Bacillus velezensis, antibacterial lipopeptide and application thereof

Abstract

The invention relates to the technical field of microorganism application, in particular to bacillus velezensis, antibacterial lipopeptide and application of the bacillus velezensis and the antibacterial lipopeptide. The invention provides bacillus velezensis BGMW2 separated from a mangrove forest water area in Qinzhou city in Guangxi Zhuang Autonomous District, and the preservation number of the bacillus velezensis BGMW2 is GDMCC No: 64790. The strain BGMW2 has broad-spectrum antibacterial activity, and can significantly inhibit pathogenic bacteria such as escherichia coli, salmonella typhimurium, shigella, bacillus licheniformis and staphylococcus aureus. According to the invention, an antibacterial lipopeptide is separated from a metabolite of the strain BGMW2, and the antibacterial lipopeptide is separated, purified and identified as C12surfactin. The antibacterial effect of the C12surfactin is superior to that of a common bacteriostatic agent Nisin, and can be comparable with that of gentamicin sulfate. The C12surfactin also has the characteristic of stable antibacterial property at the temperature of between 40 and 80 DEG C and the pH value of between 2.0 and 12.0, and has hydrolase resistance and low hemolytic activity. The bacterial strain BGMW2 and a metabolite antibacterial lipopeptide thereof provided by the invention have important application prospects in the fields of food, pharmacy, disinfection, protection, health care products, cosmetics and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to Bacillus velez, antimicrobial lipopeptide and application thereof. Background Art

[0002] Improper use of antibiotics may lead to drug resistance in microorganisms. Microbial resistance has been identified by the World Health Organization (WHO) as one of the three major public health threats. Fortunately, research and development of new antimicrobial agents is one of the key strategies to reduce the burden of microbial resistance, but progress in developing new effective antimicrobial agents is still insufficient. Therefore, discovering more new antimicrobial compounds is an area that urgently needs research.

[0003] Bacillus is known for producing a variety of metabolites with antimicrobial activity. About 5% to 8% of the total genome of Bacillus is dedicated to the synthesis of biologically active secondary metabolites such as peptides and lipopeptides, polyketides, bacteriocins and aglycones. B. velezensis is one of the newest strains of Bacillus. Therefore, it is more likely to produce new antimicrobial compounds. Recently, B. velezensis, which produces potent antimicrobial compounds, has received more attention. B. velezensis was first isolated from the Velez River estuary in Malaga (southern Spain) in 2005. It is widely distributed in various environments such as plant nodules, soil, rivers, human food, animal intestines and seawater, and it is easy to isolate and culture. In recent years, various antimicrobial compounds such as bacteriocins, β-1,3-1,4-glucanases, lipopeptides, etc. have been continuously discovered in the metabolites of B. velezensis. Surfactin produced by B. velezensis SK has good antibacterial activity (1600AU / mL) against drug-resistant food-borne Bacillus cereus and human pathogen Staphylococcus aureus. A novel antimicrobial peptide YS12 isolated from B. velezensis CBSYS12 has strong antibacterial activity, with minimum inhibitory concentration values ​​of 6-12μg / mL against Gram-positive and Gram-negative bacteria. Therefore, researchers need to work harder to explore the antimicrobial compounds of B. velezensis.

[0004] Surfactin is a secondary metabolite produced by Bacillus sp. strains. It is a cyclic lipopeptide composed of a heptad and a β-hydroxy fatty acid chain. It has multiple homologues and can be divided into C 12- C 16Five components. Compared with general chemical surfactants, in addition to reducing surface tension, stabilizing emulsions and increasing foam, it also has the advantages of thermal stability, non-toxicity, biodegradability and stronger surface and interfacial activity. It has multiple biological activities and can inhibit the growth of bacteria, viruses, fungi and mycoplasma. They mainly act on the phospholipid bilayer of the pathogen cell membrane, change the permeability and functionality of the cell membrane, thereby inhibiting the growth of pathogens and not easily developing drug resistance. As a highly efficient surfactant and new antibacterial substance, antimicrobial lipopeptide surfactin has a wide range of applications in agricultural biological control, food preservation, petroleum industry, etc.

