Strain of diffusion carbon pad bacteria LUCF 4343-3, bacterial agent and application of bacterial agent in preparation of antibacterial agent

Abstract

The invention belongs to the technical field of antibacterial active microorganisms, and particularly relates to a strain of diffusion carbon pad bacteria LUCF 4343-3, a bacterial agent and application of the bacterial agent in preparation of an antibacterial preparation. According to the invention, an endophytic fungus strain with remarkable antibacterial potential is successfully separated from Parmotrema, and the endophytic fungus strain is identified as diffusion carbon pad fungus LUCF 4343-3. Experiments show that an ethyl acetate extract of the strain shows a good inhibition effect on various clinical common pathogenic bacteria, including gram-positive bacteria (staphylococcus aureus and enterococcus faecalis), gram-negative bacteria (pseudomonas aeruginosa), fungi (candida albicans and candida glabrata) and the like; the discovery provides an important basis for the application of the strain in the development of novel broad-spectrum antibacterial drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial active microorganisms, and particularly relates to a strain of Hypoxylon diffusum LUCF 4343-3, a bacterial agent and its application in the preparation of antibacterial agents. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] With the increasingly severe problem of pathogenic microorganism drug resistance, the global scientific research community is accelerating the search for antibacterial substances with innovative mechanisms of action. Microbial secondary metabolites, due to their structural diversity, have become important targets for drug development. As a special symbiont formed by fungi and photosynthetic organisms (cyanobacteria / green algae), lichens contain two types of fungal communities inside: the lichen-forming fungi (mainly ascomycetes) that constitute the symbiont and the symbiotic endophytic fungal population. The latter has formed a special metabolic network during long-term evolution.

[0004] Endophytic lichen fungi (ELF) can synthesize a variety of structurally novel active substances, including: nitrogen-containing compounds (such as alkaloids), polyketide derivatives, terpenoids, quinones. These metabolites have significant antibacterial activity, and their chemical structures are both similar to and unique compared to the substances produced by the host. As a natural compound library, ELF provides an important resource for the development of new anti-infective drugs.

[0005] Parmelia saxatilis Parmotrema tinctorum (Despr. ex Nyl.) Hale], as a lichen species with important research value, its history can be traced back to what Linnaeus recorded in "Species Plantarum" in 1753, " Lichen chinensis". This species has significant advantages such as wide distribution (covering Asia, America and other regions), large biomass (single plant dry weight can reach 200-300g), and easy collection (often epiphytic on tree trunks or rock surfaces), making it an ideal natural drug research material. However, there are still obvious deficiencies in the systematic research on endophytic fungi (ELF) of the genus Macrophylla in China: First, existing research focuses on the analysis of the chemical composition of the lichen itself, and the isolation and identification of its symbiotic fungal community and the pharmacodynamic evaluation of its metabolites are still relatively blank. The latest research shows that ELF isolated from lichens of similar genera can produce Novel antimicrobial substances (such as depsidone compounds) are produced, and their MIC values can reach 0.5-2μg / mL. This strongly suggests that ELF of Prunus armeniaca may contain untapped antimicrobial resources. Based on the special ecological niche of ELF of this genus (long-term exposure to antimicrobial substances produced by the host) and evolutionary pressure (need to compete for limited nutrients), its metabolites are likely to have innovative structures that break through existing drug resistance mechanisms. Therefore, systematic isolation and cultivation, activity screening and mechanism research of ELF of Prunus armeniaca can not only fill the knowledge gap in this field, but also have the potential to discover a new generation of antimicrobial lead compounds. Summary of the invention

[0006] Based on the previous systematic study on the medicinal potential of endophytic fungi in the genus Macrophylla, this study adopted a targeted screening strategy to select Macrophylla reticulate ( Parmotrema reticulatum ) tissues, an endophytic fungus with significant antibacterial activity was successfully isolated. ITS sequence analysis identified the fungus as Diffusive Carbon Mat Fungus ( Nemania diffusa ), numbered LUCF4343-3, the crude extract prepared after extraction with ethyl acetate showed broad-spectrum antibacterial properties. This discovery provides an important candidate material basis for the development of new antibacterial preparations.

[0007] According to the above achievements, the present invention provides the following technical solutions: In the first aspect, a diffusion carbon mat bacteria ( Nemania diffusa ) LUCF 4343-3, which was deposited on April 8, 2025 in the General Microbiological Center of the China Microbiological Culture Collection Administration, referred to as CGMCC, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and its biological deposit number is CGMCC No.41883. In this article, it is referred to as "Diffuse Carbon Mat Bacteria LUCF 4343-3" The ITS sequence of the strain is shown in SEQ ID NO:1.

