CN115386497B - Podosporium cranbergii strain, cultivation method and application thereof - Google Patents
Podosporium cranbergii strain, cultivation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract
The invention provides a strain of phellinus linteus, a cultivation method and application thereof. The invention discovers a strain YM11 (Coriopsstrongii) of the Philippine cinquefoil Kong Junxin with anti-tumor activity through tissue separation, identification and activity analysis of wild fruiting bodies of the Philippine cinquefoil, and enriches strain resources of the Philippine cinquefoil. The polysaccharide of the Philippine cinquefoil herb has the advantages of inhibiting various tumor cells, having broad-spectrum high-efficiency anti-tumor activity and having important potential application value in the aspect of anti-tumor drug development.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a bristle thick cover Kong Junxin strain YM11 with broad-spectrum efficient antitumor activity, and a cultivation method and application thereof.
Background
The large fungi are important resources for excavating medicinal active substances, the extracts in the mushrooms are found to have an obstructing effect on tumors in mice in the field of Japanese scientist in 1969, the research prologues of the edible and medicinal fungi are pulled open from the research, along with continuous exploration, the edible and medicinal fungi are found to be harmless to human bodies and provide comprehensive nutrition, and then a large amount of edible and medicinal fungi are prepared into anticancer drugs, so that more than 50 kinds of edible and medicinal fungi are used for processing at present. Compared with western medicines, the large medicinal fungi have the advantages of long curative effect and small damage to the body, and have attracted high attention in the medical community in recent years, so that the development of medicinal fungi resources is significant for the development of active medicines or medicine lead molecules.
The Philippine cinquefoil (Coriolopsis trogii) is an important large medicinal fungus belonging to Basidiomycetes, agaricales, polyporales, polyporaceae, coriolus, and is a wood rot fungus growing in temperate zone, commonly found on poplar and willow, distributed in northeast, north China, inner Mongolia, northwest, china, etc. The phellinus linteus has various biological activities, can secrete cellulase, peroxidase and laccase, and has stronger lignin degradation capability; can decolorize industrial wastewater and adsorb Hg 2+ 、 Cd 2+ And Zn 2+ Heavy metal ions have an environmental remediation function; the fruit body crude extract has various activities of improving immunity, resisting oxidation, protecting liver and the like, and has important medicinal value. However, the current report on systematic identification and biological characteristic analysis of the Philippine California is less, and no report on cultivation application of the Philippine California exists, and we have isolated and identified a wild Philippine California strain and developed domestication of the strain for the first timeThe cultivation and the antitumor activity analysis of the cultivated fruiting body enrich the resource of the sclerotium rolfsii and provide reference for the development and utilization of the strain.
Disclosure of Invention
The primary purpose of the invention is to separate, systematically classify and domesticate and cultivate the strain resources of the Inula fornix with anti-tumor activity by excavating, and provide an Inula fornix (Coriolopsis trogii) YM11 strain with tumor cell inhibition, which is preserved in China general microbiological culture Collection center (CGMCC, address: north Chen Silu No. 1, 3 in the Korean region of Beijing) on the day 10 of 2 months in 2022, with the preservation number of: CGMCC No.40067.
The fruiting body of the strain is obtained from willows collected from the roadside of Qingdao 502 of Shandong province, the wild fruiting body is subjected to tissue separation to obtain the strain YM11, the strain is relatively close to the Philippine cinquefoil herb through morphological analysis, the ITS sequences of the strain are compared and phylogenetic analysis is carried out to find that the strain has the highest homology similarity with the Philippine cinquefoil herb (Coriolopsis trogii), the highest similarity is 98%, genetic difference exists between the strain YM11 and the published Philippine cinquefoil herb, and the strain YM11 can be determined to be a new strain of the Philippine cinquefoil herb by combining morphological characteristics.
A second object of the invention is to provide the use of said Convolvulus arvensis (Coriolopsis trogii) YM11, i.e. for the preparation of antitumor drugs.
Further, the extract of the Convolvulus arvensis YM11 is used for preparing antitumor drugs. Preferably the extract comprises a polysaccharide.
