Brevibacillus laterosporus and application of antibacterial peptide Brevicidine expressed by brevibacillus laterosporus

Abstract

The invention discloses brevibacillus laterosporus and an application of an antibacterial peptide Brevicidine expressed by the brevibacillus laterosporus. And the brevibacillus laterosporus is brevibacillus laterosporus 811, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No. 32462. Experiments prove that the brevibacillus laterosporus 811 can inhibit bacteria, and the antibacterial peptide Brevicidine expressed by the brevibacillus laterosporus 811 not only can inhibit the bacteria, but also can inhibit the bacteria; the composition can also reduce the level of pro-inflammatory cytokines, enhance the intestinal barrier function, maintain the balance of intestinal microbiota, increase the abundance of beneficial microbiota, maintain animal intestinal homeostasis, maintain animal health and reduce bacterial adhesion and / or invasion. Therefore, the brevibacillus laterosporus 811 and the antibacterial peptide Brevicidine expressed by the brevibacillus laterosporus 811 can be used for preventing and / or treating diseases caused by bacterial infection. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of Brevibacillus laterosporus and application of an antimicrobial peptide Brevicidine expressed therein. Background Art

[0002] Given the relatively slow pace of development and approval of new antibiotics, the growing problem of antibiotic resistance poses a serious challenge to many fields. Resistant bacterial strains are becoming increasingly difficult to treat, leading to an increase in mortality from resistant infections. Many infectious diseases caused by bacterial infections remain one of the main threats to public health. Therefore, the global demand for new classes of antibiotics is becoming more urgent. The development of anti-inflammatory and anti-pathogen drugs is essential to combat serious resistant bacterial infections.

[0003] The continued emergence of multidrug-resistant pathogens further highlights the need to find new antibiotic alternatives to manage bacterial infections. Natural antimicrobial peptides (AMPs) produced by soil microorganisms are a reliable source of antimicrobial compounds and drug candidate molecules. With the advancement of bioinformatics, the tools for predicting biosynthetic gene clusters (BGCs) encoding novel natural AMPs have become more and more sophisticated, making genome mining a key method to reveal AMPs with important medical and agricultural application prospects. Bacillus microorganisms can produce many well-known AMPs, such as bacitracin and polymyxin, which have been approved by the FDA for the treatment of multidrug-resistant infections. In addition, these species can also produce a variety of other types of antimicrobial substances. Therefore, AMPs discovered from Bacillus have become a potential solution to the antibiotic resistance crisis with their broad-spectrum antimicrobial activity, unique mechanism of action, low risk of resistance and immunomodulatory effects, and are of great significance in anti-infective treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a bacterial strain capable of inhibiting bacteria and to separate an effective component capable of inhibiting bacteria, namely, an antimicrobial peptide, from the bacterial strain.

[0005] The present invention first protects a strain of Brevibacillus laterosporus, named Brevibacillus laterosporus 811, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​November 1, 2024, with a deposit number of CGMCC No. 32462. Brevibacillus laterosporus 811 CGMCC No. 32462 is referred to as Brevibacillus laterosporus 811.

[0006] The present invention also protects a bacterial agent, which may contain Brevicidine 811. The bacterial agent may be used to prepare the antimicrobial peptide Brevicidine or inhibit bacteria. The bacteria may be at least one of Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Salmonella typhimurium, Pseudomonas aeruginosa and Shigella dysenteriae.

[0007] The present invention also protects a method for preparing the bacterial agent, which may include the following steps: inoculating Brevibacillus laterosporus 811 into a bacterial culture medium and culturing the culture medium, and the obtained bacterial liquid is the bacterial agent.

[0008] The bacterial culture medium can be NB liquid culture medium or LB liquid culture medium.

[0009] In addition to the active ingredient, the bacterial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymer compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth. The plant material may be at least one of bran, soybean meal, corn flour, soybean flour and starch. The polymer compound may be polyvinyl alcohol and / or polyglycol. The liquid carrier may be an organic solvent, vegetable oil, mineral oil or water. The organic solvent may be decane and / or dodecane. In the bacterial agent, the active ingredient may be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the composition may be a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0010] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0011] The present invention also protects the use of Brevibacillus laterosporus 811CGMCC No. 32462 or any of the above-mentioned bacterial agents in preparing the antimicrobial peptide Brevicidine or inhibiting bacteria.

[0012] In the above application, the fermentation supernatant of Brevibacillus laterosporus 811 contains the antimicrobial peptide Brevicidine.

[0013] In the above application, any of the above bacterial agents is centrifuged to separate the supernatant; the supernatant contains the antimicrobial peptide Brevicidine.

[0014] The present invention also protects the use of the antimicrobial peptide Brevicidine, which can be at least one of B1)-B11); B1) reducing the level of pro-inflammatory cytokines; B2) enhancing the intestinal barrier function; B3) maintaining the balance of intestinal microbiota; B4) increasing the abundance of beneficial bacteria; B5) resisting bacterial infection; B6) maintaining animal intestinal homeostasis; B7) maintaining animal health; B8) inhibiting bacteria; B9) promoting bacterial death; B10) reducing bacterial adhesion and / or invasion; B11) preventing and / or treating diseases caused by bacterial infection.

[0015] In the above application, the antimicrobial peptide Brevicidine can be prepared by using Brevibacillus laterosporus 811CGMCC No.32462 or any of the above bacterial agents.

[0016] In the above application, in B9), promoting bacterial death can be manifested as destroying the membrane structure of bacteria.

[0017] In the above applications, the protection against bacterial infection may be manifested as inhibiting bacterial colonization in the intestinal tract of an animal and / or reducing systemic spread of bacteria.

[0018] In the above application, the maintaining of animal health may be manifested as increasing the survival rate of animals infected with bacteria and / or reducing the degree of diarrhea of ​​animals infected with bacteria.

[0019] The present invention also protects a method for inhibiting bacteria, which comprises treating bacteria with Brevibacillus laterosporus 811CGMCC No.32462, any of the above-mentioned bacterial agents, or the antimicrobial peptide Brevicidine prepared from Brevibacillus laterosporus 811CGMCC No.32462.

[0020] Any of the above-mentioned bacteria is at least one of Staphylococcus aureus, Escherichia coli, Salmonella Gallinarum, Salmonella Typhimurium, Pseudomonas aeruginosa and Shigella dysenteriae.

[0021] Any of the above-mentioned Staphylococcus aureus can be specifically Staphylococcus aureus ATCC 43300. Any of the above-mentioned Escherichia coli can be specifically Escherichia coli ATCC 25922, Escherichia coli PN16, Escherichia coli K88 or Escherichia coli O157:H7. Any of the above-mentioned Salmonella typhi can be specifically Salmonella typhi CVCC 534. Any of the above-mentioned Salmonella typhimurium can be specifically Salmonella typhimurium SL1344. Any of the above-mentioned Pseudomonas aeruginosa can be specifically Pseudomonas aeruginosa ATCC 27853. Any of the above-mentioned Shigella dysenteriae can be specifically Shigella dysenteriae CGMCC 1.1869.

[0022] In the above, the method for preparing the antimicrobial peptide Brevicidine using any of the above-mentioned bacterial agents can be as follows:

[0023] (1) Centrifuging any of the above bacterial agents to collect the supernatant; then freeze-drying the supernatant to obtain a freeze-dried powder.

[0024] (2) After completing step (1), weigh the lyophilized powder and dissolve it in water to obtain a lyophilized solution. Add n-butanol to the lyophilized solution for extraction to obtain an n-butanol extract.

[0025] (3) After completing step (2), an organic solvent is added to the n-butanol extract, mixed, and white floccules are precipitated. Then, the mixture is allowed to stand, centrifuged, and the precipitate is collected; the precipitate is the crude extract of Brevicidine.

[0026] The preparation method of any of the above-mentioned bacterial agents specifically comprises the following steps: inoculating Brevibacillus laterosporus 811 into a bacterial culture medium (NB liquid culture medium or LB liquid culture medium) and culturing, and the obtained bacterial liquid is the bacterial agent. The culture conditions can be 35-39°C, 160-220r / min, and culture for 10-14 hours. The culture can also be expanded as needed.

[0027] In the above step (2), the extraction using n-butanol may be performed two or more times.

[0028] In the above step (2), the step of "adding n-butanol to the lyophilized liquid for extraction to obtain an n-butanol layer" can specifically include: adding n-butanol to the lyophilized liquid, shaking, and obtaining a water layer 1 and an n-butanol layer 1; transferring the n-butanol layer 1 to a new centrifuge tube, continuing to add n-butanol to the water layer 1, shaking, and obtaining a water layer 2 and an n-butanol layer 2; transferring the n-butanol layer 2 to the centrifuge tube containing the n-butanol layer 1, continuing to add n-butanol to the water layer 2, shaking, and obtaining a water layer 3 and an n-butanol layer 3; transferring the n-butanol layer 3 to the centrifuge tube containing the n-butanol layer 1 and the n-butanol layer 2, and obtaining an n-butanol liquid. The n-butanol liquid is the final n-butanol extract.

[0029] In the above step (3), the organic solvent may be ether or acetone.

[0030] In the above step (3), the standing may be at 4° C. for 2 hours.

[0031] In the above step (3), the centrifugation can be performed at 2-6°C and 8000-16000 rpm for 10-20 min.

[0032] The method for preparing the antimicrobial peptide Brevicidine using any of the above-mentioned bacterial agents may further include step (4): filtering the solution of the crude extract of Brevicidine with a filter membrane, separating and purifying the filtrate by high performance liquid chromatography, removing acetonitrile from the collected fractions with a rotary evaporator, and freeze-drying to obtain pure Brevicidine.

[0033] The pore size of the filter membrane may specifically be 0.22 μm.

[0034] The molecular formula of any of the above antimicrobial peptides Brevicidine is specifically C 74 H 106 N 18 O 17The CAS number of any of the above antimicrobial peptides Brevicidine is: 2676889-05-5. The SMILES expression of the chemical formula of any of the above-mentioned antimicrobial peptide Brevicidine is CC[C@H](C)[C@H]1C(=O)NCC(=O)N[C@H](C(=O)O[C@@H]([C@@H](C(=O)N1)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@@H](CCCN)NC(=O)CNC(=O)[C@H](CCCN)NC(=O)[C@@H](CCCN)NC(=O)[C@@H](CCCN)NC(=O)[C@@H](CC4=CNC5=CC=CC=C54)NC(=O)[C@@H](CC6=CC=C(C=C6)O)NC(=O)[C@@H](CC(=O)N)NC(=O)CCC(C)CC)C)CO.

