Novel polymyxin compound as well as preparation method and application thereof

Abstract

The invention aims to provide a novel polymyxin compound as well as a preparation method and application thereof. The compound provided by the invention is shown as a formula (IV), r1 is (CH3) 2CH or CH3CH2CH (CH3) or CH3CH2CH2, and R2 is CH3CH2CH3 or CH3CH2CH2; and R2 is H or CH. The invention also protects the application of the compound or the pharmaceutically acceptable salt thereof in preparation of a bacterial inhibitor. The invention further provides the paenibacillus polymyxa CPCC 101041 and the application of the paenibacillus polymyxa. The compound is obtained by being separated from a fermentation product of paenibacillus polymyxa CPCC 101041. The invention further discloses a preparation method of the compound. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a new polymyxin compound and a preparation method and application thereof. Background Art

[0002] Paenibacillus polymyxa is a spore-forming Gram-positive bacterium belonging to the genus Paenibacillus of the family Bacillaceae. Paenibacillus polymyxa is widely distributed in nature and is commonly found in soil, water, plant surfaces, roots, stems and other habitats. It is a common endophytic bacterium in plants. Paenibacillus polymyxa is not pathogenic to humans, animals and plants. It is one of the commercially applicable microorganisms announced by the U.S. Environmental Protection Agency and is also a first-level strain exempted from safety identification announced by the Ministry of Agriculture and Rural Affairs of my country.

[0003] Paenibacillus polymyxa can produce a variety of physiologically active substances, such as antimicrobial peptides, enzymes, flocculants, and plant hormones. Polymyxin is a cationic antimicrobial peptide produced by Paenibacillus polymyxa. The most studied mechanism of action of polymyxins is that their cations and fatty acid side chains bind to the lipopolysaccharide and phospholipids of the outer membrane of Gram-negative bacteria, thereby destroying the integrity of the outer membrane, increasing the permeability of the cell membrane, and causing bacterial death. In addition, there are also reports that the vesicle contact pathway causes bacterial swelling and dissolution, oxidative stress response, and inhibition of the respiratory chain of the bacterial inner membrane. After entering this century, with the widespread emergence of multidrug-resistant Gram-negative bacteria and the lack of effective therapeutic drugs in clinical practice, polymyxin has been valued in clinical application and is regarded as the last line of defense against Gram-negative bacterial infections. Summary of the invention

[0004] The purpose of the present invention is to provide a novel polymyxin compound and a preparation method and application thereof.

[0005] The novel polymyxin compound provided by the present invention is as shown in formula (IV);

[0006]

[0007] R1 is (CH3)2CH or CH3CH2CH(CH3) or CH3CH2CH2 or (CH3)2CHCH2 or CH3CH2;

[0008] R2 is H or CH3.

[0009] Specifically, the compound is represented by formula (I), formula (II) or formula (III).

[0010]

[0011] The present invention also protects the use of any of the above compounds or pharmaceutically acceptable salts thereof in the preparation of bacterial inhibitors.

[0012] The present invention also protects a bacterial inhibitor, which contains any of the above compounds or pharmaceutically acceptable salts thereof.

[0013] The present invention also provides a strain of Paenibacillus polymyxa, namely Paenibacillus polymyxa (Paenibacillus polymyxa) CPCC 101041, which was deposited on September 21, 2023 in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and the deposit registration number is CGMCC No. 28502.

[0014] The present invention also protects a bacterial agent, which comprises the Paenibacillus polymyxa and / or a culture of the Paenibacillus polymyxa and / or a metabolite of the Paenibacillus polymyxa.

[0015] The culture of Paenibacillus polymyxa is a substance obtained by culturing Paenibacillus polymyxa CPCC 101041. The culturing may be specifically carried out by culturing in a culture medium. The culture medium may specifically be a microbial culture medium. The culture medium may specifically be a fermentation culture medium. The culture may specifically be a fermentation product obtained by fermenting Paenibacillus polymyxa CPCC 101041 in a culture medium. When the culture medium is a liquid culture medium, the fermentation product is a fermentation liquid. When the culture medium is a solid culture medium, the fermentation product is a solid fermentation product. The fermentation product may include Paenibacillus polymyxa CPCC 101041 and substances secreted into the culture medium during the culturing process of Paenibacillus polymyxa CPCC 101041.

[0016] The metabolite of Paenibacillus polymyxa can be a fermentation liquid or a fermentation product of Paenibacillus polymyxa CPCC 101041. The fermentation liquid of Paenibacillus polymyxa CPCC 101041 can be prepared according to the following method: culturing Paenibacillus polymyxa CPCC 101041 in a liquid fermentation medium, and collecting the fermentation liquid (containing Paenibacillus polymyxa CPCC 101041 and substances secreted into the liquid medium). The fermentation product of Paenibacillus polymyxa CPCC 101041 can be prepared according to the following method: culturing Paenibacillus polymyxa CPCC 101041 in a solid fermentation medium, and collecting the fermentation product (containing Paenibacillus polymyxa CPCC 101041 and substances secreted into the solid medium).

[0017] The active ingredients of the above-mentioned bacterial agent may also contain other biological components or non-biological components. Those skilled in the art may determine the other active ingredients of the above-mentioned bacterial agent based on the effects of the bacterial agent.

[0018] In the above-mentioned bacterial agent, the bacterial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0019] The above-mentioned bacterial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0020] The present invention also protects the use of Paenibacillus polymyxa CPCC 101041 in preparing any of the above compounds.

