CN102876587A - Cordyceps militaris strain for producing cordycepin with high yield - Google Patents

Cordyceps militaris strain for producing cordycepin with high yield Download PDF

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Publication number
CN102876587A
CN102876587A CN2012103690137A CN201210369013A CN102876587A CN 102876587 A CN102876587 A CN 102876587A CN 2012103690137 A CN2012103690137 A CN 2012103690137A CN 201210369013 A CN201210369013 A CN 201210369013A CN 102876587 A CN102876587 A CN 102876587A
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Prior art keywords
cordyceps militaris
cordycepin
bacterial strain
link
fermentation
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Inventor
蔡宇杰
丁彦蕊
岳翠翠
陈涛
金大勇
朱建航
廖祥儒
张大兵
李枝玲
罗军侠
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Jiangnan University
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Jiangnan University
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Abstract

The invention discloses a cordyceps militaris strain for producing cordycepin with high yield and belongs to the technical field of bioengineering. The cordyceps militaris strain is named as cordyceps militaris JN168 by classification and is preserved with CCTCC No: M2011333 inChinaCenter for Type Culture Collection. The cordyceps militaris strain which is stable in character is obtained by isolation and screening of wild cordyceps militaris sporocarps collected from Antu, Jilin Province. The cordyceps militaris strain is capable of producing the cordycepin by liquid-state fermentation at the fermentation temperature of 30 DEG C, and the final concentration of cordycepin can be 6g/L after fermentation for 5 days.

