Bacillus amyloliquefaciens, bacteriostatic lipopeptide and application thereof

Abstract

The invention provides bacillus amyloliquefaciens, bacteriostatic lipopeptide and application of the bacillus amyloliquefaciens, the bacillus amyloliquefaciens and the bacteriostatic lipopeptide, the bacillus amyloliquefaciens is named as BA0001 and is preserved in China Center for Type Culture Collection (CCTCC) on July 26th, 2024, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20241691, and the bacillus amyloliquefaciens is classified and named as bacillus amyloliquefaciens. In-vitro bacteriostasis experiments show that the lipopeptide obtained through purification has a good bacteriostasis effect and has an inhibition effect on six pathogenic bacteria, namely, Aeromonas dakara, Aeromonas hydrophila, Aeromonas veronii, Pseudomonas aeruginosa, Edwardsiella and Streptococcus agalactiae, and the lipopeptide has a good application prospect in the aspects of agriculture and fishery.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and relates to Bacillus amyloliquefaciens, antibacterial lipopeptide and applications thereof. Background Art

[0002] Bacillus amyloliquefaciens is a Gram-positive bacterium widely found in the natural environment. It is similar to Bacillus Velez and Bacillus subtilis in morphology and structure, but there are certain differences in physiological and biochemical characteristics between the two, and the requirements for nutrients are also different. A variety of basic culture media can culture this bacterium, such as LB culture medium, and form light yellow or white opaque colonies. By naked eye observation, it is found that its surface is relatively dry. Its rod-shaped cells can form endogenous spores, and these spores are usually oval with blunt ends, which also provides conditions for its survival under extreme conditions. The bacterium has certain motility and can produce a variety of α-amylases and proteases.

[0003] Bacillus amyloliquefaciens is considered a probiotic. During its growth, it can produce a variety of antibacterial substances including polypeptides, lipopeptides and antibacterial proteins, which indicates that it has the possibility of application in the fields of agriculture and medicine. It can not only resist stress and prevent aging, promote plant growth, improve soil and increase fertility, but also reduce agricultural product residues and increase yields. In practical applications, Bacillus amyloliquefaciens has been used to produce a variety of biological products such as enzymes, antibacterial agents, pesticides, etc. Its metabolites, such as surfactants, fenmucin and sporangiocin D, have a significant inhibitory effect on pathogenic fungi. Bacillus amyloliquefaciens not only plays an important role in nature, but also has a positive impact in various fields of human society. Summary of the invention

[0004] The purpose of the present invention is to provide a Bacillus amyloliquefaciens, an antibacterial lipopeptide and an application thereof, wherein a strain of Bacillus amyloliquefaciens is isolated from the intestinal tract of fish. In an in vitro antibacterial experiment, it is found that the purified lipopeptide has a good antibacterial effect and has a good application prospect in agriculture and fishery.

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

[0006] The present invention provides a Bacillus amyloliquefaciens, named BA0001, which was deposited in the China Center for Type Culture Collection (CCTCC) on July 26, 2024, with a deposit address of Wuhan University, Wuhan, China, a deposit number of CCTCC NO: M20241691, and a classification name of Bacillus amyloliquefaciens.

[0007] Furthermore, the gene sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO:7.

[0008] The invention also provides an antibacterial lipopeptide prepared from the above-mentioned Bacillus amyloliquefaciens.

[0009] Furthermore, the antibacterial lipopeptide has molecular ion peaks of eight lipopeptides, namely, C13-Surfactin, C14-Surfactin, C15-Surfactin, C16-Fengycin A, C16-Fengycin B, C13-Bacillomycin D, C14-Bacillomycin D, and C15-Bacillomycin D.

[0010] Furthermore, the antibacterial lipopeptide is prepared by the following method: the amyloliquefaciens Bacillus is inoculated into LB liquid culture medium, shaken and cultured at 37°C and 180r / min for 60h, and then the fermentation broth is centrifuged at 4°C and 7000r / min for 20min, and the supernatant is collected after the fermentation is completed. The pH value of the supernatant is adjusted to 2.0, kept at 4°C overnight, centrifuged at 5000r / min and 4°C for 20min, and the sediment at the bottom of the centrifuge tube is collected, dissolved in methanol with the assistance of ultrasound, filtered through a hydrophilic membrane with a pore size of 0.22mm, and then subjected to rotary vacuum evaporation.

[0011] The present invention also provides the use of the Bacillus amyloliquefaciens in the production of antibacterial lipopeptides.

[0012] The present invention also provides an antibacterial agent, comprising the antibacterial lipopeptide.

