Nitrosporium citrinum LCUF 4067-1, microbial agent and application thereof in field of broad-spectrum bacteriostasis

Abstract

The invention belongs to the technical field of antibacterial active microorganisms, and particularly relates to a melanospora citrina strain LCUF 4067-1, a fungicide and application of the melanospora citrina strain LCUF 4067-1 in the field of broad-spectrum bacteriostasis. According to the invention, an endophytic fungus is obtained by screening from large-leaf plum, the endophytic fungus is named as nigrospora citrina LCUF 4067-1, and an ethyl acetate extract of the fungus shows inhibitory activity on a plurality of harmful microorganisms such as enterococcus faecalis, staphylococcus aureus, pseudomonas aeruginosa, bacillus subtilis, candida glabrata and candida albicans; the compound is expected to be applied to development of novel antibiotics or antibacterial preparations, and the broad-spectrum antibacterial effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial active microorganisms, and particularly relates to a Nigrospora aurantiaca LCUF 4067-1, a microbial agent and its application in the field of broad-spectrum antibacterial. Background Art

[0002] Disclosing the information of this background art section is only intended to increase 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] The world is making efforts to discover new antibiotics to address the increasingly severe problem of microbial resistance to existing therapeutic drugs. Screening microbial drugs with high efficiency, low toxicity and novel mechanisms of action has become an important research focus. Fungi, as key producers of bioactive substances, are important sources of microbial drugs. Research shows that endolichenic fungi exhibit broad antibacterial activity. Lichens are symbiotic organisms composed of symbiotic fungi and photosynthetic symbionts. The symbiotic fungi in them are mainly ascomycetes, and a few are basidiomycetes. The photosynthetic symbionts are cyanobacteria or green algae. The symbiotic fungi that make up lichens are also called lichen-forming fungi, while the non-lichen-forming fungi existing in lichens are called endolichenic fungi. The species and quantities of endolichenic fungi are very rich. During the long-term co-evolution process with the host, in addition to producing active substances identical or similar to those of the host, they also derive unique metabolic pathways and produce a large number of compounds with complex structures, novel skeletons and rich biological activities, such as alkaloids, polyketides, terpenoids, quinones and other compounds, becoming an important source of natural medicinal chemistry. These endolichenic fungi (ELF) provide opportunities for discovering emerging natural drugs because they are promising biological resources capable of producing bioactive metabolites covering unique and diverse structural categories.

[0004] What Linnaeus described in the first edition of Species Plantarum in 1753 as " Lichen chinensis " is exactly what is now widely known as Parmelia saxatilis Parmotrema tinctorum (Despr. ex Nyl.) Hale]. And in view of the advantages of the wide geographical distribution, easy discovery, convenient collection and rich biomass of the lichens of this genus, domestic researchers have conducted less research on the endolichenic fungi of the genus Parmelia. Therefore, the inventor believes that taking the genus Parmelia as the screening object is expected to obtain microbial preparations with high antibacterial activity. Summary of the Invention

[0005] Based on the above research background, the present invention uses Parmelia saxatilis ( Parmotrema tinctorumAs a screening object, an endophytic fungus was screened and obtained, named Nigrospora aurantiaca LCUF 4067-1. The ethyl acetate extract of the above strain was confirmed to have inhibitory activities against various bacteria and fungi, and is expected to be developed as a broad-spectrum antibacterial active ingredient in the follow-up.

[0006] Based on the above technical effects achieved, the present invention provides the following technical solutions: In the first aspect, a strain of Nigrospora aurantiaca ( Nigrospora aurantiaca ) LCUF 4067-1 is provided. This strain was deposited on April 8, 2025 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, abbreviated as CGMCC, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its biological deposit number is: CGMCC No. 41884. In this article, it is abbreviated as "Nigrospora aurantiaca LCUF 4067-1".

[0007] The above Nigrospora aurantiaca LCUF 4067-1 was isolated from the genus Parmelia and belongs to lichen endophytic fungi. DNA was extracted by scraping hyphae, and through genomic sequencing alignment, it has a high identity with the genus Nigrospora and was named Nigrospora aurantiaca LCUF4067-1. Its ITS sequence is as shown in SEQ ID NO:1.

