Strain of lindneria diffusa lucf 4343-3, microbial inoculum and use thereof in the preparation of an antimicrobial preparation

Abstract

The present invention belongs to the field of antibacterial active microorganism technology, and specifically relates to a diffusion carbon mat fungus LUCF 4343-3, a bacterial agent and its application in the preparation of antibacterial preparations. Parmotrema ) were successfully isolated from a strain of endophytic fungus with significant antimicrobial potential and identified as LUCF 4343‑3. Experiments showed that the ethyl acetate extract of this strain exhibited potent inhibitory effects against a variety of common clinical pathogens, including Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis), Gram-negative bacteria (Pseudomonas aeruginosa), and fungi (Candida albicans and Candida glabrata). These findings provide important evidence for the application of this strain in the development of new broad-spectrum antimicrobial drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antimicrobial active microorganisms, and particularly relates to a strain of dispersing carbon pad fungus LUCF 4343-3, a microbial agent and application thereof in preparation of an antimicrobial preparation. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the background of the application without admitting that such information forms any part of prior art.

[0003] With the increasingly serious problem of pathogenic microbial drug resistance, the global scientific research community is accelerating the search for antimicrobial substances with innovative mechanisms. Microbial secondary metabolites have become important targets for drug development due to their structural diversity. Lichens are special symbiotic organisms formed by fungi and photosynthetic organisms (cyanobacteria / green algae), and there are two types of fungal communities inside them: lichen-type fungi (mainly ascomycetes) that constitute the symbiotic body and symbiotic endophytic fungi. The latter has formed a special metabolic network in long-term evolution.

[0004] Endolichenic fungi (ELF) can synthesize a variety of active substances with novel structures, including nitrogen-containing compounds (such as alkaloids), polyketide derivatives, terpenoids, and quinone substances. These metabolites have significant antimicrobial activity, and their chemical structures are both similar to and unique from those produced by the host. ELF serves as a natural compound library and provides an important resource for the development of new anti-infective drugs.

[0005] Lobaria xishanensis Parmotrema tinctorum As a lichen species with important research value, Lobaria xishanensis (Despr. ex Nyl.) Hale has a history dating back to 1753 when Linnaeus recorded it in Flora Svecica as "Lichen xishanensis". It is a species of lichen that is endemic to China and is distributed in the subtropical and tropical regions of the world. Lichen chinensis". This species has significant advantages such as wide distribution (throughout 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 medicine research material. However, the current systematic research on endophytic fungi (ELF) of the genus Macrophylla in China still has obvious shortcomings: First, existing research mostly focuses on the analysis of the chemical composition of the lichen itself, and research on the isolation and identification of its symbiotic fungal community and the pharmacological evaluation of its metabolites is relatively blank. The latest research shows that ELF isolated from lichens of related genera can produce Novel antimicrobial compounds (such as depsidone compounds) have been generated, with MIC values ​​reaching 0.5-2 μg / mL. This strongly suggests that ELFs from the genus Prunus may harbor untapped antimicrobial resources. Given the unique ecological niche (long-term exposure to host-produced antimicrobial substances) and evolutionary pressure (competition for limited nutrients) of this genus of ELFs, their metabolites are likely to possess innovative structures that could overcome existing resistance mechanisms. Therefore, systematic isolation, culture, activity screening, and mechanistic studies of ELFs from the genus Prunus will not only fill knowledge gaps in this field but also hold promise for the discovery of a new generation of lead antimicrobial compounds. Summary of the Invention

[0006] Based on the previous systematic study on the medicinal potential of endophytic fungi of the genus Macrophylla, this study adopted a targeted screening strategy to select the fungi from Macrophylla reticulate ( Parmotrema reticulatum ) tissues, an endophytic fungus with significant antibacterial activity was successfully isolated. ITS sequence analysis identified the fungus as Diffuse Carbonaceous Mat ( Nemania diffusa The crude extract prepared from the 1,000-mg chlorinated ...