Description

One plant height produces the cordyceps militaris link bacterial strain of cordycepin
Technical field
One strain cordyceps militaris link bacterial strain belongs to technical field of bioengineering.The present invention relates to a strain can be at the cordyceps militaris link bacterial strain of 30 ℃ of lower high yield cordycepins of higher leavening temperature, and its bacterium colony and fruit-body color are yellow, and this bacterial strain is for the suitability for industrialized production important in inhibiting of cordycepin.
Background technology
Cordyceps militaris (L.) Link. is one of famous and precious Chinese medicinal materials of China, has very high pharmaceutical use and nutritive value.The main chemical composition of Cordyceps militaris (L.) Link. is very similar to wild cordyceps, mainly comprises nucleosides material (such as cordycepin, adenosine etc.), Cordyceps polysaccharide, cordycepic acid, sterols material, superoxide-dismutase and amino acid etc.Modern study shows, contains a lot of activeconstituentss in the Cordyceps militaris (L.) Link., and especially cordycepin has multiple biological activity.Cordycepin is a kind of active substance that antitumor, antiviral, antibiotic and antifungic action are arranged, and studies show that recently cordycepin also has the effect of reducing blood-fat.The content of cordycepin in wild cordyceps is atomic, and the cordycepin that present institute uses generally extracts from Cordyceps militaris (L.) Link..
The structural formula of cordycepin
Liu Yanfang (the optimization of Cordyceps militaris (L.) Link. submerged fermentation high yield cordycepin technique, the Shanghai Agricultural journal, 2010,26 (3), 26-30) reported that fermentation produces the cordyceps militaris link bacterial strain of cordycepin, leave standstill under 25 ℃ temperature in this article and cultivate 28d, the cordycepin content in the gained fermented liquid reaches 2.05g/L.
But the bacterial strain cordycepin output of having reported height is uneven, and leavening temperature is not identical yet, and bacterial strain Morphological Shape color is also different.It is stable that separation screening of the present invention has obtained a strain proterties, can be under 30 ℃ fermentation high temperature the cordyceps militaris link bacterial strain of high yield cordycepin, it produces xanthein, bacterium colony and fruit-body color are deep yellow.
Patent CN102220397A provides a kind of method that improves cordycepin output.This invention improves cordycepin output by add organic solvent oleic acid in fermention medium, and its leavening temperature is 26 ℃, and fermentation period is 7d, and obtaining final cordycepin concentration is 5.621mg/L.
Patent CN101554121A has invented a kind of method that improves cordyceps militaris biomass and cordycepin content, and its leavening temperature is 20 ℃, and fermentation period is 8d, and cordycepin content is 10.93mg/g in the thalline that obtains.
Patent CN101245361A has introduced a kind of the produce method of cordycepin and seed selection and the application that a plant height produces cordyceps militaris link bacterial strain, this patent obtains a plant height by mutagenic and breeding and produces cordyceps militaris link bacterial strain, this strain fermentation temperature is controlled at 23~27 ℃, stop fermentation after leaving standstill top fermentation 16~20d, the concentration that obtains cordycepin in the fermented liquid is 3028mg/L.
Patent CN101492706A provides a kind of method that improves Cordyceps militaris ferment cordyceps sinensis element output, and the leavening temperature of the method is 23 ℃, and fermentation period is 11d, and the concentration of cordycepin is 1.8g/L in the fermented liquid that finally obtains.
The report that also has has been found the albefaction bacterial strain of Cordyceps militaris (L.) Link., such as woods heroes (Cordyceps militaris (L.) Link. albefaction Biological Characteristics of Strain and sporophore composition analysis thereof, the edible mushrooms journal, 2011,18 (1), 1-4) and Jiang Qianting (Cordyceps militaris (L.) Link. albefaction bacterial strain anti-oxidant activity, the edible mushrooms journal, 2011,18 (2), 62-64) about the albefaction cordyceps militaris link bacterial strain.
Patent CN102210255A has found the cultivating method of a kind of Cordyceps militaris (L.) Link. albefaction strain and sporophore thereof.Its culture temperature is at 20 ℃~30 ℃.
These reports are not all carried out follow-up further research to the cordyceps militaris link bacterial strain that can produce cordycepin under comparatively high temps, and fermentation period is longer, can't be applied to industrialized production.The present invention has obtained the stable new bacterial strain of Cordyceps militaris (L.) Link. of proterties by the wild cordyceps militaris sporophore that gathers from the Antu, Jilin is carried out separation screening Cordyceps militarisJN168, this bacterial strain can be cultivated 5 days under 30 ℃ leavening temperature, and the cordycepin concentration in the fermented liquid can reach maximum value.
Summary of the invention
The purpose of this invention is to provide a strain can be under 30 ℃ leavening temperature the cordyceps militaris link bacterial strain of high yield cordycepin, stable through obtained proterties from open-air separation screening, can be under 30 ℃ leavening temperature the cordyceps militaris link bacterial strain of high yield cordycepin, can produce cordycepin by liquid state or solid state fermentation, and fermentation time is short, and output is high.
Technical scheme of the present invention: a strain can be produced the cordyceps militaris link bacterial strain of cordycepin, Cordyceps militaris (L.) Link. under 30 ℃ leavening temperature Cordyceps militarisJN168 has been preserved in Chinese Typical Representative culture collection center, and preserving number is for being CCTCC NO:M2011333, September 28 2011 preservation time.
Further, the molecular biology 26S rDNA sequence of this bacterial strain is SEQ ID NO:1.
Further, this bacterial strain main metabolites is cordycepin, reaches 6g/L.
Further, the available carbon source of this bacterial strain is one or more the combination in glucose, fructose, maltose, sucrose, lactose, D-semi-lactosi, seminose, sorbose, Zulkovsky starch, the yam starch.
