CN103283482B - Cordyceps militaris, cultivation method and separation method - Google Patents
Cordyceps militaris, cultivation method and separation method Download PDFInfo
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- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 49
- 238000000926 separation method Methods 0.000 title claims abstract description 12
- 238000012364 cultivation method Methods 0.000 title abstract 4
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 claims abstract description 11
- 230000004913 activation Effects 0.000 claims abstract description 11
- 230000003213 activating effect Effects 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 21
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- -1 0.5 gram Chemical compound 0.000 claims description 12
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of microorganisms, in particular to cordyceps militaris paecilomyces varioti, a cultivation method and a separation method. The collection number of the cordyceps militaris GZU-19 is CCTCC (China Center for Type Culture Collection) NO (Number): M2013056. The cultivation method of the cordyceps militaris comprises the steps of activating a culture: inoculating the collected cordyceps militaris onto an oblique surface of a Somogyi culture medium for activation cultivation, dibbling the activated cordyceps militaris onto a flat plate of the Somogyi culture medium for morphological identification growth cultivation, and obtaining an aerial mycelium. The cordyceps militaris paecilomyces varioti, the cultivation method and the separation method have higher cordycepin synthesis ability, and a foundation is laid for the fermentation industry of artificial cultivation fruit bodies and microorganisms of high-yield cordycepin.
Description
Technical field
The present invention relates to microorganism field, in particular to Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19), cultural method and separation method.
Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19), preserving number is CCTCC NO:M2013056, is deposited in Chinese Typical Representative culture collection center (CCTCC), address: China. Wuhan. and Wuhan University, postcode: 430072, the preservation time is on February 4th, 2013.
Background technology
Cordyceps militaris (L.) Link. claims again Cordyceps militaris, Cordyceps militaris (L.) Link..Cordyceps militaris (L.) Link. anamorph is Cordyceps militaris (L.) Link. Paecilomyces varioti, and anamorph is in single full daughter bacteria strain level, and colony characteristics, sexual fruiting body form the aspects such as ability, conidial fructification and differ greatly.Cordyceps militaris (L.) Link. contains abundant active substance, as cordycepin, polysaccharide, nucleosides, carotenoid etc., has the physiologically active widely such as antitumor, antiviral, anti-inflammatory, antibacterial, anti-oxidant, immunomodulatory.
Cordycepin is one of most important active substance of Cordyceps militaris (L.) Link., is a kind of nucleoside antibiotics, has a series of pharmacologically active, as effects such as immunoregulation effect, antitumor action, antibiosis and antiviral functions, anti-inflammatory action, leukemias.As medicament for treatment of leukemia, in the U.S., entered phase iii clinical trial.Also there are some researches show, cordycepin also has and suppresses active HIV-I C-type virus C.Cordyceps militaris (L.) Link. is Chinese medicine and the food of medicine-food two-purpose, being in great demand of market.Content requirement as its cordycepin of food is not high, but just very high to the requirement of cordycepin as medicine source or direct drug injection.But at present the Cordyccps-militaris-(L.)-link. Sporophore cordycepin content of artificial growth is not high, generally at 0.5%(, accounts for sporophore dry weight) below, demand that far away can not fulfilling medicinal, also far can not meet the needs in market.Therefore the key, addressing this problem is to obtain the bacterial strain of the synthetic cordycepin of high-performance bio.
Summary of the invention
The object of the present invention is to provide Cordyceps militaris (L.) Link. Paecilomyces varioti, cultural method and separation method, to solve the above problems.
Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 is provided in an embodiment of the present invention), its preserving number is CCTCC NO:M2013056.
A kind of described Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 is provided in an embodiment of the present invention) cultural method of CCTCC NO:M2013056, comprising:
Actication of culture, described actication of culture comprises the Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 of preservation) CCTCC NO:M2013056 is transferred to sabouraud medium inclined-plane and carries out activation culture;
Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 after will activate) dibbling of CCTCC NO:M2013056 is carried out grown cultures to sabouraud medium flat board, obtains aerial hyphae.
Optimize, in every liter of described sabouraud medium, contain: maltose 10-20 gram, glucose 10-30 gram, peptone 8-23 gram, yeast extract paste 4-15 gram, agar 15-20 gram, magnesium sulfate 0.1-1 gram, ferrous sulfate 0.1-1 gram, Repone K 0.5-2 gram.
