An antibacterial peptide basbpin derived from bacillus subtilis and application thereof

Abstract

The application discloses an antibacterial peptide Basbpin derived from Bacillus subtilis and application thereof. The application has a wide antibacterial spectrum and unique cross-border double antibacterial activity, the bactericidal mechanism of the application is not prone to induce drug resistance, the application has excellent thermal stability, can be well adapted to extensive feed processing technology, and has high biological safety and no cytotoxicity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and feed additive technology, specifically relating to an antimicrobial peptide Basbpin derived from Bacillus subtilis and its application. Background Technology

[0002] The discovery and widespread use of antibiotics is a significant milestone in the development of modern medicine and modern aquaculture. However, with the long-term, excessive, and even abuse of antibiotics in human clinical medicine, animal husbandry, veterinary medicine, and aquaculture, the problem of antimicrobial resistance (AMR) has become increasingly prominent globally. The rapid emergence and spread of drug-resistant pathogens not only significantly reduces the clinical efficacy of conventional antibiotics, leading to a dramatic increase in the difficulty and cost of infection control, but also poses a major threat to global public health security and the ecological environment.

[0003] In the aquaculture sector, the overuse and dependence on antibiotics is a particularly serious problem. Modern aquaculture is typically characterized by high density and open technology, making it easy for drugs introduced into the aquaculture system, within the animals, and in the surrounding aquatic ecosystem to accumulate and proliferate. This not only directly promotes the accumulation and cross-transmission of drug-resistant bacteria and genes in the water and related environment, but also leads to widespread drug residues in aquatic products, seriously threatening food safety and public health for end consumers. In actual aquaculture production, Streptococcus agalactiae, Aeromonas schuberis, and Vibrio fluvialis are common and prevalent aquatic pathogens that can easily cause large-scale outbreaks of disease and mortality in economically important aquatic animals such as fish, shrimp, and crabs. Long-term reliance on traditional chemical antibiotics not only fails to eradicate the recurring outbreaks of these opportunistic pathogens but also accelerates the evolution of super-resistant strains, posing a serious risk of economic losses to the aquaculture industry.

[0004] Furthermore, aquatic and livestock feeds are highly susceptible to fungal contamination (such as Cryptococcus neoformans, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Aspergillus niger, and Aspergillus ochraceus) during storage, transportation, and use due to changes in environmental humidity and temperature. Fungal growth not only depletes the nutrients in feed and reduces feed efficiency, but also produces highly toxic mycotoxins during metabolism. Long-term ingestion of mold-contaminated feed by animals can lead to significantly weakened immunity, organ damage, stunted growth, and even mass mortality. Therefore, developing novel green biological control agents with both antibacterial and antifungal activities to achieve "dual effects with one agent" has become an urgent and practical need to ensure public health safety, food safety, and the sustainable development of modern aquaculture.

[0005] Antimicrobial peptides (AMPs), as an important component of the organism's innate immune system, are considered one of the most promising candidates to replace traditional chemical antibiotics due to their unique advantages such as broad antibacterial spectrum, rapid bactericidal action, unique mechanism of action, and low likelihood of inducing drug resistance. Bacillus subtilis, a highly adaptable and widely used non-pathogenic beneficial microorganism in agriculture and animal husbandry, secretes a variety of peptide antimicrobial active substances, making it a natural resource for discovering and screening novel antimicrobial peptides.