[0005] Although there are many studies on the inhibitory effect of antimicrobial lipopeptides produced by Bacillus Velez on pathogens, most of the studies are still mainly focused on agriculture, biological control and aquaculture, while there are few reports on food preservation, antimicrobial agents and public health safety. Antimicrobial lipopeptides have advantages such as low toxicity, good physical and chemical properties such as biodegradability and stability, as well as high biological activity, which makes Bacillus Velez and its antimicrobial lipopeptides have potential application value and huge market demand in food, medicine, health care products, etc. Summary of the invention

[0006] In view of this, the present invention proposes a Bacillus velezensis BGMW2, which has a broad-spectrum antibacterial activity. The present invention also separates and extracts antimicrobial lipopeptides from the metabolites of strain BGMW2, which are further purified and identified as C 12 surfactin. C 12 Surfactin also has high antibacterial properties, and is acid- and alkali-resistant, high-temperature-resistant, protease-resistant, amylase-resistant, and non-hemolytic. The Bacillus velezensis BGMW2 and its metabolites have important application prospects in the fields of food, pharmaceuticals, disinfection, protection, health products, cosmetics, etc.

[0007] The technical solution of the present invention is achieved in this way:

[0008] In the first aspect, the present invention provides a Bacillus velezensis BGMW2, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 24, 2024, with the deposit number GDMCC No: 64790.

[0009] In a second aspect, the present invention provides a bacterial agent, which includes the Bacillus velezensis BGMW2 or its fermentation broth, and the fermentation broth contains the Bacillus velezensis BGMW2.

[0010] In a third aspect, the present invention provides the use of the Bacillus velezensis BGMW2 or the bacterial agent in inhibiting bacteria, wherein the use is not for the purpose of diagnosing or treating a disease.

[0011] Furthermore, the bacteria include Escherichia coli, Salmonella typhi, Shigella, Bacillus licheniformis, and Staphylococcus aureus.

[0012] In some preferred embodiments, the effective viable count of the bacterial agent is ≥ 10 5 CFU / mL.

[0013] In a fourth aspect, the present invention further provides an antibacterial product, wherein the product contains the Bacillus velezensis BGMW2 or the bacterial agent.

[0014] In a fifth aspect, the present invention further provides an antimicrobial lipopeptide, wherein the preparation method of the antimicrobial lipopeptide comprises: inoculating the Bacillus velezensis BGMW2 into a culture medium for fermentation to obtain a fermentation broth, and separating and extracting the antimicrobial lipopeptide from the fermentation broth.

[0015] In some preferred embodiments, the culture medium is LB liquid culture medium, whose components include: 5 g / L yeast powder, 10 g / L sodium chloride, and 10 g / L peptone.

[0016] In some preferred embodiments, the preparation method of the antimicrobial lipopeptide specifically further includes: centrifuging the fermentation broth at 8000-12000rpm to obtain a fermentation supernatant, adjusting the pH of the fermentation supernatant to 2.0 with 6M hydrochloric acid, and after precipitation at 4°C overnight, centrifuging the fermentation supernatant at 8000-12000rpm, discarding the supernatant, resuspending the precipitate with distilled water, freeze-drying, extracting with methanol solvent, removing methanol by vacuum rotary evaporation at 45°C, and then dissolving it with methanol and freeze-drying it to obtain the antimicrobial lipopeptide produced by Bacillus Velez.

[0017] In a sixth aspect, the present invention also provides the use of the antimicrobial lipopeptide in the preparation of antimicrobial products, wherein the antimicrobial products include food preservatives, antimicrobial drugs, antimicrobial disinfectants, antimicrobial protective products, cosmetics, skin care products or health care products.

[0018] In a seventh aspect, the present invention provides the use of the antimicrobial lipopeptide in inhibiting bacteria, wherein the use is not for the purpose of diagnosing or treating a disease.

