CN101215527A - Method for cultivating silkworm chrysalis Cordyceps sinensis - Google Patents
Method for cultivating silkworm chrysalis Cordyceps sinensis Download PDFInfo
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- CN101215527A CN101215527A CNA2008100138317A CN200810013831A CN101215527A CN 101215527 A CN101215527 A CN 101215527A CN A2008100138317 A CNA2008100138317 A CN A2008100138317A CN 200810013831 A CN200810013831 A CN 200810013831A CN 101215527 A CN101215527 A CN 101215527A
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Abstract
A cultivation method for a silkworm chrysalis aweto is that wild and natural aweto strain spore originated from tibetan plateau is inoculated to organism silkworm chrysalis and is cultivated into the silkworm chrysalis aweto artificially, which comprises the following procedures: (1) screening pupa cordyceps fungus strains; (2) making silkworm chrysalis organic culture medium; (3) making spore suspension; (4) preparing host; (5) infecting pupa cordyceps fungus; (6) protecting infected pupas; (7) cultivating stroma; (8) culturing stroma directionally; (9) collecting and keeping the silkworm chrysalis aweto. The invention employs the silkworm chrysalis as the host, inoculates the pupa cordyceps fungus artificially, simulates the natural ecologic environment of pupa cordyceps fungus and cultivates artificially. The inoculating success ratio and the inoculated rigidity ratio are more than 95%, the growth ratio of fruiting body is more than 90%, the culture time is about 35-45 days from inoculating to mature of fruiting body, the chemical composition has the same property as the aweto, wherein cordycepin and cordycepic acid of the active composition are 2 to 3 times as much as the aweto.
Description
Technical field
The invention belongs to the fungus growing technique field, the method of cultivation that relates to a kind of silkworm chrysalis Cordyceps sinensis relates in particular to the method for cultivation that wild natural cordyceps bacterial strain spore inoculating artificial culture to organic living silkworm chrysalises that a kind of usefulness originates in the Qinghai-Tibet Platean forms silkworm chrysalis Cordyceps sinensis.
Background technology
Cordyceps sinensis and genseng, pilose antler are the same, are one of " Chinese medicine three are big precious ".Along with the raising of people's living standard with to the attention of health, Cordyceps sinensis and auxiliary product thereof become one and develop industry rapidly.Cordyceps sinensis is classified national second class protection species as, and various places government also appearance policy taboo is dug, limit and dig, but not excavating of Cordyceps sinensis is curbed.Cordyceps sinensis is a kind of rare Chinese medicine of China, because it needs special growth conditions, vegetative period is longer, thereby very rare in occurring in nature quantity, along with the change of envrionment conditions, output is downtrending gradually, and social demand is increasing, so very famous and precious, price is also high, reaches in the market about 40,000 yuan every kilogram.
Silkworm chrysalis Cordyceps sinensis (Cordyceps militaris) compares with Cordyceps sinensis (Cordyceps): Chinese caterpillar fungus is famous and precious Chinese medicinal materials, has very high tonic and is worth and pharmaceutical use.Kind China Chinese caterpillar fungus has kind more than 200, and what wherein using value was the highest is to originate in the Cordyceps sinensis in Tibet and the Cordyceps militaris (L.) Link. that originates in the Changbai Mountain.The two belongs to Ascomycetes Hypocreales Clavicipitaceae Cordyceps in classification of organisms.Cordyceps sinensis originates from natural, is that Cordyceps fungus parasitizes the bat moth larvae and forms.Up to the present, artificial large-scale planting not success as yet.Silkworm chrysalis Cordyceps sinensis is that Cordyceps militaris (L.) Link. fungus is inoculated in the silkworm chrysalis body, forms through artificial culture.The cultivation of silkworm chrysalis Cordyceps sinensis has at present formed mass-producing.Silkworm chrysalis Cordyceps sinensis and Cordyceps sinensis medicine change into branch and pharmacological function is all very close.Studies show that, inorganic elements kind basically identical in silkworm chrysalis Cordyceps sinensis and the Cordyceps sinensis, the content of the two selenium is all higher, and is higher 3.3 times and 5.8 times than selenium-rich anticancer Chinese medicine astragalus respectively.Silkworm chrysalis Cordyceps sinensis contains identical ucleosides composition with Cordyceps sinensis, has only the cordycepin content silkworm chrysalis Cordyceps sinensis to exceed much than Cordyceps sinensis, and the cordycepic acid content silkworm chrysalis Cordyceps sinensis is lower slightly, and the two is suitable for Cordyceps polysaccharide content.Silkworm chrysalis Cordyceps sinensis and Cordyceps sinensis pharmacodynamic feature basically identical, all have antitumor, antiviral, antifatigue, male hormone sample, regulate effect such as lipid metabolism, immunity system there are promotion or dual regulation, can protect the liver, protect liver, resistance of hepatitis B and antibechic, eliminate the phlegm, relieving asthma, spasmolysis, can treat heart disorder, chronic nephritis and renal failure.
Silkworm chrysalis Cordyceps sinensis and Cordyceps sinensis all are the combinations of insect and fungi, and the synergy of worm and grass makes them have the magical effect of tonifying both YIN and YANG.The Cordyceps sinensis wild resource is in great shortage now, and price is constantly soaring, and silkworm chrysalis Cordyceps sinensis can be used as a new health care treasure fully and is widely used.
Cordyceps sinensis is valuable natural resources of Chinese medicinal materials; the main producing region is district, provinces such as Tibet, Qinghai, Sichuan, Yunnan, Gansu; pay attention to its protection and exploitation from strategic height; strengthen the fundamental research dynamics; seek to promote the new way of its existence procreation by human intervention; to satisfy ever-increasing health care and medicinal demand, become extremely urgent task.At present; this precious wild resource atrophy of Cordyceps sinensis, resource resources are unclear, regularity of occurrence and development not clear, accelerate research abroad; and silkworm chrysalis Cordyceps sinensis is the host with the silkworm pupa; the artificial inoculation Cordyceps militaris (L.) Link. fungus; simulation Cordyceps militaris (L.) Link. natural ecological environment, the success of artificial organic cultivation is strong protection and the exploitation to Cordyceps sinensis; also be the promotion that silkworm is already developed, especially to the expansion of plate cocoon industry.