[0021] The present invention also protects the use of Paenibacillus polymyxa CPCC 101041 or / and the bacterial agent in preparing products.

[0022] The present invention also protects a product, which contains Paenibacillus polymyxa CPCC 101041 and / or the bacterial agent.

[0023] The present invention also protects a method for preparing a product, comprising the following steps: using Paenibacillus polymyxa CPCC101041 or / and the bacterial agent as components of the product to obtain the product.

[0024] Any of the above products is the following (a) and / or (b) and / or (c): (a) bacterial inhibitor; (b) agricultural fertilizer; (c) biological pesticide.

[0025] The invention also protects a preparation method of the compound, comprising the following steps: fermenting and culturing Paenibacillus polymyxa CPCC 101041 to obtain the compound.

[0026] Specifically, the preparation method comprises the following steps: fermenting and culturing Paenibacillus polymyxa CPCC101041 with a culture medium to obtain a fermentation product, and obtaining the compound from the fermentation product.

[0027] The culture medium may specifically be a microbial culture medium.

[0028] The culture medium may specifically be a fermentation medium.

[0029] The method for culturing Paenibacillus polymyxa CPCC 101041 by fermentation with a culture medium specifically comprises the following steps:

[0030] ① Inoculate Paenibacillus polymyxa CPCC 101041 into the fermentation medium and culture by shaking;

[0031] ② After completing step ①, add macroporous adsorption resin into the system, shake and culture, and obtain the fermentation product.

[0032] The method for culturing Paenibacillus polymyxa CPCC 101041 by fermentation with a culture medium specifically comprises the following steps:

[0033] ① Inoculate Paenibacillus polymyxa CPCC 101041 into the fermentation medium and culture with shaking for 48 hours;

[0034] ② After completing step ①, add macroporous adsorption resin into the system, shake and culture for 4 hours to obtain the fermentation product.

[0035] The method for culturing Paenibacillus polymyxa CPCC 101041 by fermentation with a culture medium specifically comprises the following steps:

[0036] ① Inoculate 100 mL of Paenibacillus polymyxa CPCC 101041 seed solution into 1 L of fermentation medium and culture with shaking for 48 h;

[0037] ② After completing step ①, add 100 mL of macroporous adsorption resin into the system and shake culture for 4 hours to obtain the fermentation product.

[0038] The method for culturing Paenibacillus polymyxa CPCC 101041 by fermentation with a culture medium specifically comprises the following steps:

[0039] ① Inoculate 100 mL of Paenibacillus polymyxa CPCC 101041 seed solution into 1 L of fermentation medium and culture at 30°C and 200 r / min for 48 h;

[0040] ② After completing step ①, add 100 mL of macroporous adsorption resin into the system, shake and culture at 30° C. and 200 r / min for 4 h to obtain a fermentation product.

[0041] The method for preparing any of the above seed solutions comprises the following steps: picking a single colony of Paenibacillus polymyxa CPCC101041, inoculating it into a seed culture medium, and culturing it by shaking to obtain a seed solution.

[0042] The method for preparing any of the above seed solutions comprises the following steps: picking a single colony of Paenibacillus polymyxa CPCC101041, inoculating it into a seed culture medium, and shaking and culturing for 16 hours to obtain a seed solution.

[0043] The method for preparing any of the above seed solutions comprises the following steps: picking a single colony of Paenibacillus polymyxa CPCC101041, inoculating it into 100 mL of seed culture medium, and shaking and culturing at 30° C. and 200 r / min for 16 hours to obtain seed solution.

[0044] Specifically, any of the above seed culture media (TSB culture media) may contain 15 g / L tryptone, 5 g / L soy peptone and 5 g / L sodium chloride, with the remainder being water; and the pH value is 7.0-7.4.

[0045] Specifically, any of the above fermentation medium may contain: 30 g / L corn flour, 10 g / L soluble starch, 1 g / L yeast powder, 5 g / L ammonium sulfate and 10 g / L calcium carbonate, with the remainder being water; and the pH value is 7.0-7.4.

[0046] The method for obtaining the compound from the fermentation product comprises the following steps:

[0047] (1) taking the fermentation product, collecting the macroporous adsorption resin and transferring it to a chromatography column, then eluting the chromatography column with deionized water, 20% methanol aqueous solution and 50% methanol aqueous solution in sequence, and collecting the eluate after passing through the column when eluting with 50% methanol aqueous solution;

[0048] (2) taking the post-column eluate obtained in step (1), loading the solid sample and performing chromatographic separation and purification to obtain a C6 fraction and a C7 fraction;

[0049] (3) taking the C6 fraction obtained in step (2), loading the solid sample and performing chromatographic separation and purification to obtain the D5 fraction;

[0050] (4) taking the D5 fraction obtained in step (3), loading the solid sample and performing chromatographic separation and purification to obtain the E4 fraction;

[0051] (5) taking the E4 fraction obtained in step (4), separating and purifying it by liquid chromatography to obtain the compound represented by formula (I);

[0052] (6) taking the C7 fraction obtained in step (2), loading the solid sample and performing chromatographic separation and purification to obtain the D11 fraction;

[0053] (7) The D11 fraction obtained in step (6) is separated and purified by liquid chromatography to obtain the compound represented by formula (II) and the compound represented by formula (III).

