Enshi L77 for producing polyether antibiotics and application of Enshi L77

Abstract

The invention relates to the technical field of fermentation engineering, and in particular relates to Enshi L77 for producing polyether antibiotics and application of the Enshi L77. The preservation number of the Ennuli L77 is CGMCC (China General Microbiological Culture Collection Center) No.36394. The The application comprises the following steps: fermenting by adopting the Enshi L77, extracting and separating from a fermentation product and thalli to obtain a polyether compound, and using the polyether compound as an antibiotic. According to the invention, a strain of Enshi L77 is obtained through research and screening, functional verification of the Enshi L77 shows that a polyether compound with relatively good antibacterial activity can be separated from a fermentation product of the Enshi L77, and the Enshi L77 can be used for preparing human or livestock anti-infection drugs and has a relatively good application prospect.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to Embryobacter L77, a polyether antibiotic producer, and its applications. Background Technology

[0002] Streptomycetaceae strains within the phylum Actinobacteria are a major source of natural antibiotics. With the application of genomics technology in microbial taxonomy, the classification system of actinomycetes has been revised and improved. In 2018, based on genomic data, researchers revised the Streptomycetaceae family, establishing a new genus—*Embrylium*. Embleya ), and originally belonged to the Streptomyces genus Streptomyces scabrisporus Revised to Embleya scabrispora Currently, the number of validly published species in the genus *Embryonicus* is extremely small, including only a limited number... Embleya scabrispora and Embleya hyalina Two species.

[0003] Polyether antibiotics are a class of ion carriers with important pharmacological activities. Their structural characteristic is the presence of one or more polyether fragments, enabling them to react with Na+. + Ca 2+ The binding of isocations promotes the transport of these ions across the cell membrane, thereby exerting biological effects. Calcimycin (also known as A23187) is a typical representative of this class of compounds, initially isolated from... Streptomyces chartreusis Structurally, cathinone is mainly composed of... α It consists of three core components: a ketopyrrole ring, a spirocyclic ketal ring, and a benzoxazole ring. The number of existing naturally derived cathinone analogs is limited, mainly including... N -demethyl calcimycin, cezomycin, AC7230, demethyl (C-11)cezomycin, X-14885A, routiennocin, precezomycin, and phytohabimicin, etc. Among them, precezomycin is a cezomycin derivative with an open oxazole ring, while phytohabimicin is a benzoxazole ring replaced by a thiazole ring.

[0004] Existing research indicates that cathinone compounds possess broad biological activities and significant application value. This is due to their ability to specifically chelate calcium... 2+ Mg 2+ Divalent metal cations are often used as tool drugs to study cell membrane biological properties and calcium.2+ Mediated biochemical pathway research. In terms of pharmacological activity, cassiopum and its analogues exhibit antibacterial, anti-insect, anti-tumor and other activities. For example, compound X-14885A has activity against pig dysentery spirochetes (Leptospira interrogans) (Leptospira interrogans) ; generally, such compounds are mainly directed against gram-positive bacteria and fungi, but structural modification (such as replacement of the thiazole ring in phytohabimicin) can enable them to obtain the ability to resist gram-negative bacteria (such as Pseudomonas aeruginosa). In addition, such compounds also have the functions of inducing mammalian sperm acrosome reaction, activating oocytes, etc., and have application potential in the field of assisted reproduction. Treponema hyodysenteriae

[0005] However, although cassiopum compounds have significant prospects for medical use, the types of natural analogues discovered so far are rare, and Embrikia as a potential secondary metabolite resource library, its chemical diversity has not been fully explored. Therefore, finding new Embrikia strains and isolating novel cassiopum derivatives with unique activity from them is of great significance for enriching antibiotic chemical structure types and developing new drugs. SUMMARY

[0006] In order to solve the problems existing in the prior art, the present application provides an Embrikia L77 producing polyether antibiotics and its application.

[0007] In a first aspect, the present application provides an Embrikia sp. L77, the preservation number of which is CGMCC No.36394. Embleya sp. L77, the preservation number of which is CGMCC No.36394.

[0008] The Embrikia L77 of the present application is isolated from the soil of Talatang in Gonghe County, Qinghai Province, China. The soil diluent is spread on the isolation medium by gradient dilution method to obtain the strain by isolation and purification. The present application further carries out biological preservation, and the preservation information is as follows: Preservation number: CGMCC No.36394. Classification and naming: Embleya sp. L77. Preservation unit: China General Microbiological Culture Collection Center. Preservation address: No.3, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101. Preservation date: October 29, 2025.

[0009] In a second aspect, the present application provides a fermentation product of the aforementioned Embrikia L77.

[0010] In a third aspect, the present application provides a microbial agent, which comprises the aforementioned Embrikia L77 or the aforementioned fermentation product.

[0011] ​In a fourth aspect, the present application provides a polyether compound, which comprises any one of the following structural formulae: 、 ; wherein R1-R3 represent H or alkyl, and R4 and R5 represent H or OH.

[0012] Preferably, R1-R3 represent H, methyl or ethyl.

[0013] Further, the polyether compound comprises any one of the following structural formulae: 、 、 、 、 、 、 、 .

[0014] The compounds shown in the above formulae I-VIII are eight new polyether compounds isolated from the study of the active secondary metabolites of Embleya L77, which are named as Embleyamycin A-F, 15-demethyl precezomycin and iso-15-demethyl precezomycin, respectively.

[0015] wherein the molecular formula of Embleyamycin A is C 26 H 31 N3O6, and the molecular weight is 481.55; the molecular formula of Embleyamycin B is C 26 H 31 N3O6, and the molecular weight is 481.55; the molecular formula of Embleyamycin C is C 26 H 31 N3O6, and the molecular weight is 481.55; the molecular formula of Embleyamycin D is C 27 H 33 N3O6, and the molecular weight is 495.58; the molecular formula of Embleyamycin E is C 28 H 35 N3O6, and the molecular weight is 509.60; the molecular formula of Embleyamycin F is C 28 H 35 N3O6, and the molecular weight is 509.60; the molecular formula of 15-demethyl precezomycin is C 27 H34 N2O7, having a molecular weight of 498.58; iso-15-desmethyl precezomycin has a molecular formula of C 27 H 34 N2O7, having a molecular weight of 498.58.

[0016] In a fifth aspect, the present application provides a medicament comprising the aforementioned polyether compound, or a pharmaceutically acceptable salt thereof. Preferably, the medicament further comprises a pharmaceutically acceptable carrier and / or excipient.