Zhengjiang City's Dantu District silkworm and mulberry stand in the help of University Of Suzhou and instruct down, utilize silkworm pupa by inoculating Cordyceps at the beginning of 2005 6 months, under conditions such as certain temperature, humidity, illumination, through surplus 40 days take pains to foster, have successfully cultivated silkworm chrysalis Cordyceps sinensis.
2005, the Cordyceps militaris (L.) Link. y3 bacterial strain of professor Jiang Xiaolu of Department of Food Engineering of Qingdao Marine University screening, system is by getting with tissue isolation from the wild cordyceps militaris of Jilin hada, tieback experiment through the host, successfully grow the stroma identical (in full accord) through microscopy ascoma and thecaspore and natural pupa grass with wild-type, separate again obtain of the same race purebred.Compare through the Chemical Composition analysis to tame Cordyceps militaris (L.) Link. and wild cordyceps, the result is that the various Chemical Composition and the content of tame Cordyceps militaris (L.) Link. and wild cordyceps is basic identical, and some effective component content is higher than wild cordyceps.
Application number is 93102809.4, denomination of invention for " patent of a kind of " method of using baked silkworm chrysalis for artificially breeding Chinese caterpillar fungus ", its method be with inoculation on slant medium, carry out the cultivation in mycelia stage; Using baked silkworm chrysalis is clean and dry with flushing with clean water; The processing of on using baked silkworm chrysalis, punchinging; With cleaning the using baked silkworm chrysalis bottling of punchinging, add suitable quantity of water, carry out autoclave sterilization; A little mycelia piece of picking is put on the pupal cell of bottle in inoculation tank, the envelope flask culture, and temperature is 15 ℃～30 ℃, natural lighting can grow Cordyceps sporophore in about two months.Using baked silkworm chrysalis helps a large amount of purchases and long-term storage, and the using baked silkworm chrysalis after punchinging can improve the success ratio of artificially breeding Chinese caterpillar fungus through autoclave sterilization.And this moment usefulness silkworm chrysalis be mostly purchase come the silkworm chrysalis produced behind traditional cocoon filature, contain pharmaceutical chemicalss such as highly basic, be not organic silkworm chrysalis, do the host with this and can't produce organic silkworm chrysalis Cordyceps sinensis.
Summary of the invention
The purpose of this invention is to provide the method for cultivation that wild natural cordyceps bacterial strain spore inoculating artificial culture to organic living silkworm chrysalises that a kind of usefulness originates in the Qinghai-Tibet Platean forms silkworm chrysalis Cordyceps sinensis.
For achieving the above object, the method for cultivation of a kind of silkworm chrysalis Cordyceps sinensis of the present invention is characterized in that, may further comprise the steps:
(1) screening of Cordyceps militaris (L.) Link. fungus bacterial strain
To originate in the wild fresh Cordyceps strain spore of Qinghai-Tibet Platean, the isolating method of application organizes is obtained the pure bacterial strain of Cordyceps sinensis; Utilize this bacterial strain to carry out rejuvenation, and then filter out the Cordyceps militaris (L.) Link. fungus bacterial strain that the silkworm chrysalis virulence by force, is easily formed spore by the silkworm chrysalis body of living;
(2) the organic substratum of silkworm chrysalis is made
Live body protein 10 0 gram, 200 milliliters in dense rice soup, vitaminB10 milligram/1000 grams, glucose 25 grams, potassium primary phosphate 2 grams with living silkworm chrysalises or the cured matter of live body silkworm removal epidermis, put into 1000 milliliters beaker, it is miscible to add 20～50 milliliters in water and heated and stirred, stop heating behind the complete miscibility, the packing test tube, jump a queue, cool off, promptly make the organic substratum of silkworm chrysalis of semi-liquid-like;
(3) making of spore suspension liquid
On the organic substratum of silkworm chrysalis that the Cordyceps militaris (L.) Link. fungus inoculation that (1) step was cultivated was made in (2) step, put under 20 ℃ of temperature and cultivate, after waiting conidium to occur, a picking part is placed on that to make the spore suspension liquid in the sterilized water stand-by;
(4) host prepares
Live body silkworm, pupa or traditional cocoon are gone cocoon shell live body pupa, silkworm, about 5～10 minutes or amino acid iodine sterilization about 1 minute, do the host with 75% alcohol disinfecting;
(5) infection of Cordyceps militaris (L.) Link. fungus
In inoculation tank, dip in the spore suspension liquid with the inoculating needle needle point and thrust above-mentioned silkworm, pupal cell joint, by 0.2～0.3 milliliter/only inject bacterium liquid;
(6) protection of infection pupal cell
Silkworm after the microbiological contamination, pupal cell are divided at the bottom of the bottle or on silkworm plaque or the bamboo mat, protect to pupal cell stiff under the room temperature;
(7) cultivation of stroma
The natural ecological environment of simulation Cordyceps militaris (L.) Link., cultivating stiff pupal cell grows in seat: method is to cover one deck porous material on stiff pupal cell, as broken sponge, thin cinder, gac, place under 10～30 ℃ of alternating temperatures, relative air humidity is that 85～95% natural lighting envrionment conditions is cultivated;
(8) post-directed training of stroma
For making the stroma that bears long hard, can adopt post-directed training: method is, with stiff pupa head upwards or pupal cell keep flat, insert or put into the material of porous thermal insulating such as the coal briquette slag after sponge or the burning, be placed on cultivation aforementioned temperature, humidity and illumination condition under;
(9) collection of silkworm chrysalis Cordyceps sinensis is preserved
After the stroma maturation, dig out cleaning, carry out the body surface sterilization, be placed on again under 60 ℃ of environment and dried 1～5 hour, put shady and cool dry place sealing preservation with edible ethanol.
