Wild Pleurotus fungus and culture method thereof

Abstract

The invention discloses a wild bamboos fungus and a culture method thereof. The Gymnopus sp.lhf1475 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on July 11, 2024, the address is the 5th floor of the building 59, No. 100 courtyard, Xianlie Middle Road, Guangzhou, the postal code is 510070, and the preservation number is GDMCC No: 64861. According to the invention, the new species, namely the fungus Gymnopus sp.lhf1475, of the gymnopus is separated from a Guizhou gymnopus specimen (HGASMF01-23515), hyphae can be rapidly produced under an artificial culture condition, and the fungus has certain active substances and bacteriostatic action and has a relatively good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and provides a new resource of Pleurotus ostreatus fungi and a liquid fermentation culture method of mycelium. Background Art

[0002] Gymnopus sp. belongs to the phylum Basidiomycota, class Agaricomycetes, order Agricales, family Omphalataceae. Gymnopus sp. is a polyphyletic group in the order Agaricales, characterized by brown fruiting bodies, white spore prints, flat hyphae in the cortex of the cap, and difficult to distinguish between genus and neighboring genera. There are more than 300 species. In recent years, it has been found that the number of Gymnopus sp. species is huge and it is extremely difficult to accurately identify them. This type of fungus has certain active substances and antibacterial effects, but the types of wild strains that can be used are limited, and the separation and purification technology is backward, so it has not yet been realized in production and application.

[0003] Klein J (1990) used YMG medium to culture liquid strains of Gymnopus sp., and then used M2A medium for fermentation to obtain mycelium and extract active substances; Mitsuhashi et al. (2015) isolated an autophagy inducer that can be used for cancer treatment from the mycelium of Gymnopus sp.; Chai Xinyi et al. (2017) found that the secondary metabolites of Gymnopus confluens had inhibitory effects on both Bacillus subtilis and Escherichia coli; PAN et al. (2017) isolated cyclic peptides A and B with anti-cancer effects from Gymnopus fusipes; Wu Sheng et al. (2016) found that the secondary metabolites of Gymnopus sp. strains had a strong inhibitory effect on the green mold pathogen that causes post-harvest rot in citrus, and achieved a significant yield increase effect by optimizing the fermentation medium.

[0004] It can be seen that this type of resource has application prospects, but lacks corresponding research.

[0005] Through the above analysis, the problems and defects of the prior art are as follows:

[0006] (1) The identification of existing Pleurotus ostreatus species is unclear and their species names cannot be determined.

[0007] (2) The mycelium of Pleurotus ostreatus grows slowly and has a long growth cycle, making it difficult to apply in production. Summary of the invention

[0008] The present invention provides a method for identifying wild Gymnopus sp. fungi and a new strain. The strain is isolated from the wild in Guizhou and can produce mycelium under artificial culture conditions. It is named Gymnopus sp.lhf1475. The strain is isolated from a Guizhou Gymnopus sp. specimen (HGASMF01-23515) and was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on July 11, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the collection number is: GDMCC No: 64861.

[0009] The second object of the present invention is to provide a method for culturing Gymnopus sp.lhf1475, which comprises inoculating Gymnopus sp.lhf1475 into a culture medium for culturing.

[0010] The culture medium contains carbon source, nitrogen source, inorganic salts required by Gymnopus sp.lhf1475

[0011] The most suitable carbon source is maltose, followed by glucose and sucrose.

[0012] The best nitrogen source is peptone, followed by yeast extract and yeast powder.

[0013] The inorganic salt can be MgSO4, CaCO3, KCl, MgCl 2、 NaCl, preferably MgSO4.

[0014] Preferably, the optimum culture medium contains 12 g potato extract powder, 20 g soluble starch, 20 g agar powder, 5 g peptone, 2.5 g MgSO4 per liter of water, and a natural pH.

[0015] The culture is carried out under light conditions, the culture temperature is 16-28°C, the optimal temperature is 28°C, and the pH value of the culture medium is between 7 and 8.

[0016] The invention also provides the use of the Gymnopus sp. lhf1475 in preparing antioxidant cosmetics or medicines.