[0035] Experiments have shown that Brevibacillus laterosporus 811CGMCC No.32462 can inhibit bacteria such as Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Salmonella typhimurium, Pseudomonas aeruginosa and / or Shigella dysenteriae. The antimicrobial peptide Brevicidine was isolated and purified from the fermentation supernatant of Brevibacillus laterosporus 811CGMCC No.32462. The antimicrobial peptide Brevicidine can not only inhibit bacteria in the same way, but also reduce the level of pro-inflammatory cytokines, enhance intestinal barrier function, maintain the balance of intestinal microbiota, increase the abundance of beneficial bacteria, resist bacterial infection, maintain animal intestinal homeostasis, maintain animal health, promote bacterial death, and reduce bacterial adhesion and / or invasion. It can be seen that Brevibacillus laterosporus 811CGMCC No.32462, especially the antimicrobial peptide Brevicidine separated and purified from its fermentation supernatant, can prevent and / or treat diseases caused by bacterial infection. The present invention has important application value.

[0036] Collection Instructions

[0037] Bacteria name: Brevibacillus laterosporus

[0038] Latin name: Brevibacillus laterosporus

[0039] Strain ID: 811

[0040] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0041] Abbreviation of depository institution: CGMCC

[0042] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0043] Date of deposit: November 1, 2024

[0044] CGMCC Registration Number: CGMCC No.32462 BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 These are the results of the antibacterial ability test of strains 13-11, 631, 632, 621, 622, 811, 812 and 13-1X against Escherichia coli.

[0046] Figure 2 The results show the antibacterial ability of strain 811 against different tested strains.

[0047] Figure 3 Phylogenetic tree of 811 strains based on 16S rDNA sequencing.

[0048] Figure 4 This is the circular genome map of Brevibacillus laterosporus 811.

[0049] Figure 5 GOG and KEGG annotation analysis of the genome of Brevibacillus laterosporus 811.

[0050] Figure 6 Analysis of secondary metabolite biosynthesis gene clusters in Brevibacillus laterosporus 811 for antiSMASH.

[0051] Figure 7 The high performance liquid chromatogram of the Brevicidine crude extract solution.

[0052] Figure 8 The purity results of pure Brevicidine were tested by high performance liquid chromatography.

[0053] Fig. 9 This is the primary mass spectrum of the antimicrobial peptide Brevicidine.

[0054] Fig.10 This is the secondary mass spectrum of the antimicrobial peptide Brevicidine.

[0055] Fig.11 A standard curve was drawn based on the concentration and peak area of ​​the antimicrobial peptide Brevicidine.

[0056] Fig.12 The chromatograms are of Brevicidine standard solution and Brevibacillus laterosporus 811 fermentation broth.

[0057] Fig.13 The time-killing curves of the antimicrobial peptide Brevicidine against Escherichia coli (left) and Salmonella (right) at different concentrations.

[0058] Fig.14 Scanning electron micrographs of the bacterial surface of Escherichia coli (top) and Salmonella (bottom) after treatment with different concentrations of the antimicrobial peptide Brevicidine.

[0059] Fig.15 The MIC changes of Escherichia coli (left) and Salmonella (right) induced by sublethal concentrations of the antimicrobial peptide Brevicidine and various traditional antibiotics.

[0060] Fig.16 The content and antibacterial activity of the antimicrobial peptide Brevicidine in different temperatures (A and B), different pH values ​​(C and D) and artificial gastrointestinal fluid (E and F) change.

[0061] Fig.17 The effects of the antimicrobial peptide Brevicidine on the proliferation activity of IPEC-J2 cells (A), metabolic activity LDH (B), porcine blood cell hemolysis (C) and mouse blood cell hemolysis (D). Among them, the porcine ileal intestinal epithelial cells IPEC-J2 were treated with different concentrations of the antimicrobial peptide Brevicidine for 24 hours, and the effects of Brevicidine on cell proliferation (A) and its cytotoxicity (B) were detected using CCK-8 and LDH kits, respectively. The hemolysis of different concentrations of Brevicidine was detected using porcine and mouse blood cells (C and D), and PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively.

[0062] Fig.18 The protective effect of the antimicrobial peptide Brevicidine on Escherichia coli adhesion (A) and Salmonella (B) invasion of IPEC-J2 cells.

[0063] Fig.19Pretreatment with the antimicrobial peptide Brevicidine effectively alleviated the clinical symptoms of mice challenged with Escherichia coli and Salmonella. A is the experimental design of the mouse model of infection with Escherichia coli and Salmonella. B is the weight change of mice after oral administration of Brevicidine for 7 days during the pretreatment period (n=8, 10, 8, 8, 8). C is the weight change of mice 3 days after infection (n=8). D is the survival rate of mice in the infection mouse model experiment (n=8, 10, 8, 8, 8). E is the body temperature of mice within 3 days after infection (n=8). Diarrhea scores were recorded on the first day of infection in F and the third day of infection in G (n=8). All data are expressed as mean ± standard error (SEM), and different lowercase letters within each group indicate significant differences (P<0.05); *** indicates P<0.001. H is the bacterial count in feces, I is the bacterial count in the liver and J is the bacterial count in the spleen. The difference in pathogen colonization and metastasis in feces and organs was evaluated using the non-parametric Mann-Whitney test (n=8). All data are expressed as mean ± standard error (SEM). Different symbols between the two groups indicate significant differences; * indicates P < 0.05 vs. control group, # indicates P < 0.05 vs. challenge group, ^ indicates P < 0.05 vs. treatment group 1, + indicates P < 0.05 vs. treatment group 2, and - indicates P < 0.05 vs. treatment group 3.

[0064] Fig. 20 Pretreatment with the antimicrobial peptide Brevicidine modulated the immunoglobulin levels and inflammatory response of challenged mice. A was the determination of immunoglobulins such as IgA, IgG and IgM in serum, as well as cytokines IL-1β and TNF-α in serum (n=8). B was the determination of the relative mRNA expression levels of TNF-α, IL-1β and IL-6 genes in the ileum and colon by qPCR (n=8). C was the determination of the cytokine levels of TNF-α, IL-1β and IL-6 in the ileum and colon by ELISA (n=6). Data are expressed as mean ± SEMs. Different lowercase letters within the group indicate significant differences (P<0.05).

[0065] Fig.21The effects of pretreatment with the antimicrobial peptide Brevicidine on intestinal morphology and barrier function. A (H&E staining) and B (PAS staining) are observations of histopathological changes in the ileum and colon. C is the histopathological score of the ileum and colon, including inflammatory cell infiltration, lamina propria edema, glandular alveolar expansion, decreased goblet cell number, and tissue fibrosis (n=8). The mRNA expression levels of intestinal epithelial E-cadherin and tight junction proteins ZO-1, Claudin-1, and Occludin in D (ileum) and E (colon) were determined by RT-qPCR (n=8). F and G are representative images of E-cadherin, ZO-1, Claudin-1, and Occludin protein expression in the ileum and colon. H is the relative abundance of related proteins (n=3). Bre_7.5, Bre_15, and Bre_30 represent treatment group 1, treatment group 2, and treatment group 3, respectively. The data are expressed as mean ± SEMs, and different lowercase letters within each group indicate significant differences (P < 0.05).

[0066] Fig. 22 The effect of the antimicrobial peptide Brevicidine on the diversity and species richness of the intestinal microbiota of challenged mice. A is the community coverage index; α diversity index, including (B) Shannon index and (C) Simpson index; and (D) community richness index (ACE) display. (E) PCoA analysis of microbial β diversity and (F) box plots of five groups. (G) Treatment group 2 showed a clear clustering pattern compared with the control group and the challenge group. (H) Venn diagram comparison of OTU abundance in the five groups. Bre_7.5, Bre_15 and Bre_30 represent treatment group 1, treatment group 2 and treatment group 3, respectively. Data are presented as mean ± SEMs of 6 replicates per group.

[0067] Fig.23 Effects of pretreatment with the antimicrobial peptide Brevicidine on the abundance and taxonomic differences of intestinal microbial communities in challenged mice. Three days after infection, the taxonomic distribution of intestinal bacterial composition of mice at the (A) phylum level and (B) genus level was shown, and the results were expressed as the average relative abundance of the classification within each group. (C) Cluster heat map of the intestinal microbiota of mice at the phylum level and (D) genus level. (E) Differential abundance analysis of the intestinal microbiota of mice at the phylum level and (F) genus level. Bre_7.5, Bre_15, and Bre_30 represent treatment group 1, treatment group 2, and treatment group 3, respectively. Data are expressed as the average of 6 replicates per group. * indicates P < 0.05, and ** indicates P < 0.01. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0069] Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Brevibacillus laterosporus ATCC 9141 and Enterococcus faecalis ATCC 29212 were purchased from the American Type Culture Collection (ATCC), and the public can obtain the above strains from the American Type Culture Collection.

[0070] Shigella dysenteriae CGMCC 1.1869 was purchased from China General Microbiology Center (CGMCC).

[0071] Escherichia coli PN16 is recorded in the following literature: Qiue Yang, Mei Li, et al. Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms. Nature Communications volume 8, Article number: 2054 (2017) (https: / / www.nature.com / articles / s41467-017-02149-0). The Escherichia coli PN16 in this application was donated by Shen Jianzhong's team from the College of Veterinary Medicine, China Agricultural University.

[0072] Salmonella enterica serovar Gallinarum CVCC 534 is recorded in the following document: Haitao Yu, Ning Li, et al. A Comprehensive Antimicrobial Activity Evaluation of the Recombinant Microcin J25 Against the Foodborne Pathogens Salmonella and E. coli O157:H7 by Using a Matrix of Conditions. Front. Microbiol., 27 August 2019 Sec. Antimicrobials, Resistance and Chemotherapy (https: / / doi.org / 10.3389 / fmicb.2019.01954).

[0073] Escherichia coli K88 is described in the following literature: Haitao Yu, et al. Biosynthetic Microcin J25 Exerts Strong Antibacterial, Anti-Inflammatory Activities, Low Cytotoxicity Without Increasing Drug-Resistance to Bacteria Target. Front. Immunol., 18 February 2022. Sec. Microbial Immunology. Volume 13-2022

[0074] (https: / / doi.org / 10.3389 / fimmu.2022.811378).