[0013] Furthermore, the pathogens inhibited by the antibacterial agent include Aeromonas dakar, Aeromonas hydrophila, Aeromonas vermifuge, Pseudomonas aeruginosa, Edwardsiella and Streptococcus agalactiae.

[0014] The present invention further provides the use of the antibacterial lipopeptide in inhibiting pathogenic bacteria.

[0015] Furthermore, the pathogenic bacteria include Aeromonas dakar, Aeromonas hydrophila, Aeromonas vermifuge, Pseudomonas aeruginosa, Edwardsiella and Streptococcus agalactiae.

[0016] The beneficial effects of the present invention are:

[0017] A probiotic strain was isolated from the intestinal tract of fish, and its DNA sequence was amplified and detected by PCR technology, confirming that it was Bacillus amyloliquefaciens. The antibacterial substance secreted by it, the lipopeptide, was separated and purified by chemical means. After LC-MS analysis, the molecular ion peaks of eight lipopeptides, C13-Surfactin, C14-Surfactin, C15-Surfactin, C16-Fengycin A, C16-Fengycin B, C13-Bacillomycin D, C14-Bacillomycin D, and C15-Bacillomycin D, were found. In the in vitro antibacterial experiment, it was found that the purified lipopeptide had a good antibacterial effect, and had an inhibitory effect on six pathogens, including Aeromonas dakar, Aeromonas hydrophila, Aeromonas vermiformis, Pseudomonas aeruginosa, Edwardsiella and Streptococcus agalactiae, showing that it has good application prospects in agriculture and fisheries. In addition, crude lipopeptides have strong resistance to acid, alkali, high temperature and light, can overcome extreme environmental conditions, and have strong stability. Lipopeptides are also expected to become a substitute for antibiotics. The analysis of lipopeptide structure based on modern chemical methods will also help to further explore the binding sites of lipopeptides on pathogens, and explore methods and pathways for artificial synthesis, and are expected to become a substitute for antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The Bacillus amyloliquefaciens obtained after purification in the embodiment of the present invention.

[0019] Figure 2 BLAST result diagram of BA0001 PCR product in the embodiment of the present invention.

[0020] Figure 3 Agarose gel electrophoresis diagram of PCR production in the embodiment of the present invention.

[0021] Figure 4 Liquid chromatogram in the embodiment of the present invention.

[0022] Figure 5 Mass spectrometry in the embodiments of the present invention Figure 1 .

[0023] Figure 6 Mass spectrometry in the embodiments of the present invention Figure 2 .

[0024] Figure 7 Mass spectrometry in the embodiments of the present invention Figure 3 .

[0025] Figure 8 Mass spectrometry in the embodiments of the present invention Figure 4 .

[0026] Fig. 9 Mass spectrometry in the embodiments of the present invention Figure 5 .

[0027] Fig.10 Mass spectrometry in the embodiments of the present invention Figure 6 .

[0028] Fig.11 Mass spectrometry in the embodiments of the present invention Figure 7 .

[0029] Fig.12 Mass spectrometry in the embodiments of the present invention Figure 8 .

[0030] Fig.13 Lipopeptide antibacterial experiment in the examples of the present invention.

[0031] Fig.14 Methanol antibacterial experiment in the embodiment of the present invention.

[0032] Fig.15 Antibacterial experiments after lipopeptides were treated at different temperatures in the examples of the present invention.

[0033] Fig.16 Antibacterial experiment after lipopeptides were treated with different pH in the examples of the present invention.

[0034] Fig.17 Antibacterial experiment after lipopeptide was treated with different light exposure times in the examples of the present invention. DETAILED DESCRIPTION

[0035] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0036] Example

[0037] 1. Isolation and identification of Bacillus amyloliquefaciens

[0038] 1.1 Sampling and inoculation

[0039] Dead fish were obtained from Dongpo Lake in Haidian Campus of Hainan University, dissected next to an alcohol lamp, their intestines were cut out, and samples were taken with an inoculation loop burned on an alcohol lamp and cooled. The samples were then streaked on LB culture medium and placed in an incubator at 37°C for inversion culture.

[0040] LB (Luria-Bertani) medium: Take 5 g of tryptone, 2.5 g of yeast extract, 5 g of sodium chloride (NaCl), and 7.5 g of agar powder, make up to 500 mL, and sterilize.

[0041] 1.2 Separation and purification

[0042] After 20 hours of inoculation, it was removed from the water-tight constant temperature incubator, and a single colony was picked up with a sterilized disposable pipette tip and placed in a 100 mL conical flask containing 30 mL LB liquid culture medium for isolation and culture, and placed in a constant temperature shaker at 37°C and 180 r / min for 14 hours.