[0017] Preferably, the Gymnopus sp.lhf1475 is an aqueous extract of mycelium of Gymnopus sp.lhf1475.

[0018] The present invention separates a new species of Gymnopus sp. lhf1475 from a Gymnopus specimen (HGASMF01-23515) from Guizhou Province. The fungus can quickly produce mycelium under artificial culture conditions. The fungus has certain active substances and antibacterial effects and has good application prospects.

[0019] Gymnopus sp.lhf1475 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on July 11, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCCNo: 64861. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the characteristic of Gymnopus sp.lhf1475. Ac fruiting body macroscopic morphology, d. culture hyphae characteristics, e. cap cortex, -f. folded capsule, i. basidia, j. basidiospore;

[0021] Figure 2 This is the ML phylogenetic tree constructed by ITS of Gymnopus sp.lhf1475;

[0022] Figure 3 This is a graph showing the effect of different carbon sources on the mycelial growth of Gymnopus sp.lhf1475;

[0023] Figure 4 This is a graph showing the effect of different nitrogen sources on the mycelial growth of Gymnopus sp.lhf1475;

[0024] Figure 5 This is a graph showing the effects of different inorganic salts on the mycelial growth of Gymnopus sp.lhf1475;

[0025] Figure 6 It is a graph showing the effect of different carbon source addition amounts on the growth of mycelium of Gymnopus sp.lhf1475;

[0026] Figure 7 It is a graph showing the effect of different nitrogen source addition amounts on the growth of mycelium of Gymnopus sp.lhf1475;

[0027] Figure 8 It is a graph showing the effect of different inorganic salt addition amounts on the growth of mycelium of Gymnopus sp.lhf1475;

[0028] Fig. 9 The effect of different light conditions on the mycelial growth of Gymnopus sp.lhf1475;

[0029] Fig.10 This is a graph showing the effect of different temperatures on the mycelial growth of Gymnopus sp.lhf1475;

[0030] Fig.11 This is a graph showing the effect of different pH on the mycelial growth of Gymnopus sp.lhf1475. DETAILED DESCRIPTION

[0031] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0032] Example 1

[0033] The wild Gymnopus strain of the present invention is named: Gymnopus pseudoandrosaceus Chun Y. Deng, JP Li & Antonín, sp. nov., that is, Gymnopus sp. lhf1475. The strain is isolated from the Gymnopus specimen of Guizhou (HGASMF01-23515) and was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on July 11, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the collection number is: GDMCC No: 64861.

[0034] The strain used in the present invention was discovered by the inventor when investigating fungal resources in the wild and was obtained by adopting a tissue separation method.

[0035] 1.1 The Gymnopus fungus used in the present invention has been identified as a new species, named Gymnopus pseudoandrosaceus Chun Y. Deng, JP Li & Antonín, sp. nov. Its main characteristics are:

[0036] Morphological identification: Cap 8–15 mm wide, broadly convex to nearly flat, with stripes, membranous, brown, dark brown, dark in the center. Flesh 0.5 mm thick, brown. Gills straight to shallowly extended, 1–1.5 mm wide, sometimes bifurcated or slightly transversely veined, light brown, dense, 24–25 complete gills, 4–5 small gills, gill edges and gills of the same color, brown. Stipe 7–14×0.5–1 mm, mesogenous, cylindrical, dark brown, shiny. Growing from substrate or cords. Cords well developed, branched or unbranched, 0.5–1 mm wide, with a lacquer-like luster, shiny. Basidiospores 6.4–9.8(–10.9)×3.1–4.7μm (average=8.13×3.63μm, E=(1.84–)1.87–2.55(–2.78), Q=2.23), elliptical, oblong, transparent. Basidiospores 24.5–38.2×5–7.6μm, 2-spore or 4-spore. Pleated cysts 10–38×2.5–9μm, irregularly rod-shaped, with tubercles at the top, branched or unbranched. Lateral cysts are absent. The hyphae of the cap cortex are flat, composed of pigment hyphae, occasionally with tubercles. The hyphae of the stipe cortex are composed of parallel transparent hyphae. Lock-like union is common. Morphologically, the cap of G.brunneiniger is lighter in color, the stipe is thin and linear, and the hyphae of the cap cortex have no pigment cells. G.androsaceus usually grows on coniferous trees, with pink gills and cap cortical hyphae with sac-like cells.