[0007] Based on the above results, the present invention provides the following technical solutions:

[0008] In the first aspect, a diffusion carbon mat bacteria ( Nemania diffusa LUCF 4343-3 was deposited on April 8, 2025, at the General Microbiology Center of the China Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its biological deposit number is CGMCC No. 41883. This article will refer to it as "Diffuse Carbon Mat Bacteria LUCF 4343-3."

[0009] The ITS sequence of the strain is shown in SEQ ID NO: 1.

[0010] Observed under a microscope, the morphological characteristics of the diffusive carbon mat fungus LUCF 4343-3 are as follows:

[0011] Mycelial characteristics: microscopic observation showed that the mycelium was white, smooth surface, with branches and septum. After the culture time was extended to 25 days, black material was observed on the colony, but no spore formation was found on the PDA medium.

[0012] Colony characteristics: when cultured on PDA medium, white cottony colonies were formed, the mycelium was dense and grew radially, with irregular wavy edges; the back of the colony was white at the edge and yellowish in the middle. Under the culture condition of 25℃, the colony diameter could reach 5 cm within 5 days.

[0013] In a second aspect, a bacterial agent is provided, containing the aforementioned L. diffusa LUCF 4343-3 strain ， or / and metabolites, culture or / and extracts of L. diffusa LUCF 4343-3.

[0014] In the above third aspect, the "metabolites" cover all chemical substances secreted by the mycelium during liquid / solid culture, including but not limited to antibiotics, enzymes and other bioactive secondary metabolites.

[0015] The "culture" refers to the mycelium-medium complex system formed by fermentation under controlled conditions (e.g., suitable temperature of L. diffusa LUCF 4343-3 28±2℃, pH 6.0-7.0) for a period of time (e.g., 5-7 days).

[0016] The "extract" refers to the active substances separated from the mycelium or medium using polar solvents with the mycelium and / or its growth carrier as the extraction object, and the growth carrier includes solid medium and liquid medium.

[0017] In a preferred embodiment of the present application, an ethyl acetate extract of the aforementioned L. diffusa LUCF 4343-3 is provided, and the preparation method is as follows: L. diffusa LUCF 4343-3 is inoculated into liquid PDB medium, and cultured for 5-7 days at an inoculation amount of 8-12%; ethyl acetate is added to the medium and soaked for 20-25h, the solid part is filtered out, and the ethyl acetate is evaporated, to obtain the ethyl acetate extract. The extract has been verified to have inhibitory activity against multiple harmful microorganisms such as Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata and Candida albicans.

[0018] In a third aspect, the aforementioned L. diffusa LUCF 4343-3 of the first aspect is provided 、 The application of the bacterial agent of the second aspect in the preparation of an antibacterial preparation.

[0019] The application mode of the antibacterial preparation of the third aspect includes but is not limited to any one of the following:

[0020] 1) Environmental disinfection: control of microbial pollution in object surface, water body, living environment and public place;

[0021] 2) Pharmaceutical development: inhibition of related diseases caused by harmful microorganisms;

[0022] 3) Daily chemical products: can be added as a bacteriostatic agent to cleaning agents, cosmetics and other products to achieve daily cleaning effect.

[0023] Further, the harmful microorganisms include, but are not limited to, one or more of Streptococcus, Staphylococcus, Pseudomonas, Bacillus, Candida; further, the harmful microorganisms are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata and Candida albicans.

[0024] Compared with the prior art, the beneficial effects of the present application are:

[0025] The present application is based on the unique niche advantage of lichen symbiotic system. The special microenvironment formed by algae and fungi in the symbiotic layer, through long-term coevolution selection pressure, promotes the endophytic fungi to evolve novel secondary metabolites. The present application first carries out systematic screening on lichens of the genus Melanelia, Parmotrema ) and successfully isolates an endophytic fungus with significant antibacterial activity: Diffugia carbonata LUCF 4343-3, the ethyl acetate extract of which shows broad-spectrum antibacterial properties, and shows inhibitory effect on a variety of clinically common pathogenic bacteria. This discovery not only expands the lichen endophytic fungus resource library in China, but also provides an innovative source for the discovery of antifungal drug lead compounds. BRIEF DESCRIPTION OF DRAWINGS

[0026] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0027] Figure 1 is a colony morphology diagram of Diffugia carbonata LUCF 4343-3 described in Example 1;

