CN115386497A - Aphroporus rugoso-annulata and cultivation method and application thereof - Google Patents

Abstract

The invention provides a chaetomium globosum and a cultivation method and application thereof. According to the invention, through tissue separation, identification and activity analysis of wild fruiting bodies of the chaetomium fortunei, a new strain YM11 (Coriopsistogrii) of the chaetomium fortunei with anti-tumor activity is discovered, and the strain resource of the chaetomium fortunei is enriched. The polysaccharide of the Geotrichum furiosum is found for the first time to be capable of inhibiting various tumor cells, has broad-spectrum and efficient anti-tumor activity and has important potential application value in the development of anti-tumor drugs.

Description

Aphroporus rugoso-annulata and cultivation method and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a new strain YM11 of Geotrichum bristlegrass with broad-spectrum efficient antitumor activity, and a cultivation method and application thereof.

Background

The macro fungi are important resources for excavating active substances of medicines, the extract in the shiitake mushrooms is found to have a barrier effect on tumors in mice by Japanese scientist Tianchi in 1969, research orders of edible and medicinal fungi are drawn from the research, with continuous exploration, the edible and medicinal fungi are found to be harmless to human bodies and can provide comprehensive nutrition, and then a large amount of edible and medicinal fungi are prepared into anti-cancer medicines, and more than 50 kinds of the edible and medicinal fungi are used for processing at present. Compared with western medicines, the large-scale medicinal fungi have the advantages of long curative effect and small damage to bodies, and have attracted high attention of the medical community in recent years, so that the development of medicinal fungi resources is of great significance for developing active medicines or medicine lead molecules.

The Coriolus versicolor (Coriolus trogii) is an important large medicinal fungus, belongs to Basidiomycota (Basidiomycota), agaricaceae (Agaricaceae), polyporales (Polyporales), polyporaceae (Polyporaceae) and Coriolus (Coriolus), is a wood rot fungus growing in temperate zone, is commonly found on poplar trees and willow trees, and is distributed in northeast, northwest and China, and the like. The Geotrichum furiosum has various biological activities, can secrete cellulase, peroxidase and laccase, and has strong lignin degradation capability; can decolorize industrial wastewater, andcan adsorb Hg ²⁺ 、 Cd ²⁺ And Zn ²⁺ Heavy metal ions, which have an environmental remediation function; the crude extract of the fruit body has multiple activities of improving immunity, resisting oxidation, protecting liver and the like, and has important medicinal value. However, at present, there are few reports on systematic identification and biological characteristic analysis of the chaetomium globosum, and there is no report on cultivation application of the chaetomium globosum, a wild chaetomium globosum is separated and identified, domestication cultivation of the strain and antitumor activity analysis of cultivated fruit bodies are carried out for the first time, resources of the chaetomium globosum are enriched, and references are provided for development and utilization of the strain.

Disclosure of Invention

The invention mainly aims to provide a coriolus versicolor (Coriolus trogii) YM11 with tumor cell inhibition function, which is preserved in China general microbiological culture Collection center (CGMCC for short, address: beijing city, chaoyang district, north Cheng West Lu No. 1 Hospital No. 3) in 2022, 10 days by excavating and separating, systematically classifying and domesticating the coriolus versicolor strain resource with anti-tumor activity, wherein the preservation number is as follows: CGMCC NO.40067.

The fruiting body of the strain is collected from willow on the roadside of 502 province of Qingdao city in Shandong province, wild fruiting bodies are subjected to tissue isolation to obtain a strain YM11, morphological analysis is closer to that of the Geotrichum briggo, and the strain has the highest homology similarity with the Geotrichum briggo (Coriolopsis trogii) and is found to have genetic difference with published Geotrichum briggo by comparison and phylogenetic analysis, so that the strain YM11 can be determined to be a new strain of the Geotrichum briggo by combining morphological characteristics.

The second purpose of the invention is to provide the application of the mannheimia pilosa (Coriolopsis trogii) YM11, namely the application of the mannheimia pilosa (Coriolopsis trogii) YM11 in preparing antitumor medicines.

Further, an extract from Geotrichum furiosum YM11 is used for preparing an antitumor drug. Preferably the extract comprises polysaccharides.