[0008] The morphological characteristics of the above Nigrospora aurantiaca LCUF 4067-1 are as follows: Mycelium characteristics: Observation under an optical microscope shows that the hyphae are brown, smooth on the surface, with branches and septa, and the width ranges from 2.3 to 7.5 μm; the conidiophores are degenerate and directly form sporogenous cells. The conidia are mostly solitary, mainly nearly round in shape, smooth and shiny on the surface, black in color, and about 12 to 13 μm in diameter.

[0009] Colony characteristics: When growing on PDA medium, the colony morphology is flat, with a neat edge. Initially, it presents orange and secretes orange pigment; as the culture time extends, a tufted structure gradually forms in the center of the colony, and the color turns black. When cultured at 25°C for 5 days, the colony diameter can expand to 5.5 cm.

[0010] The optimal culture conditions are as follows: PDA medium, 25°C, 5 - 7 days.

[0011] In the second aspect, a bacterial agent is provided, containing the aforementioned Nigrospora aurantiaca LCUF 4067-1, and / or metabolites, cultures or / and extracts of Nigrospora aurantiaca LCUF 4067-1.

[0012] In the third aspect described above, the "metabolite" refers to the chemical substances produced by the strain during growth and reproduction, including primary metabolites and secondary metabolites; the "culture" refers to the general term for the active fungal population and its growth carrier obtained through separation, inoculation, and cultivation under certain culture conditions; the "extract" refers to the single or mixed components separated and concentrated from the bacterial cells and / or their growth carrier by physical and chemical methods. The above-mentioned growth carrier includes solid medium and liquid medium.

[0013] In a preferred embodiment of the present invention, an ethyl acetate extract of the aforementioned Nigrospora aurantia LCUF 4067-1 is provided. This extract has been verified to exhibit inhibitory activity against various harmful microorganisms such as Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata, and Candida albicans. In this embodiment, the preparation method of the above-mentioned ethyl acetate extract is as follows: Nigrospora aurantia LCUF 4067-1 is inoculated into a 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 20-25 h, the solid part is filtered off, and the ethyl acetate is evaporated to obtain the ethyl acetate extract.

[0014] In the third aspect, there is provided the Nigrospora aurantia LCUF 4067-1 described in the first aspect 、 The use of the bacterial agent described in the second aspect in the field of broad-spectrum antibacterial

[0015] The purposes of the use described in the third aspect include but are not limited to any one of the following: (1) For controlling harmful microorganisms in the environment, water bodies, and on the surfaces of articles; (2) For preparing products that can be used to control harmful microorganisms in the environment, water bodies, and on the surfaces of articles; (3) For preventing, improving, or treating diseases caused by harmful microorganisms; (4) For preparing an antibacterial preparation for preventing, improving, or treating diseases caused by harmful microorganisms.

[0016] In the above uses (1)-(4), the harmful microorganisms include but are not limited to one or more of the genera Streptococcus, Staphylococcus, Pseudomonas, Bacillus, and Candida; further, the harmful microorganisms are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata, and Candida albicans.

[0017] Compared with the prior art, the beneficial effects of the present invention are: The special structure of the lichen symbiont (algal-fungal symbiotic layer) forms a unique microecological environment. Long-term coevolution has promoted the development of unique secondary metabolic pathways in endophytic fungi, which theoretically can give birth to a more diverse microbial community than single plants. In this invention, the genus Parmelia was used as the screening object, and the endophytic fungus Nigrospora oryzae LCUF 4067-1 with broad-spectrum antibacterial activity was screened therefrom, which first fills the research gap of domestic researchers on endophytic fungi of Parmelia lichens. Secondly, the invention also provides an ethyl acetate extract of the above-mentioned fungus. After verification, the extract of the above-mentioned fungus shows inhibitory activity against a variety of different types of harmful microorganisms, and has particularly prominent inhibitory activity against specific Candida glabrata and Candida albicans, providing a research basis for the development and screening of new antibiotic compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 It is a colony morphology diagram of Nigrospora oryzae LCUF 4067-1 in Example 1; Figure 2 It is an antibacterial effect diagram of Nigrospora oryzae LCUF 4067-1 in Example 1; Among them, Figure 2 A in it is the inhibitory effect on Staphylococcus aureus CMCC26003; Figure 2 B in it is the inhibitory effect on Candida glabrata ATCC15126; Figure 2 C in it is the inhibitory effect on Candida albicans CMCC98001; Figure 2 D in it is the inhibitory effect on Bacillus subtilis BC.CS136; Figure 2 E in it is the inhibitory effect on Pseudomonas aeruginosa CMCC10104; Figure 2 F in it is the inhibitory effect on Enterococcus faecalis ZDZA0109; Description of the deposit: The fungus is named Nigrospora oryzae LCUF 4067-1, deposited in the China General Microbiological Culture Collection Center, abbreviated as CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 8, 2025, and the registration number in the deposit center is: CGMCC No. 41884. DETAILED DESCRIPTION OF THE INVENTION