[0043] In this experiment, a bacterium similar in morphology to Bacillus amyloliquefaciens was isolated from the intestine of fish. Figure 1 , named BA0001.

[0044] 1.3 Identification of Bacillus amyloliquefaciens

[0045] (1) Extraction of genomic DNA

[0046] 2 mL of bacterial solution after overnight culture was taken and DNA was extracted using the Novezan genome extraction kit. The product was stored in a refrigerator at -20°C for later use.

[0047] (2) PCR amplification

[0048] Using the extracted DNA as a template, PCR reactions were performed using specific primers for the γ-mannanase genes of Bacillus subtilis and Bacillus amyloliquefaciens and 16s rDNA primers. PCR reaction system 50μL: 1μL DNA template, 2×Taq MasterMix (Dye Plus) 25μL, 1μL each of forward and reverse primers, and double distilled water added to 50μL. Add the above system to a 2mL sterile centrifuge tube and mix well for PCR amplification. PCR reaction program: 94.0℃5min; 94.0℃30s, 50.0℃30s, 72.0℃1min30s; 35 cycles; 72.0℃10min. The primer sequences are as shown in Table 1:

[0049] Table 1 Primer sequences and annealing temperatures

[0050]

[0051] (3) Agarose gel electrophoresis

[0052] The above PCR products were subjected to electrophoresis test experiments to ensure successful PCR amplification. The 16s-RNA products successfully amplified by PCR were purified using a product purification kit and sent to Nanshan Biological Company for sequencing.

[0053] The obtained DNA sequences were entered into the NCBI database and sequence alignment was performed using Blast. Figure 2 The gene sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO: 7.

[0054] The comparison results showed that the similarity between the strains was high with Bacillus subtilis and Bacillus amyloliquefaciens, and the species could not be determined. Then specific PCR was performed, and the product was observed under ultraviolet light after agarose gel electrophoresis. Figure 3 As shown, a clear band was obtained after amplification with the specific primers of Bacillus amyloliquefaciens, but no band was obtained after amplification with the primers of Bacillus subtilis, thus proving that the strain is Bacillus amyloliquefaciens.

[0055] Bacillus amyloliquefaciens BA0001 was deposited in the China Center for Type Culture Collection (CCTCC) on July 26, 2024. The deposit address is Wuhan University, Wuhan, China. The deposit number is CCTCC NO:M 20241691, and the classification name is Bacillus amyloliquefaciens.

[0056] 2. Isolation and identification of lipopeptides secreted by Bacillus amyloliquefaciens

[0057] A single colony identified as Bacillus amyloliquefaciens was picked and inoculated into 30 mL of LB liquid culture medium, and cultured at 37°C and 180 r / min for 60 h, and then the fermentation broth was placed in a centrifuge for centrifugation at 4°C and 7000 r / min for 20 min, and the supernatant was collected after the end. The pH value of the supernatant was adjusted with 6 mol / L HCl, and the solution was gently stirred and mixed while adding HCl. The acidity of the solution was tested at any time with a pH meter, and the pH value was adjusted to 2.0, kept at 4°C overnight, centrifuged at 5000 r / min and 4°C for 20 min, and the sediment at the bottom of the centrifuge tube was collected, dissolved in methanol under ultrasound assistance, filtered through a hydrophilic membrane with a pore size of 0.22 mm, and then rotary vacuum evaporated. The residue after evaporation was dissolved in methanol for subsequent determination of pathogen inhibition activity and LC-MS detection.

[0058] The crude lipopeptide extract was sent to the Hainan University Analysis and Testing Center for LC-MS analysis. The conditions are shown in Table 2.

[0059] Table 2 Liquid chromatography conditions

[0060]

[0061] Liquid phase diagram Figure 4 The types and m / z of lipopeptides are shown in Table 3.

[0062] Table 3 Identification of lipopeptides in LC-MS spectra

[0063]

[0064]

[0065] From mass spectrometry Figures 5 to 12It can be seen that there are eight lipopeptides of three categories, among which the molecular ion peak of C13-Surfactin is 1030.6460m / z, and other fragment ion peaks appear at 1017.6307m / z. The molecular ion peak of C14-Surfactin is 1044.6620m / z, and no other fragment ion peaks are found. The molecular ion peak of C15-Surfactin is 1058.6827m / z, and other fragment ion peaks appear at 742.4072 and 814.9935m / z. The molecular ion peak of C16-Fengycin A is 1463.7466m / z, and the fragment ion peak appears at 996.6581m / z. The molecular ion peak of C16-Fengycin B is 1491.7916m / z, and the fragment ion peak appears at 994.6509m / z. The molecular ion peak of C13-Bacillomycin D is 1017.6373m / z, but other fragment ion peaks appear at 791.5884 and 740.3997m / z. The molecular ion peak of C14-Bacillomycin D is 1031.6485m / z, and other fragment ion peaks appear at 754.4148m / z. The molecular ion peak of C15-Bacillomycin D is 1045.5626m / z, and no other fragment ion peaks are found.