[0019] The beneficial effects of the present invention include at least the following:

[0020] (1) The Bacillus Velezii BGMW2 provided by the present invention has the effect of significantly inhibiting the activity of pathogenic bacteria Escherichia coli, Salmonella typhi, Shigella, Bacillus licheniformis and Staphylococcus aureus. In particular, the antibacterial activity of the strain BGMW2 is significantly superior to that of the antibacterial agent Nisin against Staphylococcus aureus and Bacillus licheniformis.

[0021] (2) Metabolite C of Bacillus velezine BGMW2 of the present invention 12 The antibacterial effect of surfactin is better than that of the commonly used antibacterial agent Nisin (nisin), and is comparable to the antibacterial effect of the commonly used antibiotic gentamicin sulfate. 12 Surfactin also has the characteristics of antibacterial stability in the range of 40-80℃ and 2.0-12.0pH, and has hydrolase resistance and low hemolytic activity. 12 Surfactin has great application potential in the fields of food and pharmaceutical industries, and can be used as a safe and effective natural antibacterial agent and food preservative.

[0022] (3)C 12 Surfactin also has significant antibacterial effects on Escherichia coli and Staphylococcus aureus, with MICs of 7.5 μg / mL and 1.87 μg / mL for Escherichia coli and Staphylococcus aureus, respectively. 12 Surfactin has important practical value in food safety and public health fields such as milk preservation.

[0023] Bacteria biological preservation

[0024] The Bacillus velezensis BGMW2 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 24, 2024, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the deposit number is GDMCC No: 64790; the strain was received and registered by the collection center on June 24, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 The colony morphology of Bacillus velezensis BGMW2 of the present invention;

[0027] Figure 2 This is a SEM electron microscope image of Bacillus velezensis BGMW2 of the present invention;

[0028] Figure 3 The metabolite C of Bacillus velezensis BGMW2 of the present invention 12 The inhibitory effect of surfactin on bacteria, the experimental group is C 12 The surfactin treatment and the blank group were treated with methanol solvent; the indicator bacteria in Figure (1) were Escherichia coli; the indicator bacteria in Figure (2) were Salmonella typhi; the indicator bacteria in Figure (3) were Pseudomonas aeruginosa; the indicator bacteria in Figure (4) were Staphylococcus aureus; the indicator bacteria in Figure (5) were Bacillus subtilis; the indicator bacteria in Figure (6) were Bacillus licheniformis;

[0029] Figure 4 C 12 Comparison of the antibacterial effect of surfactin with the positive control (gentamicin sulfate or Nisin) and blank control (PBS) of the same concentration; the indicator bacteria in Figure A is Escherichia coli, and the indicator bacteria in Figure B is Staphylococcus aureus;

[0030] Figure 5 C 12 The antibacterial effect of surfactin under different temperatures (Figure A), different pH (Figure B) and different hydrolases (Figure C);

[0031] Figure 6 C 12 The results of the hemolytic activity test of surfactin on sheep erythrocytes;

[0032] Figure 7 C 12 The test results of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of surfactin against Escherichia coli and Staphylococcus aureus;

[0033] Figure 8 C 12 The bactericidal effect of surfactin on Escherichia coli and Staphylococcus aureus; Figure A shows the bactericidal effect of surfactin on Escherichia coli and Staphylococcus aureus at different concentrations. 12 The bactericidal time curve of surfactin against Escherichia coli; Figure B shows the different concentrations of C 12 The bactericidal time curve of surfactin against Staphylococcus aureus;

[0034] Fig. 9 C 12The effect of surfactin on the growth of Escherichia coli and Staphylococcus aureus in milk; Figure A shows the growth of C at 25℃ 12 The effect of surfactin on the growth of Escherichia coli in milk; Figure B shows the growth of Escherichia coli at 4°C 12 The effect of surfactin on the growth of Escherichia coli in milk; Figure C shows the ... 12 Effect of surfactin on the growth of Staphylococcus aureus in milk; Figure D shows the growth of Staphylococcus aureus at 4°C 12 Effect of surfactin on the growth of Staphylococcus aureus in milk; in the figure, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in this application are all conventional reagents and can be obtained from commercial sources; the methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by technicians in the technical field of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention.

