Bacillus velezensis Scm3 and application thereof

Abstract

The invention discloses bacillus velezensis Scm3 and application thereof.The bacillus velezensis Scm3 is obtained by screening a strain Scm with an inhibition effect on aeromonas hydrophila from turbot intestinal contents and then conducting ultraviolet mutagenesis on the strain to obtain a strain Scm3 with an enhanced antagonism effect on aeromonas hydrophila, and the strain Scm3 is applied to the preparation of the bacillus velezensis Scm3 and the application of the bacillus velezensis Scm3 to prepare the bacillus velezensis Scm3. 16SrDNA (deoxyribonucleic acid) sequence identification and genome sequencing prove that the strain is bacillus velezensis, and the inhibition mechanism of the bacillus velezensis Scm3 on aeromonas hydrophila is researched by a non-contact co-culture method.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus Velezii Scm3 and application thereof. Background Art

[0002] Aeromonas hydrophila belongs to the genus Aeromonas of the family Vibrio, a Gram-negative short rod with a single flagellum, no spores, and no capsule. It is widely distributed in nature, especially in freshwater environments, and is one of the main pathogens that cause hemorrhagic disease in aquaculture. Aquatic animals with hemorrhagic disease will have inflammation and bleeding in organs such as the intestines, with a very high mortality rate. For a long time in the past, antibiotics have been widely used to treat diseases caused by Aeromonas hydrophila, but with the abuse of antibiotics, Aeromonas hydrophila has become less and less sensitive to antibiotics, and in actual application, it has developed extremely serious cross-resistance, affecting the prevention and treatment effect of antibiotics, and bringing greater difficulties to the prevention and treatment of aquaculture diseases. In the breeding process, once drug resistance is generated, it will be difficult to disappear, and it will cause more complex drug resistance mechanisms, with far-reaching effects.

[0003] In order to ensure the sustainable and healthy development of aquaculture and solve the problem of resistance pollution caused by the abuse of antibiotics, biological control methods are one of the more effective methods at present, which can be divided into methods such as using bacteria to control bacteria, using insects to control insects, and using bacteria to control insects. This method uses the relationship between biological species to inhibit another or another type of organism with one or one type of organism, which is incomparable to non-biological control and has the characteristics of environmental friendliness and sustainability. Bacillus is ubiquitous in nature and has advantages such as strong resistance to stress. It can survive stably in harsh environments and is usually selected as a bacterium for human probiotics.

[0004] Bacillus Velez is a new species of the genus Bacillus, which has excellent growth characteristics, strong antibacterial activity and a broad antibacterial spectrum. In addition, its spores have significant resistance and dormancy ability, so Bacillus Velez has extremely high development and utilization value. Summary of the invention

[0005] The invention screens a Velez subtilis strain having an inhibitory effect on Aeromonas hydrophila from the intestinal contents of turbot, and obtains a Velez subtilis Scm3 strain having an obviously enhanced antagonistic effect on Aeromonas hydrophila by ultraviolet mutagenesis of the strain.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first object of the present invention is to provide a Bacillus velezensis Scm3 strain, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 30, 2024, with a deposit number of CGMCC No.31790.

[0008] The second object of the present invention is to use the Bacillus velezensis Scm3 strain in inhibiting pathogenic bacteria for purposes other than disease diagnosis and treatment.

[0009] Furthermore, the pathogen is specifically Edwardsiella piscicida.

[0010] Furthermore, the pathogenic bacteria include one or more of Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, Salmonella typhi, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio anguillarum.

[0011] The third object of the present invention is to provide an antibacterial agent, which comprises Bacillus velezensis Scm3 and / or a bacterial solution of Bacillus velezensis Scm3.

[0012] Furthermore, the bacterial liquid is the fermentation supernatant.

[0013] The following beneficial effects can be obtained through the above technical solution:

[0014] The invention involves screening a strain Scm with an inhibitory effect on Aeromonas hydrophila from the intestinal contents of turbot, then subjecting the strain to ultraviolet mutagenesis to obtain a strain Scm3 with enhanced antagonism to Aeromonas hydrophila, proving that the strain is Bacillus Velezii through 16SrDNA sequence identification and genome sequencing, and studying the inhibitory mechanism of Bacillus Velezii Scm3 on Aeromonas hydrophila through a non-contact co-culture method.

