A strain of Melanosporium citrinum LCUF 4067-1, a microbial agent, and its application in broad-spectrum antibacterial applications

Abstract

The present invention belongs to the field of antibacterial active microorganism technology, and specifically relates to a strain of Citrus citriodora LCUF 4067-1, a bacterial agent, and its application in the field of broad-spectrum antibacterial. The present invention screened an endophytic fungus from Prunus armeniaca and named Citrus citriodora LCUF 4067-1. Black spore orange )LCUF 4067‑1 ， The ethyl acetate extract of this fungus shows inhibitory activity against a variety of harmful microorganisms such as Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida glabrata and Candida albicans, and is expected to be used in the development of new antibiotics or antibacterial preparations to achieve broad-spectrum antibacterial effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial active microorganisms, and particularly relates to a strain of Nigrospora citrinum LCUF 4067-1, a bacterial agent and an application thereof in the field of broad-spectrum antibacterial activity. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Global efforts are underway to discover new antibiotics to address the increasing problem of microbial resistance to existing therapeutics. Screening for microbial drugs with high efficacy, low toxicity, and novel mechanisms of action has become a key research focus. Fungi, as key producers of bioactive compounds, are an important source of microbial drugs. Studies have shown that endophytic fungi in lichens exhibit a wide range of antimicrobial activities. Lichens are symbiotic associations composed of symbiotic fungi and photosynthetic symbionts. The symbiotic fungi are primarily ascomycetes, with a few being basidiomycetes, and the photosynthetic symbionts are cyanobacteria or green algae. The symbiotic fungi that make up lichens are called lichen-forming fungi, while the non-lichen-forming fungi that reside within lichens are called lichen endophytes. Lichen endophytes are extremely diverse in species and abundance. Through long-term coevolution with their hosts, they not only produce the same or similar active compounds as their hosts but also develop unique metabolic pathways, producing a large number of compounds with complex structures, novel scaffolds, and rich bioactivities, such as alkaloids, polyketides, terpenes, and quinones, making them an important source of natural medicinal chemicals. These endophytic fungi (ELFs) from lichens offer opportunities for the discovery of emerging natural medicines as they are promising bioresources capable of producing bioactive metabolites covering unique and diverse structural classes.

[0004] In 1753, Linnaeus described the " Lichen chinensis "It is the now well-known big-leaf plum Parmotrema tinctorum (Despr. ex Nyl.) Hale]. Given the advantages of this genus, such as its widespread geographical distribution, ease of discovery and collection, and abundant biomass, research on endophytic fungi in the genus Hale is relatively limited in China. Therefore, the inventors believe that using Hale as a screening target may offer the potential for obtaining microbial preparations with high antimicrobial activity. Summary of the Invention

[0005] According to the above research background, the present invention uses Prunus mume ( Parmotrema tinctorum) were selected as the target for screening, and an endophytic fungus strain, named Melanosporium citrinum LCUF 4067-1, was isolated. The ethyl acetate extract of this strain has been shown to exhibit inhibitory activity against a variety of bacteria and fungi, and is expected to be developed as a broad-spectrum antimicrobial active ingredient.

[0006] Based on the above technical effects, the present invention provides the following technical solutions:

[0007] In the first aspect, a strain of black spore mold ( Nigrospora aurantiaca ) LCUF 4067-1, which 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 accession number is CGMCC No. 41884. It is referred to herein as "Melanosporium citrinum LCUF 4067-1."

[0008] The above-mentioned black spore mold LCUF 4067-1 was isolated from the genus Macrophylla and belongs to the lichen endophytic fungus. DNA was extracted by scraping mycelium. After genome sequencing, it showed high identity with the genus Black spore mold and was named as black spore mold LCUF4067-1. Its ITS sequence is shown in SEQ ID NO: 1.

[0009] The morphological characteristics of the orange black spore mold LCUF 4067-1 are as follows:

[0010] Fungal characteristics: Light microscopy reveals brown hyphae with smooth surfaces, branches, and septa, ranging in width from 2.3 to 7.5 μm. Conidiophores are degenerate, forming conidial cells directly. Conidia are mostly solitary, predominantly nearly round, smooth and glossy, black in color, and approximately 12 to 13 μm in diameter.

[0011] Colony Characteristics: When grown on PDA medium, the colonies are flat with neat edges, initially appearing orange and secreting orange pigment. Over time, a tufted, hair-like structure gradually forms in the center of the colony, turning black. After 5 days of cultivation at 25°C, the colonies can expand to 5.5 cm in diameter.

[0012] The optimal culture conditions are as follows: PDA medium at 25°C for 5-7 days.

[0013] In a second aspect, a bacterial agent is provided, comprising the aforementioned Nigrospora citrinum LCUF 4067-1, and / or metabolites, cultures, or / and extracts thereof.

[0014] In the third aspect above, the term "metabolites" refers to chemical substances produced during the growth and reproduction of a strain, including primary and secondary metabolites; the term "culture" refers to the collective term for the active fungal population and its growth medium obtained through isolation, inoculation, and cultivation under specific culture conditions; and the term "extract" refers to a single or mixed component obtained by separating and concentrating the fungus and / or its growth medium through physical or chemical methods. The aforementioned growth medium includes solid culture media and liquid culture media.

