Bacillus subtilis and screening method and application thereof

Abstract

This invention discloses a strain of Bacillus subtilis, its screening method, and its application. The Bacillus subtilis strain is named... Bacillus subtilis BS61, deposited on September 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 35815. The screening method included phenotypic and genomic screening. Phenotypic screening tested the strain's resistance to acid, bile salts, heat, and artificial gastrointestinal fluids. It revealed that strain BS61 exhibited significantly better antibacterial effects against various pathogens, including *Escherichia coli* K88 and *Salmonella typhimurium*, compared to the standard strain *Bacillus subtilis* BS168. It contains antibacterial secondary metabolic gene clusters such as Fengycin, Thurincin H, and Bacillibactin, but lacks aggressive virulence factors and contains only three low-abundance drug resistance genes. This invention can be applied to the preparation of livestock and poultry feed additives, with no risk of widespread drug resistance. Its use does not affect animal health or food safety, and it can replace antibiotics, improving the intestinal health of livestock and poultry.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Bacillus subtilis that inhibits a variety of pathogens, its screening methods and applications, and especially to strain screening methods and applications based on a phenotypic-genomic dual screening system, as well as the application of this strain in the preparation of natural antibacterial agents, the production of antibacterial secondary metabolites, and the replacement of antibiotics, which are applicable to scenarios such as green and healthy livestock and poultry farming. Background Technology

[0002] Pathogen contamination is a significant problem in the food industry and environmental governance. Pathogens such as Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7 can easily cause food spoilage and environmental microbial imbalance. Traditional antibiotic control methods lead to the spread of drug resistance and pose ecological safety risks. With the advancement of green development concepts, finding safe and effective antibiotic alternatives has become an urgent need for the industry.

[0003] Bacillus subtilis, as an important class of probiotics, has attracted widespread attention due to its advantages such as strong environmental tolerance, production of various antibacterial metabolites, and no residue risk. However, existing Bacillus subtilis screening methods, such as CN202510909010.5 and CN202410260140.6, largely rely on single phenotypic characteristics (such as tolerance and inhibition zone diameter), lacking precise validation at the genomic level. This leads to several defects in the screened strains: some strains appear phenotypically superior, but their genomes contain aggressive virulence factors, posing potential safety risks; some strains are rich in drug resistance genes, and long-term use may induce drug resistance in pathogens; and some strains have unstable tolerance and are easily inactivated by environmental factors (such as acid and bile salts) in practical applications. Furthermore, existing screening systems have not established a systematic comparison with standard strains, making it difficult to objectively assess the advantages of the strains. The standard strain *Bacillus subtilis* BS168 is a commonly used model strain in microbial research, with well-defined characteristics such as tolerance and antibacterial activity. However, current technologies do not comprehensively compare screened strains with BS168, failing to highlight the performance advantages of the target strain. Furthermore, traditional screening methods do not link phenotype and genotype, resulting in unclear antibacterial mechanisms and easy attenuation of antibacterial efficacy during mass production. Therefore, there is an urgent need to establish a dual screening system that considers both phenotypic advantages and genomic safety to screen for *Bacillus subtilis* strains with broad antibacterial spectrum, low safety and toxicity, low risk of drug resistance, and stable application effects. This would provide support for solving pathogen contamination problems and promoting the development of green antibacterial technologies. Currently, there are no published patents on screening *Bacillus subtilis* and its applications using phenotypic results combined with genomic analysis. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide Bacillus subtilis that inhibits a variety of pathogens, as well as its screening method and application.

