CN100469866C - Mountain Tai aweto and artificial culture method thereof - Google Patents
Mountain Tai aweto and artificial culture method thereof Download PDFInfo
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- CN100469866C CN100469866C CNB2005101337206A CN200510133720A CN100469866C CN 100469866 C CN100469866 C CN 100469866C CN B2005101337206 A CNB2005101337206 A CN B2005101337206A CN 200510133720 A CN200510133720 A CN 200510133720A CN 100469866 C CN100469866 C CN 100469866C
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Abstract
The invention relates to a new microbiological species, the phorozoon is Paecilomyces suffultus (Petch) Samson. The method consists of inoculating the said germ to parasite body or subculture medium, cultivating them in a proper condition to obtain a yellow-while fruiting body of Cordyceps. The said germ has advantages that it is suitable for artificial culture in a large number, the effective composition content is high, the adaptive capacity for environment is strong, the production velocity of bacterium is rapid, the stress is strong, the yield rate is high, the cultivated cycle is short and the germ can not degenerate.
Description
Technical field
The present invention relates to a kind of new microorganism, relate in particular to a kind of mountain Tai aweto bacterium and method of cultivation thereof.
Background technology
Cordyceps militaris (L.) Link. has another name called Cordyceps militaris, is a kind of entomogenous fungi, colonize on various insects pupa (worm) body, and be the treasure of a kind of food, medicine dual-purpose.In recent years,, further recognize the importance of Cordyceps militaris (L.) Link. nourishing and medical dual function, measured Cordyceps militaris (L.) Link. and contained cordycepin, Cordyceps polysaccharide and superoxide dismutase material along with further investigation both domestic and external.Cordycepin all has obvious antagonism or restraining effect to Bacillus subtilus, mycobacterium tuberculosis, human nasopharyngeal carcinoma KB cell, people's epiderm-like mole etc.Cordycepin also is used as RNA synthetic inhibitor in cell research.In addition, the biological control that is used for several lepidoptera pests with mycelia and the spore goods of Cordyceps militaris (L.) Link. has also obtained significant effect.But the Chinese caterpillar fungus wild resource is limited now, it is reported, the Qinghai Province county is driven by sudden huge profits every year in the Cordyceps sinensis season of growth, the hundreds of thousands of people pours into the meadow, Chinese caterpillar fungus is excavated without restraint, badly damaged vegetation, the predation worm grass resources destroys the local eubiosis, cause soil erosion, desertification of land is serious; Natural cordyceps growth cycle length is arranged again, yield poorly, far can not satisfy growing consumers demand, the distributed areas of natural cs are narrow, self-sow needed 1 year or the longer time, output is extremely low, because of its outstanding curative effect and fancy price cause unauthorized and wasteful mining, resource is on the verge of exhaustion, is badly in need of the exploitation substitute products.
Summary of the invention
One of purpose of the present invention be to provide a kind of be suitable for artificial culture, environment-adapting ability strong, send out that bacterium speed is fast, strong stress resistance, output height, active constituent content height, the short cordyceps species of cultivation period.
Two of purpose of the present invention is to provide and is suitable for the method for cultivation that above-mentioned Cordyceps is produced in commercialization.
Below be main contents of the present invention:
Mountain Tai aweto bacterial strain, this bacterial strain are preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 4th, 2005, and preserving number is CGMCC No.1341.Its anamorph is holding Paecilomyces varioti (Paecilomycessuffultus (Petch) Samson), does not appear in the newspapers at present, belongs to the new record kind.
Bacterial classification of the present invention is to parasitize the Cordyceps of lepidopteran (Lepidoptera) Pyralidae (Pyralidae) or Arctiidae (Arctiidae) from the area, Mount Taishan, obtains with tissue isolation, the separation of basic thing partition method, purifying.