Further, the available nitrogenous source of this bacterial strain is one or more the combination in inorganic ammonium salt, inorganic nitrate, yeast powder, yeast extract paste, peptone, extractum carnis, soybean cake powder, urea, the wort.
Gather the wild cordyceps militaris sporophore from the Antu, Jilin.Select individual hypertrophy, the appearance color is entirely the Cordyccps-militaris-(L.)-link. Sporophore of darker yellow.At first use the sterilized water washes clean, then the spirituous solution with 75% concentration for preparing in advance soaked 1-2 minute, after the alcohol volatilization is done, Cordyccps-militaris-(L.)-link. Sporophore is carried out surface sterilization, then with aseptic scalper the particle that its interior tissue cuts into the mung bean size is placed the separation agar plate surface, under aseptic condition, it is pressed in the agar gently and cultivates.Fungi separates to diffusion growth all around centered by implanting point after cultivating.The isolation medium that primary dcreening operation is used is: potato 200g, glucose 10g, agar 20g, water 1000mL, penicillin 20mg, pH nature.The culture condition of primary dcreening operation is 30 ℃ of constant temperature, cultivates 4-5 days.Select the darker bacterium colony of color and luster and carry out multiple sieve cultivation, sieve again cultural method: the bacterial classification that will just sift out carries out fermentation test, selects and produces the highest bacterial classification of cordycepin, passes through separation and purification again, finally obtains the new bacterial strain of Cordyceps militaris (L.) Link. Cordyceps militarisJN168.
The measuring method of cordycepin concentration in the fermented liquid: with the fermenation raw liquid that obtains with the centrifugal 20min of 8000rpm, get supernatant liquor, then measure the content of cordycepin in the supernatant liquor with the HPLC method, the C18 post (250mm * 4.6mm), the mensuration wavelength is 260nm, moving phase is 20% methyl alcohol, flow velocity 1mL/min, 30 ℃ of column temperatures.
Bacterial classification of the present invention has following feature
1, Cordyceps militaris (L.) Link. Cordyceps militarisThe JN168 culture condition
The cultivation normal condition of this bacterial classification is aerobic cultivation.The saccharine material that is used for the cultivation bacterial classification can be the one of glucose, fructose, maltose, sucrose, lactose, D-semi-lactosi, seminose, sorbose, Zulkovsky starch, yam starch or several persons' combination.The nitrogenous source of cultivating usefulness can be one in inorganic ammonium salt, inorganic nitrate or yeast powder, yeast extract paste, peptone, extractum carnis, the wort or several persons' combination.The growth temperature range of this bacterial strain is 15 ℃~35 ℃, and optimum growth temp is 28 ℃~31 ℃.In the culturing process of bacterial classification, can also add the inorganic salt such as potassium primary phosphate, dipotassium hydrogen phosphate, sal epsom.
2, Cordyceps militaris (L.) Link. Cordyceps militarisThe form of JN168 is described
The dull and stereotyped form of cultivating: Cordyceps militarisJN168 cultivated after 2-3 days, begin to have yellow bacterium colony to produce on the flat board, had formed diameter after 5 days and had been about bacterium colony about 5cm, and bacterium colony be ellipse, neat in edge; Mycelium has covered with flat board fully after 10 days, even has also formed the shape of similar sporophore, and this moment, growth stopped substantially.Mycelium is in the process of the dull and stereotyped growth of PDA solid medium, and it is yellow cotton-shaped that mycelium is, and the bacterium colony densification is flocculence, and aerial mycelium is very flourishing, is easy to picking.If illumination cultivation, bacterium colony are pure yellow, if dark the cultivation, bacterium colony then is white in color, and does not have coloured variation.Find by the observation under Electronic Speculum, the mutual weave in of mycelium forms a kind of similar netted structure ascus and is not specification slender type, and smooth surface is smooth.
3, the new bacterial strain of Cordyceps militaris (L.) Link. Cordyceps militarisThe molecular biological characteristics of JN168 is seen sequence table.
4, the Cordyceps militaris (L.) Link. that obtains from open-air separation screening Cordyceps militarisThe character of JN168
This bacterium is to obtain by the wild cordyceps militaris sporophore that gathers from the Antu, Jilin is carried out separation screening, has the ability of good product cordycepin.The ability that under 30 ℃ leavening temperature, has equally good product cordycepin through the bacterial strain that separation and purification obtains.
5, can adopt conventional liquid state fermentation method, utilize above-mentioned bacterial classification to produce cordycepin.Substratum can be with containing the ordinary culture mediums such as different carbon sources, nitrogenous source, mineral ion.
Also can add the nutrition such as the necessary amino acid of an amount of microorganism growth, VITAMIN in the substratum, generalized case, these nutrition are present in the above-mentioned natural nutrition source.
The optimum carbon source that cordycepin is produced in this bacterial strain liquid state fermentation is maltose, and optimum nitrogen source is yeast powder, and inorganic salt are sal epsom.Can reach 6g/L with this substratum in the output of 30 ℃ of lower cordycepins.Begin to produce cordycepin after 1 day, reached the climax on the 5th day.
Beneficial effect of the present invention: this bacterial strain is by the wild cordyceps militaris sporophore that gathers from the Antu, Jilin is carried out separation screening, obtained the stable cordyceps militaris link bacterial strain of proterties, this bacterial strain can be produced cordycepin by liquid state fermentation or solid state fermentation, and fermentation time is short, and output is high.Leavening temperature is 30 ℃, begins to produce cordycepin after 1 day, reaches the climax on the 5th day, and can obtain the final concentration of cordycepin through 5 days fermentations is 6 g/L.