[0028] Figure 2 is a bacteriostatic effect diagram of Diffugia carbonata LUCF 4343-3 described in Example 1;

[0029] wherein, Figure 2 A is the inhibitory effect on Staphylococcus aureus CMCC26003;

[0030] Figure 2 B is the inhibitory effect on Candida glabrata ATCC15126;

[0031] Figure 2C is the inhibition effect of Candida albicans CMCC98001;

[0032] Figure 2 D is the inhibition effect of Bacillus subtilis BC.CS136;

[0033] Figure 2 E is the inhibition effect of Pseudomonas aeruginosa CMCC10104;

[0034] Figure 2 F is the inhibition effect of Enterococcus faecalis ZDZA0109;

[0035] Preservation condition:

[0036] The fungus is Dischidia grisaille LUCF 4343-3, preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Huayuancun, West Beichen Road, Beijing, China, on April 8, 2025, and registered as CGMCC No. 41883. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0038] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components and / or combinations thereof.

[0039] In the context of the present specification, the word "comprise" is to be interpreted as meaning "consist at least of". It should not be interpreted as meaning "consist only of".

[0040] In order to enable a person skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0041] Example 1

[0042] I. Strain isolation and identification

[0043] In this study, 32 strains were isolated from the genus Dischidia. The crude extracts of all strains were obtained by fermentation and rotary evaporation, and were preliminarily screened for inhibition zones. One strain with broad-spectrum antibacterial activity was screened for MIC determination and identified asNemania diffusa, Lichen endophytic fungus LUCF 4343-3 。

[0044] 1. Identification and morphological characteristics of the fungus

[0045] The genomic DNA of the lichen endophytic fungus was obtained by rapid extraction. The nucleic acid extraction was completed according to the standard process using the REDExtract-N-Amp™ (Sigma-Aldrich) kit. The total DNA of the endophytic fungus was used as the template, and the ITS1F / ITS4 primer pair was selected for the amplification reaction, and the sequences thereof are as follows:

[0046] Forward: 5'-CTTGGTCATTTAGAGGAAGTAA-3' (SEQ ID NO: 2)

[0047] Reverse: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 3).

[0048] The PCR system was configured as follows: ddH2O 19 µL, 2×Taq PCR MasterMix 25 µL, upstream primer 2 µL, downstream primer 2 µL, DNA template 2 µL, and a total of 50 µL system. The PCR reaction process was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 52℃ for 30 s, extension at 72℃ for 1 min, 34 cycles; and finally extension at 72℃ for 10 min. After the target product was confirmed by 1% agarose gel electrophoresis, the sequence determination was completed by entrusting a professional sequencing agency.

[0049] For microscopic observation of the LUCF 4343-3 strain of the lichen endophytic fungus, the morphological characteristics of the strain are as follows:

[0050] Fungal body characteristics: microscopic observation showed that the mycelium was white, smooth in 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.

[0051] Colony characteristics: when cultured on the PDA medium, white cottony colonies were formed, the mycelium was dense and grew radially, and the edge was irregularly wavy; the back of the colony was white at the edge and yellowish in the middle. Under the culture condition of 25℃, the colony diameter could reach 5 cm within 5 days.

[0052] 2. Fungal extract

[0053] (1) Activation and expansion culture of the strain

[0054] The preserved strain was inoculated to PDA plates and incubated at 25°C for 7 days. The activated mycelium was inoculated to PDB liquid medium, and after 48 hours, it was transferred to fresh PDB medium at a 10% inoculation amount.

[0055] (2) Oscillating culture

[0056] It was placed in a constant temperature shaker and incubated at 25°C and 150 r / min for 5-7 days until the mycelium grew fully.

[0057] (3) Metabolite extraction

[0058] After the culture ended, ethyl acetate was added at a volume ratio of 1:1, and the extraction was allowed to stand for 24 hours. The mycelium was removed by sterile gauze filtration, and the organic phase was collected.

[0059] (4) Crude extract preparation

[0060] The organic phase was concentrated using a rotary evaporator to obtain a solid extract. Methanol was used to dissolve and prepare a crude extract solution with a final concentration of 100 mg / mL.