The separation of bombys batryticatus body (being sclerotium): before the separation, brush away polypide surface earth gently with the fine, soft fur brush earlier, clean 2～3 times with 75% ethanol again, put into the inoculation tank sterilization, behind the 0.5h, aseptic technique, the bombys batryticatus body is separated from the centre, get the hypha body in the haemocoele, insert slant tube, 23～25 ℃ of cultivations, when treating that mycelia grows, the purifying of in time ruling on flat board, switching go in the new slant medium to cultivate, and preserve.
Stroma separates: fresh stroma takes off clean from bombys batryticatus (pupa) body, put into 0.1% mercuric chloride sterilization 3min, with sterilized water washing, in cutting, the tissue of top, choose in the slant medium, carry out 23～25 ℃ of cultivations, after treating mycelium germination, timely picking colony front end mycelia, line purifying on the flat board, switching goes in the new slant medium to cultivate, and selects the mycelia stalwartness, does not have the inclined-plane mycelia preservation that assorted bacterium is infected.
The mycelium proterties:
(1) microstructure: opticmicroscope is observed down, and hyphae colorless is level and smooth, branch, tool tabula, wide 1.5um～3.2um.Conidiophore Dan Sheng is on mycelia, and give birth to or verticillate ampuliform conidiogenous cell on the top.Conidiogenous cell, Dan Sheng on mycelia, 2～5 give birth on the conidiophore top or verticillate on conidiophore, colourless, base portion expands, and is upwards tapered, 6.5～7.5um * 2.5～4um.Conidium is oval or cylindrical, colourless, unicellular, and directly or crooked slightly, level and smooth, chain is given birth to, 4.3～8.5 (-11) um * 2.3～3.2um, and indivedual spores can reach 17um * 3.9um.
(2) cultivate proterties: on the PDA plate culture medium, cultivated 10 days for 25 ℃, the bacterium colony circle, tiling, white, mycelia is rare, is the light fleece shape, the radial rill of tool, inoculation point in the back side is tawny, the yellow-white that other position is very shallow, diameter 4cm～5.2cm.
On the PDA slant medium, mycelia is dense white, and aerial hyphae is vigorous, and it is strong to climb wall energy power, the easy blackening of mycelia on the test tube wall of aging back.Long-term storage easily produces oidium.
Liquid nutrient medium is cultivated, and mycelia is that down-like is uniformly distributed in the substratum, after the maturation, and the substratum flavescence, but as clear as crystal.
The sporophore shape feature:
This bacterial strain is just faint yellow, careless bud shape, and list is given birth to or is grown thickly; Ripe back is bar-shaped, and base portion is filbert, produces the spore place and expands, and forms the bottle-neck type gleba, produces a large amount of conidiums, is white in color powdery.Conidium is straight or crooked, and size is 3.2～6.5um * 2.2～2.6um, and perithecium is not seen in sporophore section microscopic examination.
A kind of method of cultivation of above-mentioned mountain Tai aweto, the bacterial classification inoculation that will be made by above-mentioned bacterial strains continue to cultivate until growing the mountain Tai aweto sporophore on living silkworm chrysalises, cocoon chrysalis, cicada nymph under optimum conditions; Above-mentioned vaccination ways can adopt injection, pricks the hole, the solid kind directly inserts, a kind of in the natural infection method.
The method of cultivation of another kind of mountain Tai aweto, the bacterial classification inoculation that will be made by above-mentioned bacterial strains are to solid medium, and the nutritive medium of quality such as adding, continue under optimum conditions to cultivate until growing the mountain Tai aweto sporophore; Described solid medium can be wheat, rice or their mixture.
In the mycelium culture stage, temperature is controlled at 18～24 ℃, the dark cultivation, and incubation time is 4-7 days, PH6-9;
Sporophore forms phase temperature should be controlled at 18-22 ℃, and humidity is at 70%-90%, and the cultivation that adds lustre to, until gathering.
Description of drawings
Figure is the influence test of pH value to the liquid mycelial growth.