[0017] The medicament of the present application can be prepared into any dosage form to be used. The dosage form can be tablets, capsules, pills, granules, powders, pastes, boluses, dripping pills, oral solutions, suspensions, solutions, injections, suppositories, creams, sprays or patches. The pharmaceutical composition can be prepared into common formulations, sustained-release formulations, controlled-release formulations, targeted formulations and various microparticle drug delivery systems.

[0018] The medicament can use various carriers and / or excipients known in the art, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, low-substituted hydroxypropyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc. The medicament or pharmaceutical composition of the present application can be administered by any known administration method in order to achieve the administration purpose and enhance the therapeutic effect.

[0019] In the present application, the pharmaceutically acceptable salt of the polyether compound also falls within the protection scope of the present application. The polyether compound of the present application can be used in the form of a pharmaceutically acceptable salt derived from an inorganic acid or an organic acid. The term "pharmaceutically acceptable salt" refers to a salt which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response and the like, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al., "Pharmaceutical Salts", Pharmaceutical Research, 3(6), 1986, pp. 114-131. et alPharmaceutically acceptable salts are described in detail in J. Pharmaceutical Sciences, 1977, 66: 1. The salts can be prepared by reacting the compounds of the present application with an organic or inorganic base.

[0020] In a sixth aspect, the present application provides a method for preparing the aforementioned polyether compound, comprising: The aforementioned Emblic fungus L77 is used for fermentation, and the aforementioned polyether compound is extracted and separated from the fermentation product and the mycelium.

[0021] Further, the fermentation comprises seed culture and fermentation culture. The seed culture comprises culturing in a seed culture medium at 28-30 ℃ and 150-300 rpm for 3-5 days. The fermentation culture comprises culturing in a fermentation culture medium at 28-30 ℃ and 150-300 rpm for 7-8 days.

[0022] In the present application, the culture medium used in the method for preparing the aforementioned polyether compound can be a culture medium capable of culturing actinomycetes, which can be a solid culture medium, a semi-solid culture medium or a liquid culture medium. The actinomycete culture medium can be International Streptomyces Project Medium (ISP 2, ISP 3, ISP 4, etc.), Gause No. 1 Medium, Trypticase Soy Broth Medium (TSB) or other actinomycete fermentation culture media well known to those skilled in the art. The ISP 2 medium can be made of glucose, malt extract, yeast extract and water, or made of glucose, malt extract, yeast extract, inorganic salt and water, or made of glucose, malt extract, yeast extract, inorganic salt, vitamin complex and water. The Gause No. 1 Medium can be made of starch, inorganic salt and water, or made of starch, inorganic salt, vitamin complex and water. The Trypticase Soy Broth Medium can be made of tryptophan peptone, soy peptone, glucose and water, or made of tryptophan peptone, soy peptone, glucose, inorganic salt and water, or made of tryptophan peptone, soy peptone, glucose, inorganic salt, vitamin complex and water. The other actinomycete fermentation culture media well known to those skilled in the art can be made of one or more types of readily available carbon sources, one or more types of readily available nitrogen sources, water and / or inorganic salts, etc. The carbon source is a nutrient for microbial growth, which is a carbon-containing compound, including sugars, oils, organic acids, organic acid esters and small molecule alcohols as readily available and delayed available carbon sources. The nitrogen source refers to a substance that provides nitrogen elements required for microbial nutrition, including peanut meal, soybean meal, yeast powder, peptone, ammonia water, ammonium salt and nitrate as readily available and delayed available nitrogen sources.

[0023] Preferably, the fermentation is liquid fermentation, the seed culture medium comprises glucose, yeast powder, malt extract powder and water; and the fermentation culture medium comprises soluble starch, soybean powder, glycerol, peptone, calcium carbonate and water.

[0024] Further preferably, the seed culture medium comprises, in parts by weight, glucose 2-6 parts, yeast powder 2-6 parts, malt extract powder 8-12 parts; pH=7-7.5; and / or, The fermentation culture medium comprises, in parts by weight, soluble starch 8-12 parts, soybean powder 3-7 parts, glycerol 8-12 parts, peptone 12-18 parts, calcium carbonate 1-3 parts; pH=7-7.5.

[0025] Further, the extraction and separation comprise: For the supernatant and the bacterial body obtained by the fermentation, the supernatant is subjected to extraction treatment to obtain a supernatant extract, and the bacterial body is subjected to ultrasonic extraction using a solvent to obtain a bacterial body extract, and the supernatant and the bacterial body crude extract are combined into a total crude extract; The total crude extract is preliminarily separated by a reverse-phase silica gel column chromatography, and then separated by HPLC to obtain the polyether compound.

[0026] Further, the extraction solvent comprises one or more of chloroform, dichloromethane and ethyl acetate; in an embodiment of the present application, the extraction solvent is ethyl acetate.

[0027] Further, the medium used by the reverse-phase silica gel column comprises C18 reverse-phase silica gel or phenyl reverse-phase silica gel; in an embodiment of the present application, the medium used by the reverse-phase silica gel column is C18 reverse-phase silica gel.

[0028] Further, the chromatographic column used in the HPLC preparative separation comprises a C18 chromatographic column, a C8 chromatographic column, a C3 chromatographic column or a phenyl-bonded column; in an embodiment of the present application, the chromatographic column used in the HPLC preparative separation is a C18 chromatographic column.

[0029] Further, the solution used in the reverse-phase silica gel column chromatographic separation comprises one or more of an acetone aqueous solution, an alcohol solution with a carbon chain length ≤4, an acetonitrile aqueous solution and methanol; in an embodiment of the present application, the solution used in the reverse-phase silica gel column chromatographic separation is a methanol aqueous solution.

[0030] Further, the mobile phase used in the HPLC preparative separation comprises one or more of an acetone aqueous solution, an alcohol solution with a carbon chain length ≤4 and an acetonitrile aqueous solution; in an embodiment of the present application, the mobile phase used in the HPLC preparative separation is an acetonitrile aqueous solution.

[0031] As a preferred embodiment of the present application, the reversed-phase silica gel column can be C18 reversed-phase silica gel, gradient elution is performed using 20% to 100% methanol aqueous solution to obtain 18 components (Fr. 1~18), Fr. 12~13 are combined and then subjected to HPLC preparation to obtain iso-15-demethyl precezomycin and enniatin A, Fr. 14 is subjected to HPLC preparation to obtain component 77-14-3, enniatin C and enniatin D. Component 77-14-3 is subjected to HPLC preparation to obtain enniatin B. Fr. 17 is subjected to HPLC purification to obtain 15-demethyl precezomycin, enniatin E and enniatin F. The mobile phase used in the HPLC preparation separation can be an acetonitrile and water system.