[0061] II. Verification of antibacterial activity

[0062] 1. Selection of test strains

[0063] Six representative microorganisms were selected for the experiment, including Gram-positive bacteria (Enterococcus faecalis ZDZA0109, Staphylococcus aureus CMCC26003), Gram-negative bacteria (Pseudomonas aeruginosa CMCC10104), Bacillus (Bacillus subtilis BC.CS136), and yeast (Candida glabrata ATCC15126, Candida albicans CMCC98001).

[0064] 2. Experimental operation process

[0065] (1) Under sterile conditions, use a sterile spreader to evenly spread each test bacterial suspension on the surface of MH agar plates;

[0066] (2) Use a sterile puncher to prepare a 8mm diameter sample hole;

[0067] (3) Accurately add 100μL of the crude extract solution to be tested to each hole;

[0068] (4) Set up a methanol solvent control, and set 3 replicates for each experiment.

[0069] 3. Culture and result determination

[0070] The treated culture dish was sealed and incubated in a 37℃, 60% humidity incubator for 24 hours. The precision measuring tool was used to record the size of the inhibition ring, with a measurement accuracy of 0.1mm. The specific experimental results are shown in Table 1. Figure 2 .

[0071] 4. Measurement of MIC value of strain antibacterial

[0072] Take 1.5mL of the concentrated crude extract solution, centrifuge at 12000r / min for 5 minutes to remove the precipitate. Take 50.00, 25.00, 12.50, 6.25, 3.12μL of the supernatant in turn and add to a 96-well plate, and each well is diluted to 50μL with CAMHB medium. The OD600 value of the test bacterial suspension is measured by spectrophotometer, and after adjusting to 1.0, it is diluted 2500 times for standby. Add 50μL of diluted bacterial solution to each well, and after incubation at 37℃ for 12-18 hours, observe the results and determine the minimum drug concentration (MIC) that inhibits bacterial growth. The specific data are shown in Table 1.

[0073] Table 1 Antifungal activity of two kinds of crude extracts

[0074]

[0075] In the above Table 1, "4343-3" refers to the diffusing carbon pad fungus LUCF 4343-3 in the present embodiment, and "4067-1" represents another strain of fungus isolated from the same batch.

[0076] According to the results of Figure 2 and Table 1, the crude extract of the above strain shows inhibitory effect on a variety of different species of fungi and bacteria, and is a broad-spectrum antibacterial active ingredient.

[0077] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of diffusing carbon mat bacteria ( Nemania diffusa ) Application of LUCF 4343-3 ethyl acetate extract in the preparation of antibacterial preparations, characterized in that: The strain was deposited on April 8, 2025, at the General Microbiology Center of China Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its biological deposit number is CGMCC No. 41883. The preparation method of the ethyl acetate extraction is as follows: diffuse carbon mat bacteria ( Nemania diffusa LUCF 4343-3 is inoculated into liquid PDB medium and cultured for 5 to 7 days at an inoculum size of 8 to 12%. Ethyl acetate is added to the medium and soaked for 20 to 25 hours. The solid portion is filtered out and the ethyl acetate is evaporated to obtain the ethyl acetate extract. The harmful microorganisms acted by the antibacterial preparation are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata and Candida albicans; the number of the Staphylococcus aureus is CMCC26003.

2. The carbon mat bacteria according to claim 1 ( Nemania diffusa ) Application of LUCF 4343-3 ethyl acetate extract in the preparation of antibacterial preparations, characterized in that: The application method of the antibacterial preparation is selected from any one of the following: (1) Environmental disinfection: Control of microbial contamination on surfaces, water bodies, living environments, and public places; (2) Pharmaceutical development: inhibiting diseases caused by harmful microorganisms; (3) Daily chemical products: Added as an antibacterial agent to detergents and cosmetics to achieve daily cleaning effects; The microorganisms described in (1), the harmful microorganisms described in (2), or the bacteria inhibited by the antibacterial agent described in (3) are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata, and Candida albicans; the number of Staphylococcus aureus is CMCC26003.

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