A strain of marasmius rotula dcy6478, its culture medium and application

Abstract

The application discloses a strain of Marasmiellus sp. DCY6478, a culture medium thereof and an application, and belongs to the technical field of microorganisms. The application discloses a strain of Marasmiellus sp. DCY6478 with a preservation number of GDMCC No. 65883. Experimental verification shows that the most suitable culture medium formula for the growth of the Marasmiellus sp. is 40% apple sawdust, 36% corn cob, 14% cotton seed hull, 1% lime and 1% gypsum in percentage by weight. It is verified that the fermentation liquor has inhibitory effects on a plurality of pathogenic bacteria. It is verified that the DPPH free radical scavenging capacity, superoxide dismutase activity and catalase activity of the fermentation mycelium are higher than those of the fruiting body, and the fermentation mycelium or the fruiting body can be used as a natural antioxidant source. It is verified that the fermentation mycelium or the fruiting body has the abilities of secreting amino acids, polysaccharides and producing crude protein, and provides a new raw material selection for food, medicine and other industries.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of white Michelia champaca DCY6478, its culture medium, and its applications. Background Technology

[0002] Marasmiellus candidus, belonging to the genus Marasmiellus of the family Omphalotaceae within the phylum Basidiomycota, order Agaricales, is a group of large fungi commonly found in subtropical regions. As important pathogens and medicinal fungi of subtropical plants, they have potential research and application value in the fields of ecosystems and medicine. However, research on the genus Marasmiellus started late, received insufficient attention, and suffers from numerous problems such as confusion regarding scientific names and serious misidentification.

[0003] In its natural environment, *Micropignum moss* typically decomposes in decaying wood and plant humus, making its growth environment highly susceptible to external disturbances. In recent years, human activities have led to ecological damage, with deforestation and habitat fragmentation becoming increasingly severe, significantly reducing the natural growing space of *Micropignum moss*. Simultaneously, over-collection is rampant. On the one hand, some individuals, lacking sufficient understanding of the ecological value of *Micropignum moss*, collect it indiscriminately in the wild to satisfy needs for collection or medicinal purposes based on misconceptions. On the other hand, the increasing demand for specimens and research materials as research on the *Micropignum* genus further exacerbates the pressure of wild collection. This has resulted in a sharp decline in wild *Micropignum moss* resources, posing a survival crisis and making it difficult to maintain a stable population size. It may even have a cascading impact on the material cycle and energy flow of its ecosystem.

[0004] From the perspective of artificial cultivation, research and practice on the artificial cultivation of *Marasmiellus hupehensis* are severely lagging behind. Because naturally collected *Marasmiellus hupehensis* often grows saprophytically in complex environments, identifying its specific host is extremely difficult, making it hard to find suitable culture media for cultivating artificial fruiting bodies. Furthermore, *Marasmiellus hupehensis* often appears as an endophytic fungus, and does not naturally form fruiting bodies in its endophytic state, which further poses a significant challenge to artificial cultivation. Currently, there are almost no publicly available reports on artificial cultivation methods for *Marasmiellus hupehensis*. Existing research on the *Marasmiellus* genus includes only a few explorations of cultivation for other species, such as the domestication of *Marasmiellus inoderma* as a pathogen on oil palm trees, and the domestication of *Marasmiellus scandens*, an endophytic fungus isolated and purified from *Aquilaria sinensis*. Under these circumstances, research on artificial cultivation methods for *Marasmiellus hupehensis* is urgently needed. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a Marasmiellus sp. DCY6478 plant. Another technical problem to be solved by the present invention is to provide a culture medium for cultivating Marasmiellus sp. yet another technical problem to be solved by the present invention is to provide an application of Marasmiellus sp. DCY6478 for cultivating Marasmiellus fruiting bodies.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A white microbark umbrella (Marasmiellus sp.) DCY6478 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 65883, on February 14, 2025.

[0008] Application of Marasmiellus sp. DCY6478 in the cultivation of Marasmiellus sp. fruiting bodies.

[0009] A culture medium for cultivating white spores DCY6478, the culture medium formula being: 40% sawdust, 36% corn cob, 14% cottonseed hulls, 1% lime, and 1% gypsum by weight.

[0010] The wood chips include apple wood chips, small-leaved oak wood chips, or agarwood wood chips.

[0011] Application of fermentation broth of Marasmiellus sp. DCY6478 in inhibiting the growth of pathogens.

[0012] The pathogens are Fusarium solani, Fusarium oxysporum, Penicillium aurantiogriseum, and Phona bellidis.

[0013] Application of fermentation mycelium of Marasmiellus sp. DCY6478 in the preparation of antioxidants.

[0014] Application of fermentation mycelia or fruiting bodies of Marasmiellus sp. DCY6478 in the secretion of amino acids.

[0015] Application of fermentation mycelia or fruiting bodies of Marasmiellus sp. DCY6478 in the secretion of polysaccharides.