Optimize, in every liter of described sabouraud medium, contain: 5 grams of maltose, 20 grams of glucose, 10 grams of peptones, 5 grams of yeast extract pastes, agar 15-20 gram, 0.5 gram, magnesium sulfate, 0.5 gram, ferrous sulfate, 1 gram, Repone K.
Optimize, in described actication of culture, activation culture temperature is 24 ℃-26 ℃, and the activation culture time is 5-7 days.
Optimize, in described grown cultures, grown cultures temperature is 25 ℃, and the grown cultures time is 14 days.
Optimize, the form of described aerial hyphae in sabouraud medium flat board is: bacterium colony is circular, 34 millimeters of diameters, and bacterium colony central uplift, has 2 or 3 concentric rings outside it, and abundant is velvet-like, and the conidial fructification of described aerial hyphae is Paecilomyces varioti type.
Optimize, it is orange-yellow to chrome yellow look that described aerial hyphae is, and the aerial hyphae at edge is white, and the bacterium colony back side is light yellow.
Optimize, described method also comprises: Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19) rDNA of CCTCC NO:M2013056 transcribes; RDNA transcribes district's DNA sequencing fragment:
ACATGCTTAGGTTGGGCGTTTTACGGCGTGGCCACGTCGGGTTCCCGGTGCGAGTTGGAGTACTACGCAGAGGTCGCCGCGGACGGGCCGCCACTTCATTTCGGGGGCGGCGGTGTGCTGCCGGTCCCCAACGCCGACATCCCCCAGGGGACGTCGAGGGTTGAAATGAACGCTCGAACAGGCATGCCCGCCAGAATGCTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAGCCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTGTTTTGCCTTGCGGCGGATTCAGAAAAACTGGTAGATACAGTGTTTGGGGCCCCCGACGGCCGCCGCCCAGGCCGGCGTCCAGGCGCTGGGCGAGTCCGCCGAAGCAACGATAGGTATGTTCACAAAGGGTTGGGAGTTGGAAAACTCGTTAATGATCCCTCCGCTGGTTCACCAAGGGAGACCTTGTTACGACTTTTTCTTCCAGGA。
The present invention also provides a kind of described Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19) separation method of CCTCC NO:M2013056, comprising:
The Cordyccps-militaris-(L.)-link. Sporophore collecting is carried out to surface sterilization with 75% ethanol, then with aseptic scalper, cutting stroma can pregnant part, Qu Yi little block organization or be with pyrenocarpic tissue to be inoculated on Sa Shi flat board, cultivate 4-5 days for 24 ℃-26 ℃, switching Sa Shi inclined-plane, cultivate 6-7 days, take out 4 ℃ of preservations for 24 ℃-26 ℃.
The Cordyceps militaris (L.) Link. Paecilomyces varioti that the above embodiment of the present invention provides, cultural method and separation method, Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19), its preserving number is CCTCC NO:M2013056, be deposited in Chinese Typical Representative culture collection center (CCTCC), address: China. Wuhan. Wuhan University, postcode: 430072, after actication of culture and grown cultures, possessed sequence signature between certain morphological specificity and rDNA transcriptional domain, there is the ability of synthetic cordycepin more by force, artificial culture sporophore for high yield cordycepin, the microorganism fermentation industry industry of high yield cordycepin lays the foundation.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
In the embodiment of the present invention, provide Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19), its preserving number is CCTCC NO:M2013056, being deposited in Chinese Typical Representative culture collection center (is called for short: CCTCC), the preservation time is on February 4th, 2013.
Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 is provided in the embodiment of the present invention) cultural method of CCTCC NO:M2013056, comprising:
Actication of culture, described actication of culture comprises the Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 of preservation) CCTCC NO:M2013056 is transferred to sabouraud medium inclined-plane and carries out activation culture;
Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 after will activate) dibbling of CCTCC NO:M2013056 is carried out grown cultures to sabouraud medium flat board, obtains aerial hyphae.
The present invention also provides a kind of described Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19) separation method of CCTCC NO:M2013056, comprising:
The Cordyccps-militaris-(L.)-link. Sporophore collecting is carried out to surface sterilization with 75% ethanol, then with aseptic scalper, cutting stroma can pregnant part, Qu Yi little block organization or be with pyrenocarpic tissue to be inoculated on Sa Shi flat board, cultivate 4-5 days for 24 ℃-26 ℃, switching Sa Shi inclined-plane, cultivate 6-7 days, take out 4 ℃ of preservations for 24 ℃-26 ℃.
