CN109832085A - Double spore cordyceps sinensis and its artificial cultivation method - Google Patents
Double spore cordyceps sinensis and its artificial cultivation method Download PDFInfo
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- CN109832085A CN109832085A CN201711185747.9A CN201711185747A CN109832085A CN 109832085 A CN109832085 A CN 109832085A CN 201711185747 A CN201711185747 A CN 201711185747A CN 109832085 A CN109832085 A CN 109832085A
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Abstract
The invention belongs to microorganisms technical fields, are related to a kind of cordyceps sinensis New Records --- double spore cordyceps sinensis [Ophiocordyceps bispora(Stifler) G.H.Sung, J.M.Sung, Hywel-Jones&Spatafora], and a kind of artificial method for cultivating double spore cordyceps militaris sporocarps.This method with Yunnan odontoterme (Odontotermesyunnanensis) it is culture medium carrier, using double spore Anamorph of Cordyceps as strain, main operational steps are as follows: A, actication of culture；B, liquid shaking bottle seed culture；C, cordyceps militaris sporocarp culture.The double spore cordyceps militaris sporocarps manually cultivated are in rodlike, milky or brown, it is mature after blackening grey, length is similar with wild double spore cordyceps sinensis with size.The present invention be it is pioneering, can be stablized using this method, cultivate double spore cordyceps militaris sporocarps continuously, in bulk, the edible and medicinal study for double spore cordyceps sinensis lays the foundation.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of entomogenous fungi --- the phorozoon strain of double spore cordyceps sinensis
With a kind of method of the double spore cordyceps militaris sporocarps of artificial cultivation.
Background technique
Double spore cordyceps sinensis (Ophiocordyceps bispora) distribution site be detected in African Tanzania Tanzania and
Kenya Kenya belongs to Asia new record close to Semi-arid Grassland and Yunnan Province's phosphorus pools area of interior Robbie Nairobi
Kind.Double spore cordyceps sinensis are under the jurisdiction of Eumycota, Ascomycetes, nematode grass section, line Cordyceps.The host of double spore cordyceps sinensis is the white of Isoptera
Ant, this seminar purify to obtain its phorozoon strain by separating for several times.Through literature survey, such fungi is planted not yet both at home and abroad
The report and patent of training.Double spore cordyceps sinensis wild resources are rare, realize that its artificial cultivation is this kind of worm grass resources needs of development and utilization
The major issue of solution.
Chinese medicine thinks that termite is mild-natured, and taste is salty, has special medical value.In the Compendium of Material Medica of Li Shizhen of the Ming Dynasty, just
The record that useful termite is cured the disease.In recent years it has been found that termite protein rich in, amino acid, DNA and
Other medicinal ingredients.According to surveying and determination, the amino acid content of termite is higher by 11.1% than rare invigorant bird's nest, is also rich in microelement
Iron and Vitamin A, vitamin B2.Existing disease-resistance substance steroidal (mainly has cholesterine and its derivative, paddy steroid in termite body
Alcohol, stigmasterol etc.), there is certain inhibiting effect to cancer cell.
The present invention by the phorozoon strain of double spore cordyceps sinensis be seeded to Yunnan odontoterme (Odontotermes yunnanensis), then it adds other nutrient solutions and is cultivated, by the strict control to temperature, humidity, illumination, ventilation etc.,
The nutritional condition and environmental condition better than wild environment are built, double spore Cordyceps Militaris mycelia differentiation, fruit-body formation are finally made,
After drying, crushing, it can be used for developing strengthen immunity and health food for relieving physical fatigue or medicine.
Summary of the invention
The cordyceps sinensis that one of the objects of the present invention is to provide a kind of suitable for manually cultivating, yield is high, active constituent content is high
Strain.
The second object of the present invention is to provide a kind of breeding method suitable for the above-mentioned Cordyceps Militaris of large-scale production.