[0016] Application of fermentation mycelia or fruiting bodies of Marasmiellus sp. DCY6478 in the production of crude protein.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1) This invention successfully establishes a method for the artificial cultivation of white umbrella trees, enabling their mass production and breaking the limitation of relying solely on wild collection. While protecting wild resources and alleviating resource pressure caused by over-collection, it provides a stable source of raw materials for the subsequent development and utilization of white umbrella trees, promoting sustainable resource utilization and ensuring the long-term development of related industries.

[0019] 2) Through comparative experiments on different cultivation substrates, the results of this invention show that apple sawdust is the most suitable substrate for white Michelia champaca, and the mycelium of white Michelia champaca is more suitable for soil covering cultivation, providing a precise basis for substrate selection in actual production and improving the cultivation success rate and yield.

[0020] 3) This invention verifies that the fermentation broth of Marasmiellus sp. DCY6478 has an inhibitory effect on pathogens such as Fusarium solani, Fusarium oxysporum, Penicilliumaurantiogriseum, and Phoma bellidis, and has potential application value in the prevention and control of agricultural diseases.

[0021] 4) This invention verifies that the fermentation mycelium of Marasmiellus sp. DCY6478 exhibits excellent antioxidant properties. Its DPPH free radical scavenging ability, superoxide dismutase activity, and catalase activity are all higher than those of the fruiting body, and it can be developed as a source of natural antioxidants.

[0022] 5) This invention verifies that the fermentation mycelia or fruiting bodies of *Marasmiellus* sp. DCY6478 possess the ability to secrete amino acids, polysaccharides, and produce crude protein. Among these, the fermentation mycelia are a more efficient source of polysaccharides and have a greater advantage in accumulating umami substances. Although the amino acid composition of the mycelia and fruiting bodies does not perfectly meet the ideal protein requirements, there is still room for research and development in the field of amino acid production, providing new raw material options for the food, pharmaceutical, and other industries. Attached Figure Description

[0023] Figure 1 The images show colonies of DCY6478 on petri dishes (a is the front view, B is the back view);

[0024] Figure 2The images show the primordia formed by the domestication and cultivation of *Michelia champaca* in oak sawdust cultivation bags (A), the primordia formed by the domestication and cultivation of *Michelia champaca* in apple wood sawdust cultivation bags (B), the fruiting bodies cultivated in oak sawdust cultivation bags (C, D), the fruiting bodies cultivated in apple wood sawdust cultivation bags (E, F, G), the growth of *Michelia champaca* in cultivation bags of formulas 1, 2, and 3 (H, from left to right are formulas 1, 2, and 3), and the growth of *Michelia champaca* in tissue culture bottles of formula 1 (I).

[0025] Figure 3 The graph shows the inhibitory activity of crude extracts against Fusarium solani (A1-A3 represent the concentrations of crude extracts in the upper layer containing methanol as 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; B1-B3 represent the concentrations of crude extracts in the lower layer containing methanol as 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; CK is the blank).

[0026] Figure 4 The graph shows the inhibitory activity of crude extracts against Fusarium oxysporum (A1-A3 represent the concentrations of crude extracts in the upper layer containing methanol of 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; B1-B3 represent the concentrations of crude extracts in the lower layer containing methanol of 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; CK is the blank).

[0027] Figure 5 The graph shows the inhibition of crude extracts against Penicillium aurantiogriseum (A1-A3 represent the concentrations of the upper crude extract containing methanol as 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; B1-B3 represent the concentrations of the lower crude extract containing methanol as 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; CK is the blank).

[0028] Figure 6 The graph shows the inhibitory activity of the crude extract against Phoma bellidis (A1-A3 represent the concentrations of the upper crude extract containing methanol as 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; B1-B3 represent the concentrations of the lower crude extract containing methanol as 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, respectively; CK is the blank). Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0030] The PDA solid medium formula is as follows: 200g potato, 20g glucose, 20g agar, 2g peptone, 1.5g magnesium sulfate, 3g potassium dihydrogen phosphate, and distilled water to a final volume of 1000mL, with a natural pH. This medium is used for mycelial activation and propagation.

[0031] The preparation method for PDA liquid culture medium is as follows: Add 25g of PDA reagent (purchased from Shanghai Bowei Microbial Technology Co., Ltd.) to 1000mL of distilled water, heat to boiling to dissolve, sterilize at 121℃ for 20min, and then cool naturally.

[0032] The wild fruiting bodies of *Marasmiellus* sp. used in this application are deposited in the Herbarium of Macrofungi (HGASMF) of the Guizhou Provincial Institute of Biology.

[0033] The Fusarium solani, Fusarium oxysporum, Penicillium aurantiogriseum, and Phona bellidis used in this application were provided by the Guizhou Institute of Biology.

[0034] Example 1

[0035] 1. Separation of Marasmiellus sp. DCY6478

[0036] Wipe the surface of the gills of the collected wild-type Marasmiellus sp. fruiting bodies with 75% alcohol and wait for the alcohol to completely evaporate. Then, using tweezers cooled by burning alcohol near an alcohol lamp, tear open the cap epidermis of the specimen, take a small amount of flesh and gills, place them on a PDA solid plate, and seal the plate interface with sealing film. Repeat the same operation to obtain two more isolation plates for subsequent purification.