[0054] The step (1) is as follows: taking 100 L of the fermentation product, collecting the macroporous adsorption resin and transferring it to a chromatography column (column volume 2 L), then eluting the chromatography column with 10 L of deionized water, 20 L of 20% methanol aqueous solution and 30 L of 50% methanol aqueous solution in sequence, and collecting the eluate after passing through the column when eluting with 50% methanol aqueous solution.

[0055] In step (1), the elution flow rate is 2 times column volume / h.

[0056] The step (2) is: taking the post-column eluate collected in step (1), concentrating it, then adding the swollen YMCODS-AQ-HG filler and mixing well, then concentrating and evaporating to obtain a solid sample; filling the solid sample into a sample column, and then connecting it to a chromatographic column for separation and purification.

[0057] The step (2) comprises: taking the eluate collected in step (1), concentrating it to 100 ml using a rotary evaporator, adding 10 ml of swollen YMC ODS-AQ-HG filler, mixing well, concentrating and evaporating to dryness to obtain a solid sample; filling the solid sample into a sample column, and then connecting the solid sample to a chromatographic column for separation and purification.

[0058] In the step (2), the chromatographic column is Flash Spherical C18; 20-35 μm; 100A; 120 g.

[0059] In the step (2), the chromatograph is a Combiflash Rf200 preparative chromatograph produced by TELEDYNE ISCO, USA.

[0060] In the step (2), the elution procedure is as follows: from 0 to 20 min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; from 20 to 60 min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 40%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 60%; from 60 to 80 min, the mobile phase consists of 40% mobile phase A and 60% mobile phase B; from 80 to 100 min, the volume fraction of mobile phase A in the mobile phase increases linearly from 40% to 100%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 60% to 0%; from 100 to 120 min, the mobile phase is entirely mobile phase A.

[0061] Mobile phase A: consists of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.04%.

[0062] Mobile phase B: consists of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.04%.

[0063] In the step (2), the flow rate of the mobile phase is 30 mL / min.

[0064] In the step (2), the detection wavelength is 210 nm.

[0065] The C6 fraction corresponds to the elution peak with a retention time of 32-45 min.

[0066] The C7 fraction corresponds to the elution peak with a retention time of 45-52 min.

[0067] The step (3) is: taking the C6 fraction, concentrating it, then adding the swollen YMC ODS-AQ-HG filler, and then evaporating to dryness to obtain a solid sample; filling the solid sample into a sample column, and then connecting it to a chromatographic column for separation and purification.

[0068] The step (3) is as follows: taking the C6 fraction, concentrating it to 100 ml by rotary evaporation, then adding 10 ml of swollen YMC ODS-AQ-HG filler and mixing well, concentrating and evaporating to dryness to obtain a solid sample; filling the solid sample into a sample column, and then connecting it to a chromatographic column for separation and purification.

[0069] In the step (3), the chromatographic column is Flash Spherical C18; 20-35 μm; 100A; 80 g.

[0070] In the step (3), the chromatograph is a Combiflash Rf200 preparative chromatograph produced by TELEDYNE ISCO, USA.

[0071] In the step (3), the elution procedure is as follows: from 0 to 30 min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; from 30 to 60 min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 30%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 70%; from 60 to 90 min, the mobile phase consists of 30% mobile phase A and 70% mobile phase B; from 90 to 100 min, the volume fraction of mobile phase A in the mobile phase increases linearly from 30% to 100%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 70% to 0%; from 100 to 120 min, the mobile phase is entirely mobile phase A.

[0072] Mobile phase A: consists of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.04%.

[0073] Mobile phase B: consists of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.04%.

[0074] In the step (3), the flow rate of the mobile phase is 30 mL / min.

[0075] In the step (3), the detection wavelength is 210 nm.

[0076] The D5 fraction corresponds to the elution peak with a retention time of 41-46 min.

[0077] The step (4) is: taking the D5 fraction, concentrating it, then adding the swollen YMC ODS-AQ-HG filler, and then evaporating to dryness to obtain a solid sample; filling the solid sample into a sample column, and then connecting it to a chromatographic column for separation and purification.

[0078] The step (4) is as follows: taking the D5 fraction, concentrating it to 100 ml by rotary evaporation, then adding 5 ml of swollen YMCODS-AQ-HG filler and mixing well, concentrating and evaporating to dryness to obtain a solid sample; filling the solid sample into a sample column, and then connecting it to a chromatographic column for separation and purification.

[0079] In the step (4), the chromatographic column is Flash Spherical C18; 20-35 μm; 100A; 80 g.

[0080] In the step (4), the chromatograph is a Combiflash Rf200 preparative chromatograph produced by TELEDYNE ISCO, USA.

[0081] In the step (4), the elution procedure is as follows: 0-20 min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; 20-90 min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 20%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 80%; 90-100 min, the mobile phase is entirely mobile phase A.

[0082] Mobile phase A: composed of formic acid and acetonitrile, with a formic acid concentration of 0.15%.

[0083] Mobile phase B: consists of formic acid and water, with a formic acid concentration of 0.15%.

[0084] In the step (4), the flow rate of the mobile phase is 30 mL / min.

[0085] In the step (4), the detection wavelength is 210 nm.

[0086] The E4 fraction corresponds to the elution peak with a retention time of 52-63 min.

[0087] The step (5) is as follows: using a rotary evaporator to concentrate and evaporate the E4 fraction, dissolving it in an acetonitrile aqueous solution to obtain an E4 concentrate, and separating and purifying it by liquid chromatography to obtain the compound represented by formula (I).