[0020] 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 pertains.

[0021] It should be noted that the terms used herein are only for describing specific embodiments 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 forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] 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.

[0023] Example 1 I. Strain isolation and identification In this study, 32 strains were isolated from the genus Parmelia. The crude extracts were obtained by fermenting and rotary evaporating all the strains, and the antibacterial circle screening was initially carried out. One strain with broad-spectrum antibacterial activity was selected for MIC determination and was identified as Nigrospora aurantiaca ， Named as 。

[0024] 1. Identification and morphological characteristics of the fungus Scrape the mycelium for DNA extraction and amplification and sequencing of ITS sequences: The Sigma-Aldrich REDExtract-N-AmpTM kit was used to extract the genome of lichen endophytic fungi according to the instructions. Using the total DNA of the endophytic fungi as a template, PCR amplification was carried out using the fungal universal primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO: 3).

[0025] ​The PCR reaction system is as follows: 19 μL of ddH2O, 25 μL of 2×Taq PCR MasterMix, 2 μL of upstream primer, 2 μL of downstream primer, and 2 μL of DNA template, with a total volume of 50 μL. The PCR reaction procedure: 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; finally, extension at 72 °C for 10 min. The target product was sequenced by Beijing Tsingke Biotechnology Co., Ltd. after confirmation by 1% agarose gel electrophoresis. A phylogenetic tree was constructed and combined with morphology to identify the strains.

[0026] Observation of Nigrospora aurantia LCUF 4067-1 under an optical microscope showed that: the hyphae were brown, smooth on the surface, branched and septate, with a width range of 2.3 - 7.5 μm; the conidiophores were degenerate and directly formed sporogenous cells. The conidia were mostly solitary, mainly nearly round in shape, smooth and shiny on the surface, black in color, with a diameter of about 12 - 13 μm. When this strain grew on PDA medium, the colony morphology was flat, with neat edges, initially orange in color and secreting orange pigment; as the culture time extended, a tufted structure gradually formed in the center of the colony, and the color turned black. After culturing at 25 °C for 5 days, the colony diameter could expand to 5.5 cm. The morphology is shown in Figure 1 。

[0027] 2. Bacterial extracts After activating the above strain, it was inoculated on PDA solid medium and cultured at 25 °C for 7 d. The hyphae of Nigrospora aurantia LCUF4067-1 were scraped and inoculated into PDB liquid medium. Two days later, it was transferred to another new PDB medium according to an inoculation amount of 10%, and cultured by shaking flask at 25 °C and 150 r / min for 5 - 7 d. Ethyl acetate was added to the above liquid medium at a volume ratio of 1:1, and the fungal culture was soaked for 24 h and then filtered through sterile gauze to discard the mycelial balls. The organic layers were collected and combined, and the extract was concentrated under reduced pressure using a rotary evaporator to obtain a solid product, which was dissolved in methanol to obtain a crude extract, and the concentration of the crude extract was prepared to be 100 mg / mL.

[0028] III. Verification of antibacterial activity The antibacterial activity of the above crude extract was verified by the inhibition zone method. The test strains were: Enterococcus faecalis ZDZA0109, Staphylococcus aureus CMCC26003, Pseudomonas aeruginosa CMCC10104, Bacillus subtilis BC.CS136, Candida glabrata ATCC15126, and Candida albicans CMCC98001.

