CN109055230B - Method for obtaining wild strain of rare medicinal Marasmius androsaceus - Google Patents
Method for obtaining wild strain of rare medicinal Marasmius androsaceus Download PDFInfo
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- CN109055230B CN109055230B CN201810741466.5A CN201810741466A CN109055230B CN 109055230 B CN109055230 B CN 109055230B CN 201810741466 A CN201810741466 A CN 201810741466A CN 109055230 B CN109055230 B CN 109055230B
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- 241000233062 Gymnopus androsaceus Species 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 12
- 229940079593 drug Drugs 0.000 claims description 11
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 9
- 244000061456 Solanum tuberosum Species 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000004098 Tetracycline Substances 0.000 claims description 7
- 229960002180 tetracycline Drugs 0.000 claims description 7
- 229930101283 tetracycline Natural products 0.000 claims description 7
- 235000019364 tetracycline Nutrition 0.000 claims description 7
- 150000003522 tetracyclines Chemical class 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000011691 vitamin B1 Substances 0.000 claims description 6
- 230000003115 biocidal effect Effects 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
- WLJVXDMOQOGPHL-PPJXEINESA-N 2-phenylacetic acid Chemical compound O[14C](=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-PPJXEINESA-N 0.000 claims description 3
- 229930091371 Fructose Natural products 0.000 claims description 3
- 239000005715 Fructose Substances 0.000 claims description 3
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 3
- 241000018650 Pinus massoniana Species 0.000 claims description 3
- 235000011610 Pinus tabuliformis Nutrition 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 229930003270 Vitamin B Natural products 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 3
- 239000000920 calcium hydroxide Substances 0.000 claims description 3
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 229920002545 silicone oil Polymers 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000011720 vitamin B Substances 0.000 claims description 3
- 235000019156 vitamin B Nutrition 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000002023 wood Substances 0.000 claims description 2
- 241000894007 species Species 0.000 claims 3
- 230000000694 effects Effects 0.000 abstract description 5
- 208000004296 neuralgia Diseases 0.000 abstract description 4
- 208000014674 injury Diseases 0.000 abstract description 3
- 230000008736 traumatic injury Effects 0.000 abstract description 3
- 238000000855 fermentation Methods 0.000 abstract description 2
- 230000004151 fermentation Effects 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 241000233866 Fungi Species 0.000 description 3
- 230000002538 fungal effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 206010072132 Fracture pain Diseases 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 241001237200 Marasmiellus Species 0.000 description 1
- 208000019695 Migraine disease Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000008765 Sciatica Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 206010027599 migraine Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 206010044652 trigeminal neuralgia Diseases 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention relates to a method for obtaining wild strains of rare medicinal Marasmius androsaceus, which comprises the following steps: collecting wild Marasmius androsaceus rhizomes; separating strains; and (5) purifying the strains. The invention utilizes the wild Marasmius androsaceus funk rhizomes to obtain the Marasmius androsaceus funk strain for the first time, has the advantage of simple operation, can utilize the obtained wild Marasmius androsaceus funk high-activity strain to carry out the research and development of more effective medicaments for treating neuralgia, traumatic injury and the like, such as cultivation, fermentation and the like, and has good application prospect.
Description
Technical Field
The invention belongs to the field of medicinal fungi, and particularly relates to a method for obtaining rare medicinal Marasmius androsaceus wild strains.
Background
The wild Marasmiellus androsaceus Singer is a rare medicinal fungus, has the effect of relieving pain and the like, has the efficacy of treating traumatic injury, trigeminal neuralgia, migraine, supraorbital neuralgia, fracture pain, leprosy neuralgia, sciatica and rheumatoid arthritis, and has related medicaments in China. However, most of the related drugs produced in China are collected from wild fruit bodies. Wild Marasmius androsaceus is extremely rare, has little wild distribution and extremely little quantity, and most people do not know the wild Marasmius androsaceus at all, even many people engaged in the classification of wild large fungi do not know the wild Marasmius androsaceus, so that the wild Marasmius androsaceus is difficult to develop and utilize on a large scale. Therefore, finding the marasmius androsaceus strain with high activity and carrying out corresponding development and research are the premise for developing marasmius androsaceus and carrying out important drug production.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for obtaining wild strains of a rare medicinal Marasmius androsaceus, and solving the problem that the wild strains of the Marasmius androsaceus are difficult to obtain in the prior art.