[0015] In one embodiment of the present invention with better effects, an ethyl acetate extract of the aforementioned black spore mold LCUF 4067-1 is provided, which 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; in this embodiment, the preparation method of the above-mentioned ethyl acetate extract is as follows: black spore mold LCUF 4067-1 is inoculated into liquid PDB culture medium and cultured for 5 to 7 days at an inoculum size of 8 to 12%; ethyl acetate is added to the culture 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.

[0016] In the third aspect, the orange black spore mold LCUF 4067-1 described in the first aspect is provided. 、 The second aspect is the application of the bacterial agent in the field of broad-spectrum antibacterial.

[0017] The purpose of the application in the third aspect is not limited to any of the following:

[0018] (1) Used to prevent and eliminate harmful microorganisms in the environment, water bodies, and surfaces of objects;

[0019] (2) Used to prepare products that can be used to prevent and eliminate harmful microorganisms in the environment, water bodies, and surfaces of objects;

[0020] (3) Used to prevent, improve or treat diseases caused by harmful microorganisms;

[0021] (4) Used to prepare an antibacterial preparation for preventing, improving or treating diseases caused by harmful microorganisms.

[0022] In the above applications (1)-(4), the harmful microorganisms include but are not limited to one or more of 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.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The unique structure of lichen symbionts (the algal-fungal symbiotic layer) creates a unique microecological environment. Long-term coevolution has driven the development of unique secondary metabolic pathways in endophytic fungi, theoretically enabling a richer microbial community than a single plant. This study screened the genus Macrophylla and identified the broad-spectrum antimicrobial endophytic fungus, Melanosporium citrinum LCUF 4067-1, filling a gap in domestic research on endophytic fungi in Macrophylla lichens. Furthermore, the present invention provides an ethyl acetate extract of this fungus, which has been shown to exhibit inhibitory activity against a variety of harmful microorganisms, with particularly strong activity against Candida glabrata and Candida albicans. This provides a research foundation for the development and screening of new antibiotic compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 This is a colony morphology diagram of the orange black spore mold LCUF 4067-1 in Example 1;

[0027] Figure 2 This is a diagram showing the antibacterial effect of the orange black mold LCUF 4067-1 in Example 1;

[0028] in, Figure 2 A in the middle is the inhibitory effect of Staphylococcus aureus CMCC26003;

[0029] Figure 2 Middle B is the inhibitory effect of Candida glabrata ATCC15126;

[0030] Figure 2 Middle C is the inhibitory effect of Candida albicans CMCC98001;

[0031] Figure 2 D in the middle is the inhibitory effect of Bacillus subtilis BC.CS136;

[0032] Figure 2 E in the middle represents the inhibitory effect of Pseudomonas aeruginosa CMCC10104;

[0033] Figure 2 F in the middle represents the inhibitory effect of Enterococcus faecalis ZDZA0109;

[0034] Description of preservation:

[0035] The fungus is named Melanosporium citrinum LCUF 4067-1, and is deposited in the General Microbiology Center of the China Culture Collection Administration, abbreviated as CGMCC, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 8, 2025, and the registration number of the collection center is: CGMCC No. 41884. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and 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 skilled in the art to which the present invention belongs.

[0037] 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 form is 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.

[0038] 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.

[0039] Example 1

[0040] 1. Strain Isolation and Identification

[0041] In this study, 32 strains were isolated from the genus Macrophylla. All the strains were fermented and evaporated to obtain crude extracts. A preliminary inhibition zone screening was performed and one strain with broad-spectrum antibacterial activity was screened and subjected to MIC determination. It was identified as Nigrospora citrinum ( Nigrospora aurantiaca ) ， Named as Melanosporium citrinum LCUF 4067-1 。

[0042] 1. Identification and morphological characteristics of bacteria

[0043] Mycelial DNA was scraped for extraction. ITS sequence amplification and sequencing: The genome of the endophytic fungus in lichen was extracted using the Sigma-Aldrich REDExtract-N-Amp™ kit according to the manufacturer's instructions. PCR amplification was performed using the universal fungal primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO: 3) using total endophytic fungal DNA as a template.

[0044] The PCR reaction system was as follows: 19 µL ddH₂O, 25 µL 2× Taq PCR MasterMix, 2 µL upstream primer, 2 µL downstream primer, and 2 µL DNA template, for a total of 50 µL. The PCR process included initial denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The target product was confirmed by 1% agarose gel electrophoresis and sequenced by Beijing Qingke Biotechnology Co., Ltd. A phylogenetic tree was constructed and bacterial species were identified based on morphology.