The biological material specimens preservation: a plant height produces the cordyceps militaris link bacterial strain of cordycepin Cordyceps militarisJN168, preservation and Chinese Typical Representative culture collection center, be called for short CCTCC, the address: Wuhan, China Wuhan University, preserving number is CCTCC NO:M2011333, preservation date is on September 28th, 2011.
Description of drawings
The relation of Fig. 1 culture temperature and cordycepin output.
The relation of Fig. 2 pH and cordycepin output.
The relation of Fig. 3 carbon source and cordycepin output.
The relation of Fig. 4 nitrogenous source and cordycepin output.
Embodiment
Embodiment 1
Determining of optimum temperuture
With bacterial strain Cordyceps militarisJN168 is inoculated in the PDA slant medium, and 30 ℃ of illumination cultivation 7d wash spore with sterilized water, and making spore count is 10 6Spore suspension doubly.Get the spore suspension that makes, be connected in the PDA seed culture medium with 5% inoculum size, 30 ℃, 200rpm, shake-flask culture 3d obtains the female kind of liquid; In 5% inoculum size access fermention medium, the 5d secondary fermentation finishes.The composition of seed culture medium and fermention medium is glucose 40g/L, yeast extract paste 10g/L, sal epsom 0.5g/L.Use single factor to investigate the optimum temperuture of produced from Cordyceps militaris L cordycepin, respectively leavening temperature is adjusted to 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, measure the concentration of cordycepin.By data among Fig. 1 as can be known, the optimum temperuture of produced from Cordyceps militaris L cordycepin is 30 ℃.
Embodiment 2
Determining of optimal pH
With bacterial strain Cordyceps militarisJN168 is inoculated in the PDA slant medium, and 30 ℃ of illumination cultivation 7d wash spore with sterilized water, and making spore count is 10 6Spore suspension doubly.Get the spore suspension that makes, be connected in the PDA seed culture medium with 5% inoculum size, 30 ℃, 200rpm, shake-flask culture 3d obtains the female kind of liquid; In 5% inoculum size access fermention medium, 30 ℃ of leavening temperatures, the 5d secondary fermentation finishes.Seed culture medium and fermention medium consist of glucose 40g/L, yeast extract paste 10g/L, sal epsom 0.5g/L.Use single factor to investigate the best pH of produced from Cordyceps militaris L cordycepin, the pH that will ferment respectively adjusts to 4.5,5.0,5.5,6.0,6.5,7.0,7.5, measures the concentration of cordycepin.By data among Fig. 2 as can be known, the optimal pH of produced from Cordyceps militaris L cordycepin is 7.0.
Embodiment 3
Determining of optimum carbon source
With bacterial strain Cordyceps militarisJN168 is inoculated in the PDA slant medium, and 30 ℃ of illumination cultivation 7d wash spore with sterilized water, and making spore count is 10 6Spore suspension doubly.Get the spore suspension that makes, be connected in the PDA seed culture medium with 5% inoculum size, 30 ℃, 200rpm, shake-flask culture 3d obtains the female kind of liquid; In 5% inoculum size access fermention medium, 30 ℃ of leavening temperatures, the 5d secondary fermentation finishes.Seed culture medium and fermention medium consist of glucose 40g/L, yeast extract paste 10g/L, sal epsom 0.5g/L, pH is 7.0.Use single factor to investigate the optimum carbon source of produced from Cordyceps militaris L cordycepin, the glucose that uses respectively fructose, maltose, sucrose, lactose, D-semi-lactosi, Zulkovsky starch to substitute in the basic fermention medium carries out liquid state fermentation, measures the concentration of cordycepin.By data among Fig. 3 as can be known, the optimum carbon source of produced from Cordyceps militaris L cordycepin is maltose, and effect and the maltose of glucose are similar.
Embodiment 4
Determining of optimum nitrogen source
With bacterial strain Cordyceps militarisJN168 is inoculated in the PDA slant medium, and 30 ℃ of illumination cultivation 7d wash spore with sterilized water, and making spore count is 10 6Spore suspension doubly.Get the spore suspension that makes, be connected in the PDA seed culture medium with 5% inoculum size, 30 ℃, 200rpm, shake-flask culture 3d obtains the female kind of liquid; In 5% inoculum size access fermention medium, 30 ℃ of leavening temperatures, the 5d secondary fermentation finishes.Seed culture medium and fermention medium consist of maltose 40g/L, yeast extract paste 10g/L, sal epsom 0.5g/L, pH is 7.0.Use single factor to investigate the optimum nitrogen source of produced from Cordyceps militaris L cordycepin, the yeast extract paste that uses respectively ammonium nitrate, ammonium sulfate, ammonium tartrate, yeast powder, peptone, extractum carnis or wort to substitute in the basic fermention medium carries out liquid state fermentation, measures the concentration of cordycepin.By data among Fig. 4 as can be known, the optimum nitrogen source of produced from Cordyceps militaris L cordycepin is yeast powder, the effect of peptone and extractum carnis and yeast powder similar.
Embodiment 5
Fermentation strain be Cordyceps militaris (L.) Link. ( Cordyceps militaris) JN168, adopting solid state fermentation, substratum forms (% by weight): 30 purpose Semen Maydis powder 60, wheat bran 16, rice bran 22, glucose 1.5, KH 2PO 40.45, MgSO 47H 2O 0.05; Add the water spice wetting, in the wide-necked bottle of packing into, the 0.1MPa 45-60min that sterilizes, the access liquid seeds is mixed all after the cooling.30 ℃ of leavening temperatures, pH7, after 6 days fermentation ends, the content of cordycepin is 2.1% in the fermention medium.
<120〉plant height produces the cordyceps militaris link bacterial strain of cordycepin
<160> 2
<210> 1
<211> 602
<212> DNA
<213〉Cordyceps militaris (L.) Link. ( Cordyceps militaris) JN168
<400> 1
gcatatcaat aagcggagga aacgaaacca acagggattg ccccagtaac ggcgagtgaa 60
gcggcaacag ctcaaatttg aaatctggcc cccgggtccg agttgtaatt tgcagaggat 120
gctttgggcg aggtgccttc cgagttccct ggaacgggac gccacagagg cgtctggtcg 180
gacaccgagc gagtcgagta ccgtgtaaag ctccttcgac gtgagagccc gtttgggaat 240
gctgctcaaa atgggaggta tatgtcttct aaagctaaat attggccaga gaccgatagc 300
gcacaagtag agtgatcgaa agatgaaaag cactttgaaa agagggttaa acagtacgtg 360
aaattgttga aagggaagcg cctatgacca gacttgcgcc cggtgaatca tccagcgttc 420
tcgatggtgc actttgccgg gcacaggcca gcatcagttt ggcgcggggg acaaaggctt 480
cgggaatgtg gctcccccgg gagtgttata gcccgctgcg taataccctg cgccggactg 540
aggtacgcgc atcgcaagga tgctggcgta atggtcatca gcgacccgtc ttgaaacacg 600
ga 602