[0032] In a seventh aspect, the present application provides use of the aforementioned enneabacterium L77, or the aforementioned fermentation product, or the aforementioned microbial inoculum in the preparation of a product for preventing or treating microbial infection.

[0033] Further, the microorganism is a fungus or a bacterium; Preferably, the bacterium comprises one or more of Staphylococcus, Enterococcus, Bacillus or Micrococcus; and / or, The fungus comprises Candida.

[0034] The present application has the following beneficial effects: The present application provides a strain of enneabacterium L77 from the soil of Talatang in Gonghe County, Qinghai Province, China, and polyether antibiotics produced by the strain and related applications. The polyether compounds produced by fermentation of enneabacterium L77 have good antibacterial activity and can effectively inhibit various bacteria and fungi, and can be used for preparing drugs for preventing microbial infection, which has good application prospects in the field of human or animal anti-infection drug development. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0036] Figure 1 is the colony morphology of enneabacterium L77 provided in Example 2 of the present application in ISP 2 medium.

[0037] Figure 2 is the NJ phylogenetic tree constructed based on the 16S rRNA gene sequence of enneabacterium L77 provided in Example 2 of the present application.

[0038] Figure 3 is a high resolution electrospray mass spectrum of the compound of Formula I provided in Example 6 of the present invention. 1 H- 1 H COSY, HMBC correlation signals.

[0039] Figure 4 is a high resolution electrospray mass spectrum of the compound of Formula I provided in Example 6 of the present invention.

[0040] Figure 5 is a high resolution electrospray mass spectrum of the compound of Formula I provided in Example 6 of the present invention. 1 H NMR spectrum.

[0041] Figure 6 is a high resolution electrospray mass spectrum of the compound of Formula I provided in Example 6 of the present invention. 13 C NMR spectrum.

[0042] Figure 7 is a high resolution electrospray mass spectrum of the compound of Formula II provided in Example 6 of the present invention.

[0043] Figure 8 is a high resolution electrospray mass spectrum of the compound of Formula II provided in Example 6 of the present invention. 1 H NMR spectrum.

[0044] Figure 9 is a high resolution electrospray mass spectrum of the compound of Formula II provided in Example 6 of the present invention. 13 C NMR spectrum.

[0045] Figure 10 is a high resolution electrospray mass spectrum of the compound of Formula III provided in Example 6 of the present invention.

[0046] Figure 11 is a high resolution electrospray mass spectrum of the compound of Formula III provided in Example 6 of the present invention. 1 H NMR spectrum.

[0047] Figure 12 is a high resolution electrospray mass spectrum of the compound of Formula III provided in Example 6 of the present invention. 13 C NMR spectrum.

[0048] Figure 13 is a high resolution electrospray mass spectrum of the compound of Formula IV provided in Example 6 of the present invention.

[0049] Figure 14 is a high resolution electrospray mass spectrum of the compound of Formula IV provided in Example 6 of the present invention. 1 H NMR spectrum.

[0050] Figure 15 is a high resolution electrospray mass spectrum of the compound of Formula IV provided in Example 6 of the present invention. 13 C NMR spectrum.

[0051] Figure 16 The high-resolution electrospray mass spectrometry of the compound shown in Formula V provided in Example 6 of this invention.

[0052] Figure 17 It is the compound of formula V provided in Example 6 of this invention. 1 H NMR spectrum.

[0053] Figure 18 It is the compound of formula V provided in Example 6 of this invention. 13 C NMR spectrum.

[0054] Figure 19 The high-resolution electrospray mass spectrometry of the compound shown in Formula VI provided in Example 6 of this invention.

[0055] Figure 20 It is the compound of formula VI provided in Example 6 of this invention. 1 H NMR spectrum.

[0056] Figure 21 It is the compound of formula VI provided in Example 6 of this invention. 13 C NMR spectrum.

[0057] Figure 22 This is a high-resolution electrospray mass spectrometry of the compound shown in Formula VII provided in Example 6 of the present invention.

[0058] Figure 23 It is the compound of formula VII provided in Example 6 of this invention. 1 H NMR spectrum.

[0059] Figure 24 It is the compound of formula VII provided in Example 6 of this invention. 13 C NMR spectrum.

[0060] Figure 25 This is a high-resolution electrospray mass spectrometry of the compound shown in Formula VIII provided in Example 6 of the present invention.

[0061] Figure 26 It is the compound of formula VIII provided in Example 6 of this invention. 1 H NMR spectrum.

[0062] Figure 27 It is the compound of formula VIII provided in Example 6 of this invention. 13 C NMR spectrum.

[0063] Figure 28 The compound of Formula IV provided in Example 9 of this invention inhibits the biofilm of Candida albicans. Detailed Implementation

[0064] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0065] The experimental methods involved in the following examples are all conventional methods in the art if not specifically mentioned, for example, refer to the experimental manuals in the art, or follow the suggested conditions according to the manufacturer's instructions.

[0066] The experimental materials and reagents involved in the following examples can be obtained from commercial channels if not specifically mentioned.

[0067] Example 1 Isolation of Enhydrobacter L77 The soil sample collected from Talatan, Gonghe County, Qinghai Province, China was air-dried (about 16 h) on a clean bench, and then ground into powder using a sterile mortar. 1.00 g of the soil sample was added into 10 mL of culture solution (yeast extract powder 60.0 g, Na4P2O7 1.0 g, NaCl 7.5 g, KCl 0.1 g, CaCl2 0.1 g, NaHCO3 0.2 g, deionized water 1.0 L, pH 7.2, sterilized at 121 ℃ for 15 min), and cultured at 28 ℃ and 180 rpm for 4 h. The soil suspension was diluted to 10 -2 200 μL of the diluted solution was evenly spread on ISP 5 medium (L-asparagine 1.0 g, glycerol 10.0 g, K2HPO4·7H2O 1.0 g, ZnSO4·7H2O 0.001 g, MnCl2·4H2O 0.001 g, FeSO4·7H2O 0.002 g, agar 15.0 g, deionized water 1.0 L, pH 7.2, sterilized at 121 ℃ for 15 min), and incubated at 28 ℃. Single colonies were picked under sterile conditions, and purified by three-zone streaking on ISP 2 medium to obtain a pure culture colony, which was named as L77.