[0005] The objective of this invention is achieved through the following technical solution: A strain of Bacillus subtilis, a. for Bacillus subtilis BS61, deposited on September 4, 2025 at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 35815; b. The survival rates under the conditions of pH 3.0-5.0, bile salt concentration 0.1%-0.3%, artificial gastrointestinal fluid, and 37℃-60℃ are not less than 40%, 30%, and 35%, respectively, and the viability is still more than 50% after treatment at 60℃; c. It can form an inhibition zone diameter ≥12 mm in the double-layer agar diffusion experiment, and the inhibition zone diameters for Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7 are 15.8±0.5 mm, 15.2±0.8 mm, 13.0±0.6 mm, and 20.8±0.6 mm, respectively. d. The genome size is 5,336,017 bp, the GC content is 35.27%, it contains 24 scaffolds, the N50 is 824,466 bp, and the sequencing coverage is ≥98%; e. The genome contains three major antibacterial secondary metabolic gene clusters: Fengycin (MIBiG BGC0001095), Thurincin H (MIBiGBGC0000600), and Bacillibactin (MIBiG BGC0000309); f. The genome contains no genes for aggressive virulence factors with VFDB similarity ≥70%, and only contains genes for defensive virulence factors; g. The genome contains only three drug resistance genes, FosB, BcII, and lsaB, all with relative homology ≥80%, and none of them involve extensive antibiotic resistance.

[0006] A method for screening Bacillus subtilis includes two steps: phenotypic screening and genomic screening. Phenotypic screening includes: (i) Tolerance screening: The test strain and the Bacillus subtilis standard strain BS168 were cultured separately under the following conditions, and the survival rate was determined: Fermentation media with pH 3.0, 3.5, 4.0, 4.5, and 5.0 were cultured at 37°C and 180 rpm for 24 hours. Fermentation media with bile salt concentrations of 0.1% and 0.3% were cultured at 37°C and 180 rpm for 4 hours. In artificial gastric fluid (pH 2.0, containing pepsin) and artificial intestinal fluid (pH 8.0, containing trypsin), cultured at 37°C and 180 rpm for 4 hours; After being treated in a 60℃ water bath for 10 minutes, the mixture was incubated at 37℃ and 180 rpm for 24 hours. If the survival rate of the test strain is higher than that of BS168 under any of the above conditions, then proceed to the next stage; (ii) Security screening: The blood agar streak method was used, and the plates were incubated at 37°C for 48 hours. The presence of hemolytic zones was then observed. Inoculate into nitrate broth medium and incubate at 37°C for 24 hours. If the result is negative, add zinc powder and observe whether a red precipitate is produced. If the hemolysis test and the nitro reduction test of the test strain are negative, proceed to the next stage; (iii) Screening for antibacterial activity: The double-layer agar diffusion method was used, with enterotoxigenic Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7 as indicator bacteria (concentration 1×10⁻⁶). 8 CFU / mL), punch holes (6 mm in diameter) in LB agar plates, add 100 μL of supernatant from the fermentation broth of the test strain, and incubate at 37°C for 16–24 hours; Measure the diameter of the inhibition zone. If the diameter of the inhibition zone of the test strain against at least one indicator bacterium is ≥12.0 mm and is higher than that of the inhibition zone of BS168, then proceed to the next stage. Genome screening includes: (i) Perform whole-genome sequencing on the test strain to obtain genome size, GC content, Scaffold number and N50 value; (ii) Using the antiSMASH database analysis, it was confirmed that its genome contains at least two of the following secondary metabolic gene clusters: Fengycin (MIBiG number: BGC0001095); Thurincin H (MIBiG number: BGC0000600); Bacillibactin (MIBiG number: BGC0000309); (iii) Using the VFDB database analysis, it was confirmed that there were no genes in its genome with a similarity of ≥70% to the attacking virulence factors, and only defensive virulence factors, including T7SS (VFG040867), ClpP (VFG000077), EF-Tu (VFG0460), and Bacillibactin (VFG050005). (iv) Analysis using the CARD database confirmed that its genome contained only ≤3 antibiotic resistance genes, and the amino acid similarity between each gene and the resistance protein was ≥80% but <95%; If the test strain passes all the above screening steps, the target strain is obtained.

[0007] The method wherein the test strain is derived from intestinal contents samples of healthy livestock and poultry.

[0008] The method wherein the control strain for safety screening is Staphylococcus aureus.

[0009] Compared with BS168, the method described above shows that BS61 has a survival rate that is more than 100% higher under pH 3.0 conditions, a survival rate that is more than 150% higher under 0.3% bile salt conditions, a survival rate that is more than 50% higher after high temperature treatment at 60°C, and a survival rate that is more than 25% higher in artificial gastrointestinal fluid. Compared with BS168, BS61 has an inhibition zone diameter that is at least 100% larger against Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7.