[0088] The step (5) is as follows: using a rotary evaporator to concentrate and evaporate the E4 fraction, dissolving it in 5 ml of 20% acetonitrile aqueous solution to obtain the E4 concentrate, and separating and purifying it by liquid chromatography to obtain the compound represented by formula (I).

[0089] In the step (5), the chromatographic column is Japanese YMC-Pack ODS-AQ, 10 mm*250 mm, with a pore size of 5 μm.

[0090] In the step (5), the chromatograph is Shimadzu LC-20AD.

[0091] In the step (5), the mobile phase contains 0.15% formic acid and 10% acetonitrile, and the balance is water.

[0092] In the step (5), the flow rate of the mobile phase is 3.5 mL / min.

[0093] In the step (5), the detection wavelength is 210 nm.

[0094] The retention time corresponding to the peak value of the target peak (t R ):7.8min.

[0095] The solution of the target peak after passing through the column is collected, concentrated and evaporated to dryness using a rotary evaporator to obtain the compound represented by formula (I).

[0096] The step (6) is: taking the C7 fraction, concentrating it, then adding the swollen YMC ODS-AQ-HG filler, and then evaporating to dryness to obtain a solid sample; filling the solid sample into a sample column, and then connecting it to a chromatographic column for separation and purification.

[0097] The step (6) is as follows: taking the C7 fraction, concentrating it to 100 ml by rotary evaporation, then adding 10 ml of swollen YMC ODS-AQ-HG filler and mixing well, concentrating and evaporating to dryness to obtain a solid sample; filling the solid sample into a sample column, and then connecting it to a chromatographic column for separation and purification.

[0098] In the step (6), the chromatographic column is Flash Spherical C18; 20-35 μm; 100A; 80 g.

[0099] In the step (6), the chromatograph is a Combiflash Rf200 preparative chromatograph produced by TELEDYNE ISCO, USA.

[0100] In the step (6), the elution procedure is as follows: 0-20 min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; 20-90 min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 20%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 80%; 90-100 min, the mobile phase is entirely mobile phase A.

[0101] Mobile phase A: composed of formic acid and acetonitrile, with a formic acid concentration of 0.15%.

[0102] Mobile phase B: consists of formic acid and water, with a formic acid concentration of 0.15%.

[0103] In the step (6), the flow rate of the mobile phase is 30 mL / min.

[0104] In the step (6), the detection wavelength is 210 nm.

[0105] The D11 fraction corresponds to the elution peak with a retention time of 72-77 min.

[0106] The step (7) is: using a rotary evaporator to concentrate and evaporate the D11 fraction, dissolving it in an acetonitrile aqueous solution to obtain a D11 concentrate, and separating and purifying it by liquid chromatography to obtain the compound represented by formula (II) and the compound represented by formula (III).

[0107] The step (7) is as follows: using a rotary evaporator to concentrate and evaporate the D11 fraction, dissolving it in 8 ml of 20% acetonitrile aqueous solution to obtain D11 concentrate, and separating and purifying it by liquid chromatography to obtain the compound represented by formula (II) and the compound represented by formula (III).

[0108] In the step (7), the chromatographic column is Japanese YMC-Pack ODS-AQ, 10 mm*250 mm, with a pore size of 5 μm.

[0109] In the step (7), the chromatograph is Shimadzu LC-20AD.

[0110] In the step (7), the mobile phase contains 0.04% trifluoroacetic acid and 19% acetonitrile, and the balance is water.

[0111] In the step (7), the flow rate of the mobile phase is 3.5 mL / min.

[0112] In the step (7), the detection wavelength is 210 nm.

[0113] The retention time corresponding to the peak value of the first target peak (t R ):14.5min.

[0114] The solution of the first target peak after passing through the column is collected, concentrated and evaporated to dryness using a rotary evaporator to obtain the compound represented by formula (II).

[0115] The retention time corresponding to the peak value of the second target peak (t R ):16.1min.

[0116] The solution of the second target peak after passing through the column is collected, concentrated and evaporated to dryness using a rotary evaporator to obtain the compound represented by formula (III).

[0117] Any of the above mentioned bacteria may be Gram-negative bacteria or Gram-positive bacteria.

[0118] Any of the above-mentioned bacteria may be sensitive bacteria or resistant bacteria.

[0119] The Gram-negative bacteria include, but are not limited to, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Enterobacter aerogenes, Salmonella typhi, Serratia marcescens, Citrobacter freundii, Proteus raderii, Proteus vulgaris, Proteus mirabilis, Stenotrophomonas maltophilia or Shigella flexneri.

[0120] Any of the above mentioned bacteria may specifically be the bacteria listed in Table 4.

[0121] In the above, the bacterial inhibitor, in addition to containing the compound of the general formula or its pharmaceutically acceptable salt, may also contain a suitable carrier or excipient. The carrier material here includes but is not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred. Using these materials, a variety of dosage forms can be made, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc.In addition, in order to prepare isotonic injections, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. can also be added. In addition, if necessary, colorants, preservatives, spices, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal; respiratory tract administration, such as nasal cavity; mucosal administration.