[0068] Example 2 Identification of Enhydrobacter L77 (1) Morphology and culture characteristics The Enhydrobacter L77 described in the present application does not produce water-soluble pigments on ISP 2 medium, and can form a small amount of white aerial hyphae. As shown in FIG. 1, the colony is round to oval, and light yellow. The colony center is protruding, showing radial grooves and concentric ring structures. The aerial hyphae are branched, and have no rupture. Figure 1

[0069] ​(2) Molecular identification To determine the taxonomic status of Embrikia L77, its 16S rRNA gene was uploaded to Ezbiocloud database (https: / / www.ezbiocloud.net / ) for comparison, and the sequence of the model strain with high similarity was obtained, and then the MEGA 7.0.26 software was used to construct a phylogenetic tree based on the Neighbour-joining method. As shown in Fig. 1, strain L77 was clustered in the same branch with Embrikia strains, indicating that strain L77 was an Embrikia strain. At present, there are only two species in Embrikia, and through whole genome comparison, the average nucleotide identity (ANI) values of Embrikia strain L77 with Embrikia Lautus DSM41855 (GCA_000372745.1) and Embrikia sp. NBRC 13850 (GCA_003967355.1) were 90.96% and 84.99% respectively, which were lower than the new species definition value of 95%; the digital DNA-DNA hybridization (dDDH) values were 46.2% and 31.5% respectively, which were lower than the new species definition value of 70%. Therefore, strain L77 is a new species of Embrikia. Figure 2 Embleya scabrispora DSM41855 T (GCA_000372745.1) and Embleya hyalina NBRC 13850 T (GCA_003967355.1) were 90.96% and 84.99% respectively, which were lower than the new species definition value of 95%; the digital DNA-DNA hybridization (dDDH) values were 46.2% and 31.5% respectively, which were lower than the new species definition value of 70%. Therefore, strain L77 is a new species of Embrikia.

[0070] Example 3 Fermentation of Embrikia L77 Strain L77 was inoculated on ISP 2 medium plate and incubated at 28 ℃ for 7 d, and about 1 cm 2 of the agar block containing bacteria was inoculated in a 500 mL flask containing 100 mL of ISP 2 liquid medium, and incubated at 28 ℃, 180 rpm for 5 d to prepare seed liquid. 100 mL of seed liquid was transferred to a 5 L conical flask containing 1 L of liquid fermentation medium (soluble starch 10.0 g, soybean powder 5.0 g, glycerol 10.0 g, peptone 15.0 g, calcium carbonate 2.0 g, deionized water 1.0 L, pH 7.2, 121 ℃ sterilization for 15 min), a total of 35 L, and incubated at 28 ℃, 180 rpm for 7 d to obtain the fermentation liquid.

[0071] Example 4 Extraction of Embrikia L77 Crude Extract ​The fermentation broth was centrifuged, and the supernatant was extracted with an equal volume of ethyl acetate three times (35 L x 3). The ethyl acetate was removed by concentration under reduced pressure at 30°C to obtain the supernatant crude extract. The mycelium was soaked in acetone and ultrasonicated for 20 min, then centrifuged. The acetone aqueous solution was concentrated under reduced pressure to obtain an aqueous solution, which was extracted with an equal volume of ethyl acetate three times, and the ethyl acetate was removed by concentration under reduced pressure to obtain the mycelium crude extract. The supernatant and the mycelium crude extract were combined and weighed (12.9 g).

[0072] Example 5 Isolation of Enterobacter L77 metabolites The crude extract was eluted on a Flash C18 column (20%–100% methanol) to obtain 18 fractions (Fr. 1~18). Fr. 12~13 were combined and separated by HPLC (Capcell Pak C18 MGⅡ, 5 μm, 10 mm × 250 mm, 70% acetonitrile-0.01% trifluoroacetic acid, 2.0 mL / min) to obtain a compound of formula VIII (66.51 mg, t R = 20.4 min) and a compound of formula I (9.15 mg, t R = 25.1 min), and Fr. 14 was separated by a semi-preparative Capcell Pak C18 MGⅡ column (70% acetonitrile-0.01% trifluoroacetic acid, 2.0 mL / min) to obtain component 77-14-3, a compound of formula III (28.5 mg, t R = 28.8 min) and a compound of formula IV (combined with Fr. 15~16, 6.1 g, t R = 34.6 min). Component 77-14-3 was purified by a semi-preparative YMC-Pack ODS-A column (5 μm, 10 mm × 250 mm, 80% acetonitrile-0.01% trifluoroacetic acid, 2.0 mL / min) to obtain a compound of formula II (40.32 mg, t R = 16.1 min). Fr. 17 was purified by a semi-preparative YMC-Pack ODS-A column (75% acetonitrile-0.01% trifluoroacetic acid, 2.0 mL / min) to obtain a compound of formula VII (14.07 mg, t R = 20.7 min), a compound of formula V (4.46 mg, t R = 31.8 min) and a compound of formula VI (2.96 mg, t R = 35.2 min).

[0073] Example 6 Structural identification of compounds of Formula I-VIII Structural identification of compound of Formula I, enniatin A: high resolution electron spray mass spectrum (HRESIMS) shows a quasi-molecular ion peak m / z 482.2285 [M + H] + (482.2286 in theory, Figure 4 ), in combination with nuclear magnetic resonance (NMR) data, determines its molecular formula as C 26 H 31 N3O6, with 13 unsaturations. The H NMR spectrum (DMSO- 1 H NMR spectrum (DMSO- d 6, Figure 5 , Table 1-1) shows 5 aromatic proton signals in the low field region δ H 6.12~7.60 and 2 oxymethylene hydrogen signals in the high field region δ H 4.23 (td, H-10), 3.24 (ddd, H-18), 14 aliphatic methylene or methine hydrogen signals δ H 0.98~3.03 and 2 methyl hydrogen signals δ H 0.91 (d, H-11'), 0.71 (d, H-19'). 13 C NMR spectrum (DMSO- d 6, Figure 6 , Table 2) shows 26 carbon signals, including 2 carbonyl carbons δ C 191.8, 167.6), 11 other sp 2 hetero hybrid carbons (including 1 oxocarbonyl carbon sp 2 hetero hybrid carbon), 1 ketal quaternary carbon δ C 95.5 (C-14), 2 oxymethylene δ C 70.7 (C-18), 68.6 (C-10) and 10 aliphatic carbon signals δ C 10.9~45.5). The above NMR data are similar to those of calcimycin analog N -demethylcalcimycin (see Wu, Q., Gou, L., Lin, S., et alCharacterization of the N -methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882 [J] . Biochimie , 2013, 95(7): 1487-1493.), and N Compared to -demethylcalcimycin, the compound of formula I has a molecular weight 28 Da less. The main difference lies in the fact that the compound of formula I lacks two methyl and two methine signals, but adds two methylene signals, suggesting that it is -demethylcalcimycin. N -demethyl calcimycin demethylated derivatives. 2D NMR of compound I ( 1 H- 1 The H COSY, HSQC, and HMBC related signal indication structure contains a α - Ketopyrrole ring, a benzoxazole ring and a spiroketal fragment ( 1 H- 1 The results of H COSY and HMBC related signals are as follows: Figure 3 As shown (and so on). In the HMBC spectrum, H-9 is correlated with C-8 / C-10 / C-11, and H-10 is correlated with C-8, indicating that the spirocyclic ketal fragment is linked to the C-8 position of benzoxazole via a methylene group. H-19 is correlated with C-17 / C-18 / C-20, indicating... α - Ketopyrrole and spirocyclic ketal fragment via methine C-19 ( δ C 45.5) are connected. CH3-11' is associated with C-10 / C-11 / C-12, and CH3-19' is associated with C-18 / C-19 / C-20, determining the connection positions of the two methyl groups. This confirms the planar structure of the compound of formula I.