Strain and characteristic:
Double spore cordyceps sinensis provided by the invention belong to Asia New Records, and anamorphic strain was preserved on August 10th, 2017
Chinese herbal medicine biological the Study on Resources institute, Yunnan University fungal cultures collection, deposit number are YFCC 170801.The bacterial strain exists
It is cultivated 21 days, colony diameter 14-16mm for 25 DEG C in PDA culture medium, white, villiform is round, and tufted is commonly formed in neat in edge
Fructification (coremium)；The not chromogenic element in the back side.Mycelia is transparent, separates, smooth, mycelia is 3.3-8.1 μm wide.Conidiogenous cell bottle stalk
Shape is divided into A type bottle stalk and Type B bottle stalk.A type bottle stalk is longer, typically occurs in mycelia top, Dan Sheng, taper or cylinder,
15.1-26.8 μm long, base portion is 2.3-5.1 μm wide, is tapered upwards, and top is 0.9-2.1 μm wide, generates single spore.Type B
Bottle stalk typically occurs in aerial hyphae, and Dan Sheng or verticillate, 9.7-14.7 μm long, base portion expands, ellipse or spherical, wide 2.1-
4.8 μm, attenuate to form elongated bottleneck upwards suddenly, bottleneck is 1.99-5.33 μm long, 0.8-1.5 μm wide.Usually proliferation is formed bottle stalk
Many small bottlenecks of thread form.Conidium monospore, it is smooth, it is subsphaeroidal or oval, 3.8-6.4 × 3.4-5.2 μm.
Cultural method:
Double spore Anamorph of Cordyceps strains are inoculated in the PDA solid medium of improvement, carry out actication of culture.Then by the bacterium of activation
The preparation of seed liquor is carried out on kind inoculation liquid medium within.A can is taken, the Yunnan 110-130g odontoterme and 40- is added
60mL nutrient solution, moist heat sterilization and after cooling down are inoculated with strain liquid by the inoculum concentration of 5-10%, carry out fructification under optimum conditions
Culture.Fructification Initial stage of culture is carried out in the dark under 23-27 DEG C of temperature, humidity 30-75%, grows a large amount of mycelium to 10-20 days
After go to growth cabinet culture.Followed by the fruit-body formation stage, divide Low- temperature culture, Fluctuation temperature culture, three step of constant temperature incubation.
Firstly, Low- temperature culture 5-10 days, temperature should be controlled at 17-22 DEG C, humidity 40-60%, intensity of illumination 10-50Lx.Then alternating temperature
Culture 5-10 days keeps humidity constant (50-70%), cultivates 16h under 20-25 DEG C of temperature, intensity of illumination 100-200Lx；Then
Adjusting temperature is 10-15 DEG C, is protected from light culture 8h.Finally, constant temperature incubation 15-20 days, condition of culture is 22-27 DEG C of temperature, humidity
75-100%, illumination 300-700 Lx.It is similar with wild collecting sample to cultivate mature fructification, rodlike, black gray expandable has obvious
Ascus shell structure, and rich in various nutritional ingredients possessed by wild double spore cordyceps sinensis.
Specific embodiment
Following embodiment can be with the invention will be further described:
1. solid medium prepare: take do not germinate, the potato of paleness, clean peeling, weigh 200g, be cut into 1 centimetre or so
The fritter of square.The potato fritter cut is put into about 1000mL water, is kept for 30 minutes after boiling with warm fire.After use
Filtered through gauze, obtaining filtrate is potato juice, and filtrate is complemented to 1000mL.Then glucose 20g, peptone 5g, fine jade is added
Rouge 20g, pours into test tube respectively after mixing evenly, sterilizes 30 minutes at 121 DEG C, takes out rear-inclined and places, can after solidification to be cooled
For transferred species.
2. fluid nutrient medium prepare: take do not germinate, the potato of paleness, clean peeling, weigh 200g, be cut into 1 centimetre
The fritter of left and right square.The potato fritter cut is put into about 1000mL water, is kept for 30 minutes after boiling with warm fire.Terminate
Filtered through gauze is used afterwards, and obtaining filtrate is potato juice, and filtrate is complemented to after 1000mL and is distributed into 500mL/ bottles (triangular flask is
1000mL specification).Every bottle of addition glucose 10g, corn flour 10g, soybean powder 5g, konjaku flour 5g, peptone 5g, yeast extract
3g, magnesium sulfate 1.0g, potassium dihydrogen phosphate 0.5g are stirred evenly, beyond the Great Wall tampon, natural cooling, standby after sterilizing 30 minutes at 121 DEG C
With.