The present invention is further illustrated below in conjunction with embodiment:
Embodiment
Existing technical approach can be used for reference or adopt to the technology contents of below not addressing all:
1, bacterial classification obtains
The mountain Tai aweto standard specimen that parasitizes lepidopteran (Lepidoptera) Pyralidae (Pyralidae) or Arctiidae (Arctiidae) to area, Mount Taishan pollution-free ecological environment is extensively gathered, obtain the good wild cordyceps militaris sporophore of economical character, carry out preliminary classification and identify that sample is preserved.
Adopt conventional spore separation method, tissue isolation, basic thing partition method, separate, the purifying wild type strain, strain identification, preserve standby.In the fact-finding process, the Cordyceps militaris (L.) Link. that stroma is sturdy, fresh is taken out from soil, packs in the previously prepd template triangle bag, takes back the laboratory, and it is to be separated to put into refrigerator (4 ℃).
The separation of bombys batryticatus body (being sclerotium): before the separation, brush away polypide surface earth gently with the fine, soft fur brush earlier, clean 2～3 times with 75% ethanol again, put into the inoculation tank sterilization, behind the 0.5h, aseptic technique, the bombys batryticatus body is separated from the centre, get the hypha body in the haemocoele, insert slant tube, 23～25 ℃ of cultivations, when treating that mycelia grows, the mycelia that picking meets the bacterial classification proterties purifying of in time ruling on flat board, switching goes in the new slant medium to cultivate, and preserves.
Stroma separates: fresh stroma takes off clean from bombys batryticatus (pupa) body, put into 0.1% mercuric chloride sterilization 3min, with sterilized water washing, in cutting, the tissue of top, choose in the slant medium, carry out 23～25 ℃ of cultivations, after treating mycelium germination, timely picking colony front end mycelia, line purifying on the flat board, switching goes in the new slant medium to cultivate, and selects the mycelia stalwartness, does not have the inclined-plane mycelia preservation that assorted bacterium is infected.
Above-mentioned bacterial classification can be made liquid spawn or solid spawn as required.
(1) the preparation solid spawn of solid spawn is cultivated and can adopt following substratum:
1 wheat 1000 of filling a prescription restrains, peptone 10 grams, sal epsom 1 gram, potassium primary phosphate 1.5 grams, glucose 10 grams, 0.5 milligram of vitamins B, 1000 milliliters in water.
By the wheat bottling of every bottle 40 gram, other composition dissolves in the water, every bottle of water of pouring into wheat equivalent, and polypropylene film seals, autoclaving.The aseptic technique inoculation, every female kind in inclined-plane connects the 6-7 bottle.After the inoculation, under 20 ~ 22 ℃ of temperature, secretly cultivate.500 milliliters of seed bottle cover with in bottle by 15 ~ 20 days mycelia.
(2) preparation of liquid spawn
In actual production, the artificial culture of Cordyceps militaris (L.) Link. adopts liquid-spawn inoculation mostly, and its nutrient solution can have following prescription:
1 potato 200 of filling a prescription restrains, glucose 20 grams, and peptone 10 grams, potassium primary phosphate 1.5 grams, sal epsom 1 gram, SODIUM PHOSPHATE, MONOBASIC 1 restrains, and VB is an amount of, 1000 milliliters in water, pH value 6.5.
2 potatoes 200 of filling a prescription restrain, glucose 20 grams, peptone 10 grams, potassium primary phosphate 1 gram, sal epsom 0.5 gram, 1000 milliliters in water; PH value 6.5.
2 potatoes 200 of filling a prescription restrain, Semen Maydis powder 30 grams, and glucose 20 grams, peptone 3 grams, potassium primary phosphate 1.5 grams, sal epsom 0.5 restrains 1000 milliliters in water; PH value 6.5.