[0008] Observed under a microscope, the morphological characteristics of the diffuse carbon mat bacteria LUCF 4343-3 are as follows: Morphological characteristics of the strain: Microscopic observation shows that the hyphae are white, smooth on the surface, with branches and septa. After the culture time is extended to 25 days, black substances can be observed on the colony, but no spore formation is found on the PDA medium.

[0009] Colony characteristics: When cultured on the PDA medium, a white cottony colony is formed. The mycelium is dense and grows radially, with an irregular wavy edge; the back of the colony is white at the edge and light yellow in the middle area. Under the culture condition of 25 °C, the colony diameter can reach 5 cm within 5 days.

[0010] In a second aspect, a microbial agent is provided, which contains the aforementioned Dictyophora effusa LUCF 4343-3 strain ， or / and metabolites, cultures or / and extracts of Dictyophora effusa LUCF 4343-3.

[0011] In the above third aspect, the "metabolites" cover all chemical substances secreted by the strain during liquid / solid culture, including but not limited to secondary metabolites with biological activities such as antibiotics and enzymes.

[0012] The "culture" refers to a mycelium-medium composite system formed by fermentation for a period of time (such as 5-7 days) under controlled conditions (for example, at the appropriate temperature of 28±2 °C and pH 6.0-7.0 for Dictyophora effusa LUCF 4343-3).

[0013] The "extract" refers to the active substances separated from the strain or the medium by using polar solvents with the strain and / or its growth carrier as the extraction object, and the growth carrier includes solid medium and liquid medium.

[0014] In a preferred embodiment of the present invention, an ethyl acetate extract of the aforementioned Dictyophora effusa LUCF 4343-3 is provided, and the preparation method is as follows: Inoculate Dictyophora effusa LUCF 4343-3 into a liquid PDB medium and culture for 5-7 days according to an inoculum size of 8-12%; add ethyl acetate to the medium and soak for 20-25 h, filter out the solid part, and evaporate the ethyl acetate to obtain the ethyl acetate extract. This extract has been verified to have inhibitory activities against various harmful microorganisms such as Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata, and Candida albicans.

[0015] In a third aspect, the application of the Dictyophora effusa LUCF 4343-3 described in the first aspect 、 in the preparation of an antibacterial preparation of the microbial agent described in the second aspect is provided.

[0016] The application methods of the antibacterial preparation described in the third aspect include but are not limited to any one of the following: 1) Environmental disinfection: control of microbial contamination on object surfaces, water bodies, living environments, and public places; 2) Pharmaceutical development: inhibition of diseases caused by harmful microorganisms; 3) Daily chemical products: can be added as a bacteriostatic agent to products such as cleaners and cosmetics to achieve daily cleaning effects.

[0017] Furthermore, the harmful microorganisms include, but are not limited to, one or more of the genera Streptococcus, Staphylococcus, Pseudomonas, Bacillus, and Candida; furthermore, the harmful microorganisms are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata, and Candida albicans.

[0018] Compared with the prior art, the beneficial effects of the present invention are: The present invention explores based on the unique ecological niche advantages of the lichen symbiotic system. The special microenvironment formed by algae and fungi in the symbiotic layer, through the long-term co-evolutionary selection pressure, promotes the endophytic fungi to evolve into secondary metabolites with novel structures. The present invention first conducts a systematic screening on the lichens of the genus Parmelia ( Parmotrema ), and successfully isolates an endophytic fungus with significant antibacterial activity: Hypoxylon bullatum LUCF 4343-3. The ethyl acetate extract thereof exhibits broad-spectrum antibacterial properties, and it shows inhibitory effects on various common clinical pathogenic bacteria. This discovery not only expands the resource library of endophytic fungi in lichens in China, but also provides an innovative source for the discovery of lead compounds for antifungal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0020] Figure 1 It is the colony morphology diagram of Hypoxylon bullatum LUCF 4343-3 described in Example 1; Figure 2 It is the antibacterial effect diagram of Hypoxylon bullatum LUCF 4343-3 described in Example 1; Among them, Figure 2 A in it is the inhibitory effect of Staphylococcus aureus CMCC26003; Figure 2 B in it is the inhibitory effect of Candida glabrata ATCC15126; Figure 2 C in it is the inhibitory effect of Candida albicans CMCC98001; Figure 2 D in it is the inhibitory effect of Bacillus subtilis BC.CS136; Figure 2 Among them, E is the inhibitory effect on Pseudomonas aeruginosa CMCC10104; Figure 2 Among them, F is the inhibitory effect on Enterococcus faecalis ZDZA0109; Description of preservation situation: The fungal name is Coltricia cinnamomea LUCF 4343-3, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, abbreviated as CGMCC, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is April 8, 2025, and the registration number in the preservation center is: CGMCC No. 41883. Specific implementation manners