Silkworm chrysalis Cordyceps sinensis of the present invention is the host with the silkworm pupa, the artificial inoculation Cordyceps militaris (L.) Link. fungus, and simulation Cordyceps militaris (L.) Link. natural ecological environment, artificial culture is succeedd.Success ratio of inoculation, postvaccinal rigid rate reach more than 95%, and the rate of growing of sporophore reaches more than 90%, from being inoculated into ripe about 35～45 days of sporophore.The silkworm chrysalis Cordyceps sinensis of property of medicine analysis revealed artificial culture does not have the difference of essence matter with Cordyceps sinensis on Chemical Composition, active ingredient cordycepin and cordycepic acid are 2～3 times of Cordyceps sinensis.Toxicity test shows that silkworm chrysalis Cordyceps sinensis belongs to nontoxic substantially level, and no teratogenesis, mutagenesis positive effect do not have influence to the mouse Bone marrow cells micronucleus occurrence rate.Pharmacological testing shows at aspect silkworm chrysalis Cordyceps sinensis such as anti-hypoxia, calmness, anti-inflammatory and is similar to Cordyceps sinensis, then is better than Cordyceps sinensis at anti-tumor aspect.Cultivate silkworm chrysalis Cordyceps sinensis as the substitute or the new protective foods of Cordyceps sinensis or further therefrom extract effective ingredient and prepare new drug vast market will be arranged.
The silkworm chrysalis Cordyceps sinensis that method of cultivation of the present invention is produced is the entomogenous fungi combination that forms with the wild natural cordyceps bacterial strain spore inoculating that originates in Qinghai-Tibet Platean artificial culture to organic living silkworm chrysalises.Chemical examination detects through domestic authoritative analyzing and testing mechanism: mineral elements such as the contained cordycepin of silkworm chrysalis Cordyceps sinensis, N.F,USP MANNITOL, SOD and phosphorus, potassium, zinc, selenium all meet or exceed natural cordyceps.Bacterial classification is pure, nothing is degenerated, effective component content is stable.Silkworm chrysalis Cordyceps sinensis has antifatigue, anti-ageing, antitumor, antiviral, raise immunity, irregular pulse, accent lipid metabolism, antitussive and antiasthmatic, the spasmolysis of eliminating the phlegm there is special effect, protect the liver and protect anti-hepatitis b, ten big functions such as treatment chronic nephritis, renal failure and sexual function improvement, acute, subacute toxicity test is nontoxic, takes safety.
Silkworm chrysalis Cordyceps sinensis is famous and precious medicinal material, be again to have a very health care tonic of high nutritive value, can with chicken and duck with stewing, the effect that have tonifying Qi, replenishing essence, improves the health has the merit of mending the stomach establishing-Yang with the peeled shrimp Baoshang.Steeping in wine infuses tea drinks improving immunocompetence, and body-building is anti-ageing.
Significantly reduce and can't carry out under commercialization artificial culture, the market situation that there is a serious shortage in the supply at the Cordyceps sinensis wild resource, silkworm chrysalis Cordyceps sinensis has become the emphasis of domestic and international every profession and trade (subject) research and development.The success of artificial culture silkworm chrysalis Cordyceps sinensis has positive effect to the development of sericulture industry and the lifting of benefit.Because silkworm chrysalis Cordyceps sinensis is very strict to the requirement of temperature, humidity, illumination, special facilities and equipment (must adopt sterilization, temperature control and control facilities and equipments such as wet) also will be arranged, therefore, the cultivation difficulty is very big, cultivates basis and empirical people and is difficult to successfully.
Embodiment
The invention will be further described below in conjunction with specific embodiment:
Living silkworm chrysalises among the present invention, live body silkworm can be adopted byproduct-living silkworm chrysalises, live body silkworm after plate cocoon (" a kind of plate cocoon production technology " of my invention, the patent No. is: the ZL 02135193.7) production.
Silkworm chrysalis Cordyceps sinensis of the present invention is cultivated technical scheme:
During the artificial culture silkworm chrysalis Cordyceps sinensis, earlier Cordyceps militaris (L.) Link. fungus is inoculated on the substratum, forms a large amount of conidiums, the picking conidium is made the spore suspension liquid with sterilized water again, and being inoculated in the host is on the pupal cell, and the protection pupal cell is until stiff.Then stiff silkworm chrysalis body is placed on simulation Cordyceps militaris (L.) Link. natural ecological condition and cultivates (ecological factors such as the weather of Cordyceps sinensis living environment, landform, soil, vegetation and other associated biomolecules) down, just from silkworm chrysalis, grow with the same stroma of wild silkworm chrysalis.Technical scheme is as follows:
1. the screening of Cordyceps militaris (L.) Link. fungus bacterial strain
To originate in the wild fresh Cordyceps strain spore of Qinghai-Tibet Platean, the isolating method of application organizes is obtained the pure bacterial strain of Cordyceps sinensis.Utilize this bacterial strain to carry out rejuvenation, and then filter out the Cordyceps militaris (L.) Link. fungus bacterial strain that the silkworm chrysalis virulence by force, is easily formed spore by the silkworm chrysalis body of living.