After actication of culture and grown cultures, possessed sequence signature between certain morphological specificity and rDNA transcriptional domain, the ability more by force with synthetic cordycepin, for the artificial culture sporophore of high yield cordycepin, the microorganism fermentation industry industry of high yield cordycepin lay the foundation.
Next, by several specific embodiments, describe the cultural method of Cordyceps militaris (L.) Link. Paecilomyces varioti in detail:
Embodiment mono-:
The Cordyccps-militaris-(L.)-link. Sporophore collecting is carried out to surface sterilization with 75% ethanol, then with aseptic scalper, cutting stroma can pregnant part, Qu Yi little block organization or be with pyrenocarpic tissue to be inoculated on Sa Shi flat board, cultivate 4-5 days for 24 ℃-26 ℃, switching Sa Shi inclined-plane, cultivate 6-7 days, take out the Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 that 4 ℃ of preservations obtain for 24 ℃-26 ℃) CCTCC NO:M2013056.
Prepare sabouraud medium, first take the various raw materials of sabouraud medium: 5 grams of maltose, 20 grams of glucose, 10 grams of peptones, 5 grams of yeast extract pastes, 15 grams, agar, 0.5 gram, magnesium sulfate, 0.5 gram, ferrous sulfate, 1 gram, Repone K; Then by maltose, glucose, peptone, yeast extract paste, agar, magnesium sulfate, ferrous sulfate, Repone K adds in the distilled water of 600ml successively prepares mixing solutions, and the solution constant volume of drifting along or through is to 1000ml, sterilizing at 210 ℃.
By the Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 of preservation), its preserving number is that CCTCC NO:M2013056 is transferred on sabouraud medium inclined-plane and carries out activation culture, and activation culture temperature is 24 ℃-26 ℃, and the activation culture time is 5-7 days;
Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 after will activate) dibbling of CCTCC NO:M2013056 is carried out grown cultures to sabouraud medium flat board, obtains aerial hyphae, and grown cultures temperature is 25 ℃, and the grown cultures time is 14 days.
The morphological specificity of the aerial hyphae obtaining after above-mentioned grown cultures is: bacterium colony is circular, 34 millimeters of diameters, bacterium colony central uplift, central uplift is the shape description mode that microorganism field is conventional, outside it, there are 2 or 3 concentric rings, abundant is velvet-like, velvet-like is the shape description language that microorganism field is conventional, those skilled in the art can understand the velvet-like mode that defines completely, the conidial fructification of described aerial hyphae is Paecilomyces varioti type, collection of illustrative plates in the color of aerial hyphae and streptomycete identification handbook is compared, it is orange-yellow to chrome yellow look that described aerial hyphae is, the aerial hyphae at edge is white, the bacterium colony back side is light yellow.
Next carry out rDNA and transcribe, concrete steps are:
(1) cultivation of bacterial strain: Cordyceps militaris (L.) Link. Paecilomyces varioti spore liquid (can buy from market) is coated the Sa Shi flat board that covers one deck glassine paper, cultivated 2 days for 25 ± 1 ℃, collect mycelia, proceed in centrifuge tube, standby.
(2) extraction of genomic dna
A. in the centrifuge tube of mycelia is housed, add a little quartz sand, add cracked solution 200 μ l, fully grind to form homogenate.
B. add beta-mercaptoethanol 2 μ l, Proteinase K solution 20 μ l, concussion mixes, 56 ℃ of water-bath 1h.
C. add PE damping fluid 100 μ l, fully mix ,-20 ℃ of refrigerators are placed 5min.
D. room temperature 10, and the centrifugal 5min of 000rpm moves on to supernatant liquor in new 1.5ml centrifuge tube.
E. add BD damping fluid 200 μ l, dehydrated alcohol 200 μ l, fully mix, and proceed in the adsorption column being contained in centrifuge tube.
F. standing 2min, at the centrifugal 1min of 10,000rpm room temperature, outwells the waste liquid in collection tube.
G. adsorption column is put back to collection tube, add PW solution 500 μ l, the centrifugal 30s of 10,000rpm outwells the waste liquid in collection tube.
H. adsorption column is put back to collection tube, add Wash solution 500 μ l, the centrifugal 30s of 10,000rpm outwells the waste liquid in collection tube.