Further, the tumor includes: cervical cancer cells, breast cancer cells, and liver cancer cells.
The invention extracts crude polysaccharide from YM11 strain cultivated sporophore, and discovers that polysaccharide components in the Inula fordii can obviously inhibit cervical cancer cells (Hela), breast cancer cells (MCF-7) and liver cancer cells (Hep-3B) tumor cells for the first time through in vitro cell experimental study, and half inhibition concentrations of the polysaccharide components are respectively 2.14mg/mL, 1.37mg/mL and 2.43mg/mL, so that the polysaccharide components have broad-spectrum efficient antitumor activity and important potential application values in the aspect of antitumor drug development.
The process of extracting YM11 strain cultivated sporophore extract according to polysaccharide extraction method is as follows: drying YM11 strain cultivated fruiting body, grinding the dried fruiting body to obtain fruiting body powder, adding distilled water into the powder, heating and extracting, concentrating the obtained filtrate, removing protein to obtain water extract, slowly adding the water extract into absolute ethanol, standing at 4deg.C, centrifuging at high speed to collect bottom precipitate, and freeze drying the collected precipitate.
Further, 2kg of YM11 strain cultivated fruiting body is taken and dried at 40 ℃, the dried fruiting body is ground and filtered by a 200-mesh filter screen to obtain fruiting body powder, distilled water is added into the powder, then leaching is carried out for 12 hours at 100 ℃, the obtained filtrate is concentrated by rotary evaporation for 10 times, the 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, standing is carried out for 8-10 hours at 4 ℃, centrifugation is carried out for 10 minutes at 10000rpm and 4 ℃, bottom sediment is collected, and the collected sediment is frozen and dried.
The third object of the present invention is to provide a cultivation method of the Convolvulus arvensis (Coriolopsis trogii) YM11, comprising 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) Mother culture: inoculating YM11 strain on GYM solid culture medium, and culturing in dark to obtain cultivation mother strain;
2) Stock culture: inoculating the obtained culture mother strain into a culture bag containing an original strain culture medium by a punching method, performing light-proof culture, shaking up fungus blocks to enhance ventilation after hyphae germinate, and promoting hyphae growth until the hyphae grow to be full of the bag;
3) And (3) manufacturing a cultivation bag: bagging materials by using a cultivation bag, sterilizing at 121 ℃, and inoculating when cooling after sterilization is finished; inoculating a crude strain of the porus rupestris into each bag, and culturing the fungus bags under a light-proof condition until mycelia grow fully;
4) Fruiting body management, namely after hypha is fully distributed in a cultivation bag, a side opening is used for promoting bud fruiting body, before forming buds, the fruiting body is cultivated in a dark place, and after forming the buds, scattered light cultivation is provided for a certain time every day.
The formula of the GYM solid medium comprises: glucose 30g/L, peptone 15g/L, mgSO 4 ·7H 2 O 1g/L，KH 2 PO 4 1.5g/L, 15g/L of agar powder.
