Bacillus megaterium WSY10 for producing bacteriocin, and product and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to bacillus megatherium WSY10 for producing bacteriocin as well as a product and application thereof. The bacillus megatherium WSY10 for producing bacteriocin is screened and separated, the bacteriocin is named as Megasin WSY10, and the bacillus megatherium WSY10 has efficient bactericidal activity on methicillin-resistant staphylococcus aureus, can be used for products such as drugs, feed additives and bacteriostatic agents, and has further development and utilization prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to bacteriocin-producing Bacillus megaterium WSY10, its products and applications. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) has developed varying degrees of resistance to antibacterial drugs such as β-lactam antibiotics, macrolides, fluoroquinolones, aminoglycosides, and tetracyclines, which has brought great challenges to clinical anti-infection treatment. The antibiotics currently used in the clinical treatment of MRSA infections mainly include vancomycin, linezolid, teicoplanin, daptomycin, etc. However, with the annual increase in cases of methicillin-resistant Staphylococcus aureus infection and the widespread use of vancomycin in clinical practice, vancomycin-resistant MRSA has emerged. In addition, the toxic and side effects of vancomycin, such as ototoxicity and nephrotoxicity, are also quite obvious. In recent years, new antibiotics such as linezolid, daptomycin, and teicoplanin, which have good efficacy against MRSA infections, have also been detected in clinically resistant strains to the above drugs, and the toxic and side effects also limit the application of these drugs.

[0003] Bacteriocins are a class of proteins or polypeptides produced by bacteria through ribosome synthesis mechanism with antibacterial activity and high safety. Given the multidrug resistance of MRSA, it is urgent to discover more bacteria that can produce anti-MRSA bacteriocins. Summary of the invention

[0004] In order to solve the above technical problems, the object of the present invention is to provide bacteriocin-producing Bacillus megaterium WSY10, its products and applications.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a bacteriocin-producing Bacillus megaterium WSY10, which was deposited in the China Center for Type Culture Collection on November 27, 2023, with a deposit number of CCTCC NO: M20232361.

[0007] The present invention collects soil samples from Xiangshan Mountain Range in Huaibei City, Anhui Province, and screens and isolates a bacteriocin-producing Bacillus megaterium WSY10. After fermenting Bacillus megaterium WSY10 for 12 to 72 hours, the supernatant is taken and it is found that the fermentation supernatant has obvious antibacterial activity against methicillin-resistant Staphylococcus aureus.

[0008] The present invention also provides bacteriocin Megasin WSY10 produced from the above-mentioned Bacillus megaterium WSY10. The molecular weight of the bacteriocin Megasin WSY10 including a single isotope is 2064.8256 Da, and the amino acid sequence of its precursor peptide is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for preparing the bacteriocin Megasin WSY10, comprising the following steps:

[0010] S1. Fermenting Bacillus megaterium WSY10 to obtain a fermentation broth, centrifuging the fermentation broth, adsorbing the supernatant with a macroporous adsorption resin column, and then eluting to obtain an eluate;

[0011] S2. The eluate is concentrated and freeze-dried to obtain a dry powder, the dry powder is dissolved in an acetonitrile solution with a volume percentage concentration of 40% to 60%, and the solution is centrifuged to obtain a crude extract. The crude extract is separated by high performance liquid chromatography, and the chromatographic peaks appearing within 1 to 25 minutes are collected. The antibacterial activity of the effluent corresponding to the chromatographic peak against methicillin-resistant Staphylococcus aureus is detected by an agar diffusion method. The effluent with antibacterial activity is dried and freeze-dried to obtain the bacteriocin Megasin WSY10.

[0012] Further, the conditions of the high performance liquid chromatography are:

[0013] Chromatographic column: Agilent C18 reverse phase column;

[0014] Mobile phase: Phase A is ddH2O containing TFA, the mass fraction of TFA in phase A is 0.1% to 0.2%, and phase B is acetonitrile;

[0015] Mobile phase conditions: 0-25 min, 10%-90% acetonitrile gradient elution;

[0016] Flow rate: 1~2mL / min.

[0017] The present invention also provides use of the bacteriocin Megasin WSY10 in the preparation of an antibacterial product, wherein the antibacterial product is used to inhibit methicillin-resistant Staphylococcus aureus.

[0018] Furthermore, the antibacterial product is a medicine or an antibacterial agent.