I. take out adsorption column, put into a new 1.5ml centrifuge tube, add TE damping fluid 50 μ l, standing 3min, in the centrifugal 2min of 12,000rpm room temperature, collects DNA solution, and-20 ℃ save backup.
Cordyceps militaris (L.) Link. Paecilomyces varioti rDNA transcribes the pcr amplification of a district (internal transcribed spacer, ITS) DNA sequence dna
By the rDNA ITS sequence of primer I TS4 and ITS5 amplification bacterial strain, primer sequence is as follows:
ITS5：GGAAGTAAAAGTCGTAACAAGG，ITS4：TCCTCCGCTTATTGATAGC。
Amplification system: PCR reaction system is 20ul, comprising:
Amplification condition:
Sequence measurement
Adopt PCR product direct Sequencing.Use Sanger zymetology method to check order.
RDNA transcribes district's DNA sequencing fragment:
ACATGCTTAGGTTGGGCGTTTTACGGCGTGGCCACGTCGGGTTCCCGGTGCGAGTTGGAGTACTACGCAGAGGTCGCCGCGGACGGGCCGCCACTTCATTTCGGGGGCGGCGGTGTGCTGCCGGTCCCCAACGCCGACATCCCCCAGGGGACGTCGAGGGTTGAAATGAACGCTCGAACAGGCATGCCCGCCAGAATGCTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAGCCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTGTTTTGCCTTGCGGCGGATTCAGAAAAACTGGTAGATACAGTGTTTGGGGCCCCCGACGGCCGCCGCCCAGGCCGGCGTCCAGGCGCTGGGCGAGTCCGCCGAAGCAACGATAGGTATGTTCACAAAGGGTTGGGAGTTGGAAAACTCGTTAATGATCCCTCCGCTGGTTCACCAAGGGAGACCTTGTTACGACTTTTTCTTCCAGGA。
Next the Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 of this embodiment is provided), its preserving number is the separation method of CCTCC NO:M2013056:
The Cordyccps-militaris-(L.)-link. Sporophore collecting is carried out to surface sterilization with 75% ethanol, then with aseptic scalper, cutting stroma can pregnant part, Qu Yi little block organization or be with pyrenocarpic tissue to be inoculated on Sa Shi flat board, cultivate 4-5 days for 24 ℃-26 ℃, switching Sa Shi inclined-plane, cultivate 6-7 days, take out 4 ℃ of preservations for 24 ℃-26 ℃.
For the Cordyceps militaris (L.) Link. GZU-19(Cordycepes militaris GZU-19 that further proof is preserved), its preserving number is that CCTCC NO:M2013056 possesses high yield entomogenous fungi, adopts cultural method and test below, can obtain cordycepin content 1.3%:
One, slant strains is cultivated
By the Cordyceps militaris spawn Sa Shi inclined-plane of transferring, cultivate 7d for 27 ℃ ± 1 ℃.
Two, the preparation of spore suspension
With the sterile distilled water that contains 0.05% tween 80, wash lower inclined plane bacterial classification spore, proceed in the 50ml triangular flask with granulated glass sphere, vibration is prepared into the spore suspension of homogeneous, and final concentration is 106cfu/ml.
Three, Cordyceps sporophore cultivation
1, substratum
Rice 30g, peptone 0.10g, potassium primary phosphate, 0.030g, magnesium sulfate 0.035g, glucose 0.1g, vitaminB10 .40mg, water 3,5ml, pH value 6, packs in culturing bottle, polypropylene film sealing.121 ℃ of autoclaving 60min.
2, inoculation
Spore suspension is inoculated in the culturing bottle of sterilization zone substratum to stirring and evenly mixing by 3ml/ bottle.
3, secretly cultivate
Vaccinated culturing bottle is placed into and is sterilized in black out room, 22 ℃ ± 2 ℃ of temperature, humidity, cultivate 1 week 65% left and right, and culturing bottle covers with mycelia.
5, mycelia annesl is cultivated
The cultivation bottle transposition health of mycelia, clean, room with good ventilation, 22 ℃ of left and right of temperature will be covered with; Humidity keeps 75% left and right; The 3h that ventilates every day, illumination 12h, cultivates 7 days, and annesl completes, and starts to form the former base of millet shape (being called again button) in media surface.