Further, the cultivation method is more specifically as follows:
1) Mother culture: inoculating YM11 strain on GYM solid culture medium, and culturing at 25-28deg.C in dark for 8-12d to obtain cultivation mother strain;
2) Stock culture: inoculating the obtained cultivation mother seeds into culture bags containing stock culture media by a punching method, inoculating 3-6 fungus blocks in each bag, performing light-proof cultivation under the conditions of 25-28 ℃ and 50-70% humidity, and shaking the fungus blocks every 8-12d to enhance ventilation after hyphae germinate, so as to promote hyphae growth until the hyphae grow into bags, wherein the stock culture media comprises the following formula: 85% of wheat grains, 11% of willow sawdust, 3% of glucose and 1% of gypsum; adding 1/7 of the water decoction filtrate of ramulus Salicis Babylonicae (1 kg/1) of the total weight of the culture medium, adding water until the humidity of the culture medium is 50-70%, weighing the wheat grains, soaking in water for 1-3 days, cutting every 1kg of ramulus Salicis Babylonicae, boiling with 7kg of water at 100deg.C for 45min, and filtering with three layers of gauze;
3) And (3) manufacturing a cultivation bag: a 17cm multiplied by 33cm polypropylene plastic cultivation bag is adopted, the evenly stirred culture materials are filled into bags, the bagging amount is 0.8-1.0kg of each bag, and a plastic ring and a sealing cover are sleeved after the material surface is flattened; sterilizing the bag material at 121 ℃, cooling to 25-28 ℃ after sterilization, and inoculating; inoculating 8-12g of Podospora crassa stock strain into each bag, inoculating, and culturing the fungus bags at 25-28deg.C under dark condition until mycelia grow fully; the formula of the culture material is as follows: 75% of willow sawdust, 10% of cotton seed hulls, 10% of bran, 4% of glucose, 1% of gypsum and 60% -65% of water content;
4) Fruiting body management, namely after hypha is fully distributed in a cultivation bag, transversely cutting a 3-5cm opening on the side edge of the bag by using a surgical knife to promote bud fruiting body, controlling the environmental temperature to be 25-28 ℃ and the environmental humidity to be 50-70% before forming buds, culturing in a dark place, 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 hours per day.
The invention has the advantages that:
1. the invention discovers a strain YM11 (Coriolopsis trogii) of the Philippine cinquefoil herb Kong Junxin with anti-tumor activity through tissue separation, identification and activity analysis of wild fruit bodies of the Philippine cinquefoil herb, enriches strain resources of the Philippine cinquefoil herb, and has important potential application value in the aspect of drug development.
2. The invention discovers that the polysaccharide of the stropharia rugosa can inhibit cervical cancer cells (Hela), breast cancer cells (MCF-7) and liver cancer cells (Hep-3B) tumor cells for the first time, and the half inhibition concentrations are respectively as follows: 2.14mg/mL, 1.37mg/mL and 2.43mg/mL, has broad-spectrum high-efficiency anti-tumor activity and has important potential application value in the aspect of anti-tumor drug development.
3. According to the invention, the artificial cultivation of the phellinus linteus is realized for the first time, the fruiting body yield reaches 1.2 kg/bag, the biological conversion rate reaches 80%, the polysaccharide content reaches 5.1%, the polysaccharide active ingredient is extracted from the cultivated fruiting body, and the domestication cultivation of the phellinus linteus YM11 is realized: the strain is subjected to mother seed production, stock seed production, cultivar production and cultivation management in sequence to obtain a culture fruiting body of the phellodendron amurense; domesticated cultivation of Convolvulus arvensis was reported for the first time.
Drawings
Fig. 1: wild Conium roseum fruiting body
A, wild fruiting body original habitat; b: a fruiting body; c: the fruiting body is separated into mycelium.
Fig. 2: fruiting body shape characteristics of wild Conoplarum crudus YM11
A1, observing the lower surface of the fruiting body by a microscope (50X); b: microscopic observation of the upper surface of the fruiting body (50X); c: the fruiting body was observed with a split microscope (50X).
Fig. 3: scanning electron microscope observation of the morphology of Conoclavia crudus YM11
A, mycelium germination stage morphology; b: mycelium ageing morphology; C. d: spore morphology.
Fig. 4: phellinus linteus YM11 phylogenetic tree based on ITS sequence.
Fig. 5: cultivation of Convolvulus arvensis YM 11.
A. B, C the tender stage of fruiting body; D. e, F: and (5) cultivating fruiting body mature stage.
Fig. 6: the polysaccharide of the George crassifolium inhibits tumor cells.
Fig. 7: MTT assay for antitumor Activity of polysaccharide from fruiting body of Calophyllum Instrongylum cultivated fruiting body.
The specific embodiment is as follows:
the invention will be further illustrated with reference to the following examples without limiting the invention.