3. the solid culture production of hybrid seeds: taking double spore Anamorph of Cordyceps strains, the solid that access step 1 makes under aseptic condition is oblique
Face culture medium, 25 DEG C are protected from light culture.When mycelia covers surface about 70%, mycelia can be accessed the fluid nutrient medium production of hybrid seeds.Production of hybrid seeds process
It is as follows: 1 fritter (size about 3-5mm × 3-5mm) mycelia being taken to be put into the triangular flask equipped with fluid nutrient medium from slant medium
It is interior, it is cultivated at 100rpm, 25 DEG C about 5 days, it is long to suitable size to bacterium ball, it can be used to be inoculated with.
4. termite culture medium is prepared: the Yunnan 120g odontoterme and 50mL nutrient solution are added in a can, at 121 DEG C
Sterilizing 30 minutes.The formula of nutrient solution are as follows: glucose 20g, corn flour 20g, soybean powder 10g, konjaku flour 10g, magnesium sulfate 2.0g,
Potassium dihydrogen phosphate 1.0g, vitamin B1 5mg, water 1000mL adjust pH8.
5. inoculation: after the termite culture medium cooling after sterilizing, being inoculated with strain liquid by 8% inoculum concentration.
6. the can after inoculation is placed in progress fructification culture under suitable condition.Fructification Initial stage of culture is in temperature
25 DEG C, be carried out in the dark under humidity 60%, go to growth cabinet culture after 20 days grow a large amount of mycelium.Followed by sub real
Body formation stages divide Low- temperature culture, temperature difference culture, three step of constant temperature incubation.Low- temperature culture 10 days, temperature should be controlled at 18 DEG C, wet
Degree 50%, intensity of illumination 50Lx；Then it carries out.Fluctuation temperature culture 5 days, keep humidity constant (70%), 25 DEG C of temperature, intensity of illumination
16h is cultivated under 300Lx, then adjusting temperature is 15 DEG C, is protected from light culture 8h；Finally, constant temperature incubation 20 days, condition of culture is changed to temperature
27 DEG C of degree, humidity 90%, 500 Lx of illumination.
7. harvesting mature double spore cordyceps sinensis.It is close after being collected after double spore cordyceps sinensis in can are smashed with mechanical force, being dry
Envelope is used for further deep processing.Every bottle can harvest fresh double spore cordyceps sinensis 135-145g, every bottle of dry product weight about 40-50g after drying.
8. the double spore cordyceps sinensis analysis of effective component manually cultivated
Through detecting, above-mentioned drying, crush double spore cordyceps sinensis in, every 1g contain Cordyceps sinensis polysaccharide, cordycepic acid, thymidine, 2`- desoxyadenossine,
Uracil, adenine are respectively 63.48mg, 114.33mg, 51.47 μ g, 785.96 μ g, 819.28 μ g, 344.57 μ g, hence it is evident that excellent
In wild double spore cordyceps sinensis and wild cordyceps (see Table 1).
Multiple batches of cultivation is carried out according to the method described above, it is as a result stable.
Double spore cordyceps sinensis analysis of effective component (n=3) that table 1 is manually cultivated
| Species | Cordyceps sinensis polysaccharide mg/g | Cordycepic acid mg/g | Thymidine μ g/g | 2`- desoxyadenossine μ g/g | Uracil μ g/g | Adenine μ g/g |
| The double spore cordyceps sinensis manually cultivated | 63.48±3.06 | 114.33±11.61 | 51.47±2.62 | 785.96±54.74 | 819.28±82.94 | 344.57±29.48 |
| Wild double spore cordyceps sinensis | 16.11±4.54 | 68.90±3.29 | 0 | 376.69±8.48 | 115.24±18.98 | 251.24±5.33 |
| Wild cordyceps | 44.79±4.58 | 89.20±0.63 | 28.57±1.53 | 24.33±0.96 | 75.43±4.17 | 46.80±1.14 |
Claims (5)
1. a kind of entomogenous fungi, for double spore cordyceps sinensis [Ophiocordyceps bispora (Stifler) G.H.Sung,
J.M.Sung, Hywel-Jones & Spatafora] phorozoon, culture presevation YFCC 170801.
