Bacillus atrophaeus NB65 and method for preparing lipopeptide from bacillus atrophaeus NB65

Abstract

The invention discloses bacillus atrophaeus NB65, which is characterized in that the strain has relatively strong inhibitory activity on pathogenic bacteria of northern corn leaf blight and corn stalk rot, is separated from a deep-sea sediment sample in South China Sea, is preserved in China Center for Type Culture Collection on May 25, 2023, is called CCTCC for short, and has a preservation number of CCTCC M 2023819. The invention discloses a preparation method for producing two novel cyclic lipopeptides, i.e., iso-C16 [Ile7] surfactin (1) and iso-C14 [MeGlu1, Ile7] surfactin (2), by virtue of the novel cyclic lipopeptides, i.e., iso-C16 [Ile7] surfactin (1) and iso-C14 [MeGlu1, Ile7] surfactin (2). The bacillus atrophaeus NB65 is subjected to fermentation, acid precipitation, rapid column chromatography, high performance liquid chromatography preparation and other methods to separate six surfactin (1-6), and then the structure of the compound is determined through the technologies of spectral analysis, improved Marfe's, quantum chemistry calculation and the like. The bacillus atrophaeus NB65 and the two new compounds generated by the bacillus atrophaeus NB65 have the potential of serving as biological agents, surfactants and biological active pesticides for inducing plant disease resistance.

Description

Technical Field

[0001] The present invention relates to the marine bacterium Bacillus atrophaeus NB65 and the extraction and separation of surface-active cyclic lipopeptide compounds from the fermentation broth of Bacillus atrophaeus NB65. Specifically, two new cyclic lipopeptides are separated and purified from the fermentation broth of Bacillus atrophaeus NB65: iso-C 16 [Ile 7 ]surfactin(1) and iso-C 14 [MeGlu 1 ,Ile 7 ]surfactin(2), and the potential application of the lipopeptide, the producing bacteria Bacillus atrophaeus NB65 and the fermentation broth in biopharmaceuticals or biocontrol agents, and surfactants. Background Art

[0002] Bacillus is a key component of plant disease biocontrol microorganisms. Since 2014, Bacillus has become a globally registered and marketed new microbial pesticide, becoming increasingly mainstream. In the 2020 list of microbial pesticides released in China, Bacillus accounted for over 80% of bacterial microbial pesticides. Bacillus, as a biocontrol agent, controls plant diseases through various mechanisms: antibiotics, nutrient competition, spatial competition, hyperparasitism, and the induction of systemic resistance in plants. Its primary biocontrol mechanism is the production of antagonistic substances that inhibit the growth of harmful pathogens or kill them. These substances are mostly peptide antibiotics, typically including lipopeptides from the iturin, fengycin, and surfactin families, which offer advantages such as high efficacy, low residues, and environmental friendliness. Marc Ongena et al. have found that plants such as beans and tomatoes can sense compounds such as surfactin and fengycin, which act as signals to activate defense mechanisms—the development of systemic resistance (ISR). Maurice E. Snook et al. isolated and identified Leu from the endophyte Bacillus mojavensis RRC 101. 7 -Surfactin has a good antagonistic effect on Fusarium verticillioides (Fusarium monilioides) in corn and other plants, and can reduce the production of Fusarium monilioides toxins.

[0003] Surfactin lipopeptides were first isolated in 1968 from the fermentation supernatant of Bacillus subtilis. Surfactin exhibits various physiological activities, including as a fibrin coagulation inhibitor and a cell lysate. Besides antiviral and antitumor properties, it also exhibits antibacterial, antifungal, antimycoplasmal, and anti-inflammatory properties, with a broad antimicrobial spectrum. Furthermore, it is not susceptible to developing drug resistance, can be degraded by animal digestive enzymes, and produces no residue, demonstrating its potential for pharmaceutical applications. To date, the amino acid configuration of the surfactin family heptapeptides is LLDLLDL, with D-Leu at positions 3 and 6 of the peptide chain. Currently, approximately 20 lipopeptides exist, and with the exception of esperin, all consist of a heptapeptide linked to a β-OH fatty acid. Table 1 shows a selection of compounds in the surfactin family.