[0010] The application of Bacillus subtilis in the preparation of livestock and poultry feed additives. It is used to inhibit one or more of the following enteric pathogens: Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7. It is used in livestock and poultry farming as an alternative to antibiotics.

[0011] The application of Bacillus subtilis BS61 in the preparation of natural antibacterial agents involves obtaining a fermentation broth of BS61 through fermentation, followed by centrifugation and filtration to obtain a sterile fermentation broth.

[0012] The beneficial effects of this invention are as follows: A phenotypic-genomic dual screening system was disclosed, which not only ensures that the strain meets the phenotypic standards in terms of tolerance and antibacterial activity, but also clarifies the antibacterial mechanism, safety characteristics, and drug resistance risk through genomic validation. The screening accuracy is significantly higher than that of traditional phenotypic screening methods. The BS61 strain is resistant to acid, bile salts, high temperature, and artificial gastrointestinal fluid, and can stably colonize the gastrointestinal tract of piglets. It has a broad antibacterial spectrum and significant antibacterial effect against a variety of intestinal pathogens. The inhibition zone diameter is 12.97-20.83 mm. The genome contains a clear antibacterial secondary metabolic gene cluster, the antibacterial mechanism is clear, and the application effect is stable. The strain is non-hemolytic and non-nitro-reducing, and the genome contains no aggressive virulence factors, only defensive virulence factors. The number of drug resistance genes is only 3, with no risk of widespread drug resistance. It does not affect animal health or food safety after use. Attached Figure Description

[0013] Figure 1 This study compares the survival rates of Bacillus subtilis BS61 and BS168 in culture media with different pH values.

[0014] Figure 2 This study compares the survival rates of Bacillus subtilis BS61 and BS168 in different concentrations of bile salts.

[0015] Figure 3 This study compares the survival rates of Bacillus subtilis BS61 and BS168 in artificial gastrointestinal fluid.

[0016] Figure 4 This is a comparison of the survival rates of Bacillus subtilis BS61 and BS168 at different temperatures.

[0017] Figure 5 The results of the hemolytic test of the strains are as follows: (A) Bacillus subtilis BS61; (B) Staphylococcus aureus.

[0018] Figure 6 This is the result of the nitro reduction test.

[0019] Figure 7 The comparison of antibacterial effects is as follows: (A) Enterotoxigenic Escherichia coli K88; (B) Salmonella Typhimurium; (C) Staphylococcus aureus; (D) Enterohemorrhagic Escherichia coli O157:H7. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Example 1: Whole genome sequencing analysis of Bacillus subtilis BS61 1) Genomic characterization of Bacillus subtilis BS61 Table 1. Genomic characteristics of Bacillus subtilis in Example 1 of the present invention

[0022] Whole-genome sequencing analysis revealed that the BS61 genome has 24 scaffolds, a genome size of 5,336,017 bp, an N50 (sequences longer than N50 account for 50% of the total genome length) of 824,466 bp, and a sequencing completion rate of 98.92%.

[0023] 2) Analysis of secondary metabolite synthesis in Bacillus subtilis BS61 Table 2. Secondary metabolites predicted by the antiSMASH database for probiotics in Example 1 of the present invention.

[0024] AntiSMASH analysis of the secondary metabolite gene clusters of candidate probiotic strains revealed three highly similar secondary metabolite clusters in BS61: Fengycin (fentycin lipopeptide), Thurincin H (bacteriocin produced by Bacillus thuringiensis), and Bacillibactin (catechol-type heparin). The products of these secondary metabolite clusters generally exhibit antibacterial, antifungal, antiviral, antibiofilm, and biocontrol activities.