[0036] Example 1 Screening, separation and identification of strains

[0037] The inventors isolated the BGMW2 strain from mangrove waters in Qinzhou City, Guangxi Zhuang Autonomous Region, China.

[0038] After activating the strain BGMW2 stored at -80℃ in LB solid medium for 24h, pick a single colony and inoculate it into LB liquid medium, and culture it at 37℃ shaker (220rpm) for 12h to obtain the seed liquid of strain BGMW2. Dip the seed liquid with a sterile inoculation loop and streak it on the slant medium. After culturing at 37℃ for 18h, it can be stored at 4℃ for 3 months. At the same time, mix the seed liquid with 50% (v / v) sterile glycerol at a ratio of 1:1, place it at 4℃ for 2h, then transfer it to -20℃ for 12h, and store it at -80℃ for a long time. The formula of LB liquid medium is: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L, add water to 1000mL, initial pH 7.0; the formula of LB solid medium is: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L, agar powder 15g / L, add water to 1000mL, initial pH 7.0.

[0039] (1) Morphological identification

[0040] Pick a small amount of BGMW2 bacteria, streak it on a solid LB plate, culture it at 37℃ for 24h, observe the colony morphology of the strain, pick the colony for Gram staining and observe the bacterial morphology under a microscope. The results are as follows: Figure 1 As shown in Figure 2, the colonies of the strain are light yellow, opaque, round, and have rough edges. Figure 2 As shown, the cell morphology is short rod-shaped. Gram staining results in purple, indicating that it is a Gram-positive bacterium.

[0041] (2) 16S rDNA sequence homology identification

[0042] DNA was extracted, and the 16S rDNA gene was amplified by PCR using universal primers 27F-1492R. The forward primer for amplification was 27F (sequence: TACGGYTACCTTGTTACGACTT), and the reverse primer was 1492R (sequence: AGAGTTTGATCMTGGCTCAG). The amplified product was detected by 0.1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing result is shown in SEQ ID NO: 1. The sequencing result was compared with the known 16S rDNA sequence in GenBank and identified as Bacillus velezensis, belonging to Bacillus velezensis, and named Bacillus velezensis BGMW2.

[0043] (3) Preservation of strains:

[0044] The Bacillus velezensis BGMW2 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 24, 2024, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the deposit number is GDMCC No: 64790; the strain was received and registered by the collection center on June 24, 2024.

[0045] Example 2 Antibacterial detection

[0046] The seed liquid of strain BGMW2 was inoculated into LB liquid medium at a 1% (V / V) inoculation rate, and cultured at 37°C and 220 rpm for 24 h to obtain the fermentation liquid. The antibacterial activity was then identified by agar diffusion method. The specific steps were as follows: the indicator bacteria obtained from the culture in the logarithmic growth phase were inoculated at a final concentration of 10 6CFU / mL was added to LB solid medium cooled to below 50°C, transferred to a sterile culture dish, and after solidification, a hole (6 mm in diameter) was punched, and 100 μL of BGMW2 fermentation broth or control antibacterial agent (Nisin) was added to each well. Incubated at 37°C overnight, the diameter of the antibacterial zone around the well was measured.

[0047] Table 1

[0048]

[0049] Note: “-” indicates no inhibitory effect on indicator bacteria; different letters indicate significant differences among groups (p<0.05).

[0050] As can be seen from Table 1, strain BGMW2 has the excellent property of broad-spectrum inhibition of pathogenic bacteria, especially has a significant antibacterial activity against Staphylococcus aureus and Bacillus licheniformis. Under the same concentration conditions, compared with the antibacterial agent Nisin commonly used in the food industry, the antibacterial activity of the fermentation broth of strain BGMW2 has a significant advantage, indicating that the strain BGMW2 provided by the present invention has important development potential in the application of sterilization and disinfection in the food industry.