[0004] Table 1 Structural characteristics of peptides of surfactin-like compounds

[0005]

[0006] Summary of the Invention

[0007] The present invention aims to clarify the production of two novel cyclic lipopeptide compounds iso-C by Bacillus atrophaeus NB65. 16 [Ile 7 ]surfactin(1) and iso-C 14 [MeGlu 1 ,Ile 7 ]Separation and purification of surfactin (2) and its potential applications. The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description progresses. However, these examples are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements fall within the scope of protection of the present invention.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] Two surfactant lipopeptide compounds iso-C 16 [Ile 7 ]surfactin(1) and iso-C 14 [MeGlu 1 ,Ile 7]surfactin(2), two new cyclic lipopeptide compounds obtained by extraction and separation from the fermentation broth of Bacillus atrophaeus NB65 isolated from deep-sea sediment samples in the South China Sea.

[0010] Bacillus atrophaeus NB65 was deposited in the China Center for Type Culture Collection (CCTCC) on May 25, 2023. The address is: Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number being CCTCC NO: M 2023819.

[0011] Bacillus atrophaeus NB65 was isolated from sediment samples of the South China Sea.

[0012] Culture medium:

[0013] TSB medium: casein peptone (trypsin digest) 15 g, soy peptone 5 g, glucose 2.5 g, NaCl 5 g, K2HPO4 2.5 g, agar 20 g, water 1 L, natural pH.

[0014] LB medium: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 20 g of agar, 1 L of distilled water, pH 7.0.

[0015] Streak the NB65 strain from the LB medium slant onto TSB medium, separating the strains by three streaks, and incubate the strain upside down in a 28°C incubator. Select a single, uniform colony in the NB65 medium and observe its morphology. Also, select a colony and examine its microscopic morphology under a microscope.

[0016] Within 3 days of culture on TSB plates, NB65 colonies were white or yellow, irregularly round, sticky and moist, transparent, with smooth edges, and a colony diameter of 2.0 to 2.6 mm ( Figure 8 After 4 days, it gradually becomes turbid and turns yellow-brown. After 10 days of culture, the colony turns dark brown, the surface shrinks, and becomes opaque, and a large amount of black-brown pigment is produced in the culture medium. Figure 7 ), NB65 was named Bacillusatrophaeus NB65.

[0017] Table 2 Physiological and biochemical characteristics of strain NB65

[0018]

[0019] Bacillus atrophaeus NB65 was cultured in a lipopeptide-producing fermentation medium to obtain 120 L of fermentation broth. Crude lipopeptides were prepared by centrifugation, acid precipitation, and methanol extraction. The crude lipopeptides were then loaded onto a silica gel column for RP-HPLC analysis and preparation to yield the target compounds. Target compound 1 (10.2 mg) and compound 2 (10.2 mg) were obtained, along with other compounds 3 (11.3 mg), 4 (7.0 mg), 5 (15.3 mg), and 6 (7.3 mg).

[0020]

[0021] Structural analysis of compound 1:

[0022]