[0074] Structural identification of compound II, embryomycin B: HRESIMS showed a quasi-molecular ion peak. m / z 482.2285[M + H] + (The theoretical value is 482.2286,) Figure 7 Based on NMR data, its molecular formula was determined to be C64. 26 H 31 N3O6, identical to compound I. 1D NMR data (DMSO-) for compound II. d 6, Figure 8 , Figure 9, Table 1-1, Table 2) are similar to that of the compound of Formula I, with the main difference being the spiroketal and related structural fragments, and the methyl substitution position of the spiroketal is presumed to be different. Analysis of 2D NMR (H COSY, HSQC and HMBC) data indicates that the methyl substitution of the spiroketal of enniastatin B is at the C-17 position, rather than at the C-11 position of enniastatin A. The planar structure of the compound of Formula II is thus confirmed. 1 H- 1 HCOSY, HSQC and HMBC) data analysis, the methyl substitution of the spiroketal of enniastatin B is at the C-17 position, rather than at the C-11 position of enniastatin A. The planar structure of the compound of Formula II is thus confirmed.

[0075] Structure identification of the compound of Formula III, enniastatin C: HRESIMS shows the quasi-molecular ion peak m / z 482.2285[M + H] + (the theoretical value is 482.2286, Figure 10 ), in combination with NMR data, the molecular formula of the compound is determined to be C 26 H 31 N3O6, which is the same as that of the compound of Formula I. The 1D NMR data (CDCI3, Figure 11 , Figure 12 , Table 1-1, Table 2) of the compound of Formula III are similar to those of the compound of Formula I, in combination with 2D NMR ( 1 H- 1 H COSY, HSQC and HMBC) data analysis, the difference is determined to be the methyl substitution position. The two methyl groups of enniastatin C are connected at the C-11 and C-17 positions, respectively. The planar structure of the compound of Formula III is thus confirmed.

[0076] Structure identification of the compound of Formula IV, enniastatin D: HRESIMS shows the quasi-molecular ion peak m / z 496.2444[M + H] + (the theoretical value is 496.2442, Figure 13 ), in combination with NMR data, the molecular formula of the compound is determined to be C 27 H 33 N3O6. The 1D NMR data (CDCI3, Figure 14 , Figure 15 , Table 1-1, Table 2) of the compound of Formula IV are similar to those of the compound of Formula III, and the difference is analyzed by 2D NMR ( 1 H- 1 HCOSY, HSQC and HMBC) data. Enniastatin D has one more methyl substitution at the C-19 position, and the planar structure of the compound of Formula IV is thus determined.

[0077] Table 1-1 H NMR data of the compounds shown in Formulas I-IV 1 H NMR data of the compounds shown in Formulas I-IV

[0078] a Measured in DMSO- d 6. b Measured in CDCl3 Table 1-2 1D NMR data of compounds of Formula V-VIII 1 H NMR data

[0079] a Measured in DMSO- d 6. b Measured in CDCl3 Table 2 1D NMR data of compounds of Formula I-VIII 13 C NMR data

[0080] a Measured in DMSO- d 6. b Measured in CDCl3. Structure identification of compound of Formula V, enniatin E: HRESIMS shows the quasi-molecular ion peak m / z 510.2575 [M + H] + (theoretical value is 510.2599, Figure 16 ), combined with NMR data, the molecular formula of C 28 H 35 N3O6 is determined. By comparison of 1D NMR data (DMSO- d 6, Figure 17 , Figure 18 , Table 1-2, Table 2), the 1D NMR data of compound of Formula V is similar to that of compound of Formula IV, and by analysis of 2D NMR ( 1 H- 1 H COSY, HSQC and HMBC) data, the difference is that enniatin E is substituted with ethyl at C-19, instead of methyl of enniatin D. Thus the planar structure of compound of Formula V is determined.

[0081] Structure identification of compound of Formula VI, enniatin F: HRESIMS shows the quasi-molecular ion peak m / z 510.2575 [M + H] + (theoretical value is 510.2599,Figure 19 ), in combination with NMR data, the molecular formula of enbiliafungin F was determined to be C 28 H 35 N3O6, the same as that of the compound of formula V. Analysis of the 1D NMR (CDC13, Figure 20 , Figure 21 , Table 1-2, Table 2) and 2D NMR ( 1 H- 1 H COSY, HSQC and HMBC) data, the chemical structure of enbiliafungin F was determined to be similar to that of enbiliafungin D, with the difference that enbiliafungin F is ethyl substituted at C-11 and methyl substituted at C-19. Thus the structure of the compound of formula VI was determined.

[0082] Structure determination of the compound of formula VII, 15-desmethyl precezomycin: HRESIMS shows the quasi-molecular ion peak m / z 499.2444 [M + H] + (499.2439 in theory, Figure 22 ), in combination with NMR data, the molecular formula of enbiliafungin F was determined to be C 27 H 34 N2O7, with 12 degrees of unsaturation. Analysis of the 1D NMR data of the compound of formula VII, except for the benzoxazole ring moiety, is basically the same as that of the compound of formula IV. The 1 H NMR spectrum (DMSO- d 6, Figure 23 , Table 1-2) in the low field region, in addition to the pyrrole ring, there are three aromatic proton signals δ H 7.42 (d, H-3), 7.05 (t, H-4), 6.97 (d, H-5), in combination with the molecular formula, it is speculated that the C-3 position of the benzene ring is not substituted with an amino group. And 13 C NMR spectrum (DMSO- d 6, Figure 24 , Table 2) in the low field region, in addition to the pyrrole ring, there are three aromatic proton signals δ C 149.8) and C-8 ( δ C 170.7) shift to the low field, C-7 ( δ C 127.1) shift to the high field, in combination with the degrees of unsaturation, it is speculated that the benzene ring is connected to C-8 through an amide bond. Through 2D NMR analysis, in combination with the NMR data of the casimycin analog precezomycin in the literature (see Mao, D., Yu, P., Shinzato, N. et alPrecezomycin, a novel antibiotic biosynthetic precursor of cezomycin, from actinomycete Kitasatospora putterlickiae 10–13[J]. J Antibiot The structure of compound VII was determined (Wu, X., et al., J. Nat. Prod. 2019, 82, 1877–1886; Wu, X., et al., J. Nat. Prod. 2020, 83, 1898–1907; Wu, X., et al., J. Nat. Prod. 2021, 84, 1877–1886; Wu, X., et al., J. Nat. Prod. 2022, 85, 1898–1907; Wu, X., et al., J. Nat. Prod. 2023, 86, 1877–1886; Wu, X., et al., J. Nat. Prod. 2024, 77, 189–192.).