[0025] 3) Analysis of Bacillus subtilis BS61 virulence factor database Table 3. Virulence factor genes with a similarity greater than 70% to Bacillus subtilis in Example 1 of the present invention

[0026] The virulence factor database (VFDB) and the comprehensive antibiotic resistance database (CARD) were used to analyze the strain genomes. Table 3 shows the genes with a similarity greater than 70% in VFDB. No aggressive virulence genes with a similarity greater than 70% were found in this whole-genome analysis. The main virulence factors obtained through database analysis were defensive virulence factors, namely delivery systems and stress survival, and their main functions include regulating interbacterial signaling, nutrition, and metabolism.

[0027] 4) Analysis of the Bacillus subtilis BS61 antibiotic resistance gene database Table 4. Antibiotic resistance genes with a similarity of more than 80% to Bacillus subtilis in Example 1 of the present invention

[0028] *ARO means Antibiotic Resistance Ontology Table 4 shows the antibiotic resistance genes with a similarity greater than 80% found in the BS61 genome according to the Comprehensive Antibiotic Resistance Database (CARD). The main antibiotic resistance genes corresponding to BS61 are... FosB , BcI and lsaB The corresponding resistant antibiotics are mainly phosphonic acid antibiotics, cephalosporins, lincosamide antibiotics, pleuromutilin antibiotics, and streptogramin antibiotics.

[0029] In summary, the gene-level analysis of Bacillus subtilis BS61 indicates that it has strong antibacterial potential. Therefore, this probiotic was selected as the strain for subsequent feeding experiments to explore its function.

[0030] Example 2: Isolation and Phenotypic Screening of Bacillus subtilis BS61 Test materials Intestinal contents of healthy pigs; standard strain Bacillus subtilis BS168; indicator pathogens (Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, enterohemorrhagic Escherichia coli O157:H7); LB medium, fermentation medium, blood agar medium, nitrate broth medium; artificial gastric fluid (pH 2.0, containing pepsin), artificial intestinal fluid (pH 8.0, containing trypsin); constant temperature incubator, clean bench, centrifuge, vernier calipers, pH meter.

[0031] Experimental methods Acid tolerance test: The strain and BS168 were inoculated into fermentation media at pH 3.0, 3.5, 4.0, 4.5, and 5.0, respectively, and cultured at 37℃ and 180 rpm for 24 h. OD was then measured. 600 The value is used to calculate the survival rate.

[0032] Bile salt tolerance test: The strain and BS168 were inoculated into fermentation medium with bile salt concentrations of 0.1% and 0.3%, respectively, and cultured at 37℃ and 180 rpm for 4 h. OD was then measured. 600 The value is used to calculate the survival rate.

[0033] Tests for resistance to artificial gastrointestinal fluid: The strain and BS168 were inoculated into artificial gastric fluid and artificial intestinal fluid, respectively, and incubated at 37°C and 180 rpm for 4 hours. OD was then measured. 600 The value is used to calculate the survival rate.

[0034] High-temperature resistance test: The strain and BS168 bacterial solution were treated in water baths at 37℃ and 60℃ for 10 min respectively, and then incubated at 37℃ and 180 rpm for 24 h. OD was then measured. 600 The value is used to calculate the survival rate.

[0035] Hemolytic test: Streak the strain on blood agar plates and incubate at 37°C for 48 hours. Observe whether a hemolytic zone appears.

[0036] Nitro reduction test: Inoculate the strain into nitrate broth medium and incubate at 37°C for 24 hours. Observe the color change. If the result is negative, add zinc powder and observe again.

[0037] 3) Results and Analysis Acid resistance: such as Figure 1As shown, at pH 3.0, the survival rate of BS61 was 42%, while that of BS168 was only 18%; at pH 4.5, the survival rate of BS61 was 82%, while that of BS168 was 65%. The survival rate of BS61 was significantly higher than that of BS168 at all pH gradients.

[0038] Tolerance to bile salts: such as Figure 2 As shown, at a bile salt concentration of 0.1%, the survival rate of BS61 was 78%, while that of BS168 was 62%; at a bile salt concentration of 0.3%, the survival rate of BS61 was 23%, while that of BS168 was only 9%, indicating that BS61 has a significantly better tolerance to bile salts than BS168.