3 glucose 10 of filling a prescription restrain, peptone 10 grams, and dried silkworm chrysalis meal 10 grams, milk powder 12 grams, Sodium phosphate dibasic 1 gram, potassium primary phosphate 1.5 restrains 1000 milliliters in water; PH value 6.5.
4 Semen Maydis powder 30 of filling a prescription restrain, potassium primary phosphate 1 gram, SODIUMNITRATE 1 gram, 1000 milliliters in water; PH value 6.5.
In 500 milliliters of erlenmeyer flasks, 150 ~ 200 milliliters of dress nutrient solutions are pricked the envelope bottleneck with polypropylene film, and sterilization is 30 minutes under 1.3 kilograms of pressure.Every female kind is inoculated 5 ~ 6 bottles, and static cultivation placed on the reciprocating type shaking table after 24 hours, and under frequency 110 ~ 120 times/minute, 7 ~ 9 centimetres and 21 ~ 25 ℃ temperature condition of amplitude, shaking culture is standby after 4 ~ 6 days.If no shaking table can manually shake bottle, every day twice, each 30 minutes.Cultured liquid spawn has a large amount of mycelium pellets, and nutrient solution is limpid, and strong Chinese caterpillar fungus fragrance is arranged.If find the nutrient solution muddiness, should ban use of.
2, bacterial strain is cultivated
Method 1 living worm body artificial culture (herb plantation)
To the live body host, the live body host can select cocoon chrysalis, silkworm chrysalis, cicada nymph etc. for use with bacterial classification inoculation, preferred cocoon chrysalis or silkworm chrysalis.
The polypide inoculation is optional in order to following method: the one, and injection.In liquid spawn for inoculation usefulness, add the granulated glass sphere or the glass fragment of sterilization, fully shake, mycelium pellet is broken into the mycelia fragment.Draw bacterium liquid with asepsis injector, inject the intersegmental membrane of body surface sterilization silkworm chrysalis, each pupa injects 0.2～0.5 milliliter of bacterium liquid, puts into the Cans of sterilization immediately, cultivates with film seal.The 2nd, tissue block method.A long osculum of 0.2～0.3 centimetre is severed at pupal cell intersegmental membrane place after body surface sterilization with the sterilization scalper, and the bacterium piece of picking grain of rice size is filled in pupal cell wound place and wrapped then, puts into the sterilization Cans and seals cultivation.The 3rd, streak method.Dip in writing brush or cotton balls and to get bacterium liquid, brushing is coated with for several times repeatedly through disinfectant pupal cell surface, puts into the cultivation of sterilization Cans.
The different polypide inoculation tests of table 1
Annotate: going out the original hase time is from being inoculated into out original hase
Postvaccinal Cans are emitted in the clean culturing room.10 ~ 25 ℃ of room temperatures, relative humidity 60% ~ 85%, unglazed, keep cultivating under the airy condition.Infected silkworm chrysalis needs 10 ~ 15 days from being inoculated into death.Inoculate about 20 days, thrust appears in the pupal cell intersegmental membrane, and is granular as millet, faint yellow, i.e. fruit body primordium.Should provide the envrionment conditions of suitable fruit body development this moment, promotes sporophore growth to grow.After 10 days, sporophore is extended gradually, and will add forced ventilation this moment, increases illumination, keeps relative air humidity between 85% ~ 90%, 18 ~ 25 ℃ of room temperatures, and the bottleneck plastics film unclamped ventilation, prevent to go rotten.
Cultivated in 35 ~ 45 days the inoculation back, and the sporophore maturation is high 3 ~ 8 centimetres, is yellowish white, and uncork is gathered during outsourcing one deck white powder.Oven dry preservation below low temperature (30 ℃).
Method 2 generations material substratum is cultivated
Nutrient solution prescription; Add potato 20g, KH in each thousand ml water
2PO
43g, MgSO
41.5g, VB
10.1mg the pH value is 6-9; Also should add an amount of carbon source and organic nitrogen source, above-mentioned carbon source can be selected one or more in glucose, sucrose, the maltose for use, and organic nitrogen source can be selected one or more in peptone, dried silkworm chrysalis meal, the wheat bran for use.