EXAMPLE 1 isolation of wild Confucius crassa fruiting bodies
The fruiting body of the strain is obtained from willow at road side of Qingdao 502 in Shandong province, and after surface sterilization, fruiting body blocks of soybean size are dug from middle part of fruiting body with sterile inoculating hook, and placed in culture dish containing GYM solid culture medium. Placing the culture dish in a constant temperature incubator at 26 ℃, transferring the culture dish into a fresh GYM solid culture medium for culture after the hyphae germinate, wherein the obtained hyphae are pure hyphae culture of the strain YM11 of the Philippine cinnaria, and the GYM solid culture medium is prepared according to the following formula: glucose 30g/L, peptone 15g/L, KH 2 PO 4 1.5g/L， MgSO 4 ·7H 2 O1.0 g/L, agar powder 15g/L.
Example 2 identification of YM11 Strain
Morphological identification: the fruiting body has no handle, is covered with tile-shaped growth, has a wooden structure, extends out a fungus cover for 12-16cm, has fungus meat with a thickness of 3cm, has yellow brown upper surface, dense bristles for growth, has dense holes on the lower surface, has yellow brown holes of 2-4 holes per millimeter, has saw-tooth edges, has a loose longitudinal section structure, has obvious microtubes, has the same color as the fungus meat, and has visible white hyphae in the microtubes. Hypha is white on GYM solid culture medium, the hypha structure is branched and has no diaphragm, basidiomycete is round, the diameter is 2-3 mu m, obvious thorns are arranged on the surface, and the shape basically accords with the characteristics of the thick cover porium of the bristle.
Molecular biology identification: the genome of YM11 strain was extracted using a fungal genome kit. The extracted genome is used as template, and the common primers ITS1 (5 '-TCC GTAGGTGGTGAACCTGCGG-3') and ITS4 (5 '-TCCTCCGCTTATTGA TATGC-3') are used for PCR amplification of the ITS sequence of the strain. Reaction system 30.0 μl:10 XPCR Buffer 5.0. Mu.L, dNTP 3.0. Mu.L, 1.0. Mu.L each of the forward and reverse primers, 2.0. Mu.L of DNA template, 2U of Taq DNA polymerase, and 30.0. Mu.L of sterile deionized water. Amplification procedure: pre-denatured at 94℃for 4min,94℃for 45s,50℃for 50s,72℃for 90 s; 30 cycles were performed, extending at 72℃for 10min and ending at 10 ℃. The ITS sequences obtained by PCR amplification were sent to the engineering (Shanghai) Co., ltd for sequencing.
Blast comparison is carried out on the results of the ITS sequence sequencing in NCBI database, the ITS sequence closest to the ITS sequence of the Geotrichum arvense C.trogii (MG 779615.1) is found, the ITS sequence with similar comparison of YM11 strain is selected, and a phylogenetic tree (replication=1000, bootstrap value percentage) is constructed by adopting MEGA3.1 and Clustal X1.81 and adopting an adjacent method (N-J). Phylogenetic analysis of YM11 strain also showed closest affinity to Calophyllum Instrongylus, but the highest similarity of YM11 strain ITS sequence to Calophyllum Instrongylus C.trogii (MG 779615.1) was only 98%, and genetic differences with published Calophyllum Instrongylus were seen, so that in combination with morphological analysis YM11 strain could be determined to be a new strain of Calophyllum Instrongylus.
The ITS sequence of Convolvulus arvensis YM11 is as follows.