[0083] The structure of compound VIII iso-15-demethyl precezomycin was determined: HRESIMS showed a pseudomolecular ion peak at m / z 499.2444 [M + H] m / z 499.2444 [M + H] + (499.2439, Figure 25 calcd.), combined with NMR data, determined its molecular formula as C 27 H 34 N2O7, with 12 degrees of unsaturation. The 1D NMR data of compound VIII was basically identical to that of compound VII except for the benzene ring moiety. The 1H NMR spectrum of compound VIII (DMSO- 1 d6, 400 MHz) was shown in Table 1-2. d 6, Figure 26 There was an AMX system in the low field region of the 1H NMR spectrum of compound VIII (DMSO- δ H 6.76 (d, J = 8.9 Hz, H-6), 7.12 (1H, dd, J = 8.9, 2.8 Hz, H-5), 7.50 (1H, d, J = 2.8 Hz, H-3). In the HMBC spectrum, H-3 was correlated with C-1 / C-2 / C-4 / C-7, H-5 was correlated with C-3 / C-4 / C-7, and H-6 was correlated with C-2 / C-7. Combined with compound VIII and compound VII, it was determined that the enniatin series of compounds should have a common core skeleton biosynthetic pathway, C-2 was substituted with a carboxyl group, C-4 was substituted with a hydroxyl group, and C-7 was connected with an amide bond. Thus, the structure of compound VIII was determined. The 13C NMR spectrum of compound VIII (DMSO- 13 d6, 100 MHz) was shown in Table 1-2. Figure 27

[0084] The relative configuration of compounds I-VIII was determined by ROESY spectrum and coupling constants. The ROESY spectrum of compound I showed that H-10 was correlated with H-18, indicating that the two tetrahydropyran rings were perpendicular to each other, and H-10 and H-18 were on the same side. H-9 was correlated with H-11', H-10 was correlated with H-11 / H-12, indicating that H-10 and H-11 were on the same side. Because 3 ​J H-18,19 = 10.3 Hz (9-12 Hz) and H-17 is correlated with H-19' in the ROESY spectrum, so the configuration of C-18 and C-19 is cis ( erythro ). Therefore, the relative configuration of the compound of formula I is (10 R* , 11 R* , 14 R* , 18 S* , 19 S* ). The ROESY spectrum of the compound of formula II shows that H-10 is correlated with H-18, indicating that the two tetrahydropyran rings are perpendicular to each other and H-10 and H-18 are on the same side. H-18 is correlated with H-16 / H-17 / H-19', and H-19 is correlated with H-17', indicating that H-18 and H-17 are on the same side. Because 3 J H-18,19 = 10.3 Hz (9-12 Hz) and H-17 is correlated with H-19' in the ROESY spectrum, so the configuration of C-18 and C-19 is cis ( erythro ). Therefore, the relative configuration of the compound of formula II is (10 S* , 14 S* , 17 R* , 18 S* , 19 S* ). The ROESY spectrum of the compound of formula III shows that H-10 is correlated with H-18, indicating that the two tetrahydropyran rings are perpendicular to each other and H-10 and H-18 are on the same side. H-9 is correlated with H-11', and H-10 is correlated with H-11 / H-12, indicating that H-10 and H-11 are on the same side. H-18 is correlated with H-16 / H-17, and H-19 is correlated with H-17', indicating that H-18 and H-17 are on the same side. Therefore, the relative configuration of the compound of formula III is (10 R* , 11 R* , 14 S* , 17 R* , 18 R* ). The ROESY spectrum of the compound of formula IV shows that H-10 is correlated with H-18, indicating that the two tetrahydropyran rings are perpendicular to each other and H-10 and H-18 are on the same side. H-9 is correlated with H-11', and H-10 is correlated with H-11 / H-12, indicating that H-10 and H-11 are on the same side. H-18 is correlated with H-16 / H-17 / H-19', and H-19 is correlated with H-17', indicating that H-18 and H-17 are on the same side. Because 3 J H-18,19= 10.3 Hz (9~12 Hz), and ROESY spectrum H-17 and H-19' are related, so the configuration of C-18 and C-19 is erythro erythro ). Therefore, the relative configuration of the compound of formula IV is (10 R* ,11 R* ,14 S* ,17 R* ,18 S* ,19 S* ). The ROESY correlation and 3 J H-18,19 Similar to formula IV, it is determined that the relative configuration of the compound of formula V is (10 R* ,11 R* ,14 S* ,17 R* ,18 S* ,19 S* ), the relative configuration of the compound of formula VI is (10 R* ,11 R* ,14 R* ,17 R* ,18 S* ,19 S* ), the relative configuration of the compound of formula VII is (10 R* ,11 R* ,14 S* ,17 R* ,18 S* ,19 S* ), and the relative configuration of the compound of formula VIII is determined to be (10 R* ,11 R* ,14 S* ,17 R* ,18 S* ,19 S* ).

[0085] The absolute configuration of the compounds represented by formulas I to VIII is determined by comparing the experimental values of electronic circular dichroism (ECD) with the calculated values. The absolute configuration of the compound of formula I is (10 R ,11 R ,14 R ,18 S ,19 S ), the absolute configuration of the compound of formula II is (10 S ,14 S ,17 R ,18 S ,19 S ), the absolute configuration of the compound of formula III is (10 R ,11 R ,14 S,17 R ,18 R ), the absolute configuration of the compound of formula IV is (10 R ,11 R ,14 S ,17 R ,18 S ,19 S ), the absolute configuration of the compound of formula V is (10 R ,11 R ,14 S ,17 R ,18 S ,19 S ), the absolute configuration of the compound of formula VI is (10 R ,11 R ,14 R ,17 R ,18 S ,19 S ), the absolute configuration of the compound of formula VII is (10 R ,11 R ,14 S ,17 R ,18 S ,19 S ), and the absolute configuration of the compound of formula VIII is (10 R ,11 R ,14 S ,17 R ,18 S ,19 S ).