[0023] The compound is a white powder (methanol). HR-ESI-MS gives a molecular ion peak [M+Na] + m / z 1072.6864( Figure 5 ), the molecular formula of the compound is determined to be C 54 H 95 N7O 13 , the unsaturation is 11. 1 H-NMR (DMSO-d6, 600 MHz) gives the hydrogen proton signals of 7 amide bonds δ H 8.32 (1H, br s), 8.26 (1H, d, J = 8.0 Hz), 8.18 (1H, d, J = 7.0 Hz), 8.04 (1H, br s), 7.83 (1H, br s), 7.78 (1H, br s) and 7.66 (1H, br s), which were speculated to be 7 peptides (Table 3). 1 H NMR (DMSO-d6, 600 MHz) observed that H The large signal at 1.22 indicates the presence of fatty acid chains in the molecule. 13 C-NMR (DMSO-d6, 150 MHz) gave 10 carbonyl carbon signals δ C 174.1, 172.1, 172.1, 171.8, 171.8, 171.1, 170.7, 170.6, 169.9 and 169.4. The above 1D NMR signal characteristics suggest the presence of surfactin derivatives (Table 3). 1 H– 1 H COSY, HSQC and HMBC connection analysis showed that ( Figure 2), 7 amino acids form one glutamic acid, one aspartic acid, one valine, one isoleucine and 3 leucines. The above amino acids have a total of 9 carbonyl carbon signals, and the degree of unsaturation is 9. According to the molecular formula, the degree of unsaturation is 11, which suggests the presence of an ester bond and that the surfactin derivative is cyclic. The connection sequence of the 7 amino acids is N-Glu-Leu-Leu-Val-Asp-Leu-Ile-C based on the two-dimensional HMBC spectrum. According to the molecular formula of the compound, it is speculated that there is a hydroxy fatty acid chain with a length of 16 carbon atoms. Due to 13 C-NMR (DMSO-d6, 150 MHz) gave δ C The carbon signals at 38.5, 27.4, 22.5 and 22.5 (C-13 to C-16) indicate that the terminal methyl group is in iso mode. The absolute configuration of the amino acids of compound 1 was determined by a modified Marfey's method. The retention times of the L-FDLA-derivatives of compound 1 and the L-FDLA-derivatives of the standard amino acids were consistent, and it can be inferred that the absolute configuration of the amino acids of compound 1 is L1L2D3L4L5D6L7( Figure 3 The absolute configuration of β-hydroxyl in compound 1 was determined to be R (94.41%) by calculation of NMR and DP4+ analysis ( Figure 4 ). Based on the above data analysis, the chemical structure of compound 1 was determined to be a new compound not reported in the literature and was named iso-C 16 [Ile 7 ]surfactin.

[0024] Table 3 1D NMR data of compound 1-2 (DMSO-d6)

[0025]

[0026]

[0027]

[0028] Superscripts ah indicate overlapping.

[0029] The structural analysis of compound 2 is as follows:

[0030]

[0031] Compound 2 is a white powder (methanol). HR-ESI-MS gives a molecular ion peak [M+Na] + m / z 1058.6703( Figure 6 ), the molecular formula of the compound is determined to be C53 H 93 N7O 13 , the unsaturation is 11. 1 H-NMR (DMSO-d6, 600 MHz) gives the hydrogen proton signals of 7 amide bonds δ H 8.35 (1H, d, J = 6.7 Hz), 8.27 (1H, d, J = 8.0 Hz), 8.18 (1H, d, J = 7.3 Hz), 8.04 (1H, br s), 7.81 (1H, d, J = 5.8 Hz), 7.74 (1H, d, J = 8.3 Hz), 7.64 (1H, d, J = 8.4 Hz), which is speculated to be a 7-peptide. 1 H NMR (DMSO-d6, 600 MHz) observed that H The large signal at 1.22 indicates the presence of fatty acid chains in the molecule. 13 C-NMR (DMSO-d6, 150 MHz) gave 10 carbonyl carbon signals δ C 172.9, 172.2, 172.1, 171.8, 171.7, 170.9, 170.7, 170.6, 169.9, 169.4. The above 1D NMR signal characteristics suggest the presence of surfactin derivatives (Table 3). 1 H– 1 H COSY, HSQC and HMBC connection analysis showed that the seven amino acids consist of one methylated glutamic acid, one aspartic acid, one valine, one isoleucine and three leucines. The above amino acids have a total of 9 carbonyl carbon signals, and the degree of unsaturation is 9. According to the molecular formula, the degree of unsaturation is 11, which suggests the presence of an ester bond and that the surfactin derivative is cyclic. The connection sequence of the seven amino acids is N-Glu-Leu-Leu-Val-Asp-Leu-Ile-Cl based on the two-dimensional HMBC spectrum. According to the molecular formula of the compound, it is speculated that there is a hydroxy fatty acid chain with a length of 14 carbon atoms. Due to 13 C-NMR (DMSO-d6, 150 MHz) gave δ C The carbon signals at 38.5, 27.4, 22.5 and 22.5 (C-11 to C-14) indicate that the terminal methyl group is in iso mode. Based on the modified Marfey's results and by comparing them with the corresponding NMR data and optical rotation, the absolute configuration of compound 2 was determined to be the same as that of compound 1 ( Figure 3 ). Based on the above data analysis, the chemical structure of compound 2 was determined to be a new compound not reported in the literature and was named iso-C 14 [MeGlu 1 ,Ile 7 ]surfactin.