2. the organic substratum of silkworm chrysalis is made
Plate cocoon is produced back byproduct-living silkworm chrysalises, live body silkworm (or traditional cocoon goes the cocoon shell to make kind use living silkworm chrysalises) remove that live body protein 10 0 gram, 200 milliliters in dense rice soup, vitaminB10 milligram/1000 grams, the glucose 25 of the cured matter of epidermis restrain, potassium primary phosphate 2 restrains, put into 1000 milliliters beaker, it is miscible to add 20～50 milliliters in water and heated and stirred, stop heating behind the complete miscibility, the packing test tube, jump a queue, the organic substratum of silkworm chrysalis (semiliquid) is promptly made in cooling.Its advantage is that the Cordyceps mycelium utmost point of being turned out is beneficial to infection live body silkworm, pupa.
3. the making of spore suspension liquid
Inoculation is put under 20 ℃ of left and right sides temperature and is cultivated on " the organic substratum of silkworm chrysalis ", and after waiting conidium to occur, a picking part is placed on that to make the spore suspension liquid in the sterilized water stand-by.
4. the host prepares
With plate cocoon produce back byproduct-live body silkworm, pupa or traditional cocoon go cocoon shell live body pupa, silkworm with 75% alcohol disinfecting about 5～10 minutes or amino acid iodine sterilized about 1 minute, do the host.
5. the infection of Cordyceps militaris (L.) Link. fungus
In inoculation tank, dip in the spore suspension liquid with the inoculating needle needle point and thrust above-mentioned silkworm, pupal cell joint, and inject bacterium liquid (0.2～0.3 milliliter /), infecting success ratio can be more than 95%.
6. infect the protection of pupal cell
Silkworm after the microbiological contamination, pupal cell are divided at the bottom of the bottle or on silkworm plaque or the bamboo mat, protect to pupal cell stiff under the room temperature.
7. the cultivation of stroma
The natural ecological environment of simulation Cordyceps militaris (L.) Link. is cultivated stiff pupal cell and is grown in seat.Method is to cover one deck porous material on stiff pupal cell, as broken sponge, thin cinder, gac etc., places under 10～30 ℃ of alternating temperatures, relative air humidity is that 85～95% natural lighting envrionment conditions is cultivated.
8. the post-directed training of stroma
For making the stroma that bears long hard, can adopt post-directed training.Method is, with stiff pupa head upwards or pupal cell keep flat, insert or put into the material of porous thermal insulating such as the coal briquette slag after sponge or the burning, be placed on cultivation aforementioned humiture and illumination condition under.
9. the collection of silkworm chrysalis Cordyceps sinensis is preserved
After the stroma maturation, dig out cleaning, carry out the body surface sterilization, be placed on again under 60 ℃ of environment and dried 1～5 hour, put shady and cool dry place sealing preservation with edible ethanol.
Cordyceps militaris (L.) Link. by above-mentioned approach is cultivated needs 35 days～45 days approximately from being inoculated into the stroma maturation, cultivates area for every square metre and can gather in the crops 200 grams～300 grams.According to detection, the Cordyceps militaris (L.) Link. of artificial culture contains a large amount of cordycepic acids, cordycepin, amino acid, trace quantity mineral substance element and alkaloid, can mend marrow, antitumor, calm anti-inflammatory, hemostasis and phlegm, diseases such as ephritis, hepatitis, asthma, neurasthenia are had obvious curative effects.
Embodiment one:
With 4～5 age silkworm or living silkworm chrysalises be organic silkworm chrysalis Cordyceps sinensis method of cultivation of major ingredient.
To originate in the wild fresh Cordyceps sinensis of Qinghai-Tibet Platean, the isolating method of application organizes is obtained the pure bacterial strain of Chinese caterpillar fungus.Utilize this bacterial strain to carry out rejuvenation, and then filter out the bacterial strain that the silkworm chrysalis virulence by force, is easily formed spore, carry out organic silkworm chrysalis Cordyceps sinensis cultivation again by the pupal cell of living.Technical scheme is as follows:
(1) screening of Cordyceps militaris (L.) Link. fungus bacterial strain
1, bacterial classification is made
(1) breeding.Technological method: choose high yield, high-quality, precocity, originate in Qinghai-Tibet wild fresh Cordyceps sporophore, alcohol with 75% or 10% amino acid iodine solution carry out surface sterilization, fall surperficial soup with sterile water wash, place when filling the synthetic medium surface and asterism shape entomogenous fungi occurring and fall, the single or multiple bacterium colonies of picking are put the test tube slant culture medium culturing in inoculation tank.Treat to purify again after the Cordyceps militaris (L.) Link. mycelia is covered with the inclined-plane.The female kind of the spore that obtains must be chosen the high-quality Cordyceps sporophore and carry out the primary structure separation again after go out careless comparison test, can be used for commentaries on classics through the screening rear and expands plantation.
(2) species test.After mother planted enlarged culturing, put on the rice medium, cultivate 20-30d down in 18 ℃-25 ℃, observe growing state, if see that bacterium or certain kind of berries fungi pollution are arranged, the female kind of reply is further purified; If no living contaminants can continue to cultivate, promptly there is orange red sporophore to produce after one month, illustrate that female kind is reliable.
(3) secondary seed is cultivated.Liquid nutrient medium is sub-packed in the 500ml triangular flask, after the sterilization in 15-30 minute, uses the inoculation clasp joint under aseptic condition in triangular flask mother's kind, every inclined-plane can connect 8～20 bottles.Inoculation is placed under 18～22 ℃ of conditions, shakes about kind of cultivation 6d, promptly can be used for cultivar behind the even bead to be formed.It should be noted that: mix if nutrient solution becomes, two kinds of possibilities are arranged, the one, bacterial contamination be should give superseded; The 2nd, because due to the undue thickness of mycelial growth.Suffering can be carried out microscopy to judge.