[0006] Although there have been some sporadic reports on antimicrobial peptides derived from Bacillus subtilis in existing technologies, and attempts have been made to develop them as green feed additives or anti-infective agents, existing natural or artificially designed antimicrobial peptides still face many insurmountable technical bottlenecks when it comes to practical industrialization and large-scale application. First, the antimicrobial spectrum of most known antimicrobial peptides is relatively limited, often showing good activity only against specific types of Gram-positive bacteria, and lacking the ability to simultaneously kill Gram-positive bacteria, Gram-negative bacteria, and filamentous fungi with a broad-spectrum dual-killing resistance. Second, the higher-order spatial structure of natural peptides is usually quite sensitive to the physicochemical environment. The rigid high-temperature, high-pressure conditioning and extrusion granulation processes in modern aquaculture and livestock feed processing can easily cause peptides to denature, degrade, and become completely inactive. Furthermore, some peptides that exhibit high antimicrobial activity in vitro are often accompanied by significant cytotoxicity or hemolytic activity, causing varying degrees of damage to mammalian cells or non-target aquatic animal cells, making it difficult to guarantee their biosafety and feasibility for in vivo application. The aforementioned defects and uncertainties greatly limit the practical large-scale application of existing Bacillus subtilis antimicrobial peptides in pharmaceutical preparation, modern feed industry, and green aquaculture. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antimicrobial peptide, Basbpin, derived from Bacillus subtilis.

[0008] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptide Basbpin.

[0009] The technical solution of the present invention is as follows:

[0010] Basbpin, an antimicrobial peptide derived from Bacillus subtilis, has the amino acid sequence shown in SEQ ID NO.01.

[0011] The application of the aforementioned antimicrobial peptide Basbpin in the preparation of antibacterial products.

[0012] In a preferred embodiment of the present invention, it has inhibitory and bactericidal effects on Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus gasseri, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Acinetobacter baumannii, Aeromonas schubert, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, and Vibrio fluvialis.

[0013] An antibacterial product whose active ingredient includes the aforementioned antimicrobial peptide Basbpin.

[0014] The application of the aforementioned antimicrobial peptide Basbpin in the preparation of antifungal products.

[0015] In a preferred embodiment of the present invention, it has inhibitory and killing effects on Cryptococcus neoformans, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Aspergillus niger and Aspergillus ochraceus.

[0016] An antifungal product whose active ingredient includes the aforementioned antimicrobial peptide Basbpin.

[0017] The application of the aforementioned antimicrobial peptide Basbpin in the preparation of aquatic feed additives.

[0018] An aquatic feed additive whose active ingredient includes the aforementioned antimicrobial peptide Basbpin.

[0019] The beneficial effects of this invention are:

[0020] 1. The antimicrobial peptide Basbpin of this invention not only exhibits excellent inhibitory and killing capabilities against a variety of Gram-positive and Gram-negative bacteria, but also efficiently kills a variety of complex fungi and filamentous fungi, including Cryptococcus, Fusarium, and Aspergillus. This broad-spectrum dual-antibacterial and antifungal property makes it extremely valuable for addressing infections in complex environments and controlling feed mold.

[0021] 2. The antimicrobial peptide Basbpin of this invention exerts its bactericidal effect primarily through a physical mechanism that directly targets and disrupts the integrity of the pathogenic bacterial cell membrane, leading to the release of intracellular substances. This non-specific membrane disruption mechanism differs from the protein or nucleic acid target mechanism of traditional antibiotics, thereby fundamentally reducing the risk of pathogenic microorganisms developing drug resistance.

[0022] 3. The antimicrobial peptide Basbpin of this invention has excellent high-temperature resistance. After undergoing severe high-temperature heating treatment, its spatial structure and biological activity remain stable without significant inactivation. This characteristic enables it to perfectly withstand the high-temperature processes that aquatic feeds must undergo during industrial extrusion and granulation.

[0023] 4. Cytotoxicity evaluation confirmed that the antimicrobial peptide Basbpin of this invention, at doses far exceeding its effective bactericidal concentration, did not exhibit any cytotoxicity against mammalian cells or non-target aquatic animal cells, and cell viability remained good. This indicates that the antimicrobial peptide has extremely high biosafety, providing a solid guarantee for its safe application in in vivo animals and in human clinical medicine. Attached Figure Description

[0024] Figure 1 This is the bactericidal kinetic curve of the antimicrobial peptide Basbpin against Streptococcus agalactiae in Example 1 of the present invention.