[0037] 2. Strain identification and preservation

[0038] 1) Morphological identification: In PDA solid plates, the colonies of this strain initially appear pure white with a smooth surface. With prolonged incubation, the area from the center outwards gradually turns into a pale yellow or orange-white film. The colony edges are wavy or irregular in shape. Figure 1 ).

[0039] 2) Molecular biological identification: Molecular phylogenetic identification was performed using ITS rDNA sequencing analysis. Genomic DNA of the strain was extracted and preserved using the TSINGKE Plant DNA Universal Extraction Kit (TSINGKE). ITS universal primer pairs were used: ITS 4(5) ′ -TCCGTAGGTGA ACCTGCGG-3′ ) / ITS 5(5 ′ -TCCTCCGCTTATTGATATGC-3 ′ Amplification was performed. PCR primer synthesis and sequencing of the amplified products were completed by Shanghai Sangon Biotech Co., Ltd., and the ITS rDNA amplified sequence fragment size was 730 bp. The obtained sequence was compared with the NCBI database using BLAST sequence comparison, and high similarity sequences were downloaded. Multiple sequence alignment was performed using software such as Clustalx and DNA Star, and it was determined that the sequence similarity between this strain and Marasmiellus candidus with sequence number OP364581 in the NCBI database was 99.70%. Based on morphological characteristics and ITS rDNA sequence alignment results, the isolated strain was identified as Marasmiellus sp. DCY6478, which has been deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number: GDMCC No. 65883, deposit date: February 14, 2025.

[0040] Example 2

[0041] The *Trichoderma hyacinthus* strain DCY6478, stored at 4℃, was allowed to activate at room temperature for two days. Afterward, it was inoculated onto PDA medium in 9cm petri dishes and cultured at 25℃ until the mycelia reached approximately 8cm in length. Then, holes were punched at the edge of the colonies with a 7mm inner diameter punch to create small holes of the same radius. The optimal growth conditions for *Trichoderma hyacinthus* strain DCY6478 were determined as follows:

[0042] 1. Screening using different carbon sources

[0043] Glucose, fructose, lactose, starch, sucrose, and maltose were used as different carbon sources, with no added carbon source serving as a blank control (CK). Uniformly sized mycelial discs were inoculated into the center of agar plates and incubated in the dark at 25°C. Five replicates were performed for each carbon source. Mycelial growth was observed and recorded every 24 hours, starting 24 hours after inoculation. Colony morphology, color, and mycelial vigor were observed and recorded every 24 hours, and colony growth was measured and recorded using the cross-crossing method. Incubation was terminated when the colony diameter reached 80% of the plate's height. The number of days to full plate coverage (T) and the colony diameter at this point (D) were recorded. The mycelial growth rate was calculated using the following formula:

[0044] Mycelial growth rate V (mm / d) = (Dd) / 2T, where D is the last measured colony diameter (mm), d is the diameter of the punch (mm), and T is the incubation time. Analysis of variance was performed on the obtained data using SPSS 27.0 software.

[0045] The results are shown in Table 1. *Amanita muscaria* hyphae can grow in all seven culture media. The fastest growth rate (5.52 ± 0.07 mm / d) and densest hyphae were observed with maltose as the carbon source. Sucrose, glucose, and fructose followed, with no significant difference in growth rate among these three carbon sources (P < 0.05). However, the hyphae grew slowly in the lactose-added medium, and their diameter was smaller than the control group (4.03 ± 0.23 mm / d). This indicates that maltose is the optimal carbon source for *Amanita muscaria* hyphae growth. The growth rate of hyphae from fastest to slowest among different carbon sources was: maltose > sucrose > glucose > fructose > starch > blank control (CK) > lactose.

[0046] Table 1 Effects of different carbon sources on the mycelial growth of *Amanita muscaria*

[0047] carbon source Mycelial growth rate (mm / d) hyphae growth maltose 5.52±0.07a White, flocculent ++++ sucrose 5.07±0.07b White, flocculent +++ glucose 4.78±0.06bc White, flocculent +++ fructose 4.59±0.19cd White, flocculent ++ starch 4.31±0.07de White, flocculent ++ Blank control (CK) 4.03±0.23e White, flocculent ++ lactose 3.25±0.03f White, flocculent +

[0048] Note: Different letters in the table indicate significant differences (P<0.05). "++++" indicates very strong mycelial growth, "+++" indicates relatively strong mycelial growth, "++" indicates moderate mycelial growth, and "+" indicates weak mycelial growth.

[0049] 2. Screening using different nitrogen sources

[0050] Potassium nitrate, urea, beef extract, yeast extract, malt extract powder, and ammonium sulfate were used as different nitrogen source treatments, with no nitrogen source added as a blank control. The inoculation and recording methods were the same as above.