Further, tumors include: cervical cancer cells, breast cancer cells, and liver cancer cells.

The invention extracts crude polysaccharide from YM11 strain culture fruiting body, finds for the first time through in vitro cell experiment research that polysaccharide component in Geotrichum bristlensis can obviously inhibit cervical cancer cell (Hela), breast cancer cell (MCF-7) and liver cancer cell (Hep-3B) tumor cells, half of inhibition concentration of the polysaccharide component is respectively 2.14mg/mL, 1.37mg/mL and 2.43mg/mL, has broad-spectrum and efficient antitumor activity, and has important potential application value in the development of antitumor drugs.

The process of extracting the extract of the cultured fruit body of YM11 strain according to the polysaccharide extraction method is as follows: drying YM11 strain cultured fruiting body, grinding the dried fruiting body to obtain fruiting body powder, adding distilled water into the powder, heating and leaching, concentrating the obtained filtrate, removing protein to obtain water extract, slowly adding the water extract into anhydrous ethanol, standing at 4 deg.C, centrifuging at 4 deg.C at high speed, collecting bottom precipitate, and freeze drying the collected precipitate.

Further, 2kg of YM11 strain cultured fruit body is dried at 40 ℃, the dried fruit body is ground and filtered by a 200-mesh filter screen to obtain fruit body powder, distilled water is added into the powder, then the powder is leached for 12h at 100 ℃, the obtained filtrate is concentrated by rotary evaporation for 10 times, protein is removed by a sevag method to obtain a water extract, the water extract is slowly added into 4 times volume of absolute ethyl alcohol, the mixture is kept stand for 8 h-10 h at 4 ℃, then the mixture is centrifuged for 10min at 10000rpm and 4 ℃, bottom sediment is collected, and the collected sediment is frozen and dried.

The third purpose of the invention is to provide the cultivation method of the chaetomium globosum (Coriolopsis trogii) YM11, which comprises the following steps:

1) Culturing mother seeds;

2) Stock culture;

3) Manufacturing a cultivation bag;

4) And (5) fruiting body management.

The cultivation method further comprises the following steps:

1) Culturing mother seeds: inoculating YM11 strain on GYM solid culture medium, and culturing in dark to obtain culture mother strain;

2) Stock culture: inoculating the obtained mother culture in culture bag containing original culture medium by punching method, culturing in dark place, shaking off the blocks after mycelia germinate to enhance ventilation and promote mycelia growth until the bags are full of mycelia;

3) Manufacturing a cultivation bag: adopting cultivation bag material, sterilizing at 121 deg.C, and inoculating when sterilization is finished and cooling; inoculating the stock strain of the hard-bristle coarse-covered fungus into each bag, and culturing the fungus bags under the condition of keeping out of the sun after inoculation until the bags are full of hyphae;

4) And (3) fruiting body management, namely, after hyphae are fully distributed in the cultivation bag, opening the side edge to promote bud formation to obtain a fruiting body, culturing in a dark place before a bud is formed, and after the bud is formed, providing scattered light for a certain time every day for culturing.

The formula of the GYM solid culture medium is as follows: 30g/L glucose, 15g/L peptone, mgSO ₄ ·7H ₂ O 1g/L，KH ₂ PO ₄ 1.5g/L and 15g/L of agar powder.

Further, the cultivation method is more specifically as follows:

1) Mother seed culture: inoculating YM11 strain on GYM solid culture medium, and culturing at 25-28 deg.C in dark for 8-12 days to obtain culture mother strain;

2) Stock culture: inoculating the obtained mother culture into culture bags containing original culture medium by punching method, inoculating 3-6 pieces of bacteria in each bag, culturing at 25-28 deg.C and humidity of 50-70% in dark place, shaking off the pieces of bacteria every 8-12 days after hypha germinates to enhance ventilation and promote hypha growth until the hypha grows into full bags, wherein the original culture medium comprises the following formula: 85% of wheat grains, 11% of willow sawdust, 3% of glucose and 1% of gypsum; adding willow branch decoction filtrate accounting for 1/7 of the total weight of the culture medium, adding water until the humidity of the culture medium is 50-70%, weighing the wheat kernels according to the amount, soaking the wheat kernels in water for 1-3 days, and filtering the willow branch decoction filtrate by using three layers of gauze after boiling 7kg of water at 100 ℃ for 45min after each 1kg of willow branches are cut off;