[0037] Strain characteristics: Mycelium grows on solid culture medium, pure white ( Figure 1 ).

[0038] Molecular phylogenetic analysis: Based on the comparison of ITS sequences and the phylogenetic tree analysis and identification of ITS gene fragments, the results obtained are as follows Figure 2 As shown in the figure, it can be seen that the two specimens of Gymnopus sp. 5702 and LHF1475 and the strain sequences of LHF1475 are clustered in a single branch, with a support rate of 100% ( Figure 2 ), this branch is located at the base of Gymnopus sect.andrasacei, forming a sister group with other species in the Gymnopus sect. G.androsaceus, G.pseudoandraxaceus and G.pseudoandraxaceus are most closely related, and belong to different branches with G.pseudoandraxaceus, which also received 100% support. Based on comprehensive morphological identification, it is believed that this strain is a new species of Gymnopus.

[0039] Example 2: A method for rapidly growing Pleurotus ostreatus hyphae

[0040] A production method for rapid growth of Pleurotus ostreatus hyphae. A culture medium for protecting the hyphae: by weight, comprising the following raw materials: 12 g potato powder, 20 g glucose, 20 g agar powder, natural PH, pure water to 1000 ml,

[0041] 1. Screening of carbon sources

[0042] The basal culture medium was used as the blank control group (CK): 12g potato powder, 20g glucose, 20g agar powder, natural pH, and pure water was used to make the volume 1000ml. On the basis of the CK group, the carbon source glucose was changed to 20g each of starch, sucrose, maltose, fructose, and lactose, and 5 replicates were set for each treatment. A 0.5mm puncher was used to inoculate the bacterial block on the 90mm above-mentioned different carbon source culture medium, and cultured under the optimal temperature conditions. The mycelial growth rate was measured when the mycelium was full.

[0043] 2. Selection of nitrogen sources

[0044] The optimal carbon source medium screened out was used as CK, and 15g of beef extract, peptone, yeast powder, yeast extract, and urea were added as nitrogen sources, and the volume was fixed to 1000ml with pure water. Five replicates were set for each treatment, and the culture was placed at the optimal temperature after inoculation. The project determination method, inoculation method, and determination method were the same as above.

[0045] 3. Screening of different inorganic salt single factors

[0046] The screened carbon and nitrogen source culture medium was used as CK, and 1.5g of potassium chloride (KCl), magnesium chloride (MgCl2), sodium chloride (NaCl), calcium chloride (CaCl2), magnesium sulfate (MgSO4), and calcium carbonate (CaCO3) were added respectively. Five replicates were set for each treatment. After inoculation, the culture was cultured under the selected appropriate temperature conditions. The project measurement method, inoculation method and measurement method were the same as above.

[0047] 4. Screening of the amount of carbon and nitrogen sources and inorganic salts added

[0048] The most basic culture medium was used as the blank control (CK): 12g potato powder, 20g agar powder, natural pH, and pure water to 1000mL. The optimal carbon source addition amount was set to 0, 10, 20, 30, and 40g, and 5 replicates were set for each treatment. After inoculation, the full plate was cultured under the selected suitable temperature and rotation speed conditions. The project determination method, inoculation method, and determination method were the same as above.

[0049] The selected suitable carbon source culture medium was used as CK, and the selected optimal nitrogen source addition amounts were set to 0, 5, 15, 25, and 35 g, respectively. Each treatment was repeated 5 times, and the project determination method, inoculation method, and determination method were the same as above.

[0050] The screened suitable carbon and nitrogen source culture medium was used as CK, and the screened suitable inorganic salt addition amounts were set to 0, 0.5, 1.5, 2.5, and 3.5 g, respectively. Each treatment was set with 5 replicates, and the project determination method, inoculation method, and determination method were the same as above.

