Laricifomes officinalis strain, fermentation liquor thereof and application thereof

Abstract

The application discloses a strain of Flammulina velutipes, a fermentation liquor of the strain and application thereof, and belongs to the technical field of microorganisms.The Flammulina velutipes strain R12 is preserved in the Guangdong Provincial Microbial Culture Collection Center on August 18, 2025, the address of the preservation center is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100, and the preservation number is GDMCC NO. 66855.The fermentation liquor of the Flammulina velutipes strain R12 disclosed by the application has a specific bacteriostatic effect on vancomycin moderately drug-resistant Staphylococcus aureus XN108, and provides an important candidate substance for development of a new type of anti-Staphylococcus aureus drug.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Flammulina salsa*, its fermentation broth, and its applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus is an opportunistic pathogen widely found in the natural environment. Its pathogenicity is closely related to the various virulence factors it secretes and its strong environmental adaptability, including its resistance to high temperatures, high salts, and a broad pH range (4.8–9.4). Furthermore, Staphylococcus aureus can form biofilms, further evading host immunity and antibiotics, leading to chronic or refractory infections.

[0003] The widespread use and even abuse of antibiotics has accelerated the evolution of drug-resistant strains, with methicillin-resistant Staphylococcus aureus (MRSA) being particularly prominent. Currently, the resistance rate of MRSA to commonly used antibiotics such as β-lactams and macrolides exceeds 80%, posing a major challenge to global public health. Vancomycin, as the last line of defense against MRSA infection, has seen its treatment options further limited by the emergence of moderately resistant strains (VISA) and completely drug-resistant strains (VRSA). The transmission routes of drug-resistant strains are complex and diverse, including human-to-human contact, contaminated food, medical procedures, and environmental biofilms. Furthermore, the overuse of antibiotics in the livestock industry has exacerbated the evolution and spread of resistance, significantly increasing the difficulty of infection control. Therefore, while strictly controlling infection routes and standardizing antibiotic use, it is urgent to develop novel antimicrobial agents and find highly effective, safe, and specific antibacterial substances. This has significant practical and clinical application value in addressing the growing threat of drug resistance in Staphylococcus aureus. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a strain of *Flammulina velutipes*, its fermentation broth, and its application, so as to solve the technical problem that existing preparations have little effect on the control of multidrug-resistant *Staphylococcus aureus*.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a *Enoki mushroom* (also known as *Symplocos edulis*). Flammulina rossica ) strain R12, Flammulina salina strain R12 ( Flammulina rossica It was deposited on August 18, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO. 66855.

[0006] The present invention also discloses a fermentation broth containing the above-mentioned Flammulina velutipes strain R12. The fermentation broth is prepared by inoculating Flammulina velutipes strain R12 into a culture medium, culturing it at 20-30℃ and 150-170r / min for 13-15 days, filtering, and concentrating to obtain the fermentation broth of Flammulina velutipes strain R12.

[0007] Based on the above technical solution, the present invention can be further improved as follows:

[0008] Furthermore, the culture medium is a fungal fermentation medium or a potato glucose broth medium.

[0009] The present invention also discloses a microbial inoculant comprising the above-mentioned *Flammulina velutipes* strain R12 or fermentation broth.

[0010] This invention also discloses the application of *Flammulina salsa* strain R12, fermentation broth of *Flammulina salsa* strain R12, or microbial inoculants in the preparation of agents to inhibit pathogens.

[0011] Based on the above technical solution, the present invention can be further improved as follows:

[0012] Furthermore, the pathogen is multidrug-resistant Staphylococcus aureus.

[0013] Furthermore, the pathogen is VISA.