[0032] Bacillus atrophaeus NB65 produces six surfactins, including two new cyclic lipopeptide compounds 1 and 2. Figure 1 ) Furthermore, the crude extract of the fermentation broth of the NB65 strain can inhibit the growth of various maize pathogens, such as Exserohilum turcicum, Fusarium verticillium, and Fusarium graminearum, exhibiting both biological and surface bioactivity against plant pathogenic fungi. This novel lead compound and its producing strain, Bacillus atrophaeus NB65, have potential as biological agents and biopharmaceuticals, and can be used as active ingredients in biopharmaceuticals, biocontrol agents, and surfactants. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the chemical structure of compound 1-6 of the present invention.

[0034] Figure 2 The key to compound 1-2 of the present invention 1 H- 1 H COSY, HMBC and ROESY connections.

[0035] Figure 3 This is the analysis result of the compound 1-2 according to the present invention by the Marfey's method.

[0036] Figure 4 Calculated NMR and DP4+ analysis of compound 1 of the present invention.

[0037] Figure 5 HR-ESI-MS of compound 1 of the present invention.

[0038] Figure 6 This is the HR-ESI-MS of compound 2 of the present invention.

[0039] Figure 7 The diagram is a phylogenetic tree of the 16SrRNA gene sequence of Bacillus atrophaeus NB65 of the present invention.

[0040] Figure 8 This is the colony culture morphology of Bacillus atrophaeus NB65 of the present invention.

[0041] Figure 9 The invention discloses that the Bacillus atrophaeus NB65 can inhibit the corn leaf blight Exserohilum turcicum.

[0042] Figure 10 It is the surface activity (oil-repelling ring) of compound 1-2 of the present invention. DETAILED DESCRIPTION

[0043] Example:

[0044] The purified Bacillus atrophaeus NB65 strain was propagated on TSB solid medium (TSB medium: 15 g casein peptone (tryptic digest), 5 g soy peptone, 2.5 g glucose, 5 g NaCl, 2.5 g K2HPO4, 20 g agar, 1 L water, pH natural). The culture was then inoculated using the cloning method into a lipopeptide-producing liquid medium (medium formulation: 2% sucrose, 0.3% KH2PO4, 1% Na2HPO4, 0.2% NH4NO3, 0.02% MgSO47H2O, 0.02% yeast extract, 0.02% CaCl2, 0.7 μg / 100 mL, 1 μg / 100 mL MnSO4·H2O, pH 7.2). The medium was sterilized in an autoclave at 115°C, 0.1 MPa, for 30 min. 50 mL of seed solution per flask was shaken in a shaker at 28°C, 180 rpm, for 24 h. The cultured seed liquid was inoculated into a 3L large shake flask at a volume ratio of about 5% (i.e., 1 small bottle of seed liquid was inoculated into a 1L fermentation shake flask), placed in a shaker at 28°C and shaken at 180 rpm for 48 hours. A total of 138 bottles were fermented. At the end of the fermentation, 120L of fermentation liquid was obtained, which can be used to extract crude lipopeptides.