3) Manufacturing a cultivation bag: adopting 17cm multiplied by 33cm polypropylene plastic cultivation bags, filling the uniformly stirred culture materials into the bags, wherein the bagging amount is 0.8-1.0kg per bag, and covering a plastic ring and a sealing cover after the material surface is flattened; sterilizing the bag material at 121 deg.C, cooling to 25-28 deg.C, and inoculating; inoculating 8-12g of Inonotus obliquus stock strain into each bag, and culturing the bags at 25-28 deg.C in the dark until the bag is full of mycelia; the formula of the culture material is as follows: 75% of willow sawdust, 10% of cottonseed hulls, 10% of bran, 4% of glucose, 1% of gypsum and 60% -65% of water content;

4) And fruiting body management, namely after the cultivation bags are fully covered with hypha, transversely cutting 3-5cm openings on the side edges of the cultivation bags by using a scalpel to perform bud forcing to obtain fruiting bodies, controlling the environmental temperature to be 25-28 ℃ and the environmental humidity to be 50-70% before forming buds, performing light-shielding cultivation, controlling the environmental temperature to be 23-26 ℃ and the environmental humidity to be 85-95% after forming the buds, and providing 1000Lx-1500 Lx scattered light for 2-3 h every day.

The invention has the advantages that:

1. according to the invention, through tissue separation, identification and activity analysis of wild fruiting bodies of the fomes fomentarius, a new strain YM11 (Coriolups trogii) of the fomes fomentarius with anti-tumor activity is discovered, the strain resource of the fomes fomentarius is enriched, and the application value of the novel strain is important in the aspect of drug development.

2. The invention discovers for the first time that the polysaccharide of the chaetomium fortunei can inhibit the tumor cells of cervical cancer cells (Hela), breast cancer cells (MCF-7) and liver cancer cells (Hep-3B), and the half inhibition concentrations are respectively as follows: 2.14mg/mL, 1.37mg/mL and 2.43mg/mL, has broad-spectrum and efficient antitumor activity and has important potential application value in the development of antitumor drugs.

3. The invention realizes the artificial cultivation of the fomes fomentarius for the first time, the yield of the fruit body reaches 1.2 kg/bag, the biological conversion rate reaches 80 percent, the polysaccharide content reaches 5.1 percent, the polysaccharide active ingredients are extracted from the cultivated fruit body, and the acclimatization cultivation of the fomes fomentarius YM11 comprises the following steps: the strain is subjected to mother strain preparation, stock strain preparation, cultivated species preparation and cultivation management in sequence to obtain a chaetomium fortunei cultivated sporocarp; domestication and cultivation of fomes fomentarius is reported for the first time.

Drawings

FIG. 1: fruiting body of wild Acorus robustus

A, the original habitat of the wild sporocarp; b: fruiting bodies; c: separating hypha from sporophore.

FIG. 2: morphological characteristics of fruiting body of wild Acorus pachyrhizi YM11

A, observing the surface of the fruiting body under a microscope (50X); b: observing the upper surface of the fruiting body with microscope (50X); c: longitudinal section of fruiting body and microscope observation (50X).

FIG. 3: scanning electron microscope observation of shape of Geotrichum furiosum YM11

A, mycelium germination stage morphology; b: mycelium aging morphology; C. d: spore morphology.

FIG. 4: geotrichum bristlensis YM11 is an ITS sequence-based phylogenetic tree.

FIG. 5: cultivation of Geotrichum chaetosa YM 11.

A. B, C cultivating young and tender fruiting body; D. e, F: and (5) cultivating the fruiting body at a mature stage.

FIG. 6: the polysaccharide of Fomitopsis furiosa inhibits tumor cells.

FIG. 7 is a schematic view of: MTT (methyl thiazolyl tetrazolium) analyzes the antitumor activity of polysaccharide of the sporocarp cultivated by the chaetomium fortunei.