TAGAGGAAGTAAAAGTCGTATCAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCA TTATCGAGTTTTGAAATGGGTTGTAGCTGGCTTCTCCGCAGGCATCTGCACGCCCTGCT CATCCACTCTACACCTGTGCACTTACTGTGGGTATCGGGAGGTGTCGCGTCGTTTACGG CGAGGCGTTAACCGTGCCTACGTTTTACTACAAACCATTCAGTATCAGAATGTGTATTG CGATGTAACGCATCTATATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATG AAGAACGCAGCGAAATGCGATAAGTAATGTGATTTGCAGAATTCTGTGAATCATCGAAT CTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATG AAATTCTCAAACCCATAAGTCTTTGCGGGCTTACGGGCTTTCGACTTGGAGGCTTGTCG GCGACCCTGAGGTCACATCGACTCCTCTCAAATGCATTAGCTTGATTCCTTGCGGATCG GCTCTCGGTGTGATAATTGTCTACGCCGTGACCGTGAAGCGTTTTGGCAAGCTTCTAAC CGTCTCTAACGAGACAGCTTACTTTGACCTCTGACCTCAAATCAGGTAGGACTACCCGC TGAACTTAAGCATATCAAA
EXAMPLE 3 cultivation of YM11 Strain
Mother culture: after the YM11 strain is inoculated on a culture dish containing GYM solid culture medium, the culture dish is placed in a 26 ℃ incubator to be subjected to light-proof culture for 10d, and a cultivation mother strain is obtained.
Stock culture: inoculating the obtained cultivation mother seeds into culture bags containing stock culture media by a punching method, inoculating 5 fungus blocks in each bag, performing light-proof cultivation under the conditions of 26 ℃ and 60% humidity, shaking the fungus blocks every 10d after hypha germinates to enhance ventilation and promote hypha growth until the hypha grows into the bags, wherein the stock culture media comprises the following formula: 85% of wheat grains, 11% of willow wood dust, 3% of glucose, 1% of gypsum and water decoction filtrate of willow branches (1/7 of the total weight of the culture medium), adding water until the humidity of the culture medium is 60%, weighing the wheat grains according to the weight, soaking the wheat grains in water for 2 days, wherein the willow branch water decoction filtrate is obtained by cutting every 1kg of willow branches, boiling the willow branches with 7kg of water at 100 ℃ for 45min, and filtering the willow branches with three layers of gauze.
And (3) manufacturing a cultivation bag: a17 cm multiplied by 33cm polypropylene plastic cultivation bag is adopted, the evenly stirred culture materials are filled into bags, 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 121deg.C for 4 hr, cooling to 26deg.C after sterilization, and inoculating. 10g of crude porus rupestris seeds are inoculated into each bag, and after inoculation, the fungus bags are placed at 26 ℃ and are cultivated under the dark condition until mycelia grow into full bags. The formula of the culture material is as follows: 75% of willow sawdust, 10% of cotton seed hulls, 10% of bran, 4% of glucose, 1% of gypsum and 60% -65% of water content;
and (3) fruiting body management, namely after hyphae are fully distributed in a cultivation bag, transversely cutting a port of 4 cm on the side edge of the bag by using a surgical knife to promote bud fruiting bodies, controlling the ambient temperature to 26 ℃ and the ambient humidity to 60% before forming buds, culturing in a dark place, controlling the ambient temperature to 26 ℃ and the ambient humidity to 90% after forming the buds, and providing 1000Lx-1500 Lx scattered light for 2-3 hours per day.
The fruit bodies of the sclerotium rolfsii are normally discharged after the steps are sequentially carried out, hyphae of a cultivation bag are white, the fruit bodies are white at the initial stage and are yellow brown after being ripe, the morphological characteristics of wild fruit bodies are basically met, the yield of the fruit bodies of the sclerotium rolfsii 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 sclerotium rolfsii with high yield, high conversion rate and high active ingredients is realized.
EXAMPLE 4 extraction of polysaccharide from fruiting body of YM11 Strain
2kg of YM11 strain cultivated fruiting body is taken and dried at 40 ℃, the dried fruiting body is ground and filtered by a 200-mesh filter screen to obtain fruiting body powder, distilled water is added into the powder, the powder is leached for 12 hours at 100 ℃, the obtained filtrate is concentrated by rotary evaporation for 10 times, the protein is removed by a sevag method to obtain a water extract, the water extract is slowly added into absolute ethyl alcohol with 4 times of volume, the water extract is kept stand for 10 hours at 4 ℃, and then the sediment at the bottom is collected after centrifugation for 10 minutes at 10000rpm and 4 ℃, and the collected sediment is frozen and dried to obtain polysaccharide.