[0066] 3. Antibacterial Experiment

[0067] 3.1 Pathogen culture

[0068] Aeromonas dakar, Aeromonas hydrophila, Aeromonas vermifuge, Pseudomonas aeruginosa and Streptococcus agalactiae stored in the laboratory were taken out from the ultra-low temperature refrigerator. In an ultra-clean workbench sterilized by ultraviolet irradiation for 30 minutes, 5 mL of sterilized LB liquid culture was pipetted into a disposable shaking tube with a pipette. 20 μL of bacterial liquid was pipetted from the glycerol frozen bacteria and inoculated into the shaking tube respectively, and cultured at 37°C and 180 r / min for 16 hours.

[0069] 3.2 Determination of lipopeptide inhibitory activity against pathogens

[0070] The revived pathogens were measured with a UV-visible spectrophotometer at a wavelength of 600 nm using LB liquid culture medium to adjust the absorbance value OD 600 To 0.5, LB liquid culture medium was used as blank control for absorbance detection. 100 μL of the adjusted concentration of pathogens was drawn with a sterilized pipette tip and evenly spread on the LB culture medium plate, and 8 μL of crude extracted lipopeptides were added as a positive experiment. At the same time, an equal amount of methanol was used instead of crude extracted lipopeptides as a negative control. The size of the inhibition zone was measured after 16 hours, and the determination was repeated three times.

[0071] Antibacterial test Fig.13 The diameters of the inhibition zones are shown in Table 4.

[0072] Table 4 Diameter of lipopeptide inhibition zone

[0073]

[0074]

[0075] The results showed that the crude lipopeptides extracted from Bacillus amyloliquefaciens had an inhibitory effect on the growth of Aeromonas dakar, Aeromonas hydrophila, Aeromonas vermifuge, Pseudomonas aeruginosa, Edwardsiella and Streptococcus agalactiae.

[0076] Among them, methanol, the solvent for crude lipopeptide extraction, was used as a blank control. Fig.14 The diameters of the inhibition zones are shown in Table 5.

[0077] Table 5 Diameter of methanol inhibition zone

[0078]

[0079] In Pseudomonas aeruginosa, the average diameter of the inhibition zone of crude lipopeptide was 12.98 mm, and the average diameter of methanol inhibition was 9.60 mm. In Streptococcus agalactiae, the average diameter of the inhibition zone of crude lipopeptide was 13.46 mm, and the average diameter of methanol inhibition was 7.68 mm. In Aeromonas daka, the average diameter of the inhibition zone of crude lipopeptide was 14.10 mm, and the average diameter of methanol inhibition was 9.93 mm. In Aeromonas hydrophila, the average diameter of the inhibition zone of crude lipopeptide was 12.35 mm, and the average diameter of methanol inhibition was 9.53 mm. In Aeromonas vernix, the average diameter of the inhibition zone of crude lipopeptide was 13.08 mm, and the average diameter of methanol inhibition was 9.17 mm. In Edwardsiella, the average diameter of the inhibition zone of crude lipopeptide was 10.41 mm, and the average diameter of methanol inhibition was 5.38 mm. It can be seen that the solvent methanol of crude lipopeptide has an antibacterial effect on pathogens, but the diameter of the inhibition zone is smaller than that of the crude lipopeptide. It can be seen that crude lipopeptide has an antibacterial effect on pathogens, and the antibacterial effect on Streptococcus agalactiae is the best.

[0080] 3.3 Analysis on the stability of antibacterial substances of Bacillus amyloliquefaciens

[0081] (1) Sensitivity to temperature

[0082] The crude lipopeptide extracts secreted by Bacillus amyloliquefaciens were treated at 40, 60, 80, 100, and 121°C for 25 min and then tested for antibacterial effect. 600After 0.5, 100 μL was taken up with a sterilized pipette tip, and the dry bacterial solution was spread on the LB medium with a disposable coating stick, and 8 μL of the crude lipopeptide extract treated at different temperatures was added, and the determination was repeated three times.