[0029] In a laminar flow hood, the test strain was evenly spread on an MH plate using a disposable spreading rod. An 8-mm hole was punched in the center of the MH plate using a hole puncher, and 100 μL of the crude extract solution of the strain was pipetted into the hole. Methanol solvent was set as the negative control, and independent biological replicates were performed three times. After sealing the culture dish, it was incubated in a constant temperature incubator at 37 °C for 24 h (relative humidity 60%). Subsequently, the diameter of the inhibition zone was measured using a vernier caliper (accurate to 0.1 mm), and the results are as Figure 2 shown.

[0030] Measurement of the MIC value of the strain against bacteria: The crude extract solution after rotary evaporation was added to a 1.5-mL centrifuge tube and centrifuged at 12,000 r / min for 5 min. Then, 50.00, 25.00, 12.50, 6.25, and 3.12 μL of the supernatant were respectively pipetted into a 96-well plate and made up to 50 μL with CAMHB medium. The absorbance value of the test bacterial suspension was measured at a wavelength of 600 nm using a spectrophotometer. After the absorbance value reached 1.0, it was diluted 2500 times for standby. 50 μL of the test bacterial suspension was added to the 96-well plate, and the minimum inhibitory concentration (MIC) of the crude extract solution was detected after incubation at 37 °C for 12 - 18 h. The results are shown in Table 1.

[0031] Table 1 Antibacterial activities of two crude extracts In Table 1 above, "4067-1" refers to Nigrospora aurantia LCUF 4067-1 in this example, and "4343-3" represents another strain of fungus isolated from the same batch.

[0032] According to Figure 2 and the results in Table 1, the crude extracts of the above two strains showed inhibitory effects on various fungi and bacteria of different genera and species, and are a broad-spectrum antibacterial active ingredient.

[0033] The above are only the preferred embodiments of the present invention and are not used 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 Nigrospora oryzae Nigrospora aurantiaca LCUF 4067-1, which was deposited on April 8, 2025 at the General Microbiological Center of the China National Culture Collection Center, abbreviated as CGMCC, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its biological deposit number is: CGMCC No. 41884.

2. The Nigrospora oryzae Nigrospora aurantiaca LCUF 4067-1 as claimed in claim 1, characterized in that The ITS sequence of the strain is shown in SEQ ID NO:

1.

3. The Nigrospora oryzae Nigrospora aurantiaca ) LCUF 4067-1 as claimed in claim 1, characterized in that The morphological characteristics of the orange black spore mold LCUF 4067-1 are as follows: Bacterial characteristics: Observation under an optical microscope showed that the hyphae were brown, smooth, branched and septate, with a width ranging from 2.3 to 7.5 μm; the conidiophores were degenerate and directly formed spore-forming cells; conidia were mostly solitary, with a nearly round shape, smooth and shiny surface, black in color, and a diameter of 12 to 13 μm; Colony characteristics: When grown on PDA medium, the colonies are flat in morphology, with neat edges, initially appearing orange and secreting orange pigments; as the culture time increases, a tufted hair-like structure gradually forms in the center of the colonies, and the color turns black.

4. A bacterial agent, characterized in that, containing the metabolite, culture or extract of Nigrospora oryzae ( Nigrospora aurantiaca ) LCUF 4067-1, or Nigrospora oryzae ( Nigrospora aurantiaca ) LCUF 4067-1.

5. The bacterial agent according to claim 4, characterized in that, The microbial agent is the ethyl acetate extract of Nigrospora aurantiaca Nigrospora aurantiaca Nigrospora aurantiaca LCUF 4067-1, and the preparation method is as follows: inoculate Nigrospora aurantiaca Nigrospora aurantiaca LCUF 4067-1 into the liquid PDB medium, culture for 5-7 days according to the 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 Nigrospora oryzae ( Nigrospora aurantiaca ) LCUF 4067-1, and the microbial agent according to claim 4 or 5, in the field of broad-spectrum antibacterial 7. The application according to claim 6, characterized in that, The application is selected from any one of the following: (1) Used to prevent and eliminate harmful microorganisms in the environment, water bodies, and surfaces of objects; (2) Used to prepare products that can be used to prevent and eliminate harmful microorganisms in the environment, water bodies, and surfaces of objects; (3) Used to prepare an antibacterial preparation.

8. The application according to claim 7, wherein The harmful microorganisms are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata and Candida albicans.

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