CN113913359B - Solid culture medium for promoting growth of antrodia mycelium and synthesis of triterpene - Google Patents
Solid culture medium for promoting growth of antrodia mycelium and synthesis of triterpene Download PDFInfo
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- CN113913359B CN113913359B CN202111366387.9A CN202111366387A CN113913359B CN 113913359 B CN113913359 B CN 113913359B CN 202111366387 A CN202111366387 A CN 202111366387A CN 113913359 B CN113913359 B CN 113913359B
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- 239000001963 growth medium Substances 0.000 title claims abstract description 31
- 150000003648 triterpenes Chemical class 0.000 title claims abstract description 26
- 239000007787 solid Substances 0.000 title claims abstract description 21
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 15
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 15
- 230000001737 promoting effect Effects 0.000 title claims abstract description 13
- 241000123370 Antrodia Species 0.000 title abstract description 10
- 241001486992 Taiwanofungus camphoratus Species 0.000 claims abstract description 35
- 241001563035 Clinopodium polycephalum Species 0.000 claims abstract description 26
- 239000000284 extract Substances 0.000 claims abstract description 17
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 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 abstract description 8
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims abstract description 8
- 239000001888 Peptone Substances 0.000 claims abstract description 8
- 108010080698 Peptones Proteins 0.000 claims abstract description 8
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 8
- 239000008103 glucose Substances 0.000 claims abstract description 8
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims abstract description 8
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims abstract description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 8
- 235000019319 peptone Nutrition 0.000 claims abstract description 8
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012138 yeast extract Substances 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 12
- 238000002386 leaching Methods 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 238000007873 sieving Methods 0.000 claims description 3
- 229930003270 Vitamin B Natural products 0.000 claims description 2
- 235000019156 vitamin B Nutrition 0.000 claims description 2
- 239000011720 vitamin B Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 6
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 abstract description 6
- 230000035784 germination Effects 0.000 abstract description 2
- 241001062954 Clinopodium Species 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- 238000012364 cultivation method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 241000368424 Plumbago scandens Species 0.000 description 2
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 2
- 229960002227 clindamycin Drugs 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000723346 Cinnamomum camphora Species 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 244000166675 Cymbopogon nardus Species 0.000 description 1
- 235000018791 Cymbopogon nardus Nutrition 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000219071 Malvaceae Species 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 235000021438 curry Nutrition 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000010979 ruby Substances 0.000 description 1
- 229910001750 ruby Inorganic materials 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- -1 triterpene compound Chemical class 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
Classifications
-
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- 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
-
- 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
- C12P33/00—Preparation of steroids
Abstract
The invention discloses a solid culture medium capable of promoting growth of Antrodia camphorata mycelia and synthesis of triterpenes, and belongs to the field of biotechnology. The solid culture medium contains glucose, peptone, maltose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and vitamin B 1 And a Clinopodium polycephalum extract or a Clinopodium polycephalum extract. The method has the characteristics of quick germination and quick growth of the Antrodia mycelium, the triterpene content is 23.6 percent higher than that of the traditional culture medium, and the method is easy to obtain in China, has simple preparation method and strong repeatability, can be used as the same raw material to prepare different culture mediums according to actual production conditions, and can provide a large number of novel methods for the Antrodia mycelium in actual production.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a solid culture medium capable of promoting growth of Antrodia camphorate mycelia and synthesis of triterpenes.
Background
Antrodia camphorataAntrodia cinnamomea) Is a rare medicinal fungus variety in Taiwan area in China, and the triterpene compound is a main functional component thereof, has various effects of protecting liver, detoxification, anti-inflammation, antioxidation and the like, and is researched to the prior art that Taiwan secondary-stage care tree camphor tree is preparedCinnamomum kanehirai) Is the only host of Antrodia camphorata, and is known as 'ruby in forest' due to the extremely small amount of the host in the natural world and the slow growth speed of Antrodia camphorata. The clinopodium polycephalum is a plantCymbopogon citratus(DC.) Stapf) is a perennial dense-cluster type fragrant herb of Gramineae, citronella, and is widely planted in tropical areas, and is widely seen in Guangdong, fujian, taiwan, etc. of China. The stem and leaf can extract lemon essential oil for preparing perfume and soap, and can be eaten, and the tender stem and leaf is a raw material for preparing curry seasoning; the medicine has the effects of dredging collaterals and dispelling wind. Researches show that the Antrodia mycelium can effectively replace fruit bodies under partial conditions and has very similar health care function, in order to solve the increasing demands of people on Antrodia, the invention screens the Clinopodium polycephalum leaching solution and the like as a culture medium, can greatly and rapidly promote the growth of the Antrodia mycelium and the synthesis of triterpene content, improves the content of active ingredients of the Antrodia mycelium, has correct guiding effect on the development of Antrodia market, and generates great economic and social benefits for the fungus industry of China.