[0075] Escherichia coli O157:H7 is recorded in the following literature: Haitao Yu, Ning Li, et al. A Comprehensive Antimicrobial Activity Evaluation of the Recombinant Microcin J25 Against the Foodborne Pathogens Salmonella and E.coli O157:H7 by Using a Matrix of Conditions. Front. Microbiol., 27 August 2019 Sec. Antimicrobials, Resistance and Chemotherapy (https: / / doi.org / 10.3389 / fmicb.2019.01954).

[0076] Salmonella typhimurium SL1344 is described in the following literature: Yuxin Fan, et al. Protective Role of Indole-3-Acetic Acid Against Salmonella Typhimurium: Inflammation Moderation and Intestinal Microbiota Restoration. Microorganisms 2024, 12(11), 2342.

[0077] (https: / / doi.org / 10.3390 / microorganisms12112342).

[0078] Bacillus cereus CVCC 4101 was purchased from the National Veterinary Microbiology and Virus Collection Center.

[0079] Example 1. Isolation, identification and preservation of Brevibacillus laterosporus 811CGMCC No.32462

[0080] 1. Separation

[0081] 1. Select 20 soil samples with a depth of 0-30 cm from different areas of Chongqing with little human activity, no antibiotic and pesticide pollution, and vegetation. After removing impurities such as grass roots, put them into sterile self-sealing bags, record the sampling information, mark them, and store them at 4°C.

[0082] 2. Each soil sample was subjected to the following experiments:

[0083] (1) Add 10 g of soil sample and an appropriate amount of glass beads to 90 mL of sterile saline and shake to obtain the soil stock solution (the dilution degree is recorded as 10). -1 ). Pipette 1 mL of the supernatant of the soil stock solution into a test tube containing 9 mL of sterile saline and mix thoroughly (the dilution is 10 at this time). -2 ), then draw 1mL from this test tube and add it to another test tube containing 9mL of sterile saline and mix well. Repeat this process to make dilution 1 (dilution degree is 10 -3 ), diluent 2 (dilution is 10 -4 ) and dilution 3 (dilution 10 -5 ).

[0084] (2) Use a pipette to draw 100 μL of diluent 1, diluent 2, or diluent 3 and evenly spread it on NB solid medium. Incubate at 30°C for 7 days, observe the growth status of the colonies, and count the number of colonies on the medium.

[0085] NB solid medium was obtained by adding 2% agar powder (Beijing Solebow Technology Co., Ltd., product catalog number: A8190) to NB liquid medium (Beijing Solebow Technology Co., Ltd., product catalog number: N8300).

[0086] (3) After completing step (2), take the NB solid culture medium with 30-100 single colonies, mark the colonies respectively, and pick them with a toothpick into 3 mL of NB liquid culture medium, and culture them at 37°C and 180 r / min for 16 h to obtain a culture solution.

[0087] (4) After completing step (3), take the culture solution, centrifuge it at 10,000 rpm for 20 min, and collect the supernatant; the supernatant is the fermentation supernatant of the strain.

[0088] About 1500 single colonies, i.e. 1500 strains, were obtained from 20 soil samples, and finally the fermentation supernatant of 1500 strains was obtained.

[0089] 3. Detection of the antibacterial ability of fermentation supernatants of 1500 strains against Escherichia coli ATCC 25922

[0090] The strain to be tested is Escherichia coli ATCC 25922.

[0091] The experiment was repeated three times, and the steps for each repetition were as follows:

[0092] (1) The strain to be tested was inoculated into NB solid medium by streak method and cultured at 37°C overnight (for activation).

[0093] (2) A single clone of the strain to be tested obtained in step (1) was inoculated into 4 mL of NB liquid culture medium and cultured overnight at 37° C. and 180 rpm to obtain a bacterial suspension of the strain to be tested.

[0094] (3) Pour 20 μL of the test strain suspension into 100 mL of NB solid culture medium at about 45°C and mix well to obtain a mixed solution.

[0095] (4) Take 8 Oxford cups and place them in a culture dish, with the distance between the centers of the Oxford cups being more than 25 mm and the distance from the edge of the culture dish being more than 15 mm, and the distances between the centers of the Oxford cups being uniform; then pour the mixed solution in, and remove the Oxford cups after cooling.

[0096] (5) After completing step (4), add 100 μL of the fermentation supernatant obtained in step 2 or sterile water (as a negative control) to the wells, and blow dry the liquid in the wells in a clean bench. Finally, place the culture dish at 37°C for 20 h, and then measure the diameter of the inhibition ring with a vernier caliper.

[0097] (6) After completing step (5), make the following judgment:

[0098] If the diameter of the antibacterial ring is greater than 0 mm, it is judged to have an antibacterial effect; otherwise, it is judged to have no antibacterial effect;

[0099] The three repeated tests all showed antibacterial effects, and it was judged that the tested strain had antibacterial function;

[0100] There should be no inhibition zone in the negative control, otherwise the test is invalid.

[0101] Some test results can be found in Figure 1 The results showed that only 8 bacterial strains among 1500 strains had fermentation supernatants that could inhibit Escherichia coli ATCC 259228. The names of these 8 bacterial strains were 13-11 strain, 631 strain, 632 strain, 621 strain, 622 strain, 811 strain, 812 strain and 13-1X strain.

[0102] 4. Detect the antibacterial ability of the fermentation supernatant of 8 strains

[0103] (1) A single clone of the 13-11 strain, the 631 strain, the 632 strain, the 621 strain, the 622 strain, the 811 strain, the 812 strain or the 13-1X strain was inoculated into 3 mL of NB liquid culture medium, and cultured at 37° C. and 180 rpm for 16 h to obtain a culture solution; the culture solution was then centrifuged at 10,000 rpm for 20 min, and the supernatant was collected; i.e., the fermentation supernatant.

[0104] (2) Detect the antibacterial ability of the fermentation supernatant against the strains to be tested by referring to the method of step 3. The strains to be tested are Staphylococcus aureus ATCC 43300, Shigella dysenteriae CGMCC 1.1869, Escherichia coli ATCC 25922, Escherichia coli PN16, Salmonella gallinarum CVCC 534 and Pseudomonas aeruginosa ATCC 27853.

[0105] The test results showed that ( Figure 2 ), the fermentation supernatant of strain 811 can effectively inhibit Staphylococcus aureus, Shigella dysenteriae, Escherichia coli, Escherichia coli PN16 (polymyxin-resistant Escherichia coli), Salmonella gallinarum and Pseudomonas aeruginosa. It can be seen that strain 811 is a candidate strain that can produce effective antibacterial peptides and has the potential to inhibit bacterial pathogens.

[0106] 2. Identification

[0107] The genomic DNA of strain 811 was extracted using a bacterial genomic DNA rapid extraction kit (Shanghai Biotech) and used as a template for PCR amplification using a primer pair consisting of primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain the 16S rDNA sequence of strain 811.

[0108] The 16S rDNA sequence of strain 811 is shown in SEQ ID NO:1.

[0109] The 16S rDNA sequence of strain 811 was subjected to BLAST in the NCBI Standard Nucleotide database, and the phylogenetic tree of strain 811 was constructed by the neighbor-joining method based on the 16S rDNA sequence, and the genus and species of strain 811 were finally determined.

[0110] The phylogenetic tree of strain 811 is shown in Figure 3 The results showed that strain 811 was closely clustered with strains of the genus Brevibacillus laterosporus and had 99.87% homology with the 16S rDNA sequence of Brevibacillus laterosporus DSM 25 (GenBank No.: GCA_002706795.1). Therefore, strain 811 was identified as Brevibacillus laterosporus.

[0111] 3. Preservation

[0112] The 811 strain isolated in step 1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​November 1, 2024, with a deposit number of CGMCC No. 32462. The full name of the 811 strain is Brevibacillus laterosporus 811CGMCC No. 32462, abbreviated as Brevibacillus laterosporus 811.

[0113] Example 2: Genome sequencing and antiSMASH analysis of Brevibacillus laterosporus 811

[0114] 1. Inoculate a single clone of Brevibacillus laterosporus 811 into 3 mL NB liquid culture medium, culture at 37°C and 180 r / min to obtain a culture solution; during the culture period, sample every 2 hours. Centrifuge the culture solution at 10,000 rpm for 20 min, and collect the supernatant; that is, the fermentation supernatant. Detect the antibacterial ability of the fermentation supernatant against Escherichia coli ATCC 25922 by referring to the method 3 in step 1 of Example 1. End the culture when the antibacterial ability reaches the maximum.

[0115] 2. After completing step 1, take the culture solution, centrifuge it, and collect the bacteria.

[0116] 3. After completing step 2, the genomic DNA of the bacteria collected in step 2 was extracted using the SDS method, and then the purity and integrity of the DNA were detected by agarose gel electrophoresis and quantified using Qubit.

[0117] 4. After completing step 3, the qualified DNA samples detected by electrophoresis were randomly broken into fragments of about 350 bp in length using a Covaris ultrasonic crusher. Ultra TMDNA Library Prep Kit for Illumina (NEB, USA) was used to complete the entire library construction through end repair, A-tailing, sequencing adapter addition, purification, PCR amplification and other steps. The library was first quantified using Qubit 2.0, and the library was diluted to 2ng / L. Then, the insert fragment of the library was detected using Agilent 2100. After the insert size met the expectations, the effective concentration of the library was accurately quantified using the Q-PCR method to ensure the quality of the library. After the library inspection was qualified, different libraries were sequenced by Illumina NovaSeq PE150 according to the effective concentration and target data volume. After obtaining the genome information of the selected strain, the secondary metabolite gene cluster of the selected strain was analyzed using antiSMASH (https: / / antismash.secondarymetabolites.org / ).

[0118] The results showed that B. laterosporus 811 had a single circular chromosome of 5.44 Mbp in size (see Figure 4 ), the guanine-cytosine (GC) content in the genome is 40.3%. It is predicted that there are 6792 coding genes in the genome, accounting for 82.01% of the genome. In addition, it is predicted that the genome of B. laterosporus 811 contains 36 rRNAs, 111 tRNAs, 24 genomic islands, 4 phage precursors and 1 CRISPR-Cas system.