[0039] Tolerance to artificial gastrointestinal fluids: such as Figure 3 As shown, in artificial gastric fluid, the survival rate of BS61 was 72%, while that of BS168 was 51%; in artificial intestinal fluid, the survival rate of BS61 was 85%, while that of BS168 was 70%, indicating that BS61 has a stronger tolerance to gastrointestinal fluid.

[0040] High temperature resistance: such as Figure 4 As shown, after treatment at 60℃, the survival rate of BS61 was 68%, while that of BS168 was 45%, indicating that BS61 has better high-temperature stability.

[0041] Security results: such as Figure 5 As shown, BS61 showed no hemolytic zone (negative hemolytic test); as Figure 6 As shown, the nitro reduction test was negative, ruling out the risk of pathogenicity.

[0042] Example 3: Validation of the antibacterial activity of Bacillus subtilis BS61 (including comparison with BS168) 1) Test materials Experimental materials: BS61 pure culture, standard strain BS168, indicator pathogens (Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, enterohemorrhagic Escherichia coli O157:H7); LB medium, fermentation medium; constant temperature shaker, clean bench, vernier calipers.

[0043] Fermentation broth preparation: BS61 and BS168 were inoculated into fermentation medium, cultured at 37℃ and 180 rpm for 24 h, centrifuged at 6500 rpm for 10 min, the supernatant was collected, and sterilized by 0.22 μm filtration to obtain sterile fermentation broth.

[0044] 2) Test methods The double-layer agar diffusion method was used, with 10 mL of LB medium at the bottom and 10 mL of medium containing indicator bacteria (1×10⁻⁶) at the top. 8LB medium (CFU / mL) was prepared, and 100 μL of BS61 fermentation broth, BS168 fermentation broth (control), or sterile fermentation medium (blank control) were added to the wells (6 mm in diameter). The medium was incubated at 37°C for 16-24 h, and the diameter of the inhibition zone was measured. Each group was set up with 3 replicates, and the mean ± standard deviation was calculated.

[0045] Results and Analysis

[0046] As shown in Table 5 and Figure 7 As shown, the results of the antibacterial activity comparison showed that BS168 had no obvious inhibition zone (diameter <7.5mm) against the four indicator bacteria; BS61 had the largest inhibition zone diameter against enterohemorrhagic Escherichia coli O157:H7, reaching 20.83±0.59mm, and the smallest inhibition zone diameter against Staphylococcus aureus, at 12.97±0.57mm. The inhibition zone diameters against Escherichia coli K88 and Salmonella typhimurium were 15.77±0.47mm and 15.20±0.75mm, respectively, all of which were superior to BS168, indicating that BS61 has a stronger broad-spectrum antibacterial advantage.

[0047] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations and equivalent substitutions are possible. All variations and equivalent substitutions that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention are within the protection scope of this invention.

Claims

1. A strain of Bacillus subtilis, characterized by: a. for Bacillus subtilis BS61, deposited on September 4, 2025 at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 35815; b. The survival rates under the conditions of pH 3.0-5.0, bile salt concentration 0.1%-0.3%, artificial gastrointestinal fluid, and 37℃-60℃ are not less than 40%, 30%, and 35%, respectively, and the viability is still more than 50% after treatment at 60℃; c. It can form an inhibition zone diameter ≥12 mm in the double-layer agar diffusion experiment, and the inhibition zone diameters for Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7 are 15.8±0.5 mm, 15.2±0.8 mm, 13.0±0.6 mm, and 20.8±0.6 mm, respectively. d. The genome size is 5,336,017 bp, the GC content is 35.27%, it contains 24 scaffolds, the N50 is 824,466 bp, and the sequencing coverage is ≥98%; e. The genome contains three major antibacterial secondary metabolic gene clusters: Fengycin (MIBiG BGC0001095), Thurincin H (MIBiG BGC0000600), and Bacillibactin (MIBiG BGC0000309); f. The genome contains no genes for aggressive virulence factors with VFDB similarity ≥70%, and only contains genes for defensive virulence factors; g. The genome contains only three drug resistance genes, FosB, BcII, and lsaB, all with relative homology ≥80%, and none of them involve extensive antibiotic resistance.