[0086] The physicochemical property data of the compounds shown above are as follows: The compound of formula I: white powder, easily soluble in methanol, chloroform, DMSO. α ]20 D +24.8 ( c 0.50, MeOH);ECD (MeOH) λ max (Δ ε ) 206 (-1.42), 230 (+1.99), 270 (-0.56), 296 (+0.92) nm;UV (MeOH) λ max (log ε ) 224 (4.41), 275 (4.20), 287 (4.21), 361 (3.84) nm; 1 The HNMR data are shown in Table 1-1, 13 The C NMR data are shown in Table 2; (+)-HRESIMS m / z482.2285 [M + H] + C 64.63 H 4.48 N 10.48 26 H 32 N3O6 + , 482.2286).

[0087] Compound of formula II: white powder, easily soluble in methanol, chloroform, DMSO. α ]20 D +38.3 ( c 0.71, MeOH); ECD (MeOH) λ max (Δ ε ) 207 (-1.71), 231 (+1.13), 255 (-0.48), 293 (+1.99) nm; UV (MeOH) λ max (log ε ) 224 (4.35), 274 (4.14), 288 (4.16), 359(3.80) nm; 1 H NMR data see Table 1-1, 13 C NMR data see Table 2; (+)-HRESIMS m / z 482.2285 [M +H] + C 64.63 H 4.48 N 10.48 26 H 32 N3O6 + , 482.2286).

[0088] Compound of formula III: white powder, easily soluble in methanol, chloroform, DMSO. α ]20 D +21.1 ( c 0.15, MeOH); ECD (MeOH) λ max (Δ ε ) 208 (-1.08), 230 (+2.00), 279 (-4.27), 312 (+1.14) nm; UV (MeOH) λ max (log ε ) 224 (4.56), 274 (4.34), 287 (4.36), 359(3.98) nm; 1 H NMR data see Table 1-1, 13 C NMR data see Table 2; (+)-HRESIMS m / z 482.2285 [M +H] + C 64.63 H 4.48 N 10.4826 H 32 N3O6 + , 482.2286)。

[0089] Compound of formula IV: white powder, easily soluble in methanol, chloroform, DMSO. α ]20 D +33.3 ( c 0.26, MeOH); ECD (MeOH) λ max (Δ ε ) 207 (-1.86), 231 (+2.59), 266 (-1.08), 298 (+1.79), 348 (-0.85) nm; UV (MeOH) λ max (log ε ) 224 (4.55), 274 (4.36), 287(4.38), 359 (3.99) nm; 1 H NMR data see Table 1-1, 13 C NMR data see Table 2; (+)-HRESIMS m / z 496.2444 [M + H] + (theoretical value C 27 H 34 N3O6 + , 496.2442)。

[0090] Compound of formula V: white powder, easily soluble in methanol, chloroform, DMSO. α ]20 D +45.0 ( c 0.16, MeOH); ECD (MeOH) λ max (Δ ε ) 202 (-6.02), 232 (+2.23), 290 (+3.28) nm; UV(MeOH) λ max (log ε ) 223 (4.33), 275 (4.09), 290 (4.13), 357 (3.76) nm; 1 H NMR data see Table 1-2, 13 C NMR data see Table 2; (+)-HRESIMS m / z 510.2575 [M + H] + (theoretical value C 28 H 36 N3O6+ , 510.2599).

[0091] Compound of formula VI: white powder, easily soluble in methanol, chloroform, DMSO. α ]20 D +30.1 c 0.19, MeOH); ECD (MeOH) λ max (Δ ε ) 204 (-2.35), 231 (+1.52), 266 (-0.17), 296 (+1.54) nm; UV (MeOH) λ max (log ε ) 223 (4.41), 275 (4.18), 287 (4.19), 359(3.84) nm; 1 H NMR data are reported in Tables 1-2, 13 C NMR data are reported in Table 2; (+)-HRESIMS m / z 510.2575 [M + H] + (calcd C 28 H 36 N3O6 + , 510.2599).

[0092] Compound of formula VII: white powder, easily soluble in methanol, chloroform, DMSO. α ]20 D +124.4 c 0.14, MeOH); ECD (MeOH) λ max (Δ ε ) 206 (-5.33), 224 (+2.17), 286 (+14.27), 315 (-2.08) nm; UV (MeOH) λ max (log ε ) 217 (4.68), 289 (4.51) nm; 1 H NMR data are reported in Tables 1-2, 13 C NMR data are reported in Table 2; (+)-HRESIMS m / z 499.2444 [M + H] + (calcd C 27 H 35 N2O7 + ,499.2439).

[0093] Compound of formula VIII: White powder, readily soluble in methanol, chloroform, and DMSO. α ]20 D +32.4 ( c 0.28,MeOH); ECD (MeOH) λ max (Δ ε ) 206 (-0.88), 222 (+2.88), 254 (+1.29), 277 (-1.89), 305 (+0.47) nm; UV (MeOH) λ max (log ε ) 217 ​​(4.49), 255 (4.13), 288 (4.25), 337 (3.71) nm; 1 The H NMR data are shown in Table 1-2. 13 C NMR data are shown in Table 2; (+)-HRESIMS m / z 499.2444 [M + H] + (Theoretical value C) 27 H 35 N2O7 + , 499.2439).

[0094] Example 7: Determination of the antibacterial activity of compounds of formulas I to VIII against bacteria The antibacterial activity was determined using a 96-well plate microdilution method, and compounds of formulas I to VIII were tested against six Gram-positive bacteria. Enterococcus faecium 310682, Enterococcus faecalis ATCC 33186, Staphylococcus aureus ATCC 33591, Staphylococcus aureus ATCC 29213, Bacillus subtilis CPCC 100029, Micrococcus luteus CMCC 28001) and 4 Gram-negative strains ( Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700721, Acinetobacter baumannii The antibacterial activity of 2799 was assessed. The 10 tested bacteria were cultured in Mueller-Hinton (MH) hydrolyzed casein medium to the logarithmic growth phase, and the bacterial suspensions were diluted to 5 × 10⁻⁶ with MH medium. 5CFU / mL was prepared for use. The diluted bacterial suspension was added to sterile 96-well plates, 198 μL per well in column 1 and 100 μL per well in the remaining wells. In the first column of wells, 2 μL of the test sample and the positive control drug levofloxacin (6.4 mg / mL) were added sequentially, mixed, and then serially diluted column by column to obtain 11 concentration gradients: 64–0.0625 μg / mL. In addition, four negative control wells (containing only bacterial suspension) and four blank control wells (containing only MH liquid medium) were set up in the 96-well plate. The 96-well plate was incubated at 37 °C for 16 h, and the minimum drug concentration (MIC) that completely inhibited bacterial growth was observed and recorded. The results are shown in Table 3. Compounds I–VI all showed significant inhibitory effects on the tested Gram-positive bacteria (MIC = 0.25–8 μg / mL). However, the MICs of compounds I–VIII for the tested Gram-negative bacteria were all greater than 64 μg / mL.