[0051] Experimental Results

[0052] 1. Effects of different carbon sources on the growth of Mycelium of Microdermabrasion

[0053] like Figure 3 As shown, the zero carbon source was used as the control group (CK). Gymnopus sp.lhf1475 was cultured in a dark environment at 28℃ on different carbon source media, and the mycelial growth rates were ranked from large to small as follows: soluble starch>maltose>glucose>sucrose>fructose>CK>lactose. Among them, when soluble starch was used as the carbon source, the mycelial growth rate was the fastest (1.33±0.003) mm / d, but the mycelium was thin and sparse, with neat edges. The second was maltose (1.23±0.004) mm / d. There was no significant difference in growth rate between glucose and sucrose, but when glucose was used as the carbon source, the mycelium was stronger than sucrose. When lactose was used as the carbon source, the mycelial growth rate was even lower than that of the control group (1.00±0.04) mm / d. The mycelium was white and dense, but the edges were sparse, weak and uneven. Comprehensively considering the mycelial growth rate and mycelial growth state, the most suitable carbon source was maltose, followed by glucose and sucrose.

[0054] 2. Effects of different nitrogen sources on the growth of Mycelium of Microdermabrasion

[0055] like Figure 4 As shown, zero nitrogen source addition was used as the control (CK). Gymnopus sp.lhf1475 can grow in all nitrogen sources except ammonium sulfate. The growth rates are in the following order: peptone> yeast extract> yeast powder> CK> beef extract. When urea was used as the nitrogen source medium, the mycelium did not grow; when peptone was used as the N source, the mycelium growth rate was the fastest, which was (1.42±0.002) mm / d; the mycelium was white, grew vigorously, and the mycelium at the edges was thick and neat. When beef extract was used as the nitrogen source, the mycelium growth rate was the slowest, which was (1.22±0.001) mm / d; the mycelium was dense, thick, white, and had neat edges. According to the mycelium growth potential, growth rate, color, etc., the best nitrogen source is peptone, followed by yeast extract and yeast powder.

[0056] 3. Effects of different inorganic salts on the growth of Mycelium of Microdermabrasion

[0057] like Figure 5As shown in the figure, Gymnopus sp.lhf1475 can grow normally in 5 inorganic salt culture media. The growth rates are MgSO4>CaCO3>KCl>MgCl2>NaCl>CK>K2HPO4>CaCl2. Among them, the fastest growth was in the culture medium with MgSO4 as inorganic salt, which was (1.46±0.05)mm / d; the mycelium was white, dense and strong. KCl, CaCO3, MgCl2 and NaCl were followed, with mycelium growth rates of (1.43±0.005)mm / d, (1.43±0.002)mm / d and (1.43±0.001)mm / d; among them, the growth rates when K2HPO4 and CaCl2 were used as inorganic salts were even lower than those of the blank group (1.41±0.003)mm / d. When K2HPO4 was used as inorganic salt, the hyphae were sparse and the edge hyphae were weak; when CaCl2 was used as inorganic salt, the hyphae were strong and neat. The medium with calcium carbonate as inorganic salt grew the slowest, at (1.29±0.003) mm / d.

[0058] 4. Effects of different carbon and nitrogen sources and inorganic salt additions on the mycelial growth of Microdermabrasion

[0059] like Figure 6 As shown in the figure, with the increase of the amount of carbon source maltose added, the mycelial growth rate first increased and then decreased. When the addition amount was 0-20g / L, the mycelial biomass increased exponentially, and decreased after exceeding 20g / L. When the addition amount was 20g / L, the mycelial biomass was the largest (1.48±0.005)mm / d, followed by the addition amount of 30g / L (1.44±0.005)mm / d; it can be seen that the optimal carbon source addition amount is 20g / L.

[0060] like Figure 7 As shown in the figure, with the increase of nitrogen source peptone addition, the mycelial biomass generally showed a trend of first increasing and then decreasing. When the addition amount was 5g / L, the mycelial biomass was the largest (1.35±0.005)mm / d, followed by 15g / L (1.30±0.005)mm / d. It can be seen that the appropriate addition amount can promote mycelial growth, and the optimal nitrogen source addition amount is 5g / L.

[0061] like Figure 8 As shown in the figure, with the increase of the addition amount of inorganic salt MgSO4, the mycelial biomass first increased and then decreased. When the addition amount was 2.5g / L, the mycelial biomass was the largest (2.5±0.005) and the bacterial density was high, followed by 3.5g / L (1.38±0.005). It can be seen that high concentrations of inorganic salts may have an inhibitory effect on mycelial growth, and the optimal addition amount of inorganic salt source is 2.5g / L.