[0014] The beneficial effects of this invention are as follows: By screening the fermentation broth of wild macrofungi, this invention obtained the *Flammulina velutipes* strain R12, which has significant antibacterial activity against vancomycin-resistant Staphylococcus aureus (VISA). The fermentation broth of R12 has a specific antibacterial effect against vancomycin-resistant Staphylococcus aureus XN108, providing an important candidate substance for the development of novel anti-Staphylococcus aureus drugs. Attached Figure Description

[0015] Figure 1 The results of the clustering phylogenetic tree of the strains;

[0016] Figure 2 The results of different concentrations of oxacillin inhibiting the activity of indicator bacteria XN108, N315 and NCTC8325.

[0017] Figure 3 The results of different concentrations of vancomycin inhibiting the activity of indicator bacteria XN108, N315 and NCTC8325.

[0018] Figure 4 The results show the antibacterial effects of the fermentation broth of *Enoki mushroom* R12 on indicator bacteria XN108, N315, and NCTC8325. Detailed Implementation

[0019] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0020] Example 1: Isolation and Identification of Enoki Mushroom R12

[0021] 1. Separation

[0022] Mycelial cultures were isolated from the fruiting bodies of *Flammulina salsa*. The tissue isolation steps were as follows: The surface of the fungal fruiting body was gently wiped with a cotton ball soaked in 75% alcohol. The fruiting body was then longitudinally cut open with a scalpel sterilized by an alcohol lamp to expose the internal mycelium. A 3-5 mm section was cut from the junction of the cap and stipe. 3 Small pieces of tissue were placed in the center of a PDA plate medium and cultured at 25°C until mycelia grew. The tips of the mycelia were cut off with a sterile scalpel and placed in the center of a PDA slant medium. The culture was cultured at 25°C until mycelia were fully grown, and the mycelial culture was obtained. The culture was stored at 4°C, and the selected strains were subjected to subsequent molecular identification.

[0023] 2. Identification (ITS sequencing)

[0024] A small piece of agar was picked from a mycelium tube stored at 4℃ and placed onto a PDA plate. The activated mycelium was obtained by static culture at 25℃ for 7 days. The surface mycelium was collected, and the genome was extracted using a DNA extraction kit (omega, D3390-01). PCR amplification was performed using ITS universal primers ITS4 and ITS5, and the sample was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis.

[0025] The obtained ITS sequence was BLAST-aligned in the NCBI database. The ITS sequence of the strain is shown below (SEQ ID NO.1):

[0026] Sequencing results showed that the amplified ITS sequence of this strain was 664 bp in length. Homology comparison in GenBank showed that the strain shared 100% homology with the ITS sequence of *Flammulina salsa*. A phylogenetic tree was constructed using the maximum likelihood method in MEGA 12 software, and the results are as follows: Figure 1 As shown, the strain is similar to *Flammulina salina* (…). Flammulina rossica The bacteria aggregated together, therefore, the strain can be identified as *Flammulina salina*. Flammulina rossica .

[0027] The obtained strain was named R12 and deposited on August 18, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO. 66855.

[0028] Example 2: Antibacterial effect of Enoki mushroom R12 against drug-resistant bacteria

[0029] 1. Laboratory supplies

[0030] The sources of the indicator bacteria and antibiotics used are shown in Table 1.

[0031] Table 1. Source Information of Indicator Bacteria and Antibiotics

[0032]

[0033] (1) Preparation of indicator bacterial suspension

[0034] Pick one loopful of indicator bacteria preserved at -80℃ using a sterile inoculation loop and streak it onto an LA plate. Incubate at 37℃ until visible colonies appear (approximately 15 hours). Pick the activated bacterial colonies into a 12 mL test tube containing 2 mL of LB medium and incubate overnight at 37℃ with shaking at 200 rpm (approximately 12 hours). Transfer the cultured bacterial solution at a 1:100 (V:V) ratio into a new 12 mL test tube containing 2 mL of LB medium and incubate at 37℃ with shaking at 200 rpm until the logarithmic growth phase (approximately 5 hours). Measure the absorbance (OD) of the bacterial solution at 600 nm using a spectrophotometer. 600 Use sterile water to adjust OD 600 The value is 0.05, at which point the bacterial concentration is 2.5 × 10⁻⁵. 7 CFU / mL.