[0019] Furthermore, the antibacterial product contains bacteriocin Megasin WSY10 as the only active ingredient.

[0020] Furthermore, the minimum inhibitory concentration of the bacteriocin Megasin WSY10 against methicillin-resistant Staphylococcus aureus is 0.2 μM.

[0021] Furthermore, the antibacterial agent is obtained by dissolving bacteriocin Megasin WSY10 in sterile water.

[0022] Storage Instructions:

[0023] Bacterial species name: Bacillus megaterium WSY10;

[0024] Latin name: Bacillus megaterium WSY10;

[0025] Deposit number: CCTCC NO: M 20232361;

[0026] Depository: China Center for Type Culture Collection;

[0027] Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0028] Date of preservation: November 27, 2023.

[0029] The present invention has the following beneficial effects:

[0030] The present invention determines that the Bacillus megaterium WSY10 strain can produce a substance with high antibacterial activity against methicillin-resistant Staphylococcus aureus through antibacterial activity screening. The antibacterial active substance produced by the Bacillus megaterium WSY10 strain is extracted and identified as a new type of bacteriocin, named Megasin WSY10. Megasin WSY10 has high bactericidal activity against methicillin-resistant Staphylococcus aureus, can be used in medicines, feed additives, food antibacterial agents, etc., and has the prospect of further development and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A is a colony morphology of Bacillus megaterium WSY10; B is a test result of the antibacterial activity of the supernatant of Bacillus megaterium WSY10 fermented at different times against methicillin-resistant Staphylococcus aureus. The outer circle adds the fermentation supernatant at different fermentation time points, and the inner circle adds the supernatant treated with mixed protease.

[0032] Figure 2 It is a sequence analysis diagram, wherein A is the homology analysis of the proteins encoded by each gene in the synthetic gene cluster of bacteriocin MegasinWSY10 in the genome of Bacillus megaterium WSY10; B is the homology analysis of the amino acid sequence of bacteriocin Megasin WSY10.

[0033] Figure 3 HPLC analysis of crude extract of bacteriocin Megasin WSY10.

[0034] Figure 4 This is the primary mass spectrometry analysis diagram of bacteriocin Megasin WSY10, where the arrow points to the peak diagram after the target peak is magnified.

[0035] Figure 5 This is the secondary mass spectrometry analysis of bacteriocin Megasin WSY10.

[0036] Figure 6 Schematic diagram of the structure of bacteriocin Megasin WSY10.

[0037] Figure 7 This is a graph showing the determination of the minimum inhibitory concentration of bacteriocin Megasin WSY10 against methicillin-resistant Staphylococcus aureus.

[0038] Figure 8 The figures are bacteriocin action determination diagrams, wherein A is the growth curve of methicillin-resistant Staphylococcus aureus under the action of different concentrations of bacteriocin Megasin WSY10; B is the determination of the number of live methicillin-resistant Staphylococcus aureus under the action of different concentrations of bacteriocin Megasin WSY10; C is the determination of the turbidity change of methicillin-resistant Staphylococcus aureus liquid after the action of different concentrations of bacteriocin Megasin WSY10 for 2 hours; D is the determination of the morphological change of methicillin-resistant Staphylococcus aureus after the action of different concentrations of bacteriocin Megasin WSY10 for 2 hours. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0040] Example 1: Isolation and classification and identification of Bacillus megaterium WSY10.

[0041] Soil samples were collected from Xiangshan Mountain Range, Huaibei City, Anhui Province, at latitude and longitude: 116.80324161823273, 33.98120252976587. Longitude E: 116°48′11.67″ Latitude N: 33°58′52.33″; 10 g of soil sample was put into a 250 mL sterile triangular flask, 30 mL of sterile water was added, and the mixture was thoroughly mixed; the triangular flask was placed in an 80°C water bath for 3 min, and then allowed to stand for 10 min; 50 μL of the supernatant was applied to TSB solid medium in a clean bench; the applied plate was placed at 25°C for 18 h; a single colony was picked and streaked on a new TSB solid medium; strain WSY10 was isolated and obtained, such as Figure 1 As shown in A. It was used for the next step of anti-methicillin-resistant Staphylococcus aureus activity screening.