[0015] The Bacillus Velezii Scm3 provided by the present invention has the characteristics of a safe strain. The strain does not form a hemolytic ring on a blood plate and is sensitive to 18 antibiotics such as erythromycin, kanamycin, ampicillin, and cefoperazone. In addition, analysis of virulence factors at the genetic level also proves that the strain does not contain invasive putative virulence factors.

[0016] The Bacillus Velezii Scm3 provided by the present invention has a wide antibacterial spectrum, and can not only inhibit Aeromonas hydrophila, but also has a certain inhibitory effect on Edwardsiella tarda, Edwardsiella piscicida, Vibrio alginolyticus, Salmonella typhi, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio anguillarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the antagonistic effect of strain Scm3 on Aeromonas hydrophila.

[0018] Figure 2 This is a graph showing the antagonistic effect of strain Scm3 on pathogenic bacteria preserved in this laboratory.

[0019] Figure 3 A phylogenetic tree was constructed for phylogenetic analysis of strain Scm3 based on 16S rDNA sequence.

[0020] Figure 4 This is the species distribution map of strain Scm3 based on NR database analysis.

[0021] Figure 5 This is the growth curve of Bacillus velez Scm3.

[0022] Figure 6 This is the growth curve of Aeromonas hydrophila.

[0023] Figure 7 This is a graph showing the effect of Bacillus Velez Scm3 on the survival rate of Aeromonas hydrophila.

[0024] Figure 8 This is the result of PI staining.

[0025] Fig. 9 This is a graph showing the effect of Bacillus Velez Scm3 on the biofilm formation ability of Aeromonas hydrophila.

[0026] Fig.10 This is a graph showing the effect of Bacillus Velez Scm3 on the metabolic activity of Aeromonas hydrophila cells.

[0027] Fig.11 This is a graph showing the growth results of Bacillus Velez Scm3 on blood agar plates.

[0028] Fig.12This is the analysis result of antibacterial active substances of Bacillus velez Scm3.

[0029] Fig.13 The graph shows the antibacterial results of ammonium sulfate precipitation with different saturations.

[0030] Fig.14 This is a graph showing the effect of the antibacterial substances of Bacillus velezensis Scm3 on the survival rate of Edwardsiella fish-killing bacteria.

[0031] Fig.15 This is a graph showing the effect of the antibacterial substances of Bacillus velezensis Scm3 on the biofilm formation ability of Edwardsiella fish-killing bacteria.

[0032] Fig.16 This is a graph showing the effects of the antibacterial substances of Bacillus velezensis Scm3 on the metabolic activity of Edwardsiella fish-killing cells. DETAILED DESCRIPTION

[0033] The following is combined with Figure 1-16 The present invention is further described as follows:

[0034] Example 1

[0035] Breeding and identification of Bacillus velez Scm3

[0036] (1) Culture of turbot intestinal contents

[0037] Under sterile conditions, the intestinal contents of turbot were taken and chopped, and placed in a conical flask containing 50 mL of LB liquid culture medium, and cultured at 30°C and 180 rpm in a shaking incubator for 72 h.

[0038] (2) Isolation and purification of strains

[0039] Heat shock the culture solution in a 70°C water bath for 15 minutes. Under sterile conditions, use a pipette to aspirate 0.1 mL of the culture solution after heat shock onto the LB solid medium. Then, invert it in a constant temperature incubator and incubate it at 37°C for 12 hours before observing. Select a culture medium with clear colonies and a colony count of 30-300 cfu, pick out single colonies of different sizes and shapes, spot them on the LB solid medium, and then invert it at 37°C for 20 hours.

[0040] (3) Primary and secondary screening of strains

[0041] Adjust the OD of Aeromonas hydrophila suspension with sterile water. 600=1, collect the fermentation supernatant of the strain to be tested, and use the agar diffusion method to observe the size of the inhibition zone. The larger the diameter of the inhibition zone, the stronger its antibacterial ability. During the initial screening, a strain with antagonistic effect on Aeromonas hydrophila was determined for UV mutagenesis, and the strain was named Scm. Collect the fermentation supernatant of the mutated strain, use the agar diffusion method to observe the size of the inhibition zone, select the strain with the largest inhibition zone, and conduct subsequent experiments. The strain was named Scm3.

[0042] Depend on Figure 1 It can be seen that the strain Scm before mutation has a significant antibacterial effect on Aeromonas hydrophila, with an inhibition diameter of about 1.2 cm; the strain Scm3 after mutation has a significantly enhanced antibacterial effect on Aeromonas hydrophila, with an inhibition diameter of about 2 cm.