[0045] After fermentation, the pH of the fermentation liquid was adjusted to 8.0 with a 2% NaOH solution, and then centrifuged at 4000 rpm for 30 minutes at room temperature to remove the bacteria and other solids. The fermentation supernatant was taken and the pH of the fermentation supernatant was adjusted to 2.0 with 6 mol / L hydrochloric acid. It was placed in a 4°C refrigerator overnight to facilitate the precipitation of crude lipopeptides. The fermentation supernatant after overnight was centrifuged at 4000 rpm for 30 minutes in a centrifuge, the supernatant was discarded, and the precipitate was collected. The precipitate was washed at least twice with hydrochloric acid at pH 2.0 to remove the residual supernatant on the surface, and then extracted with methanol in small amounts multiple times. After repeated extraction three times, the extract was evaporated to dryness under reduced pressure at 35°C in a rotary evaporator to remove the methanol solvent. After drying, 9.5 g of crude lipopeptide was obtained.

[0046] The crude extract was first separated by silica gel chromatography using dichloromethane:methanol (v:v) at 100:0, 100:2, 100:5, 100:7, 100:10, 100:15, 100:30, and 0:100 as eluent. Eight fractions (Fa-h) were collected and analyzed by HPLC-DAD. Surfactin-like compounds were primarily concentrated in the Fb and Fc fractions.

[0047] Fraction Fb was separated by Sephadex LH-20 gel column chromatography, and the obtained subfractions were analyzed by HPLC-DAD. It was confirmed that the surfactin-like compound was contained in subfraction Fb1. Subfraction Fb1 was subjected to semi-preparative liquid phase separation (95:5, MeOH:H2O, v:v) to obtain compound 6 (7.3 mg, 31.27 min);

[0048] Fraction Fc was separated by Sephadex LH-20 gel column chromatography, and the obtained subfractions were analyzed by HPLC-DAD, confirming that the surfactin-like compounds were in subfraction Fc1. Subfraction Fc1 was subjected to semi-preparative liquid phase separation (95:5, ACN:H2O, v:v) to obtain compound 1 (10.2 mg, 32.68 min), compound 2 (10.2 mg, 30.39 min), compound 3 (11.3 mg, 32.21 min), compound 4 (7.0 mg, 27.10 min), and compound 5 (15.3 mg, 29.05 min).

[0049] Structural analysis of compound 1:

[0050]

[0051] The compound is a white powder (methanol). HR-ESI-MS gives a molecular ion peak [M+Na] + m / z 1072.6864, confirming that the molecular formula of the compound is C 54 H 95 N7O 13 ( Figure 5 ), with an unsaturation of 11. 1 H-NMR (DMSO-d6, 600 MHz) gives the hydrogen proton signals of 7 amide bonds δ H 8.32 (1H, br s), 8.26 (1H, d, J = 8.0 Hz), 8.18 (1H, d, J = 7.0 Hz), 8.04 (1H, br s), 7.83 (1H, br s), 7.78 (1H, br s) and 7.66 (1H, br s), which were speculated to be 7 peptides (Table 3). 1 H NMR (DMSO-d6, 600 MHz) observed that H The large signal at 1.22 indicates the presence of fatty acid chains in the molecule. 13 C-NMR (DMSO-d6, 150 MHz) gave 10 carbonyl carbon signals δ C174.1, 172.1, 172.1, 171.8, 171.8, 171.1, 170.7, 170.6, 169.9 and 169.4. The above 1D NMR signal characteristics suggest the presence of surfactin derivatives (Table 3). 1 H– 1 H COSY, HSQC and HMBC connection analysis showed that ( Figure 2 ), 7 amino acids form one glutamic acid, one aspartic acid, one valine, one isoleucine and 3 leucines. The above amino acids have a total of 9 carbonyl carbon signals, and the degree of unsaturation is 9. According to the molecular formula, the degree of unsaturation is 11, which suggests the presence of an ester bond and that the surfactin derivative is cyclic. The connection sequence of the 7 amino acids is N-Glu-Leu-Leu-Val-Asp-Leu-Ile-C based on the two-dimensional HMBC spectrum. According to the molecular formula of the compound, it is speculated that there is a hydroxy fatty acid chain with a length of 16 carbon atoms. Due to 13 C-NMR (DMSO-d6, 150 MHz) gave δ C The carbon signals at 38.5, 27.4, 22.5 and 22.5 (C-13 to C-16) indicate that the terminal methyl group is in iso mode. The absolute configuration of the amino acids of compound 1 was determined by a modified Marfey's method. The retention times of the L-FDLA-derivatives of compound 1 and the L-FDLA-derivatives of the standard amino acids were consistent, and it can be inferred that the absolute configuration of the amino acids of compound 1 is L1L2D3L4L5D6L7( Figure 3 The absolute configuration of β-hydroxyl in compound 1 was determined to be R (94.41%) by calculation of NMR and DP4+ analysis ( Figure 4 ). Based on the above data analysis, the chemical structure of compound 1 was determined to be a new compound not reported in the literature and was named iso-C 16 [Ile 7 ]surfactin.