[0045] Observation of the orange black spore mold LCUF 4067-1 under an optical microscope showed that the hyphae were brown, with a smooth surface, branches and septa, and a width ranging from 2.3 to 7.5 μm; the conidiophores degenerated and directly formed spore-forming cells. Conidia were mostly solitary, with a nearly round shape, a smooth and shiny surface, black in color, and a diameter of about 12 to 13 μm. When the strain grew on PDA culture medium, the colony morphology was flat, with neat edges, initially orange in color, and secreted orange pigments; as the culture time increased, a tufted hair-like 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 can expand to 5.5 cm. See the morphology. Figure 1 .

[0046] 2. Bacterial extract

[0047] After activation, the strain was inoculated onto PDA solid medium and cultured at 25°C for 7 days. Mycelia of LCUF4067-1 were scraped and inoculated into PDB liquid medium. Two days later, the inoculation was transferred to fresh PDB medium at a 10% inoculum and cultured in shake flasks at 25°C and 150 rpm for 5-7 days. Ethyl acetate was added to the liquid medium at a volume ratio of 1:1. The fungal culture was soaked for 24 hours before being filtered through sterile gauze and the mycelial pellets discarded. The organic layers were collected and combined, and the extracts were concentrated under reduced pressure on a rotary evaporator to obtain a solid product. This solid product was dissolved in methanol to obtain a crude extract at a concentration of 100 mg / mL.

[0048] 3. Antimicrobial Activity Verification

[0049] The antibacterial activity of the crude extracts 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.

[0050] In the clean bench, use a disposable coating rod to evenly spread the test strain on the MH plate. Use a hole puncher to punch an 8 mm hole in the center of the MH plate, and use a pipette to draw 100 μL of the crude strain extract solution into the hole. Set methanol solvent as the negative control, and repeat the experiment three times. After sealing the culture dish, incubate it in a constant temperature incubator at 37 ° C for 24 h (relative humidity 60%), and then measure the diameter of the inhibition zone (accurate to 0.1 mm) with a vernier caliper. The results are as follows: Figure 2 shown.

[0051] To determine the antibacterial MIC of the strains, add the crude extract after rotary evaporation to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 5 minutes. Then, aliquots of 50.00, 25.00, 12.50, 6.25, and 3.12 μL of the supernatant were added to a 96-well plate. The volume was then made up to 50 μL with CAMHB medium. The absorbance of the test bacterial suspension at 600 nm was measured using a spectrophotometer. When the absorbance reached 1.0, the suspension was diluted 2500-fold for later use. Fifty μL of the test bacterial suspension was added to a 96-well plate and incubated at 37°C for 12–18 hours. The minimum inhibitory concentration (MIC) of the crude extract was determined. The results are shown in Table 1.

[0052] Table 1 Antibacterial activity of two crude extracts

[0053]

[0054] In the above Table 1, "4067-1" refers to the Citrullus citrinum LCUF 4067-1 in this example, and "4343-3" refers to another fungus isolated from the same batch.

[0055] in accordance with Figure 2 As shown in Table 1, the crude extracts of the two strains mentioned above showed inhibitory effects on a variety of fungi and bacteria of different genera, and are broad-spectrum antibacterial active ingredients.

[0056] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Orange black mold ( Nigrospora aurantiaca ) The application of the LCUF 4067-1 bacterial agent in the field of broad-spectrum antibacterial, characterized in that: The orange black spore mold ( Nigrospora aurantiaca ) LCUF 4067-1, which 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. 41884. The bacterial agent is black spore mold ( Nigrospora aurantiaca ) The ethyl acetate extract of LCUF 4067-1 was prepared as follows: Nigrospora aurantiaca ) LCUF 4067-1 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 application in the field of broad-spectrum antibacterial is selected from any one of the following: (1) Used to remove harmful microorganisms in the environment, water bodies, and surfaces of objects; (2) Used to prepare products that can be used to remove harmful microorganisms from the environment, water bodies, and surfaces of objects; (3) Used to prepare an antimicrobial preparation for inhibiting harmful microorganisms; In (1)-(3), the harmful microorganisms are one or more of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida glabrata and Candida albicans, and the Candida albicans is numbered CMCC98001.

2. Orange black spore mold as claimed in claim 1 ( Nigrospora aurantiaca ) The application of the LCUF 4067-1 bacterial agent in the field of broad-spectrum antibacterial, characterized in that: The ITS sequence of the strain is shown in SEQ ID NO:

1.

3. Orange black spore mold as claimed in claim 1 ( Nigrospora aurantiaca ) The application of the LCUF 4067-1 bacterial agent in the field of broad-spectrum antibacterial, characterized in that: The morphological characteristics of the orange black spore mold LCUF 4067-1 are as follows: Fungal characteristics: Optical microscopy revealed brown hyphae with smooth surfaces, branches, and septa, ranging in width from 2.3 to 7.5 μm. Conidiophores degenerate, forming conidial cells directly. Conidia are mostly solitary, nearly round in shape, smooth and shiny, black, and 12 to 13 μm in diameter. Colony characteristics: When grown on PDA medium, the colonies are flat with neat edges, initially appearing orange and secreting orange pigment. As the culture time increases, a tufted hair-like structure gradually forms in the center of the colony, and the color turns black.

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