2. a kind of artificial cultivation method of double spore Cordyceps Militaris, it is characterised in that:
A, actication of culture is carried out in the PDA solid medium of improvement with strain inoculated described in claim 1；
B, the preparation of seed liquor will be carried out on the strain inoculation liquid medium within of activation；
C, in a can be added the Yunnan 110-130g odontoterme (Odontotermesyunnanensis) and 40-60mL battalion
Nutrient solution, moist heat sterilization and after cooling down, by 5-10%(w/w) inoculum concentration strain liquid is added, carry out fructification training under optimum conditions
It is feeding that (fructification Initial stage of culture is carried out in the dark under 23-27 DEG C of temperature, humidity 30-75%；The fruit-body formation stage, firstly, low temperature
Culture 5-10 days, temperature should be controlled at 17-22 DEG C, humidity 40-60%, intensity of illumination 10-50Lx.Then alternating temperature temperature difference culture
It 5-10 days, keeps humidity constant (50-70%), cultivates 16h under 20-25 DEG C of temperature, intensity of illumination 100-200Lx daily；Then
Adjusting temperature is 10-15 DEG C, is protected from light culture 8h；Finally, constant temperature incubation 15-20 days, condition of culture is changed to 22-27 DEG C of temperature, wet
Spend 75-100%, illumination 300-700Lx).
3. according to the method described in claim 2, it is characterized in that improveing PDA solid medium raw material proportioning described in a are as follows: Ma Ling
Potato juice 1000mL, glucose 5-25g, peptone 5-15g, agar 15-20g.
Here potato juice is obtained by following step: into the water by the peeled potatoes being cut into small pieces, potato and water
Weight ratio is 1:5-1:6, boils rear warm fire and keeps 25-40min, filters off slag, and filtrate is potato juice, following potato juice with
This is identical.
4. according to the method described in claim 2, it is characterized in that liquid medium starting material described in b matches are as follows: potato juice
1000mL, glucose 5-25g, corn flour 5-25g, soybean powder 5-15g, konjaku flour 5-15g, peptone 5-15g, yeast extract 5-
10g, magnesium sulfate 1.0-3.5g, potassium dihydrogen phosphate 1.0-2.0g.
5. according to the method described in claim 2, it is characterized in that the formula of nutrient solution described in c are as follows: glucose 5-25g, corn
Powder 5-25g, soybean powder 5-15g, konjaku flour 5-15g, magnesium sulfate 1.0-3.5g, potassium dihydrogen phosphate 1.0-3.0g, vitamin B1 5-
10mg, water 1000mL adjust pH6-9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111615992A (en) * | 2020-06-02 | 2020-09-04 | 石河子大学 | Artificial cultivation method of Sinkiang cordyceps sinensis fruiting bodies |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102138437A (en) * | 2011-01-13 | 2011-08-03 | 安徽农业大学 | Artificial cultivation method for Taiwan cordyceps fruiting bodies |
| CN102742454A (en) * | 2012-06-05 | 2012-10-24 | 安徽农业大学 | Artificial culture method of cordyceps longissima sporocarp |
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2017
- 2017-11-24 CN CN201711185747.9A patent/CN109832085A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102138437A (en) * | 2011-01-13 | 2011-08-03 | 安徽农业大学 | Artificial cultivation method for Taiwan cordyceps fruiting bodies |
| CN102742454A (en) * | 2012-06-05 | 2012-10-24 | 安徽农业大学 | Artificial culture method of cordyceps longissima sporocarp |
Non-Patent Citations (4)
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| BENJAMIN H. CONLON等: "Draft genome of the fungus-growing termite pathogenic fungus Ophiocordyceps bispora (Ophiocordycipitaceae, Hypocreales, Ascomycota", 《JOURNAL DATE IN BRIEF》 * |
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| 河北省农业广播电视学校 等: "《农博士有问必答 （种植篇）》", 28 February 1997, 中国农业大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111615992A (en) * | 2020-06-02 | 2020-09-04 | 石河子大学 | Artificial cultivation method of Sinkiang cordyceps sinensis fruiting bodies |
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