[0119] Gene function annotation was performed using GO, KEGG, COG, NR, Pfam, TCDB, Swiss-Prot, effector molecules, etc. The results are shown in Figure 5 The results are as follows:

[0120] Overall, 72.26% of the genes were annotated by COG (Clusters of Orthologous Groups); COG classification showed that most genes were involved in basic cellular processes such as metabolism, cellular processes and signal transduction, and information storage and processing. Specifically, genes involved in amino acid transport and metabolism (520 genes), carbohydrate transport and metabolism (329 genes), transcription (452 ​​genes), translation, ribosome structure and biogenesis (306 genes) occupied an important position, indicating that B. laterosporus 811 has strong metabolic capacity and adaptability.

[0121] KEGG pathway analysis further revealed the metabolic pathways and biosynthetic capabilities of B. laterosporus 811. Major metabolic pathways, including amino acid metabolism (341 genes), carbohydrate metabolism (362 genes), lipid metabolism (143 genes), and metabolism of cofactors and vitamins (304 genes), were well represented, indicating that B. laterosporus 811 possesses the necessary mechanisms for efficient energy production and nutrient absorption. In addition, 81 biosynthetic pathways for secondary metabolites were identified, highlighting the potential of B. laterosporus 811 to produce bioactive compounds.

[0122] At the same time, through the online submission of genome data by antiSMASH, 20 gene clusters related to the biosynthesis of secondary metabolites, including non-ribosomal peptides (NRPs), polyketides, and other bioactive compounds, were identified in the genome of B. laterosporus 811. Seven of these gene clusters showed high similarity to known compounds (see Figure 6 ), one of the gene clusters has a 100% similarity with the antimicrobial peptide Brevicidine. The antimicrobial peptide Brevicidine is a lipopeptide substance with broad-spectrum antimicrobial activity, with a molecular weight of about 1530Da. It has a good inhibitory and killing effect on a variety of Gram-negative pathogens and has the potential to replace antibiotics and resist bacterial infections in animals.

[0123] The functional gene annotation and secondary metabolite analysis results of Brevicidine 811 showed that there were multiple secondary metabolite gene clusters, including the synthesis gene cluster of the antimicrobial peptide Brevicidine. Brevicidine is a lipopeptide with strong antibacterial activity against Gram-negative bacteria and a potential alternative to antibiotics.

[0124] Example 3. Preparation and identification of antimicrobial peptide Brevicidine

[0125] 1. Preparation of the antimicrobial peptide Brevicidine

[0126] 1. Inoculate a single clone of Brevibacillus laterosporus 811 into 3 mL LB liquid culture medium (product of Beijing Solebow Technology Co., Ltd., catalog number L1010), and culture at 37° C. and 180 r / min for 12 hours to obtain culture solution 1; inoculate 3 mL of culture solution 1 into 300 mL LB liquid culture medium, and culture at 37° C. and 180 r / min for 12 hours to obtain culture solution 2; centrifuge the culture solution 2 and collect the supernatant; finally, freeze-dry the supernatant to obtain a lyophilized powder.

[0127] 2. After completing step 1, accurately weigh 1g of lyophilized powder and dissolve it with 5mL of ultrapure water to obtain a lyophilized solution. Add 5mL of n-butanol to the lyophilized solution and shake it slightly (for the purpose of extracting the antimicrobial peptide Brevicidine) to obtain water layer 1 and n-butanol layer 1. Transfer n-butanol layer 1 to a new centrifuge tube, continue to add 5mL of n-butanol to water layer 1, shake it slightly, and obtain water layer 2 and n-butanol layer 2. Transfer n-butanol layer 2 to a centrifuge tube containing n-butanol layer 1, continue to add 5mL of n-butanol to water layer 2, shake it slightly, and obtain water layer 3 and n-butanol layer 3. Transfer n-butanol layer 3 to a centrifuge tube containing n-butanol layer 1 and n-butanol layer 2 to obtain n-butanol liquid.

[0128] 3. After completing step 2, add 5 mL of ether or acetone to the n-butanol solution, mix well, and precipitate white flocs. Then, let stand at 4°C for 2 hours, centrifuge at 4°C and 12000 rpm for 15 minutes, and collect the precipitate; the precipitate is the crude extract of Brevicidine.

[0129] The crude Brevicidine extract was dissolved in ultrapure water to obtain a crude Brevicidine extract solution with a concentration of 200 mg / mL.

[0130] 4. After completing step 3, the crude extract of Brevicidine was filtered through a 0.22 μm filter membrane, and the filtrate was separated and purified by high performance liquid chromatography.

[0131] The chromatographic column was an Agilent ZORBAX StableBond 300C18 reversed-phase semi-preparative column (9.4×250 mm, 5 μm); the instrument parameters were set as follows: diode array detector (DAD) detection wavelength 220 nm; column temperature 25°C; flow rate 5 mL / min; mobile phase A: ultrapure water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid. The mobile phase gradient elution conditions are shown in Table 1.

[0132] The sample loading volume was set to 500 μL each time, and the instrument automatically collected fractions into glass test tubes according to the set fraction receiving trigger conditions. The collected fractions were lyophilized after removing acetonitrile using a rotary evaporator to obtain pure Brevicidine.

[0133] Table 1

[0134] Time, min A，％ B，％ Flow rate, mL / min 0.00 95.00 5.00 5.00 5.00 95.00 5.00 5.00 7.00 70.00 30.00 5.00 36.00 52.00 48.00 5.00 37.00 5.00 95.00 5.00 39.00 5.00 95.00 5.00 40.00 95.00 5.00 5.00 52.00 95.00 5.00 5.00

[0135] 5. After completing step 4, pure Brevicidine was dissolved in ultrapure water to obtain a Brevicidine solution with a concentration of 200 μg / mL. The Brevicidine solution was subjected to mass spectrometry analysis.

[0136] Mass spectrometry analysis was performed using an ultra-high performance liquid chromatography (UPLC) coupled to a Thermo Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer. The UPLC analysis was performed using a Fenome Aeris Peptide XB-C18 column (150×2.1 mm, 2.6 μm). Mobile phase A was 0.1% formic acid in water, and mobile phase B was pure acetonitrile. The flow rate was set at 0.3 mL / min. The gradient elution program was: initial 5% mobile phase B, mobile phase B increased from 5% to 95% within 5-60 minutes, and mobile phase B decreased from 95% to 5% within 60-65 minutes. The mass spectrometer was equipped with an electrospray ionization (ESI) source and operated in positive ion mode. The spray voltage was set to 3.5 kV, and the sheath gas and auxiliary gas flow rates were set to 40 and 10 arbitrary units, respectively. The capillary temperature was maintained at 320°C, and the radio frequency of the S-Lens was set to 60%. Full scan mass spectra were performed in the mass range of m / z 200 to 2000, with a resolution of 70,000 (m / z 200) and a scan rate of 3 Hz. In the dd-MS2 scan, the NCE energy was set to 25 eV, the isolation width was 2.0 Da, the resolution was 17500 (m / z 200), and the scan rate was 2 Hz. Data dependent acquisition (DDA) was used to select the 10 most responsive ions from the full scan for MS / MS analysis, and dynamic exclusion was enabled (repeat count of 1 and exclusion duration of 30 s). Data were acquired and processed using ThermoScientific Xcalibur 3.1Qual Browser software.

[0137] The HPLC chromatogram of the crude Brevicidine solution is shown in Figure 7 , where 2 is the chromatographic peak corresponding to the antimicrobial peptide Brevicidine. The fractions corresponding to the chromatographic peak are collected and freeze-dried to obtain pure Brevicidine.

[0138] The purity results of pure Brevicidine by HPLC are shown in Figure 8 The results showed that the purity of pure Brevicidine was 97.7%. Samples of this purity can be used for subsequent detection and biological activity tests of the antimicrobial peptide Brevicidine.

[0139] The MS1 and MS2 mass spectra of pure Brevicidine are shown in Fig. 9 and Fig.10 Under the set parameters, two quasi-molecular ions of Brevicidine were detected: [M+3H] 3+ m / z 570.2724 and [M+2H] 2+m / z 760.4046. The mass-to-charge ratio shows that the exact molecular weight of Brevicidine is 1518.8092Da. This result is completely consistent with the existing literature (Yong-XinLi, Zheng Zhong, Wei-Peng Zhang, Pei-Yuan Qian. Discovery of cationic nonribosomal peptides as Gram-negative antibiotics through global genomemining. Nature Communications volume 9, Article number: 3273 (2018); https: / / doi.org / 10.1038 / s41467-018-05781-6). The m / z of 760.4046 ([M+2H] 2+ ) as the precursor ion, and perform secondary mass spectrometry scanning of Brevicidine molecules. The results are as follows Fig.10 shown. Fig.10 The fragment ion peaks at m / z 227.1386 and m / z 1293.6588 are the paired N-terminal (b2) and C-terminal (y11) peptide molecules after the Brevicidine molecule is broken. The experimental results are completely consistent with the existing literature (Yong-Xin Li, Zheng Zhong, Wei-Peng Zhang, Pei-Yuan Qian. Discovery of cationic nonribosomal peptides as Gram-negative antibiotics through global genome mining. Nature Communications volume 9, Article number: 3273 (2018); https: / / doi.org / 10.1038 / s41467-018-05781-6).

[0140] It can be seen that the fermentation supernatant of Brevibacillus laterosporus 811 in the present application contains the antimicrobial peptide Brevicidine. The molecular formula of the antimicrobial peptide Brevicidine is C 74 H 106 N 18 O 17, CAS No.: 2676889-05-5, the SMILES expression of the chemical structure is CC[C@H](C)[C@H]1C(=O)NCC(=O)N[C@H](C(=O)O[C@@H]([C@@H](C(=O)N1)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@@H](CCC N)NC(=O)CNC(=O)[C@H](CCCN)NC(=O)[C@@H](CCCN)NC(=O)[C@@H](CC4=CNC5=CC=CC=C 54)NC(=O)[C@@H](CC6=CC=C(C=C6)O)NC(=O)[C@@H](CC(=O)N)NC(=O)CCC(C)CC)C)CO.