[0122] Paenibacillus polymyxa CPCC 101041 is isolated from the medicinal plant Thalictrum sphaerocephalum, and the fermentation product of the strain has obvious antibacterial activity against multiple drug-resistant bacteria. The inventors conducted liquid fermentation of Paenibacillus polymyxa CPCC 101041, and separated and identified three new polymyxin compounds (101041-3, 101041-4 and 101041-5) from the liquid fermentation product by using separation methods such as macroporous adsorption resin chromatography, ODS column chromatography and liquid phase preparation, combined with physical and chemical determination and spectroscopic identification methods, which have obvious antibacterial activity against multiple Gram-negative drug-resistant bacteria. DETAILED DESCRIPTION

[0123] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0124] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0125] The macroporous adsorption resin is DIAION HP20 macroporous adsorption resin (a product of Mitsubishi Chemical Corporation, Japan); DIAION HP20 macroporous adsorption resin is a non-polar macroporous adsorption resin with a water content (%) of 55-65, a wet apparent density (g / LR) of 680, a pore volume (ml / g) of 1.3, and a specific surface area (m 2 / g) is 590, good frequency radius 290, particle size distribution (≥0.25mm) ≥90%, effective particle size (mm) ≥0.25, uniformity coefficient ≤1.6, use temperature (℃) ≤130. YMC ODS-AQ-HG filler: product of Japan YMC Co., Ltd., item number AQG12S50.

[0126] Unless otherwise specified, % in the examples refers to volume percentage.

[0127] Example 1. Acquisition, identification and preservation of Paenibacillus polymyxa CPCC 101041

[0128] 1. Isolation of strains

[0129] The strain was isolated from the root tissue of Thalictrum speciosissimum Wall. Thalictrum speciosissimum was collected from Yadong County, Shigatse City, Tibet Autonomous Region. The surface-sterilized tissue was crushed under aseptic conditions, inoculated into a starch inorganic salt separation medium, and cultured at 28°C for 21 days. Colonies were picked from the culture medium and streaked to purify and obtain pure culture strains, one of which was named strain CPCC 101041.

[0130] 2. Identification of strains

[0131] The identification of morphological and physiological and biochemical characteristics was carried out according to the conventional methods, referring to the "Bergey's Manual of Systematic Bacteriology" (Garrity, 2001) and "Common Identification Methods of General Bacteriology" (Dong Xiuzhu et al., 2001). The morphological characteristics of strain CPCC 101041: the colonies are smooth, sticky, convex, milky white, and the colonies adhere to the surface of the culture medium without soluble pigments; under the microscope, the bacteria are rod-shaped, (2.4-3.2)×(0.8-1.0)μm, and the spores are spindle-shaped, 3.4×1.6μm; Gram staining is positive. Physiological and biochemical characteristics of strain CPCC101041: it can decompose lactose, galactose, maltose, mannitol, and mannose, and produce acid and gas; it can decompose glucose to produce acid but not gas.

[0132] The bacterial cells of CPCC 101041 were collected, genomic DNA was extracted, 16s rDNA was amplified by PCR reaction, the amplified product was recovered and purified, sequenced, and then sequenced in the GenBank database. The amplified product is shown in Sequence 1 of the sequence table, and has a similarity of 99.57% with Paenibacillus polymyxa ATCC 842.

[0133] Based on the morphological, physiological and biochemical characteristics and 16s rDNA sequence homology analysis results, strain CPCC 101041 belongs to Paenibacillus polymyxa.

[0134] 3. Preservation of strains

[0135] Paenibacillus polymyxa (Paenibacillus polymyxa) CPCC 101041 was deposited on September 21, 2023 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with the deposit registration number CGMCC No.28502.

[0136] Example 2: Fermentation of Paenibacillus polymyxa CPCC 101041 to prepare compounds

[0137] 1. Cultivation and fermentation

[0138] Seed culture medium (TSB medium): contains 15 g / L tryptone, 5 g / L soy peptone and 5 g / L sodium chloride, the balance is water; pH is 7.0-7.4; sterilize at 121°C for 15 min.

[0139] Fermentation medium: contains 30g / L corn flour, 10g / L soluble starch, 1g / L yeast powder, 5g / L ammonium sulfate and 10g / L calcium carbonate, the balance is water; pH is 7.0-7.4, sterilize at 121℃ for 15min.

[0140] 1. Pick a single colony of Paenibacillus polymyxa CPCC 101041 and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium. Incubate at 30 °C and 200 r / min for 16 h to obtain seed solution.

[0141] 2. Inoculate 100 mL of the seed solution obtained in step 1 into a 5 L Erlenmeyer flask containing 1 L of fermentation medium and culture at 30 °C and 200 r / min for 48 h.

[0142] 3. After completing step 2, add 100 mL of macroporous adsorption resin sterilized by wet heat into the system, and culture at 30°C and 200 r / min for 4 h to obtain the fermentation product.

[0143] Multiple repeated treatments were performed simultaneously to obtain a total of 100 L of fermentation product.

[0144] 2. Separation and purification of compounds

[0145] 1. Take 100L of the fermentation product from step 1, pass it through a 60-mesh sieve, and repeatedly rinse the macroporous adsorption resin in the sieve with water until the washing liquid is clear. Then, transfer the macroporous adsorption resin to a chromatography column (column volume 2L). Then, elute the chromatography column with 10L of deionized water, 20L of 20% methanol aqueous solution, 30L of 50% methanol aqueous solution, and 30L of 80% methanol aqueous solution in sequence, with a flow rate of 2 times column volume / h. Collect the eluate (about 30L) after the column is eluted with 50% methanol aqueous solution.

[0146] 2. The post-column eluate (about 30 L) collected in step 1 was rotary evaporated to about 100 ml, and then 10 mL of swollen YMC ODS-AQ-HG filler was added and mixed well. The solid sample was obtained after concentration and evaporation.

[0147] 3. Fill the solid sample obtained in step 2 into the sample column, and then connect it to the chromatographic column for separation and purification.