[0043] (4) Antibacterial spectrum experiment

[0044] The present invention analyzes the inhibition of strain Scm3 on other pathogens stored in the laboratory, and adjusts the OD of the pathogenic bacteria suspension with sterile water. 600 =1, collect the fermentation supernatant of the strain to be tested, and use the agar diffusion method to observe the size of the inhibition zone. The larger the diameter of the inhibition zone, the stronger its antibacterial ability.

[0045] Depend on Figure 2 It can be seen that the antibacterial diameter of the supernatant of strain Scm3 against Salmonella typhi is about 1.8 cm; the antibacterial diameter against Pseudomonas aeruginosa is about 1.6 cm; the antibacterial diameter against Listeria monocytogenes is about 1.7 cm; the antibacterial diameter against Edwardsiella tarda is about 1.8 cm; the antibacterial diameter against Edwardsiella piscicola is about 2 cm, the antibacterial diameter against Vibrio anguillarum is about 1 cm, the antibacterial diameter against Staphylococcus aureus is about 1.2 cm, and the antibacterial diameter against Vibrio alginolyticus is about 2 cm.

[0046] (5) Identification of strain Scm3

[0047] The 16S rDNA sequence of strain Scm3 was amplified using bacterial universal primers (16s-27F / 16S-1492R), and the amplified product was sent to Anshengda Biotechnology Co., Ltd. for sequencing. The sequencing results are as follows:

[0048] TGGCTCCTAAAAGGTTACCTCAGCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGG

[0049] GCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAG

[0050] CGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGT

[0051] GGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGT

[0052] AGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA

[0053] CCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCT

[0054] CGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCT

[0055] GTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACC

[0056] TGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCC

[0057] CCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGC

[0058] GTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGC

[0059] GTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGT

[0060] TACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCG

[0061] CTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCT

[0062] CCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTAC

[0063] GCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGT

[0064] TAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTT

[0065] CTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTC

[0066] CGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGC

[0067] CGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTG

[0068] GTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCC

[0069] GAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGT

[0070] TTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGT。

[0071] The phylogenetic tree constructed based on the sequencing results (see Figure 3), the results showed that the 16S rDNA sequence of strain Scm3 and Bacillus velezensis was 100% similar. The 16S rDNA sequence of strain Scm3 has been submitted to GenBank with the accession number PQ471301. In addition, the whole genome homology results further confirmed that strain Scm3 can be attributed to the species Bacillus velezensis (see Figure 4 ). The strain Scm3 was deposited in the China General Microbiological Culture Collection on August 30, 2024, with the address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the deposit number is CGMCC No.31790.

[0072] Example 2

[0073] Investigating the antibacterial mechanism of Bacillus Velezii Scm3 against Aeromonas hydrophila based on non-contact co-culture

[0074] (1) Growth curve determination

[0075] Bacillus Velezii Scm3 and Aeromonas hydrophila were inoculated into 50 mL of sterilized LB liquid medium at 1% inoculum, and then cultured in a shaking incubator at 30°C and 180 rpm. The bacterial liquid was taken at 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, and 48h, and the OD was measured using a spectrophotometer. 600 The data were used to draw the growth curve of the corresponding strain using Origin software (see Figure 5 , Figure 6 ). Each group of experiments was set up with 3 parallels.

[0076] (2) Effect of Bacillus Velezii Scm3 on the survival rate of Aeromonas hydrophila

[0077] At a 1% inoculation rate, Bacillus Velez Scm3 and Aeromonas hydrophila were cultured in a shaking incubator (30° C., 180 rpm) to mid-logarithmic phase, respectively, and reserved as seed solution for later use.

[0078] The non-contact co-culture of Bacillus Velez Scm3 and Aeromonas hydrophila was completed using a transwell 6-well plate. Experimental group: lower chamber: 20 μL pathogenic bacteria solution was mixed with 2 mL LB liquid medium, upper chamber: 20 μL antagonistic bacteria solution was mixed with 1.3 mL LB liquid medium; control group: lower chamber: 20 μL pathogenic bacteria solution was mixed with 2 mL LB liquid medium, upper chamber: 1.32 mL LB liquid medium was added; the transwell 6-well plate was placed in a 37°C incubator and cultured for 3h, 6h, 9h, 12h, 15h, and 18h ​​respectively. At the corresponding time points, the bacterial suspensions of the experimental group and the control group were aspirated, diluted and spread, and the survival rate of Aeromonas hydrophila was calculated.