[0083] After treatment at different temperatures, the diameter of the inhibition zone remained basically unchanged at 40-80°C, but decreased after treatment at 100°C, approaching the inhibition diameter of the methanol negative control. It is believed that the antibacterial activity of the lipopeptide disappeared after treatment at 100°C. The diameters of the inhibition zones under different temperature conditions are shown in Table 6. Fig.15 .

[0084] Table 6 Diameters of lipopeptide inhibition zones under different temperature conditions

[0085]

[0086] (2) Acid and alkali resistance

[0087] The crude lipopeptide extracts secreted by Bacillus amyloliquefaciens were adjusted to pH 3.0, 5.0, 7.0, 9.0, and 11.0 with NaOH and HCl solutions, and then the antibacterial effect was tested. 600 After 0.5, 100 μL was taken up with a sterilized pipette tip and evenly spread on the LB medium plate with a disposable sterile coating rod, and 8 μL of the crude lipopeptide extract under different pH conditions was added, and the measurement was repeated three times.

[0088] After treatment at different pH values, the diameter of the inhibition zone remained basically unchanged in the pH range of 3-11, and was larger than the negative control inhibition diameter of methanol. The diameters of the inhibition zones under different pH conditions are shown in Table 7. Fig.16 .

[0089] Table 7 Diameters of lipopeptide inhibition zones under different pH conditions

[0090]

[0091] (3) Light stability

[0092] The crude lipopeptide extracts secreted by Bacillus amyloliquefaciens were irradiated under ultraviolet light for 4, 6, 8, 12, and 24 hours before testing the antibacterial effect. 600 After 0.5, 100 μL was taken up with a sterilized pipette tip and evenly spread on an LB medium plate, and 8 μL of crude lipopeptide extracts treated with different UV irradiation times were added, and the determination was repeated three times.

[0093] After irradiation under UV light for different time periods, the diameter of the inhibition zone did not change significantly and was larger than the negative control inhibition diameter of methanol. The diameters of the inhibition zones after treatment with different illumination time periods are shown in Table 8. Fig.17 .

[0094] Table 8 Diameters of lipopeptide inhibition zones after treatment with different illumination times

[0095]

[0096] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens, named BA0001, was deposited in the China Center for Type Culture Collection (CCTCC) on July 26, 2024, with the deposit address at Wuhan University, Wuhan, China, the deposit number is CCTCC NO:M 20241691, and the classification name is Bacillus amyloliquefaciens.

2. The Bacillus amyloliquefaciens according to claim 1, characterized in that The gene sequence of Bacillus amyloliquefaciens is shown in SEQ ID NO:

7.

3. An antibacterial lipopeptide prepared from the Bacillus amyloliquefaciens according to claim 1.

4. The antibacterial lipopeptide according to claim 3, characterized in that The antibacterial lipopeptide has molecular ion peaks of eight lipopeptides, namely, C13-Surfactin, C14-Surfactin, C15-Surfactin, C16-Fengycin A, C16-Fengycin B, C13-Bacillomycin D, C14-Bacillomycin D and C15-Bacillomycin D.

5. The antibacterial lipopeptide according to claim 3, characterized in that The antibacterial lipopeptide is prepared by the following method: inoculating the Bacillus amyloliquefaciens into LB liquid culture medium, shaking and culturing at 37°C and 180r / min for 60h, then centrifuging the fermentation broth at 4°C and 7000r / min for 20min, collecting the supernatant after completion, adjusting the pH value of the supernatant to 2.0, keeping at 4°C overnight, centrifuging at 5000r / min and 4°C for 20min, collecting the sediment at the bottom of the centrifuge tube, dissolving it in methanol with the assistance of ultrasound, filtering it through a hydrophilic membrane with a pore size of 0.22mm, and then performing rotary vacuum evaporation.

6. Use of the Bacillus amyloliquefaciens according to claim 1 or 2 in the production of antibacterial lipopeptides.

7. An antibacterial agent comprising the Bacillus amyloliquefaciens according to claim 1 or 2.

8. The antibacterial agent according to claim 7, characterized in that The pathogens inhibited by the antibacterial agent include Aeromonas dakar, Aeromonas hydrophila, Aeromonas vermifuge, Pseudomonas aeruginosa, Edwardsiella and Streptococcus agalactiae.

9. Use of the antibacterial lipopeptide according to any one of claims 3 to 5 in inhibiting pathogenic bacteria.

10. The use according to claim 9, characterized in that: The pathogenic bacteria include Aeromonas dakar, Aeromonas hydrophila, Aeromonas vernix, Pseudomonas aeruginosa, Edwardsiella and Streptococcus agalactiae.

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