[0148] Chromatographic column: Flash Spherical C18; 20-35μm; 100A; 120g.

[0149] Chromatograph: Combiflash Rf200 preparative chromatograph from TELEDYNE ISCO, USA.

[0150] Mobile phase A: composed of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.04%. Mobile phase B: composed of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.04%. Mobile phase flow rate: 30 mL / min.

[0151] Detection wavelength: 210nm.

[0152] Elution program: 0-20min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; 20-60min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 40%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 60%; 60-80min, the mobile phase consists of 40% mobile phase A and 60% mobile phase B; 80-100min, the volume fraction of mobile phase A in the mobile phase increases linearly from 40% to 100%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 60% to 0%; 100-120min, the mobile phase is all mobile phase A.

[0153] A total of 12 fractions were obtained during the entire elution process (each fraction corresponds to a post-column solution of an elution peak, and the 12 fractions are named C1 to C12 in sequence), among which C6 corresponds to an elution peak with a retention time of 32-45 min, and C7 corresponds to an elution peak with a retention time of 45-52 min.

[0154] The C6 fraction was rotary evaporated to about 100 ml, and then 10 mL of swollen YMC ODS-AQ-HG filler was added and mixed well. After concentration and evaporation, a solid sample (named C6 solid sample) was obtained.

[0155] The C7 fraction was rotary evaporated to about 100 ml, and then 10 mL of swollen YMC ODS-AQ-HG filler was added and mixed well. After concentration and evaporation, a solid sample (named C7 solid sample) was obtained.

[0156] 4. Fill the C6 solid sample into the sample column and then connect it to the chromatographic column for separation and purification.

[0157] Chromatographic column: Flash Spherical C18; 20-35μm; 100A; 80g.

[0158] Chromatograph: Combiflash Rf200 preparative chromatograph from TELEDYNE ISCO, USA.

[0159] Mobile phase A: composed of trifluoroacetic acid and acetonitrile, with a trifluoroacetic acid concentration of 0.04%. Mobile phase B: composed of trifluoroacetic acid and water, with a trifluoroacetic acid concentration of 0.04%. Mobile phase flow rate: 30 mL / min.

[0160] Detection wavelength: 210nm.

[0161] Elution program: 0-30min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; 30-60min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 30%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 70%; 60-90min, the mobile phase consists of 30% mobile phase A and 70% mobile phase B; 90-100min, the volume fraction of mobile phase A in the mobile phase increases linearly from 30% to 100%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 70% to 0%; 100-120min, the mobile phase is all mobile phase A.

[0162] A total of 7 fractions were obtained during the entire elution process (each fraction was a post-column solution corresponding to an elution peak, and the 7 fractions were named D1 to D7 in sequence), among which D5 corresponded to an elution peak with a retention time of 41-46 min.

[0163] The D5 fraction was rotary evaporated to about 100 ml, and then 5 mL of swollen YMC ODS-AQ-HG filler was added and mixed well. After concentration and evaporation, a solid sample (named as D5 solid sample) was obtained.

[0164] 5. Fill the D5 solid sample into the sample column and then connect it to the chromatographic column for separation and purification.

[0165] Chromatographic column: Flash Spherical C18; 20-35μm; 100A; 80g.

[0166] Chromatograph: Combiflash Rf200 preparative chromatograph from TELEDYNE ISCO, USA.

[0167] Mobile phase A: composed of formic acid and acetonitrile, formic acid concentration is 0.15%. Mobile phase B: composed of formic acid and water, formic acid concentration is 0.15%. Mobile phase flow rate: 30mL / min.

[0168] Detection wavelength: 210nm.

[0169] Elution program: 0-20min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; 20-90min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 20%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 80%; 90-100min, the mobile phase is all mobile phase A.

[0170] A total of 10 fractions were obtained during the entire elution process (each fraction was a post-column solution corresponding to an elution peak, and the 10 fractions were named E1 to E10 in sequence), among which E4 corresponded to an elution peak with a retention time of 52-63 min.

[0171] The E4 fraction was concentrated and evaporated to dryness using a rotary evaporator, and dissolved in 5 ml of a 20% acetonitrile aqueous solution to obtain the E4 concentrate.

[0172] 6. Take 5 ml of E4 concentrate and separate and purify it using HPLC.

[0173] Chromatographic column: Japan YMC-Pack ODS-AQ, 10mm*250mm, pore size 5μm.

[0174] Chromatograph: Shimadzu LC-20AD.

[0175] Mobile phase: contains 0.15% formic acid and 10% acetonitrile, the balance is water; mobile phase flow rate: 3.5mL / min.

[0176] Detection wavelength: 210nm.

[0177] The retention time corresponding to the peak value of the target peak (t R ):7.8min.

[0178] The solution of the target peak after passing through the column was collected, concentrated and evaporated to dryness using a rotary evaporator to obtain compound 101041-3.

[0179] 4.7 mg of compound 101041-3 was obtained.

[0180] 7. Fill the C7 solid sample into the sample column and then connect it to the chromatographic column for separation and purification.

[0181] Chromatographic column: Flash Spherical C18; 20-35μm; 100A; 80g.

[0182] Chromatograph: Combiflash Rf200 preparative chromatograph from TELEDYNE ISCO, USA.

[0183] Mobile phase A: composed of formic acid and acetonitrile, formic acid concentration is 0.15%. Mobile phase B: composed of formic acid and water, formic acid concentration is 0.15%. Mobile phase flow rate: 30mL / min.