Disclosure of Invention
The invention aims to provide a solid culture medium for rapidly and efficiently promoting growth of Antrodia camphorata mycelia and synthesis of triterpenes, and provides a scientific means for industrially producing Antrodia camphorata mycelia and accumulating triterpenes in a large amount.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a solid culture medium capable of promoting growth of Antrodia camphorate mycelia and synthesis of triterpenes comprises, by mass, 2.5% of glucose, 0.5% of peptone, 0.3% of maltose, 0.3% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate heptahydrate and 0.1% of vitamin B 1 0.1% of agar powder 1.5% dissolved in 1000 mL% of the waterThe pH of the clinopodium polycephalum leaching solution is natural, and the clinopodium polycephalum leaching solution is sterilized for 20min at 121 ℃.
The preparation method of the clindamini leaching solution comprises the following steps:
selecting fresh and pest-free clinopodium polycephalum, drying, pulverizing, and sieving with 40 mesh sieve to obtain clinopodium polycephalum powder; decocting 50. 50g Clinopodium polycephalum powder with 500-1000. 1000mL water at 70-90deg.C for 30-60min, filtering, and fixing volume of the filtrate to 1000. 1000mL with water to obtain Clinopodium polycephalum extract.
A solid culture medium capable of promoting growth of Antrodia camphorate mycelium and synthesis of triterpene is characterized in that: the culture medium comprises 2.5% of glucose, 0.5% of peptone, 0.3% of maltose, 0.3% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate heptahydrate and vitamin B in percentage by mass 1 0.1% of the clindamycin water extract 0.05% -0.25% is dissolved in 1000mL water, the pH is natural, and the sterilization is carried out for 20min at 121 ℃.
Wherein, the preparation method of the clindamycin extract comprises the following steps:
fresh and pest-free devil's plant is selected, broken, crushed and dried, then 20 g is weighed and dissolved in 1000mL water, water bath heating and vacuum reflux are carried out at 80-90 ℃ for 3 h, then filtration is carried out, filtrate is concentrated to 30 mL, freezing is carried out at-80 ℃, and then the obtained product is put into a vacuum drying oven for drying or a spray drying mode is adopted for removing solvent, thus obtaining tan gel solid or powder solid, namely the devil's plant water extract.
The solid culture medium is applied to promoting the growth of Antrodia camphorata mycelia and the synthesis of triterpenes.
The method for culturing Antrodia camphorate by the culture medium prepared by the invention comprises the following steps: culturing at 28deg.C in the dark for 14-28 d.
The mycelium of Antrodia camphorate cultivated by the culture medium prepared by the invention starts to grow a large amount of mycelium rapidly after inoculating and cultivating 2-3 d, and the whole plate (diameter 10 cm) grows at 20-28 d, and the triterpene content in the mycelium of Antrodia camphorate can be up to 7.79+/-0.13 mg/g measured by using a vanillin-glacial acetic acid method.
The invention has the remarkable advantages that:
antrodia camphorata is a rare medicinal fungus variety in Taiwan in China, and the artificial cultivation method comprises a basswood cultivation method, a solid cultivation method and a liquid fermentation method, but the method is high in cost, long in cultivation period and low in mycelium biomass and intracellular triterpene yield. The method has the advantages that the number of the clinopodium polycephalum in China is large, the sources are wide, the price is low, the solid culture medium prepared by using the clinopodium polycephalum leaching solution or the clinopodium polycephalum extract can efficiently and quickly promote the germination and growth of the mycelium of Antrodia camphorata, and the culture time is shortened by about 30 percent compared with that of the traditional culture medium; meanwhile, the solid culture medium prepared by the invention can also effectively promote the synthesis of Antrodia camphorata triterpene substances, so that the triterpene content in Antrodia camphorata mycelia is up to 7.79+/-0.13 mg/g, which is 23.6% higher than that of the traditional culture medium. The facts indicate that the traditional culture medium can be replaced, so that the bottleneck in the artificial production and cultivation of Antrodia camphorata is solved, and the fast industrialization of Antrodia camphorata mycelia is realized.
Drawings
FIG. 1 shows the growth of Antrodia camphorata in example 1. Wherein YDC is a solid medium capable of promoting growth of Antrodia camphorata mycelia and synthesis of triterpene as described in example 1, and CK is a control medium.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
Selecting fresh and pest-free clinopodium polycephalum, drying, pulverizing, and sieving with 40 mesh sieve to obtain clinopodium polycephalum powder; decocting 50. 50g Clinopodium polycephalum powder with 500-1000. 1000mL water at 70-90deg.C for 30-60min, filtering, and fixing volume of the filtrate to 1000. 1000mL with water to obtain Clinopodium polycephalum extract. The following raw materials in percentage by mass are adopted: 2.5% glucose, 0.5% peptone, 0.3% maltose, 0.3% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 0.1% vitamin B 1 And 1.5% of agar powder is dissolved in 1000mL clinet grass leaching solution, the pH is natural, and sterilization is carried out for 20min at 121 ℃, so that the solid culture medium capable of promoting the growth of Antrodia camphorata mycelia and the synthesis of triterpenes is obtained.
The formula of the control culture medium isThe following raw materials in percentage by mass are adopted: 2.5% glucose, 0.5% peptone, 0.3% maltose, 0.3% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 0.1% vitamin B 1 And 1.5% agar powder is dissolved in 1000mL water, the pH is natural, and the sterilization is carried out for 20min at 121 ℃.