Solid-based can have following prescription (proportioning is weight proportion):
1 wheat 98% of filling a prescription, peptone (pupa albumen) 2%, vitaminize B trace in addition.
2 rice 98% of filling a prescription, peptone (pupa albumen) 2%, vitaminize B trace in addition.
3 rice 68.5% of filling a prescription, dried silkworm chrysalis meal 10%, wheat 15%, sucrose 5%, peptone 1.5%; Vitaminize B trace in addition.
4 rice 50% of filling a prescription, wheat 35%, sucrose 2%, dried silkworm chrysalis meal 6%, sal epsom 1%., 0.1% vitamins B.
5 rice 90% of filling a prescription, wheat 10%.0.2% peptone, 0.1% potassium primary phosphate, 0.05% sal epsom.
6 rice 70% of filling a prescription, wheat 30%.Other adds the glucose of gross weight 1%, the peptone of 1% dried silkworm chrysalis meal 1%, 1%, 0.1% potassium primary phosphate, 0.05% sal epsom, vitamins B trace.
Bottling, sterilization are feeded with 500 milliliters of Cans, about 35 ~ 45 grams of every bottled siccative, and auxiliary material adds in the entry to be mixed well, and every bottle adds water 45 ~ 50 grams.Bottleneck seals with the polypropylene film bundling, and sterilization is 45 minutes under 0.12 MPa pressure, or keeps 4 ~ 6 hours under 100 ℃ of temperature normal pressures.Sterilization back substratum requires humidity unanimity up and down, and the space is arranged between substratum, can not be in the pasty state.
When inoculation medium is cooled to below 30 ℃, inoculation (gnotobiosis is planted inoculation with reference to mother) under aseptic condition.Every bottle graft kind solid spawn 10 grams, or 10 milliliters of liquid spawns.
With the wheat is main matrix, adds other ancillary components, is used to cultivate mycelium and sporophore, and the production cycle is 35 ~ 50 days, shifts to an earlier date about 10 days than other substratum, can gather 2 times.Except that sporophore, the mycelium of culture medium culturing also contains bioactive ingredients similar to sporophore and VITAMIN, but hyoscine or make raw materials of food processing.
After cultivating inoculation, place the environment of cleaning lucifuge secretly to cultivate culture bottle.Keep room temperature at 16 ~ 21 ℃, relative air humidity is controlled at about 65%, when treating that the charge level mycelia is covered with 90% left and right sides, indoorly should increase illumination, utilizes natural scattering light daytime, remains on about 200 luxs; Use the daylight lamp as light source night.Every day, light application time was no less than 10 hours, to promote the mycelia annesl and to stimulate original hase to form.When media surface and around have faint yellow pigment to occur, and when cima shape protuberance not of uniform size is arranged, be about to the omen that forms for sporophore.This moment, room temp should remain between 19 ~ 25 ℃, and improved relative air humidity to 85% ~ 90%.Cordyceps militaris (L.) Link. has tangible phototaxis, therefore, after sporophore forms, should according to circumstances suitably adjust the relative direction of culturing bottle and light source, or adjust indoor light source direction, makes to be subjected to light even, to guarantee the normal growth of sporophore, improves output and quality.To suitably ventilate during the sporophore growth, replenish fresh air.Whole incubation period is not thrown off and is sealed plastics film, can use the needle penetration aperture on plastics film, in order to gaseous interchange in the bottle.
Gathering and processing to grow tall when sporophore stops growing 5 ~ 8 centimetres the time, the visible white powder thing in surface, and should in time gather this moment, in case it is slow to gather, sporophore is withered.When gathering, sporophore is taken from substratum with the aseptic operation tweezers.After gathering, can grow second batch of sporophore after 10 ~ 20 days.