[0184] Detection wavelength: 210nm.

[0185] Elution program: 0-20min, the mobile phase consists of 10% mobile phase A and 90% mobile phase B; 20-90min, the volume fraction of mobile phase A in the mobile phase increases linearly from 10% to 20%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 90% to 80%; 90-100min, the mobile phase is all mobile phase A.

[0186] A total of 12 fractions were obtained during the entire elution process (each fraction was a post-column solution corresponding to an elution peak, and the 12 fractions were named D8 to D19 in sequence), among which D11 corresponded to an elution peak with a retention time of 72-77 min.

[0187] The D11 fraction was concentrated and evaporated to dryness using a rotary evaporator, and dissolved in 8 ml of a 20% acetonitrile aqueous solution to obtain the D11 concentrate.

[0188] 8. Take 8 ml of D11 concentrate and separate and purify it using HPLC.

[0189] Chromatographic column: Japan YMC-Pack ODS-AQ, 10mm*250mm, pore size 5μm.

[0190] Chromatograph: Shimadzu LC-20AD.

[0191] Mobile phase: contains 0.04% trifluoroacetic acid and 19% acetonitrile, the balance is water; mobile phase flow rate: 3.5mL / min.

[0192] Detection wavelength: 210nm.

[0193] The retention time corresponding to the peak value of the first target peak (t R ):14.5min.

[0194] The solution of the first target peak after passing through the column was collected and concentrated and evaporated to dryness using a rotary evaporator to obtain compound 101041-4.

[0195] 6.7 mg of compound 101041-4 was obtained.

[0196] The retention time corresponding to the peak value of the second target peak (t R ):16.1min.

[0197] The solution of the second target peak after passing through the column was collected and concentrated and evaporated to dryness using a rotary evaporator to obtain compound 101041-5.

[0198] 6.9 mg of compound 101041-5 was obtained.

[0199] 3. Identification of Compounds

[0200] 1. 13 C-NMR spectrum and 1 H-NMR spectrum

[0201] The three compounds prepared in step 2 (compound 101041-3, compound 101041-4, compound 101041-5) were dissolved in DMSO-d6 and subjected to 600 MHz 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, NOESY, HSQC and HMBC NMR spectra analysis 13 C-NMR spectrum and 1 The attributions of the peaks in the H-NMR spectrum are shown in Tables 1, 2 and 3.

[0202] Table 1 Compound 101041-3 13 C-NMR and 1 H-NMR spectrum peaks attributed (DMSO-d6, 600MHz)

[0203]

[0204] Table 2 Compound 101041-4 13 C-NMR and 1 H-NMR spectrum peaks attributed (DMSO-d6, 600MHz)

[0205]

[0206] Table 3 Compound 101041-5 13 C-NMR and 1 H-NMR spectrum peaks attributed (DMSO-d6, 600MHz)

[0207]

[0208]

[0209] 2. Marfey method to determine the stereo configuration of compounds

[0210] ① Weigh 50 μg of the compound prepared in step 2 (compound 101041-3, compound 101041-4, compound 101041-5), dissolve in 500 μL 6M hydrochloric acid solution, heat in a metal bath at 100°C for 2 h, and concentrate under reduced pressure to dryness to obtain the acid hydrolyzate.

[0211] ② Dissolve the acid hydrolyzate in 50 μL pure water, add 20 μL 1M NaHCO3 solution and 100 μL 0.5% 2,4-dinitrofluorobenzene solution (solvent: acetone) in sequence, mix well and react in a metal bath at 40°C for 50 min.

[0212] ③After completing step ②, cool to room temperature, add 20 μL 1M HCl solution to neutralize to neutrality, concentrate and evaporate to dryness under reduced pressure to obtain an amino acid derivative product.

[0213] ④ The amino acid derivative product was dissolved in 100 μL of 50% acetonitrile aqueous solution and analyzed by HPLC.

[0214] The chromatographic column is: Japanese YMC-Pack ODS-AQ, 10 mm*250 mm, pore size 5 μm.

[0215] The chromatograph is: DIONEX Ultimate 3000 from the United States.

[0216] Detection wavelength: 210nm.

[0217] The mass spectrometer is: Thermo Scientific LTQ XL, USA.

[0218] Mobile phase A: composed of formic acid and acetonitrile, formic acid concentration is 0.15%. Mobile phase B: composed of formic acid and water, formic acid concentration is 0.15%. Mobile phase flow rate: 1mL / min; split flow 0.3mL / min into the mass spectrometer.

[0219] Elution program: 0-60min, the volume fraction of mobile phase A in the mobile phase increases linearly from 25% to 85%, and the corresponding volume fraction of mobile phase B in the mobile phase decreases linearly from 75% to 15%.

[0220] Polymyxin B1 (containing 6 L-Dab residues, 2 L-Thr residues, 1 D-Phe residue, and 1 L-Leu residue) and polymyxin B1-type (containing 5 L-Dab residues, 1 D-Dab residue, 2 L-Thr residues, 1 D-Phe residue, and 1 L-Leu residue) were used as references. The retention times of the amino acid derivatives were: L-Thr (m / z 414 [M+H] + , t R 16.7min)、L-Ser(m / z 398[M+H]+ , t R 17.0min), L-Leu (m / z 426[M+H] + , t R 26.8min), L-Dab(m / z 707[2M+H] + , t R 35.2min)、D-Phe(m / z 460[M+H] + , t R 32.5min), D-Dab(m / z 707[2M+H] + , t R 37.4min).