[0062] The optimal culture medium formula found by this method is: 12g potato powder, 20g soluble starch, 20g agar powder, natural pH, 5g peptone, 2.5g MgSO4 and pure water to 1000ml.

[0063] Example 3: Optimization of mycelium culture process:

[0064] (1) Effect of light on mycelial growth

[0065] Use a 0.5mm puncher to inoculate Gymnopus sp.lhf1475 blocks on 90mm PDA culture medium, and culture them under light, dark, and light-dark alternating environments, with 5 replicates for each treatment. When the mycelium fills the plate, the mycelium growth rate is measured to obtain the optimal lighting conditions.

[0066] (2) Screening of culture temperature

[0067] The prepared solid culture medium (potato powder 12g, glucose 20g, agar powder 20g, natural PH, pure water to 1000ml, sterilized) was divided into 250ml shaking bottles, 100ml for each shaking bottle, and sterilized at 121℃ for 30 minutes. The culture medium was divided into 90mm diameter culture dishes on the ultra-clean workbench with UV turned on 25 minutes in advance, 20ml for each culture dish. After the culture medium was cooled, the Gymnopus sp.lhf1475 strain that had been cultured in advance was taken out and the mycelium with the best growth potential along the edge of the mycelium was evenly punched with a 0.5mm diameter puncher. The bacterial block was picked up with an inoculation needle and inoculated in the center of the new culture medium. After the inoculation, the culture medium was placed in a constant temperature incubator at 16℃, 19℃, 22℃, 25℃, 28℃, 31℃, 34℃, and 37℃, respectively, in the dark, and the mycelium growth rate and mycelium growth potential were recorded.

[0068] (3) Screening of pH of culture conditions

[0069] Prepare 1mol / L NaOH solution and HCl solution to adjust the pH of the prepared culture medium (12g potato extract powder, 20g glucose, 20g agar powder, natural pH, made up to 1000ml with pure water and sterilized). The pH was 4, 5, 6, 7, 8, 9, 10 respectively and then sterilized. The inoculated culture medium was cultured at the optimal temperature. The project measurement method, inoculation method and measurement method were the same as above.

[0070] Experimental results: Effects of different light conditions, temperature, and pH on the growth of Microdermabrasion mycelium

[0071] like Fig. 9As shown in the figure, Gymnopus sp.lhf1475 can grow normally under light, dark, and light-dark alternation conditions, and the fastest growth is (1.27±0.04) mm / d under light conditions, and the hyphae are dense and strong. Considering the hyphae growth rate and hyphae growth state, Gymnopus sp.lhf1475 is suitable for cultivation under light conditions.

[0072] like Fig.10 As shown in the figure, Gymnopus sp.lhf1475 can grow in the temperature range of 16-37℃, and the growth rate is 28℃>31℃>34℃>25℃>22℃>37℃>19℃>16℃. In the range of 16-28℃, the mycelium growth rate gradually increases with the increase of temperature, and the mycelium edges are relatively neat; at 28℃, the mycelium growth rate is the fastest at (1.06±0.06)mm / d. At 16℃, the mycelium is thick but grows extremely slowly, at (0.33±0.04)mm / d. When the temperature rises from 16℃ to 28℃, the mycelium thickness and growth rate increase with the increase of temperature. When the temperature rises from 28℃ to 37℃, the mycelium growth rate slows down and the mycelium edges are irregular. Considering the mycelium growth rate and mycelium growth state, the optimum temperature is 28℃.

[0073] like Fig.11 As shown in the figure, the mycelial growth rate of Gymnopus sp.lhf1475 is positively correlated with the increase of pH at pH 4-7. When pH is 7-8, the mycelial growth rate is the fastest, which is (1.11±0.03) mm / d, and the mycelium is dense and has neat edges; there is no significant difference under pH conditions of 5, 6, 9, and 10. When pH is 4, the mycelial growth rate is the slowest, which is (0.88±0.02) mm / d. Considering the mycelial growth rate and mycelial growth state, the most suitable pH value is between 7 and 8.