[0042] In addition, the 16S rDNA sequence primers used for bacterial species identification are shown in the following table:

[0043] Table 1 16S rDNA primer sequences

[0044] Primer name Sequence 5′-3′ SEQ ID NO. 27F AGAGTTTGATCCTGGCTCAG 2 1492R TACGGCTACCTTGTTACGACTT 3

[0045] BlastN analysis showed that the 16SrDNA sequence of strain WSY10 was 100% identical to the 16SrDNA sequence of strain Bacillus megaterium KNU-01, so it can be determined that strain WSY10 belongs to Bacillus megaterium, so it was named Bacillus megaterium WSY10. Bacillus megaterium WSY10 was deposited in the China Center for Type Culture Collection on November 27, 2023, with the deposit number CCTCC NO: M 20232361.

[0046] Example 2: Detection of the activity of Bacillus megaterium WSY10 against methicillin-resistant Staphylococcus aureus.

[0047] Pick a single colony of Bacillus megaterium WSY10 and inoculate it in 5 mL of TSB liquid culture medium and activate it overnight at 25°C to obtain an inoculum. Transfer 1 mL of the inoculum to 100 mL of TSB liquid culture medium and culture it at 220 rpm / min and 25°C for 72 hours. Take 1 mL of the culture solution in the clean bench every 4 hours from the start of inoculation, and take samples continuously for 18 times. After the culture solution is centrifuged at 12000 rpm / min for 5 minutes, the supernatant is transferred to a new centrifuge tube. Trypsin with a final concentration of 1 mg / mL, 1 mg / mL of α-chymotrypsin, and 1 mg / mL of pepsin were added to the obtained fermentation supernatant and treated at 30°C for 2 hours. The antibacterial activity of the fermentation supernatant and the fermentation supernatant after enzymatic hydrolysis was detected by agar diffusion method. The method is as follows: Add 5×10 5 cfu / mL of methicillin-resistant Staphylococcus aureus, mix well, and pour into a plate. After solidification, use a 6mm hole puncher to punch holes. Add 50μL of fermentation supernatant to each well in the outer circle, and add fermentation supernatant after enzymatic hydrolysis to the inner circle. Place the plate at 4℃ for about 2h to allow the sample to fully diffuse, and then place it at 30℃ for 12h. Figure 1 As shown in B, the fermentation supernatant of Bacillus megaterium WSY10 at 12 to 72 h has obvious antibacterial activity against methicillin-resistant Staphylococcus aureus, and the fermentation supernatant does not produce antibacterial activity after enzymatic hydrolysis, indicating that its active ingredient is a polypeptide or protein substance.

[0048] Example 3: Whole genome sequencing and bacteriocin analysis of Bacillus megaterium WSY10.

[0049] The whole genome of Bacillus megaterium WSY10 strain was sequenced by Illuminate Hiseq2500 sequencer. The sequencing used double-end sequencing, the sequencing read length was 125bp, and the sequencing quantity was 1G. The final sequencing data was assembled and annotated by the genome assembly software PGCGAP.

[0050] The antimicrobial active substances of Bacillus megaterium WSY10 strain genome were analyzed by online software antiSMATH. Figure 2 As shown, the bacteriocin biosynthesis gene cluster encodes a bacteriocin product, which we named MegasinWSY10. The amino acid sequence of the precursor peptide of Megasin WSY10 has the highest homology with the reported lantibiotic bacteriocin Gallidermin, which is 56%. Therefore, Megasin WSY10 is a novel, unidentified bacteriocin.

[0051] Example 4: Identification of bacteriocin Megasin WSY10.

[0052] (1) Crude extraction of bacteriocin Megasin WSY10.

[0053] Pick a single colony of Bacillus megaterium WSY10 and add it to 5mL TSB liquid culture medium for overnight activation. Transfer the inoculation amount of 1% by volume to 10 bottles of 200mL TSB culture medium, and culture it in a 25℃ shaking incubator at 220r / min for 40h; centrifuge the culture medium at 12000r / min for 10min, and use 200g of macroporous adsorption resin AmberliteXAD-7HP to pass the obtained 2L supernatant for column adsorption; the column material after adsorption is first rinsed with 1L of pure water, then rinsed with 0.5L of 30% ethanol solution, and finally eluted with 0.5L of 90% ethanol; the eluate is concentrated by a rotary evaporator at 42℃, and after being spin-dried to 10mL, it is collected and freeze-dried; the obtained dry powder is dissolved in acetonitrile with a volume fraction of 50%, centrifuged at 12000r / min for 10min, and the obtained supernatant is the crude extract of antibacterial substances.