[0051] The results are shown in Table 2. *Amanita muscaria* mycelia grew on all six culture media, but not on urea medium. The fastest growth rate (5.03 ± 0.07 mm / d) was observed with malt extract as the nitrogen source; followed by yeast extract, beef extract, and the control (CK). There was no significant difference in growth rate among these three nitrogen sources (P < 0.05). This indicates that malt extract is the optimal nitrogen source for *Amanita muscaria* mycelia growth, and also demonstrates that *Amanita muscaria* utilizes organic nitrogen sources more readily than inorganic nitrogen sources. The growth rate of mycelia in different carbon sources, from fastest to slowest, was: malt extract > yeast extract > beef extract > control (CK) > potassium nitrate > ammonium sulfate > urea.

[0052] Table 2 Effects of different nitrogen sources on the growth of *Amanita muscaria* mycelium

[0053]

[0054]

[0055] Note: Different letters in the table indicate significant differences (P<0.05). "++++" indicates very strong mycelial growth, "+++" indicates relatively strong mycelial growth, "++" indicates moderate mycelial growth, and "+" indicates weak mycelial growth. No "+" indicates no growth.

[0056] 3. Screening using different pH values

[0057] Prepare 1 mol / L sodium hydroxide solution and 1 mol / L hydrochloric acid solution, and adjust the pH of the sterilized PDA medium to 5, 6, 7, 8, and 9, respectively, using a pH gradient of 1. Incubate the inoculated plates at 25°C, repeating each treatment five times. Use a pHS-3C laboratory pH meter for measurement. Inoculation and recording methods are the same as above.

[0058] The results are shown in Table 3. *Amanita muscaria* mycelium can grow in pH ranges from 5 to 9. However, different pH values ​​affect the growth rate of *Amanita muscaria* mycelium. At pH 5, the mycelium grows fastest and most vigorously, at 5.07 ± 0.08 mm / d. The growth rate of mycelium in different carbon sources, from fastest to slowest, is: pH 5 > pH 6 > pH 7 > pH 8 > pH 9. Therefore, although *Amanita muscaria* can also grow in neutral and alkaline environments, it is more suitable for growth in acidic environments.

[0059] Table 3 Effects of different nitrogen sources on the growth of *Amanita muscaria* mycelium

[0060] pH Mycelial growth rate (mm / d) hyphae growth 5 5.07±0.08d White, flocculent ++++ 6 4.62±0.01c White, flocculent ++++ 7 3.99±0.05b White, flocculent +++ 8 3.19±0.08a White, flocculent ++ 9 3.03±0.07a White, flocculent ++

[0061] 4. Use different temperatures for screening.

[0062] PDA medium was used for temperature experiments, with five different temperatures set in a 5°C gradient within the range of 15-40°C: 15°C, 20°C, 25°C, 30°C, and 35°C. After inoculation, the samples were incubated at different temperatures, with each treatment repeated five times. Inoculation and recording methods were the same as above.

[0063] The results are shown in Table 4. The mycelial growth of *Amanita muscaria* is significantly affected by temperature. Mycelia can grow within the temperature range of 15-35℃, but the growth varies considerably at different temperatures. Between 15-30℃, the mycelial growth rate increases with increasing temperature, resulting in stronger growth. However, between 30-35℃, the growth rate decreases with further increases in temperature, leading to weaker growth. The fastest growth rate (4.23±0.09 mm / d) is observed at 30℃, exhibiting the strongest growth. At 15℃, no mycelial growth occurs, indicating that low temperatures are not suitable for its growth. In conclusion, the optimal temperature for *Amanita muscaria* mycelial growth is 30℃. Growth at 15℃ is very slow, indicating that excessively low or high temperatures are unsuitable for mycelial growth. The growth rate of mycelia at different temperatures is ranked as follows: 30℃ > 25℃ > 20℃ > 35℃ > 15℃.

[0064] Table 4. Effects of different temperatures on the growth of *Amanita muscaria* mycelium.

[0065] Temperature (°C) Mycelial growth rate (mm / d) hyphae growth 15 1.47±0.11d White, fluffy + 20 3.22±0.09b White, fluffy +++ 25 4.22±0.07a White, fluffy ++++ 30 4.23±0.09a White, fluffy +++ 35 2.42±0.04d White, fluffy ++

[0066] Example 3

[0067] 1. Preparation of cultivation bags

[0068] Three formulations of culture medium for *Ulva spp.* were prepared: ① 500g of culture medium for *Ulva spp.* consisted of 40% (200g) apple sawdust, 36% (180g) corn cob, 14% (70g) cottonseed hulls, and 1% each of lime and gypsum; ② 500g of culture medium for *Ulva spp.* consisted of 40% (200g) oak sawdust, 36% (180g) corn cob, 14% (70g) cottonseed hulls, and 1% each of lime and gypsum; ③ 500g of culture medium for *Ulva spp.* consisted of 40% (200g) agarwood sawdust, 36% (180g) corn cob, 14% (70g) cottonseed hulls, and 1% each of lime and gypsum. After soaking and filtering the above materials, they were weighed according to the formula, mixed evenly, bagged, sterilized at 121℃ for 2 hours, and inoculated after cooling.