[0051] Example 3 Isolation, purification and identification of antimicrobial lipopeptides from strain BGMW2

[0052] (1) After activating strain BGMW2 in LB solid medium for 24 hours, pick a colony and inoculate it into LB liquid medium, and culture it at 37°C and 220 rpm until the logarithmic growth phase. Then inoculate it into LB liquid medium (600 mL) at 1% (V / V). Culture it at 37°C and 220 rpm for 72 hours, and then centrifuge it at 10000g at 4°C to retain the supernatant.

[0053] (2) Add HCl (6 M) to adjust the pH to 2.0 and allow to precipitate overnight at 4°C. Collect the precipitate by centrifugation at 10,000 g at 4°C, wash twice, adjust the pH to 7.0, resuspend the precipitate in distilled water, freeze-dry, and extract with methanol solvent. Remove the methanol by vacuum rotary evaporation at 45°C. Add a small amount of methanol to dissolve the extract, freeze-dry again, and weigh.

[0054] (3) The extract was purified and separated using a Sephadex G-25 gel chromatography column and the absorbance was measured at 280 nm. The eluate collected from each tube was identified for antibacterial activity by the agar well diffusion method in Example 2. The collection tubes with significant antibacterial activity were selected and further separated and purified using semi-preparative high performance liquid chromatography. The elution peak was detected at 280 nm, the extracts were collected and freeze-dried to test the antibacterial activity, the components with the strongest antibacterial activity were separated, and the purity was tested. The results showed a purity of 95%, indicating that the compound was pure. The LC-MS / MS analyzer was then used for identification. The test results determined that the antibacterial substance isolated from the fermentation broth of strain BGMW2 was an antibacterial lipopeptide connected by a C12β-fatty acid chain, namely C 12 surfactin.

[0055] Example 4 Antimicrobial Lipopeptide C 12 Surfactin antibacterial assay

[0056] (1) The antimicrobial lipopeptide C purified in Example 3 was purified by the agar well diffusion method in Example 2. 12 Surfactin was used for antibacterial testing. The indicator bacteria included Staphylococcus aureus, Bacillus subtilis, Bacillus licheniformis, Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa. The test results were as follows Figure 3 As shown, C 12 Surfactin exhibited a broad-spectrum antibacterial activity and showed good inhibitory effects against Staphylococcus aureus, Bacillus subtilis, Bacillus licheniformis, Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa.

[0057] (2) Then the same concentration (12 μg / mL) of C 12 The antibacterial effect of surfactin was compared with that of gentamicin sulfate and the antibacterial agent Nisin. The test results are as follows Figure 4 As shown, compared with the positive control at the same concentration, C 12 Surfactin has a significantly better antibacterial effect on Staphylococcus aureus (S. aureus) than Nisin. At a concentration of 12 μg / mL, Nisin has basically no antibacterial effect on S. aureus, while C 12 surfactin still has a good antibacterial effect on S. aureus. Compared with gentamicin sulfate, 12μg / mL C 12 The antibacterial effect of surfactin on E.coli is basically the same as that of gentamicin sulfate.

[0058] Example 5 Antimicrobial Lipopeptide C 12 Surfactin stability and hemolytic activity assay

[0059] (1) Thermal stability

[0060] Antimicrobial lipopeptide C 12 Surfactin (5 μg / mL) solution was treated in a water bath at 40, 60, 80, 100 and 121°C for 30 min, respectively. After the temperature treatment, the volume of the sample was restored to the original volume, so that the final sample concentration was equal to the initial concentration, and then the antibacterial activity was determined (the antibacterial activity determination in this example was the same as that in Example 2, and the antibacterial activity was identified by agar well diffusion method, and the indicator bacteria were all Escherichia coli). 12 Surfactin (5 μg / mL) solution was used as blank control.

[0061] (2) Enzyme stability

[0062] In C 12 Pepsin, alkaline protease, amylase and trypsin were added to the surfactin solution to a final concentration of 1 mg / mL, and then reacted at 37°C and a suitable pH for 2 h, and then heated in a 100°C water bath for 5 min to inactivate the enzymes, and then the C 12 The antibacterial activity of surfactin solution was determined by using C 12 Surfactin solution was used as control.