[0021] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0022] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] In the context of this specification, the word "comprise" is considered to mean "include in particular". It should not be construed as "consisting only of...".

[0024] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1 I. Strain isolation and identification In this study, 32 strains were isolated from the genus Monostroma. The crude extracts were obtained by fermentation and rotary evaporation of all strains. Preliminary screening of the inhibition zone was carried out, and 1 strain with broad-spectrum antibacterial activity was selected for MIC determination and identified as Nemania diffusa, Named Coltricia cinnamomea LUCF 4343-3 。

[0026] 1. Identification and morphological characteristics of the strain The genomic DNA of lichen endophytic fungi was obtained by a rapid extraction method. The REDExtract-N-Amp™ (Sigma-Aldrich) kit was used to complete nucleic acid extraction according to the standard procedure. Using the total DNA of endophytic fungi as a template, the ITS1F / ITS4 primer pair was selected for the amplification reaction, and their sequences are as follows: Forward: 5'-CTTGGTCATTTAGAGGAAGTAA-3' (SEQ ID NO:2) Reverse: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO:3).

[0027] The PCR system was configured as follows: 19 µL of ddH2O, 25 µL of 2×Taq PCR MasterMix, 2 µL of the upstream primer, 2 µL of the downstream primer, and 2 µL of the DNA template, for a total of 50 µL. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, for 34 cycles; and finally extension at 72°C for 10 min. After the target product was confirmed by 1% agarose gel electrophoresis, the sequence determination was completed by entrusting a professional sequencing institution.

[0028] Regarding the microscopic observation of the strain LuCF 4343-3 of Hypoxylon diffusum, the morphological characteristics of this strain are as follows: Mycelium characteristics: Microscopic observation showed that the mycelium was white, smooth on the surface, with branches and septa. After the culture time was extended to 25 days, black substances could be observed on the colony, but no spore formation was found on the PDA medium.

[0029] Colony characteristics: When cultured on PDA medium, a white cottony colony was formed, the mycelium was dense, growing radially, and the edge was irregularly wavy; the back of the colony was white at the edge and light yellow in the middle area. Under the culture condition of 25°C, the colony diameter could reach 5 cm within 5 days.

[0030] 2. Bacterial extract (1) Activation and subculture of the strain The preserved strain was inoculated onto a PDA plate and cultured statically at 25°C for 7 days. The activated mycelium was inoculated into a PDB liquid medium, and after 48 hours, it was transferred to a fresh PDB medium at an inoculation amount of 10%.

[0031] (2) Shaking culture It was placed in a constant temperature shaker and cultured at 25°C and 150 r / min for 5-7 days until the mycelium grew sufficiently.

[0032] (3) Extraction of metabolites After the cultivation was completed, ethyl acetate was added at a volume ratio of 1:1, and the mixture was allowed to stand for extraction for 24 hours. The mycelium was removed by filtration through sterile gauze, and the organic phases were collected and combined.

[0033] (4) Preparation of crude extract The organic phase was concentrated using a rotary evaporator to obtain a solid extract. The crude extract solution with a final concentration of 100 mg / mL was prepared by dissolving it in methanol.

[0034] II. Verification of antibacterial activity 1. Selection of test strains Six representative microorganisms were selected as test objects in the experiment, including Gram-positive bacteria (Enterococcus faecalis ZDZA0109, Staphylococcus aureus CMCC26003), Gram-negative bacteria (Pseudomonas aeruginosa CMCC10104), Bacillus (Bacillus subtilis BC.CS136), and yeasts (Candida glabrata ATCC15126, Candida albicans CMCC98001).

[0035] 2. Experimental operation procedure (1) Under sterile conditions, use a sterilized spreader to evenly spread each test bacterial suspension on the surface of an MH agar plate; (2) Use a sterile puncher to prepare a sampling hole with a diameter of 8 mm; (3) Precisely add 100 μL of the crude extract solution to be tested to each hole; (4) Set up a methanol solvent control, and set 3 replicates for each group of experiments.