[0069] 2. Preparation of bacterial balls

[0070] The *Micrococcus pluvialis* strain was inoculated into PDA solid medium and activated by dark incubation at 20-28℃ for 10-15 days. It was then stored in a refrigerator at approximately 4℃ for later use. 200 mL of PDB liquid medium was added to a 500 mL Erlenmeyer flask, and six 5 mm diameter pieces of the activated strain were inoculated. The flasks were then incubated on a shaker at 25℃ and 150 rpm in the dark for 6 days to obtain bacterial pellets.

[0071] 3. Preparation of mushroom sticks

[0072] The cultured mycelial pellets were homogenized under aseptic conditions using a homogenizer to obtain a suspension. This suspension was then mixed with the material in the culture bags at a mass-to-volume ratio of 1:50 and incubated in the dark at 25°C. Growth was observed and recorded periodically. Twenty bags of agarwood chips, and 15 bags each of apple wood and small-leaved oak were inoculated and incubated in the dark at 25°C.

[0073] The results are as follows Figure 2 As shown, after the mycelium of *Amanita muscaria* fully colonizes the cultivation bag, a mycelial film will form on the surface. At this time, the mycelium has stronger overall resistance. The mycelial film can be gently torn open with tweezers sterilized with alcohol. Primordia will form within 20-30 days. Figure 2 (A, B), then child bodies grow out in 7-10 days ( Figure 2 In the early stages of domesticated cultivation, the cap of the fruiting body is light gray, while the stipe is milky white. During the middle stage of fruiting body growth, the fruiting body is white with a depression in the center of the cap. As the fruiting body matures, it changes from pure white to a pale yellow. The size of the fruiting body is not significantly different from that of the wild species *Amanita muscaria*. Sequencing and comparison of the mycelium, fruiting bodies, and isolated and purified strains from the fruiting bodies showed a sequence similarity of over 98% with *Amanita muscaria* sequences on NCBI. Mycelium could grow and produce fruiting in all three formulations. There were no significant differences in the time to full coverage, primordia appearance, and harvest time between formulations 1 and 2. In contrast, formulation 3 showed significantly lower scores in all indicators compared to formulations 1 and 2, indicating that agarwood chips are not suitable as a growth substrate for *Amanita muscaria*. Furthermore, the mycelium in apple sawdust filled the bag earlier than that in *Quercus glauca* sawdust, indicating that apple sawdust is the most suitable substrate for *Amanita muscaria*. Figure 2 H).

[0074] The results are shown in Table 5. Formula 1 took 23-30 days to reach full size, Formula 2 took 25-30 days, and Formula 3 took as long as 42-50 days, with Formula 3 taking significantly longer than the other two. In terms of the time to start forming primordia, Formulas 1 and 2 took 20-30 days, while Formula 3 took 30-40 days, making Formula 3 relatively delayed. The first harvest time was 7-10 days for Formulas 1 and 2, and 8-15 days for Formula 3, making Formula 3 slightly later. All three formulas could produce a second flush, but the harvest time for the second flush was 5-7 days for Formulas 1 and 2, and 15-20 days for Formula 3, with Formula 3 having a longer interval. Formulas 1 and 2 had relatively shorter and similar times required for each stage of white Michelia champaca cultivation, making them suitable for scenarios requiring rapid output and multiple batches of production. In contrast, Formula 3 generally took longer for each stage.

[0075] Table 5 Results of domestication and cultivation of *Malus spectabilis*

[0076]

[0077] Example 4

[0078] 1. Preparation of primary liquid bacterial strains

[0079] Marasmiellus sp DCY6478 was inoculated onto PDA medium and incubated in the dark at 25°C for 5-7 days, then stored in a refrigerator at approximately 4°C for later use. PDB liquid medium was prepared, with a volume of 200mL / 500mL, and sterilized at 121°C for 20 minutes. After cooling, each Erlenmeyer flask was inoculated with 6 pieces of activated inoculum (approximately 0.5cm in size). 2 Incubate at 25℃ and 150 rpm on a shaker in the dark. They will be ready on day 6.

[0080] 2. Preparation of fermentation broth

[0081] After sterilizing 25L of PDB in a fermenter (120℃, 40min), 2.5L of *Micrococcus pluvialis* liquid inoculum was inoculated. An alcohol-soaked cotton ball was ignited at the inoculation port to create a sterile environment. Fermentation was carried out at 25℃ with an aeration rate of 6-10L / min and a stirring speed of 150-180 rpm. A small amount of fermentation broth was released daily through the fermenter's porthole and the discharge port to observe mycelial growth and any contamination. On day 14, microscopic examination confirmed the mycelium was uncontaminated. The fermentation broth was then discharged, separating the mycelium from the broth. The mycelium was rinsed 2-3 times with water to remove any remaining culture medium. The mycelium was then dried at 45℃, yielding a mycelial yield of 7.2g / L.

[0082] After extracting the obtained mycelia in an appropriate amount of methanol solvent for 45 days, the methanol solution was filtered to remove mycelial impurities. The solvent was then evaporated at 45°C using a rotary evaporator to obtain a concentrated solution. An appropriate amount of dimethyl sulfoxide solution was added for rinsing, and the solution was dried at 45°C to obtain approximately 90 mL of the concentrated solution in two layers, with an upper layer of approximately 50 mL and a lower layer of approximately 40 mL.