[0063] (3) pH stability

[0064] C 12 The pH value of surfactin solution was adjusted to 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 with 0.1M HCl solution and maintained for 2 h, and then adjusted to 7.0 with 0.1M NaOH solution, and then the antibacterial activity was measured. 12 Surfactin solution was used as control.

[0065] (4) Hemolytic activity

[0066] Take 2 mL of sheep blood, centrifuge at 1000 g for 5 min at room temperature, and remove the plasma. Then wash with PBS 3 times. Prepare a 2% red blood cell suspension. Mix 600 μL of the 2% red blood cell suspension with 600 μL of C 12 The surfactin solution was mixed, incubated at 37°C for 30 min, and then centrifuged at 1000 g for 10 min. 100 μL of supernatant was added to a 96-well plate for each sample, and the absorbance was measured at a wavelength of 570 nm. 1% TritonX-100 and PBS were used as positive and negative controls. The results of hemolytic activity were calculated as a percentage relative to the positive control (TritonX-100).

[0067] (5) Results analysis

[0068] Stability studies have shown that C 12 The antibacterial activity of surfactin against E. coli is relatively stable in the temperature range of 40-80°C. When the temperature exceeds 80°C, the antibacterial activity gradually weakens, and when the temperature exceeds 100°C, the antibacterial activity is lost (e.g. Figure 5 C 12 The antibacterial activity of surfactin against E. coli is stable in the pH range (2.0-12.0) Figure 5 B). In the presence of various hydrolases such as pepsin, trypsin, alkaline protease and amylase, C 12 The antibacterial activity of surfactin remains stable (e.g. Figure 5 C). Studies have shown that C 12 Surfactin has a wide range of applications in the food and pharmaceutical industries, including its oral administration, as its resistance to digestive system enzymes such as gastric enzymes, pancreatic enzymes, and amylases makes it a suitable candidate for this purpose.

[0069] Hemolytic activity test results (such as Figure 6 As shown in the figure, when C 12 The hemolysis rates of surfactin were 1.75% and 1.88% at 7.5 and 15 μg / mL, respectively. 12 The hemolysis rate of surfactin was 5.91%, which was less than 10%, indicating that C 12 The low hemolytic activity of surfactin confirms its weak toxicity to mammals, further supporting its low toxicity and its potential use as an antimicrobial agent and food preservative.

[0070] Example 6 Antimicrobial Lipopeptide C 12 Surfactin minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) detection

[0071] (1) MIC detection:

[0072] In this embodiment, C 12 The MIC values ​​of surfactin were determined by broth microdilution method in sterile 96-well plates.

[0073] The indicator bacteria (E. coli and S. aureus) were grown to the logarithmic phase and resuspended to a final cell density of about 5×10 5 CFU / mL. In a 96-well plate, 12Surfactin was diluted 2-fold from 200 μg / mL to 1.56 μg / mL in a volume of 20 μL. Subsequently, 180 μL of bacterial suspension was added to each well along with appropriate control groups and incubated at 37°C for 24 h. The OD was measured using a microplate reader. 600 The bacterial growth status was monitored by MIC. The MIC was determined as the lowest concentration of the antimicrobial agent that completely inhibited visible bacterial growth. All MICs were determined in three independent experiments, each in duplicate.

[0074] (2) MBC test:

[0075] After determining the growth status of the bacteria, take 10 μL of the treated bacterial suspension from each group and streak it on an LB agar plate. Let the bacteria grow at 37°C and monitor the number of colonies on the plate. The MBC value refers to the lowest concentration at which no bacterial colonies are observed on the plate.