(4) substratum batching.Culture material: make major ingredient (70%～80%) with rice flour, Semen Maydis powder etc., other adds the dried silkworm chrysalis meal of 20%-30% or milk powder to improve the quality of Cordyccps-militaris-(L.)-link. Sporophore.Nutritive medium: 200 milliliters in dense rice soup, vitaminB10 milligram/1000 grams, glucose 25 grams, potassium primary phosphate 2 grams.Culture material and nutritive medium are mixed thoroughly with 1: 1 ratio, put into 1000 milliliters beaker, it is miscible to add 20～50 milliliters in water and heated and stirred, stops heating behind the complete miscibility, and pH value is controlled at 5.5-7.0.Bottling then, autoclaving (or the full-automatic heavy vertical steam sterilizing pot of HL-380, Hung Lin Medical Instruments Co., Ltd. produces, and is warming up to 100～125 ℃, keeps 5～50 minutes), inoculate.
(5) mycelium is cultivated.Postvaccinal material bottle should place the environment of cleaning, lucifuge to cultivate, atmospheric moisture 60%～70%.Starting stage be to reduce living contaminants, and room temp should remain between 15 ℃-18 ℃, treat that charge level is covered with Cordyceps mycelium after, temperature is mentioned 20 ℃-25 ℃, continue about 15d, mycelia can be had thorough grasp culture material, finishes and nourishes and grows.
(6) sporophore is cultivated.After the mycelium maturation, transfer orange gradually to by white, at this moment, indoorly should increase illumination, utilize natural scattering light daytime, keep 150～300 luxs, can utilize night fluorescent lamp to make power supply, should be no less than 10h illumination every day, to impel the mycelium annesl and to stimulate original hase to form.Treat the charge level projection, and when forming the granular original hase of millet, suitably ventilate, replenish fresh air, keep 18 ℃-22 ℃ of room temps, and improve relative air humidity to 80%-85%.Too big as humidity, easily bring out aerial hyphae in the base, unfavorable to sporophore growth; Humidity is too little, easily makes substratum do sth. in advance dehydration and influences output.Cordyceps militaris (L.) Link. has stronger phototaxis, therefore after sporophore forms, should according to circumstances adapt to the relative direction of adjusting culturing bottle and light source, or adjust indoor light source direction, with the normal growth form of assurance sporophore, thereby improves output.Treat not regrowth of sporophore, when many thorns appears in its top, show maturation.When gathering, clamp out sporophore, and then add a little nutritive medium, bandage bottleneck again, continue to cultivate, about 20d, can grow second batch of stroma again with aseptic nipper.Sporophore under gathering dry or oven drying at low temperature after sell.Wouldn't sell, answer good seal to put cryodrying place and store.
Make spore suspension with Cordyceps militaris (L.) Link. fungus and make bacterial classification.
2, silkworm (pupa) Chinese caterpillar fungus organic farming
(1) inoculation.Make object of inoculation with silkworm, select 4-5 silkworm in age or plate cocoon to produce the pupa alive that the back produces, the decortication back is inoculated with 108/milliliter spore suspension liquid spray silkworm body or wound, put under the 20-22 ℃ of condition and cultivate, after 3～5 days, silkworm body (pupal cell) ossifys gradually, and body surface intersegmental membrane, valve director go out white aerial hyphae, on the aerial hyphae give birth to circular conidium.The silkworm body tarnishes, and brown roundlet spot appears in body surface, and the pupal cell melanism bears the aerial hyphae conidium after stiff gradually.
(2) cultivation of silkworm chrysalis Cordyceps sinensis sporophore.Before silkworm body, pupal cell sclerosis back and mycelia grew, the natural ecological environment of simulation Cordyceps militaris (L.) Link. growth (temperature 20-22 ℃, atmospheric moisture is more than 90%, the indoor natural light line) was protected.The hypha body of safran, sesame seed size appears in the pupal cell surface after 3～5 days, and after 3-5 days, hypha body changes over to and is the thorn-like pimple, along with the elongation gradually of thorn-like pimple, and becomes high 2-8 centimetre, thick 0.2-0.8 centimetre sporophore.Whole growth process is 25-35 days, and each polypide can a long 1-3 sporophore.Any one position of polypide all can grow sporophore.The rate of growing of sporophore reaches more than 95%.The bright pupa of every double centner can be done about 24 kilograms of Cordyceps militaris (L.) Link..
(3) proterties of silkworm chrysalis Cordyceps sinensis sporophore.The Cordyceps sporophore that grows from silkworm chrysalis, its outward appearance and wild Chinese caterpillar fungus are just the same.Orange, the thick shape of sporophore, long 3-8 centimetre, wherein stroma head length 1-2 centimetre, the long 2-6 of stroma handle centimetre, the thick 0.2-0.8 of sporophore centimetre, generally do not have bifurcated, mostly be cylindric, minority flats.The stroma head expands slightly, and there is papilla on the surface.It is linear that thecaspore becomes, slightly the extruding radial ejection.Transverse section shows: there is the fuse of a similar Radix Dauci Sativae core stroma head inside, stroma handle hollow.
3, gather
Gather after the stroma maturation, put shady and cool dry place sealing and preserve.
4, the toxicity test of silkworm chrysalis Cordyceps sinensis.Get 10 of 17-20 gram Kunming kind small white mouses, male and female half and half are irritated stomach by 3000 mg/kg once a day, connect and irritate 3 days, and semi-invariant is 9000 mg/kg, observes 7 days, does not see death and other abnormal responses.Prompting can clinically be used for human body.Toxicity test shows that silkworm chrysalis Cordyceps sinensis belongs to nontoxic substantially level, and no teratogenesis, mutagenesis positive effect do not have influence to the mouse Bone marrow cells micronucleus occurrence rate.
Promptly finished the bacterial strain screening that the silkworm chrysalis virulence by force, is easily formed spore by above technical scheme.