[0083] 3. Inhibits pathogenic fungi

[0084] 6g of PDA powder was added to 150mL of pure water to prepare culture medium. 0.75g, 0.15g, and 0.075g of the upper and lower layer crude extracts were weighed and dissolved in 600μL of dimethyl sulfoxide (DMSO) to prepare plates with concentrations of 5mg / mL, 1mg / mL, and 0.5mg / mL. 600μL of DMSO was added to the culture medium as a blank control (CK). After PDA sterilization, when the temperature dropped to approximately 65℃, the dissolved extract was poured into an Erlenmeyer flask and mixed thoroughly, then poured into 90mm Petri dishes. In a clean bench, the activated pathogenic fungus was punched into holes using a 7mm punch. Using a sterile inoculation needle, pathogenic fungal agar blocks were picked up and inoculated into each concentration of PDA. Three parallel experiments were performed for each concentration. The plates were sealed with sealing film and incubated at 25℃. The diameter of the colonies was recorded using the cross-hatching method, and the average value was taken. The inhibitory effect of the crude extract on the growth of the pathogenic fungus was determined by the mycelial growth rate. The mycelial growth inhibition rate was calculated as follows: inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%. Note: When measuring and recording the colony diameter, the original mycelial cake diameter of 7 mm should be subtracted.

[0085] 1) Inhibitory activity against Fusarium solani (a type of solanaceous fungus)

[0086] The results are shown in Table 6 and Figure 3 As shown, 5 days after inoculation, the upper layer of the concentrated solution (0.5 mg / mL) exhibited inhibitory effects against *Fusarium solani*, while the 1 mg / mL and 5 mg / mL solutions showed almost no inhibitory effect. The lower layer of the concentrated solution showed a significant inhibitory effect on *Fusarium solani*, with an inhibition rate of 40.54% at a concentration of 5 mg / mL, but a -4.3% inhibition rate at a concentration of 0.5 mg / mL, which actually promoted its growth. The inhibitory effect increased with increasing crude drug concentration, indicating that the compounds in the lower layer were more effective in controlling *Fusarium solani*.

[0087] Table 6. Inhibitory activity of crude extracts against Fusarium solani (a type of solanaceous fungus).

[0088]

[0089] 2) Inhibitory activity against Fusarium oxysporum

[0090] The results are shown in Table 7 and Figure 4As shown, after 5 days of inoculation, the upper layer of the concentrated solution, with drug concentrations of 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, exhibited inhibitory effects against *Fusarium solani*, with 0.5 mg / mL showing the best inhibitory effect at 24.6%. In the upper layer, as the concentration increased from 0.5 to 1 to 5 mg / mL, the inhibition rate showed a non-linear trend of first decreasing and then slightly increasing, from 24.6% to 15.11% to 16.24%, which is inconsistent with the expected dose-response relationship. This may be because the active ingredient reaches its solubility limit at a specific concentration, leading to partial precipitation and ineffectiveness at higher concentrations. Alternatively, it may be due to a bidirectional regulatory effect at different concentrations. The lower layer of the concentrated solution showed inhibitory effects against *Fusarium oxysporum* only at a drug concentration of 0.5 mg / mL, with an inhibition rate of 19.85%; however, no inhibitory effect was observed at drug concentrations of 1 mg / mL and 5 mg / mL. This indicates a concentration threshold of 0.5-1 mg / mL, beyond which the antibacterial activity saturates or becomes ineffective.

[0091] Table 7. Inhibitory activity of crude extract against Fusarium oxysporum

[0092]

[0093]

[0094] 3) Inhibitory activity against Penicillium aurantiogriseum

[0095] The results are shown in Table 8 and Figure 5 As shown, after 5 days of inoculation, the upper layer of the concentrate showed the best antibacterial effect at a concentration of 5 mg / mL, with an inhibition rate of 13.39%. At a drug concentration of 1 mg / mL, the upper layer of the concentrate showed an inhibition rate of -4.1%, and the colony diameter exceeded that of the control group, indicating growth promotion. The lower layer of the concentrate showed good inhibitory effects against *Penicillium aureus*, with the best antibacterial effect at a drug concentration of 5 mg / mL, an inhibition rate of 22.74%, higher than the optimal inhibition rate of the upper layer. The bottom layer of the concentrate, with drug concentrations of 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, all showed inhibitory effects against *Fusarium solani*, and the inhibition rate showed a dose-dependent relationship with the drug concentration.

[0096] Table 8. Inhibitory activity of crude extract against Penicillium aurantiogriseum

[0097]

[0098] 4) Inhibitory activity against Phoma bellidis on water chestnut stems

[0099] The results are shown in Table 9 and Figure 6As shown, after 5 days of inoculation, the upper layer of the concentrated solution with a drug concentration of 5 mg / mL exhibited the best antibacterial effect, with an inhibition rate of 13.04%. The trend of the upper layer inhibition rate was 0.5 mg / mL (3.34%) → 1 mg / mL (5.5%) → 5 mg / mL (13.04%). The inhibition rate at 1 mg / mL was higher than that at 0.5 mg / mL, but there was no linear increase at 5 mg / mL, indicating that there was multi-component antagonism among the compounds in the upper layer. Different components exhibited synergistic inhibition at low concentrations and competitive inhibition at high concentrations (e.g., the difference in sensitivity of *Acetus erythrorhizon* to sterol inhibitors (Long et al., 2023)). The lower layer of the concentrated solution showed the best antibacterial effect with a concentration of 5 mg / mL, with an inhibition rate of 20.2%, which was better than the optimal inhibition rate of the upper layer and showed a clear positive correlation with the concentration effect.