[0036] 3. Cultivation and result determination Seal the treated petri dishes and incubate them in an incubator at 37 °C and 60% humidity for 24 hours. Use a precision measuring tool to record the size of the inhibition zone, with a measurement accuracy of 0.1 mm. See the specific experimental results in Figure 2 .

[0037] 4. Measurement of the MIC value of antibacterial activity of strains Take 1.5 mL of the concentrated crude extract solution, centrifuge it at 12000 r / min for 5 minutes to remove the precipitate. Sequentially take 50.00, 25.00, 12.50, 6.25, 3.12 μL of the supernatant and add it to a 96-well plate, and make up the volume to 50 μL per well with CAMHB medium. Use a spectrophotometer to measure the OD600 value of the test bacterial suspension, dilute it 2500 times after adjusting it to 1.0 for standby. Add 50 μL of the diluted bacterial solution to each well, observe the results after culturing at 37 °C for 12 - 18 hours, and determine the lowest drug concentration (MIC) that inhibits the growth of bacteria. See the specific data in Table 1.

[0038] Table 1 Antibacterial activities of two crude extracts In Table 1 above, "4343-3" refers to the diffused charcoal cushion mushroom LUCF 4343-3 in this embodiment, and "4067-1" represents another strain of fungus isolated from the same batch.

[0039] According to Figure 2 and the results in Table 1, the crude extracts of the above strains show inhibitory effects on various fungi and bacteria of different genera and species, and are a broad-spectrum antibacterial active ingredient.

[0040] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A strain of Hypoxylon bullatum ( Nemania diffusa ) LUCF 4343-3, which was deposited on April 8, 2025 at the General Microbiology Center of the China National Center for Culture Collection of Microorganisms, abbreviated as CGMCC, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its biological deposit number is: CGMCC No. 41883.

2. The spreading charcoal cushion mushroom ( Nemania diffusa ) LUCF 4343-3 as claimed in claim 1, characterized in that The ITS sequence of the strain is shown in SEQ ID NO:

1.

3. The spreading charcoal cushion fungus ( Nemania diffusa ) LUCF 4343-3 as claimed in claim 1, characterized in that The morphological characteristics of the said Nemania diffusa LUCF 4343-3 are as follows: Mycelium characteristics: Microscopic observation shows that the mycelium is white, smooth on the surface, with branches and septa; after the culture time is extended to 25 days, black substances can be observed on the colony, but no spore formation is found on the PDA medium; Colony characteristics: When cultured on the PDA medium, it forms a white cottony colony, with a dense mycelium, growing radially, and the edge is irregularly wavy; the back of the colony is white at the edge and light yellow in the middle area; under the culture condition of 25 °C, the colony diameter can reach 5 cm within 5 days.

4. A bacterial agent, characterized in that, Comprising the diffused charcoal cushion fungus ( Nemania diffusa ) LUCF 4343-3, or the metabolite, culture or extract of the diffused charcoal cushion fungus ( Nemania diffusa ) LUCF 4343-3.

5. The microbial agent according to claim 4, characterized in that, The microbial agent is the ethyl acetate extract of Antrodia diffusa ( Nemania diffusa ), LUCF 4343-3, and the preparation method is as follows: inoculate Antrodia diffusa ( Nemania diffusa ), LUCF 4343-3 into a liquid PDB medium, and culture it for 5-7 days at an inoculation amount of 8-12%; add ethyl acetate to the medium and soak for 20-25 h, filter out the solid part, and evaporate the ethyl acetate to obtain the ethyl acetate extract.

6. Use of the Diffusocarpon sp. ( Nemania diffusa ) LUCF 4343-3, the bacterial agent according to claim 4 or 5, in the preparation of an antibacterial preparation.

7. The use according to claim 6 in the preparation of an antibacterial preparation, characterized in that, The application method of the said antibacterial preparation is selected from any one of the following: (1) Environmental disinfection and sterilization: for controlling microbial contamination on the surface of objects, in water bodies, living environments, and public places; (2) Pharmaceutical development: inhibiting related diseases caused by harmful microorganisms; (3) Daily chemical products: added as a bacteriostatic agent to cleaning agents and cosmetic products to achieve daily cleaning effects.

8. The use according to claim 7 in the preparation of an antibacterial preparation, characterized in that, The said harmful microorganisms are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata, and Candida albicans.

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