[0079] Survival rate of Aeromonas hydrophila = (number of colonies in the experimental group / number of colonies in the control group) × 100%

[0080] Depend on Figure 7 It can be seen that as the co-culture time increases, the survival rate of pathogens decreases. When the co-culture time is 18h, the survival rate of pathogens is close to 0.

[0081] (3) PI staining

[0082] Propidium iodide (PI) is a fluorescent dye that stains DNA and does not cross intact cell membranes. However, if the cell membrane is damaged, PI can cross the cell membrane and intercalate into DNA, releasing red fluorescence. Therefore, PI staining can be used to determine the integrity of the cell membrane of a strain.

[0083] At a 1% inoculation rate, Bacillus Velez Scm3 and Aeromonas hydrophila were cultured in a shaking incubator (30° C., 180 rpm) to mid-logarithmic phase, respectively, and reserved as seed solution for later use.

[0084] Experimental group: lower chamber: mix 20 μL pathogenic bacteria solution with 2 mL LB liquid culture medium, upper chamber: mix 20 μL antagonistic bacteria solution with 1.3 mL LB liquid culture medium; control group: lower chamber: mix 20 μL pathogenic bacteria solution with 2 mL LB liquid culture medium, upper chamber: add 1.32 mL LB liquid culture medium; place the transwell 6-well plate in a 37°C incubator and culture for 12 h and 24 h, respectively.

[0085] At the end of the culture, the upper chamber of the transwell 6-well plate was removed with tweezers, and the bacterial suspension in the lower chamber of the transwell 6-well plate was carefully pipetted into a 2 mL EP tube. The cells were centrifuged at 5000 rpm and 4°C for 3 min, and the supernatant was discarded. Then, the cells were washed twice with 1 mL PBS (pH = 7.0, 10 mmol / L). Finally, the cells were resuspended with PBS buffer and the OD was calculated. 600= 0.2 bacterial resuspension. Pipette 600 μL of bacterial resuspension and 6 μL of PI staining solution (PI staining solution specification: 1 mg / mL) into the confocal culture dish, place the confocal culture dish in a 37°C biochemical incubator for 30 minutes, and observe the fluorescence under a fluorescence microscope.

[0086] Depend on Figure 8 It can be seen that compared with the control group, a large amount of red fluorescence appeared in the experimental group after co-culture for 12h or 24h, indicating that Bacillus Velezii Scm3 can destroy the cell membrane integrity of Aeromonas hydrophila.

[0087] (4) Effect of Bacillus velezensis Scm3 on the biofilm formation ability of Aeromonas hydrophila

[0088] At a 1% inoculation rate, Bacillus Velez Scm3 and Aeromonas hydrophila were cultured in a shaking incubator (30° C., 180 rpm) to mid-logarithmic phase, respectively, and reserved as seed solution for later use.

[0089] Experimental group: lower chamber: mix 20 μL pathogenic bacteria solution with 2 mL LB liquid culture medium, upper chamber: mix 20 μL antagonistic bacteria solution with 1.3 mL LB liquid culture medium; control group: lower chamber: mix 20 μL pathogenic bacteria solution with 2 mL LB liquid culture medium, upper chamber: add 1.32 mL LB liquid culture medium; place the transwell 6-well plate in a 37°C incubator and culture for 6 h, 12 h, and 24 h, respectively.

[0090] At the end of the culture, the upper chamber of the transwell 6-well plate was removed with tweezers, and the culture medium in the lower chamber of the transwell 6-well plate was carefully aspirated with a pipette, and the supernatant was discarded. Then, the cells were washed 3 times with 1 mL PBS (pH=7, 10 mmol / L); then, the transwell 6-well was placed in a 60°C oven for about 15 min.

[0091] Stain with 1 mL 1% crystal violet for 30 min, aspirate the staining solution, and then wash the cells 3 times with PBS (pH = 7.0, 10 mmol / L). Then, place the transwell 6-well plate in 37 ° C to dry for 15 min; finally, add 1 mL 95% ethanol to the lower chamber of the transwell 6-well plate, incubate in a 37 ° C incubator for 40 min, and then aspirate 200 μL of the liquid of the experimental group and the control group at the corresponding time point into a 96-well plate, and use an enzyme-labeled instrument to measure the OD 595 Absorbance.