(2) the organic substratum of silkworm chrysalis is made
Plate cocoon is produced back byproduct-living silkworm chrysalises, live body silkworm or traditional cocoon goes the cocoon shell to make kind of live body protein 10 0 gram with the cured matter of living silkworm chrysalises removal epidermis, 200 milliliters in dense rice soup, vitaminB10 milligram/1000 grams, glucose 25 grams, potassium primary phosphate 2 grams, put into 1000 milliliters beaker, it is miscible to add 20～50 milliliters in water and heated and stirred, stop heating behind the complete miscibility, the packing test tube, jump a queue, the organic substratum of silkworm chrysalis (semiliquid) is promptly made in cooling.Its advantage is that the Cordyceps mycelium utmost point of being turned out is beneficial to infection live body silkworm, pupa.
(3) making of spore suspension liquid
Inoculation after the screening on " the organic substratum of silkworm chrysalis ", is put under 20 ℃ of left and right sides temperature and cultivated, and after waiting conidium to occur, a picking part is placed on that to make the spore suspension liquid in the sterilized water stand-by.
(4) host prepares
With plate cocoon produce back byproduct-live body silkworm, pupa or traditional cocoon go cocoon shell live body pupa, silkworm with 75% alcohol disinfecting about 5～10 minutes or amino acid iodine sterilized about 1 minute, do the host.
(5) infection of Cordyceps militaris (L.) Link. fungus
In inoculation tank, dip in the spore suspension liquid with the inoculating needle needle point and thrust above-mentioned silkworm, pupal cell joint, and inject bacterium liquid (0.2～0.3 milliliter /), infecting success ratio can be more than 95%.
(6) protection of infection pupal cell
Silkworm after the microbiological contamination, pupal cell are divided at the bottom of the bottle or on silkworm plaque or the bamboo mat, protect to pupal cell stiff under the room temperature.
(7) cultivation of stroma
The natural ecological environment of simulation Cordyceps militaris (L.) Link. is cultivated stiff pupal cell and is grown in seat.Method is to cover one deck porous material on stiff pupal cell, as broken sponge, thin cinder, gac etc., places under 10～30 ℃ of alternating temperatures, relative air humidity is that 85～95% natural lighting envrionment conditions is cultivated.
(8) post-directed training of stroma
For making the stroma that bears long hard, can adopt post-directed training.Method is with stiff pupa head upwards, in the coal briquette slag after the material of insertion porous thermal insulating such as sponge or the burning, to be placed under aforementioned humiture and the illumination condition and to cultivate.
(9) collection of silkworm chrysalis Cordyceps sinensis is preserved
After the stroma maturation, dig out cleaning, carry out the body surface sterilization, be placed on again under 60 ℃ of environment and dried 1～5 hour, put shady and cool dry place sealing preservation with edible ethanol.
Embodiment two:
With 4～5 age silkworm or living silkworm chrysalises be simple and easy organic silkworm chrysalis Cordyceps sinensis cultivating method of major ingredient.
(1) strain separating is cultivated
1, breeding: specific practice is, choose high yield, high-quality, precocious Cordyccps-militaris-(L.)-link. Sporophore, alcohol with 75% carries out surface sterilization, use aseptic water washing again, place then that to fill synthetic medium container top unsettled, leave standstill cultivation under 21 ℃～23 ℃, when treating that asterism shape Chinese caterpillar fungus bacterium colony appears in media surface, the single or multiple bacterium colonies of picking are put the test tube slant culture medium culturing in inoculation tank.Treat to purify again after the Cordyceps militaris (L.) Link. mycelia is covered with the inclined-plane.The female kind of the spore that obtains must be chosen the high-quality Cordyceps sporophore and carry out the primary structure separation again after go out careless comparison test, can be used for commentaries on classics through the screening rear and expands cultivar.
2, species test: after mother planted enlarged culturing, put on the rice medium, cultivate 20～30d down in 18 ℃～20 ℃, observe growing state.If see that bacterium or mould contamination are arranged, the female kind of reply is further purified; If no living contaminants can continue to cultivate, promptly there is orange red sporophore to produce after one month, illustrate that female kind is reliable.
3, secondary seed is cultivated: liquid nutrient medium is sub-packed in the 500ml triangular flask, and every bottle adds nutrient solution 200ml, adds one deck kraft paper with 12 layers of gauze and seals.After the sterilization in 13～30 minutes, mother is planted to go in the triangular flask with the inoculation clasp joint under aseptic condition, every inclined-plane can connect more than 10 bottles.Cultivate about 5d in 20 ℃ of shaking tables the inoculation back, promptly can be used for cultivar behind the even bead to be formed.It should be noted that: mix if nutrient solution becomes, two kinds of possibilities are arranged, the one, bacterial contamination be should give superseded; The 2nd, because due to the undue thickness of mycelial growth.If be difficult to judge, can carry out microscopy.
(2) cultivation management technology
1, substratum preparation: make major ingredient with rice, corn etc., other adds the quality that 10%～25% dried silkworm chrysalis meal or milk powder mother improve Cordyccps-militaris-(L.)-link. Sporophore.Culture material and nutritive medium are mixed thoroughly with 1: 1 ratio, and the pH value is grasped 5.5～7.0.Bottling routinely, autoclaving (or the full-automatic heavy vertical steam sterilizing pot of HL-380, Hung Lin Medical Instruments Co., Ltd. produces, and is warming up to 100～125 ℃, keeps 5～50 minutes), inoculate.
2, mycelium is cultivated: postvaccinal material bottle should place the environment of cleaning, lucifuge to cultivate, protection atmospheric moisture 60%.Starting stage be to reduce living contaminants, and indoor greenhouse should remain between 15 ℃～18 ℃, treat that charge level is covered with Cordyceps mycelium after, temperature is mentioned 20 ℃～23 ℃, continue about 15d, mycelia can be had thorough grasp culture material, finishes and nourishes and grows.