[0054] (2) Preparation of pure antimicrobial active substances.

[0055] The obtained crude extract of antibacterial substances was analyzed by high pressure liquid chromatography Dionex Ultimate 3000 system.

[0056] Main technical parameters:

[0057] Chromatographic column: Agilent C18 reverse phase column (250 mm × 4.6 mm, 5 μm);

[0058] Mobile phase: ultrapure water and chromatographic grade acetonitrile containing 0.1% to 0.2% TFA;

[0059] Mobile phase conditions: 0-25 min, 10%-90% acetonitrile gradient elution;

[0060] Detection wavelength: 220nm;

[0061] Flow rate: 2mL / min;

[0062] Collect all the chromatographic peaks that appear within 25 minutes, and use agar diffusion method to detect the antibacterial activity of the collected solution against methicillin-resistant Staphylococcus aureus to determine the retention time of the antibacterial substance. Figure 3 As shown, the chromatographic peak at 18.4 minutes has antibacterial activity against Staphylococcus aureus. The above liquid phase process was repeated and the chromatographic peaks were collected multiple times. The collected samples were placed in a -80°C refrigerator for 2 hours and then freeze-dried in a vacuum freeze dryer for 12 hours. The powder obtained after freeze-drying was weighed and dissolved in acetonitrile with a volume fraction of 50%.

[0063] (3) Primary mass spectrometry analysis of antimicrobial active substances.

[0064] The molecular weights of the active substances in the 18.4 minute chromatographic peak prepared above were determined using a mass spectrometer Agilent Technologies 6540UDH Accurate-Mass Q-TOF LC / MS.

[0065] Primary mass spectrometry analysis conditions:

[0066] Capillary voltage: 3500V;

[0067] Drying gas flow rate: 9 liters / min;

[0068] Temperature: 350℃;

[0069] Q-TOF scanning range: 100~3000m / z;

[0070] Data acquisition rate: 1spectrum / s.

[0071] like Figure 4 As shown in Figure 1, the primary mass spectrometry analysis showed that the molecular weight of bacteriocin Megasin WSY10 was 2064.8256Da. Figure 5As shown, the detected series of polypeptide fragments and the predicted amino acid sequence of the precursor peptide of Megas in WSY10 were cleaved by the leading peptide MNNVKNLFDLDVQVTTASSDVDP Q, and the four serines (S) and three threonines (T) in the generated core peptide ITSVSLCTPGCGDTGSWNSFCC underwent dehydration reaction to form dehydroalanine (Dha) and dehydrotyrosine (Dhb), and dehydroalanine (Dha), dehydrotyrosine (Dhb) and cysteine ​​(C) further formed four pairs of lanthio bonds, and the carboxyl terminal cysteine ​​(C) was decarboxylated and reduced to finally form the structure as shown in Figure 6 The compound shown. Megasin WSY10 is different from all identified and reported bacteriocins, indicating that it is a novel bacteriocin and is identified and reported for the first time. The amino acid sequence of the precursor peptide of Megasin WSY10 is shown in SEQ ID NO.1.

[0072] SEQ ID NO.1:MNNVKNLFDLDVQVTTASSDVDPQITSVSLCTPGCGDTGSWNSFCC.

[0073] Example 5: Determination of the minimum inhibitory concentration of bacteriocin Megasin WSY10 against methicillin-resistant Staphylococcus aureus.

[0074] The pure Megasin WSY10 obtained above was prepared into test samples with final concentrations of 0.05μM, 0.1μM, 0.2μM, 0.4μM, 0.8μM, 1.6μM and 3.2μM. The minimum inhibitory concentration of MegasinWSY10 at different concentrations against methicillin-resistant Staphylococcus aureus was detected by the agar diffusion method described above. Figure 7 As shown, the minimum inhibitory concentration of Megasin WSY10 against methicillin-resistant Staphylococcus aureus was determined to be 0.2 μM.

[0075] Example 6: Determination of the bactericidal activity of bacteriocin Megasin WSY10 against methicillin-resistant Staphylococcus aureus.