The specific implementation mode is as follows:

the present invention will be further illustrated with reference to the following examples, which should not be construed as limiting the invention thereto.

Example 1 isolation of fruiting bodies of wild Acorus robustus

The strain has fruiting body obtained from willow on roadside of 502 province of Qingdao, shandong province, and is prepared by sterilizing surface of fruiting body, digging semen glycines-sized fruiting body block from middle part of fruiting body with sterile inoculating hook, and placing in culture dish containing GyM solid culture medium. Placing the culture dish in a constant-temperature incubator at 26 ℃, transferring the culture dish to a fresh GYM solid culture medium for culture after hyphae germinate to obtain hyphae, namely a pure hyphae culture of the hirsutella trogii YM11 strain, wherein the formulation of the GYM solid culture medium is as follows: glucose 30g/L, peptone 15g/L, KH ₂ PO ₄ 1.5g/L， MgSO ₄ ·7H ₂ O1.0 g/L and agar powder 15g/L.

Example 2 identification of YM11 Strain

Morphological identification: the fruiting body has no handle, grows in tile shape, has wood structure, 12-16cm of pileus extending outward, 3cm of flesh thickness, has yellowish brown upper surface, dense hard bristles, dense orifices on the lower surface, 2-4 orifices per millimeter, jagged edge, loose longitudinal section structure, obvious microtubules, and white hypha in the microtubules. The mycelium is white on a GyM solid culture medium, the mycelium structure is branched, no diaphragm exists, basidiospore is circular, the diameter is 2-3 mu m, the surface has obvious small thorns, and the shape basically accords with the characteristics of chaetomium globosum.

Molecular biological identification: the genome of YM11 strain was extracted using a fungal genome kit. The extracted genome is taken as a template, and the ITS sequence of the strain is subjected to PCR amplification by adopting universal primers ITS1 (5 '-TCC GTAGGTGGTGAACCTGCGG-3') and ITS4 (5 '-TCCTCCGCTTATTGA TATGC-3'). Reaction system 30.0 μ L: 5.0 μ L of 10 XPCR Buffer, 3.0 μ L of dNTP, 1.0 μ L of each of the forward primer and the reverse primer, 2.0 μ L of DNA template, 2U of Taq DNA polymerase, and the balance of sterile deionized water to 30.0 μ L. And (3) amplification procedure: pre-denaturation at 94 ℃ for 4min, at 94 ℃ for 45s, at 50 ℃ for 50s, at 72 ℃ for 90 s; 30 cycles were performed, extension at 72 ℃ for 10min and termination at 10 ℃. The ITS sequences obtained by PCR amplification are sent to Biotechnology engineering (Shanghai) GmbH for sequencing.

The sequencing result of the ITS sequence is subjected to Blast comparison in an NCBI database, the ITS sequence is closest to the ITS sequence of the Geotrichum chaetoli C.trogii (MG 779615.1), the ITS sequence with the alignment similarity close to the YM11 strain is selected, MEGA3.1 and Clustal X1.81 are used, and an adjacency method (N-J) is adopted to construct a phylogenetic tree (Replications =1000, bootstrap value is taken as percentage). Phylogenetic analysis results of the YM11 strain also show that the genetic relationship with the Acorus chaetomium is the closest, but the ITS sequence of the YM11 strain has the highest similarity of only 98% with the C.trogii (MG 779615.1) of the Acorus chaetomium, and the genetic difference with the published Acorus chaetomium, so that the YM11 strain can be determined to be a new strain of the Acorus chaetomium by combining morphological analysis.

The ITS sequence of Geotrichum bristlegrass YM11 is as follows.