And (5) inoculating Antrodia camphorata strains after the culture medium is cooled. The culture method comprises the following steps: culturing at 28deg.C in the dark for 16-28 d. The growth condition of Antrodia camphorate is shown in figure 1.
According to measurement, under the culture of a control culture medium, the growth rate of Antrodia camphorate mycelia is 1.13+/-0.04 mm/d, and the triterpene content is 6.30+/-0.10 mg/g; under the culture of the culture medium containing the clinopodium extract, the growth rate of the Antrodia camphorata mycelium is 1.39+/-0.03 mm/d, the triterpene content is 7.74+/-0.11 mg/g, and the growth rate and the triterpene content of the mycelium are obviously different from those of a control group (P is less than 0.05).
Example 2
Fresh and pest-free clinopodium polycephalum is selected, crushed, dried in the sun, then weighed 20 g and dissolved in 1000mL water, heated in water bath at 90 ℃ for vacuum reflux for 3 h, filtered, concentrated to 30 mL, frozen at-80 ℃ and placed in a vacuum drying oven to obtain tan gel solid, namely the clinopodium polycephalum water extract. The following raw materials in percentage by mass are adopted: 2.5% glucose, 0.5% peptone, 0.3% maltose, 0.3% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 0.1% vitamin B 1 And the clinopodium polycephalum extract is dissolved in 1000mL water, the pH is natural, and the sterilization is carried out for 20min at 121 ℃ to obtain the solid culture medium capable of promoting the growth of Antrodia camphorata mycelia and the synthesis of triterpenes. Wherein the mass fractions of the clinopodium polycephalum extract are respectively 0.05%, 0.1%, 0.15%, 0.2% and 0.25%.
The formula of the control culture medium comprises the following raw materials in percentage by mass: 2.5% glucose, 0.5% peptone, 0.3% maltose, 0.3% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 0.1% vitamin B 1 And 1.5% agar powder is dissolved in 1000mL water, the pH is natural, and the sterilization is carried out for 20min at 121 ℃.
And (5) inoculating Antrodia camphorata strains after the culture medium is cooled. The culture method comprises the following steps: culturing at 28deg.C in the dark for 16-28 d. The growth conditions of Antrodia camphorate are shown in Table 1.
According to measurement, under the culture of a control culture medium, the growth rate of Antrodia camphorate mycelia is 1.13+/-0.04 mm/d, and the triterpene content is 6.3+/-0.10 mg/g; under the culture of the culture medium containing 0.2% of the clinopodium water extract, the growth rate of Antrodia camphorata mycelia is 1.43+/-0.05 mm/d, the triterpene content is 7.79+/-0.13 mg/g, and the Antrodia camphorata mycelia and the triterpene content have obvious differences (P is less than 0.05) compared with the control group.
TABLE 1 growth Rate of Antrodia camphorate mycelium
The fact shows that the preparation of the Antrodia camphorata solid culture mycelium by utilizing the clinopodium water extract and the clinopodium leaching solution can remarkably shorten the culture time, improve the production efficiency in production and reduce the production cost.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (2)
1. A solid culture medium capable of promoting growth of Antrodia camphorate mycelium and synthesis of triterpene is characterized in that: the culture medium comprises 2.5% of glucose, 0.5% of peptone, 0.3% of maltose, 0.3% of yeast extract, 0.1% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate heptahydrate and vitamin B in percentage by mass 1 0.1% of agar powder 1.5% is dissolved in 1000mL of clinopodium polycephalum extract, the pH is natural, and sterilization is carried out for 20min at 121 ℃;
the preparation method of the clindamini leaching solution comprises the following steps: selecting fresh and pest-free clinopodium polycephalum, drying, pulverizing, and sieving with 40 mesh sieve to obtain clinopodium polycephalum powder; decocting 50g of Clinopodium polycephalum powder with 500-1000mL of water at 70-90deg.C for 30-60min, filtering, and fixing volume of the filtrate to 1000mL with water to obtain Clinopodium polycephalum extract.
2. The use of the solid medium according to claim 1 for promoting the growth of Antrodia camphorata mycelium and the synthesis of triterpenes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130948A (en) * | 2014-07-11 | 2014-11-05 | 福建农林大学 | Preparation method and application of Antrodia camphorate mycelium medium |
| CN105001009A (en) * | 2015-08-13 | 2015-10-28 | 福建农林大学 | Antrodia cinnamomea terpene-rich mycelium culture medium and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104130948A (en) * | 2014-07-11 | 2014-11-05 | 福建农林大学 | Preparation method and application of Antrodia camphorate mycelium medium |
| CN105001009A (en) * | 2015-08-13 | 2015-10-28 | 福建农林大学 | Antrodia cinnamomea terpene-rich mycelium culture medium and application |
Non-Patent Citations (2)
| Title |
|---|
| 不同中药提取物对牛樟芝生长和胞内三萜产物形成的影响;冯路瑶等;中国食用菌;第第37卷卷(第第2期期);第42-46页 * |
| 忧遁草活性成分的提取及其功能评价;于群;于群(第2018 年 第02期期);B024-599 * |
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