that The invention comprises the Bacillus subtilis as claimed in claim 1.

5. The microbial agent according to claim 4, characterized in that In the bacterial agent, the concentration of Bacillus subtilis is 1×10 9 ~3×10 9 CFU / mL.

6. A feed, characterized in that The method comprises the bacterial agent according to claim 4 or 5.

7. The feed according to claim 6, wherein In the feed, the concentration of the bacterial agent is 1~3 L / t.

8. An antibacterial agent, characterized in that The antibacterial agent comprises the Bacillus subtilis BS02 as claimed in claim 1.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Bacillus subtilis BS02 according to claim 1.

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