[0141] 2. Detection of antimicrobial peptide Brevicidine

[0142] 1. HPLC parameter setting

[0143] The antimicrobial peptide Brevicidine was quantitatively analyzed using an analytical reverse phase high performance liquid chromatography system (Agilent 1260 Infinity II). The separation process used an Agilent Poroshell 120SB-C18 column (2.1×250 mm, 4 μm), the column temperature was controlled at 35°C, the injection volume was 10 μL, and the DAD detector wavelength was set at 220 nm. The mobile phase consisted of phase A (ultrapure water + 0.1% trifluoroacetic acid) and phase B (acetonitrile + 0.1% trifluoroacetic acid), and the gradient elution program is detailed in Table 2.

[0144] Table 2

[0145]

[0146]

[0147] 2. Drawing of standard curve

[0148] Accurately weigh pure Brevicidine and dissolve it in mobile phase A (0.1% trifluoroacetic acid aqueous solution) to obtain a Brevicidine standard stock solution (concentration of 5000 μg / mL). Then dilute the Brevicidine standard stock solution to obtain a series of Brevicidine working standard solutions (concentration range of 1.5625 to 1600 μg / mL). Using ultrapure water as a control, each concentration was tested 3 times, and the peak area was obtained by liquid phase.

[0149] The peak area obtained by HPLC was used as the ordinate (Y) and the sample concentration was used as the abscissa (X). The Brevicidine standard curve was drawn to obtain the linear regression equation: Y = 17.173X + 46.623, R 2 =0.9999 (n=3). The linear relationship between the peak area and the concentration of the antimicrobial peptide Brevicidine was good when it was 0-1.6 mg / mL (see Fig.11 ).

[0150] 3. Methodological validation

[0151] To verify the precision and accuracy of the detection method, a fermentation broth without Brevicidine was used as a matrix. The inventors of the present application found through a large number of experiments that the metabolites of Brevibacillus laterosporus ATCC9141 are similar to those of Brevibacillus laterosporus 811, but do not produce Brevicidine.

[0152] A single clone of Brevibacillus laterosporus ATCC 9141 was inoculated into 3 mL LB liquid medium, cultured at 33°C and 200 rpm for 24 hours to obtain culture solution a; 3 mL culture solution a was inoculated into 300 mL LB liquid medium, cultured at 33°C and 200 rpm for 24 hours to obtain culture solution b; culture solution b was centrifuged at 4°C and 12000 rpm for 10 minutes, and the supernatant was collected. A known amount of Brevicidine standard solution was added at three different concentrations, and the recovery rate was measured three times a day for 3 consecutive days. The actual sample solution was prepared by centrifuging the fermentation broth of Brevibacillus laterosporus 811 at 4°C and 12000 rpm for 10 minutes, then collecting the supernatant and filtering it through a 0.22 μm filter membrane, measuring the peak area on the machine, and calculating the relative standard deviation.

[0153] Chromatograms of Brevicidine standard solution and Brevibacillus laterosporus 811 fermentation broth (actual sample) are shown in Fig.12 The results showed that the detection method exhibited high precision, with intra-day relative standard deviation (RSD) values ​​ranging from 0.46% to 1.15% and inter-day RSD values ​​ranging from 1.25% to 2.31%, confirming its consistent reproducibility and accuracy; the deviation values ​​ranged from -2.95% to 2.29% (Table 3), further verifying the reliability of this method in the quantitative analysis of Brevicidine.

[0154] Table 3

[0155]

[0156] Note: Recovery ratea) : Mean ± standard error; precision b) RSD: relative standard deviation; accuracy c) Bias: (detection value - spiked value) / spiked value × 100%. Intra-day experiment n=3, inter-day experiment n=9.

[0157] The above results show that the pure product of the antimicrobial peptide Brevicidine with a purity of 97.7% was obtained from the fermentation broth of Brevibacillus laterosporus 811 by HPLC separation and purification, and the structure was identified. In addition, a corresponding analytical HPLC method was established, which can effectively and stably quantitatively detect the content of the antimicrobial peptide Brevicidine.

[0158] Example 4: Study on the biological activity and physicochemical properties of the antimicrobial peptide Brevicidine

[0159] 1. Minimum inhibitory concentration (MIC) and time-kill curve

[0160] 1. Minimum inhibitory concentration (MIC)

[0161] The antibacterial activity of the antimicrobial peptide Brevicidine was evaluated using the Clinical Laboratory and Standards Institute (CLSI) broth microdilution method using a sterile 96-well plate. The specific details are as follows: The indicator bacteria were cultured to the logarithmic phase and diluted with MH broth medium (Beijing Solebold Technology Co., Ltd., product catalog number M8556) to obtain a concentration of 10 5 -10 6 CFU / mL indicator bacteria dilution. The pure Brevicidine was dissolved in MH broth medium to obtain a Brevicidine stock solution with a concentration of 512μg / mL. The Brevicidine stock solution was prepared into the test concentration by the two-fold dilution method, and then 50μL of the continuously two-fold diluted Brevicidine solution and 50μL of the indicator bacteria dilution were added to a sterile 96-well plate, three parallels for each concentration, cultured at 37°C for 18h, and the OD value was read at 600nm. MIC is the minimum immunomodulatory peptide concentration that inhibits the growth rate by more than 50%. MH broth medium was used as a negative control and polymyxin was used as a positive control. All experiments were repeated three times.

[0162] As shown in Table 4, the antimicrobial peptide Brevicidine exhibited strong antibacterial activity against a variety of Gram-negative pathogens, including Escherichia coli (such as Escherichia coli ATCC 25922, Escherichia coli K88, Escherichia coli O157:H7, Escherichia coli PN16), Salmonella typhimurium CVCC 534, Salmonella typhimurium SL1344, Pseudomonas aeruginosa ATCC 27853 and Shigella dysenteriae CGMCC 1.1869, with a minimum inhibitory concentration (MIC) of 1-4 μg / mL. It is worth noting that the MIC of Brevicidine against colistin-resistant Escherichia coli is 1 μg / mL, while the MIC of colistin is 8 μg / mL. However, Brevicidine has no significant inhibitory effect on Gram-positive bacteria (such as Staphylococcus aureus ATCC 43300 and Enterococcus faecalis ATCC 29212), with MIC>256μg / mL.

[0163] Table 4. MIC of antimicrobial peptide Brevicidine against various indicator bacteria

[0164] Indicator bacteria Strain code Brevicidine MIC, μg / mL Colistin MIC, μg / mL Escherichia coli ATCC25922 1 0.5 Escherichia coli K88 1 0.5 Escherichia coli O157:H7 1 1 Escherichia coli PN16 1 8 Salmonella Gallinarum CVCC534 2 0.5 Salmonella Typhimurium SL1344 2 1 Pseudomonas aeruginosa ATCC27853 2 1 Shigella dysenteriae CGMCC1.1869 4 1 Bacillus cereus CVCC4101 32 8 Staphylococcus aureus ATCC43300 >256 >256 Enterococcus faecalis ATCC29212 >256 >256

[0165] 2. Time sterilization curve

[0166] (1) A single colony of Escherichia coli K88 or Salmonella enterica serovar Gallinarum CVCC 534 was inoculated into 5 mL of MHB medium (product of Beijing Solebaugh Technology Co., Ltd., catalog number M8556) and cultured overnight at 37°C and 200 rpm. The next day, the culture was inoculated into fresh MHB medium at a ratio of 1:100 and cultured at 37°C and 200 rpm until the logarithmic growth phase (for the purpose of antibacterial activity test).

[0167] (2) After completing step (1), approximately 5×10 5 or 10 8 CFU / mL of Escherichia coli K88 or Salmonella enterica serovar Gallinarum CVCC 534 were inoculated into 2 mL of MHB medium containing different Brevicidine concentrations (0, 1×, 2×, 4× and 8× MIC). At different time points, samples were taken and serially diluted with sterile saline, then spread on MH agar plates. After overnight incubation at 37°C, colonies were counted to determine CFU / mL.

[0168] MH agar plate: add 2% agar powder (product of Beijing Solebow Technology Co., Ltd., catalog number A8190) to MHB medium, cool and pour into a plate.

[0169] The effectiveness of Brevicidine was further confirmed by time-kill curve analysis, showing significant time-dependent inhibition of bacterial populations of Escherichia coli K88 and Salmonella enterica serovar Gallinarum CVCC 534 (see Fig.13 In the E. coli group (10 5 CFU / mL), a significant reduction was observed within 2 hours, and the 2× and 4× MICs achieved complete bactericidal effects within 6-12 hours. 5 CFU / mL), 4×MIC eliminated the bacteria within 6 hours, while 1× and 2×MIC reduced the bacterial count by 3 and 7 log units, respectively, within 24 hours.

[0170] 2. Scanning electron microscopy and drug resistance accumulation experiments

[0171] (1) Scanning electron microscopy experiment

[0172] Escherichia coli K88 or Salmonella enterica serovar Gallinarum CVCC 534 were treated with Brevicidine and then analyzed by scanning electron microscopy (SEM). The SEM samples were prepared as follows: Escherichia coli K88 or Salmonella enterica serovar Gallinarum CVCC 534 cultured overnight were inoculated into fresh MHB medium and cultured at 37°C for 8 hours until the mid-logarithmic growth phase. The cells were then collected, washed with PBS and resuspended in PBS buffer. Approximately 2×10 7 CFU / mL cells were incubated with 1×, 2× or 4× MIC concentrations of Brevicidine at 37°C for 1 hour. After incubation, the cells were centrifuged and washed, fixed with 2.5% glutaraldehyde at 4°C overnight, then fixed with 1% osmium tetroxide for 1 hour, and dehydrated in gradient ethanol and acetone. The samples were dried using a critical point dryer (K850, Quorum), gold-palladium plated, and observed under a scanning electron microscope (SU8100, HITACHI).

[0173] Scanning electron microscopy analysis further revealed significant morphological changes on the surface of Escherichia coli K88 or Salmonella enterica serovar Gallinarum CVCC 534 treated with Brevicidine (see Fig.14), specifically manifested as damaged bacterial membranes, irregular surfaces, and lysis, which showed dose-dependent changes; among them, Escherichia coli cells showed shrinkage and deformation, while Salmonella cells showed extensive membrane rupture and content leakage. These results provide direct evidence that Brevicidine destroys the integrity of bacterial cell membranes and causes cell death.

[0174] (2) Drug resistance accumulation experiment

[0175] (2-1) In order to determine whether sublethal concentrations of Brevicidine would increase the MIC value, Escherichia coli K88 or Salmonella enterica serovar Gallinarum CVCC 534 were inoculated into MHB medium and cultured overnight at 37°C and 220 rpm to obtain a culture. The culture was then diluted to an OD of 600nm The concentration of the diluted microorganism was 0.5, and then inoculated into fresh MHB medium at a ratio of 1:100 to obtain a bacterial suspension.