[0052] The structural analysis of compound 2 is as follows:

[0053]

[0054] Compound 2 is a white powder (methanol). HR-ESI-MS gives a molecular ion peak [M+Na] + m / z 1058.6703( Figure 6 ), the molecular formula of the compound is determined to be C 53 H 93 N7O 13 , the unsaturation is 11. 1H-NMR (DMSO-d6, 600 MHz) gives the hydrogen proton signals of 7 amide bonds δ H 8.35 (1H, d, J = 6.7 Hz), 8.27 (1H, d, J = 8.0 Hz), 8.18 (1H, d, J = 7.3 Hz), 8.04 (1H, br s), 7.81 (1H, d, J = 5.8 Hz), 7.74 (1H, d, J = 8.3 Hz), 7.64 (1H, d, J = 8.4 Hz), which is speculated to be a 7-peptide. 1 H NMR (DMSO-d6, 600 MHz) observed that H The large signal at 1.22 indicates the presence of fatty acid chains in the molecule. 13 C-NMR (DMSO-d6, 150 MHz) gave 10 carbonyl carbon signals δ C 172.9, 172.2, 172.1, 171.8, 171.7, 170.9, 170.7, 170.6, 169.9, 169.4. The above 1D NMR signal characteristics suggest the presence of surfactin derivatives (Table 3). 1 H– 1 H COSY, HSQC and HMBC connection analysis showed that the seven amino acids consist of one methylated glutamic acid, one aspartic acid, one valine, one isoleucine and three leucines. The above amino acids have a total of 9 carbonyl carbon signals, and the degree of unsaturation is 9. According to the molecular formula, the degree of unsaturation is 11, which suggests the presence of an ester bond and that the surfactin derivative is cyclic. The connection sequence of the seven amino acids is N-Glu-Leu-Leu-Val-Asp-Leu-Ile-Cl based on the two-dimensional HMBC spectrum. According to the molecular formula of the compound, it is speculated that there is a hydroxy fatty acid chain with a length of 14 carbon atoms. Due to 13 C-NMR (DMSO-d6, 150 MHz) gave δ C The carbon signals at 38.5, 27.4, 22.5 and 22.5 (C-11 to C-14) indicate that the terminal methyl group is in iso mode. Based on the modified Marfey's results and by comparing them with the corresponding NMR data and optical rotation, the absolute configuration of compound 2 was determined to be the same as that of compound 1 ( Figure 3 ). Based on the above data analysis, the chemical structure of compound 2 was determined to be a new compound not reported in the literature and was named iso-C 14 [MeGlu 1 ,Ile 7 ]surfactin.