Claims (6)

1. a strain cordyceps militaris link bacterial strain, its Classification And Nomenclature be Cordyceps militaris (L.) Link. ( Cordyceps militaris) JN168, being preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC NO:M2011333.
2. cordyceps militaris link bacterial strain according to claim 1, its 26S rDNA sequence is SEQ ID NO:1.
3. cordyceps militaris link bacterial strain according to claim 1 is characterized in that it can at 30 ℃ of lower high yield cordycepins of higher leavening temperature, reach 6g/L.
4. cordyceps militaris link bacterial strain according to claim 1 is characterized in that it can produce xanthein, and bacterium colony and fruit-body color are yellow.
5. cordyceps militaris link bacterial strain according to claim 1 is characterized in that available carbon source is one or more the combination in glucose, fructose, maltose, sucrose, lactose, D-semi-lactosi, seminose, sorbose, Zulkovsky starch, the yam starch.
6. cordyceps militaris link bacterial strain according to claim 1 is characterized in that available nitrogenous source is one or more the combination in inorganic ammonium salt, inorganic nitrate, yeast powder, yeast extract paste, peptone, extractum carnis, the wort.
CN2012103690137A 2012-09-27 2012-09-27 Cordyceps militaris strain for producing cordycepin with high yield Pending CN102876587A (en)