[0100] Table 9. Inhibitory activity of crude extract against Phoma bellidis on water chestnut stem.

[0101]

[0102]

[0103] 4. Antioxidant activity

[0104] 0.1 g of dried fermentation mycelium and fruiting body mycelium were weighed separately for later use. Enzyme activity was measured using the kit according to the instructions, with each enzyme activity treatment repeated three times. The antioxidant activity of *Amanita muscaria* fermentation mycelium and fruiting body was evaluated using the kit to detect total antioxidant capacity (ABTS method, kit purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number S0119), peroxidase (POD) activity (kit purchased from Beijing Solarbio Science & Technology Co., Ltd.), superoxide dismutase (SOD) activity (kit purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number S0109), and catalase (CAT) activity (kit purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number S0051) (Zhou Pengcheng et al., 2021).

[0105] The results are shown in Table 10. The DPPH free radical scavenging capacity of the fermentation mycelium of *Micrococcus pumila* was 55.05%, while that of the fruiting body was only 17.85%, indicating that the fermentation mycelium has a stronger antioxidant capacity. The peroxidase activity of the fruiting body (356.65 U / g) was significantly higher than that of the mycelium (197.65 U / g). The SOD activity of the fermentation mycelium (282.70 U / g) was more than 10 times that of the fruiting body (26.83 U / g), indicating that the fermentation mycelium is more effective in coping with superoxide oxidative stress. The catalase (CAT) activity of the fermentation mycelium (243.40 U / g) was higher than that of the fruiting body (165.33 U / g). In summary, the fermentation mycelium of *Micrococcus pumila* generally showed better antioxidant capacity, especially in DPPH free radical scavenging capacity, superoxide dismutase activity, and catalase activity, all of which were higher than those of the fruiting body.

[0106] Table 10 Results of antioxidant capacity of fermentation mycelia and fruiting bodies of *Micrococcus pumila*

[0107]

[0108]

[0109] Example 5

[0110] The dried fermentation mycelia and fruiting bodies were placed in sealed bags and the bags were sealed. The Guangdong Academy of Sciences Testing and Analysis Institute was then commissioned to determine the protein, free amino acids, vitamin B2 and crude polysaccharides.

[0111] 1. Crude protein content

[0112] The determination method referenced the standard: crude protein, GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food".

[0113] The difference rate (%) represents the relative percentage difference in crude protein content between mycelium and fruiting body, used to quantify the proportion by which mycelium contains more crude protein than fruiting body. It is calculated as follows:

[0114] Difference rate = (crude protein content of mycelium - crude protein content of fruiting body) / crude protein content of fruiting body * 100%.

[0115] The results are shown in Table 11. The crude protein content of the fermentation mycelium was 31.9 g / 100 g, while that of the fruiting body was 28.2 g / 100 g, a difference of 3.7 g / 100 g. The crude protein content of the fermentation mycelium and fruiting body of *Amanita muscaria* was higher than that of general edible fungi, but the difference rate (+13.1%) was lower than that of general edible fungi, which is related to fermentation conditions and metabolic efficiency.

[0116] Table 11 Comparison of crude protein content of fermentation mycelia and fruiting bodies of *Amanita muscaria* with other macrofungi.

[0117] sample Crude protein in mycelium (g / 100g) Crude protein content of fruiting body (g / 100g) Difference rate (%) White micro-plaster umbrella 31.9 28.2 +13.1 Lotus leaf pleated umbrella 28.3 21.4 +32.2 Bamboo fungus 24.82 17.87 +38.9

[0118] 2. Crude polysaccharide content

[0119] The determination method refers to the standard: crude polysaccharides, NY / T1676-2023 "Determination of crude polysaccharides in standard foods".

[0120] The results are shown in Table 12. The crude polysaccharide content of the fermentation mycelium was 3.31 g / 100 g, while the crude protein and crude polysaccharide content of the fruiting body was 0.651 g / 100 g. The crude polysaccharide content of the fermentation mycelium was 5.1 times that of the fruiting body, indicating that the mycelium is a more efficient source of polysaccharide production.

[0121] Table 12 Crude protein content of fermentation mycelia and fruiting bodies of *Amanita muscaria*

[0122]

[0123] 3. Amino acid content

[0124] The determination method referenced the standard: Amino acids, GB5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food".