[0055] Application examples:

[0056] The anti-plant pathogenic fungal activity of Bacillus atrophaeus NB65 fermentation broth was tested using the filter paper method. The following procedures were used: Bacillus atrophaeus NB65 was inoculated into TSB liquid medium using the cullet method, with two cullets placed in each flask. Each 60 mL flask was shaken for 72 hours at 28°C and 180 rpm. Post-fermentation treatment: After the fermentation of 60 mL of fermentation broth, 10 mL was transferred to a 50 mL beaker, refrigerated at -20°C overnight, freeze-dried, and dissolved in 1 mL of methanol for use. The remaining 50 mL of fermentation broth was centrifuged (8000 rpm for 30 minutes) and the supernatant returned to the original flask. 2 g of HP-20 resin was added to each flask, shaken at 28°C and 180 rpm for 2 hours, then poured onto filter paper and washed three times with distilled water. After draining, the resin was transferred to a 15 mL glass bottle and incubated overnight with 5 mL of methanol.

[0057] Exserohilum turcicum, Fusarium verticillium, and Fusarium graminearum, pathogens of the disease, were inoculated into the center of a PSA plate using the slug method and incubated at 28°C for 2-3 days. Colonies were used when they reached a diameter of approximately 4 cm. Filter paper discs were added with 10 μL of a 20 mg / mL methanol-dissolved crude lipopeptide extract of Bacillus atrophaeus NB65. The methanol was evaporated on a sterile workbench, and the filter paper discs were placed symmetrically around the indicator fungus. The filter paper discs had a diameter of 8 mm. Filter paper discs containing anhydrous methanol served as a negative control, and 1 mg / mL of brefeldin A served as a positive control. The plates were incubated at 28°C for 2 days. Figure 9 The results show the inhibitory activity of Bacillus atrophaeus NB65 against the pathogen of corn leaf blight.

[0058] The antibacterial activity of compounds 1 and 2 was tested using the above method. The results showed no antibacterial activity against the tested bacteria. The surface activity was tested using the oil ring method. 15 mL of distilled water was added to a 9 cm culture dish, and 100 μL of liquid paraffin was dropped into it. When the paraffin oil spread evenly, 50 μL of the sample solution of the tested compounds 1 and 2 was gently dotted into the center of the paraffin oil. The paraffin oil was expelled to the surroundings, forming a circle in the center, indicating a certain surface activity. Figure 10 shown.

[0059] Therefore, the inhibitory activity of Bacillus atrophaeus NB65 against corn leaf blight pathogens can be used to develop microbial pesticides for the prevention and control of corn leaf blight. The rich surfactin lipopeptide compounds produced by it can induce plants to produce systemic resistance, help resist fungal diseases, and improve the disease resistance of corn.

Claims

1. A Bacillus atrophaeus NB65, characterized in that: The strain is Bacillusatrophaeus NB65, which was deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on May 25, 2023. The address is: Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC M 2023819.

2. A lipopeptide compound, characterized in that: The lipopeptide compound was named iso-C 16 [Ile 7 ]surfactin or compound 2 named iso-C 14 [MeGlu 1 ,Ile 7 ]surfactin, the chemical structure is shown below: The other four compounds are: anteiso-C 15 [MeGlu 1 ,Ile 7 ]surfactin(3),iso-C 13 [Ile 7 ]surfactin(4),iso-C 14 [Ile 7 ]surfactin(5) and 3-L-isoleucine-dimethyl estersurfactin B1(6).

3. A method for preparing the lipopeptide compound according to claim 2, characterized in that: The lipopeptide compounds were compound 1 or 2 isolated from deep-sea sediment samples in the South China Sea; Bacillus atrophaeus NB65 was fermented and cultured, and the fermentation broth was subjected to acid precipitation to obtain crude lipopeptides, which were eluted and separated on a silica gel chromatography column, and the eluted components were separated by high performance liquid chromatography to obtain compounds 1 and 2.