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Cited By (8)

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CN103098648A (en) * 2013-01-30 2013-05-15 广东省微生物研究所 Cordyceps ramose bacterial strains, cordyceps ramose bacterial strain sporocarps, and artificial cultivation method
CN103283482A (en) * 2013-05-07 2013-09-11 熊艳 Cordyceps militaris, cultivation method and separation method
CN103539498A (en) * 2013-09-29 2014-01-29 台州康晶生物科技有限公司 Cordyceps militaris culture medium and application thereof
CN104086305A (en) * 2014-06-30 2014-10-08 桂林丰茂源农业技术开发有限公司 Culture medium for cordyceps militaris fruiting body
CN106148198A (en) * 2016-07-05 2016-11-23 青海珠峰冬虫夏草工程技术研究有限公司 A kind of Cordyceps fluid medium and preparation method thereof
CN106148200A (en) * 2016-07-22 2016-11-23 青海珠峰冬虫夏草工程技术研究有限公司 The low lead of a kind of selenium-rich, the culture medium of Cordyceps mycelium of arsenic and inoculation method thereof
CN108384726A (en) * 2018-04-25 2018-08-10 杭州中美华东制药有限公司 Aweto mycelium and its fermentation medium and fermentation process
CN109355211A (en) * 2018-12-13 2019-02-19 蒋涛 A kind of Moringa Cordyceps militaris and its cultural method and bacteriostasis spray

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103098648A (en) * 2013-01-30 2013-05-15 广东省微生物研究所 Cordyceps ramose bacterial strains, cordyceps ramose bacterial strain sporocarps, and artificial cultivation method
CN103283482A (en) * 2013-05-07 2013-09-11 熊艳 Cordyceps militaris, cultivation method and separation method
CN103283482B (en) * 2013-05-07 2014-07-16 熊艳 Cordyceps militaris, cultivation method and separation method
CN103539498A (en) * 2013-09-29 2014-01-29 台州康晶生物科技有限公司 Cordyceps militaris culture medium and application thereof
CN104086305A (en) * 2014-06-30 2014-10-08 桂林丰茂源农业技术开发有限公司 Culture medium for cordyceps militaris fruiting body
CN104086305B (en) * 2014-06-30 2016-03-30 桂林丰茂源农业技术开发有限公司 A kind of Cordyccps-militaris-(L.)-link. Sporophore substratum
CN106148198A (en) * 2016-07-05 2016-11-23 青海珠峰冬虫夏草工程技术研究有限公司 A kind of Cordyceps fluid medium and preparation method thereof
CN106148198B (en) * 2016-07-05 2019-12-13 青海珠峰冬虫夏草工程技术研究有限公司 Cordyceps sinensis liquid culture medium and preparation method thereof
CN106148200A (en) * 2016-07-22 2016-11-23 青海珠峰冬虫夏草工程技术研究有限公司 The low lead of a kind of selenium-rich, the culture medium of Cordyceps mycelium of arsenic and inoculation method thereof
CN106148200B (en) * 2016-07-22 2019-12-17 青海珠峰冬虫夏草工程技术研究有限公司 culture medium of selenium-rich low-lead and arsenic cordyceps sinensis mycelia and cultivation method of culture medium
CN108384726A (en) * 2018-04-25 2018-08-10 杭州中美华东制药有限公司 Aweto mycelium and its fermentation medium and fermentation process
CN109355211A (en) * 2018-12-13 2019-02-19 蒋涛 A kind of Moringa Cordyceps militaris and its cultural method and bacteriostasis spray

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Application publication date: 20130116