TAGAGGAAGTAAAAGTCGTATCAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCA TTATCGAGTTTTGAAATGGGTTGTAGCTGGCTTCTCCGCAGGCATCTGCACGCCCTGCT CATCCACTCTACACCTGTGCACTTACTGTGGGTATCGGGAGGTGTCGCGTCGTTTACGG CGAGGCGTTAACCGTGCCTACGTTTTACTACAAACCATTCAGTATCAGAATGTGTATTG CGATGTAACGCATCTATATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATG AAGAACGCAGCGAAATGCGATAAGTAATGTGATTTGCAGAATTCTGTGAATCATCGAAT CTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATG AAATTCTCAAACCCATAAGTCTTTGCGGGCTTACGGGCTTTCGACTTGGAGGCTTGTCG GCGACCCTGAGGTCACATCGACTCCTCTCAAATGCATTAGCTTGATTCCTTGCGGATCG GCTCTCGGTGTGATAATTGTCTACGCCGTGACCGTGAAGCGTTTTGGCAAGCTTCTAAC CGTCTCTAACGAGACAGCTTACTTTGACCTCTGACCTCAAATCAGGTAGGACTACCCGC TGAACTTAAGCATATCAAA

EXAMPLE 3 cultivation of YM11 Strain

Mother seed culture: the YM11 strain is inoculated on a culture dish containing a GYM solid culture medium, and then placed in an incubator at 26 ℃ for light-shielding culture for 10 days to obtain a culture mother strain.

Stock culture: inoculating the obtained mother culture into culture bags containing stock culture medium by punching, inoculating 5 bacterial blocks into each bag, culturing in dark at 26 deg.C and humidity of 60%, shaking off the bacterial blocks every 10 days after hypha germinates to enhance ventilation and promote hypha growth until the hypha grows full of the bags, wherein the stock culture medium comprises the following components: 85% of wheat grains, 11% of willow sawdust, 3% of glucose, 1% of gypsum and 1/7 of willow branch decoction filtrate (the total weight of the culture medium is 1/7), water is added until the humidity of the culture medium is 60%, the wheat grains are weighed according to the amount and then soaked in water for 2 days, and the willow branch decoction filtrate is obtained by cutting every 1kg of willow branches, boiling the cut willow branches for 45min by using 7kg of water at the temperature of 100 ℃, and filtering the boiled filtrate by using three layers of gauze.

Manufacturing a cultivation bag: the culture material which is uniformly stirred is filled into bags by adopting polypropylene plastic cultivation bags with the specification of 17cm multiplied by 33cm, the bagging amount is 0.9kg per bag, and a plastic ring and a sealing cover are sleeved after the material surface is flattened. Sterilizing the bag material at 121 deg.C for 4 hr, and cooling to 26 deg.C for inoculation. Inoculating 10g of the original strain of Geotrichum furiosum into each bag, and culturing the bags at 26 deg.C in the dark until the bags are full of mycelia. The formula of the culture material is as follows: 75% of willow sawdust, 10% of cottonseed hulls, 10% of bran, 4% of glucose, 1% of gypsum and 60% -65% of water content;

and (3) fruiting body management, namely after the cultivation bag is fully covered with hypha, transversely cutting a port of 4 cm on the side edge of the bag by using a scalpel to perform bud forcing and fruiting body generation, controlling the environmental temperature to be 26 ℃ and the environmental humidity to be 60% before forming a bud, performing dark cultivation, controlling the environmental temperature to be 26 ℃ and the environmental humidity to be 90% after forming the bud, and providing 1000Lx-1500 Lx scattered light for 2 h-3 h every day.

The fruiting bodies of the chaetomium globosum normally grow after the steps are sequentially carried out, the hypha of the cultivation bag is white, the fruiting bodies are white at the initial stage and are yellow brown after being matured, and the cultivation method basically accords with the morphological characteristics of wild fruiting bodies, the yield of the chaetomium globosum fruiting bodies cultivated by the method reaches 1.2 kg/bag, the biological conversion rate reaches 80%, the polysaccharide content reaches 5.1%, and the cultivation of the chaetomium globosum with high yield, high conversion rate and high active ingredients is realized.

Example 4 extraction of polysaccharides from cultured fruiting bodies of YM11 Strain

Drying 2kg of YM11 strain cultured fruiting body at 40 deg.C, grinding the dried fruiting body, sieving with 200 mesh sieve to obtain fruiting body powder, adding distilled water into the powder, leaching at 100 deg.C for 12h, concentrating the filtrate by rotary evaporation for 10 times, removing protein by sevag method to obtain water extract, slowly adding the water extract into 4 times volume of anhydrous ethanol, standing at 4 deg.C for 10h, centrifuging at 10000rpm and 4 deg.C for 10min, collecting bottom precipitate, and freeze drying the collected precipitate to obtain polysaccharide.