EXAMPLE 5 antitumor Activity of polysaccharide from fruiting body of YM11 Strain cultivation
The polysaccharide is proportioned into different concentration gradients by sterile water: 0. 0.5, 1, 1.5, 2, 2.5, 3, 3.5mg/mL, sterile water as control, hela, MCF-7, hep-3B tumor cells were seeded in 96-well plates at a cell number of 1X 10 per well 3 cells, 100. Mu.L, 10. Mu.L of polysaccharide solution with different concentration gradients are added after the cells are attached, and the mixture is placed in 5% CO 2 After 48h incubation in an incubator, the morphological changes of the cells were observed (see FIG. 6), and the inhibition of the tumor cells by the active ingredient was calculated by MTT method (see FIG. 7), and the semi-lethal concentration IC of the active ingredient was calculated by SPSS13.0 software 50 . As a result, it was found that after treatment of Hela, MCF-7 and Hep-3B tumor cells with YM11 strain polysaccharide, the cell morphology was contracted and the cell number was decreased, and the cells were significantly inhibited, half of which was inhibitedThe preparation concentration is respectively as follows: 2.14mg/mL, 1.37mg/mL and 2.43mg/mL, has broad-spectrum high-efficiency anti-tumor activity and has important potential application value in the aspect of anti-tumor drug development.
Sequence listing
<110> Hunan province institute of microorganisms
<120> A strain of Podospora crassa and cultivation method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tccgtaggtg gtgaacctgc gg 22
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 664
<212> DNA
<213> Confucius (Coriolopsis trogii) of the bristle
<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 (7)
1. Acidovorax faciens strainCoriolopsis trogii) YM11 with the preservation number of CGMCC NO.40067.
2. The thick california of claim 1Coriolopsis trogii) The application of YM11 is characterized in that polysaccharide extract of the Convolvulus arvensis YM11 is used for preparing antitumor drugs; the tumors are: cervical cancer cells, breast cancer cells, and liver cancer cells.
3. Use according to claim 2, characterized in that the extraction process of the extract is as follows: drying YM11 strain cultivated fruiting body, grinding the dried fruiting body to obtain fruiting body powder, adding distilled water into the powder, heating and extracting, concentrating the obtained filtrate, removing protein to obtain water extract, slowly adding the water extract into absolute ethanol, standing at 4deg.C, centrifuging at high speed to collect bottom precipitate, and freeze drying the collected precipitate.
4. Use according to claim 3, characterized in that 2kg YM11 strain cultivated fruiting bodies are taken and dried at 40 ℃, the dried fruiting bodies are ground and filtered by a 200 mesh filter to obtain fruiting body powder, distilled water is added to the powder, then 12h is leached at 100 ℃, the obtained filtrate is concentrated by rotary evaporation to 10 times, the protein is removed by sevag method to obtain water extract, the water extract is slowly added into 4 times volume of absolute ethanol, and then is left to stand at 4 ℃ for 8 h-10 h, and then is centrifuged at 10000rpm for 10min at 4 ℃, the bottom sediment is collected, and the collected sediment is freeze-dried.
5. The thick california of claim 1Coriolopsis trogii) The cultivation method of YM11 is characterized by comprising the following steps: 1) Culturing mother seeds; 2) Stock culture; 3) Manufacturing a cultivation bag; 4) And (5) fruiting body management.
6. The cultivation method according to claim 5, wherein 1) the mother culture: inoculating YM11 strain on GYM solid culture medium, and culturing in dark to obtain cultivation mother strain; 2) Stock culture: inoculating the obtained culture mother strain into a culture bag containing a culture medium by a punching method, performing light-proof culture, shaking up fungus blocks to enhance ventilation after hyphae germinate, and promoting hyphae growth until the hyphae grow to be full of the bag; 3) And (3) manufacturing a cultivation bag: bagging materials by using a cultivation bag, sterilizing at 121 ℃, and inoculating when cooling after sterilization is finished; inoculating a crude strain of the porus rupestris into each bag, and culturing the fungus bags under a light-proof condition until mycelia grow fully; 4) Fruiting body management, namely after hypha is fully distributed in a cultivation bag, a side opening is used for promoting bud fruiting body, before forming buds, the fruiting body is cultivated in a dark place, and after forming the buds, scattered light cultivation is provided for a certain time every day.