[0095] Table 3. Antibacterial activity (MIC, μg / mL) of compounds represented by formulas I to VIII

[0096] Example 8: Determination of the antifungal activity of compounds I to VIII. The antifungal activity was determined using the CLSI standard 96-well plate microdilution method to detect the activity of compounds of formulas I-VIII against Candida albicans. Candida albicans Growth inhibitory activity of ATCC 10231 was assessed. The test bacteria were cultured in Yeast Extract Peptone Dextrose (YPD) liquid medium to the logarithmic growth phase. After centrifugation, the cells were washed with PBS and resuspended in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (pH 6.0). 100 μL of bacterial culture and 100 μL of different concentrations of RPMI-1640 dilutions were added to each well of a 96-well plate. The final concentrations of the compound were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL, and the bacterial count was 5 × 10⁻⁶. 3 CFU / mL was used, with a DMSO (1%) control also included. The 96-well plates were incubated at 35 °C for 24 h, and the minimum drug concentration (MIC) required for complete bacterial inhibition was observed and recorded. The results of the anti-Candida albicans activity assay are shown in Table 4. Compounds II-VI exhibited significant anti-Candida albicans activity (MIC ≤ 8 μg / mL), while compound I showed moderate inhibitory activity (MIC = 16 μg / mL).

[0097] Table 4. Anti-Candida albicans ATCC 10231 activity (MIC, μg / mL) of compounds represented by formulas I to VIII.

[0098] Example 9: Inhibition of Candida albicans biofilm by compound of formula IV Inhibition of biofilm formation by compound IV: Candida albicans ( Candida albicans ATCC10231 cells were cultured in YPD liquid medium to the logarithmic growth phase, centrifuged, washed with PBS, and then adjusted to 2.0 × 10⁻⁶ medium with RPMI-1640 (pH 6). 6 CFU / mL. 100 μL of bacterial culture and 100 μL of different concentrations of RPMI-1640 dilution buffer were added to each well of a 96-well plate. The final concentrations of the compound were 16, 8, 4, 2, and 1 μg / mL. A DMSO (1%) control was included in the 96-well plate. The plates were incubated at 35 °C for 24 h and 48 h, respectively. The culture medium was removed, and the plates were washed three times with PBS. 100 μL of methanol was added to each well for fixation for 15 min. After drying at room temperature, 100 μL of 0.1% crystal violet solution was added to each well, and staining was performed at room temperature for 30 min. After washing three times with PBS, 100 μL of 100% ethanol was added to each well, and the plates were incubated at room temperature for 1 h. The absorbance at 595 nm was measured using a multi-mode microplate reader. RPMI-1640 culture medium served as a blank control. Microscopic observation and photographic recording were performed simultaneously.

[0099] Inhibition of biofilms at different growth stages by compound IV: The diluted bacterial culture was added to 96-well plates at 100 μL per well and incubated at 35 °C. At 2 h, 12 h, and 24 h of incubation, non-adhering cells were removed with PBS, and compound IV was added to final concentrations of 8, 4, 2, and 1 μg / mL, respectively. A 1% DMSO control was also included. After static incubation at 35 °C for 24 h, the culture medium was removed, and the cells were washed three times with PBS. The cells were then fixed, stained with crystal violet, and quantitatively analyzed according to the above steps. Microscopic observation and photographic recording were also performed.

[0100] The results are as follows Figure 28 As shown, compound IV significantly inhibited biofilm formation. When added simultaneously and after 2 hours of adhesion, biofilm formation showed a dose-dependent decrease. Figure 28 (A) in the compound; however, in the later stages of biofilm formation (12 h and 24 h), although the compound still inhibited biofilm formation, its inhibitory activity decreased significantly. Figure 28 (B in the middle).

[0101] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A type of Embryobacter ( Embleya sp.)L77, characterized in that, The preservation number of Embryobacter L77 is CGMCC No. 36394.

2. The fermentation product of Embryobacter L77 as described in claim 1.

3. A microbial agent, characterized in that, The microbial agent includes Embryobacter L77 as described in claim 1, or the fermentation product as described in claim 2.

4. A polyether compound, characterized in that, The polyether compound includes any of the following structural formulas: , ; In this context, R1~R3 ​​represent H or alkyl groups, and R4 and R5 represent H or OH groups.

5. The polyether compound according to claim 4, characterized in that, The polyether compound includes any of the following structural formulas: 、 、 、 、 、 、 、 。 6. A drug, characterized in that, The drug comprises the polyether compound of claim 4 or 5, or a pharmaceutically acceptable salt thereof; Preferably, it further includes: a pharmaceutically acceptable carrier and / or excipient.

7. The method for preparing the polyether compound according to claim 4 or 5, characterized in that, include: Fermentation was carried out using the Embryobacter L77 as described in claim 1, and the polyether compound described in claim 4 or 5 was extracted and separated from the fermentation product and bacterial cells.

8. The preparation method according to claim 7, characterized in that, The fermentation includes: seed culture and fermentation culture; The seed culture includes: culturing in a seed culture medium at 28-30 °C and 150-300 rpm for 3-5 days; The fermentation culture includes: culturing in a fermentation medium at 28-30 °C and 150-300 rpm for 7-8 days; Preferably, the seed culture medium comprises glucose, yeast powder, malt extract powder and water; the fermentation culture medium comprises soluble starch, soybean flour, glycerol, peptone, calcium carbonate and water.

9. The use of Embryobacter L77 as described in claim 1, or the fermentation product as described in claim 2, or the microbial agent as described in claim 3 in the preparation of products for the prevention or treatment of microbial infections.

10. The application according to claim 9, characterized in that, The microorganism is a fungus or a bacterium; Preferably, the bacteria include one or more of the following: Staphylococcus spp., Enterococcus spp., Bacillus spp., or Micrococcus spp.; and / or, The fungi include Candida.

Images