[0092] Depend on Fig. 9It can be seen that after 12h and 24h of co-culture, the amount of biofilm formed by Aeromonas hydrophila decreased by 14.9% and 21.8%, respectively. As the co-culture time increases, the amount of biofilm formed gradually decreases, showing a negative correlation. These results indicate that Bacillus Velezii Scm3 can effectively destroy the biofilm formation of Aeromonas hydrophila.

[0093] (5) Effects of Bacillus velezensis Scm3 on the metabolic activity of Aeromonas hydrophila cells

[0094] At a 1% inoculation rate, Bacillus Velez Scm3 and Aeromonas hydrophila were cultured in a shaking incubator (30° C., 180 rpm) to mid-logarithmic phase, respectively, and reserved as seed solution for later use.

[0095] Experimental group: lower chamber: 20 μL pathogenic bacteria solution was mixed with 2 mL LB liquid culture medium, upper chamber: 20 μL antagonistic bacteria solution was mixed with 1.3 mL LB liquid culture medium; control group: lower chamber: 20 μL pathogenic bacteria solution was mixed with 2 mL LB liquid culture medium, upper chamber: 1.32 mL LB liquid culture medium was added; blank group only added LB liquid culture medium, and the transwell 6-well plate was placed in a 37°C incubator and cultured for 6 h, 12 h, and 24 h, respectively.

[0096] At the end of the culture, the upper chamber of the transwell 6-well plate was removed with tweezers, and the medium in the lower chamber of the transwell 6-well plate was carefully aspirated with a pipette, and the supernatant was discarded, and then the cells were washed twice with 1mL PBS (pH=7.0, 10mmol / L). Finally, a bacterial suspension was prepared with 1mL PBS buffer, and 200μL of the liquid of the experimental group, control group, and blank group at the corresponding time point was aspirated into a 96-well plate, and then 10μL of CCK8 solvent was added to each well. Finally, the 96-well plate was placed in a 37℃ incubator, and the culture was continued for 4h. The OD value of each well was measured at 450nm, and the result was repeated 3 times. The cell metabolic activity was calculated according to the following formula.

[0097] Cell viability = [(OD 实验组 -OD 空白 ) / (OD 对照组 -OD 空白 )]×100%

[0098] Depend on Fig.10 It can be seen that: with the extension of co-culture time, the metabolic activity of Aeromonas hydrophila becomes lower and lower. When the co-culture time is 24h, the metabolic activity of the cells is almost lost.

[0099] Example 3

[0100] Safety evaluation of Bacillus velez Scm3

[0101] (1) Drug sensitivity test

[0102] According to the inoculation amount of 1%, the bacterial liquid of Bacillus Velezii Scm3 was drawn into a liquid test tube containing LB, and activated in a shaking table at 30°C and 180 rpm for about 20 hours. The OD value of the strain liquid was adjusted with sterile saline. 600 = 1, soak a cotton swab in OD 600 = 1 bacterial suspension, remove excess bacterial liquid by rotating and squeezing the tube wall, and evenly apply it on the surface of LB liquid culture medium, apply it multiple times at different angles, cover the dish, and dry it at room temperature for 2-3 minutes. When the surface moisture is absorbed by agar, put the tweezers on the alcohol lamp to burn it, and use the tweezers to stick the drug-sensitive paper on the surface of the culture medium, and press the tweezers slightly to prevent it from falling off. Cover the dish with a lid and incubate it upside down in a 37℃ incubator for about 24 hours.

[0103] Table 1: Antibiotic sensitivity test of Bacillus velez Scm3

[0104]

[0105]

[0106] Note: Diameter of drug sensitive tablet is 6mm; drug resistant (R) ≤ 1.0cm; moderately sensitive (I) 1~1.5cm; sensitive (S) ≥ 1.5cm

[0107] As shown in Table 1, Bacillus velezensis Scm3 is resistant to lincomycin; moderately sensitive to polymyxin B; and sensitive to piperacillin, ceftazidime, cefoperazone, cephalexin, cefuroxime sodium, ampicillin, cefazolin, ceftriaxone, penicillin, vancomycin, erythromycin, doxycycline, tetracycline, minocycline, kanamycin, gentamicin, streptomycin, and amikacin.

[0108] (2) Hemolysis test

[0109] The blood agar plate was divided into two areas: the two areas were respectively: positive control (Staphylococcus aureus) and Bacillus Velez Scm3 bacterial solution, and then lines were drawn in each area to observe whether there was hemolysis.