7. The cultivation method according to claim 6, wherein 1) the mother culture: inoculating YM11 strain on GYM solid culture medium, and culturing at 25-28deg.C in dark for 8-12d to obtain cultivation mother strain; 2) Stock culture: inoculating the obtained cultivation mother seeds into culture bags containing stock culture media by a punching method, inoculating 3-6 fungus blocks in each bag, performing light-proof cultivation under the conditions of 25-28 ℃ and 50-70% humidity, and shaking the fungus blocks every 8-12d to enhance ventilation after hyphae germinate, so as to promote hyphae growth until the hyphae grow into bags, wherein the stock culture media comprises the following formula: 85% of wheat grains, 11% of willow sawdust, 3% of glucose and 1% of gypsum; adding 1/7 of the water decoction filtrate of ramulus Salicis Babylonicae (1 kg/1) of the total weight of the culture medium, adding water until the humidity of the culture medium is 50-70%, weighing the wheat grains, soaking in water for 1-3 days, cutting every 1kg of ramulus Salicis Babylonicae, boiling with 7kg of water at 100deg.C for 45min, and filtering with three layers of gauze; 3) And (3) manufacturing a cultivation bag: a 17cm multiplied by 33cm polypropylene plastic cultivation bag is adopted, the evenly stirred culture materials are filled into bags, the bagging amount is 0.8-1.0kg for each bag, and a plastic ring and a sealing cover are sleeved after the material surface is flattened; sterilizing the bag material at 121 ℃, cooling to 25-28 ℃ after sterilization, and inoculating; inoculating 8-12g of Podospora crassa stock strain into each bag, inoculating, and culturing the fungus bags at 25-28deg.C under dark condition until mycelia grow fully; the formula of the culture material is as follows: 75% of willow sawdust, 10% of cotton seed hulls, 10% of bran, 4% of glucose, 1% of gypsum and 60% -65% of water content; 4) Fruiting body management, namely after hypha is fully distributed in a cultivation bag, a surgical knife is used for transversely cutting 3-5cm openings on the side edges of the bag to promote bud fruiting bodies, before forming buds, the environmental temperature is controlled at 25-28 ℃, the environmental humidity is controlled at 50-70%, light-shielding cultivation is carried out, after forming the buds, the environmental temperature is controlled at 23-26 ℃, the environmental humidity is 85% -95%, and 1000Lx-1500 Lx scattered light is provided for 2 h-3 h per day.
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| CN112625916A (en) * | 2020-12-15 | 2021-04-09 | 湖南省微生物研究院 | Fibrinella rough W01, application thereof and separation method of natural product with anti-tumor activity |
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| CN109662198A (en) * | 2017-10-16 | 2019-04-23 | 吉林农业大学 | The development and its immunocompetence of the thick cap bore bacterium compound feed additive of bristle |
| CN112625916A (en) * | 2020-12-15 | 2021-04-09 | 湖南省微生物研究院 | Fibrinella rough W01, application thereof and separation method of natural product with anti-tumor activity |
Non-Patent Citations (4)
| Title |
|---|
| Coriolopsis trogii hydrophobin genes favor a clustering distribution and are widely involved in mycelial growth and primordia formation;Lining Wang 等;Gene .;第802卷;415863 * |
| 硬毛粗盖孔菌YM11 菌株的分离鉴定与驯化栽培;唐少军等;中国食用菌;第41卷(第5期);15-22 * |
| 硬毛粗盖孔菌子实体提取物对小鼠免疫功能及抗氧化能力的影响;王洪发等;食用菌学报;第27卷(第2期);51-62 * |
| 硬毛粗盖孔菌子实体提取物抑制H22 荷瘤小鼠肿瘤活性研究;高雪阳等;菌物学报;第36卷(第9期);1278-1288 * |
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