Example 5 antitumor Activity of polysaccharides of cultured fruiting body of YM11 Strain

The polysaccharide is proportioned into different concentration gradients by using sterile water: 0. 0.5, 1, 1.5, 2, 2.5, 3, 3.5mg/mL sterile water as control, and inoculating Hela, MCF-7, hep-3B tumor cells in 96-well plate, wherein the number of cells per well is 1 × 10 ³ cells, volume 100. Mu.L, to which cells are attachedAdding 10 μ L of polysaccharide solution with different concentration gradients, and placing in 5% CO ₂ In the incubator, after 48h of culture, the cell morphology change is observed (see fig. 6), the inhibition rate of the active component on the tumor cells is calculated by using the MTT method (see fig. 7), and the half-lethal concentration IC of the active substance is calculated by using SPSS13.0 software ₅₀ . As a result, after Hela, MCF-7 and Hep-3B tumor cells are treated by YM11 strain polysaccharide, the cell morphology is shrunk, the cell number is reduced, the cells are obviously inhibited, and the half inhibition concentrations are respectively as follows: 2.14mg/mL, 1.37mg/mL and 2.43mg/mL, has broad-spectrum and efficient antitumor activity and has important potential application value in the development of antitumor drugs.

Sequence listing

<110> institute of microbiology, hunan province

<120> a chaetomium globosum and cultivation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

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tccgtaggtg gtgaacctgc gg 22

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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tcctccgctt attgatatgc 20

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<211> 664

<212> DNA

<213> Aphanomyces rugosus (Coriolopsis trogii)

<400> 3

tagaggaagt aaaagtcgta tcaaggtttc cgtaggtgaa cctgcggaag gatcattatc 60

gagttttgaa atgggttgta gctggcttct ccgcaggcat ctgcacgccc tgctcatcca 120

ctctacacct gtgcacttac tgtgggtatc gggaggtgtc gcgtcgttta cggcgaggcg 180

ttaaccgtgc ctacgtttta ctacaaacca ttcagtatca gaatgtgtat tgcgatgtaa 240

cgcatctata tacaactttc agcaacggat ctcttggctc tcgcatcgat gaagaacgca 300

gcgaaatgcg ataagtaatg tgatttgcag aattctgtga atcatcgaat ctttgaacgc 360

accttgcgct ccttggtatt ccgaggagca tgcctgtttg agtgtcatga aattctcaaa 420

cccataagtc tttgcgggct tacgggcttt cgacttggag gcttgtcggc gaccctgagg 480

tcacatcgac tcctctcaaa tgcattagct tgattccttg cggatcggct ctcggtgtga 540

taattgtcta cgccgtgacc gtgaagcgtt ttggcaagct tctaaccgtc tctaacgaga 600

cagcttactt tgacctctga cctcaaatca ggtaggacta cccgctgaac ttaagcatat 660

caaa 664

Claims (9)

1. A strain of Geotrichum furiosum (Coriolopsis trogii) YM11 with the preservation number of CGMCC NO.40067.

2. Use of Geotrichum chaetomium (Coriolus trogii) YM11 according to claim 1 for the preparation of an anti-tumor medicament.

3. Use according to claim 2, characterized in that the extract of Geotrichum bristlensis YM11 is used for the preparation of an antitumor medicament, preferably the extract comprises polysaccharides.

4. The use of claim 2 or 3, wherein the tumor comprises: cervical cancer cells, breast cancer cells, and liver cancer cells.

5. The use as claimed in claim 3, wherein the extract is extracted as follows: drying YM11 strain cultured fruiting body, grinding the dried fruiting body to obtain fruiting body powder, adding distilled water into the powder, heating and leaching, concentrating the obtained filtrate, removing protein to obtain water extract, slowly adding the water extract into anhydrous ethanol, standing at 4 deg.C, centrifuging at 4 deg.C at high speed, collecting bottom precipitate, and freeze drying the collected precipitate.