[0110] Depend on Fig.11 It can be seen that Bacillus Velez Scm3 did not form a hemolytic ring, while Staphylococcus aureus generated a hemolytic ring. This result shows that Bacillus Velez Scm3 does not have hemolytic activity.

[0111] (3) Virulence gene analysis

[0112] After culturing Bacillus Velez Scm3 to the logarithmic growth phase, the cells were collected, genomic DNA was extracted, and sent to Anshengda Biotechnology Co., Ltd. for whole genome sequencing. The protein sequence of the Bacillus Velez Scm3 gene was compared with the VFDB database (Virulence Factors of Pathogenic Bacteria) using BLAST, and the gene and its corresponding virulence factor function annotation information were combined. The annotation result with the E-value of the sequence comparison set to 1e-5 was used as a reliable comparison result, and the comparison results are shown in Table 2.

[0113] Table 2: Virulence factors contained in Bacillus velez Scm3

[0114]

[0115]

[0116] As shown in Table 2, the virulence genes contained in Bacillus velez Scm3 are related to protein synthesis (such as elongation factor), stress adaptability (such as γ-glutamyl transpeptidase), transport and synthesis of polyglutamate (such as capsular polyglutamate synthetase, capsular polyglutamate amide ligase), and do not contain invasive putative virulence factors.

[0117] Example 4

[0118] Study on the antibacterial mechanism of antibacterial active substances of Bacillus velez Scm3 against Edwardsiella piscicola

[0119] (1) Analysis of antibacterial active substances of Bacillus velezensis Scm3

[0120] Bacillus Velez Scm3 was cultured in a shaker (30°C, 180 rpm) at a 1% inoculation rate for about 20 h, then centrifuged (12000 rpm, 2 min, 4°C), filtered through a sterile 0.22 μm filter membrane, and the supernatant was collected.

[0121] Prepare 10 mg / mL proteinase K, trypsin, and catalase stock solutions, and add them to the sterile fermentation supernatant to make the final concentrations 1 mg / mL. Place the mixture in a 37°C water bath for 2 h, add an equal volume of PBS solution to replace the protease solution as a positive control, and use the agar diffusion method to detect the antibacterial activity and determine the antibacterial active components (see Fig.12 ).

[0122] Depend on Fig.12The results showed that the antibacterial active substance was not sensitive to catalase, and hydrogen peroxide was not the main antibacterial active substance in the fermentation supernatant; however, when trypsin or proteinase K was added, the antibacterial activity of the fermentation supernatant of Bacillus Velez subtilis Scm3 was significantly decreased or lost, indicating that the active substances secreted by Bacillus Velez subtilis Scm3 were mainly proteins or peptides.

[0123] (2) Crude extraction of antibacterial active substances from Bacillus velezensis Scm3

[0124] Bacillus Velez Scm3 was fermented in large quantities, centrifuged (12000rpm, 2min, 4℃) to remove the bacteria, and the supernatant was collected. 200mL of each supernatant was taken, and ammonium sulfate was slowly added to the saturation of 30%, 40%, 50%, 60%, 70% and 80%, respectively, and precipitated at 4℃ overnight. The precipitate was collected by centrifugation (12000rpm, 2min, 4℃), and the precipitate was dissolved with PBS (pH 7.0) and fixed to the same volume, and then dialyzed with a treated 3.5kD dialysis bag for about 12h (the buffer was changed every 3h), and the dialyzate was collected. Its antibacterial activity was determined by agar diffusion method. The ammonium sulfate saturation of the component with the strongest antibacterial activity was the optimal ammonium sulfate saturation required for preparing the antibacterial crude extract.

[0125] Depend on Fig.13 It is known that when ammonium sulfate with a saturation of 30% is used to precipitate antibacterial active substances, the antibacterial activity of the antibacterial substances at this saturation is the strongest. When ammonium sulfate with a saturation higher than 30% is used to extract antibacterial active substances, its antibacterial activity shows a downward trend. Therefore, ammonium sulfate with a saturation of 30% is used to crudely extract antibacterial substances from Bacillus Velezii Scm3.