[0176] (2-2) The initial Brevicidine MIC for each bacterial strain was determined using the broth microdilution method. Two-fold serial dilutions of Brevicidine in MHB medium were prepared, with the starting concentration slightly below the MIC (0.25×, 0.5×, and 1× MIC). Then 100 μL of the Brevicidine dilution was added to each well of a 96-well microtiter plate, and each well was inoculated with 100 μL of the bacterial suspension to a final bacterial concentration of approximately 5×10 5 CFU / mL. After 24 hours of incubation, OD was measured 600nm To assess bacterial growth.

[0177] (2-3) Take 100 μL of culture from the wells showing growth and transfer it to a 96-well plate containing fresh dilutions of Brevicidine at the same sub-MIC level. Amoxicillin, colistin, and ciprofloxacin at 0.25×, 0.5×, and 1× MIC were used as controls. Serial subcultures were performed daily for up to 30 days to simulate long-term exposure, during which the MIC values ​​of Brevicidine against bacterial strains were measured and recorded daily to observe whether resistance increased.

[0178] To further verify whether resistance to Brevicidine would develop during treatment, the bacteria were continuously exposed to sub-MIC levels of Brevicidine. The results showed that within 30 days, Escherichia coli K88 and Salmonella enterica serovar Gallinarum CVCC 534 did not develop significant resistance to Brevicidine, and the MIC fluctuated only within a 1- to 2-fold range (see Fig.15In contrast, sublethal levels of ciprofloxacin (Cip), amoxicillin (Amp), and colistin (Col) induced more than 4-fold resistance in Escherichia coli K88 within 3 days and in Salmonella enterica serovar Gallinarum CVCC 534 within 9-10 days, respectively.

[0179] 3. Stability experiment of anti-lipopeptide Brevicidine

[0180] 1. High temperature tolerance test

[0181] The pure Brevicidine was dissolved in ultrapure water with a pH value of 3 to obtain a Brevicidine solution with a concentration of 200 μg / mL. The Brevicidine solution was placed in an environment of 37°C, 55°C, 75°C, 100°C or 121°C, and at 0h, 2h, 4h and 6h respectively: 200 μL of the Brevicidine solution was taken to detect the chromatographic peak area of ​​Brevicidine at different time points using high performance liquid chromatography, and the degradation rate of Brevicidine was calculated; 100 μL of the Brevicidine solution was taken to detect the antibacterial activity of Brevicidine using the inhibition zone method.

[0182] 2. pH tolerance test

[0183] Ultrapure water was adjusted to an aqueous solution with a pH value of 1, 3, 5, 7, 9 or 11 using dilute hydrochloric acid and sodium hydroxide solution. Pure Brevicidine was dissolved in the above aqueous solution and prepared into a Brevicidine solution with a concentration of 200 μg / mL, which was placed at room temperature. At 0, 0.5, 1, 1.5 and 2 h respectively: 200 μL of Brevicidine solution was taken to detect the chromatographic peak area of ​​Brevicidine in aqueous solutions with different pH values ​​by high performance liquid chromatography, and the degradation rate of Brevicidine in aqueous solutions with different pH values ​​was calculated; 100 μL of Brevicidine solution was taken to detect the antibacterial activity of Brevicidine by the inhibition zone method.

[0184] 3. Artificial gastrointestinal fluid tolerance test

[0185] According to the Pharmacopoeia of the People's Republic of China (2020 edition), add 800 mL of water and 10 g of pepsin to 16.4 mL of dilute hydrochloric acid, shake well, transfer to a 1000 mL volumetric flask and dilute to 1000 mL with ultrapure water to obtain artificial gastric juice. Dissolve and dilute pure Brevicidine to a concentration of 200 μg / mL with artificial gastric juice, place in a 37°C water bath, and take samples at 0, 2, 4, and 6 h. After the sample was inactivated by 85°C water bath for 5 min, it was centrifuged at 4°C and 10,000 rpm for 15 min, and the chromatographic peak area of ​​Brevicidine at different time points was detected to calculate the degradation rate of Brevicidine; 100 μL of solution was taken to detect the antibacterial activity of Brevicidine by the inhibition zone method.

[0186] Also according to the "Pharmacopoeia of the People's Republic of China" (2020 edition), add 118mL of 0.2mol / L sodium hydroxide solution to 250mL of 0.2mol / L potassium dihydrogen phosphate solution to obtain liquid A; take another 10g of pancreatic enzyme, add appropriate amount of water to dissolve, and obtain liquid B; mix liquid A and liquid B and transfer them to a 1000mL volumetric flask and dilute to 1000mL with water to obtain artificial intestinal fluid. Dissolve and dilute the pure Brevicidine to a concentration of 200μg / mL with artificial intestinal fluid, place it in a 37℃ water bath, and take samples at 0, 2, 4, and 6h. After the sample was inactivated by 85℃ water bath for 5min, it was centrifuged at 4℃ and 10000rpm for 15min, and the chromatographic peak area of ​​Brevicidine at different time points was detected, and the degradation rate of Brevicidine was calculated; 100μL of solution was taken to detect the antibacterial activity of Brevicidine by the inhibition zone method.

[0187] In order to study the application potential of Brevicidine, this application evaluated its stability in various biochemical environments and demonstrated the remaining intact Brevicidine content through HPLC results, while the changes in Brevicidine bioactivity were demonstrated by agar diffusion method results. After heating from room temperature to 100°C for 2 hours, the structure and bioactivity of Brevicidine remained stable, with an average retention rate of 96.5%; however, after high-pressure sterilization at 121°C for 2 hours, its content dropped to 74.2% (see Fig.16 Brevicidine also maintained integrity and biological activity after treatment at pH 1.0-9.0 for 2 hours, with an average HPLC retention of 98.5% (see Fig.16In addition, in simulated gastric fluid (SGF), the intact concentration of Brevicidine and its bioactivity did not change significantly, while in simulated intestinal fluid (SIF), Brevicidine was degraded faster, decreasing to 87.6% of the minimum level after 6 h (see Fig.16 However, the diameter of the inhibition zone of Brevicidine in SIF was not significantly reduced ( Fig.16 (F), indicating that although the intact form of Brevicidine is disrupted, its core functional structure is still preserved.

[0188] 4. Cytotoxicity and hemolysis (used to detect the safety of antimicrobial active substances)

[0189] 1. Cytotoxicity test

[0190] Take a 96-well plate and add 100 μL of 1×10 5 cell / mL of intestinal epithelial cell IPEC-J2 suspension, culture the cells until the confluence is 90%. After cell starvation treatment, add the antimicrobial peptide Brevicidine solution (solvent is DMEM / F12 culture medium) and control the final concentration of Brevicidine to 0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 and 256 μg / mL. After culturing for 24 hours, add 10 μL of CCK8 solution to each well of the culture plate, continue to culture for 2 hours, and measure the absorption value at 450nm with an enzyme reader. Take another culture plate to detect the level of cell LDH release. The toxicity of the antimicrobial peptide Brevicidine to IPEC-J2 cells was judged by cell activity and LDH release level.

[0191] 2. Hemolytic test

[0192] (1) Whole blood was collected from pigs using a blood collection tube containing sodium heparin, and red blood cells were obtained after centrifugation. Under the same conditions, venous blood was collected from under the eyeball of mice, and red blood cells were obtained after centrifugation.

[0193] (2) Wash blood cells (pig red blood cells or mouse red blood cells) with 10 mM PBS (pH 7.3) until the supernatant has no obvious red color, and then prepare the blood cells into an 8% cell suspension with PBS buffer.

[0194] (3) 100 μL of cell suspension and 100 μL of 1-256 μg / mL diluted antimicrobial peptide Brevicidine solution were added to the wells of a 96-well plate, incubated at 37°C for 1 h, centrifuged at 1500 rpm / min for 5 min, the supernatant was collected and transferred to a new 96-well plate, and the OD value at 540 nm was detected using an ELISA reader.

[0195] 10 mM PBS (pH 7.3) and 0.1% Triton X-100 were used as negative and positive controls, respectively.

[0196] Test results see Fig.17 The antimicrobial peptide Brevicidine at a concentration of 0.5-256μg / mL did not produce cytotoxicity to porcine intestinal epithelial cells IPEC-J2 (P<0.05). The hemolytic activity of the antimicrobial peptide Brevicidine at a concentration of 0.5-256μg / mL on porcine blood cells was equivalent to that of the negative control. The above results show that the antimicrobial peptide Brevicidine has no cytotoxicity and hemolytic activity.

[0197] 5. Study on the antibacterial adhesion and invasion effects of the antimicrobial peptide Brevicidine

[0198] In order to further evaluate and clarify the anti-infective activity of Brevicidine, its protective ability against Escherichia coli adhesion and Salmonella invasion was studied. IPEC-J2 cells were treated with the antimicrobial peptide Brevicidine and then co-cultured with bacteria to observe the protective effect of the antimicrobial peptide Brevicidine on the anti-bacterial adhesion and invasion of IPEC-J2 cells, as follows: IPEC-J2 cells were cultured in 6-well plates until a monolayer of cells was formed. Before infection, the cells were incubated with DMEM medium or medium containing Brevicidine for 24 hours. Escherichia coli K88 or Salmonella enterica serovar Gallinarum CVCC 534 (concentration of 5×10 8 CFU / mL) were washed with PBS, resuspended in culture medium and added to cells. Different concentrations of Brevicidine were added to the treatment groups, and the control group was added with an equal volume of PBS. All groups were incubated at 37°C for 2 hours.

[0199] For the E. coli infection group, after removing the culture medium, the cells were washed three times with PBS to remove non-adherent E. coli. Subsequently, the cells were added with 1 mL of 0.1% Triton X-100 and incubated for 5-10 minutes to collect the cell suspension and perform serial dilutions. 100 μL of each dilution was inoculated on MH agar plates and cultured overnight at 37°C to count colonies.

[0200] For the Salmonella infection group, after incubation with different concentrations of Brevicidine for 2 hours, the culture medium was removed, and 200 mg / mL gentamicin solution was added to the cell culture medium for incubation for 1 hour. Subsequently, the cells were washed three times with PBS to remove the gentamicin solution and treated with 0.1% Triton X-100 for 5 minutes. Then, 800 μL PBS was added to collect the cells. The cell lysate was serially diluted, and 100 μL of each gradient dilution was inoculated on MH agar plates and cultured overnight at 37°C to count colonies.