2. A method for screening Bacillus subtilis, characterized in that, It includes two steps: phenotypic screening and genome screening. Phenotypic screening includes: (i) Tolerance screening: The test strain and the Bacillus subtilis standard strain BS168 were cultured separately under the following conditions, and the survival rate was determined: Fermentation media with pH 3.0, 3.5, 4.0, 4.5, and 5.0 were cultured at 37°C and 180 rpm for 24 hours. Fermentation media with bile salt concentrations of 0.1% and 0.3% were cultured at 37°C and 180 rpm for 4 hours. In an artificial gastric fluid containing pepsin at pH 2.0 and an artificial intestinal fluid containing trypsin at pH 8.0, the mixture was incubated at 37°C and 180 rpm for 4 hours. After being treated in a 60℃ water bath for 10 minutes, the mixture was incubated at 37℃ and 180 rpm for 24 hours. If the survival rate of the test strain is higher than that of BS168 under any of the above conditions, then proceed to the next stage; (ii) Security screening: The blood agar streak method was used, and the plates were incubated at 37°C for 48 hours. The presence of hemolytic zones was then observed. Inoculate into nitrate broth medium and incubate at 37°C for 24 hours. If the result is negative, add zinc powder and observe whether a red precipitate is produced. If the hemolysis test and the nitro reduction test of the test strain are negative, proceed to the next stage; (iii) Screening for antibacterial activity: The double-layer agar diffusion method was used, with enterotoxigenic Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7 as indicator bacteria, at a concentration of 1×10⁻⁶. 8 CFU / mL, punch 6 mm diameter holes in LB agar plates, add 100 μL supernatant of the fermentation broth of the test strain, and incubate at 37°C for 16–24 hours. Measure the diameter of the inhibition zone. If the diameter of the inhibition zone of the test strain against at least one indicator bacterium is ≥12.0 mm and is higher than that of the inhibition zone of BS168, then proceed to the next stage. Genome screening includes: (i) Perform whole-genome sequencing on the test strain to obtain genome size, GC content, Scaffold number and N50 value; (ii) Using the antiSMASH database analysis, it was confirmed that its genome contains at least two of the following secondary metabolic gene clusters: Fengycin (MIBiG number: BGC0001095); Thurincin H (MIBiG number: BGC0000600); Bacillibactin (MIBiG number: BGC0000309); (iii) Using the VFDB database analysis, it was confirmed that there were no genes in its genome with a similarity of ≥70% to the attacking virulence factors, and only defensive virulence factors, including T7SS (VFG040867), ClpP (VFG000077), EF-Tu (VFG0460), and Bacillibactin (VFG050005). (iv) Analysis using the CARD database confirmed that its genome contained only ≤3 antibiotic resistance genes, and the amino acid similarity between each gene and the resistance protein was ≥80% but <95%; If the test strain passes all the above screening steps, the target strain is obtained.

3. The method according to claim 2, characterized in that, The bacterial strains to be tested were derived from intestinal contents samples of healthy livestock and poultry.

4. The method according to claim 2, characterized in that, The control strain used in the safety screening was Staphylococcus aureus.

5. The method according to claim 2, characterized in that, Compared with BS168, BS61 showed a survival rate that was more than 100% higher at pH 3.0, more than 150% higher at 0.3% bile salts, more than 50% higher after high-temperature treatment at 60°C, and more than 25% higher in artificial gastrointestinal fluid. Compared with BS168, BS61 has an inhibition zone diameter that is at least 100% larger against Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7.

6. The application of Bacillus subtilis according to claim 1 in the preparation of livestock and poultry feed additives, characterized in that: It is used to inhibit one or more of the following enteric pathogens: Escherichia coli K88, Salmonella typhimurium, Staphylococcus aureus, and enterohemorrhagic Escherichia coli O157:H7. It is used in livestock and poultry farming as an alternative to antibiotics.

7. The application of Bacillus subtilis BS61 according to claim 1 in the preparation of natural antibacterial agents, characterized in that: The fermentation broth of BS61 was obtained through fermentation, and then sterile fermentation broth was obtained by centrifugation and filtration.

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