[0035] (2) Antibiotic preparation

[0036] Preparation of oxacillin working solution: Weigh an appropriate amount of oxacillin powder and prepare a stock solution of 64 mg / mL with sterile water. Then prepare working solutions with gradient concentrations of 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL and 16 μg / mL.

[0037] Vancomycin working solution preparation: Weigh an appropriate amount of vancomycin hydrochloride powder and prepare a stock solution of 32 mg / mL with sterile water. Then prepare working solutions with gradient concentrations of 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL and 16 μg / mL.

[0038] (3) Culture medium

[0039] LB (Luria-Bertani) medium: 1% (w / v) NaCl, 1% (w / v) tryptone, 0.5% (w / v) yeast extract, dissolved in ultrapure water and brought to a final volume of 1L. Autoclave at 121℃ for 15 min.

[0040] LA (Luria-Bertani Agar) medium: Add 1.5% (w / v) agar A to LB medium. Autoclave at 121°C for 15 min.

[0041] MH II (Mueller Hinton II) solid medium: Weigh 22g MH II powder (BD, BBLTM Mueller Hinton II Broth, 212322), dissolve in ultrapure water and bring the volume to 1L, add 1.5% (w / v) agar A. Autoclave at 121℃ for 10min.

[0042] PDA (Potato Dextrose Agar) medium: Weigh 46g of PDA powder (Solarbio, P8931), dissolve in ultrapure water and bring the volume to 1L. Autoclave at 121℃ for 15min.

[0043] Macrofungal fermentation medium: 3% (w / v) glucose, 2% (w / v) tryptone, 0.3% (w / v) KH2PO4, 0.1% (w / v) MgSO4·7H2O, dissolved in ultrapure water and brought to a final volume of 1L. Autoclave at 121℃ for 15 min.

[0044] 2. Preparation of fermentation broth for the tested macrofungi

[0045] A small piece of agar was picked from an R12 test tube stored at 4℃ and placed onto a PDA plate. Activated mycelia were obtained by static incubation at 25℃ for 7 days. Seven pieces of activated mycelia, about the size of rice grains, were picked and placed into a 500mL Erlenmeyer flask containing 300mL of macrofungi fermentation medium. The flask was then shaken at 25℃ and 160r / min for 14 days. The fungal fermentation products were collected. Mycelia were first removed by filtration through gauze, and the filtrate was then filtered through qualitative filter paper to obtain the fermentation broth. The fermentation broth was concentrated to 50mL using a rotary evaporator (70℃ water bath). The concentrate was then filtered through a 0.22μm sterile filter membrane to obtain the fermentation broth of *Flammulina velutipes* R12, which was then frozen at -20℃.

[0046] 3. Punch-hole method inhibition zone test

[0047] An antibacterial agar plate was prepared by adding 20 mL of MH II solid medium to a 90 mm sterile petri dish; 80 μL of OD was then used. 600A 0.05 μL bacterial suspension was spread evenly on a plate using a sterile triangular spreading stick, and then perforated with a 6 mm punch. Experimental group: 60 μL of filtered and sterilized fermentation broth was injected into the perforated wells; control group: 60 μL of oxacillin or vancomycin working solution was injected into the perforated wells. The plates were then incubated at 37°C for 15 hours. Finally, photographs were taken and the size of the inhibition zone was measured. The formula for calculating the radius of the inhibition zone is:

[0048] ;

[0049] Where a is the radius of the inhibition zone (mm); b is the diameter of the inhibition zone of the treated sample (mm); and c is the diameter of the circular hole (mm).

[0050] 4. Experimental Results

[0051] (1) Antibacterial effect of antibiotics

[0052] The antibacterial effect of oxacillin is as follows Figure 2 As shown in the figure, oxacillin did not form a significant inhibition zone against vancomycin-resistant Staphylococcus aureus XN108 and methicillin-resistant Staphylococcus aureus N315, indicating no significant antibacterial activity.