The invention provides a method for obtaining wild strains of rare medicinal Marasmius androsaceus, which comprises the following steps:
(1) collecting wild Marasmius androsaceus rhizomes growing on a specific substrate, shearing small segments of the Marasmius androsaceus rhizomes, placing the small segments of the Marasmius androsaceus rhizomes in a special culture medium, and culturing for 5-6 days at 18-20 ℃ in dark light to obtain heterozygosis strain containing Marasmius androsaceus hyphae;
(2) transferring the heterozygosis strain of Marasmius androsaceus mycelium into antibiotic culture medium containing tetracycline, and culturing at 18-20 deg.C under dark light for 10-11 days to obtain pure Marasmius androsaceus strain.
The specific substrate in the step (1) is Chinese pine and sapwood with the length of 2-3 cm.
The wild Marasmius androsaceus fun rhizomes in the step (1) grow in the environment with the air temperature of 15-18 ℃ and the humidity of 85-90%.
The special culture medium in the step (1) comprises the following components: 200g of potato, 10g of glucose, 10g of fructose, 5mL of wort, 0.25g of magnesium sulfate, 0.5g of dipotassium hydrogen phosphate, 20g of agar and vitamin B1100mg, 1000mL of distilled water, pH 7.5.
The preparation steps of the special culture medium are as follows: decocting potato in water, filtering, and sequentially adding vitamin B1Other medicines are added, the volume is determined to be 1000mL after the medicines are completely dissolved, and the medicines are packaged into test tubes and sterilized; after sterilization, when the culture medium is cooled to below 50 ℃, vitamin B is added into a super clean workbench1And then, adjusting the pH value of the solution to 7.5 by using a precise pH test paper, and subpackaging the solution into test tubes for later use.
The preparation step of the antibiotic culture medium with penicillin and tetracycline in the step (2) comprises the following steps: 200g of potato boiled liquid, 20g of glucose, 20g of agar, 0.2g of silicone oil, 1.0g of calcium carbonate, 0.4g of urea, 0.2g of phenylacetic acid, 0.05g of calcium hydroxide and 1000mL of purified water, sterilizing at high pressure for 20min, cooling to 50 ℃, respectively adding 10-15mg of penicillin and 10-15mg of tetracycline into a super clean workbench, stirring uniformly, subpackaging test tubes, and condensing for later use.
Advantageous effects
The invention utilizes the wild Marasmius androsaceus funk rhizomes to obtain the Marasmius androsaceus funk strain for the first time, has the advantage of simple operation, can utilize the obtained wild Marasmius androsaceus funk high-activity strain to carry out the research and development of more effective medicaments for treating neuralgia, traumatic injury and the like, such as cultivation, fermentation and the like, and has good application prospect.
Drawings
FIG. 1 is a diagram of wild Marasmius androsaceus;
FIG. 2 shows the wild Marasmius androsaceus strain obtained by the present invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
1) Preparing a special culture medium:
the components are as follows: 200g of potato, 10g of glucose, 10g of fructose, 5mL of wort, 0.25g of magnesium sulfate, 0.5g of dipotassium hydrogen phosphate, 20g of agar and vitamin B1100mg, 1000mL of distilled water, pH 7.5.
The preparation steps are as follows: decocting potato in water, filtering, and sequentially adding vitamin B1And (4) other medicines are added, the volume is determined to be 1000mL after the medicines are completely dissolved, and the medicines are packaged in test tubes and sterilized. After sterilization, when the culture medium is cooled to below 50 ℃, vitamin B is added into a super clean workbench1And then, adjusting the pH value of the solution to 7.5 by using a precise pH test paper, and subpackaging the solution into test tubes for later use.
2) Collecting wild Marasmius androsaceus fun rhizomes growing on a specific substrate: picking up Marasmius androsaceus (shown in figure 1) grown in 2-3cm length on dried wood of Chinese pine with air temperature of 15-18 deg.C and humidity of about 90%, cleaning and sterilizing the surface residues, and separating the strains.