6. The use according to claim 5, wherein 2kg of the cultured fruit body of YM11 strain is dried at 40 ℃, the dried fruit body is ground and passed through a 200 mesh sieve to obtain a fruit body powder, distilled water is added to the powder, then the powder is leached at 100 ℃ for 12 hours, the obtained filtrate is concentrated by rotary evaporation 10 times, protein is removed by sevag method to obtain an aqueous extract, the aqueous extract is slowly added to 4 times volume of absolute ethyl alcohol, the mixture is allowed to stand at 4 ℃ for 8 to 10 hours, then the mixture is centrifuged at 10000rpm and 4 ℃ for 10 minutes to collect bottom precipitate, and the collected precipitate is freeze-dried.

7. The method for cultivating Coriolus versicolor (Coriolus trogii) YM11 according to claim 1, comprising the steps of:

1) Culturing mother seeds;

2) Stock culture;

3) Manufacturing a cultivation bag;

4) And (5) fruiting body management.

8. The cultivation method as claimed in claim 7,

1) Culturing mother seeds: inoculating YM11 strain on a GYM solid culture medium, and culturing in dark to obtain a culture mother strain;

2) Stock culture: inoculating the obtained mother culture in culture bag containing culture medium by punching method, culturing in dark place, shaking off the blocks after mycelia germinate to enhance ventilation and promote mycelia growth until the bags are full of mycelia;

3) Manufacturing a cultivation bag: adopting cultivation bag material, sterilizing at 121 deg.C, and inoculating when sterilization is finished and cooling; inoculating the stock strain of the hard-bristle coarse-covered fungus into each bag, and culturing the fungus bags under the condition of keeping out of the sun after inoculation until the bags are full of hyphae;

4) And (3) fruiting body management, namely, after hyphae are fully distributed in the cultivation bag, opening the side edge to promote bud formation to obtain a fruiting body, culturing in a dark place before a bud is formed, and after the bud is formed, providing scattered light for a certain time every day for culturing.

9. The cultivation method as claimed in claim 8,

1) Mother seed culture: inoculating YM11 strain on GYM solid culture medium, and culturing at 25-28 deg.C in dark for 8-12 days to obtain culture mother strain;

2) Stock culture: inoculating the obtained cultured mother strain into culture bags containing stock culture medium by punching, inoculating 3-6 strain blocks into each bag, culturing in dark at 25-28 deg.C and humidity of 50-70%, shaking out strain blocks every 8-12 days after mycelia germinate to enhance ventilation and promote mycelia growth until the bags are full of mycelia, wherein the stock culture medium comprises: 85% of wheat grains, 11% of willow sawdust, 3% of glucose and 1% of gypsum; adding willow branch decoction filtrate accounting for 1/7 of the total weight of the culture medium, adding water until the humidity of the culture medium is 50-70%, weighing the wheat kernels according to the amount, soaking the wheat kernels in water for 1-3 days, and filtering the willow branch decoction filtrate by using three layers of gauze after boiling 7kg of water at 100 ℃ for 45min after each 1kg of willow branches are cut off;

3) Manufacturing a cultivation bag: adopting polypropylene plastic cultivation bags with the specification of 17cm multiplied by 33cm, filling the uniformly stirred culture materials into the bags, wherein the bagging amount is 0.8-1.0kg per bag, and sleeving a plastic ring and a sealing cover after flattening the material surface; sterilizing the bag material at 121 deg.C, cooling to 25-28 deg.C, and inoculating; inoculating 8-12g of coarse hirsutella sinensis stock into each bag, and culturing the bags at 25-28 deg.C in the dark until mycelia grow over the bags; the formula of the culture material is as follows: 75% of willow sawdust, 10% of cottonseed hulls, 10% of bran, 4% of glucose, 1% of gypsum and 60% -65% of water content;

4) And fruiting body management, namely after the cultivation bags are fully covered with hypha, transversely cutting 3-5cm openings on the side edges of the cultivation bags by using a scalpel to perform bud forcing to obtain fruiting bodies, controlling the environmental temperature to be 25-28 ℃ and the environmental humidity to be 50-70% before forming buds, performing light-shielding cultivation, controlling the environmental temperature to be 23-26 ℃ and the environmental humidity to be 85-95% after forming the buds, and providing 1000Lx-1500 Lx scattered light for 2-3 h every day.

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