[0053] For methicillin-sensitive Staphylococcus aureus NCTC8325, a weak inhibition zone was formed at a concentration of 1 μg / mL of oxacillin, and a distinct inhibition zone was formed at 2 μg / mL. The inhibition zone gradually increased in size with increasing concentration. When the concentration of oxacillin was increased to 8 and 16 μg / mL, the radii of the inhibition zones were 7.92±0.39 mm and 9.44±0.68 mm, respectively.

[0054] The antibacterial effect of vancomycin is as follows: Figure 3 As shown in the figure, vancomycin did not form a clear inhibition zone against vancomycin-resistant Staphylococcus aureus XN108, indicating no significant antibacterial activity.

[0055] When the concentration of vancomycin was 1 μg / mL, it formed a weak inhibition zone against N315. At 2 μg / mL, it formed a distinct inhibition zone. As the concentration increased, the inhibition zone gradually became larger. When the concentration of vancomycin increased to 8 and 16 μg / mL, the radii of the inhibition zones were 2.73±0.25 mm and 3.24±0.39 mm, respectively, indicating that vancomycin has good antibacterial activity against N315.

[0056] Vancomycin formed a weak inhibition zone against NCTC8325 at a concentration of 2 μg / mL, and the inhibition zone gradually increased in size with increasing concentration. When the concentration of vancomycin increased to 8 and 16 μg / mL, the radii of the inhibition zones were 1.8 ± 0.46 mm and 2.84 ± 0.52 mm, respectively. This indicates that both oxacillin and vancomycin have good antibacterial activity against methicillin-sensitive Staphylococcus aureus NCTC8325.

[0057] The above results indicate that the vancomycin-resistant Staphylococcus aureus XN108 exhibits strong resistance to both oxacillin and vancomycin, highlighting the urgent need to develop targeted new antibacterial agents.

[0058] (2) Antibacterial results of fermentation broth of Enoki mushroom R12

[0059] The antibacterial results of *Flammulina velutipes* R12 fermentation broth (concentration 0.37 g / mL) against different drug-resistant *Staphylococcus aureus* are as follows: Figure 4 As shown in the figure, the R12 fermentation broth formed a significant inhibition zone (inhibition zone radius 5.32±0.76 mm) against vancomycin-resistant Staphylococcus aureus XN108, but no significant inhibition zone was formed against methicillin-resistant Staphylococcus aureus N315 and methicillin-sensitive Staphylococcus aureus NCTC8325. These results indicate that the R12 fermentation broth has a specific antibacterial effect against XN108, and its fermentation broth contains highly effective antibacterial components against vancomycin-resistant Staphylococcus aureus, showing potential for development into a novel antibacterial agent.

Claims

1. A type of enoki mushroom (Vigna arvense) Flammulina rossica strain R12, characterized in that, The willow enoki mushroom ( Flammulina rossica Strain R12 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 18, 2025, with accession number GDMCC NO. 66855.

2. A fermentation broth, characterized in that, The fermentation broth is prepared as follows: the *Flammulina velutipes* strain R12 described in claim 1 is inoculated into a culture medium and cultured at 20-30℃ and 150-170 r / min for 13-15 days. The mycelium is removed by filtration and the broth is concentrated to obtain the fermentation broth of *Flammulina velutipes* strain R12. The culture medium is a fungal fermentation medium. The fungal fermentation medium consists of: 3% (W / V) glucose, 2% (W / V) tryptone, 0.3% (W / V) KH2PO4, and 0.1% (W / V) MgSO4·7H2O.

3. A microbial inoculant, characterized in that, Includes the fermentation broth as described in claim 2.

4. The application of the *Flammulina velutipes* strain R12 according to claim 1, the fermentation broth according to claim 2, or the microbial agent according to claim 3 in the preparation of pathogen-inhibiting agents, characterized in that, The pathogen is vancomycin-resistant Staphylococcus aureus XN108.

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