3) Strain separation: shearing off 0.5mm long fungal mycelia from the light color part at the top of wild Marasmius androsaceus fungal mycelia, placing in a special culture medium prepared in advance, and culturing at 18-20 deg.C under dark light for about 5d to obtain hybrid bacterial strain containing Marasmius androsaceus hyphae.
4) And (3) purification: the preliminarily obtained heterozygosis strain of the Marasmius androsaceus mycelium is transferred to an antibiotic culture medium (the components are 200g of potato cooking liquor, 20g of glucose, 20g of agar, 0.2g of silicone oil, 1.0g of calcium carbonate, 0.4g of urea, 0.2g of phenylacetic acid, 0.05g of calcium hydroxide and 1000mL of purified water, the mixture is sterilized under high pressure for 20min, cooled to 50 ℃, 10-15mg of penicillin and 10-15mg of tetracycline are respectively and sequentially added into a super clean workbench, the mixture is uniformly stirred and then is subpackaged into test tubes and is condensed for standby use), and the culture is carried out for about 10 days under the dark light of 18-20 ℃ to obtain the pure Marasmius androsaceus mycelium, as shown in figure 2.
Claims (4)
1. A method for obtaining wild strain of rare medicinal Marasmius androsaceus L.var.manshuriensis comprises:
(1) collecting wild Marasmius androsaceus fun mycelium growing on specific substrate, cutting small segments of mycelium from the light-colored top of the wild Marasmius androsaceus fun mycelium, placing in a special culture medium, and culturing at 18-20 deg.C in dark for 5-6d to obtain heterozygosis strain containing Marasmius androsaceus mycelium; wherein the specific substrate is 2-3cm long Chinese pine rotten wood; the special culture medium comprises the following components: 200g of potato, 10g of glucose, 10g of fructose, 5mL of wort, 0.25g of magnesium sulfate, 0.5g of dipotassium hydrogen phosphate, 20g of agar and vitamin B1100mg, 1000mL of distilled water, pH7.5;
(2) transferring the heterozygosis strain of Marasmius androsaceus mycelium into antibiotic culture medium containing tetracycline, and culturing at 18-20 deg.C under dark light for 10-11 days to obtain pure Marasmius androsaceus strain.
2. The method for obtaining wild species of rare medicinal Marasmius androsaceus as claimed in claim 1, wherein: the wild Marasmius androsaceus fun rhizomes in the step (1) grow in the environment with the air temperature of 15-18 ℃ and the humidity of 85-90%.
3. The method for obtaining wild species of rare medicinal Marasmius androsaceus as claimed in claim 1, wherein: the preparation steps of the special culture medium are as follows: decocting potato in water, filtering, and sequentially adding vitamin B1Other medicines are added, the volume is determined to be 1000mL after the medicines are completely dissolved, and the medicines are packaged into test tubes and sterilized; after sterilization, when the culture medium is cooled to below 50 ℃, vitamin B is added into a super clean workbench1And then, adjusting the pH value of the solution to 7.5 by using a precise pH test paper, and subpackaging the solution into test tubes for later use.
4. The method for obtaining wild species of rare medicinal Marasmius androsaceus as claimed in claim 1, wherein: the preparation steps of the antibiotic culture medium with tetracycline in the step (2) are as follows: 200g of potato boiled liquid, 20g of glucose, 20g of agar, 0.2g of silicone oil, 1.0g of calcium carbonate, 0.4g of urea, 0.2g of phenylacetic acid, 0.05g of calcium hydroxide and 1000mL of purified water, sterilizing at high pressure for 20min, cooling to 50 ℃, respectively adding 10-15mg of penicillin and 10-15mg of tetracycline into a super clean workbench, stirring uniformly, subpackaging test tubes, and condensing for later use.
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| CN102687639A (en) * | 2011-12-19 | 2012-09-26 | 河南科技大学 | Method for separating Morchella strains |
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2018
- 2018-07-05 CN CN201810741466.5A patent/CN109055230B/en not_active Expired - Fee Related
Patent Citations (7)
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