5, going out sporophore cultivates
When media surface forms the former base of millet shape in culturing bottle, temperature is adjusted into 23 ℃ of left and right, humidity is adjusted into 85% left and right, the 3h that ventilates every day, illumination 12h, cultivates 5-7 days, former base extends for stroma (being called again sporophore), at this moment at culturing bottle sealing film, pricks 5 holes.Continue to cultivate 30 days the sporophore of gathering.
6, the Cordyccps-militaris-(L.)-link. Sporophore that bacterial strain M2013056 obtains in order to upper method cultivation is golden yellow, and cordycepin content is 1.3%.
Embodiment bis-:
Be with the distinctive points of embodiment mono-: sabouraud medium: 10 grams of maltose, 10 grams of glucose, 8 grams of peptones, 4 grams of yeast extract pastes, 15 grams, agar, 0.1 gram, magnesium sulfate, 0.1 gram, ferrous sulfate, 0.5 gram, Repone K.
Embodiment tri-:
Be with the distinctive points of embodiment mono-: sabouraud medium: 20 grams of maltose, 30 grams of glucose, 23 grams of peptones, 15 grams of yeast extract pastes, 20 grams, agar, 1 gram, magnesium sulfate, 1 gram, ferrous sulfate, 2 grams, Repone K.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Sequence table
<110> bear is gorgeous
<120> Cordyceps militaris (L.) Link., cultural method and separation method
<160>1
<210>1
<211>540
<212>DNA
<213> Cordyceps militaris (L.) Link. (Cordycepes militaris)
<400>1
ACATGCTTAGGTTGGGCGTTTTACGGCGTGGCCACGTCGGGTTCCCGGTGCGAGTTGGAGTACTACGCAGAGGTCGCCGCGGACGGGCCGCCACTTCATTTCGGGGGCGGCGGTGTGCTGCCGGTCCCCAACGCCGACATCCCCCAGGGGACGTCGAGGGTTGAAATGAACGCTCGAACAGGCATGCCCGCCAGAATGCTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAGCCAAGAGATCCGTTGTTGAAAGTTTTGATTCATTTGTTTTGCCTTGCGGCGGATTCAGAAAAACTGGTAGATACAGTGTTTGGGGCCCCCGACGGCCGCCGCCCAGGCCGGCGTCCAGGCGCTGGGCGAGTCCGCCGAAGCAACGATAGGTATGTTCACAAAGGGTTGGGAGTTGGAAAACTCGTTAATGATCCCTCCGCTGGTTCACCAAGGGAGACCTTGTTACGACTTTTTCTTCCAGGA
Claims (10)
1. Cordyceps militaris (L.) Link. (Cordycepes militaris) GZU-19, its preserving number is CCTCC NO:M2013056.
2. a cultural method of Cordyceps militaris (L.) Link. claimed in claim 1 (Cordycepes militaris) GZU-19CCTCC NO:M2013056, is characterized in that, comprising:
Actication of culture, described actication of culture comprises that the Cordyceps militaris (L.) Link. of preservation (Cordycepes militaris) GZU-19CCTCC NO:M2013056 is transferred to sabouraud medium inclined-plane carries out activation culture;
The dibbling of Cordyceps militaris (L.) Link. (Cordycepes militaris) GZU-19CCTCC NO:M2013056 after activating is carried out to the grown cultures of identification of morphology to sabouraud medium flat board, obtain aerial hyphae.
3. cultural method according to claim 2, is characterized in that,
In every liter of described sabouraud medium, contain: maltose 10-20 gram, glucose 10-30 gram, peptone 8-23 gram, yeast extract paste 4-15 gram, agar 15-20 gram, magnesium sulfate 0.1-1 gram, ferrous sulfate 0.1-1 gram, Repone K 0.5-2 gram.
4. cultural method according to claim 2, is characterized in that,
In every liter of described sabouraud medium, contain: 5 grams of maltose, 20 grams of glucose, 10 grams of peptones, 5 grams of yeast extract pastes, agar 15-20 gram, 0.5 gram, magnesium sulfate, 0.5 gram, ferrous sulfate, 1 gram, Repone K.
5. cultural method according to claim 2, is characterized in that, in described actication of culture, activation culture temperature is 24 ℃-26 ℃, and the activation culture time is 5-7 days.
6. cultural method according to claim 2, is characterized in that, in described grown cultures, grown cultures temperature is 25 ℃, and the grown cultures time is 14 days.