[0076] Table 2

[0077]

[0078] Depend on Figure 7 From the test results in Table 2, we can see that antimicrobial lipopeptide C 12 The MIC values ​​of surfactin against E. coli and S. aureus were 7.5 μg / mL and 1.87 μg / mL, respectively. 12 The MBC values ​​of surfactin against E. coli and S. aureus were 30 μg / mL and 7.5 μg / mL, respectively. 12 Surfactin has strong antibacterial activity against E. coli and S. aureus.

[0079] Example 7 Antimicrobial Lipopeptide C 12 Determination of the killing time curve of surfactin

[0080] Add 50 μL of bacterial suspension (5×10 5 CFU / mL). Then PBS, 1×MIC, 2×MIC and 4×MIC C 12 50 μL of surfactin solution was incubated at 37°C. At the indicated time points, the serially diluted cultures were inoculated on LB plates and incubated at 37°C overnight. After taking the dilution factor into account, the number of viable colonies per 1 mL of the original culture was determined by colony counting.

[0081] The results of the test are as follows Figure 8 As shown, compared with the control, C 12After 30 min of treatment with surfactin at the minimum inhibitory concentrations of 1×MIC, 2×MIC, and 4×MIC, E. coli and S. aureus cells were rapidly inactivated within 30 min. After 120 min of treatment with the minimum inhibitory concentration of 4×MIC, there were almost no living cells of E. coli and S. aureus, indicating that C 12 Surfactin showed strong and rapid inactivation effect on E. coli and S. aureus. The results of dilution coating experiment showed that C 12 The antibacterial effect of surfactin on E.coli and S.aureus increased with the increase of C 12 The activity of surfactin was enhanced with the increase of surfactin concentration and was concentration-dependent. In particular, after treatment with the minimum inhibitory concentration of 4×MIC, no active colonies grew on the LB plate, which was consistent with the results of the above time-killing test. 12 The rapid bactericidal effect of surfactin shows great potential as a natural preservative in food applications.

[0082] Example 8 Antimicrobial Lipopeptide C 12 Application of surfactin in milk preservation

[0083] E. coli and S. aureus cells were diluted to about 1 × 10 with 20 mL of fresh milk. 4 CFU / mL, add 1×MICC 12 After surfactin, the cells were cultured at 25°C and 4°C. The control group did not contain C 12 The concentration of microorganisms was detected by colony counting method at 1h, 12h, 24h, 48h and 72h of culture.

[0084] The results are as follows Fig. 9 As shown in A and C, when milk was stored at 25℃, compared with the control group, at different storage times, C 12 Surfactin significantly inhibited the reproduction of E. coli and S. aureus; for example, compared with the control group, C 12 After 24h and 72h of surfactin treatment, the concentration of E. coli decreased from 7.5 to 4.8 log 10 CFU / mL and 8.5 to 5.1log 10 CFU / mL(p<0.01); 12 After 24h and 72h of surfactin treatment, the concentration of S. aureus decreased from 6.1 to 4.0 log 10 CFU / mL and 8.1 down to 5.0log 10CFU / mL (p<0.01).

[0085] On the other hand, Fig. 9 As shown in B and D, when milk was stored at 4°C, the growth of E. coli and S. aureus in the blank group was slow. 12 After surfactin treatment, the number of E. coli and S. aureus continued to decrease, indicating that C 12 Surfactin has a better antibacterial effect at 4℃.

[0086] Dairy products are essential in human life and provide health benefits in addition to basic nutrition. Milk is a nutrient medium with high water activity, which promotes the growth of undesirable microorganisms, mainly Staphylococcus aureus and Escherichia coli, and makes it unsafe for human consumption. Therefore, it is necessary to develop safe and efficient milk preservatives. Therefore, the above results can provide a reference for the application of C 12 surfactin to control pathogens transmitted by E. coli and S. aureus in dairy products provides a theoretical basis.

[0087] In summary, firstly, the Bacillus velezensi BGMW2 provided by the present invention has the excellent property of broad-spectrum inhibition of pathogenic bacteria, especially against Staphylococcus aureus and Bacillus licheniformis, and the antibacterial activity of strain BGMW2 has significant advantages over the antibacterial agent Nisin.