3, sporophore is cultivated: after the mycelium maturation, transfer orange gradually to by white, at this moment, indoor should to increase illumination white, utilizes natural scattering light daytime, keeps 200 luxs, can utilize the daylight lamp as light source night, and should be no less than 10h illumination every day, to impel the mycelium annesl and to stimulate original hase to form.Treat the charge level projection, and when forming the granular original hase of millet, suitably ventilate, replenish fresh air, 18 ℃～22 ℃ of protection room temps, and improve air relative temperature to 80%～85%.Too big as humidity, easily make substratum do sth. in advance dehydration and influence output.Cordyceps militaris (L.) Link. has stronger 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, with the normal growth form of assurance sporophore, thereby improves output.Treat not regrowth of sporophore, when many spinules appears in its top, show ripe.
4, gather: clamp out sporophore with aseptic nipper, and then add a little nutritive medium, bandage bottleneck again, continue to cultivate, about 20d, can grow second batch of stroma again.Sporophore under gathering dry or oven drying at low temperature after sell.Wouldn't sell, answer good seal to put cryodrying place and store.
Embodiment three:
With 4～5 age silkworm or living silkworm chrysalises be organic silkworm chrysalis Cordyceps sinensis cultivating method of major ingredient.
(1) starting material requirement
1, selects bacterial classification.Select that mycelia is pure white for use, adaptability is strong, see annesl behind the light, go out the fast growing and high yield high-quality silkworm chrysalis Cordyceps sinensis bacterial classification that grass is fast, proterties is stable, be cultivate successfully and key efficiently.First grower should choose the many solid original seeds of bacterial classification amount, has cultivation experience person can buy slant tube kind or liquid spawn.
2, select major ingredient.Select that fresh nothing is gone mouldy, free of contamination rice or broken corn, account for 70～80%; 4～5 age silkworm or the pupa (dewax matter epidermis) of living, account for 20～30%.The two mixes.
3, select cultivating container.Selecting the glassware of 250ml or 500ml or 1000ml or thickness 0.05ml are above, specification is 150ml * 300ml for use, (or 170ml * 300ml), thin and thick are evenly, no high-quality polypropylene plastics pocket run-down, that toughness is strong is cultivating container.
4, select stain control agent.Be the loss of preventing and remedying pollution, can select for use to integrate nutrition, the special-purpose stain control agent of Chinese caterpillar fungus antifouling, that encourage effect, as the special-purpose specific anti-fouling agent of edible mushrooms.
(2) cultivating method
1, culture medium prescription: 1 kilogram of major ingredient, special-purpose stain control agent 35 grams of Chinese caterpillar fungus, 1.5 kilograms in water.
2, compound method: major ingredient restrains by every bag 50 or 100 grams divide the cultivating container of packing into.1.5 liters 50 ℃ hot water is poured in the clean basin, the special-purpose stain control agent of Chinese caterpillar fungus is put into hot water, stir to dissolving fully nutritive medium.Restrain by material-water ratio (being the amount ratio of major ingredient and nutritive medium) 1: 1.5 milliliters take by weighing nutritive medium and pour in the cultivating container that installs major ingredient sealing into.
3, sterilization: add suitable quantity of water in the Autoclave, the cultivating container that installs culture material is put into high-pressure sterilizing pot (or HL-380 automatically weighs vertical steam sterilizing pot, Hung Lin Medical Instruments Co., Ltd.'s production), be warming up to 100～125 ℃, kept 5～50 minutes, and took out, the cooling back moves into transfer room.With high-quality aerial fog disinfectant or the airtight sterilization of ordinary method 30 minutes, promptly wearable sterilization clothes entered the transfer room inoculation.
4, inoculation: inoculating tool, bacterial classification outer wall, operator's both hands etc., with 75% alcohol wipe or dipping sterilization.With the solid original seed is embodiment:
(1) cultivation is expanded in the commentaries on classics of test tube original seed: take out behind the test tube plug 2～3 of bacterial classifications that take the beans size with disinfectant inoculation hook, insert on the basal plane of cultivating cultivating container sealing.When (2) expanding cultivating bag with the original seed commentaries on classics, earlier bacterial classification is smash into finger or beans size bulk, every bag of access 5～10 gram bacterial classifications are evenly distributed on the charge level.Can suitably increase inoculum size as conditions permit, in order to the mycelia faster growing.Inoculated the back by former sealing method sealing, can move into cultivation indoor cultivation through sterilization and moth proofing.
5, cultivate: the mycelial growth temperature scope is 5～30 ℃, and the mycelia optimum growth temperature is 15～25 ℃, and the stroma optimum growth temperature is 18～22 ℃.Keep 15～18 ℃ of shadings to cultivate temperature at first.When mycelial growth during to substratum 1/2～2/3, temperature can be risen to 20 ℃, 20 days mycelia can send out cultivating container full.After several days,, give 10～15 ℃ thermal stimulation, and increase the light stimulation annesl when the hyphal peg time-out appears in substratum top.After changeing look in 7～10 days culturing room was sterilized 30 minutes, separate then and remove sealing, for the stroma growth provides the space.When stroma grows to 1 centimetre, in time to the cultivating bag ventilation, 18～22 ℃ of temperature maintenances, relative air humidity 80～90%.
6, gather: when stroma is the bar-shaped not regrowth of orange or safran, it is adopted down from base portion, put into clean utensil and in time dry or dry, be not exposed to the sun in order to avoid fade with pocket knife or scissors.Leave in the airtight plastics bag after the drying or sale.
Embodiment four:
Major ingredient all be 4～5 age silkworm or organic silkworm chrysalis Cordyceps sinensis cultivating method of living silkworm chrysalises.
(1) selects bacterial classification.Select that mycelia is pure white for use, strong stress resistance, go out the fast growing and high yield high-quality silkworm chrysalis Cordyceps sinensis bacterial classification that grass is fast, proterties is stable, select liquid spawn.