After sporophore is gathered,, in time dry sporophore root clean it up, down oven dry or freeze-drying of low temperature.Spraying eases back it then, puts in order and is bundled into wisp after straight, uses the weather-proof dressing preservation.
3, mountain Tai aweto and Cordyceps militaris comparative variety trial:
Bacterial classification: A, Cordyceps militaris; B, mountain Tai aweto.If dull and stereotyped cultivation, liquid culture, three kinds of training methods of generation material cultivation compare.The result of watch 2,3,4 shows: it is fast that the mountain Tai aweto bacterium sends out bacterium speed than Cordyceps militaris, and mycelia is dense, sturdy; The mountain Tai aweto mycelium is featheriness under the liquid culture condition, and it is velvet-like to introduce scarlet caterpiller fungus mycelia; Under identical liquid nutrient medium and culture condition, the mountain Tai aweto growth is fast; The mountain Tai aweto mycelium is sent out bacterium than other bacterial classifications in rice medium fast, goes out original hase early, and near or surpass Cordyceps militaris, mountain Tai aweto is than other kind strong stress resistances, and is less demanding to light, cultivation success ratio height.
Table 2 Cordyceps is adding growing state on the rich PDA substratum
Growing state in the table 3 Cordyceps liquid medium within
Table 4 Cordyceps growing state in rice medium
4, artificial culture mountain Tai aweto effective constituent assay
By table as can be seen, mountain Tai aweto artificial culture sporophore cordycepin analytical proof (table 5): mountain Tai aweto contains cordycepin, and content is 2 times of natural cordyceps above natural cordyceps; Uridine content is 0.160%, and adenosine content is 0.063%, and gland fat purine content is 0.016%, and uridylic content is 0.005%, all is higher than natural cordyceps.Illustrate that mountain Tai aweto can replace natural cordyceps; develop in a large number; this is for healthcare products and pharmaceuticals industry development; alleviate expensive day by day natural cordyceps imbalance between supply and demand; the protection wild resource; maintaining ecological balance and species diversity, preserving the ecological environment all has remarkable economical and social benefit.
Table 5 nucleosides content % (n=3)
Table 6 Determination of trace elements
Trace element analysis proves (table 6), and the contained micro-kind of mountain Tai aweto surpasses natural cordyceps; Mountain Tai aweto potassium element content is 13145mg/kg, and natural cordyceps potassium element content is 9396mg/kg, and both differences are 3749mg/kg.Potassium deficiency is one of pathogenic factor of diabetes, so this bacterial strain can consider to be developed as diabetics's special food additive or medicine.Mercury element content is 0.032mg/kg, only account for the regulation of edible mushrooms hygienic standard GB 7096-1996 in the national standard 0.2mg/kg 0.16.The Cordyceps militaris (L.) Link. of artificial culture contains Trace Mercury, and this is feeds utilized in feeding process with used silkworm chrysalis, water etc. suffers environmental pollution relevant, when selecting silkworm chrysalis, is to guarantee the high-quality important measures of artificial culture mountain Tai aweto with pollution-free silkworm chrysalis.
Analysis of amino acids result's (table 7), natural cordyceps contains 16 seed amino acids, and mountain Tai aweto contains 19 seed amino acids, and total content is that 22.584mg/100mg surpasses natural cordyceps (20.12mg/100mg); Mountain Tai aweto also contains special composition such as taurine, gamma amino butyric acid except that containing 8 necessary seed amino acids of human body, taurine, gamma amino butyric acid are broad-spectrum anti-inflammatory, analgesic drug product.Therefore mountain Tai aweto has more pharmaceutical use than natural cordyceps.