7. cultural method according to claim 2, is characterized in that, the form of described aerial hyphae in sabouraud medium flat board is: bacterium colony is circular, 34 millimeters of diameters, bacterium colony central uplift, has 2 or 3 concentric rings outside it, abundant is velvet-like, and the conidial fructification of described aerial hyphae is Paecilomyces varioti type.
8. cultural method according to claim 7, is characterized in that, it is orange-yellow to chrome yellow look that described aerial hyphae is, and the aerial hyphae at edge is white, and the bacterium colony back side is light yellow.
9. cultural method claimed in claim 2, is characterized in that, described method also comprises: the rDNA of Cordyceps militaris (L.) Link. (Cordycepes militaris) GZU-19CCTCC NO:M2013056 transcribes;
RDNA transcribes district's DNA sequencing fragment:
ACATGCTTAGGTTGGGCGTTTTACGGCGTGGCCACG?TCGGGTTCCCGGTGCGAGTTGGAGTACTACGCAGAGGTC?GCCGCGGACGGGCCGCCACTTCATTTCGGGGGCGGCGG?TGTGCTGCCGGTCCCCAACGCCGACATCCCCCAGGGGA?CGTCGAGGGTTGAAATGAACGCTCGAACAGGCATGCCC?GCCAGAATGCTGGCGGGCGCAATGTGCGTTCAAAGATTC?GATGATTCACTGAATTCTGCAATTCACATTACTTATCGCAT?TTCGCTGCGTTCTTCATCGATGCCAGAGCCAAGAGATCC?GTTGTTGAAAGTTTTGATTCATTTGTTTTGCCTTGCGGCG?GATTCAGAAAAACTGGTAGATACAGTGTTTGGGGCCCCC?GACGGCCGCCGCCCAGGCCGGCGTCCAGGCGCTGGGCG?AGTCCGCCGAAGCAACGATAGGTATGTTCACAAAGGGTT?GGGAGTTGGAAAACTCGTTAATGATCCCTCCGCTGGTTC?ACCAAGGGAGACCTTGTTACGACTTTTTCTTCCAGGA。
10. a separation method of Cordyceps militaris (L.) Link. claimed in claim 1 (Cordycepes militaris) GZU-19CCTCC NO:M2013056, is characterized in that, comprising:
The Cordyccps-militaris-(L.)-link. Sporophore collecting is carried out to surface sterilization with 75% ethanol, then with aseptic scalper, cutting stroma can pregnant part, Qu Yi block organization or be with pyrenocarpic tissue to be inoculated on Sa Shi flat board, cultivate 4-5 days for 24 ℃-26 ℃, switching Sa Shi inclined-plane, cultivate 6-7 days, take out 4 ℃ of preservations for 24 ℃-26 ℃.
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| CN113348965B (en) * | 2021-07-22 | 2023-11-17 | 成都天草生物科技有限公司 | Proliferation method of Cordyceps sinensis for infecting hepialus larva |
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| CN101245361A (en) * | 2008-03-11 | 2008-08-20 | 华中农业大学 | A method for producing cordycepin and the breeding and application of high-yielding Cordyceps militaris strain BYB-08 |
| CN102210255A (en) * | 2011-04-06 | 2011-10-12 | 广东省微生物研究所 | Cordyceps militaris albino strain and cultivation method of fruit body thereof |
| CN102450157A (en) * | 2010-10-26 | 2012-05-16 | 华中农业大学 | Solid fermentation method for cordyceps militaris |
| CN102876587A (en) * | 2012-09-27 | 2013-01-16 | 江南大学 | Cordyceps militaris strain for producing cordycepin with high yield |
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| CN101245361A (en) * | 2008-03-11 | 2008-08-20 | 华中农业大学 | A method for producing cordycepin and the breeding and application of high-yielding Cordyceps militaris strain BYB-08 |
| CN102450157A (en) * | 2010-10-26 | 2012-05-16 | 华中农业大学 | Solid fermentation method for cordyceps militaris |
| CN102210255A (en) * | 2011-04-06 | 2011-10-12 | 广东省微生物研究所 | Cordyceps militaris albino strain and cultivation method of fruit body thereof |
| CN102876587A (en) * | 2012-09-27 | 2013-01-16 | 江南大学 | Cordyceps militaris strain for producing cordycepin with high yield |
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| US12161069B2 (en) | 2015-04-15 | 2024-12-10 | Ecovative Llc | High density rigid molded body of composite mycological material |
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