4. The preparation method according to claim 3, characterized in that: The specific preparation process is as follows: the weight composition of the fermentation medium of Bacillus atrophaeus NB65 is as follows: sucrose (1-5)%, KH2PO4 (0.1-1.0)%, Na2HPO4 (0.1-3), NH4NO3 (0.1-1.0)%, MgSO4.7H2O (0.01-0.05)%, yeast powder (0.01-0.05)%, CaCl2 (0.1-1.0) μg / 100mL, MnSO4.H2O (0.1-2.0) μg / 100mL, pH (5.0-9.0), and the balance is water; the above culture medium needs to be steam sterilized in a high-pressure steam sterilizer at 115-121°C and 0.08-0.1Mpa for 15-30 minutes. After the fermentation is completed, the pH of the fermentation liquid is adjusted to 7.5-8.0 with a 2% mass concentration of NaOH solution, and then centrifuged at 4000 rpm for 30 minutes at room temperature to remove the bacteria and other solids; the fermentation supernatant is taken, and the pH of the fermentation supernatant is adjusted to 2.0 with 6 mol / L hydrochloric acid, and placed in a 0-4°C refrigerator overnight (8-12 hours) to facilitate the precipitation of crude lipopeptides. The fermentation supernatant after overnight is centrifuged at 4000 rpm for 30 minutes in a centrifuge, the supernatant is discarded, and the precipitate is collected, and the precipitate is washed at least twice with hydrochloric acid at pH 2.0 to remove the residual supernatant on the surface, and then extracted with methanol in small amounts multiple times. After repeated extraction three times, the extract is evaporated to dryness under reduced pressure at 35°C in a rotary evaporator to remove the methanol solvent, and the crude lipopeptide is obtained after drying; The crude extract was first separated by silica gel chromatography using dichloromethane:methanol (v:v) at 100:0, 100:2, 100:5, 100:7, 100:10, 100:15, 100:30, and 0:100 as eluent. Eight fractions (Fa-h) were collected. HPLC-DAD analysis revealed that surfactin-like compounds were primarily concentrated in the Fb and Fc fractions. Fraction Fb was separated by Sephadex LH-20 gel column chromatography, and the obtained subfractions were analyzed by HPLC-DAD. It was confirmed that the surfactin-like compound was contained in subfraction Fb1. Subfraction Fb1 was subjected to semi-preparative liquid phase separation (95:5, MeOH:H2O, v:v) to obtain compound 6 (7.3 mg, 31.27 min); Fraction Fc was separated by Sephadex LH-20 gel column chromatography, and the obtained subfractions were analyzed by HPLC-DAD, confirming that the surfactin-like compounds were in subfraction Fc1. Subfraction Fc1 was subjected to semi-preparative liquid phase separation (95:5, ACN:H2O, v:v) to obtain compound 1 (10.2 mg, 32.68 min), compound 2 (10.2 mg, 30.39 min), compound 3 (11.3 mg, 32.21 min), compound 4 (7.0 mg, 27.10 min), and compound 5 (15.3 mg, 29.05 min).

5. A use of the lipopeptide compound according to claim 2, characterized in that: ISO-C 16 [Ile 7 ]surfactin(1) and iso-C 14 [MeGlu 1 ,Ile 7 Surfactin (2) has surface activity and plant-induced systemic disease resistance activity, and can be used as an active ingredient in biological drugs or biocontrol agents, and surfactants.

6. A use of the Bacillus atrophaeus NB65 according to claim 1, characterized in that: The strain is Bacillus atrophaeus NB65, which has been deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on May 25, 2023, with the deposit number CCTCC M 2023819.

7. The use according to claim 6, characterized in that: The bacteria, spores and fermentation liquid of Bacillus atrophaeus NB65 can be used as active ingredients in biopharmaceuticals, biocontrol agents and surfactants, especially for, but not limited to, controlling corn leaf blight caused by Exserohilum turcicum and corn stalk rot caused by Fusarium verticillium and Fusarium graminearum.

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