[0025] Figure 2 This is a thermostability graph of the antimicrobial activity of the antimicrobial peptide Basbpin against Streptococcus agalactiae in Example 1 of the present invention.

[0026] Figure 3 This is a diagram showing the morphological changes of Streptococcus agalactiae caused by treatment with the antimicrobial peptide Basbpin in Example 1 of the present invention.

[0027] Figure 4 The graph shows the results of the CCK-8 assay for detecting the cytotoxicity of the antimicrobial peptide Basbpin against ZF4 and HEK-293T cells in Example 1 of this invention. The horizontal axis represents the concentration of Basbpin (μM), and the vertical axis represents cell viability (%). Detailed Implementation

[0028] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0029] Example 1

[0030] The amino acid sequence of the antimicrobial peptide Basbpin derived from Bacillus subtilis in this embodiment is SEQ ID NO. 01: GVRLFQNIAVIRRNLLTKWTLSKK.

[0031] In this embodiment, Nanjing GenScript Co., Ltd. was commissioned to synthesize the antimicrobial peptide Basbpin with a purity of over 95%. The company provided information on the polypeptide molecular weight, HPLC and other detection data. HeliQuest was used to predict its charge and hydrophobicity, and ProtParam was used to predict other physicochemical parameters. The physicochemical parameters of the antimicrobial peptide Basbpin are shown in Table 1.

[0032] Table 1 Physicochemical parameters of antimicrobial peptide Basbpin

[0033] amino acid residues 24 molecular weight 2855.47 Da isoelectric point 12.31 Net charge +6 hydrophobic 42.7% Average hydrophilicity index -0.083

[0034] I. Determination of Basbpin's minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC)

[0035] 1. The strains involved include: Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, Vibrio fluvialis, Cryptococcus neoformans, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Aspergillus niger, and Aspergillus ochraceus. All of the above strains were purchased from the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences.

[0036] Aeromonas schubert and Streptococcus agalactiae were collected and identified in December 2023 from diseased Litopenaeus vannamei shrimp at a large-scale aquaculture farm in Guangzhou, Guangdong Province.

[0037] Lactobacillus gasseri was collected and identified in 2024 from diseased samples and water from a large-scale aquaculture farm in Guangzhou, Guangdong Province.

[0038] Bacillus cereus was collected and identified in 2024 from diseased materials and water in a large-scale aquaculture farm in Guangzhou, Guangdong Province, and is designated as Bacillus cereus GZ.

[0039] 2. The specific measurement method is as follows:

[0040] (1) Activate the bacterial strains by streaking bacteria onto NB plates, Vibrio onto 2216E plates, and yeast / fungi onto YPD plates and culturing until plump single colonies are formed; drop filamentous fungal spores onto PDA plates and culturing until a large number of spores are formed.

[0041] (2) Pick a single bacterial colony and inoculate it into a suitable liquid culture medium. Culture it to the logarithmic phase and collect the bacterial cells. Dilute the bacteria with MH liquid medium, the Vibrio with TSB+2% NaCl liquid medium, and the yeast fungus with YPD liquid medium to adjust the bacterial suspension concentration to 1×10⁻⁶. 6 CFU / mL. Filamentous fungal spores were washed away with PDW liquid medium, and the spore concentration was adjusted to 5 × 10⁻⁶. 4 CFU / mL.

[0042] (3) Dissolve Basbpin powder in sterile MilliQ water to 3 μM, 6 μM, 12 μM, 24 μM, 48 μM and 96 μM.

[0043] (4) On a 96-well cell culture plate, a blank control group (50 μL of test peptide solution and 50 μL of culture medium), a negative control group (50 μL of sterile MilliQ water and 50 μL of bacterial suspension) and a test experimental group (50 μL of test peptide solution and 50 μL of bacterial suspension) were set up for each test bacteria, and three replicates were set up for each group.