[0126] (3) Effect of antibacterial substances of Bacillus velezensis Scm3 on the survival rate of Edwardsiella piscicola

[0127] Take 400 mL of fermentation supernatant, add ammonium sulfate to the supernatant while stirring to make its saturation reach 30%, and precipitate overnight at 4°C. Centrifuge (12000 rpm, 2 min, 4°C) to collect the precipitate, dissolve the precipitate with PBS (pH 7.0) and make up to 4 mL, then dialyze with a treated 3.5 kD dialysis bag for about 12 h (change the buffer every 3 h), collect the dialysate, and make up to 8 mL with PBS to obtain a Velez-Bacillus Scm3 antibacterial solution.

[0128] According to the inoculation amount of 1%, Edwardsiella piscicida was cultured in a shaker (30℃, 180rpm) to the mid-logarithmic phase and reserved as seed solution for later use. Experimental group: 20μL of antibacterial solution was added to 2mL of Edwardsiella piscicida bacterial suspension; control group: 20μL of PBS solution was added to 2mL of Edwardsiella piscicida bacterial suspension; the experimental group and the control group were placed in a shaker (30℃, 180rpm) and cultured for 3h, 6h, 9h, and 12h respectively. At the corresponding time points, the bacterial suspensions of the experimental group and the control group were aspirated, diluted and coated, and the survival rate of Edwardsiella piscicida was calculated.

[0129] Survival rate of Edwardsiella piscicidalis = (number of colonies in the experimental group / number of colonies in the control group) × 100%

[0130] Depend on Fig.14 It can be seen that as the culture time increases, the survival rate of pathogens decreases. When the culture time is 12h, the survival rate of Edwardsiella piscicidalis is close to 0.

[0131] (4) Effect of antibacterial solution on biofilm formation ability of Edwardsiella piscicola

[0132] At a 1% inoculation rate, Edwardsiella piscicida was cultured in a shaker (30°C, 180 rpm) to mid-logarithmic phase and reserved as seed solution. Experimental group: 20 μL of antibacterial solution was added to 2 mL of Edwardsiella piscicida suspension; control group: 20 μL of PBS solution was added to 2 mL of Edwardsiella piscicida suspension; the experimental group and the control group were placed in a shaker (30°C, 180 rpm) and cultured for 3, 9, and 15 hours, respectively. The method for determining the amount of Edwardsiella piscicida biofilm formation refers to Example 2 (4).

[0133] Depend on Fig.15 It can be seen that when cultured for 9h and 15h, the amount of biofilm formed by Edwardsiella piscicida decreased by 33.6% and 48% respectively, and the amount of biofilm formed gradually decreased with the extension of co-culture time. These results show that the antibacterial substances secreted by Bacillus Velezii Scm3 can effectively destroy the biofilm formation of Edwardsiella piscicida.

[0134] (5) Effect of antibacterial solution on metabolic activity of Edwardsiella piscicola cells

[0135] The detection of metabolic activity of Edwardsiella piscicidal cells was carried out as described in Example 2 (5).

[0136] Depend on Fig.16 It can be seen that: with the extension of culture time, the metabolic activity of Edwardsiella piscicida decreases, and when the culture time is 15h, the metabolic activity of the cells is almost lost.

[0137] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.

Claims

1. A strain of Bacillus velez Bacillus velezensis ) Scm3 strain, characterized in that: The Bacillus Velez Bacillus velezensis ) The Scm3 strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on August 30, 2024, with the deposit number CGMCC No.31790.

2. The Bacillus Velezii according to claim 1 ( Bacillus velezensis ) Application of Scm3 strain in inhibiting pathogens for non-disease diagnosis and treatment purposes.

3. The use according to claim 2, characterized in that: The pathogen is specifically Edwardsiella piscicida ( Edwardsiella piscicida ).

4. The use according to claim 2, characterized in that: Pathogens include Aeromonas hydrophila ( Aeromonas hydrophila ), Edwardsiella tarda ( Edwardsiella tarda )、Vibrio alginolyticus( Vibrio alginolyticus ), Salmonella typhi ( Salmonella typhi ), Listeria monocytogenes ( Listeria monocytogenes ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Staphylococcus aureus ( Staphylococcus aureus )、Vibrio anguillarum( Vibrio anguillarum ) 5. An antibacterial agent, characterized in that The antibacterial agent includes the Bacillus Velezii as claimed in claim 1 ( Bacillus velezensis ) Scm3 and / or Bacillus velezensis ( Bacillus velezensis ) Scm3 bacterial solution.

6. The antibacterial agent according to claim 5, characterized in that: The bacterial solution is the fermentation supernatant.

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