[0201] Test results see Fig.18 Compared with the control group, Brevicidine significantly reduced the adhesion of E. coli at different MIC levels (P<0.05), while at concentrations of 2×MIC or higher, Brevicidine significantly reduced the invasion of Salmonella (P<0.05). At 8×MIC, Brevicidine completely inhibited E. coli and Salmonella.

[0202] The above results show that the antimicrobial peptide Brevicidine has strong activity against Gram-positive and Gram-negative bacteria, can quickly kill bacteria by destroying the bacterial membrane structure, and is not easy to cause bacterial resistance. At the same time, the antimicrobial peptide Brevicidine has strong tolerance to temperature, pH value and simulated digestive fluid in vitro, and 0.5-256μg / mL of the antimicrobial peptide Brevicidine is not toxic to porcine intestinal epithelial cells IPEC-J2 and is not hemolytic to pig and mouse blood cells. These results highlight the potential of the antimicrobial peptide Brevicidine as a potent and long-term antimicrobial agent in controlling animal infections caused by Escherichia coli and / or Salmonella.

[0203] Example 5: Study on the effect of antimicrobial peptide Brevicidine on antibacterial infection and maintaining intestinal homeostasis

[0204] 1. Experimental Methods

[0205] 1. Escherichia coli and Salmonella co-infection mouse model

[0206] Female C57BL / 6 mice aged 6–7 weeks used in this study were purchased from Beijing Huafukang Company (license number: SCXK2019-0008, Beijing, China). All animal experiments were performed in accordance with the guidelines of the Laboratory Animal Welfare and Animal Experiment Ethics Review Committee of China Agricultural University (approval number: AW21804202-1-2). All mice were housed in a laminar flow cabinet and housed individually in a temperature- and humidity-controlled room with a 12-h light / dark cycle. Feed and water were provided throughout the experiment.

[0207] (1) Preparation of mixed inoculum

[0208] The strains used in this study include Escherichia coli K88, Escherichia coli O157:H7, Salmonella typhi CVCC 534, and Salmonella typhimurium SL1344. The above strains were inoculated into MH broth medium, and cultured overnight at 37°C and 220 rpm to obtain cultures; then the above cultures were diluted to OD 600nm is 0.5, corresponding to about 10 for each strain 10 CFU / mL. The diluted cultures of the four strains were mixed in equal volumes to prepare a mixed inoculum; the concentration of each strain in the mixed inoculum was 2.5×10 9 CFU / mL.

[0209] (2) Before the experiment, 42 mice were randomly divided into a control group, treatment group 1, treatment group 2, treatment group 3 and a challenge group. The control group, treatment group 1, treatment group 2 and treatment group 3 each consisted of 8 mice, and the challenge group consisted of 10 mice. After a 5-day adaptation period, the mice in each group were treated as follows:

[0210] Control group: 0.3 mL PBS buffer was administered by intragastric gavage every day for 8 days.

[0211] One treatment group was given 0.3 mL of Brevicidine solution (the solvent was PBS buffer) by oral gavage every day, and the dosage of Brevicidine was 7.5 mg / kg (per kilogram of body weight); this lasted for 7 days; on the 8th day, 0.3 mL of the mixed inoculum was administered by oral gavage for virus attack.

[0212] Treatment group 2: 0.3 mL of Brevicidine solution (solvent is PBS buffer) was administered by oral gavage every day, and the dosage of Brevicidine was 15 mg / kg (per kilogram of body weight); this lasted for 7 days; on the 8th day, 0.3 mL of the mixed inoculum was administered by oral gavage for virus attack.

[0213] Three groups were treated with 0.3 mL of Brevicidine solution (the solvent was PBS buffer) by oral gavage every day, and the dosage of Brevicidine was 30 mg / kg (per kilogram of body weight); this lasted for 7 days; on the 8th day, 0.3 mL of the mixed inoculum was administered by oral gavage for virus attack.

[0214] The challenge group was given 0.3 mL of PBS buffer by oral gavage every day for 7 days; on the 8th day, 0.3 mL of mixed inoculum was given by oral gavage for challenge.

[0215] 2. Measurement indicators and methods

[0216] (1) Clinical symptom recording and sample collection

[0217] The mice were monitored daily for symptoms of infection, including weight loss, body temperature, diarrhea, and clinical signs. At the predetermined time point (72 hours after challenge), the mice were euthanized and samples were collected. Serum was collected from the mice. The liver, spleen, and feces were stored in 30% glycerol for bacterial load determination. The colon contents were collected and stored in liquid nitrogen for microbial diversity analysis. The jejunum and ileum were immediately placed in liquid nitrogen after collection and stored at -80°C for cytokine and protein analysis. Tissues and organs were fixed in 4% paraformaldehyde for histopathological evaluation, and histopathological changes were examined by hematoxylin-eosin staining (H&E) and periodic acid-Schiff (PAS) staining.

[0218] (2) Bacterial count and fecal microbial diversity analysis

[0219] After the collected tissue samples are weighed, they are homogenized with sterile PBS and serial dilutions are plated on MacConkey agar plates. Bacteria that ferment lactose (such as E. coli) will form pink colonies, while bacteria that do not ferment lactose (such as Salmonella) will form colorless or light-colored colonies. The plates are incubated overnight at 37°C and the colonies are counted to determine the bacterial load in each tissue sample.

[0220] Microbial diversity sequencing was then performed to analyze the composition of the gut microbiota. Fecal samples were collected from each group and stored at -80 °C until processing. DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The extracted DNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA) and its integrity was checked by agarose gel electrophoresis. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 341F: 5'-CCTAYGGGRBGCASCAG-3' and 806R: 5'-GGACTACNNGGGTATCTAAT-3'. PCR products were purified using AMPure XP magnetic beads (Beckman Coulter, CA, USA) and quantified using a Qubit2.0 fluorometer (Invitrogen, CA, USA). Equimolar amounts of purified amplicons were mixed and sequenced using the Illumina MiSeq platform using a 2 × 250 bp paired-end protocol. Raw sequencing data were processed using the QIIME2 pipeline and clustered into operational taxonomic units (OTUs) at 97% sequence similarity using the Greengenes database (version 13.8). Alpha diversity indices such as the Shannon and Simpson indices were calculated to assess microbial diversity within the samples. Beta diversity was analyzed by principal coordinate analysis (PCoA) based on the Bray-Curtis dissimilarity to compare the microbial community structure among the groups. The microbial composition at different taxonomic levels (phylum, genus, species) was determined to identify differences in microbial populations among the experimental groups.

[0221] (3) Assessment of intestinal barrier function and inflammation

[0222] The concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in serum were determined using commercial ELISA kits. Total protein in the jejunum and colon was extracted, and the concentrations of TNF-α, IL-1β, and IL-6 were determined using commercial ELISA kits.

[0223] The relative expression levels of target genes were quantitatively detected by RT-qPCR. The primers used are shown in Table 5. Total RNA was extracted from tissue samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. The quality and concentration of RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA), ensuring that the A260 / A280 ratio was between 1.8 and 2.0. 1 μg of RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Tokyo, Japan), and qRT-PCR was performed using SYBR Green Master Mix (Applied Biosystems, MA, USA) in a LightCycler 96 Real-Time PCR System (Roche Diagnostics, Rotkreuz, Switzerland), with three replicates for each sample. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene, and the relative expression levels of target genes were calculated using 2 -ΔΔCt Method calculation.

[0224] Table 5

[0225]

[0226]

[0227] The expression of intestinal barrier proteins, including E-cadherin, ZO-1, Claudin-1, and Occludin, was evaluated by Western blot analysis. 30–50 mg of intestinal tissue was obtained from a −80°C freezer and washed three times with sterile precooled PBS to remove blood contamination. The samples were minced and lysed in a tissue homogenizer with 200 μL of precooled RIPA lysis buffer (containing protease inhibitors, Solarbio Science and Technology Co., Ltd., Beijing) for 10 min and incubated on ice for 30 min, with pipetting every 5 min to ensure adequate lysis. The samples were then centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was collected as the total protein solution. The protein concentration was determined using a BCA protein assay kit. The protein samples were mixed with 5× loading buffer at a ratio of 4:1 and boiled in a PCR instrument for 5 min to denature the protein. The denatured protein samples were stored at −80°C. Equal amounts of protein (20-30 μg) were separated on 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membrane was blocked with 5% skim milk powder for 1 hour, followed by the addition of primary antibodies to the target protein and internal reference β-actin (Proteintech Group, Inc., IL, USA) and incubated at 4°C overnight. After washing, secondary antibodies (ThermoFisher Scientific, MA, USA) were added and incubated at room temperature for 1 hour. Bands were observed using an enhanced chemiluminescence (ECL) detection system (ThermoFisher Scientific, MA, USA) and quantified using ImageJ software. Relative protein expression levels were normalized to the internal reference and analyzed.

[0228] (4) Statistical analysis

[0229] Data were analyzed using SAS system (version 9.2, SAS Institute, Inc., NC, USA) for one-way analysis of variance (ANOVA). Tukey's multiple comparison test was used to determine the differences between treatments. All data were visualized using GraphPad Prism6 software (GraphPad Software Inc., CA, USA) and Origin software (version 2018, OriginLabCorporation, MA, USA). Microbial diversity statistical analysis was performed using R software (v3.6.1, RFoundation for Statistical Computing, Vienna, Austria). Permutation multivariate analysis of variance (PERMANOVA) was applied to assess the statistical significance of differences in microbial community structure in each group. In addition, the DESeq2 software package in R was used to perform differential abundance analysis at the phylum and genus levels. The results are expressed as mean ± standard error (SEM). Statistical significance was set at P < 0.05.