Table 7 aminoacids content is measured
The present invention compares than prior art and mainly has the following advantages and good effect:
1, the needed temperature range of mycelial growth is wider, all can grow in 10～30 ℃ of scopes; As shown in the figure, mountain Tai aweto bacterium Silk is 12 o'clock in the pH value, and the energy normal growth illustrates that this bacterial strain alkali resistance ability is strong, also can say this bacterial strain accommodative ability of environment By force;
2, mountain Tai aweto is sent out on bacterium speed, resistance, the fruiting body yield all above existing kind mycelia, easy artificial the cultivation, and 4 Still stronger through refrigerating in the term bacterial classification viability under ℃ condition.
Prove by mountain Tai aweto active ingredient assay that 3, the artificial Mount Taishan Cordyceps militaris of cultivating contains the characteristic chemical constituent one of Cordyceps sinensis Cordycepin, and content surpasses natural cordyceps. Other compositions such as adenine, adenosine, uridine, amino acid and some trace units Cellulose content all is higher than Cordyceps sinensis. Particularly the content of potassium element is higher than Cordyceps sinensis and other Cordyceps militaris, and potassium deficiency is diabetes One of pathogenic factor, so this bacterial strain can be used for the exploitation of diabetes patient's special food additive or medicine;
4, in the above-mentioned cultural method, during as the culture medium major ingredient, output is the highest and with short production cycle with wheat, be easy to reduce cost and Plant at northern area;
5, cicada nymph live body is cultivated this bacterial strain and can be gone out fructification (being cicada fungus), further research and utilization.
Claims (2)
1, a kind of method of cultivation of mountain Tai aweto, it is characterized in that: utilize preserving number for the mountain Tai aweto bacterial strain of CGMCC NO.1341 prepare bacterial classification then with this bacterial classification inoculation to solid medium, and the nutritive medium of quality such as adding, continue under optimum conditions to cultivate until growing the mountain Tai aweto sporophore; Described solid medium major ingredient is wheat, rice or their mixture;
Above-mentioned nutritive medium is made up of following component: add potato 20g, KH in each thousand ml water
2PO
43g, MgSO
41.5g, VB
10.1mg reach an amount of carbon source and organic nitrogen source, above-mentioned carbon source is selected one or more in glucose, sucrose, the maltose for use, organic nitrogen source is selected one or more in peptone, dried silkworm chrysalis meal, the wheat bran for use, and the pH value is 6-9.
2, according to the method for cultivation of the described mountain Tai aweto of claim 1, it is characterized in that: be controlled at 19-23 ℃ in the mycelium culture phase temperature, the dark cultivation, PH6-9 is cultured to mycelia and is covered with charge level 90% and begins to see light; Sporophore forms the stage, and temperature is controlled at 18-22 ℃, and atmospheric moisture is at 70-90%, and the cultivation that adds lustre to, until gathering.
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| CN102334678B (en) * | 2011-07-11 | 2013-03-06 | 范根生 | Method for producing cordyceps mycelium powder by utilizing sprouted soybeans |
| CN103858676B (en) * | 2014-03-24 | 2016-01-20 | 山东晨阳菌业有限公司 | The preparation method of a kind of Cordyceps militaris liquid spawn |
| CN104164367B (en) * | 2014-08-01 | 2017-02-08 | 沁阳市西向食用菌研究所 | Dried silkworm cordyceps militaris and culture method thereof |
| CN104871830B (en) * | 2015-06-15 | 2017-03-15 | 鲁东大学 | With bean worm as the method for carrier-planting Cordyceps militaris (L.) Link. |
| CN105746172A (en) * | 2016-02-23 | 2016-07-13 | 张家港顺泰元生物科技有限公司 | Selenium-enriched cordyceps culture method |
| 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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| CN1092105A (en) * | 1994-02-26 | 1994-09-14 | 苏州蚕桑专科学校 | The high-yield quick artificial culturing method of Cordyceps militaris (L.) Link. |
| CN1200403A (en) * | 1997-05-07 | 1998-12-02 | 李春燕 | High yield cultivating method for cordyceps |
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