[0044] (5) Place the 96-well plate in an incubator, culture bacteria for 24 h and fungi for 72 h, and observe the MIC results.

[0045] (6) Mix the liquid in the well by blowing, take an appropriate amount of bacterial solution and drop it onto the corresponding plate. Culture the bacteria for 48 h and the fungi for 96 h, and observe the MBC results.

[0046] 3. The antibacterial activity of the antimicrobial peptide Basbpin is shown in Table 2. Basbpin has broad-spectrum antibacterial activity.

[0047] Table 2. Antimicrobial activity of the antimicrobial peptide Basbpin

[0048] Gram-positive bacteria Bacillus cereus GZ Bacillus cereus 24-48 24-48 1.2135 Enterococcus faecalis Enterococcus faecalis 12-24 24-48 1.131 Enterococcus faecalis Enterococcus faecium 3-6 6-12 Lactobacillus gasseri Lactobacillus gasseri 6-12 12-24 1.10753 Listeria monocytogenes Listeria monocytogenes 3-6 6-12 1.2465 Staphylococcus aureus Staphylococcus aureus 12-24 24-48 1.426 Staphylococcus epidermidis Staphylococcus epidermidis 3-6 3-6 agalactococcus Streptococcus agalactiae 6-12 6-12 Gram-negative bacteria 1.6769 Acinetobacter baumannii Acinetobacter baumannii 3-6 3-6 Aeromonas schuberis Aeromonas schubertii 12-24 24-48 1.2421 Pseudomonas aeruginosa Pseudomonas aeruginosa 6-12 6-12 1.1859 Salmonella enterica Salmonella enterica 24-48 >48 1.1868 Shigella freundii Shigella flexneri 6-12 6-12 1.1609 Vibrio fluvialis Vibrio fluvialis 24-48 24-48 Yeast fungi 2.1563 Cryptococcus neoformans Cryptococcus neoformans 12-24 12-24 filamentous fungi 3.4521 Fusarium graminearum Fusarium graminearum 24-48 24-48 3.6785 Fusarium oxysporum Fusarium oxysporum 12-24 12-24 5840 Fusarium solani Fusarium solani 12-24 12-24 3.316 Aspergillus niger Aspergillus niger 24-48 24-48 3.583 Ochratoxin Aspergillus ochraceus 24-48 24-48

[0049] Note: MIC: Minimum inhibitory concentration (μM), denoted by ab. a: The highest peptide concentration at which bacterial growth is visible to the naked eye; b: The lowest peptide concentration at which no bacterial growth is visible to the naked eye. MBC: Minimum bactericidal concentration (μM), denoted by ab. a: The highest peptide concentration at which colonies are visible on agar plates; b: The lowest peptide concentration at which no colonies are visible on agar plates.

[0050] II. Basbpin bactericidal kinetic curve

[0051] 1. The strain involved is: Streptococcus agalactiae.

[0052] 2. The specific method is as follows:

[0053] The specific method is similar to the method for determining antibacterial activity. The antimicrobial peptide Basbpin is co-incubated with bacteria, and the mixture is taken at different time points, serially diluted, spread on plates, and incubated statically before colony counting.

[0054] 3. The bactericidal kinetics curve of the antimicrobial peptide Basbpin against Streptococcus agalactiae is as follows: Figure 1 As shown, the antimicrobial peptide Basbpin has a rapid bactericidal effect.

[0055] III. Thermal stability of Basbpin

[0056] 1. The strain involved is: Streptococcus agalactiae.

[0057] 2. The specific method is as follows:

[0058] The antimicrobial peptide Basbpin was heated in boiling water at 100°C for 10 min, 20 min, and 30 min, and then placed on ice for later use. Subsequently, the antimicrobial peptide Basbpin was co-incubated with the test bacteria for 24 h, and the OD was continuously monitored using a microplate reader. 595 .