[0125] The results are shown in Table 13. The total amino acid content of the fruiting body was 1.45 g / 100 g. The fruiting body contained 16 amino acids, with 8 essential amino acids and 8 non-essential amino acids. Glutamic acid was the most abundant non-essential amino acid, at 0.4 g / 100 g; followed by alanine, glycine, and arginine. Threonine was the most abundant essential amino acid, at 0.12 g / 100 g; followed by lysine, valine, and leucine. The essential amino acid content of the fruiting body was 0.5469 g / 100 g, while the non-essential amino acid content was 0.9031 g / 100 g, indicating that the essential amino acid content was lower than the non-essential amino acid content. The E / N% ratio of the fruiting body was above 60%, but at 37.77%, it did not meet the ideal protein conditions proposed by FAO / WHO (E / N% above 60%, E / N% between 39% and 40%).

[0126] Table 13 Amino acid composition and content of fruiting bodies

[0127] amino acids Content (g / 100g) amino acids Content (g / 100g) Glutamic acid (Glu) 0.40 Isoleucine*Ile* 0.022 Alanine (Ala) 0.38 Tyrosine*Tyr* 0.010 Glycine 0.12 Phenylalanine (Phe) 0.0099 Threonine *Thr* 0.12 Methionine (Met) 0.005 Arginine (Arg) 0.10 Proline 0.005 Serine 0.091 Essential amino acid content E 0.5469 Aspartic acid Asp 0.068 Non-essential amino acid content N 0.9031 Lysine* 0.039 Total amino acid E+N 1.45 Histidine 0.034 E / N% 60.56％ Valine* 0.028 E / (E+N)% 37.77％ Leucine*Leu* 0.025

[0128] Note: "*" in the table indicates essential amino acids.

[0129] The results are shown in Table 14. The total amino acid content of the fermentation mycelium was 2.94 g / 100 g. The fermentation mycelium contained 16 amino acids, including 7 essential amino acids and 9 non-essential amino acids, with non-essential amino acids being more abundant than essential amino acids. The most abundant non-essential amino acid was alanine, with a content of 0.99 g / 100 g; followed by glutamic acid, arginine, and glycine. The most abundant essential amino acid was lysine, with a content of 0.18 g / 100 g; followed by isoleucine, threonine, and valine. The essential amino acid content in the fruiting body was 0.456 g / 100 g, while the non-essential amino acid content was 2.475 g / 100 g, indicating that the essential amino acid content in the fruiting body was lower than that of the non-essential amino acids. The E / N% and E / (E+N)% results indicate that the fermentation mycelium is not a good source of amino acids.

[0130] Table 14 Amino acid composition and content of fermentation mycelia

[0131] amino acids Content (g / 100g) amino acids Content (g / 100g) Alanine (Ala) 0.99 Proline 0.052 Glutamic acid (Glu) 0.52 Leucine*Leu* 0.029 Arginine (Arg) 0.39 Phenylalanine (Phe) 0.022 Lysine* 0.18 Tyrosine Tyr 0.011 Glycine 0.16 Methionine (Met) 0.005 Aspartic acid Asp 0.14 Essential amino acid content E 0.456 Serine 0.11 Non-essential amino acid content N 2.475 Histidine 0.11 Total amino acid E+N 2.94 Isoleucine*Ile* 0.092 E / N% 18.42 Threonine *Thr* 0.075 E / (E+N)% 15.51 Valine* 0.062

[0132] Note: "*" in the table indicates essential amino acids.

[0133] 4. Umami amino acid content

[0134] The results are shown in Table 15. The total umami amino acid content of the fermentation mycelium (2.94 g / 100 g) was twice that of the fruiting body (1.45 g / 100 g). Furthermore, the contents of all four umami amino acids in the fermentation mycelium were higher than those in the fruiting body, indicating that the mycelium has a greater advantage in umami substance accumulation, which may be related to enhanced protein metabolism during fermentation. However, the proportion of total amino acids (TFA) was slightly lower in the fruiting body (66.76%) than in the mycelium, indicating that the fruiting body is more flavorful than the mycelium, meaning that the synthesis of umami amino acids in the fruiting body is stronger during growth than in the mycelium during fermentation.

[0135] Table 15. Umami amino acid content of white microbarkia fruiting bodies and fermentation mycelia.

[0136]

[0137]

[0138] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A white microbark umbrella (Marasmiellus sp.) DCY6478 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC No.65883, deposit date: February 14, 2025.

2. The application of Marasmiellus sp. DCY6478 as described in claim 1 in the cultivation of Marasmiellus fruiting bodies.

3. Application of fermentation broth of Marasmiellus sp. DCY6478 in inhibiting the growth of pathogens, wherein the pathogens are Fusarium solani, Fusarium oxysporum, Penicillium aurantiogriseum, and Phoma bellidis.

4. Application of fermentation mycelium of Marasmiellus sp. DCY6478 in the preparation of antioxidants.

5. Application of fermentation mycelia or fruiting bodies of Marasmiellus sp. DCY6478 in amino acid production.

6. Application of fermentation mycelia or fruiting bodies of Marasmiellus sp. DCY6478 in polysaccharide production.

7. Application of fermentation mycelia or fruiting bodies of Marasmiellus sp. DCY6478 in the production of crude protein.

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