[0074] The culture method with the fastest mycelium growth rate is: the maximum amount of mycelium is obtained under light conditions, 28°C, and pH 7-8.

[0075] Example 4 Antioxidant activity of Gymnopus sp. lhf1475

[0076] 1. Liquid seed culture

[0077] Liquid seed culture medium: glucose 20g / L, peptone 4g / L, beef extract 4g / L, yeast extract 1g / L, KH2PO41.0g / L, MgSO4·7H2O 0.5g / L, pH 6.0, 100mL per bottle. The preparation method is to put each component into solvent water and sterilize it for later use.

[0078] The mycelial blocks of Gymnopus sp.lhf1475 were picked out from the slant solid culture medium, transferred to the liquid culture medium, and cultured at 28°C, 180 r / min, with shaking for 4-5 days as liquid seeds.

[0079] 2. Extraction of mycelium

[0080] Weigh 1.0 g of Gymnopus sp.lhf1475 mycelium, grind it into powder (freeze-dried sample), add distilled water at a solid-liquid ratio of 1 g:10 ml (adjusted according to the situation), heat in a water bath at 100°C for 3 h, centrifuge at 11000 r / min for 2 min, and take the supernatant, which is the water extract of Gymnopus sp.lhf1475.

[0081] 3. In vitro antioxidant test

[0082] DPPH free radical scavenging experiment is widely used to evaluate the antioxidant capacity of plant extracts (Lin Qunying, Long Liangkun, Xiang Zhun, et al. Study on the antioxidant activity of artificially cultivated and wild lead green pleated mushrooms [J]. Journal of Fujian Agricultural Sciences, 2018, 33(5): 534-537; Peng Changlian, Chen Shaowei. Evaluation of plant antioxidant capacity by scavenging organic free radicals DPPH method. Progress in Biochemistry and Biophysics, 2000, 27(6): 658-661.).

[0083] The calculation formula of the scavenging effect of the sample on DPPH free radicals is as follows:

[0084] DPPH clearance rate = (1-(A1-A2) / A0)*100%

[0085] A0: absorbance of 0.5mL ethanol + 1.5mL DPPH solution

[0086] A1: Absorbance value of 0.5mL sample solution + 1.5mL DPPH solution

[0087] A2: Absorbance value of 0.5mL sample solution + 1.5mL water

[0088] The experimental results show that the water extract and freeze-dried samples of Gymnopus sp.lhf1475 have good scavenging effects on DPPH free radicals. The scavenging rate of the water extract of Gymnopus sp.lhf1475 on DPPH is as high as 94.25%.

Claims

1. Gymnopus sp.lhf1475, deposit number: GDMCC No: 64861.

2. A method for cultivating Gymnopus sp.lhf1475 according to claim 1, characterized in that: The method is to inoculate Gymnopus sp.lhf1475 into a culture medium for cultivation.

3. The culture method according to claim 2, characterized in that The culture medium contains the carbon source, nitrogen source and inorganic salt required by Gymnopus sp. lhf1475.

4. The culture method according to claim 2, characterized in that The carbon sources are maltose, glucose and sucrose.

5. The culture method according to claim 2, characterized in that: The nitrogen source is peptone, yeast extract and yeast powder.

6. The culture method according to claim 2, characterized in that: The inorganic salt is MgSO4, CaCO3, KCl, MgCl 2、 NaCl.

7. The culture method according to claim 2, characterized in that: The culture medium contains 12 g potato extract powder, 20 g soluble starch, 20 g agar powder, 5 g peptone, 2.5 g MgSO4 per liter of water, and the pH is natural.

8. The culture method according to claim 2, characterized in that: The culture is carried out under light conditions, the culture temperature is 16-28°C, the optimal temperature is 28°C, and the pH value of the culture medium is between 7 and 8.

9. Use of Gymnopus sp. lhf1475 according to claim 1 in the preparation of antioxidant cosmetics or medicines.

10. The use according to claim 9, characterized in that: The Gymnopus sp.lhf1475 is a water extract of mycelium of Gymnopus sp.lhf1475.

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