[0076] Methicillin-resistant Staphylococcus aureus was cultured in TSB medium until OD was 0.4, and the bacteriocin Megasin WSY10 was added at a final concentration of 0.2μM and 0.4μM, respectively, and cultured at 30℃ and 200rpm for 2 hours. The OD value of the bacterial solution was measured at 1h and 2h in the intermediate culture, and the number of viable bacteria in the bacterial solution was detected by the dilution coating plate method. In addition, the 2h bacterial solution was centrifuged at 6000rpm for 10 minutes, and the supernatant was discarded. After injecting glutaraldehyde to fix the cells for 4 hours, the cells were washed three times by centrifugation at 4500rpm with PBS buffer, and then dehydrated with ethanol gradient, with a volume fraction of 10%, 30%, 50%, 70%, and 90% ethanol once each, and 100% ethanol twice, 20min / time. Subsequently, ethanol was replaced with isoamyl acetate twice, 20min / time. Finally, the samples were frozen at -80℃ for 12 hours, and dried in a freeze dryer for 12 hours for scanning electron microscopy observation.

[0077] The results are as follows Figure 8 As shown in the figure, 0.2μM bacteriocin Megasin WSY10 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus, while 0.4μM bacteriocin Megasin WSY10 caused the turbidity of the methicillin-resistant Staphylococcus aureus liquid to continue to decrease, the number of live bacteria dropped to 0 after 1 hour, and the bacteria finally lysed and died. In addition, scanning electron microscopy results showed that 0.2μM bacteriocin Megasin WSY10 caused the methicillin-resistant Staphylococcus aureus to deform and rupture, while 0.4μM bacteriocin Megasin WSY10 caused the methicillin-resistant Staphylococcus aureus to completely lose its cell morphology and lyse and die. In summary, Megasin WSY10 is a bactericidal active substance for methicillin-resistant Staphylococcus aureus, which causes cell death by lysing cells.

[0078] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0079] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0080] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A bacteriocin-producing Bacillus megaterium WSY10, characterized in that: It was deposited in the China Center for Type Culture Collection on November 27, 2023, with the deposit number being CCTCC NO: M20232361.

2. The bacteriocin Megasin WSY10 produced from the Bacillus megaterium WSY10 according to claim 1, characterized in that: The molecular weight of the bacteriocin Megasin WSY10 including a single isotope is 2064.8256 Da, and the amino acid sequence of its precursor peptide is shown in SEQ ID NO.

1.

3. The method for preparing the bacteriocin Megasin WSY10 according to claim 2, characterized in that: The steps include: S1. Fermenting Bacillus megaterium WSY10 to obtain a fermentation broth, centrifuging the fermentation broth, adsorbing the supernatant with a macroporous adsorption resin column, and then eluting to obtain an eluate; S2. The eluate is concentrated and freeze-dried to obtain a dry powder, the dry powder is dissolved in an acetonitrile solution with a volume percentage concentration of 40% to 60%, and the solution is centrifuged to obtain a crude extract. The crude extract is separated by high performance liquid chromatography, and the chromatographic peaks appearing within 1 to 25 minutes are collected. The antibacterial activity of the effluent corresponding to the chromatographic peak against methicillin-resistant Staphylococcus aureus is detected by an agar diffusion method. The effluent with antibacterial activity is dried and freeze-dried to obtain the bacteriocin Megasin WSY10.

4. The method for preparing bacteriocin Megasin WSY10 according to claim 3, characterized in that: The conditions of the high performance liquid chromatography are: Chromatographic column: Agilent C18 reverse phase column; Mobile phase: Phase A is ddH2O containing TFA, the mass fraction of TFA in phase A is 0.1% to 0.2%, and phase B is acetonitrile; Mobile phase conditions: 0-25 min, 10%-90% acetonitrile gradient elution; Flow rate: 1~2mL / min.

5. The use of the bacteriocin Megasin WSY10 according to claim 2 in the preparation of antibacterial products, characterized in that: The antibacterial product is used for inhibiting methicillin-resistant Staphylococcus aureus.

6. The use according to claim 5, characterized in that: The antibacterial product is a medicine or a bacteriostatic agent.

7. The use according to claim 6, characterized in that: The antibacterial product contains bacteriocin Megasin WSY10 as the only active ingredient.

8. The use according to claim 5, characterized in that: The minimum inhibitory concentration of the bacteriocin Megasin WSY10 against methicillin-resistant Staphylococcus aureus is 0.2 μM.

9. The use according to claim 6, characterized in that: The antibacterial agent is obtained by dissolving bacteriocin MegasinWSY10 in sterile water.

Images