[0221] Compounds 101041-3 and 101041-4 were subjected to acid hydrolysis and amino acid derivatization, and HPLC-MS / UV analysis showed that D-Phe, L-Ser, L-Thr, L-Dab, and D-Dab were present in their derivatives, and the peak area ratio of L-Dab to D-Dab was 5: 1. Compound 101041-5 was subjected to acid hydrolysis and amino acid derivatization, and HPLC-MS / UV analysis showed that D-Phe, L-Ser, L-Thr, L-Dab, and D-Dab were present in its derivatives, and the peak area ratio of L-Dab to D-Dab was 5: 1.

[0222] Combined with NMR analysis and Marfey stereochemical analysis, the three compounds prepared in step 2 were identified as new lipopeptide compounds, which are formed into a ring by a fatty acid and ten amino acids.

[0223] The structural formula of compound 101041-3 is shown in formula (Ⅰ).

[0224]

[0225] The structural formula of compound 101041-4 is shown in formula (II).

[0226]

[0227] The structure of compound 101041-5 is shown in formula (III).

[0228]

[0229] Example 3. In vitro antibacterial activity of compounds

[0230] Test compounds: the three compounds prepared in step 2 of Example 2 (compound 101041-3, compound 101041-4, compound 101041-5) and the positive control drug (colistin). Colistin: National Drug Standard Substance Query and Ordering Platform of China Food and Drug Inspection Institutes, item number 130327. Test bacteria: internationally recognized standard strains of drug-resistant bacteria and representative drug-resistant strains isolated clinically in recent years, see Table 4 for details.

[0231] The two-fold dilution method was used to conduct drug sensitivity test on the test compound. Specific method: dilute the test compound with MH broth medium by two times, then add 1ml of dilution to the plate, then add 14ml of melted MH agar medium and mix well, then let it stand at room temperature until a plate is formed; then inoculate the test bacteria on the plate (each plate can be inoculated with multiple points, evenly set without affecting each other, the inoculation amount of the test bacteria is about 10 4 cfu / point), cultured at 35°C for 18 h, and then observed and calculated the MIC value.

[0232] The results are shown in Table 4.

[0233] Table 4 In vitro antibacterial activity test results of compounds

[0234]

[0235] E.coli 08-85, E.coli 13-43, E.coli 13-68, K.pneumoniae 09-20 are recorded in the following documents: Low Prevalence of mcr-1Among Clinical Enterobacteriaceae Isolates and Co-transfer of mcr-1and blaNDM-1from Separate Donors; MICROBIAL DRUG RESISTANCEVolume 00,Number 00, 2020.

[0236] P. aeruginosa PAO1 is described in the following literature: Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains; JOURNAL OF BACTERIOLOGY, Feb. 2010, p. 1113–1121; Vol. 192, No. 4.

[0237] E. cloacae 1000654 (Enterobacter cloacae 1000654) is described in the following document: New Delhi Metallo-β-Lactamase–producing Enterobacteriaceae, United States; Emerging Infectious Diseases·www.cdc.gov / eid·Vol.19, No.6, June 2013.

[0238] S.typhi H901 is recorded in the following documents: Evaluation of Two Salmonella typhi Strains with Reduced Virulence for Use in Teaching and Proficiency Testing; JOURNAL OFCLINICAL MICROBIOLOGY, June 1982, p.1085-1091.

[0239] P. rettgeri ATCC 31052 is recorded in the following documents: The primary structure of Providencia rettgeri penicillin C amidase gene and its relationship to othergram negative amidases; DNA Sequence-J. DNA Sequencing and Mapping, Vol. 3, pp. 195-200.

[0240] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A compound as shown in formula (IV); R1 is (CH3)2CH or CH3CH2CH(CH3) or CH3CH2CH2 or (CH3)2CHCH2 or CH3CH2; R2 is H or CH3.

2. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a bacterial inhibitor.

3. A bacterial inhibitor comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

4. Paenibacillus polymyxa, wherein the Paenibacillus polymyxa is Paenibacillus polymyxa CPCC 101041, and its deposit registration number is CGMCC No.28502.

5. A bacterial agent comprising the Paenibacillus polymyxa according to claim 4 or / and a culture of the Paenibacillus polymyxa according to claim 4 or / and a metabolite of the Paenibacillus polymyxa according to claim 4.

6. Use of the Paenibacillus polymyxa according to claim 4 in the preparation of the compound according to claim 1.

7. Use of the Bacillus polymyxa described in claim 4 and / or the bacterial agent described in claim 5 in the preparation of products; the products are the following (a) and / or (b) and / or (c): (a) bacterial inhibitors; (b) agricultural fertilizers; (c) biological pesticides.

8. A product comprising the Paenibacillus polymyxa described in claim 4 and / or the bacterial agent described in claim 5; the product is the following (a) and / or (b) and / or (c): (a) a bacterial inhibitor; (b) an agricultural fertilizer; (c) a biological pesticide.

9. A method for preparing a product, comprising the following steps: using the Bacillus polymyxa described in claim 4 and / or the bacterial agent described in claim 5 as a component of the product to obtain the product; the product is the following (a) and / or (b) and / or (c): (a) bacterial inhibitor; (b) agricultural fertilizer; (c) biological pesticide.

10. A method for preparing the compound of claim 1, comprising the following steps: fermenting and culturing the Paenibacillus polymyxa of claim 4 to obtain the compound of claim 1.

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