[0059] 3. The thermal stability of the antimicrobial peptide Basbpin against Streptococcus agalactiae is as follows: Figure 2 As shown, the antimicrobial peptide Basbpin retains its high antimicrobial activity even after treatment at 100℃ for 30 min.

[0060] IV. Scanning electron microscopy observation of morphological and structural changes in bacteria after Basbpin treatment

[0061] 1. The strains involved include: Streptococcus agalactiae.

[0062] 2. The specific experimental method is as follows:

[0063] (1) Scanning electron microscopy sample preparation: Single clones were picked and cultured in liquid culture medium until the logarithmic growth phase. The cells were collected by centrifugation, and the concentration of the bacterial suspension was adjusted to 1×10⁻⁶ with PBS. 8 CFU / mL, add an equal volume of antimicrobial peptide to make the concentration 2×MBC of the corresponding bacteria, incubate for 2 h, and then centrifuge to collect the bacterial cells.

[0064] (2) Fixation, washing, and dropping: Fix the bacterial cells with 2.5% glutaraldehyde at 4℃ for 2 h, wash the bacterial cells three times with PBS to prepare a high-concentration suspension, drop it onto a glass slide, and after the bacterial cells adhere, perform gradient dehydration with ethanol.

[0065] (3) After the critical point drying, sputter gold at a current of 10 mA for 60 s; observe and record the results using a scanning electron microscope.

[0066] The morphological and structural changes of Streptococcus agalactiae after treatment with the antimicrobial peptide Basbpin are as follows: Figure 3 As shown, Basbpin treatment disrupts the integrity of the bacterial cell membrane, leading to significant damage to the cell membrane structure and the release of intracellular substances.

[0067] V. CCK-8 assay for detecting and evaluating the cytotoxicity of Basbpin

[0068] Human embryonic kidney cells (HEK-293T) and zebrafish embryonic fibroblasts (ZF4) were selected to determine the cytotoxicity of the antimicrobial peptide Basbpin. Figure 3 As shown, the horizontal axis represents the concentration of Basbpin (μM), and the vertical axis represents cell viability (%).

[0069] The specific method is as follows:

[0070] (1) Collect cells in good growth condition and adjust the cell concentration to 1×10⁻⁶. 7 Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate at a suitable temperature until more than 80% of the cells adhere to the plate.

[0071] (2) Add different concentrations of Basbpin and incubate in a suitable temperature incubator for 24 h.

[0072] (3) Add CCK-8 mixed solution, incubate in the dark for 1-4 h, and then detect the absorbance at 450 nm to calculate cell viability.

[0073] The results are as follows Figure 4 The antimicrobial peptide Basbpin showed no cytotoxicity to ZF4 and HEK-293T cells.

[0074] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Basbpin, an antimicrobial peptide derived from Bacillus subtilis, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The use of the antimicrobial peptide Basbpin according to claim 1 in the preparation of antibacterial products.

3. The application as described in claim 2, characterized in that: It has inhibitory and bactericidal effects on Bacillus cereus, Enterococcus faecalis, Lactobacillus gasseri, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Acinetobacter baumannii, Aeromonas schubert, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, and Vibrio fluvialis.

4. An antibacterial product, characterized in that: Its active ingredient includes the antimicrobial peptide Basbpin as described in claim 1.

5. The use of the antimicrobial peptide Basbpin according to claim 1 in the preparation of antifungal products.

6. The application as described in claim 5, characterized in that: It has inhibitory and bactericidal effects on Cryptococcus neoformans, Fusarium graminearum, Fusarium oxysporum, Fusarium solani, Aspergillus niger and Aspergillus ochraceus.

7. An antifungal product, characterized in that: Its active ingredient includes the antimicrobial peptide Basbpin as described in claim 1.

8. The application of the antimicrobial peptide Basbpin according to claim 1 in the preparation of aquatic feed additives.

9. An aquatic feed additive, characterized in that: Its active ingredient includes the antimicrobial peptide Basbpin as described in claim 1.

Images