[0230] 2. Experimental Results

[0231] 1. The preventive effect of the antimicrobial peptide Brevicidine on clinical symptoms of challenged mice

[0232] Experimental design Fig.19 As shown in A. In the pretreatment stage, the control group and the challenge group were given PBS by oral gavage, while the treatment groups (treatment group 1, treatment group 2, and treatment group 3) were given 7.5-30 mg / kg of the antimicrobial peptide Brevicidine every day for 7 days. No significant difference was found in body weight ( Fig.19 B), indicating that Brevicidine at this concentration had no negative effect on the growth of mice. Three days after the challenge with E. coli and Salmonella, the body weight of the challenge group decreased significantly compared with the control group (P<0.05), with an average weight loss of 11%; while the body weight of the treatment group 1, treatment group 2, and treatment group 3 improved, especially the body weight of the treatment group 3 increased by 0.8% at a dose of 30 mg / kg ( Fig.19 The survival rate of the challenge group was 80%, with two mice dying, while no mice died in the other groups ( Fig.19 D). Three days after the challenge, the body temperature of mice in the challenge group was significantly lower by 1°C than that in the control group, treatment group 1, treatment group 2, and treatment group 3 (P<0.05) ( Fig.19 E). According to previous studies, the diarrhea scores of mouse feces were recorded daily. The diarrhea scores of the challenge group on the first and third days were significantly increased (P<0.05), and Brevicidine treatment effectively alleviated these symptoms ( Fig.19 F and G).

[0233] In addition, the antimicrobial peptide Brevicidine significantly inhibited the colonization and metastasis of Escherichia coli and Salmonella in the mice of the treatment groups (treatment group 1, treatment group 2, and treatment group 3). The number of bacteria in the fecal samples of treatment groups 1, 2, and 3 was significantly reduced compared with the challenge group ( Fig.19 H), indicating that Brevicidine limits the persistence of these pathogens in the gastrointestinal tract. In addition, Brevicidine treatment also reduced the Salmonella load in the liver and spleen ( Fig.19 I and J). Thus, Brevicidine not only prevents intestinal colonization but also reduces the systemic spread of the pathogen.

[0234] 2. Pretreatment with the antimicrobial peptide Brevicidine regulates immunoglobulin levels and inflammatory responses in challenged mice

[0235] The changes in immunoglobulin and cytokine levels in mouse serum were evaluated. The experimental results showed that the challenge significantly reduced the IgG level in mouse serum (P<0.05), while treatment with different doses of Brevicidine alleviated this decline and had no significant effect on the serum levels of IgA and IgM ( Fig. 20 ELISA analysis of serum inflammatory factors showed that the challenge significantly increased the levels of proinflammatory cytokines IL-1β and TNF-α in the serum of mice (P<0.05) ( Fig. 20 Middle A). The brevicidine-treated groups (treatment group 1, treatment group 2, and treatment group 3) maintained normal levels of these inflammatory cytokines, with no significant difference compared with the control group.

[0236] Further q-PCR analysis was used to evaluate the anti-inflammatory properties of Brevicidine. Brevicidine significantly reduced the mRNA expression levels of TNF-a, IL-1β, and IL-6 in the ileum and colon of challenged mice (P<0.05) ( Fig. 20 In order to verify these results at the protein level, proteins were extracted from the ileum and colon of mice, and the expression levels of intestinal inflammatory cytokines were quantitatively analyzed by ELISA. Fig. 20 Middle C) is consistent with the qPCR results, confirming the reliability of Brevicidine's anti-inflammatory effect on intestinal tissue.

[0237] 3. Pretreatment with the antimicrobial peptide Brevicidine protects the intestinal morphology and barrier function of challenged mice

[0238] The challenge with Escherichia coli and Salmonella significantly disrupted the intestinal morphology of mice and led to obvious histopathological changes, such as Fig.21As shown in A and B. However, the experimental results showed that Brevicidine treatment effectively protected the intestinal morphology of the challenged mice. Histological analysis showed that the intestinal tissues of mice in the Brevicidine-treated groups (treatment group 1, treatment group 2, and treatment group 3) maintained normal villus structure, crypt structure, and goblet cell number (see Table 6), and the signs of inflammation and tissue damage were significantly reduced compared with the untreated challenged group ( Fig.21 (C) This protective effect was observed in both the ileum and colon, with the integrity of the intestinal epithelium preserved, demonstrating the potential of brevicidine to mitigate the deleterious effects of pathogen infection on intestinal morphology.

[0239] The effects of Brevicidine pretreatment on key intestinal barrier proteins (E-cadherin, ZO-1, Claudin-1, Occludin) in the ileum and colon were evaluated by qPCR and Western blot analysis. Fig.21 D and E) showed that the mRNA expression levels of E-cadherin, Claudin-1, and Occludin in the challenge group were significantly lower than those in the control group (P<0.05), but the Brevicidine treatment groups (treatment group 1, treatment group 2, and treatment group 3) moderately increased the expression of these genes. Western blot analysis further confirmed that the protein levels of E-cadherin, ZO-1, Claudin-1, and Occludin in the Brevicidine treatment groups (treatment group 1, treatment group 2, and treatment group 3) were increased compared with the untreated challenge group ( Fig.21 The above results indicate that Brevicidine pretreatment upregulated the expression of key intestinal barrier proteins, highlighting its role in maintaining barrier integrity and resisting pathogen infection.

[0240] Table 6. Effects of the antimicrobial peptide Brevicidine on the intestinal morphology of challenged mice

[0241]

[0242] Note: Data are presented as the mean of 8 biological replicates. Different superscript lowercase letters in the same group indicate significant differences (P<0.05).

[0243] 4. Effect of Brevicidine pretreatment on the microbial diversity of mouse colon contents

[0244] The experiment evaluated the effect of pretreatment with the antimicrobial peptide Brevicidine on the colonic microbiota. The coverage index of all samples was greater than 0.99 ( Fig. 22Brevicidine pretreatment had no significant effect on alpha diversity (Shannon or Simpson index) or community richness (ACE index), indicating the stability of the overall microbial community ( Fig. 22 For β diversity, principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity at the OTU level showed that there were obvious clustering patterns among the control group, the challenge group, and the second treatment group, while the first and third treatment groups were closer to the control group ( Fig. 22 (EG).

[0245] Analysis of intestinal microbiota composition ( Fig.23 Middle A) At the phylum level, the main phyla are Firmicutes, Bacteroidetes, and Verrucomicrobia. The challenge treatment significantly increased the abundance of Bacteroidetes and Proteobacteria, while reducing the abundance of Firmicutes.

[0246] Brevicidine treatment reduced the abundance of Proteobacteria and restored the abundance of Firmicutes to varying degrees. Notably, treatment 2 showed an increase in the abundance of Verrucomicrobia. Fig.23 B), the main genera were unclassified Muribaculaceae, Lactobacillus, and unclassified Clostridia. Challenge significantly reduced the abundance of Lactobacillus, while increasing the abundance of unclassified Muribaculaceae and unclassified Clostridia. Further differential abundance analysis showed that Brevicidine pretreatment significantly altered the taxonomic composition of colonic contents ( Fig.23 CF). At the phylum level, the Brevicidine-treated group showed a significant increase in the abundance of Firmicutes, while the abundance of Bacteroidetes and Proteobacteria decreased compared with the challenge group. In particular, treatment group II significantly increased the abundance of Verrucomicrobia. At the genus level, challenge significantly reduced the abundance of Lactobacillus, while Brevicidine pretreatment restored the imbalance caused by challenge. Notably, treatment group II showed an increase in the abundance of Akkermansia. The above results indicate that Brevicidine pretreatment effectively regulated the composition of the intestinal microbiota, enhanced the abundance of beneficial bacteria (such as Lactobacillus and Akkermansia), and inhibited harmful bacteria. This regulation may contribute to the protective effect of Brevicidine on the intestinal health of challenged mice.

[0247] The above results show that in the in vivo experiment of mice, pretreatment with the antimicrobial peptide Brevicidine significantly alleviated the symptoms of weight loss in mice caused by mixed challenge of Escherichia coli and Salmonella, increased the survival rate, and reduced the severity of diarrhea. Further analysis showed that Brevicidine protected intestinal morphology, reduced the level of pro-inflammatory cytokines, and enhanced intestinal barrier function by upregulating key proteins. Intestinal microbiota analysis showed that Brevicidine treatment restored the balance of intestinal microbiota and increased the abundance of beneficial bacteria such as Lactobacillus and Akkermansia. Brevicidine has important potential in resisting bacterial infection, maintaining intestinal homeostasis, and improving animal health.

[0248] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Brevibacillus laterosporus 811, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.32462.

2. A bacterial agent comprising the Brevibacillus laterosporus 811CGMCC No.32462 according to claim 1.

3. Use of Brevibacillus laterosporus 811CGMCC No.32462 or the bacterial agent according to claim 2 in preparing the antimicrobial peptide Brevicidine or inhibiting bacteria.

4. The use of the antimicrobial peptide Brevicidine, which is at least one of B1) to B11); B1) reduce the levels of pro-inflammatory cytokines; B2) Enhance intestinal barrier function; B3) Maintain the balance of intestinal microbiota; B4) Increase the abundance of beneficial bacteria; B5) resist bacterial infection; B6) Maintaining intestinal homeostasis in animals; B7) Maintaining animal health; B8) inhibit bacteria; B9) promote bacterial death; B10) reduce bacterial adhesion and / or invasion; B11) Prevention and / or treatment of diseases caused by bacterial infection.

5. The use according to claim 4, characterized in that: In said B9), the promotion of bacterial death is manifested in the destruction of the bacterial membrane structure.

6. The use according to claim 4 or 5, characterized in that: The bacterium is at least one of Staphylococcus aureus, Escherichia coli, Salmonella typhi, Salmonella typhimurium, Pseudomonas aeruginosa and Shigella dysenteriae.

7. The use according to claim 4, characterized in that: The protection against bacterial infection is manifested in inhibiting bacterial colonization in the intestinal tract of the animal and / or reducing systemic spread of bacteria.

8. The use according to claim 4, characterized in that: The maintenance of animal health is manifested in improving the survival rate of animals infected with bacteria and / or reducing the degree of diarrhea of ​​animals infected with bacteria.

9. The use according to any one of claims 4 to 8, characterized in that: The antimicrobial peptide Brevicidine is prepared by using the Brevibacillus laterosporus 811CGMCC No.32462 described in claim 1 or the bacterial agent described in claim 2.

10. A method for inhibiting bacteria, comprising treating bacteria with the Brevibacillus laterosporus 811CGMCC No.32462 of claim 1, the bacterial agent of claim 2, or the antimicrobial peptide Brevicidine prepared from the Brevibacillus laterosporus 811CGMCC No.32462 of claim 1.

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