(2) major ingredient sterilization.To 4～5 age silkworm or living silkworm chrysalises carry out body surface sterilization, method with 75% alcohol: contain the cotton balls wiping 10～60 seconds of 75% alcohol or soaked 10～60 seconds.Or 10% amino acid iodine cotton balls wiping 10～60 seconds or soaked 10～60 seconds.And it is indoor to be placed on disinfection by ultraviolet light.Or add suitable quantity of water in the Autoclave, and the cultivating container that installs major ingredient is put into high-pressure sterilizing pot (or HL-380 automatically weighs vertical steam sterilizing pot, Hung Lin Medical Instruments Co., Ltd.'s production), be warming up to 100～125 ℃, kept 5～50 minutes.Take out, the cooling back moves into transfer room.With high-quality aerial fog disinfectant or the airtight sterilization of ordinary method 30 minutes, promptly wearable sterilization clothes entered the transfer room inoculation.
(3) connect bacterial classification.Make object of inoculation with silkworm, select 4-5 silkworm in age or plate cocoon to produce the pupa alives that the back produces, the sterilization back is with 108/milliliter spore suspension liquid spray silkworm body or with the inoculation of needle tubing wound, and inoculum size is sufficient.Or in inoculation tank, dip in the spore suspension liquid with the inoculating needle needle point and thrust above-mentioned silkworm, pupal cell joint, and inject bacterium liquid (0.2 milliliter /), infecting success ratio can be more than 95%.
(4) organic farming administrative skill.
1, the silkworm after the microbiological contamination, pupal cell are divided at the bottom of the bottle or on the flat implements, protect to pupal cell stiff under the room temperature.
2, nourish and grow: 15 ℃～18 ℃ in humidity 60%, greenhouse, natural lighting, 3～5d.Reproductive growth: the natural ecological environment of simulation Cordyceps militaris (L.) Link., cultivate stiff pupal cell and grow in seat.Method is to cover one deck porous material on stiff pupal cell, as broken sponge, thin cinder, gac etc., places under 10～30 ℃ of alternating temperatures, relative air humidity is that 85～95% natural lighting envrionment conditions is cultivated.Or the post-directed training of stroma--long hard for making the stroma that bears, can with stiff pupa head upwards insert in the material such as the coal briquette slag after sponge or the burning of porous thermal insulating, be placed under aforementioned humiture and the illumination condition and cultivate.
(5) collection of silkworm chrysalis Cordyceps sinensis is preserved
After the stroma maturation, dig out cleaning, carry out the body surface sterilization, be placed on again under 60 ℃ of environment and dried 1～5 hour, put shady and cool dry place sealing preservation with edible ethanol.
Claims (1)
1. the method for cultivation of a silkworm chrysalis Cordyceps sinensis is characterized in that, may further comprise the steps:
(1) screening of Cordyceps militaris (L.) Link. fungus bacterial strain
To originate in the wild fresh Cordyceps strain spore of Qinghai-Tibet Platean, the isolating method of application organizes is obtained the pure bacterial strain of Cordyceps sinensis; Utilize this bacterial strain to carry out rejuvenation, and then filter out the Cordyceps militaris (L.) Link. fungus bacterial strain that the silkworm chrysalis virulence by force, is easily formed spore by the silkworm chrysalis body of living;
(2) the organic substratum of silkworm chrysalis is made
Live body protein 10 0 gram, 200 milliliters in dense rice soup, vitaminB10 milligram/1000 grams, glucose 25 grams, potassium primary phosphate 2 grams with living silkworm chrysalises or the cured matter of live body silkworm removal epidermis, put into 1000 milliliters beaker, it is miscible to add 20～50 milliliters in water and heated and stirred, stop heating behind the complete miscibility, the packing test tube, jump a queue, cool off, promptly make the organic substratum of silkworm chrysalis of semi-liquid-like;
(3) making of spore suspension liquid
On the organic substratum of silkworm chrysalis that the Cordyceps militaris (L.) Link. fungus inoculation that (1) step was cultivated was made in (2) step, put under 20 ℃ of temperature and cultivate, after waiting conidium to occur, a picking part is placed on that to make the spore suspension liquid in the sterilized water stand-by;
(4) host prepares
Live body silkworm, pupa or traditional cocoon are gone cocoon shell live body pupa, silkworm, about 5～10 minutes or amino acid iodine sterilization about 1 minute, do the host with 75% alcohol disinfecting;
(5) infection of Cordyceps militaris (L.) Link. fungus
In inoculation tank, dip in the spore suspension liquid with the inoculating needle needle point and thrust above-mentioned silkworm, pupal cell joint, by 0.2～0.3 milliliter/only inject bacterium liquid;
(6) protection of infection pupal cell
Silkworm after the microbiological contamination, pupal cell are divided at the bottom of the bottle or on silkworm plaque or the bamboo mat, protect to pupal cell stiff under the room temperature;
(7) cultivation of stroma
The natural ecological environment of simulation Cordyceps militaris (L.) Link., cultivating stiff pupal cell grows in seat: method is to cover one deck porous material on stiff pupal cell, as broken sponge, thin cinder, gac, place under 10～30 ℃ of alternating temperatures, relative air humidity is that 85～95% natural lighting envrionment conditions is cultivated;
(8) post-directed training of stroma
For making the stroma that bears long hard, can adopt post-directed training: method is, with stiff pupa head upwards or pupal cell keep flat, insert or put into the material of porous thermal insulating such as the coal briquette slag after sponge or the burning, be placed on cultivation aforementioned temperature, humidity and illumination condition under;
(9) collection of silkworm chrysalis Cordyceps sinensis is preserved
After the stroma maturation, dig out cleaning, carry out the body surface sterilization, be placed on again under 60 ℃ of environment and dried 1～5 hour, put shady and cool dry place sealing preservation with edible ethanol.
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