[0088] Then, the present invention uses Escherichia coli as an indicator bacterium and uses gel column chromatography and high performance liquid chromatography to separate and purify the antimicrobial lipopeptide, and further uses LC-MS / MS to identify the antimicrobial lipopeptide and determine that it is C 12 surfactin. The present invention detects C 12 The antibacterial effect of surfactin was compared with that of the commonly used antibiotic gentamicin sulfate and the commonly used antibacterial agent Nisin (nisin), and it was found that its antibacterial effect was better than Nisin and equivalent to that of gentamicin sulfate. 12 Surfactin also showed stability and safety, C 12 Surfactin has stable antibacterial properties at 40-80°C and 2.0-12.0 pH, and is resistant to protease and amylase hydrolysis, and has low hemolytic activity. 12Surfactin, the antimicrobial lipopeptide, not only has a strong antibacterial effect, but also maintains stable antibacterial activity in a wide range of temperature and pH, and is resistant to protease and amylase hydrolysis and has low hemolytic activity. These characteristics ensure its stability and safety in various application environments, and also make C 12 Surfactin becomes a potential natural antibacterial agent.

[0089] Finally, the present invention found that C 12 Surfactin also has significant antibacterial effects on Escherichia coli and Staphylococcus aureus, with MICs of 7.5 μg / mL and 1.87 μg / mL for Escherichia coli and Staphylococcus aureus, respectively. 12 Surfactin added to milk can significantly reduce the number of Escherichia coli and Staphylococcus aureus in milk, indicating that C 12 Surfactin has important practical applications in the fields of food safety and public health.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus velezensis BGMW2, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 24, 2024, with the deposit number GDMCC No: 64790.

2. A bacterial agent, characterized in that The bacterial agent includes the Bacillus velezensis BGMW2 or its fermentation broth as claimed in claim 1, and the fermentation broth contains the Bacillus velezensis BGMW2.

3. Use of the Bacillus velezensis BGMW2 of claim 1 or the bacterial agent of claim 2 in inhibiting bacteria, characterized in that: The application is not intended for the diagnosis or treatment of disease.

4. The use according to claim 3, characterized in that: The bacteria include Escherichia coli, Salmonella typhi, Shigella, Bacillus licheniformis, and Staphylococcus aureus.

5. The bacterial agent according to claim 2, characterized in that The number of effective live bacteria in the bacterial agent is ≥10 5 CFU / mL.

6. An antibacterial product, characterized in that: The product contains the Bacillus velezensis BGMW2 described in claim 1 or the bacterial agent described in claim 2.

7. An antimicrobial lipopeptide, characterized in that: The method for preparing the antimicrobial lipopeptide comprises: inoculating the Bacillus velezensis BGMW2 described in claim 1 into a culture medium for fermentation to obtain a fermentation broth, and separating and extracting the antimicrobial lipopeptide from the fermentation broth.

8. The antimicrobial lipopeptide according to claim 7, characterized in that: The preparation method of the antimicrobial lipopeptide specifically further includes: centrifuging the fermentation liquid at 8000-12000rpm to obtain a fermentation supernatant, adjusting the pH of the fermentation supernatant to 2.0 with 6M hydrochloric acid, and after precipitation at 4°C overnight, centrifuging the fermentation supernatant at 8000-12000rpm, discarding the supernatant, resuspending the precipitate with distilled water, freeze-drying, extracting with methanol solvent, removing methanol by vacuum rotary evaporation at 45°C, and then dissolving with methanol and freeze-drying to obtain the antimicrobial lipopeptide produced by Bacillus Velez.

9. Use of the antimicrobial lipopeptide according to claim 7 or 8 in the preparation of antimicrobial products, characterized in that: The antimicrobial products include food preservatives, antimicrobial drugs, antimicrobial disinfectants, antimicrobial protective products, cosmetics, skin care products or health products.

10. Use of the antimicrobial lipopeptide according to claim 7 or 8 in inhibiting bacteria, characterized in that: The application is not intended for the diagnosis or treatment of disease.

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